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Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease ( S1P ) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and , hence , essential for infectivity and virus spread . Therefore , we tested in this study the concept of using S1P as a target to block efficient virus replication . We demonstrate that stable cell lines inducibly expressing S1P-adapted α1-antitrypsin variants inhibit the proteolytic maturation of GP-C . Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells . Moreover , S1P-specific α1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus . Inhibition of viral replication correlated with the ability of the different α1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor . Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies .
Lassa virus ( LASV ) belongs to the family Arenaviridae , which are enveloped , single-stranded RNA viruses distributed worldwide . Based on their antigenic relationships and geographic distribution , arenaviruses are divided into two major groups . The Old World group includes the prototype of this family , lymphocytic choriomeningitis virus ( LCMV ) , and LASV , which is endemic in West African countries and causes every year thousands of human infections with hemorrhagic fever as a severe clinical manifestation [1] . The New World group includes among others Machupo , Junin , Guanarito and Sabia viruses which can cause viral hemorrhagic fever ( VHF ) . With the exception of the New World virus Tacaribe , which was isolated from Artibeus bats , arenaviruses are rodent-borne viruses [2] . Over the past few years great efforts have been made to find potential therapeutic and vaccination approaches in the arenavirus field ( reviewed in [3] , [4] , [5] ) . Until now there is no specific and effective treatment available to combat hemorrhagic fevers caused by arenaviruses . Administration of convalescent plasma has been reported to reduce the mortality rates of patients with Argentine hemorrhagic fever , however , 10% of immune-plasma recipients developed a late neurological syndrome of unknown origin [6] . The only existing drug used to treat Lassa fever and certain South American hemorrhagic fevers is the broad-spectrum antiviral agent ribavirin , a ribonucleoside analogue , which has shown to be partially effective if given early in the course of illness [7] , [8] , [9] , [10] . Even though the drug is relatively inexpensive for patients in high-developed countries , it is still unaffordable for many of those living in West Africa and South America . Moreover , several adverse effects have been associated with ribavirin therapy in patient studies and animal models [11] , [12] , [13] , [14] , [15] . The lack of effective disease control measures as well as the discovery of new fatal arenavirus species that pose a risk of epidemic potential [16] , [17] , emphasize the need for novel therapeutic interventions . Lassa virions are pleomorphic lipid-enveloped particles that contain two single-stranded RNA segments , designated L ( large ) and S ( small ) , encoding four viral proteins in a unique ambisense coding strategy . The L segment encodes the viral RNA-dependent RNA polymerase ( L ) and the small zinc finger matrix protein ( Z ) [18]; the S segment encodes the virus nucleoprotein ( NP ) and the virus surface glycoprotein precursor ( preGP-C ) [19] . preGP-C is cleaved co-translationally into a stable signal peptide and GP-C [20] . Post-translational maturation cleavage of GP-C by the proprotein convertase site 1 protease ( S1P , [21] ) , also known as subtilisin kexin isozyme-1 ( SKI-1 , [22] ) , leads then to the generation of the distal receptor-binding subunit GP-1 and the transmembrane-spanning fusion competent subunit GP-2 [23] . Together with the signal peptide these subunits form the tripartite glycoprotein spike complex on the viral surface [24] , [25] . The glycoproteins of the Old World arenaviruses LASV and LCMV were the first viral glycoproteins that were shown to be proteolytically processed by S1P [23] , [26] , which normally plays important physiological regulatory roles in cholesterol metabolism , ER stress response , cartilage development and other cellular processes [21] , [27] , [28] , [29] , [30] , [31] . Using systematic mutational analysis of the LCMV GP cleavage site , the consensus motif R- ( R/K/H ) -L- ( A/L/S/T/F ) was determined , which is conserved in the glycoprotein sequences of the Old World viruses LASV , Mopeia and Mobala , as well as the New World virus Pichinde , suggesting that all arenavirus glycoproteins are cleaved by S1P [26] , [32] . Indeed , more recently Rojek et al . reported that glycoproteins from the New World hemorrhagic fever viruses Junin , Machupo and Guanarito are also processed by S1P , although Guanarito possesses a protease recognition motif that differs from known arenavirus GP consensus cleavage sequences , indicating a broader substrate specificity of S1P than previously anticipated [33] . Proteolytic activation of LASV GP-C by S1P is not necessary for transport of GP-C to the cell surface , where budding of arenaviruses occurs , but is essential for incorporation of the cleaved subunits into virions , and thus , for the formation of infectious viral particles . In the absence of GP-C cleavage , enveloped non-infectious LASV-like particles are released containing L , NP , Z protein and viral RNA but are devoid of viral glycoproteins [23] . Similar results were described for LCMV and New World hemorrhagic fever viruses [33] , [34] . In addition to its important role in the arenaviral life cycle , S1P is critical for the infectivity of Crimean-Congo hemorrhagic fever virus ( CCHFV ) , a member of the Bunyaviridae family , through processing of the glycoprotein Gn [35] , [36] . These findings make the inhibition of S1P particularly interesting for the development of a novel antiviral therapeutic that will target pathogenic viruses known to be processed by S1P . A successful approach to inhibit proprotein convertases involves genetically engineered antitrypsins , which are derived from α1-antitrypsin ( α1-AT ) . α1-AT is a serine protease inhibitor ( serpin ) with a characteristic exposed reactive center loop ( RCL ) , which mediates binding to the active site of its target protease . The exploration for the potential use of modified antitrypsins with an altered inhibitory spectrum has been guided by the discovery of a natural variant of α1-AT , known as Pittsburgh ( α1-AT-PIT ) , found in a patient who had a severe bleeding disorder caused by mutation of the P1 reactive center residue of antitrypsin from methionine to arginine [37] . This substitution changed its specificity from elastase to thrombin and other coagulation proteases . Due to the introduction of a second mutation from alanine to arginine at P4 of the RCL , the engineered α1-antitrypsin variant Portland ( α1-AT-PDX ) showed high affinity for furin [38] . α1-AT-PDX efficiently inhibited the formation of infectious HIV , measles virus , and human cytomegalovirus progeny by blocking furin-dependent processing of glycoproteins gp160 , F0 and gB , respectively [38] , [39] , [40] , [41] . Pullikotil and co-workers used this approach for the generation of highly selective α1-antitrypsin variants specific for S1P by introducing various S1P recognition motifs into the RCL of α1-antitrypsin [42] . The adaptation of α1-antitrypsin towards S1P efficiently inhibited the processing of the S1P substrates SREBP-2 ( sterol regulatory element binding protein ) , ATF6 ( activating transcription factor 6 ) as well as CCHFV glycoprotein [42] . However , the effect of these inhibitors on CCHFV infection was not analyzed in that study . To block cleavage of the LASV glycoprotein , we generated here recombinant α1-antitrypsin variants mimicking the S1P recognition motifs RRIL , RRVL and RRYL that exhibited the greatest inhibitory potential based on immunoblot quantification . In addition , we used an α1-AT construct that contains the LASV GP cleavage motif RRLL in its RCL . Using a doxycycline regulated expression system we demonstrate that S1P-adapted α1-antitrypsin variants efficiently block proteolytic maturation of the glycoprotein precursor GP-C , whereas a furin-specific α1-AT had no effect on GP-C processing . Virus replication of both a replication-competent recombinant vesicular stomatitis virus expressing the LASV glycoprotein GP-C ( VSVΔG/LASVGP ) and authentic LASV was significantly inhibited in the presence of S1P-specific α1-antitrypsins . The degree of inhibition of viral replication correlated with the ability of the different α1-antitrypsin variants to inhibit the processing of LASV GP-C . Since glycoprotein processing by the endoprotease S1P is not only critical for virus infectivity of LASV [23] , and other arenaviruses causing hemorrhagic fever [33] , but also for members of the Bunyaviridae family [36] , further optimization based on our findings could lead to a potent and specific S1P inhibitor with the potential treatment of certain VHFs .
cDNA of the open reading frame of rat α1-antitrypsin ( Gene Bank Accession Number NM_022519 ) ( a kind gift from Dr . G . Thomas , Vollum Institute , Oregon Health & Science University , Portland , USA ) was inserted into pSG5 and used as a template to generate S1P-specific α1-antitrypsin variants by recombinant polymerase chain reaction ( PCR ) using overlapping oligonucleotides [43] . The sequences of the oligonucleotides used are listed in Table S1 . The resulting full-length PCR products were digested with BamHI and NheI and cloned into the tetracycline ( Tet ) -controlled inducible mammalian expression vector pTRE2hyg ( Clontech ) . The accuracy of all constructs was confirmed by DNA sequencing . To generate stably expressing cell lines , Chinese hamster ovary ( CHO ) -K1 Tet-On cells ( Clontech ) were transfected with pTRE2hyg containing the α1-antitrypsin constructs using Lipofectamine 2000 ( Invitrogen ) according to manufacturer's instructions . Cells were then cultured for 2 weeks under selective pressure in the presence of 500 µg/ml Hygromycin B , the selection agent for the α1-antitrypsin expressing plasmid , and 500 µg/ml G418 , the selection agent for the rtTA ( reverse Tet-controlled transactivator ) cassette . The selective media were replaced every 3 days . Well-separated antibiotic-resistant cell clones were individually isolated with cloning cylinders ( Sigma ) . Therefore , a small volume of Trypsin-EDTA ( Sigma ) was added and the culture dish was incubated briefly at 37°C until cells detach . Cells were then collected from inside the cylinder and transferred to individual wells of a 24-well plate for further growth in selective medium . When grown to confluence , cells were transferred to larger flasks . Protein expression was induced with 1 µg/ml doxycycline ( Clontech ) and analyzed by Western Blot and immunofluorescence . Stable cell lines showing similar expression levels of the various α1-antitrypsins were chosen for further experiments . Vero E6 cells ( green monkey kidney ) were cultured in Dulbecco's modified Eagle medium ( DMEM , Gibco ) and CHO-K1 Tet-On cells in DMEM/F12 ( Gibco ) , both media containing penicillin ( 100 U/ml ) , streptomycin ( 100 µg/ml ) , and L-glutamine ( 2 mmol/l ) ( all from Invitrogen ) as well as 10% fetal bovine serum ( PAN Biotech ) . S1P-deficient SRD-12B cells ( a generous gift from Dr . J . L . Goldstein , Department of Molecular Genetics , University of Texas Southwestern Medical Center , Dallas , USA ) were maintained as CHO cells but supplemented with 5 µg/ml of cholesterol ( Sigma ) , 1 mM sodium mevalonate ( Sigma ) , and 20 µM sodium oleate ( Sigma ) [44] . The vesicular stomatitis virus reverse genetics system ( VSV , Indiana serotype ) was kindly provided by Dr . J . K . Rose ( Department of Pathology , Yale University School of Medicine , New Haven , USA ) and was described in detail earlier [45] , [46] , [47] . Recombinant VSV expressing the glycoprotein GP-C of Lassa virus ( LASV , strain Josiah ) designated as VSVΔG/LASVGP and wild-type VSV ( VSVwt ) were propagated in Vero E6 cells as described previously [48] . Influenza virus A/FPV/Rostock/34 ( H7N1 ) , designated as fowl plague virus ( FPV ) , was propagated in embryonated hen eggs and stored at −80°C until further use . Virus titration of FPV was described previously [49] . All experiments with infectious FPV were done under biological safety level 3 conditions . VSVΔG/LASVGP titration was performed using a microplate format plaque assay with subsequent immunostaining as described before [50] . In brief , virus dilutions were incubated on Vero E6 cells with an overlay of 3% carboxymethylcellulose ( CMC ) during plaque formation . Infected cells were visualized after cell fixation with paraformaldehyde ( PFA , 4% ) and permeabilization with 0 . 3% Triton-X 100 using a specific LASV GP-C/GP-2 antibody followed by incubation with horseradish peroxidase-labeled secondary anti-rabbit antibody ( DAKO ) . Finally , cells were stained with True Blue Peroxidase substrate ( KPL ) . For virus spread experiments , CHO cell lines were seeded into 96-well plates in the presence or absence of doxycycline . 24 h after induction , cells were infected with VSVΔG/LASVGP or FPV and were grown without solid overlay . Cells were fixed at different time points post-infection and immunostaining was performed as described above using rabbit sera against VSV ( kindly provided by Dr . G . Herrler , Institut für Virologie , Zentrum für Infektionsmedizin , Stiftung Tierärztliche Hochschule Hannover , Germany ) , for the detection of VSVΔG/LASVGP infected cells , and against FPV , for cells infected with FPV , respectively . Virus titration of LASV ( strain Josiah , Gene Bank Accession Number NC_004297 and NC_004296 ) was performed by defining the 50% tissue culture infectious dose ( TCID50 ) . For this , Vero cells were grown in 96-well plates to 30 to 40% confluence . Cells were inoculated with 10-fold serial dilutions of supernatants from LASV-infected CHO cell lines grown in the presence or absence of doxycycline . The assays were evaluated at 7 to 9 days post-infection . TCID50 values were calculated using the Spearman-Karber method [51] . All experiments involving LASV-infected samples were performed under biological safety level 4 conditions at the Philipps-University Marburg . At 24 h post-infection , cell culture supernatants from infected cells were cleared from cell debris and pelleted in an SW-60 rotor through a 20% sucrose cushion at 52000 rpm at 4°C for 2 h . The pellet was then resuspended in PBS buffer and mixed with SDS-PAGE sample buffer . To control the intracellular expression level , cell lysates were collected simultaneously . Samples were analyzed by SDS-PAGE and Western blotting using protein-specific antibodies as indicated . Proteins were separated by SDS-PAGE using 10% polyacrylamide gels . Immunoblotting was performed as described previously [52] . Antiserum against Lassa virus GP-C/GP-2 was also described previously [32] . Polyclonal rabbit anti-ß-tubulin antibody was purchased from Abcam ( UK ) , and monoclonal mouse anti-Flag antibody from Sigma-Aldrich . Secondary antibodies labeled with Alexa680 or IRDye800 were from Molecular Probes Invitrogen and Biomol , respectively , and were used for visualization and quantification of detected proteins using the Odyssey Infrared Imaging System ( LI-COR Biosciences ) . CHO cell lines were grown on coverslips and 24 h after doxycycline-induction , cells were washed with PBS and fixed with 4% PFA in DMEM for 30 min . The fixative was removed , and free aldehydes were quenched with 100 mM glycine in PBS . Then , samples were washed with PBS and permeabilized for 10 min with PBS containing 0 . 1% Triton X-100 . Cells were incubated in blocking solution ( 2% bovine serum albumin , 0 . 2% Tween 20 , 5% glycerol , and 0 . 05% sodium azide in PBS ) and subsequently stained with a primary mouse-anti-flag antibody ( 1∶400 ) and a secondary anti-mouse antibody coupled to rhodamine ( 1∶200 , Jackson Immunoresearch ) . Cell nuclei were stained with DAPI ( 4′ , 6′-diamidino-2-phenylindole , Sigma ) . Microscopic analysis was performed with a Zeiss ApoTome/Axiovert 200 M microscope using a magnification of 1∶40 .
Replication-competent recombinant vesicular stomatitis virus ( rVSV ) expressing foreign envelope glycoproteins has been demonstrated to be a suitable model system to study the role of viral glycoproteins in the context of virus replication [47] , [53] , [54] . In the present study , we took advantage of a rVSV expressing the LASV glycoprotein GP ( designated VSVΔG/LASVGP ) [48] . In this system biosynthesis and processing of GP was shown to be authentic compared to LASV [48] . In an initial experiment we wanted to determine whether CHO-K1 cells are susceptible to VSVΔG/LASVGP infection . The reason we chose CHO-K1 cells for our studies is the availability of a site 1 protease-deficient CHO cell line ( designated SRD-12B cells ) , in which GP maturation is abolished and only GP-deficient non-infectious LASV particles are released [23] . Thus , this cell clone provides an ideal control for inhibition studies . Vero E6 , CHO-K1 , and SRD-12B cells were infected with either VSVΔG/LASVGP or wild-type VSV ( VSVwt ) as a control . Aliquots of cell culture supernatants were collected at different times after infection and were analyzed by plaque assay . Growth kinetics revealed that VSVΔG/LASVGP grows to similar titers in CHO-K1 cells compared to Vero E6 cells which have been used in earlier studies ( Fig . 1A ) [48] . These data demonstrated that CHO-K1 cells support efficient VSVΔG/LASVGP replication , and thus are useful tools for further investigations . As expected , VSVΔG/LASVGP lacks efficient replication in SRD-12B cells , whereas virus growth of VSVwt remained unaffected in these cells ( Fig . 1A ) . The reason for the low but detectable virus titers in the supernatant of VSVΔG/LASVGP-infected SRD-12B cells is currently not known but has been also observed for LASV ( [23] and present study ) , LCMV [34] and New Word arenaviruses [33] . Glycoprotein activation by a yet unknown protease though with only very low efficiency might explain this phenomenon . To mimic the conditions of short-term treatment , we decided to use the inducible doxycycline-dependent Tet-On expression system , which allows regulated expression of the protein of interest [55] . To determine whether treatment of cells with doxycycline interferes with viral replication , we cultivated VSVΔG/LASVGP-infected CHO-K1 Tet-On cells in the presence or absence of doxycycline ( 1 µg/ml ) for 24 h and 48 h , respectively . As shown in Fig . 1B , CHO-K1 Tet-On cells treated with doxycycline produced a virus titer comparable to cells that were cultivated in the absence of doxycycline , indicating that these conditions used in our experiments have no influence on efficient virus replication . Pullikotil and colleagues recently reported that various antitrypsins mimicking S1P recognition motifs are able to block processing of the S1P substrates SREBP and ATF6 , although to different degrees [42] . In addition to the α1-AT variants shown to be most effective in that study we have chosen the LASV GP-C cleavage motif RRLL to investigate whether they also inhibit LASV GP-C cleavage . Therefore , we generated various S1P-specific α1-ATs , and as a specificity control , a furin-adapted α1-AT , by recombinant PCR technology using the rat α1-AT-PIT as a template ( Fig . 2A ) . To facilitate their detection , we introduced a flag epitope at the C-termini of the constructs . Stable cell lines were generated and individual clones were isolated and screened for α1-antitrypsin expression after doxycycline induction by immunoblotting and immunofluorescence analysis . Cell lines that showed similar expression levels of α1-antitrypsins were chosen for further experiments ( Fig . 2B and 2C ) . To test the inhibitory potential of S1P-specific α1-antitrypsins on proteolytic processing of LASV GP , stably transfected CHO-K1 Tet-On cells , and non-transfected wild-type CHO-K1 Tet-On cells as well as SRD-12B cells were infected with VSVΔG/LASVGP at an MOI of 0 . 2 in the presence or absence of doxycycline . To allow only one replication cycle , cell lysates were analyzed 10 h post-infection for detection of LASV GP cleavage by Western blot analysis using a GP-specific antiserum that recognizes both the precursor GP-C and the cleaved subunit GP-2 . In CHO-K1 Tet-On cells LASV GP was efficiently cleaved , regardless of whether doxycycline was present or not . In contrast , virtually no detectable cleavage of GP was observed in SRD-12B cells that are deficient in S1P ( Fig . 3A , lanes 1–4 ) . Without expression of the various antitrypsins efficient cleavage was detected in these stably transfected cell lines , similar to the processing of GP in wild-type CHO-K1 Tet-On cells ( Fig . 3A , lanes 1 , 5 , 7 , 9 , 11 , and 13 ) . In contrast , cells expressing the S1P-adapted α1-antitrypsins inhibited proteolytic maturation of LASV GP ( Fig . 3A , lanes 6 , 8 , 10 , and 12 ) . Furthermore , our results show that the presence of a furin-specific α1-AT did not influence LASV GP-C processing , demonstrating the specificity of the generated S1P-adapted α1-antitrypsins ( Fig . 3A , lanes 13 and 14 ) . Quantification of GP-2 cleavage revealed that the α1-AT variant RRIL exhibited the greatest inhibitory effect on GP processing ( >80% inhibition ) followed by α1-AT RRLL ( >60% inhibition ) , which possesses the amino acid cleavage motif of the LASV GP-C . Also α1-AT variants RRVL and RRYL were found to be inhibitory , although to a lesser extent ( inhibition less than 50% ) than the variants RRIL and RRLL ( Fig . 3B ) . Taken together , these data clearly demonstrate that S1P-specific α1-antitrypsins efficiently block the maturation cleavage of LASV GP , however , they differ in regard to their inhibitory potential . We have shown earlier that S1P-mediated cleavage of GP-C is absolutely required for incorporation of the glycoprotein subunits into the virion envelope and thus for production of infectious LASV [23] . Therefore , we addressed the question of whether a S1P-specific α1-AT has the potential to prevent GP incorporation by blocking glycoprotein processing . To this end , α1-AT RRIL cells were infected in the presence or absence of doxycycline with either VSVΔG/LASVGP or VSVwt as a control . At 24 h post-infection , viral particles released into the cell culture supernatant were purified over a 20% sucrose cushion and analyzed by means of immunoblotting . In viral particles from supernatants of non-induced α1-AT RRIL cells and CHO-K1 Tet-On control cells cleaved GP-2 was readily observed , whereas in the particulate material isolated from the supernatant of α1-AT RRIL expressing cells no glycoprotein was detected ( Fig . 4A ) . However , Western Blot analysis for VSV proteins revealed the release of these viral proteins into the supernatant of α1-AT RRIL expressing cells , which is consistent with our earlier findings that , in the absence of GP-C cleavage , enveloped non-infectious LASV-like particles containing the matrix protein Z and the ribonucleoprotein ( RNP ) complex , but devoid of viral glycoproteins , are still released [23] . The lower amount of VSV proteins observed in the cell lysate and supernatant of α1-AT RRIL expressing cells reflect lower levels of viral replication , which is due to less efficient virus spread ( Fig . 4A ) . In contrast to its ability to efficiently block incorporation of LASV GP into virions , the presence of α1-AT RRIL had no effect on the release of glycoprotein G containing wild-type VSV particles . The amount of VSV proteins detected in the supernatant from α1-AT RRIL expressing cells was similar to the amount of viral proteins observed in supernatants of non-induced cells and CHO-K1 cells , indicating efficient viral replication and cell-to-cell spread of VSVwt despite the presence of α1-AT RRIL ( Fig . 4B ) . Taken together , these data demonstrate that S1P-specific α1-antitrypsins have the potential to prevent LASV GP incorporation by inhibiting glycoprotein cleavage , which is an essential prerequisite for infectious progeny . Next , we wanted to know whether the observed inhibition of LASV GP processing correlates with the ability of the different α1-antitrypsin variants to inhibit virus spread . To investigate this , we established a 96-well plate assay in which infected cells are immunostained with True Blue substrate as described in Materials and Methods . Virus spread can be monitored by the appearance of characteristic comet-shaped foci , showing that the virus progeny is carried over the cell monolayer , while prevention of virus spread results in limited radial growth , due to infection of only neighbouring cells . This assay allows rapid screening of potential inhibitors [50] . To this end , doxycycline-induced and non-induced CHO cell lines , as well as CHO-K1 Tet-On cells and SRD-12B cells , were infected with VSVΔG/LASVGP . At 24 h post-infection , cells were fixed and immunostained . Under non-induced conditions efficient virus spread was observed in all CHO-K1 Tet-On α1-AT variant cell lines as well as in CHO-K1 Tet-On wild-type cells ( Fig . 5A , upper panel ) . In contrast , virus spread was significantly diminished in cells expressing α1-AT specific for S1P ( Fig . 5A , lower panel ) . These data indicate that S1P-adapted α1-antitrypsins have the potential to specifically inhibit the processing of LASV GP , which in turn is required for efficient virus spread . It should be noted that the infectious foci observed in α1-AT RRIL expressing cells were larger compared to SRD-12B cells in which virtually no virus spread of VSVΔG/LASVGP was observed , resulting in only single infected cells ( Fig . 5A ) . Although similar inhibition values were observed by means of immunoblot quantification ( Fig . 3 ) , a few remaining non-detectable cleavage events may count for this limited cell-to-cell spread in α1-AT RRIL expressing cells . Cells expressing the furin-adapted α1-AT variant RVKR did not prevent virus spread . At first glance , we rather observed an enhancement of infectivity compared to non-induced cells , which might be due to an increase in the LASV cellular receptor α-dystroglycan on the cell surface [56] . To further confirm the specificity of the α1-AT variants , we used fowl plague virus ( FPV ) , which contains a hemagglutinin with a multibasic cleavage motif recognized by furin [57] . Thus , the furin-adapted α1-AT should prevent virus spread of FPV , while virus spread in the presence of S1P-specific α1-antitrypsins should remain unaffected . Fig . 5B clearly demonstrates that the most potent S1P-specific α1-AT variant RRIL had no effect on FPV replication , and that virus spread was found to be similar to that observed in wild type CHO-K1 Tet-On cells . In contrast , in cells expressing the furin-adapted α1-AT variant RVKR virus spread of FPV was drastically reduced , whereas FPV replication occurred efficiently under doxycycline-free conditions in these cells . These results demonstrate that the generated α1-AT variants exhibit high specificity for the corresponding proteases , which are essential for virus spread in cell culture . To further elucidate the effect of the different α1-AT variants on multicycle replication , viral titers were determined . To this end , cells were infected with VSVΔG/LASVGP at an MOI of 0 . 02 in the presence or absence of doxycycline . Cell culture supernatants were collected 24 h and 48 h post-infection and virus titers were determined by plaque assay . As shown in Table 1 , non-induced S1P-specific α1-AT cell lines permitted unaffected growth of VSVΔG/LASVGP to comparable titers , whereas virus titers were reduced in cells expressing the S1P-specific α1-AT variant . At 24 h post-infection virus production decreased about 100 fold in cells expressing the α1-AT variant RRIL compared to non-induced control cultures . The presence of α1-AT variant RRLL reduced the virus titer in the supernatant about 10 fold , followed by a 6 . 2 fold reduction of virus production in α1-AT variant RRVL expressing cells . The presence of the α1-AT variant RRYL only exhibited a very moderate inhibitory effect on viral replication ( inhibition <2 fold ) . Again , the presence of the furin-adapted α1-AT variant RVKR did not affect VSVΔG/LASVGP replication compared to non-induced control cells . Our results indicate that the various S1P-adapted α1-antitrypsins exhibit different inhibitory potentials , due to their different recognition motifs . However , the degree of inhibition of virus replication correlated well with the inhibitory potential of the various S1P-adapted α1-antitrypsin variants to block LASV GP processing . Interestingly , following the inhibition of virus progeny over a time period of 48 h only the S1P-adapted α1-AT variants RRIL and RRLL sustained their inhibitory capacity , whereas in cells expressing α1-antitrypsin variants RRVL and RRYL virus production was found to recover although the initial expression levels of α1-antitrypsin variants were similar ( Table 1 ) . These data indicate that the inhibitory potential of the α1-AT variants RRVL and RRYL is not sufficient to efficiently suppress the formation of infectious particles by effectively blocking LASV GP-C cleavage , whereas the α1-AT variants RRIL and RRLL seem to be appropriate candidates for efficient inhibition of LASV propagation . Finally , we wanted to investigate the impact of blocking S1P-mediated GP processing on virus progeny of authentic LASV . Therefore , we assessed the inhibitory potential of the most potent variant , α1-AT RRIL , on the multiplication of LASV ( strain Josiah ) . For this purpose α1-AT RRIL cells and , as controls , CHO-K1-Tet-On and SRD-12B cells were infected with LASV at an MOI of 0 . 1 . To induce α1-AT expression , α1-AT RRIL cells and , as a control for off-target effects , CHO-K1 Tet-On cells were cultivated in the presence of doxycycline . To determine virus titers , infectious virions released into the cell culture supernatant were analyzed by defining the 50% tissue culture infectious dose ( TCID50 ) at various times post-infection , as indicated . In non-induced α1-AT RRIL cells , LASV revealed a growth kinetic similar to that observed in CHO-K1 Tet-On control cultures , while expression of α1-AT RRIL resulted in an average 2 log10 reduction in viral titer ( Fig . 6 ) . The difference between infectious LASV titers in the supernatant of α1-AT RRIL expressing cells and SRD-12B cells correlated with the limited virus spread observed in α1-AT RRIL expressing cells compared to single cell infections in S1P null cells ( Fig . 5 ) . Taken together , this result highlights the inhibitory activity of modified α1-antitrypsins against LASV and demonstrates that inhibition of endogenous S1P is a potent strategy to reduce the production of infectious LASV progeny .
Current drug treatment of Lassa fever and certain New World hemorrhagic fevers is limited to the guanosine analogue ribavirin [7] , [8] , [9] . Although ribavirin therapy can reduce the mortality rates of severe clinical cases , its unavailability to most patients in West Africa and South America as well as its association with severe adverse effects including anaemia [11] , [13] , teratogenicity and embryo lethality [12] , argues for the development of new alternative treatment options . In principle , every step in the viral life cycle is a potential target for antiviral inhibitors . While current antiviral strategies in the arenavirus field mainly target virus entry [58] , [59] , [60] , [61] or replication and assembly [62] , [63] , [64] , [65] , [66] , [67] , inhibition studies of the glycoprotein activating endoprotease and its impact on viral replication are largely unexploited . Due to its central role in the arenavirus life cycle [23] , [26] , [33] , [34] , S1P should be considered as a cellular target for antiviral drug development . In the present study we analyzed the inhibitory effect of S1P-adapted α1-antitrypsins on proteolytic processing of LASV GP-C and its consequences for viral replication . To our knowledge , this is the first report that addresses the impact of protein-based S1P inhibition on LASV GP-C cleavage and multicycle replication . Furin-adapted α1-ATs have been shown to efficiently inhibit the formation of infectious progeny of other viruses ( e . g . HIV , measles virus and human cytomegalovirus ) [38] , [39] , [40] , [41] , [68] , [69] . Using a replication-competent recombinant VSV pseudotyped with the LASV glycoprotein GP [48] , we demonstrate that proteolytic maturation of the precursor GP-C is sensitive to S1P-adapted α1-ATs . Mutagenesis of the reactive centre loop ( RCL ) into the S1P recognition motif RRIL resulted in an abrogation of GP-C processing similar to that observed in S1P-deficient SRD-12B cells . The inhibitory activity of the α1-AT variant RRIL on LASV GP cleavage described here is in agreement with a previous study showing its inhibitory potential on the processing of the natural S1P substrates SREBP-2 and ATF6 [42] . Also an α1-AT variant that contains the LASV GP-C cleavage motif RRLL exhibited a high S1P inhibitory potential and was found to drastically reduce GP processing . Interestingly , this variant exhibited a 100% inhibition activity on maturation cleavage of an artificial pro-PDGF ( precursor of platelet-derived growth factor ) mutant that is processed by S1P due to introduction of a RRLL cleavage site , but failed to inhibit cleavage of endogenously expressed SREBP-2 [42] . These data indicate that various substrates differ in their sensitivity towards S1P inhibition . The outcome of severe illness increased significantly with the level of viremia in Lassa fever patients [70] . Therefore , the extent of multicycle replication of LASV and thus , the load of infectious particles in its host organism have an important impact for the progress of disease . Our studies revealed that α1-AT variants RRIL and RRLL have a potency sufficient to sustain their inhibitory capacity during multicycle replication , which resulted in a significant reduction of virus infectivity . Inhibition of viral replication correlated with the ability of the α1-AT variants RRIL and RRLL to efficiently inhibit the processing of the LASV glycoprotein precursor . Although our data demonstrated that inhibition of glycoprotein cleavage by α1-AT RRIL reduced incorporation of the subunits GP-1 and GP-2 into virions to below detectable levels , the viral titer from α1-AT RRIL expressing cells was found to be greater than that obtained from S1P null cells . Based on this observation , we consider that even the most potent α1-AT variant RRIL failed to entirely inhibit S1P activity . However , given that S1P has important biological functions in the regulation of various cellular processes , a complete inhibition of the catalytic activity of S1P is not desirable . For α1-AT variants RRVL and RRYL , we observed similar inhibition values by immunoblot quantification analysis as described for CCHFV GP cleavage [42] . Though , their inhibitory activity on LASV GP-C cleavage was not sufficient to efficiently reduce virus replication of VSVΔG/LASVGP . These results should be taken into consideration for experimental setups in future studies that address the impact of S1P inhibition in arenavirus replication . The most potent α1-AT variant RRIL revealed a similar inhibitory potential on virus release of authentic LASV to that observed with the corresponding VSVΔG/LASVGP pseudotype . Therefore , this study also demonstrates that the replication-competent VSV expressing the LASV glycoprotein is an excellent surrogate model for analyzing potential antivirals that target the biological function of GP and its consequence for virus replication . These studies can be performed under biosafety level 2 laboratory conditions that would otherwise require biosafety level 4 laboratory conditions [71] . Taken together , our data indicate that S1P-adapted α1-antitrypsins may represent a promising lead compound for the development of a new class of anti-arenavirus inhibitors . In recent years improvements were made in the application of bioengineered serpins to combat bacterial and viral infections [39] , [72] . For example , the addition of exogenous α1-AT-PDX , a potent and selective furin inhibitor , was found to efficiently block human cytomegalovirus infection [39] . However , in contrast to furin , which is known to recycle between the plasma membrane and the TGN via endosomal compartments , membrane-bound S1P is localized in the secretory pathway and can be sorted to endosomal compartments but not to the cell surface [73] , [74] , [75] . Follow-up studies with small synthetic peptides , which are derived from S1P-specific α1-antitrypsins described in the present work , are currently in progress and will address cellular delivery and organelle specific targeting , as well as their inhibitory potential on authentic LASV replication . In analogy to inhibition strategies of the eukaryotic subtilase furin , we previously designed and developed a cell-permeable peptidyl chloromethylketone S1P inhibitor , which contained the LASV GP-C cleavage site , designated dec-RRLL-cmk [76] , [77] , [78] . This irreversible inhibitor efficiently blocked the processing of LASV GP at nanomolar concentrations , however , because of cell type-dependent toxicity observed by us and others , its potential in vitro use requires further investigation [79] , [80] . Due to the essential roles of S1P in cholesterol metabolism and fatty acid synthesis , this enzyme has attracted great attention by the pharmaceutical industry . Research efforts are currently directed towards the development of S1P inhibitors that may be used in the treatment of dyslipidemia and a variety of cardiometabolic risk factors associated with diabetes and obesity [81] . Identification of specific S1P inhibitors in this therapeutic area may also be beneficial in treatment of hemorrhagic fevers caused by viruses known to be processed by S1P . Future studies will have to elucidate the anti-viral efficacy of these and other novel S1P inhibitors that have been developed [82] , [83] . While most conventional antiviral drugs target proteins that are virus-encoded , cellular proteins essential for viral replication are currently considered to be alternative potential targets for antiviral therapy [84] , [85] , [86] . With the exception of Ebola virus , whose glycoprotein cleavage by the proprotein convertase furin is not essential for virus replication in cell culture and virulence in nonhuman primates [71] , [87] , [88] , [89] , maturation cleavage of surface glycoproteins of several virus species by endoproteases is a key determinant for host cell tropism and pathogenicity [90] . Thus , the emergence of viral escape mutants that confer resistance due to targeted inhibition of an endogenous protease is rather unlikely . In S1P-deficient SRD-12B cells , which were persistently infected with Junin virus vaccine strain Candid 1 , no virus escape variants possessing a cleavage motif other than a S1P recognition motif have evolved , indicating a low potential of arenaviruses to develop de novo a different glycoprotein maturation pathway [33] . This observation together with our findings that inhibition of S1P significantly affects LASV GP processing and virus infectivity should encourage the development of S1P inhibitors as a potential drug target to counteract infections caused by pathogenic arenaviruses . | The virus family Arenaviridae includes several hemorrhagic fever causing agents such as Lassa , Guanarito , Junin , Machupo , and Sabia virus that pose a major public health concern to the human population in West African and South American countries . Current treatment options to control fatal outcome of disease are limited to the ribonucleoside analogue ribavirin , although its use has some significant limitations . The lack of effective treatment alternatives emphasizes the need for novel antiviral therapeutics to counteract these life-threatening infections . Maturation cleavage of the viral envelope glycoprotein by the host cell proprotein convertase site 1 protease ( S1P ) is critical for infectious virion production of several pathogenic arenaviruses . This finding makes this protease an attractive target for the development of novel anti-arenaviral therapeutics . We demonstrate here that highly selective S1P-adapted α1-antitrypsins have the potential to efficiently inhibit glycoprotein processing , which resulted in reduced Lassa virus replication . Our findings suggest that S1P should be considered as an antiviral target and that further optimization of modified α1-antitrypsins could lead to potent and specific S1P inhibitors with the potential for treatment of certain viral hemorrhagic fevers . | [
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] | 2009 | Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants |
Calpains are Ca2+-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes . There are more than 10 well-conserved mammalian calpains , among which eutherian calpain-6 ( CAPN6 ) is unique in that it has amino acid substitutions at the active-site Cys residue ( to Lys in humans ) , strongly suggesting a loss of proteolytic activity . CAPN6 is expressed predominantly in embryonic muscles , placenta , and several cultured cell lines . We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells . The physiological functions of CAPN6 , however , are still unclear . Here , to elucidate CAPN6's in vivo roles , we generated Capn6-deficient mice , in which a lacZ expression cassette was integrated into the Capn6 gene . These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles , as well as in cartilage and the heart . Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle . In primary cultured skeletal muscle cells that were induced to differentiate into myotubes , Capn6 expression was detected in skeletal myocytes , and Capn6-deficient cultures showed increased differentiation . Furthermore , we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration . In this experimental system , Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point . These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration , suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation .
Calpains ( Clan CA-C2 , EC 3 . 4 . 22 . 17 ) constitute a family of intracellular Ca2+-regulated cysteine proteases found in almost all eukaryotes and a few bacteria [1] , [2] . They play indispensable roles in various biological processes , including cell migration , apoptosis , platelet aggregation , and myoblast fusion , through the limited proteolytic cleavage of diverse substrates [3]–[6] . The importance of calpains' physiological roles in mammals is revealed by the various phenotypes caused by calpain deficiencies , including embryonic lethality ( in Capn2- or Capns1-deficient mice ) [7]–[10] , muscular dystrophies ( human/mouse CAPN3/Capn3 ) [11]–[13] , gastropathy ( mouse Capn8 and Capn9 ) [14] , and vitreoretinopathy ( human CAPN5 ) [15] . In addition , improper calpain activation can exacerbate a disorder or even cause death [16]–[18] . However , most in vivo functions of the calpains remain unclear . Humans have 15 calpain genes that encode a calpain-like protease ( CysPc ) domain [19] , [20] and are divided into subfamilies according to their domain structures . Calpains with a domain structure similar to that of CAPN1[μCL] and CAPN2[mCL] are termed “classical” calpains . They contain C2L ( C2-domain-like ) and PEF ( penta-EF-hand ) domains in addition to the CysPc domain . The “non-classical” calpains contain only the C2L or the PEF domain , or neither , and are subclassified accordingly . Of the human calpains , nine are classical ( CAPN1-3 , 8 , 9 , 11-14 ) and six are non-classical ( CAPN5-7 , 10 , 15 , 16 ) . Calpains are also categorized according to their tissue/organ distribution . Some human calpains are ubiquitously expressed ( CAPN1 , 2 , 5 , 7 , 10 , 13-16 ) , whereas others are expressed only in specific tissues or organs ( e . g . , CAPN3 in skeletal muscle , CAPN8/9 in gastrointestinal tracts , and CAPN11 in testis ) . It is widely assumed that the ubiquitous calpains play a fundamental role in all cells , while the tissue-specific calpains have tissue-specific roles [21] . The Capn5 and Capn6 genes were identified as orthologs of tra-3 , a nematode gene for a sex-determination factor , TRA-3 [22] , [23] . The encoded two mammalian TRA-3 orthologs , CAPN5 ( also called hTRA-3 ) and CAPN6 , share more than 30% amino acid ( aa ) identity with TRA-3 . These calpains have a C2 ( not C2L ) domain at the C-terminus , i . e . , a “CysPc-C2L-C2” domain structure . CAPN5 has a Ca2+-dependent autolytic activity , is sensitive to several calpain inhibitors [24] , and is expressed at varying levels in almost all tissues [25] . It is expressed by a subset of T cells , but the analysis of Capn5−/− mice showed that it is not required for development [26] . On the other hand , eutherian CAPN6 has a naturally occurring aa substitution at the active site Cys residue ( Lys81 instead of Cys in human and mouse CAPN6 ) , probably indicating a lack of proteolytic activity [25] . In terms of molecular evolution , metatherian ( marsupial ) and avian CAPN6 retain the active-site triad residues Cys-His-Asn , and frogs and fish have three TRA-3 homologs with conserved active-site residues . Mammalian CAPN6 is predominantly expressed in embryonic muscles , placenta [27] , and several cultured cell lines [28] . Using cultured cells , we previously found that CAPN6 regulates microtubule dynamics and actin reorganization by modulating the activity of a small G-protein [28] , [29] . The function of CAPN6 in vivo , however , is still unclear . In this study , we examined CAPN6's physiological role by analyzing Capn6-deficient mice . To our surprise , the Capn6-deficient mice displayed precocious development of embryonic skeletal muscle . We also found that CAPN6 was expressed in regenerating skeletal muscle in adulthood , and that Capn6 disruption promoted regeneration in cardiotoxin-injected skeletal muscle . Our results showed that a loss of CAPN6 promotes skeletal muscle differentiation during mouse development and regeneration , and suggest a novel physiological function for CAPN6 in suppressing differentiation in skeletal muscle .
We disrupted the mouse Capn6 locus by replacing the protein-coding sequence in exon 2 with an nls-lacZ/PGKneo cassette ( Figure 1A ) , and obtained Capn6lacZ/+ heterozygous mice . The heterozygotes , which were all female , as Capn6 is on the X-chromosome , appeared normal and were fertile . Capn6-deficient ( Capn6lacZ/Y male or Capn6lacZ/lacZ female ) mice were then obtained by intercrossing Capn6lacZ/+ females with wild-type or Capn6lacZ/Y male mice , respectively . To confirm the absence of CAPN6 protein , whole embryos ( at embryonic day 10 . 5 [E10 . 5] , E12 . 5 , and E14 . 5 ) were chosen for western blot analysis , because Capn6 mRNA is reported to be expressed during embryogenesis [27] , [28] . As shown in Figure 1C , in wild-type embryo lysate , an anti-CAPN6 antibody detected the expected 74-kDa band , and this band was absent in lysates of Capn6lacZ/Y mice . Capn6lacZ/lacZ mice gave essentially the same results ( data not shown; see below and Figure 1E ) . These results indicated that the CAPN6 protein is present in wild-type mouse embryos , and that at least full-length CAPN6 was lost in the Capn6-deficient mice . The CAPN6 bands were most clearly detected in the lysates from the embryonic tongue and limb buds ( Figure 1D and E ) , which are rich in skeletal muscle . Notably , the signal intensities of the tongue CAPN6 were E14 . 5>E16 . 5>E18 . 5 ( Figure 1D ) . The CAPN6 signal in Capn6lacZ/+ ( ♀ ) embryos was weaker than that in Capn6+/Y ( ♂ ) embryos ( Figure 1E ) , even though both possessed one Capn6 gene . This was probably because the Capn6 expression was partly suppressed by X-chromosome inactivation in the female mice , as with many other X-linked genes [30] . In our constructs , the null allele , Capn6lacZ , expressed ß-galactosidase ( ß-Gal ) in the cells where Capn6 is transcribed in wild-type mice . To verify that the expression of lacZ reflected that of authentic Capn6 , the ß-Gal activity was examined in whole-mount E10 . 5 Capn6lacZ/+ embryos . As shown in Figures 2A and 2B , the mandibular arch , limb buds , somites , and heart were stained , consistent with the known Capn6 expression pattern [27] , [28] . At later stages ( E11 . 5 and E14 . 5 ) , ß-Gal was broadly expressed in the embryo ( Figure 2C and D ) . Cryosections of E16 . 5 Capn6lacZ/+ embryos showed ß-Gal expression in all the skeletal muscles and bone cartilage throughout the embryonic body ( Figure 2E ) . Capn6lacZ/lacZ ( Figures 2F , G , and S1A–C ) and Capn6lacZ/Y ( data not shown ) embryos , in which all the Capn6 locus-active cells expressed lacZ and no Capn6 , gave essentially identical results: ß-Gal expression was observed in all muscles , cartilages , and bones at the epiphysis , throughout the craniofacial area and trunk . No obviously abnormal morphology was observed in these regions . These Capn6-deficient mice were born healthily and grew to adulthood without any apparent abnormality . In adult mice , only the placenta showed ß-Gal expression ( Figure 2H ) . Given the high levels of Capn6 expression in embryonic skeletal muscles , we focused on the physiological relevance of CAPN6 in skeletal muscle development . First , the morphology of the E16 . 5 tongue was compared between Capn6lacZ/+ and Capn6lacZ/lacZ mice . As shown in Figures 3A–D , more advanced tube formation and striated structures were observed in the tongue muscle of Capn6lacZ/lacZ embryos than in their Capn6lacZ/+ littermates . In addition , the diameters of the tongue muscle fibers in the Capn6lacZ/lacZ mice were significantly larger than in their Capn6lacZ/+ littermates ( Figures 3E and S2 ) . To confirm these differences in skeletal muscle morphology , we examined the expression of desmin and α-sarcomeric actin , which are cytoskeletal components of skeletal muscle that play crucial roles in its development [31] , [32] . Western blot analysis of embryonic limb-bud lysates showed that the expressions of desmin and α-sarcomeric actin increased with development , and that they were stronger in Capn6lacZ/Y embryos than in their Capn6+/Y littermates at E16 . 5 and E18 . 5 ( Figure 3F ) . These results indicated that the deletion of CAPN6 advances the progression of skeletal muscle development during embryogenesis . To examine how the loss of CAPN6 promotes skeletal muscle development , we observed the differentiation of primary cultured skeletal muscle ( skm-primary ) cells from 7-week ( wk ) old Capn6+/Y and Capn6lacZ/Y littermates . The Capn6+/Y skm-primary cells were induced to differentiate into myotubes with horse-serum-containing medium . In these cultures , Capn6 mRNA was detected in the differentiating myocytes as well as in undifferentiated skm-primary cells ( Figure 4A ) . The CAPN6 protein was detected in the differentiating myocytes ( Figure 4B ) . Its expression was upregulated after the induction of differentiation , and then gradually decreased . Next , the differentiation of skm-primary cells was compared between Capn6+/Y and Capn6lacZ/Y littermates . DAPI staining demonstrated that the average number of nuclei in each cell derived from the Capn6lacZ/Y mice was greater than in their Capn6+/Y littermates 5–9 days after induction ( Figure 4C and D ) . These results indicate that , in myotube differentiation , the skm-primary cells from Capn6lacZ/Y mice underwent fusion more rapidly than did those from Capn6+/Y mice . In other words , the disruption of CAPN6 promotes the differentiation of myocytes to myotubes , suggesting that CAPN6 acts as a suppressor of skeletal muscle differentiation . The above experiments using skm-primary cells suggested that CAPN6 might be involved in skeletal muscle regeneration as well as its development . To examine skeletal muscle regeneration in adult mice , cardiotoxin was injected into the mouse tibialis anterior ( TA ) muscle . Cardiotoxin is a snake venom that selectively causes myofiber degeneration , but leaves nerves , blood vessels , and satellite cells morphologically intact [33] . Two days after the cardiotoxin injection , normal myofibers distinguished by marginal nuclei ( shown as in the controls in Figure 5C ) were diminished ( data not shown ) . Then , 4d after the injection , the expression of skeletal muscle embryonic and skeletal muscle perinatal myosin heavy chain genes ( Myh3 and Myh8 , respectively , in Figure 5A ) and the cells' central nucleation ( Figure 5C ) showed that the degenerated TA skeletal muscle had begun to regenerate . During this process , we examined Capn6 expression by RT-PCR , using PBS-injected TA muscle as a control . Capn6 mRNA was detected after 4d , similar to Myh3 and Myh8 , when muscle regeneration was taking place ( Figure 5A ) . The CAPN6 protein was also detected in the regenerating muscle by western blot analysis ( Figure 5B ) . Notably , histological analysis showed more nuclei in each myofiber in the regenerating skeletal muscles of the Capn6lacZ/lacZ mice than in the Capn6lacZ/+ mice ( Figure 5C and D ) . Furthermore , the cross-sectional area ( CSA ) of the regenerating skeletal muscles of the Capn6lacZ/lacZ mice was significantly larger than that of the Capn6lacZ/+ mice ( Figure 5C and E ) . Consistent with these observations , the expression of MYH8 was also higher at 7 days after cardiotoxin injection in the regenerating Capn6lacZ/Y muscles than the Capn6+/Y muscles ( Figure 5F ) . To our surprise , the expression of CAPN1 was reciprocally suppressed with significance ( P = 0 . 019; n = 3 ) . These results indicated that the loss of CAPN6 promotes both the regeneration of skeletal muscle and its development .
In this study , we have shown a physiological function for CAPN6 for the first time: CAPN6 is involved in the suppressive modulation of skeletal muscle development and regeneration . This is the second reported calpain gene ( after CAPN3/Capn3 ) to be genetically shown to be responsible for a skeletal muscle phenotype [12] , [13] , [34] . What is the physiological relevance of CAPN6's function as a skeletal muscle growth suppressor like myostatin and muscle RING-finger proteins ( MuRFs ) [35]–[37] ? One possible function of CAPN6 is to restrict the embryo size of eutherians , because an embryo that is too large could harm the mother's body ( known as “maternal-fetal conflict” [38] ) . This idea is compatible with the uniquely eutherian non-proteolytic character of CAPN6 . During embryogenesis , CAPN6 is expressed not only in skeletal muscles but also in cartilage , the mandibular arch , somites , and the myocardium from early ( E9 . 5 ) to late ( E18 . 5 ) stages . Therefore , it is likely that CAPN6 also functions as a growth suppressor in tissues other than skeletal muscles . The role of CAPN6 , however , must be accessory and/or stress-responsive , because the CAPN6 deficiency caused milder phenotype in the skeletal muscle systems under normal conditions than those of myostatin- or MuRFs-deficient mice [36] , [39] . What is the molecular mechanism underlying CAPN6's action ? We previously showed that CAPN6 modulates lamellipodial formation and cell motility by stabilizing microtubules and regulating Rac1 activity [28] , [29] . Microtubule organization and the activity of small G-proteins are also crucial regulators of skeletal myogenesis [40] , [41] . Thus , our collective results suggest that CAPN6 suppresses skeletal muscle differentiation and growth by modulating basal cellular functions such as cell division , growth , and cell migration through cytoskeletal reorganization . Molecules and a mechanism involved in these phenomena , however , may be different between skeletal muscles and cultured fibroblasts . It has been discussed that CAPN6 may antagonize other calpains to protect their substrates from cleavage [23] , [27] , as in the case of cFLIP , which is a protease-deficient caspase homolog that functions as an apoptosis inhibitor [42] . These points should be clarified in detail in future studies . The changes in the level of another muscle-differentiation-related calpain , CAPN1 , during TA muscle degeneration and regeneration may provide another hint ( Figure 5F ) . It was reported that overexpressed CAPN1 suppresses muscle cell differentiation during myogenesis [43] . During the cardiotoxin-induced muscle degeneration , CAPN1's expression decreased , and it recovered during the regeneration process ( see Figure 5F ) , but the absence of CAPN6 delayed the recovery of CAPN1 expression in the regenerating skeletal muscle ( compare CAPN1 in Figure 5F lanes 6 and 8 ) . Capn1 mRNA expression was not changed during this process ( Figure 5A ) , suggesting the involvement of a proteolytic mechanism in this alteration of CAPN1 expression . This CAPN1 change was not observed in primary cultured cell differentiation ( Figure 4 , data not shown ) , suggesting an unknown mechanism specific to in vivo . At present , however , it cannot be eliminated that the observed CAPN1 change is a result of different muscle conditions caused by Capn6 disruption . A relationship between CAPN1 and CAPN6 is another interesting subject to be addressed . In addition , Capn6 is reported to be overexpressed in the skeletal muscle of patients with limb-girdle muscular dystrophy type 2A ( LGMD2A ) , which is caused by pathological mutations of CAPN3 [44] . How the physiological role of CAPN6 in skeletal muscle development is related to the pathological condition of muscular dystrophy , where skeletal muscles are in a continuous cycle of degeneration and regeneration , is an intriguing question . In this respect , CAPN6 could be a therapeutic target for muscular dystrophies , since its downregulation is expected to enhance the de novo formation of functional skeletal muscles . In terms of tissue engineering , repressing CAPN6 could be helpful for generating skeletal muscle from embryonic or induced pluripotent stem cells in vitro . Despite the predominant expression of CAPN6 during embryogenesis , however , Capn6-deficient mice were born and grew healthily , and showed slightly but significantly higher skeletal muscle and body weight ( see Figure S3 ) . It is possible that Capn6 deficiency did not cause severe phenotypes such as lethality or dysgenesis because of a redundancy of CAPN6's non-proteolytic function among calpains . Indeed , recent genetic studies have suggested that calpain family members have non-proteolytic roles in addition to their proteolytic functions [14] , [34] , [45] . These non-proteolytic functions are ascribed to the CysPc domain , the other domains , or both , and may include the structural regulation of other molecules and/or a chaperone function . Some calpains of schistosomes , insects , and nematodes , and all of the calpains of Trypanosoma are non-proteolytic members with substitutions in one or more of the well-conserved active-site triad residues , as seen in eutherian CAPN6 [46]–[49] . A recent bioinformatics analysis revealed that non-classical calpains are considered as the majority among all eukaryotes , and that classical calpains are relatively new in the evolution of calpains , being confined to a small subset of eukaryotes [49] . As mentioned , non-classical calpains contain various non-proteolytic calpains . These findings highlight importance of the calpains' non-proteolytic functions , which are considered to be rather recent functional modifications evolved during calpains' diversification . Although calpains' proteolytic activities are important for understanding many of their physiological functions , recent reports on the non-proteolytic functions of calpains , including this one , shed light on their more elusive physiological roles , and hence may lead to important breakthroughs in biological research and medicine .
All procedures using experimental animals were approved by the Experimental Animal Care and Use Committee of Tokyo Metropolitan Institute of Medical Science , and the animals were treated according to the committee's guidelines . C57BL/6 and ICR ( a strain established in The Institute of Cancer Research in London , United Kingdom ) mice were purchased from Nihon CLEA Inc . For embryos , mice were mated in the evening and if a vaginal plug was observed the next morning , fertilization was assumed to have taken place at midnight . Pregnant mice were sacrificed by cervical dislocation , and the embryos or fetuses were dissected from the uterine decidua . Genomic clones for mouse Capn6 were obtained by screening the C57BL6/J-derived BAC libraries from the BACPAC Resource Center . The nls-lacZ cassette was made using the lacZ gene with a nuclear localization signal ( nls-lacZ ) and flanked with lox71 at the 5′-end and lox2272 at the 3′-end , to allow recombinase-mediated cassette exchange . A pKO Scrambler NTKV-1904 plasmid ( Stratagene ) was used as the backbone vector . For the targeting construct , a PCR-amplified 1 . 1-kb fragment and a 5 . 8-kb NotI-SalI fragment from intron 1 were placed on each side of the nls-lacZ cassette . The targeting vector was linearized using NotI and electroporated into the F1 hybrid ES cell line G4 [50] . Clones surviving positive-negative selection with neomycin and FIAU were screened for homologous recombination with diagnostic PCR primers ( see Table S1 ) . Of 292 ES cell clones screened , five correctly targeted clones were identified by PCR and confirmed by Southern blot analysis . Three independent ES cell clones hemizygous for the disrupted allele were injected into ICR blastocysts to generate germline chimeras . Female mice homozygous for the Capn6lacZ allele were obtained by intercrossing F1 hemizygous males ( Capn6lacZ/Y ) and heterozygous females ( Capn6lacZ/+ ) . The genotypes of offspring were determined by PCR or Southern blot analysis of tail-tip DNA ( Figure 1B ) . After backcrossing progeny with C57BL6/J mice more than 10 times , the mice were maintained on the B6 genetic background . Polyclonal antibodies against the C- and N-terminal domains of mouse CAPN6 were generated at Transgenic Inc , as described previously [28] . The anti-CAPN6 antibody ( ab38940 ) was purchased from Abcam . An anti-desmin monoclonal antibody ( clone DE-U-10 ) , anti-α-sarcomeric actin antibody ( clone 5C5 ) , and anti-β-actin monoclonal antibody ( clone AC-15 ) were purchased from Sigma . An anti-myosin heavy chain 3 ( Myosin-embryonic clone F1 . 652 ) and an anti-myosin heavy chain 8 ( Myosin-neonatal clone N1 . 551 ) were purchased from Santa Cruz . The anti-CAPN1 monoclonal antibody was a kind gift from Dr . Jiro Takano . Protease inhibitors ( E64c , AEBSF , MG-132 , PMSF , Calpeptin , and pepstatin A ) and chemical reagents were purchased from TaKaRa , Peptide Institute , Sigma , and Kanto Chemical . To prepare protein lysates , tissues , and cultured cells were homogenized with TED buffer ( 10 mM Tris/Cl [pH 7 . 5] , 1 mM EDTA , 1 mM DTT ) containing 2% SDS and protease inhibitors , as described above . The protein concentration was determined using a DCA protein assay kit ( BioRad ) , and samples containing equal amounts of protein were separated by 7 . 5 or 10% SDS-polyacrylamide gel electrophoresis ( PAGE ) and then electrotransferred to a polyvinylidene difluoride ( PVDF ) membrane . After being blocked with 5% skim milk in 0 . 1% Tween 20 in Tris-buffered saline , pH 7 . 6 , the blot was probed with primary antibody . The membrane was then washed with 0 . 1% Tween 20 in Tris-buffered saline , pH 7 . 6 , and incubated with peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G ( Dako ) . The signals were detected using the ECL chemiluminescence detection system ( Amersham Bioscience ) . LacZ expression in Capn6 mutant mice was detected by staining with X-gal ( 5-bromo-4-chloro-3-indoyl-β-D-galactoside ) . Whole embryos were isolated in ice-cold KPP buffer ( 0 . 1 M potassium phosphate ) , then fixed with 4% PFA/0 . 1% glutaraldehyde in KPP buffer , containing 5 mM EGTA and 2 mM MgCl2 . The embryos were then rinsed 3 times with wash buffer ( 0 . 01% Na deoxycholate and 0 . 02% Nonidet P-40 in KPP buffer containing 5 mM EGTA and 2 mM MgCl2 ) . Samples for histological analysis were embedded in OTC compound and cryosectioned . The embryos or sections were incubated overnight at 37°C in lacZ-staining buffer ( 10 mM potassium ferrocyanide , 10 mM potassium ferricyanide , and 2 mg/ml X-gal in wash buffer ) . Mouse skeletal muscle primary cells were prepared as previously described [51] , and maintained in F-10 medium containing 10% FBS ( fetal bovine serum ) and 5 µg/ml FGF-8 . To induce muscle differentiation , the medium was switched to differentiation medium ( 5% horse serum [Invitrogen] and 0 . 2 M ascorbic acid in Dulbecco's modified Eagle's medium ) for further culture . All media were supplemented with 100 units/ml penicillin , 0 . 1 mg/ml streptomycin , and 2 mM L-glutamine ( Invitrogen ) . To quantify the average number of nuclei per cell , nuclei and cells were labeled by DAPI and rhodamine-phalloidin , respectively , and 100 cells were randomly selected in the fixed area . 0 . 1 ml of 10 M cardiotoxin ( Wako Pure Chemical Industries ) in 0 . 9% saline was injected directly into the left TA muscle with a 27-gauge needle under ether anesthesia . Mice were killed by cervical dislocation , and the cardiotoxin-injected TA muscles ( left ) and PBS-injected contralateral TA muscles ( right ) were removed for analysis . Several of the muscles were frozen in isopentane cooled by liquid nitrogen for histological analysis , and the other muscles were frozen directly in liquid nitrogen for RNA and Protein extraction , and stored at −80°C . Total RNA was extracted using TRIzol reagent ( Invitrogen ) . The reverse transcription reaction was carried out using the First Strand cDNA Synthesis kit ( Amersham Bioscience ) according to the manufacturer's instructions . The resultant cDNAs were amplified with ExTaq polymerase ( Takara ) in a thermocycler . The amplified mouse Capn1 and Gapdh were used as internal controls . For H-E staining , sections were stained with Mayer's hematoxylin , followed by eosin staining . H-E-stained sections were mounted with Mount-Quick ( Daido Sangyo ) , and viewed with a BX60 microscope ( Olympus ) . The CSA of H-E-stained myofiber sections was determined with NIS-Elements Ar 3 . 0 software ( Nikon ) . Cells were washed with general tubulin buffer ( GTB ) ( 80 mM PIPES [piperazine-N , N′-bis ( 2-ethanesulfonic acid ) ] , pH 7 , 1 mM MgCl2 , 1 mM EGTA ) at room temperature , fixed with 4% paraformaldehyde in GTB , permeabilized with 0 . 2% Triton X-100 in GTB , and washed with GTB at room temperature . Actin and nuclei were stained with 2 . 5 units/ml rhodamine-phalloidin and DAPI ( Life Technologies ) , respectively . The cells were viewed using a LSM510 META laser-scanning confocal microscope ( Carl Zeiss ) . Student's t-test was used to test whether or not the means of two normally distributed populations were equal . P values less than 0 . 05 were considered statistically significant . | The calpains arose evolutionarily as a family of Ca2+-regulated intracellular proteases with divergent structures and functions . Unique among mammalian calpains , calpain-6 is “non-proteolytic” , and its physiological role has remained unknown . In this study , using Capn6 knock-out mice , we discovered that Calpain-6 is involved in skeletal muscle development and regeneration in vivo . Calpain-6 was predominantly expressed in embryonic muscles and bone cartilage . Capn6 knock-out promoted skeletal-muscle development as a result of increased progenitor cell differentiation . Furthermore , calpain-6 was detected in regenerating skeletal muscles , and suppressed this process . These results indicate that mammalian calpain-6 is a suppressive modulator for skeletal muscle differentiation and growth . Our findings indicate that calpain-6 might serve as a therapeutic target for muscular dystrophy/atrophy or as a useful tool in tissue engineering . For calpain studies , the physiological non-proteolytic role of calpain-6 may shed light on the elusive calpain functions that are independent of their well-studied proteolytic activities . In addition , as this structurally non-proteolytic feature is unique to the calpain-6 of placental mammals among vertebrates , our mutant mice might provide insight into the relationship between molecular and biological evolution . | [
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] | 2013 | Calpain-6 Deficiency Promotes Skeletal Muscle Development and Regeneration |
Periplasmic flagella are essential for the distinct morphology and motility of spirochetes . A flagella-specific type III secretion system ( fT3SS ) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella . Here , we deployed cryo-electron tomography ( cryo-ET ) to visualize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi . We show , for the first time , that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the “spoke and hub” structure in B . burgdorferi . This structure not only strengthens structural rigidity of the round-shaped C-ring but also appears to rotate with the C-ring . Our studies provide structural insights into the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the basis for the development of novel therapeutic strategies against several pathogenic spirochetes .
A group of bacteria named spirochetes can cause serious human diseases such as Lyme disease ( Borrelia or Borreliella species ) , syphilis ( Treponema pallidum subsp . pallidum ) , and leptospirosis ( Leptospira interrogans and other Leptospira species ) . Spirochetes are easily recognized by their distinctive wave-like or helical morphology and unique modes of motility . Recent genetic studies indicate that their motility is crucial for host infection and/or bacterial transmission [1–6] . Spirochetal motility is driven by periplasmic flagella , which reside and rotate between the outer membrane and the peptidoglycan layer . Mutant B . burgdorferi cells that lack their periplasmic flagellar filaments are nonmotile and rod shaped [1 , 2 , 7–9] . Similar to the flagella in the model organisms Escherichia coli and Salmonella enterica , periplasmic flagella are composed of the flagellar motor , the hook , and the filament . However , the periplasmic flagella are noticeably different from other bacterial flagella in several aspects . The spirochetal flagellar motor is significantly larger than those in E . coli and S . enterica ( approximately 80 nm versus approximately 45 nm in diameter ) . A periplasmic “collar” contributes significantly to the motor structures observed in B . burgdorferi [10 , 11] and all other spirochetes characterized to date [12–15] . The large flagellar motor from B . burgdorferi appears to produce the highest torque ( approximately 4 , 000 pN nm ) observed in bacteria [16] . Furthermore , spirochetes have unusual flagellar hooks in which the hook proteins are cross-linked by a covalent bond , which is required to transmit the torque from the motor to the filament [17] . Those spirochete-specific features enable the spirochetes to bore through viscous environments in their animal hosts . The filament is the largest component of the periplasmic flagella . Multiple filaments arising from both poles form flat ribbons that wrap around the spirochete cell body in a right-handed fashion [7] . The flagella filament is assembled by the flagellar-specific type III secretion system ( fT3SS ) , which is conserved across different bacterial species [14 , 18] . Additionally , the fT3SS is evolutionally related to the virulence T3SSs ( vT3SSs ) that promote bacterial virulence by delivering effector proteins into eukaryotic cells [19 , 20] . The fT3SS is powered by proton motive force [21–23] or sodium motive force [24] , with additional involvement of ATP hydrolysis [25–27] . The fT3SS consists of a membrane-bound export gate complex made up of six membrane proteins ( FlhA , FlhB , FliO , FliP , FliQ , and FliR ) and a large cytosolic ATPase complex formed by three cytoplasmic proteins ( FliH , FliI , and FliJ ) . The ATP complex promotes the export process by binding and delivering substrates to the export apparatus [28 , 29] . FliI is an ATPase and shows structural similarity with the α and β subunits of the FOF1–ATP synthase [30]; it exhibits its full ATPase activity when it self-assembles into a homohexamer [27 , 31] . FliH probably acts as a negative regulator of the FliI ATPase , and FliJ has chaperone-like activities [28 , 32] . FliH , FliI , and FliJ coordinately deliver a chaperone–substrate complex to the export gate by binding to the docking platform of the fT3SS for substrate export [33] . FliH2 binds to the FliI ATPase and localizes FliI to the bottom of the flagellar motor through the interaction with FliN on the C-ring [34 , 35] . FlhA is required for stable anchoring of the FliI6 ring to the gate [36] . FliP , FliQ , and FliR form an export gate complex with helical symmetry [37] . Cryo-electron tomography ( cryo-ET ) studies have revealed the overall structures of the fT3SS machines in intact flagella [11–15 , 38–40] . However , those studies have not yet provided sufficient details on stoichiometry or architecture to fully understand the components of the ATPase complex and its interactions with other proteins of the flagellar motor . B . burgdorferi is the best-studied spirochete model system . Recent breakthroughs in genetic manipulations allow the production of well-defined mutations without imposing any secondary alterations [2 , 10 , 39 , 41] . The small cell diameter and the highly ordered array of multiple flagellar motors at cell poles make B . burgdorferi an excellent system for in situ structural analysis of the periplasmic flagella and their fT3SS machines by cryo-ET . Our previous structural analysis of wild-type ( WT ) cells and several rod mutants of B . burgdorferi revealed the sequential assembly of the flagellar rod , hook , and filament [39] . Furthermore , disruption of the fliH and fliI genes by transposon mutagenesis was found to disrupt the assembly and placement of the cytoplasmic ATPase complex and to greatly inhibit flagellar filament formation , which were largely restored by genetic complementation [42] . In this study , we used cryo-ET and subtomogram averaging to reveal novel features of the ATPase complex in the WT B . burgdorferi periplasmic flagellar motor . The ATPase complex is attached to the C-ring by spokes , and without the spokes , the C-ring became more flexible and elliptical in shape . Furthermore , we resolved the symmetry mismatching between the stators and spokes in class averages , showing that the ATPase complex and C-ring rotate as a rigid body with respect to the stators and collar . Comparing these results with recent studies of the T3SSs in external flagella and evolutionarily related injectisomes provides new insights into these nanomachines that are structurally and functionally different while sharing a common evolutionary origin [40 , 43 , 44] .
We utilized high-throughput cryo-ET and sophisticated subtomogram classification [45] to study the structure of B . burgdorferi flagellar motors . By analyzing 7 , 242 intact motor structures extracted from tomographic data collected on a direct detection device ( DDD ) , we generated an asymmetric reconstruction that not only revealed the previously observed 16-fold symmetry of the collar and stator structures [10 , 11 , 39] but also disclosed a novel spoke-like structure underneath the C- and MS-rings ( S1 Fig and S1 Movie ) . There are 23 spokes in most WT B . burgdorferi flagellar motors , albeit this number is varied from 21 to 24 in some rare instances ( see S2 Fig ) . The spoke-like densities extend from a hexagonal “hub” to the bottom of the C-ring ( S1 Fig and S1 Movie ) . We selected the class averages exhibiting 23 spokes , combined them , and did further image alignment with focus on the region of the “hub” and the C-ring ( Fig 1A , 1B and 1C ) . The spokes extend from the central hub to the C-ring with 46-fold symmetry ( Fig 1B ) . FliI , FliH , and FliJ are known to form a large ATPase complex that was previously proposed to be centered on the FliI6 hexamer [25–27] and to correspond to the density underlying the FlhA ring based on analysis in Campylobacter jejuni [14] and in B . burgdorferi [42] . We speculate that the FliI6–FliJ complex forms the hexagonal hub and FliH is responsible for the spoke ( Fig 1A , 1B and 1C ) . Indeed , our structures derived from fliH and fliI mutants [42] show that both the hub and the spokes are absent ( Fig 1G ) , confirming that the distinct “hub and spoke” structure is dependent upon the presence of both FliI and FliH . Our data showed that the ATPase complex is directly connected to the C-ring in the WT flagellar motor . The C-ring from the WT motors mostly maintained the round shape , with the aspect ratio ranging from 1 . 005 to 1 . 048 ( S3A Fig ) . In contrast , the C-ring density in the fliI mutant is often more elliptical , with the aspect ratio ranging from 1 . 075 to 1 . 206 ( S3B Fig ) . Among the fliI mutant motors , 49 . 9% have elliptical C-rings with aspect ratios ≥1 . 129 , and 19 . 3% have more ellipse-shaped C-rings with aspect ratios ≥1 . 206 . Therefore , we propose that the FliI–FliH complex plays an essential role in stabilizing the round-shaped C-ring structure . Without the support from the ATPase complex , the C-ring would lose the rigidity and become more flexible . To better understand the interactions between the ATPase complex and the C-ring in the intact B . burgdorferi flagellar motor , we constructed a model of the ATPase complex and its surrounding C-ring complex based on the available homologous structures . The crystal structures of FliI and FliJ from Salmonella [30 , 46] fit well into the central hub ( Fig 2D and S4 Fig ) . The N- and C-termini of FliJ insert into the middle of six FliI subunits , while the middle part of FliJ inserts into the middle of the nonameric FlhAC ring ( Fig 2D and S4 Fig ) . A FliH dimer ( FliH2 ) is known to form a stable complex with the FliI ATPase [46 , 47] . The C-terminal domain of FliH is involved in binding to FliI , while a small central region of FliH is essential for formation of the FliH2 [48] . The N-terminal domain is important for FliH–FliN interactions [34 , 35] . In our map , there are three or four spokes extending from each FliI monomer to the C-ring , although only one FliH2 binding site on each FliI was reported in a crystal structure of the FliI–FliH complex [46] . Therefore , we speculate that the first FliH2 dimer directly binds to one FliI monomer , while others bind to adjacent FliH2 dimers in a parallel fashion ( S5 Fig ) ; since FliI forms a monomeric hexamer , six bundles of FliH2 are attached to the central FliI hexamer in our model ( Fig 2E and 2F , S5 Fig ) . The hydrophobic patch ( L85 , T110 , V128 , V130 , F135 ) at the C-terminus of FliN has been reported to interact with FliH [34] . Our data indicate that the FliH2 spoke is indeed attached to FliN at bottom of the C-ring ( Fig 2E ) , in which the atomic models of FliG , FliM , and FliN [49] were fitted into the C-ring density . With an additional rotation of approximately 10° from the initial model [49] , the hydrophobic residues of FliN ( labeled red in Fig 2D and S2 Movie ) are located at the interface between FliN and the FliH spoke . The C-ring is thought to rotate together with the MS-ring and the flagellar filament , although the rotation of the C-ring has never been directly visualized . Here , because FliH spokes connected to the C-ring are visible , they can be utilized to track the rotation of the C-ring . Indeed , classification of the spoke region resulted in multiple structures , in which the ATPase complex apparently adopts different spin rotation with respect to the collar and the stator ( see Fig 3 and S3 Movie ) . In the four classes shown in Fig 3 , the cross-section view on collar and stator shows that those from four classes are in a similar orientation ( Fig 3A ) ; however , the cross-section view on the ATPase complex shows the spokes in classes 03 , 05 , and 08 rotate about 7° , 13° , and 20° from class 00 ( Fig 3B ) , respectively . As the small angular change is difficult to discern , the class averages were analyzed by rotational cross correlation , and the coefficient was plotted ( Fig 3D ) , showing the angular differences ( 7° , 13° , 20° ) between class 00 and class 03 , 05 , and 08 . As the spokes are attached to the C-ring , we propose that the C-ring and the ATPase can rotate together as a rigid body ( see also in S3 Movie ) . Although the class average structures in Fig 3 were arranged as counter-clockwise ( CCW ) from left to right , the rotation can be either CCW or clockwise ( CW ) .
T3SSs in bacterial flagella and injectisomes are highly conserved and evolutionally related . The flagella are elaborate self-assembling machines that serve as the main organelles for bacterial motility . The injectisomes are specialized nanomachines deployed by many important human pathogens such as Salmonella spp . , Shigella spp . , and Pseudomonas to deliver virulence effectors into eukaryotic cells . Our previous studies revealed key intermediates of fT3SS-mediated assembly in B . burgdorferi [39] and overall architectures of the vT3SS machines in Shigella and Salmonella [43 , 44] . Here , we focus on in situ structure of the fT3SS machine in periplasmic flagella and compare it with external flagella and vT3SS machines . The overall organization of the fT3SS machine in the B . burgdorferi periplasmic flagella shares many similar features observed with the fT3SS machine in the E . coli external flagella [50] and the vT3SS machines in Shigella and Salmonella [40 , 43 , 44] ( Fig 4 ) . However , the ATPase complex of the B . burgdorferi periplasmic flagella is noticeably different from those observed in the Salmonella injectisome ( Fig 4 ) and the E . coli/Salmonella external flagellum [40 , 50] . We observed 23 spokes and one hub in the ATPase complex of the B . burgdorferi periplasmic flagella . In contrast , no spoke has been observed in E . coli/Salmonella external flagellum . Only six spokes and one hub were observed in the Salmonella injectisome . The spokes are considerably longer in the B . burgdorferi flagellar basal body than in the Salmonella injectisome ( 6 nm versus 3 nm ) , consistent with the observation that the C-ring is much larger than the six “pods” ( 62 nm versus 36nm in diameter ) ( see Fig 4 ) . Previous studies provided evidence that OrgB ( a FliH homolog ) forms the spoke-like structure and interacts with the ATPase complex and SpaO ( a FliN homolog ) of the Salmonella injectisome [43] . In the B . burgdorferi flagellar motor , the spoke between the ATPase and C-ring is likely formed by multiple FliH2 molecules . FliH of B . burgdorferi is significantly larger ( 305 amino acid residues ) than its homolog in Salmonella ( 170 residues ) ( S6 Fig ) . Thus , the ATPase complex in the B . burgdorferi periplasmic flagella not only facilitates substrate recruitment and secretion but also supports the integrity of the C-ring , which undergoes rotation and switches between CW and CCW . We observed many different orientations of the ATPase complex relative to the periplasmic structures of the motor , suggesting that the C-ring and the ATPase complex rotate together with the MS-ring . The rotation of the C-ring is driven by 16 stators that surround the C-ring and a spirochete-specific periplasmic collar [10] . In contrast , although OrgB and SpaO likely undergo high turnover with a cytoplasmic pool , the pods found in Salmonella injectisomes do not appear to rotate . The differences between the flagellar motor and injectisome underline the distinct mechanisms involved in their assembly and function . Recent studies using fluorescence recovery after photobleaching showed the copy number of the C-ring protein FliN varies with the direction of flagellar rotation [51] . It was estimated in E . coli that there are 114 ± 17 FliN molecules in motors that rotated only CW and 144 ± 26 FliN in CCW motors [51] . Our multivariate statistical analysis results suggested that the FliH spoke numbers vary from 21 to 24 in the B . burgdorferi motor , and the spokes are distributed evenly along the C-ring ( S1 Fig ) . If there are 46 FliN tetramers when there are 23 FliH spokes , there could be 42 FliN tetramers and 48 FliN tetramers when the spoke number is 21 and 24 , respectively . Considering the C-ring in B . burgdorferi is relatively larger than the E . coli motor ( 57 nm versus 44 nm in diameter ) [50] , our estimation on FliN tetramers copies fall into a reasonable range compared with the observation from the E . coli motor . Yet further study on the ATPase complexes from the motors locked in CW or CCW rotation will be needed for a better understanding of the mechanisms underlying the C-ring proteins turnover and rotation . In conclusion , our study reveals unprecedented details about the intact flagellar motor and its T3SS machine in the Lyme disease spirochete B . burgdorferi . We present the direct structural evidence that the flagellar ATPase complex is attached to the C-ring through multiple spokes likely comprised of FliH . The novel architecture of the ATPase complex not only strengthens the C-ring but also enables an optimal translocation of substrates through the ATPase complex and the export apparatus . Remarkably , the ATPase complex together with the C-ring can adopt variable orientations , implying that the fT3SS machine undergoes rotation with the flagellar C-ring . Together , our studies not only provide a structural framework for a better understanding of the fT3SSs but also underscore the striking differences between flagella and their evolutionally related bacterial injectisomes .
High-passage B . burgdorferi strain B31A ( WT ) were grown at 35°C in BSK-II liquid medium supplemented with 6% rabbit serum or on semisolid agar plates in the presence of 2 . 5% carbon dioxide , as previously described [39 , 52] . The frozen hydrated specimens were prepared as previously described [11] . Briefly , B . burgdorferi cultures were centrifuged at 5 , 000 × g for 5 min , and pellets were suspended in 1 . 0 ml phosphate buffered saline ( PBS ) . The cells were centrifuged again and suspended in approximately 50–80 μl PBS . The cell suspensions were mixed with 10 nm colloidal gold and were then deposited onto freshly glow-discharged , holey carbon grids for 1 min . Grids were blotted with filter paper and then rapidly frozen in liquid ethane using a homemade gravity-driven plunger apparatus . Frozen hydrated specimens were imaged at −170°C using a Polara G2 electron microscope ( FEI ) equipped with a field emission gun and a Gatan K2 Summit DDD . SerialEM was used to collect tilt series from WT cells in the dose fractionation mode [53] . The microscope was operated at a magnification of 15 , 400× , resulting in an effective pixel size of 2 . 5 Å without binning and a cumulative dose of approximately 60 e−/Å2 distributed over 61 stacks . Each stack contains eight images . Tomoauto was utilized to facilitate the automation of cryo-ET data processing [44] . The main executables include the following: drift correction of dose-fractionated data using Motioncorr [54] and assembly of corrected sums into the tilt-series , alignment of tilt-series and CTF correction by IMOD [55] , and reconstruction of tilt-series into tomograms by TOMO3D [56] . In total , we extracted 7 , 242 motors from 780 tomograms on WT cells . The subtomogram analysis was implemented as previously described [11 , 39 , 57] . Briefly , the initial orientation of each motor was estimated by the center coordinates of the flagellar C-ring and the collar , thereby providing two of the three Euler angles . To accelerate image analysis , 4×4×4 binned subtomograms ( 64×64×64 voxels ) were used for initial alignment . Then , the original subtomograms ( 256×256×256 voxels ) were utilized for further image analysis . Multivariate statistical analysis and hierarchical ascendant classification were then applied to analyze the intact motor [45 , 58 , 59] . Relevant voxels of the aligned subvolumes were selected by specifying a binary mask of the motor . Class averages were computed in Fourier space , so the missing wedge problem of tomography was minimized . All class averages were further aligned with each other to minimize differences in motor orientation . Because the symmetric feature of the collar and stators is predominant , initially , the averaged structure of motors showed the 16-fold symmetry of those regions , while the structure of the ATPase complex is not well resolved . Classification on the ATPase complex yielded several class averages with 6-fold symmetry . The class averages with obvious 6-fold symmetry were selected for further analysis . The 5 , 076 subtomograms in this data set were aligned on the ATPase complex region by spin alignment with step size of 22 . 5° ( 360°/16 ) . As a result , in the global average , the “hub” showed evident features with 6-fold symmetry , while the periplasmic features maintained 16-fold symmetry . The average structure of the spokes from WT cells was generated as follows: ( 1 ) the 5 , 076 selected subtomograms that give 16-fold symmetry in the stator and collar regions and 6-fold symmetry in the “hub” region of the average structure were classified based on the spoke region . This analysis generated four class averages , three of which showed spokes . One of those three class averages is presented in S1 Movie , illustrating the 3D distribution of the 16-fold symmetry at collar and stator regions , 23-fold symmetry of the spokes , and 6-fold symmetry of the “hub . ” The structure in S1 Movie shows that three regions with different symmetry can be resolved in one averaged structure from real data . ( 2 ) Classification on the spoke region was carried out using eigenimages one to 18 to generate four class averages ( see S2 Fig ) . The first 40 eigenimages of the data set indicate the presence of different symmetries of the spoke region ( see Results ) . To define the rotation angles , the previously aligned subtomograms were classified on the collar and stator regions . The new class averages that showed symmetry of the collar and stator regions were selected and aligned by the collar and stator regions with spin alignment only . The spin rotation angles were recorded and compared . UCSF Chimera [60] was used for 3D visualization of flagellar motors . The crystal structure of MxiAC ( PDB: 4A5P ) was fitted directly into the tomographic density map of the FlhA region . The FliI/FliJ model based on two crystal structures from Salmonella [30 , 46] was fitted into the hexagonal “hub . ” The atomic structure of FliI–FliH2C ( PDB:5B0O ) was initially fitted into the segmented density by rigid fitting . As there is extra density for FliH2 in the tomographic map and three to four spokes extend from the “hub , ” three more FliH2 were placed adjacent to the first FliH2; they were fitted into the density map using MDFF [61] ( see S2 Movie ) . The crystallographic structure of E . coli FliN is organized in doughnut-shaped tetramers [62] . Combined with a recent crystal structure FliMM–FliGMC complex from Thermotoga maritima ( PDB:4FHR ) [49] , the FliN–FliMM–FliGMC complex fits well into the bulge density at the bottom of the C-ring ( S2 Movie ) . As V111 , V112 , and V113 ( E . coli ) are in the hydrophobic patch and interaction with FliH [63] , we speculate that those three valine residues face toward the FliH2 spoke . Those three valines correspond to V128 , V129 , and V130 in T . maritima [63] . As a result , when we fit the FliN tetramer ring , we have V128 , V129 , and V130 ( See S2 Movie shown in red ) facing toward the FliH2 spoke . There are 46 copies of FliG–FliM–FliN , and they fit reasonably well into the B . burgdorferi C-ring density . | Type III secretion systems are widely utilized by gram-negative bacteria to assemble flagella or to transport virulence effectors into eukaryotic cells . The central component is known as a type III secretion machine , which consists of a membrane-bound export apparatus and a cytosolic ATPase complex . Powered by the proton motive force and ATP hydrolysis , the secretion machine is responsible for substrate recognition and export . Here , we use the Lyme disease spirochete B . burgdorferi as a model system to unveil unprecedented structural details of the intact flagellar secretion machine by high-throughput cryo-electron tomography ( cryo-ET ) and subtomogram averaging . We provide the first structural evidence that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the “spoke and hub” structure in B . burgdorferi . The novel architecture of the ATPase complex not only strengthens the flagellar C-ring but also enables an optimal translocation of substrates through the ATPase complex and the export apparatus . | [
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... | 2018 | Cryo-electron tomography of periplasmic flagella in Borrelia burgdorferi reveals a distinct cytoplasmic ATPase complex |
Prion diseases are fatal transmissible neurodegenerative disorders , which include Scrapie , Bovine Spongiform Encephalopathy ( BSE ) , Creutzfeldt-Jakob Disease ( CJD ) , and kuru . They are characterised by a prolonged clinically silent incubation period , variation in which is determined by many factors , including genetic background . We have used a heterogeneous stock of mice to identify Hectd2 , an E3 ubiquitin ligase , as a quantitative trait gene for prion disease incubation time in mice . Further , we report an association between HECTD2 haplotypes and susceptibility to the acquired human prion diseases , vCJD and kuru . We report a genotype-associated differential expression of Hectd2 mRNA in mouse brains and human lymphocytes and a significant up-regulation of transcript in mice at the terminal stage of prion disease . Although the substrate of HECTD2 is unknown , these data highlight the importance of proteosome-directed protein degradation in neurodegeneration . This is the first demonstration of a mouse quantitative trait gene that also influences susceptibility to human prion diseases . Characterisation of such genes is key to understanding human risk and the molecular basis of incubation periods .
Prion diseases are fatal transmissible neurodegenerative disorders of animals and humans . These include the agriculturally and economically important diseases of scrapie and Bovine Spongiform Encephalopathy ( BSE ) and the human diseases sporadic Creutzfeldt-Jakob disease ( CJD ) , variant ( vCJD ) and kuru . Sporadic CJD has no known aetiology and vCJD is thought to have arisen following exposure to BSE prions [1] . Kuru is a prion disease that reached epidemic proportions in the 1950s in the Fore linguistic region of Papua New Guinea and is thought to have been transmitted through endocannibalism by participation in mortuary feasts [2] . Following the cessation of this practice in the late 1950's , the incidence of disease has declined , however , it remains our only experience of a large epidemic of acquired human prion disease and provides a useful model for vCJD [3] . Although there was widespread population exposure in the UK and some other countries to BSE only around 200 have developed clinical vCJD to date , although the number infected remains unknown . This represents an on-going public health concern with a risk of iatrogenic transmission through blood and surgical instruments . vCJD has not been associated with any unusual pattern of dietary or occupational exposure to BSE prions and a significant genetic component to risk seems probable therefore the identification of susceptibility factors is key to estimating individual risk [1] . All prion diseases have prolonged clinically silent incubation periods which in humans span over 50 years [2] . Marked variation in incubation period occurs between inbred lines of mice and this is determined by multiple genetic loci in addition to the prion protein gene [4] , [5] Previous studies have identified several quantitative trait loci for prion disease incubation time in mice . However , the resulting regions of interest spanned many megabases and were consequently too large for individual candidate gene analysis [6]–[10] . Several different strategies are available for fine mapping [11]–[13] and we chose to use a heterogeneous stock of mice . These are produced to model an out-bred population of mice , however they have the advantage of starting with a defined number of parental alleles . Heterogeneous stocks of mice have been shown to be a useful mapping tool because they provide a high level of recombination and the development of specific mapping software allows for convenient multipoint linkage analysis [14] , [15] . This approach led to the identification of Hectd2 , an E3 ubiquitin ligase , as a quantitative trait gene for prion disease incubation time . Mouse models are extremely useful for studying human prion diseases as they faithfully recapitulate many key features of the disease and indeed rodents are naturally susceptible to prion diseases . It is expected that susceptibility genes and pathways identified in mice will also be relevant to human prion diseases . To test this hypothesis we carried out an association study with HECTD2 markers and samples from different human prion diseases and successfully found a significant association with two acquired forms of prion disease: vCJD and kuru .
To fine map regions thought to contain quantitative trait loci for prion disease incubation time we utilised the Northport heterogeneous stock [16] ( HS ) of mice ( gift of Robert Hitzemann ) , which was produced by semi-randomly mating eight inbred lines of mice ( A/J , AKR/J , BALB/cJ , C3H/HeJ , C57BL/6J , CBA/J , DBA/2J , LP/J ) . Approximately 1000 mice were inoculated intracerebrally with mouse-adapted scrapie prions ( Chandler/RML ) and incubation times ( in days ) were determined as previously described [6] , [17] . Regions of interest for fine mapping include those identified in previous crosses [6]–[10] and also from other studies . In this study we focus on a region of Mmu19 as a result of an interest in candidate genes on human chromosome 10 ( unpublished data ) . Nine microsatellite markers from chromosome 19 ( D19Mit86-D19Mit112 see Table S1 ) at approximately 1 cM intervals were genotyped in approximately 400 animals which represent the extreme 20% of both sides of the incubation time distribution . Multipoint linkage analysis was carried out using HAPPY ( http://www . well . ox . ac . uk/happy ) [18] . A peak of linkage ( −logP = 5 . 88 ) was seen between D19Mit63 and D19Mit65 , a region of approximately 2 . 9 Mb ( Figure S1A ) . Significant linkage was taken as −logP>3 as defined by a permutation test ( n = 1000 ) carried out by HAPPY . This interval explains 6 . 9% of the observed variance therefore as predicted by other QTL mapping studies [6]–[10] , other loci are expected to contribute to prion disease incubation time . Trait estimates for each strain are shown in Table S2 . Twenty seven RefSeq genes were identified within this region ( NCBI build 37 ) , 22 of which were sequenced ( Table S3 ) in the parental strains of the HS in order to identify polymorphisms . Sequencing was not exhaustive and focused primarily on the exons including 5′ and 3′UTRs , intron/exon boundaries and potential promoters as defined by the literature for each gene or PROSCAN ( http://www-bimas . cit . nih . gov/molbio/proscan ) . 177 polymorphisms were identified across the region which included single nucleotide polymorphisms ( SNPs ) , simple repeats and insert/deletion polymorphisms . Most of the variation was observed in non-coding regions , however , several non-synonymous changes were seen ( for detail see Table S4 ) . All variants were assessed using an additional function of HAPPY which assigns a probability that any polymorphism is a quantitative trait nucleotide ( QTN ) [19] . This predicts which strain distribution pattern ( SDP ) most closely fits the pattern identified by the microsatellites in the HS animals ( Figure S1B , Table S4 ) . The main candidates to emerge from this analysis are Hectd2 , Exoc6 , Cyp26c1 , Cyp26a1 , Plce1 and Lgi1 ( Table 1 ) . Some SDPs are broadly conserved across the whole gene ( e . g . −logP = 6 . 12 Hectd2 , −logP = 6 . 74 Cyp26c1 and Plce1 ) whereas others represent single polymorphisms ( e . g . −logP = 6 . 74 Cyp26a1 and Lgi1 ) . The −logP values assigned by HAPPY are predictions . We therefore tested representative polymorphisms from either each gene , or each strain distribution pattern , by genotyping the HS ( Table 2 ) . The only highly significant SNPs were seen in Hectd2 ( P = 0 . 0008 , 0 . 0013 and 0 . 0022 , ANOVA ) suggesting that Hectd2 is the most promising candidate in this region . However , these analyses are not exhaustive and it is not possible to exclude the possibility that variation in other genes or intergenic regions also contribute to prion disease incubation time . Seven SNPs were identified in the 3′UTR of Hectd2 ( Table S4 ) , however , it is not clear whether they would affect regulation . Five polymorphisms occur within the predicted promoter ( −226 to +25 ) one of which affects a potentially functional site . Sequence for the C57BL6/J allele from −216 to −210 is TGGGCGG and the insertion of 6 Gs in the alternative allele gives TGGGGGGGGGCGG . Both variants contain the consensus sequence for a Sp1 binding site ( shown in bold ) however the insertion also generates an overlapping large T antigen binding site ( underlined ) . It is unclear whether additional mouse proteins could bind to this sequence or whether Sp1 binding would be affected . The significant SDPs were spread across the whole of Hectd2 therefore we cannot exclude any of these closely linked polymorphisms either individually or collectively from a contribution to the phenotype . To determine whether the polymorphisms detected in Hectd2 have an effect on expression , RNA was extracted from whole brains of 8 week old males from the parental strains of the HS ( except LP ) . Samples were analysed by real time RT-PCR . To examine genotype-related differential expression , strains were grouped according to the major strain distribution pattern seen in Hectd2 ( Group A = A , AKR , BALB; Group B = C3H , C57 , CBA , DBA ) . Expression was ×2 . 4 greater in group A than group B ( P = 2 . 85×10−9 , unpaired t-test ) ( Figure 1A ) . Where incubation time data are available , the increase in Hectd2 expression is associated with a shorter incubation time ( R2 = 0 . 61 ) [20]–[22] ( See also Figure S2 ) . A potential role for Hectd2 in prion disease pathogenesis was explored by comparing the mRNA expression levels between normal mice and those at the end stage of disease following infection with Chandler/RML prions . For C57BL/6 , expression was ×5 . 0 greater in the prion infected mice ( P = 2 . 66×10−8 , unpaired t-test ) ( Figure 1B ) . Our data indicate that Hectd2 influences prion disease incubation time in mice . We therefore analysed HECTD2 in a hypothesis-driven association study of human prion disease . We analysed 834 samples from patients with prion disease or strong resistance to prion disease and 1162 relevant control population samples . We tested whether genetic variation at HECTD2 was associated with a phenotype of variant and sporadic CJD . In Papua New Guinea ( PNG ) we genotyped patients who died from the epidemic prion disease kuru , transmitted by endocannibalism , and compared these data with elderly women known to have had multiple exposures to kuru at mortuary feasts prior to the cessation of endocannibalism in the late 1950's , but who are long-term survivors [2] , [23] . See methods for details of the patient data , populations , phenotype ascertainment and population stratification data . We initially tested a single SNP , rs12249854 ( A/T ) , located in a HECTD2 intron , and showed that the minor allele ( A ) was significantly over-represented in vCJD ( n = 117 , 8 . 1% ) compared to controls ( n = 601 , 3 . 9% ) , P = 0 . 0049 , ( OR 2 . 11 , 95% CI 1 . 19–3 . 77 , trend test 1 d . f . ) , and between sporadic CJD ( n = 452 , 6 . 3% ) and controls , P = 0 . 012 , ( OR 1 . 65 , 95% CI 1 . 11–2 . 46 , trend test 1 d . f . ) . Given that sample sizes are necessarily small in both sporadic and variant CJD , these data are consistent with the association of rs12249854 with risk in both prion disease categories and a large effect size . We went on to test whether the risk rs12249854 allele modified the phenotype of human prion disease . Although the age of onset of sporadic CJD with rs12249854AA was younger than other genotypes ( 53 . 5 years Vs 68 . 8 years for rs12249854AT and 69 . 0 years for rs12249854TT , P = 0 . 048 t-test ) , this genotype was rare ( n = 3 ) and the finding therefore was not robust . There was no association between vCJD year of presentation , or age of onset with rs12249854 genotype . Insufficient data were available to look at any association with duration of illness . We went on to analyse a further seven SNPs in HECTD2 selected to capture global genetic diversity based on Hapmap ( http://hapmap . org ) [24] data ( Table S6 ) . In the United Kingdom ( UK ) , we found strong linkage disequilibrium ( LD ) and a simple haplotype structure across the entire gene ( Table 3 ) . Three haplotypes were >1% frequency , the most common two haplotypes ( 1 and 2 ) differed at all SNPs , a third haplotype ( 3 ) was distinguished from the most common haplotype ( 1 ) by a single SNP upstream of HECTD2 . Increased risk of vCJD was associated with haplotype 2 , possessing rs12249854A , but the extensive LD prevented us from identifying the functional SNP . In PNG , however , we found considerably more diversity with four common haplotypes , 1 , 2 and two novel haplotypes 4 and 5 ( see methods for haplotype inference ) . Haplotype 2 , most significantly associated with vCJD ( haplotype association test , P = 0 . 006 ) , showed no significance between kuru and the elderly female survivors of mortuary feasts . Rather , in PNG we found that a population specific haplotype ( designated 4 ) was strongly associated with kuru ( P = 0 . 0009 ) . Haplotype 4 differs from haplotype 2 at a single SNP , rs12247672 , which itself is significant in vCJD ( P = 0 . 0039 ) but not at all in kuru ( P = 0 . 6138 ) . Our data suggest that there is evidence for HECTD2 association in both vCJD and kuru however the functional polymorphisms are likely to be different . This is not necessarily surprising given the distinct evolutionary history and consequent genetic differences that exist between the UK and PNG populations . It should also be noted that although vCJD and kuru are both acquired human prion diseases that share many characteristics they are also derived from different sources and caused by distinct prion strains [25] , [26] therefore the mechanism of HECTD2 involvement may also be different . We sequenced the ORF and promoter of HECTD2 in 16 vCJD , multi kuru-exposure survivors , and both UK and PNG controls . Three polymorphisms were found , of which only one is potentially functional ( Table 4 ) . rs7081363 occurs in the promoter ( −247 ) and the minor allele is predicted to remove an Sp1 binding site ( GGCG/AGG ) . rs7081363 was genotyped in our samples and shown to be in complete LD with rs12249854 in the UK population ( vCJD P = 0 . 0012; sporadic CJD P = 0 . 0065 ) . We were unable to genotype the kuru samples due to poor DNA quality , however , analysis of all other samples suggest that rs7081363 is unlikely to be significant in PNG . To determine whether the susceptibility alleles in the UK population are associated with differential mRNA expression , HECTD2 expression levels in blood lymphocytes ( n = 140 , UK blood donors ) were quantified by real-time RT-PCR . Samples were grouped according to rs12249854 genotype , however , due to the low frequency of the minor allele ( A ) , no homozygotes ( AA ) were seen . The mean expression level was ×2 . 3 greater in the heterozygotes than for the major allele homozygotes ( TT ) ( P = 0 . 0008 Mann-Whitney test , Figure 1C ) . This suggests that a higher level of HECTD2 mRNA expression may be linked with vCJD in the UK population .
Our data show that HECTD2 is linked to prion disease incubation time in mouse and is associated with sporadic and variant CJD and kuru in humans and an increase in expression is associated with a susceptibility genotype and disease pathogenesis . In mouse , we cannot exclude the possibility of other nearby genes or intergenic regions also being implicated as our sequencing studies were not exhaustive . However , in human , the LD block , based on HapMap [24] data , includes only HECTD2 and does not extend into the neighbouring genes suggesting that the association observed stems from HECTD2 and not any other gene in the area . In mouse , the promoter , 3′UTR polymorphisms and the associated differential expression suggest a mechanism by which Hectd2 may influence the incubation time phenotype . Similarly , in the UK population a promoter polymorphism is also associated with a susceptibility phenotype and a resulting increase in expression level . This suggests that the mode of HECTD2 action in prion disease may be independent of host and prion strain . Due to lack of available material it has not been possible to replicate these experiments in our kuru samples , however , our haplotype study suggest that a different polymorphism is likely to be functional in the PNG population . This does not rule out the possibility that differential expression is also important in PNG , through an alternative polymorphism , although this may be difficult to determine . Our expression analysis in terminally sick mice suggest that HECTD2 is upregulated during the course of infection therefore we can speculate that a higher base line of expression reduces the time taken to reach a threshold level thereby reducing the incubation time . The ubiquitin-proteosome system has been implicated in the pathogenesis of several neurodegenerative diseases which show an accumulation of an abnormally folded protein including prion disease , Parkinson's disease and Alzheimer's disease [27]–[29] . By homology to other family members , HECTD2 is an E3 ubiquitin ligase suggesting that common pathways are involved in the neurodegenerative processes of these different diseases . Specifically , the mouse mahoganoid coat-colour mutation is found in the gene Mahogunin which is an E3 ubiquitin ligase [30] . A null mutation of Mahogunin causes an age-related progressive neurodegenerative phenotype characterised by spongiform degeneration , neuronal loss and astrocytosis . The phenotype resembles that of prion disease however there is no PrPSc accumulation . Mutations in the E3 ubiquitin ligase parkin are associated with autosomal recessive juvenile parkinsonism and loss of ubiquitin-protein ligase activity in patients has been shown to be associated with protein accumulation [31] . E3 ubiquitin ligases have also been implicated in the pathogenesis of polyglutamine diseases in particular it has been shown that mutations in the E6-AP ubiquitin ligase reduces the frequency of nuclear inclusions in mice expressing mutant ataxin-1 while accelerating the Purkinje cell pathology [32] . Further , HECTD2 maps to a region of human chromosome 10q previously linked with Alzheimer's disease [33] suggesting that HECTD2 may also be a susceptibility factor for Alzheimer's disease and other neurodegenerative disorders . Group sizes for vCJD , kuru and elderly female survivors of mortuary feasts are of necessity small , however we believe that the combined weight of data from the mouse genetic studies , expression analyses and our association study of independent human prion diseases from different populations provide sufficient evidence to support a role for HECTD2 in prion disease . This supports a significant role for the ubiquitin-proteasome system in prion pathogenesis [27] , [34] , [35] and will contribute to modelling and understanding genetic risk of developing prion disease following BSE and secondary human prion exposure .
The clinical and laboratory studies were approved by the local research ethics committee of University College London Institute of Neurology and National Hospital for Neurology and Neurosurgery and by the Medical Research Advisory Committee of the Government of PNG . Full participation of the PNG communities involved was established and maintained through discussions with village leaders , communities , families and individuals . 28 pairs of Northport HS mice were obtained from R . Hitzemann ( Portland , Oregon , USA ) at generation 35 . Offspring from these pairs were randomly mated to produce a total of 49 pairs . 1000 offspring ( generation 37 ) were used for inoculation . All other inbred lines were obtained from Harlan , UK . Mice were identified by individual transponder tags ( Trovan ) and tail biopsies were obtained for DNA extraction . Mice were anaesthetized with isofluorane/O2 and inoculated intra-cerebrally into the right parietal lobe with 30 µl Chandler/RML prions as previously described [6] . Incubation time was calculated retrospectively after a definite diagnosis of scrapie had been made and defined as the number of days from inoculation to the onset of clinical signs [17] . All procedures were conducted in accordance with UK regulations ( Local ethics approval and Home Office regulation ) and international standards on animal welfare . | Prion diseases are fatal transmissible neurodegenerative diseases of animals and humans for which there is no treatment . They include Bovine Spongiform Encephalopathy ( BSE ) , and its human equivalent , variant Creutzfeldt-Jakob Disease ( vCJD ) . Prion diseases are characterised by a long , silent incubation period before the disease emerges , and this time interval varies greatly between individuals . Differences in our genetic makeup are a key factor in this variability . We already know that natural variation within one key gene , the prion protein gene , has a major influence on incubation time , but it is now clear that a number of other genes are also important . Using a mouse model , we have identified one of these genes , Hectd2 , which is thought to be involved in the process that removes unwanted proteins from the cell . We also show that HECTD2 is associated with an increased risk of two human prion diseases—vCJD in the United Kingdom and kuru in Papua New Guinea . These data will give us a better understanding of the fundamental processes involved in these diseases and go some way to explaining why some individuals exposed to BSE have developed vCJD and others have not . | [
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"d... | 2009 | HECTD2 Is Associated with Susceptibility to Mouse and Human Prion Disease |
In Aedes mosquitoes , infections with arthropod-borne viruses ( arboviruses ) trigger or modulate the expression of various classes of viral and host-derived small RNAs , including small interfering RNAs ( siRNAs ) , PIWI interacting RNAs ( piRNAs ) , and microRNAs ( miRNAs ) . Viral siRNAs are at the core of the antiviral RNA interference machinery , one of the key pathways that limit virus replication in invertebrates . Besides siRNAs , Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs , the best studied examples being viruses from the Togaviridae or Bunyaviridae families . Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections . In addition , some viruses , mostly with a DNA genome , express their own miRNAs to regulate host and viral gene expression . Here , we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 ( DENV ) , a member of the Flaviviridae family . Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways . Interestingly , besides the well-characterized DENV-derived siRNAs , a specific population of viral piRNAs was identified in infected Aag2 cells . Knockdown of Piwi5 , Ago3 and , to a lesser extent , Piwi6 results in reduction of vpiRNA levels , providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs . In contrast , we do not find convincing evidence for the production of virus-derived miRNAs . Neither do we find that host miRNA expression is strongly changed upon DENV2 infection . Finally , our deep-sequencing analyses detect 30 novel Aedes miRNAs , complementing the repertoire of regulatory small RNAs in this important vector species .
Aedes mosquitoes are essential vectors for the transmission of important arthropod-borne viruses ( arboviruses ) , including dengue virus ( DENV ) , yellow fever virus , and chikungunya virus [1] . While several of these arboviral infections cause disease in humans , virus replication generally does not lead to severe pathology in vector mosquitoes . Infected mosquitoes thus serve as a persistent reservoir for arboviruses in the wild and they may transmit these viruses to vertebrate hosts throughout their entire lives [2] . After ingestion in a mosquito’s blood meal , arboviruses need to overcome a number of anatomical and immunological barriers to reach sufficiently high titres in the saliva . Only then can transmission to a naive vertebrate host efficiently occur . One of the most important immune responses to arboviral infection is antiviral RNA interference ( RNAi ) [3–5] . This pathway is triggered by the presence of double stranded RNA ( dsRNA ) , which is produced during the replication of RNA and DNA viruses [6 , 7] . The dsRNA is recognized and cleaved by the RNase-III enzyme Dicer-2 ( Dcr2 ) into 21 nucleotide ( nt ) small interfering RNA duplexes ( viral siRNA; vsiRNA ) [8 , 9] . One of the siRNA strands is incorporated in Argonaute-2 ( Ago2 ) , the core protein of the RNA induced silencing complex ( RISC ) [10] . The siRNA-loaded RISC complex is guided to complementary viral RNA molecules and cleaves these target RNAs using the endonuclease ( slicer ) activity of Ago2 [11] . MicroRNAs ( miRNAs ) are a distinct class of small RNAs that are produced from genome-encoded stem loop-containing transcripts known as primary miRNA ( pri-miRNAs ) . During the canonical miRNA biogenesis pathway , the stem loop structures , known as precursor miRNA ( pre-miRNA ) , are released from the pri-miRNA by the microprocessor complex with at its core the RNase-III enzyme Drosha . After translocation into the cytoplasm , pre-miRNAs are cleaved by Dicer-1 ( Dcr1 ) to produce a small RNA duplex comprised of the two mature miRNA strands . Usually , one of these strands is then preferentially incorporated into the Argonaute-1 ( Ago1 ) containing miRNA-induced silencing complex ( miRISC ) , whereas the other strand ( the passenger or miRNA* strand ) is usually discarded [12] . Loaded miRISC complexes are able to bind to specific target sites within mRNAs . This miRNA-mRNA interaction is initiated by nucleotide two to seven of the miRNA , the so-called seed sequence [13] . Stable binding of miRISC to an mRNA target , generally causes down-regulation of gene expression via translational inhibition and mRNA destabilization [14] . Importantly , infecting viruses can directly or indirectly , as a consequence of the immune response , reshape the host miRNA expression landscape . While quite a number of studies have reported on this matter in mammalian systems [15] , little is known about virus-induced changes in miRNA levels in mosquito vectors . In Aedes mosquitoes , miRNA levels or modifications have been reported to be changed upon infections with DENV , West Nile virus , and chikungunya virus [16–20] . For most of these differentially expressed miRNAs , the biological relevance as well as the targeted mRNAs still await experimental validation . Besides modulation of host miRNAs , some DNA and retroviruses encode their own miRNAs to regulate viral and host mRNAs [21] . The expression of miRNAs from cytoplasmic RNA viruses has been controversial . However , functional introduction of artificial miRNAs into the genomes of Sindbis virus ( SINV ) and tick-borne encephalitis virus provides evidence that miRNA production from cytoplasmic RNA viruses may in principle be possible [22 , 23] . Yet , the presence and biological relevance of miRNAs encoded in the genomes of flaviviruses such as DENV is still an issue of debate [24–26] . The third , most enigmatic class of small RNAs are PIWI interacting RNAs ( piRNAs ) . These are processed from long RNA precursors that are transcribed from genomic loci known as piRNA clusters . In sharp contrast to siRNAs and miRNAs , their biogenesis into mature piRNAs is Dicer-independent . In Drosophila , piRNA maturation involves endonucleolytic cleavage of precursor transcripts by the Zucchini nuclease and the three PIWI proteins Piwi , Aubergine ( Aub ) and Argonaute-3 ( Ago3 ) [27–30] . The primary function of the piRNA pathway in this model organism is the defence against transposable elements , mainly in germ-line tissues . Interestingly , piRNAs of viral origin ( vpiRNA ) have been found in somatic tissue of Aedes mosquitoes , suggesting that they contribute to the regulation of virus replication [31] . At present , vpiRNAs have been discovered upon infection with a number of Alphaviruses , Bunyaviruses and Flaviviruses , including DENV [31–39] . However , with the exception of SINV ( Alphavirus ) , their molecular biogenesis has not been investigated [37] . Here , we make use of small RNA deep-sequencing in the siRNA , miRNA , and piRNA competent Aedes aegypti Aag2 cell line to investigate the production of small RNAs during DENV infection . We find that in addition to the well-characterized vsiRNAs , specific vpiRNAs are produced from DENV , which for their biogenesis in Aag2 cells rely on Piwi5 and Ago3 and , to a lesser extent , on Piwi6 . We do not detect DENV-derived miRNAs , or prominent changes in host miRNA levels upon infection . Finally , we identify novel host miRNAs in our small RNA deep-sequencing libraries , complementing the currently annotated miRNA repertoire in Aedes aegypti vector mosquitoes .
Aag2 cells were cultured at 25°C in Leibovitz L-15 medium ( Gibco ) supplemented with 10% heat inactivated fetal calf serum ( FCS; PAA ) , 2% tryptose phosphate broth solution ( Sigma ) , 1x MEM non-essential amino acids ( Gibco ) , and 50 U/ml penicillin and 50 μg/ml streptomycin ( pen/strep; Gibco ) . U4 . 4 and C6/36 were kept in the same culture medium at 28°C . BHK-21 cells were cultured at 37°C , 5% CO2 in Dulbecco’s modified Eagles medium ( DMEM ) supplemented with 10% FCS and pen/strep . Stocks of DENV serotype 2 ( DENV2 ) , New Guinea C ( NGC ) and 16681 strains were grown on C6/36 cells and titred on BHK-15 cells as detailed in [40] . Aag2 cells were seeded one day prior to infection and infected with DENV2 at a multiplicity of infection ( MOI ) of 0 . 5 by directly adding the virus to the culture medium . Three days post infection the culture medium was removed and cells were harvested for RNA and protein isolation as detailed below . For the detection of the DENV NS1 protein in samples used for small RNA deep-sequencing , 5% of the cells were harvested in 50 μl lysis buffer ( 50 mM Tris-HCl pH 7 . 8; 150 mM NaCl; 1 mM EDTA; 0 . 5% NP-40; 1x Protease inhibitor cocktail ( Roche ) ; 1 mM DTT ) . 12 . 5 μl of 5x Laemmli buffer was added to each sample , incubated at 95°C for 5 min , and 30μl of each sample was loaded on a 12 . 5% polyacrylamide gel . After gel electrophoresis , proteins were transferred to a nitrocellulose membrane ( Bio-Rad ) using a semi-dry blotting system ( Bio-Rad ) . The membrane was blocked in 5% non-fat dry milk ( Bio-Rad ) in 0 . 1% Tween20 in PBS ( PBS-T ) for 30 min at room temperature . Mouse anti DENV NS1 antibody was kindly provided by Dr . Peter Mason [41] . The antibody was added to the membrane in a 1:1 , 000 dilution in 5% blocking buffer . After an incubation for 1 . 5 hours at room temperature , the membrane was washed three times in PBS-T . IRdye680 conjugated goat anti mouse antibody ( 1:15 , 000 dilution in PBS-T; Licor ) was then added to the membrane and incubated at room temperature for 1 . 5 hours . After three washing steps , the membrane was imaged on an Odyssey infrared image system ( Licor ) . dsRNAs targeting PIWI/AGO transcripts or luciferase as a negative control were produced by in vitro transcription from T7-promoter flanked PCR products as detailed in [37] . Primers to produce T7-flanked PCR products are indicated in S1 Table . For dsRNA transfection , 7 . 5x105 Aag2 cells were seeded in one well of a 24-well plate . For each condition , three wells were plated . The following day , transfection mixes containing 300 μl non-supplemented L-15 medium , 450 ng dsRNA and 1 . 8 μl X-tremeGENE HP ( Roche ) were prepared according to the manufacturer’s recommendations . 100 μl of the mix was added dropwise to one well . After 2–3 hours the medium was replaced with fully supplemented L-15 medium . 48 hours later , the transfection was repeated to enhance knockdown efficiencies . Where indicated , the cells were infected with DENV2 which was added to the L-15 medium used to replace the transfection medium as specified above . Aag2 cells were lysed in Isol-RNA Lysis reagent ( 5 PRIME ) as described in the manufacturer’s instructions . Briefly , 200 μl of chloroform was added to 1 ml of Lysis reagent and mixed well . After centrifugation , the aqueous phase was collected and total RNA was purified using isopropanol precipitation . RNA was quantified on a Nanodrop spectrophotometer and RNA integrity was checked by ethidium bromide staining of ribosomal RNA bands after agarose gel electrophoresis . For RT- ( q ) PCR , 1 μg of total RNA was DNaseI ( Ambion ) treated according to the manufacturer’s instructions . The RNA was subsequently reverse transcribed in a 20 μl reaction using the Taqman reverse transcription kit ( Applied Biosystems ) . Complementary DNA ( cDNA ) was diluted 5–10 times before PCR amplification . Endpoint PCR was performed using Thermoperfect DNA Polymerase . Quantitative PCR ( qPCR ) analysis was performed using the GoTaq qPCR SYBR mastermix ( Promega ) on a LightCycler 480 instrument ( Roche ) . The relative changes in gene expression were calculated using the ΔΔCt method [42] using lysosomal aspartic protease ( LAP ) as an internal normalization control . Sequences of the PCR primers are indicated in S1 Table . Sodium periodate ( NaIO4 ) oxidation and β-elimination of total RNA was performed as described previously [43] . Total RNA ( 10 μg in 47 . 5 μl nuclease-free water ) was mixed with 12 . 5 μl 200 mM NaIO4 and 40 μl 5x borate buffer . As a control , RNA was treated with water instead of NaIO4 . The reaction was incubated at room temperature for 30 min and 10 μl glycerol was added to the reaction . The reaction was incubated for another 10 min before 10 μl of 500 mM sodium hydroxide ( NaOH ) was added to induce β-elimination . The reaction was incubated at 45°C for 90 min . After these treatments , total RNA was purified by ethanol precipitation in the presence of 300 mM NaCl and 5 μg of glycogen . Electrophoretic mobility of Aedes aegypti miR-2940-3p and DENV2 piRNAs was then analyzed by small RNA northern blotting as detailed below . Small RNA northern blot was performed as described in [44] . Briefly , total RNA was size-separated on 0 . 5x TBE , 7 M Urea , 15% Polyacrylamide gels , transferred to Hybond NX nylon membranes ( Amersham ) , and cross-linked using 1-ethyl-3- ( 3-dimethylaminopropyl ) carbodiimide ( EDC; Sigma ) . Individual small RNAs were detected with DNA oligonucleotides that were 5’ end-labelled with [32P] γ-adenosine-triphosphate ( Perking Elmer ) using T4 Polynucleotide kinase ( Roche ) . Hybridization to the oligo-probes was performed overnight at 42°C in Ultrahyb Oligo hybridization buffer ( Ambion ) . Membranes were then washed three times at 42°C in 0 . 1% SDS with decreasing concentrations of SSC ( 2x , 1x , 0 . 1x ) . Membranes were exposed to X-ray films ( Carestream ) or Phosphorimager screens ( BioRad ) . Sequences of DNA oligonucleotide probes are indicated in S1 Table . Quantification of northern blot panels was performed using ImageJ software . Bands were defined using the rectangular selection tool and the pixel density ( area under the curve ) was measured and normalized to uninfected dsLuc samples . Small RNA libraries were prepared as described previously [37 , 45] . Briefly , three 25 cm2 flasks of Aag2 cells were infected in parallel with DENV2 NGC . Three additional flasks were left uninfected . Total RNA was then isolated from these six flasks as specified above and 30 μg of total RNA was size separated on a 15% Polyacrylamide , 7 M urea , 0 . 5x TBE gel . Subsequently , the small RNAs in the size range from 18 nt to 33 nt were excised from gel using radioactively-labelled RNA oligos , loaded in the adjacent lanes of the gel , as rulers . The gel was crushed and the small RNAs were eluted in 300 mM sodium acetate overnight at 4°C under constant rotation . The RNA was recovered from the elution buffer using ethanol precipitation and eluted in 10 μl of nuclease-free water . 5 μl of the sample was directly used as input for small RNA deep-sequencing library preparation using the TruSeq small RNA library preparation kit ( Illumina ) following the manufacturer’s recommendations . The RNA was ligated to 3’ and 5’ adapters , reverse transcribed , and PCR amplified . The small RNA libraries were then size-purified from 1x TBE , 6% polyacrylamide gel using overnight elution in 300 mM sodium acetate followed by ethanol precipitation . The individual small RNA libraries were pooled and sequenced on a single sequencing lane on a HighSeq2500 by Baseclear ( Leiden , The Netherlands ) . FASTQ sequence reads were generated using the Casava pipeline ( v . 1 . 8 . 3 ) and initial quality analysis was performed using the Illumina Chastity filter and an in-house filtering protocol by Baseclear . Subsequent quality assessment was based on the FASTQC quality control tool ( v . 1 . 10 . 0 ) . The individual small RNA sequencing libraries were separated based on the TruSeq indices ( no . 1 to 6 ) that were introduced during PCR amplification . The individual libraries were subsequently analyzed using the Galaxy bioinformatics tool shed [46 , 47] . For the analysis of viral small RNAs , reads were mapped to the DENV2 NGC genome ( GenBank accession: KM204118 . 1 ) using Bowtie ( v . 1 . 1 . 2 ) [48] . Size profiles were obtained from all reads that align to this reference sequence with a maximum of one mismatch . The genome distribution of 21 nt siRNAs , 22–24 nt small RNAs , or 25–30 nt piRNAs was obtained by plotting the number of 5’ ends of these reads at each position of the genome . For the pileup plots of UTR-derived miRNA-like small RNAs , the 22–24 nt small RNAs were selected from the initial FASTQ files and mapped to the DENV-NGC genome . From the resulting SAM files the reads mapping to the ( + ) strand of the virus genome were selected and used as input for the ‘Generate pileup from BAM dataset’ tool ( v . 1 . 1 . 2 ) . The values at the nucleotide positions of the DENV2 5’UTR ( 1–96 ) and 3’ UTR ( 10273–10723 ) were selected for display . Re-analysis of the data published by Hess et al . [36] was performed on the dataset with the accession number SRR921363 . The SOLiD-formatted dataset was groomed to fit the requirements for manipulation in Galaxy . After adapter clipping , reads were mapped to the DENV2-JAM1409 genome ( GenBank accession: M20558 ) using Bowtie2 [49] . Size and genome profiles were obtained as described above . Unless specified differently , all small RNA read counts were normalized against the size of the corresponding sequencing library and are expressed as ‘% of the library’ ( i . e . reads per hundred ) . Analysis of miRNA expression levels was performed using the miRDeep2 tool . Raw data were assessed for quality using FASTQC . Subsequently , adapters were removed from the raw reads , and the reads were quality trimmed using cutadapt software ( http://dx . doi . org/10 . 14806/ej . 17 . 1 . 200 ) with parameters -O 6 -m 17 -n 5 -q 20 . Within each library , the resulting reads were collapsed to generate a non-redundant set of FASTA sequences , subsequently processed to the format required for miRNA prediction with miRDeep2 software [50] . Collapsed reads longer than 17 nucleotides were aligned to the Aedes genome ( assembly AaegL3 , downloaded from vectorbase ) and the DENV2 NGC genome using the mapper . pl component of miRDeep2 ( parameters: -o 20 -l 19 -r 100 -c ) . The resulting outputs were parsed to remove alignments that were not full length and perfect match ( FLPM ) . miRDeep2 predictions were generated from the FLPM aligned sequences , with miRBase v21 Arthropoda mature miRNA and pre-miRNA sequences as templates [51] . The ‘miRNAs_expressed’ output from miRDeep2 , which comprises tallies for each known miRNA in each sample , was further processed in the R/Bioconductor environment . Briefly , miRNA read counts were normalized to the number of Aedes-specific genome reads within each sample group , using the lowest number of reads aligning as the baseline . Subsequently , the counts were converted to abundances within each sample , converted to log2 equivalent counts , and all samples quantile normalized prior to linear model fitting with the limma package [52] . MiRNA predictions from the miRDeep2 output were manually curated using the following criteria: i ) high-confidence miRNA predictions have reads mapping to both a predicted mature and star sequence , ii ) the mature sequences have a homogenous 5’ end ( 80% of reads start at same position ) , and iii ) miRNA-miRNA* duplex should resemble a Dicer product on a genomically-encoded hairpin , having a two nt ( +/-1 nt ) overhang at the 3’ end . miRNA predictions supported by >1000 reads as well as miRNA predictions with a seed match to known insect miRNAs were also kept . To be retained , these predictions required a homogenous 5’ end , but did not require the presence of reads mapping to the expected star strand . miRNA names and accession numbers were assigned by the miRBase repository . All deep sequencing libraries have been submitted to NCBI Sequence Read Archive under the accession number SRA303329 . All source data are available in S1 Dataset .
DENV is a positive ( + ) strand RNA virus belonging to the Flavivirus genus in the Flaviviridae family . Its RNA genome is approximately 10 . 7 kilobases in size and encodes a single polypeptide that is processed by proteolytic cleavage events into three structural proteins and seven non-structural proteins ( Fig 1A ) . Since various classes of small RNAs have been implicated in modulating DENV infections in its mosquito vectors , we aimed to characterize the repertoire of virus and host-derived small RNAs in Aedes aegypti Aag2 cells . To this end , we prepared three independent small RNA deep-sequencing libraries from uninfected and DENV2 ( NGC strain ) infected cells , each . The efficiency of the three infections was comparable as assessed by western blot for the DENV2 NS1 protein ( Fig 1B ) . As expected , DENV2-derived viral small RNAs showed a clear population of 21 nt vsiRNAs mapping to both the viral positive ( + ) strand and the negative ( - ) strand in roughly equal numbers ( Fig 1C ) . Interestingly , besides siRNAs , a second population of viral small RNAs was produced that resembled vpiRNAs . These were 25–30 nt in length and almost exclusively derived from the viral ( + ) strand ( Fig 1C ) . In contrast to vsiRNA , which were distributed along the entire length of the viral genome , these putative vpiRNAs were produced only from few specific positions ( Fig 1D ) . In fact , 85% of all the 25–30 nt reads were derived from four individual vpiRNA sequences , present in the NS5 gene at positions 9180 and 9985 , 9989 and 9990 of the DENV2 NGC genome . To test whether these small RNA profiles reflect those from adult mosquitoes , we re-analyzed deep sequencing data from DENV2 infected Aedes aegypti mosquitoes published by Hess et al . [36] . We analyzed the 9 days post infection sample , which showed the highest number of viral siRNAs and piRNAs . Whereas normalized vsiRNA levels were only 2 . 2-fold lower in these libraries than in our Aag2 data , vpiRNAs were about forty times lower . Yet , the viral small RNA profiles were strikingly similar , with 21 nt reads being scattered throughout the entire viral genome and piRNA-sized reads being predominantly produced from few positions located towards the 3’ end of the viral genome ( S1 Fig ) . These data suggest that similar mechanisms might produce viral piRNAs in Aag2 cells and adult mosquitoes . To exclude the possibility that the piRNA-like molecules are sequencing artefacts and to characterize this small RNA population in more detail , we performed small RNA northern blotting for the highly-abundant small RNAs starting at DENV2 genome positions 9180 or 9985–9990 . Indeed , small RNAs in the expected size range could readily be detected specifically in DENV2 infected Aag2 cells ( Fig 2A ) . In addition , these sequences were also present in DENV2-infected U4 . 4 and C6/36 cells derived from Aedes albopictus mosquitoes , which we have previously shown to be competent in producing SINV-derived vpiRNAs [32 , 37] ( Fig 2B ) . The levels of vpiRNAs did not correlate with the expression of viral genomic RNA . Whereas viral RNA levels were roughly eighteen to nineteen fold higher in U4 . 4 and C6/36 cells than in Aag2 cells , vpiRNAs were most abundant in Aag2 cells ( Fig 2B ) . These data suggest that the composition of host factors required for their biogenesis is most favourable in Aag2 cells . As expected , mammalian BHK-21 cells , which lack an active piRNA pathway , did not produce vpiRNAs ( Fig 2B ) . To exclude that viral piRNA production is an artefact of the use of the specific DENV2 strain , we analyzed piRNA accumulation in Aag2 cells infected with either the DENV NGC or DENV 16681 strain . Small RNA northern blotting revealed that infection with either strain resulted in the production of those viral piRNA sequences that we had found by deep-sequencing ( Fig 2C ) . Next , we aimed to test whether DENV2-derived piRNAs are methylated at their 3’ end . To this end , we performed sodium periodate oxidation followed by beta-elimination , which reveals modifications of the 3’ terminal nucleotide of RNA molecules [43] . Unmodified small RNAs , such as animal miRNAs , are susceptible to this treatment and will be shortened by one nucleoside resulting in increased electrophoretic mobility . In contrast to miRNAs , piRNAs are protected against this treatment by methylation of the 2’OH on the ribose of the 3’ terminal nucleotide . Indeed , beta-elimination resulted in increased electric mobility of miR-2940-3p . Yet , the migration of DENV2 piRNA bands was not affected by beta-elimination , indicating that their 3’ terminal nucleotides are modified , most likely methylated ( Fig 2D ) . Since piRNA methylation occurs after loading into PIWI protein complexes , these data suggest that the identified DENV2 piRNAs are mature piRNAs associated with a PIWI protein . To identify which PIWI proteins are required for the biogenesis of DENV2 piRNAs in Aag2 cells , we individually knocked down expression of all the eight Aedes PIWI proteins and analyzed the production of vpiRNAs by northern blot . We confirmed knockdown efficiency of roughly 90% for the four PIWI proteins that are detectable by RT-qPCR in Aag2 cells ( i . e . Piwi4 , Piwi5 , Piwi6 , Ago3; Fig 2E ) . Expression levels of Piwi1-3 and Piwi7 were too low to allow reliable quantification . DENV2 piRNAs were almost undetectable upon knockdown of Piwi5 and Ago3 and clearly reduced upon knockdown of Piwi6 ( Fig 2F ) . These data confirm that the 25–30 nt population of DENV2-derived small RNAs are bona fide piRNAs that require host PIWI proteins for their biogenesis . To test whether knockdown of PIWI expression results in enhanced DENV2 replication , we performed RT-qPCR to compare viral RNA levels in the different knockdown conditions . We found that none of the knockdowns resulted in a significant change in viral RNA levels ( S2A Fig ) . Yet , also knockdown of the well-established antiviral factor Ago2 [53] only resulted in a minor , statistically not-significant , increase of viral RNA replication although knockdown efficiency was higher than 90% ( S2B Fig ) . This suggests that , in our hands , knockdown of small silencing pathway components in Aag2 cells is not suited to uncover robust antiviral activity against DENV2 . The significance of viral miRNA production from DENV genomic RNA is heavily debated [24–26 , 54] . To investigate whether viral miRNA-like molecules are produced in DENV2 infected Aag2 cells , we filtered 22–24 nt small RNA reads that map to the DENV2 genome with a maximum of one mismatch . In general , the number of 22 to 24 nt reads was rather low ( ~5% of all DENV2 mapping reads ) when compared to 21 nt siRNAs ( ~28% ) and 25-30nt piRNAs ( ~40% ) . Furthermore , there were only four outstanding peaks that gave rise to a somewhat higher number of small RNAs ( Fig 3A ) . All of them coincided with the position of a vpiRNA peaks ( Fig 1D ) , suggesting that these small RNAs were by-products of vpiRNA production . Parallel analysis of the virus-derived reads using miRDeep2 did not identify convincing miRNA-like candidates: some reads mapped to two predicted hairpin sequences ( genome positions: 9542 ( + ) strand; 4888 ( - ) strand ) , however the mapping patterns showed heterogeneity of the 5’ start sites and did not suggest Dicer processing ( S3A Fig ) . Recently , eight miRNA-like small RNAs were computationally predicted based on hairpin structures in the DENV2 genome , but they were not experimentally validated [54] . We specifically looked for small RNA reads in our sequencing data mapping in the proximity of these predicted viral miRNAs , allowing a margin of 3nt around the start site . For each of the predicted miRNAs , we identified only very few ( <20 ) reads in the combined set of DENV2-infected small RNA libraries ( total of >3 . 7·107 reads of which >3 . 6·105 are DENV specific ) . In another publication , several ‘miRNA-like’ RNAs ( termed vsRNA-1 to 6 ) were proposed to be produced from the 5’ and 3’ UTRs of the DENV2 RNA , based on the analysis of small RNA sequencing data [24] . The DENV UTRs are indeed prone to form RNA structures and hairpins , which were suggested to be processed by the miRNA machinery into specific small RNA species [24] . We specifically looked for the proposed vsRNA sequences in our dataset but could only identify a small RNA population that resembled vsRNA-2 located at the terminal hairpin of the DENV2 genome ( Fig 3B ) . However , small RNAs mapping in that region showed a broad size distribution with the majority ranging in size from 26 to 28 nt , arguing against vsRNA-2 small RNAs being bona fide Dicer products ( S3B and S3C Fig ) . These data suggest that the proposed DENV2 vsRNAs are not an abundant class of small RNAs . Host miRNAs function as key regulators of gene expression and changes in miRNA expression have been reported during virus infections in various animal hosts , including mosquitoes . To assess host miRNA expression in response to DENV2 infection , we made use of the miRDeep2 toolkit to quantify miRNAs in the uninfected and DENV2-infected Aag2 small RNA libraries . DENV2 infection caused only minimal changes in miRNA levels ( Fig 4 ) . The expression of three and seven miRNAs was changed more than 2-fold up or down , respectively in response to DENV infection . Yet , the majority of differentially regulated miRNAs , including all up-regulated miRNAs , were poorly expressed ( mean expression levels below twenty reads ) making it hard to discriminate these expression changes from experimental noise due to low read counts . Collectively , these data suggest that miRNA expression in Aag2 cells is not heavily affected by DENV infection . The most recent version of miRBase ( version 21 ) contains 101 Aedes aegypti miRNAs , which is considerably less than for other insect species including Drosophila melanogaster ( fruitfly; 256 miRNAs ) , Apis mellifera ( honey bee; 254 miRNAs ) or Bombyx mori ( silkworm; 487 miRNAs ) . We therefore suspected that the repertoire of published mosquito miRNAs is not yet complete and we set out to identify novel Aedes aegypti miRNAs in our small RNA sequencing data using miRDeep2 . We obtained a list of 399 miRNA predictions ( S2 Table ) , 73 of which were known miRNAs annotated in miRBase [51 , 55 , 56] . We also confirmed 16 miRNAs that were recently reported by Akbari et al . or Hu et al . , but were not yet available in miRBase [57 , 58] ( S4 Fig ) . The remaining 310 predictions of miRNA hairpins were manually inspected for novel miRNAs using a similar approach as described in [59 , 60] , based on three criteria . First , only hairpin predictions that were supported by at least 1000 mature miRNA reads or those with at least one predicted miRNA* strand were retained . If a miRNA had an identical seed to a known insect miRNA , it was also retained irrespectively of read count or the presence of a miRNA* . In total 140 predictions met these criteria . Second , mature miRNAs were inspected for a homogeneous 5’ end of the supporting small RNA reads , defined as having at least 80% of the miRNA reads starting at the same nucleotide . 68 miRNA hairpin predictions met this requirement . Third , the predicted miRNA duplex was checked to resemble a ( near ) perfect Dicer product , defined as mapping to the stem of a stem-loop structure with one , two or three nucleotide overhangs at the 3’ end . Using this approach , we identified 31 unique mature miRNAs sequences mapping to 39 predicted miRNA hairpins . Nine of the 31 mature miRNA predictions did not have reads mapping to the star strand , but were supported by a homogeneous 5’ end in combination with a seed-match to an insect miRNA or having >1000 reads ( Table 1 , Fig 5 ) . Further inspection identified one of these predictions to be derived from a tRNA which has therefore been removed from the list of predicted miRNAs . During the analysis of miRNA predictions , we noted the expression of specific small RNAs adjacent to the mature miRNA and miRNA* star strands . These miRNA offset RNAs ( moRs ) have been detected in small RNA deep-sequencing data from invertebrates , simple chordates , vertebrates and even viruses [61–68] . In total , we identified moRs for 27% ( 24/89 ) of previously reported miRNA hairpins . In many of these cases ( nine out of 24 ) , the number of moRs per hairpin is below one thousandth of the number of mature miRNA reads . Others were more abundant , with moR reads accumulating up to 3 . 6% of the number of mature miRNAs . In a single instance , miR-11894b-1 , the number of moRs reached 10 . 8% of the number of mature miRNA reads . In agreement with previous findings in Drosophila [63] 5’ moRs were more abundant than 3’ moRs ( Fig 6A ) . moRs have been proposed to be the by-products of Drosha cleavage and in line with this suggestion , we found the 3’ end of 5’ moRs and the 5’ end of 3’ moRs to be fixed , reflecting potential Drosha cleavage sites ( Fig 6B and 6C ) . In contrast , the ends of moRs facing the termini of the miRNA stemloop were less well defined , suggesting that they are processed by exonuclease activity ( Fig 6B ) . Why certain miRNA hairpins are prone to accumulation of moRs remains unclear .
Small RNA pathways critically influence the outcome of virus infections in many host organisms , including plants , fungi , invertebrates , and vertebrates [3–5 , 15 , 69] . In plants and invertebrates , siRNA-mediated antiviral immunity is key to the defence against a broad range of virus infections . In Aedes mosquitoes , viral siRNAs were detected from several virus families , including Togaviridae , Flaviviridae , Bunyaviridae , and Reoviridae [5] . In line with previous reports , our analysis identified viral siRNAs derived from the entire genomic RNA of DENV2 in Aedes aegypti cells [35 , 36] . These vsiRNAs are produced in roughly equal amounts from the ( + ) strand and the ( - ) strand of the virus , indicating that the dsRNA replication intermediates serve as substrate for Dcr2 . Upon knockdown of either Dcr2 or Ago2 in whole Aedes mosquitoes , DENV2 titres and transmission are enhanced , underlining the pivotal role of RNA interference in limiting DENV2 replication [53] . Besides siRNAs , our DENV2 infected small RNA libraries contained a substantial number of virus-mapping reads in the size range of piRNAs . Their expression was confirmed using small RNA northern blotting , validating that these small RNA reads were no sequencing artefacts . Only very few DENV2 genomic locations near the 3’ end of the DENV genome give rise to these vpiRNAs but the origin of this spiky pattern remains obscure . We hypothesized that perhaps endogenous , transposon-derived piRNAs would loosely bind the DENV2 genome at these positions triggering the production of secondary vpiRNAs . A similar mechanism has been suggested to initiate piRNA production from specific mRNAs in Drosophila [28] . However , various mapping strategies allowing small RNA alignment with up to six mismatches did not uncover endogenous piRNAs that could trigger vpiRNA production at the observed positions . Previous analyses of DENV2-derived small RNAs identified vpiRNAs in Aedes aegypti mosquitoes or Aag2 cells [35 , 36] . Also in the viral small RNA population reported by Scott and colleagues [35] , a major small RNA spike is located near the 3’ end of the DENV genome . Since the entire population of viral RNA is analyzed in this study , it is however hard to assess which type of small RNA contributes to the spike . Interestingly , the spiky genome distribution of vpiRNAs is also recapitulated in adult Aedes mosquitoes . Although in this study the exact location of vpiRNA spikes differs from the positions we found in Aag2 cells , these data suggest that similar mechanisms may be responsible for piRNA biogenesis in Aag2 cells and adult mosquitoes . Yet , genetic evidence for the PIWI protein dependency of vpiRNA production in vivo is lacking . Of note , piRNAs are far less abundant in the small RNA libraries reported by Scott et al . [35] and Hess et al . [36] compared to our data from Aag2 cells . These differences might be due to different experimental conditions , including the chosen MOI , the time point of sampling , or to differences in small RNA library preparation and sequencing methodology . In addition , the specific viral strains may critically influence the accumulation of vpiRNAs . We have tested two laboratory-adapted DENV2 strains which both give rise to vpiRNAs , and it would be interesting to test if pathogenic strains from DENV2 endemic areas would show similar phenotypes . Using knockdown of PIWI proteins , we identified Piwi5 , Ago3 and , to a lesser extent , Piwi6 as responsible for the production of vpiRNAs in Aag2 cells . Therefore , DENV2 vpiRNA biogenesis in Aag2 cells relies on a similar set of PIWI proteins as SINV vpiRNAs , which also depend on Piwi5 and Ago3 [37] . We have recently proposed that Aedes PIWI proteins are specialized in producing piRNAs from various sources . Whereas in mosquito cells piRNA biogenesis from transposons directly and indirectly depends on Piwi4-6 and Ago3 , piRNA biogenesis from SINV predominantly requires Piwi5 and Ago3 only [37] . The additional involvement of Piwi6 for piRNA production from DENV2 suggests that Aedes PIWI proteins are even further specialized towards RNA substrates from different viruses . This may be caused by virus-specific sequence elements or structures that are preferentially recognized by certain PIWI proteins . Alternatively , but not mutually exclusive , differences in replication strategies or replication sites might favour recognition of viral RNA by distinct sets of PIWI proteins . The almost complete loss of DENV2 piRNAs upon knockdown of Piwi5 and Ago3 indicates that both proteins are equally important for vpiRNA biogenesis in Aag2 cells , similar to piRNA biogenesis during SINV infection [37] . SINV piRNAs are produced by a two-step amplification mechanism that resembles ping-pong amplification of transposon piRNAs in Drosophila [27 , 29] . During this process , a piRNA-loaded PIWI protein ( Piwi5 in Aedes or Aubergine in Drosophila ) slices a complementary target RNA and transfers the 3’ slicer products as the new piRNA precursor to a second PIWI protein ( Ago3 in Aedes and Drosophila ) . From this precursor , an Ago3-bound secondary piRNA is produced that in turn is able to slice a target RNA , giving rise to a new piRNA precursor . This precursor will be matured to generate the same primary piRNA sequence that initiated the amplification . Therefore , this model predicts the presence of piRNAs derived from both strands . Although also during SINV infection ( - ) strand derived piRNAs are only a minor fraction , they can be identified as the primary piRNAs by a nucleotide bias that is characteristic for Piwi5/Aub bound piRNAs ( uridine at position one ) . During DENV2 infection , 25–30 nt reads from the ( - ) strand are extremely scarce and they do not have the nucleotide bias that would classify them as primary piRNAs . Therefore , exactly how the production of the secondary , ( + ) strand vpiRNAs is triggered or whether a different , amplification-independent mechanism is responsible for their production , remains unclear . The expression of miRNAs from DENV2 genomic RNA is still debated . Based on small RNA sequencing data , Hussain and Asgari have described a set of six viral small RNAs that have miRNA-like properties [24] . Inhibition of one of them , vsRNA-5 , by complementary RNA molecules strongly enhances DENV virus replication [24] . However , expression of these viral small RNAs is generally not high and the relevance of such a lowly abundant small RNA during the exponential growth of a virus was therefore questioned [25] . In our dataset , we find a high number of specific vsRNA reads for only vsRNA-2 . For all the other predicted vsRNAs we find no or very low numbers of reads . vsRNA-2 is located on a hairpin at the very end of the DENV2 genome . This strongly resembles KUN-miRNA1 , a viral small RNA expressed in mosquito cells infected with West Nile virus , a related flavivirus [70] . KUN-miR1 is 21nt in size and its expression is Dcr1-dependent . In contrast , our data demonstrate that vsRNA-2 has a broad size distribution of primarily 26–28 nt , arguing against it being a canonical Dicer-dependent miRNA . Altogether , these data support the notion that miRNA-like small RNAs from DENV2 are extremely lowly abundant , with a questionable role in the regulation of viral replication . Modulation of host miRNAs after virus infection may be a mechanism that coordinates gene expression during the course of the immune response . Alternatively , it may be a consequence of a viral strategy to manipulate host gene expression . Comparing uninfected with DENV2-infected Aag2 cells showed that the expression of almost all miRNAs was unchanged upon infection . Only a handful of miRNAs were up or down-regulated after exposure to DENV2 . The fold changes ranged from approximately 4 fold up to 4 fold down . In whole Aedes aegypti mosquitoes , a total of 31 miRNAs were recently shown to be differentially regulated following infection at three different time points [17] . The set of differentially expressed miRNAs is inconsistent between the different analyzed time points ( 2 , 4 and 9 days post infection , dpi ) , but the number of differentially expressed miRNAs was higher at nine dpi than at two or four dpi . These data suggest that changes in miRNA expression may be more prominent in a long-term infection and we thus cannot exclude the possibility that prolonged infection of Aag2 cells may result in more pronounced changes in miRNA expression . Alternatively , the observed miRNA changes in adult mosquitoes might not directly happen in infected cells per se , but could reflect an indirect effect of homeostatic or metabolic responses during the infection . It should be remembered that miRNA expression can be highly cell-type specific; if miRNA levels are responsive to DENV2-infection only in selected cell types within the entire mosquito , we may miss those in our Aag2 cell-based assays . In summary , here we provide an in-depth analysis of small RNAs in DENV2 infected Aag2 cells in comparison to uninfected cells . Aag2 cells provide a powerful model system for studying biochemical details of small RNA biogenesis pathways , as they are fully competent in producing siRNAs , miRNAs and piRNAs originating from both virus and host . Our analyses add both DENV-derived piRNAs and novel Aedes aegypti miRNAs to the small RNA repertoire in this medically important virus-host interaction . | Mosquitoes of the Aedes family transmit many important viruses , including dengue virus , between their vertebrate hosts . In the mosquito , the growth of these viruses is limited by the antiviral RNA interference pathway . Key to this pathway is a class of small non-coding RNAs known as small interfering RNAs ( siRNAs ) . In addition , two related but distinct small RNA pathways known as the microRNA ( miRNA ) and the PIWI-interacting RNA ( piRNA ) pathway are implicated in regulating virus replication in mosquitoes . Thus , since small RNAs may critically influence the transmission of dengue virus , we set out to analyze the populations of viral and mosquito small RNAs that are produced in infected Aedes mosquito cells . We found that besides the well-known viral siRNAs , dengue virus-derived piRNAs were produced in these cells and we identified the PIWI proteins that these small RNAs rely on . In addition , we found that viral miRNAs were not expressed from the dengue virus genome and that the levels of mosquito miRNAs were barely changed upon infection . Finally , our data allowed for the identification of novel Aedes miRNAs , complementing the repertoire of these important regulatory RNAs in vector mosquitoes . | [
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"librar... | 2016 | Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs |
Mesial temporal lobe epilepsy ( TLE ) is characterized by stereotyped origination and spread pattern of epileptogenic activity , which is reflected in stereotyped topographic distribution of neuronal atrophy on magnetic resonance imaging ( MRI ) . Both epileptogenic activity and atrophy spread appear to follow white matter connections . We model the networked spread of activity and atrophy in TLE from first principles via two simple first order network diffusion models . Atrophy distribution is modeled as a simple consequence of the propagation of epileptogenic activity in one model , and as a progressive degenerative process in the other . We show that the network models closely reproduce the regional volumetric gray matter atrophy distribution of two epilepsy cohorts: 29 TLE subjects with medial temporal sclerosis ( TLE-MTS ) , and 50 TLE subjects with normal appearance on MRI ( TLE-no ) . Statistical validation at the group level suggests high correlation with measured atrophy ( R = 0 . 586 for TLE-MTS , R = 0 . 283 for TLE-no ) . We conclude that atrophy spread model out-performs the hyperactivity spread model . These results pave the way for future clinical application of the proposed model on individual patients , including estimating future spread of atrophy , identification of seizure onset zones and surgical planning .
Mesial temporal lobe epilepsy ( TLE ) is the most common form of focal epilepsy , and is characterized by seizure focus in the mesial temporal lobe from where it can spread into other neocortical regions . Based on their aspect on the structural MRI and histopathology , two subtypes of TLE are distinguished . One that is characterized by prominent hippocampal atrophy or mesial-temporal sclerosis ( TLE-MTS ) and one where the hippocampus appears completely normal ( TLE-no ) . In both cases , new morphometric analysis using MRI shows consistent evidence of extra-hippocampal and extratemporal atrophy [1–6] . It is shown by Coan et al that atrophy progression in temporal lobe epilepsy depends on lateralization [7] . In a related work , it is observed that patients with TLE show progressive neocortical damage , likely a result of seizures [8] . Atrophy distributions appear to vary significantly , and both widespread [9] and restricted distribution of gray matter ( GM ) atrophy [4 , 10] have been observed in TLE subjects . In most cases however , damaged regions tend to be functionally and anatomically connected to the hippocampus and other medial temporal structures [3 , 11–13] . Thus , both epileptogenic activity and gross atrophy in epilepsy establish themselves at networked sites , and appear to spread along fiber pathways . However , it is not clear whether activity or atrophy is the propagating event , and two hypotheses have been proposed . First , seizure activity is the primary propagating quantity , and neuronal damage results from excitotoxicity [14] . In this view , the extrahippocampal spread of seizure activity is primarily responsible for the apparent topographic distribution of atrophy [11 , 13 , 15] . Although atrophy in all regions might worsen with time , there is no progressive outward spread of atrophy . The medial temporal and limbic structures have important connections to each other and to the hippocampus; these connections provide significant feedback mechanisms [16] , leading to the spread of initially local epileptogenic activity to widespread connected regions . The process of neuronal death secondary to sustained hyperactivity is not fully understood , but likely involves mutual inter-cortical trophic exchanges , ultimately leading to long-lasting remodeling of brain networks [17] . Once epileptogenic activity is established in a region , over time it causes local atrophy via a complex cascade of neurobiological events [18] , a process called excitotoxicity . The close correspondence between the sites of epileptogenic activity and atrophy patterns observed in TLE patients described above supports this hypothesis . The second hypothesis is that loss of hippocampal neurons leads to remote deafferentation followed by gradual and progressive neuronal loss in connected regions . Impoverishment of hippocampal connections can lead to reduced complexity of remote circuitry [9] . The processes leading up to remote atrophy are also complex , and several mechanisms have been suggested [19] . Studies of the temporal and spatial sequencing of epilepsy and regional atrophy suggest that brain atrophy is a dynamic process that progresses over time [20] . In support of this hypothesis , it was noted that seizure spread and atrophy are unrelated [21] , while white matter fiber integrity was correlated with remote atrophy [9] . Hence , the spread of both activity and atrophy are partially supported by prior studies , and a definite consensus eludes the field . The medial temporal lobe is highly connected and either case could theoretically lead to significant temporal atrophy . Since both modes of spread involve the anatomic connectivity network of the brain , a purely phenomenological or statistical analysis would not be able to disambiguate between them , as both would estimate similar topography , at least in the vicinity of the hippocampus . However , the brain-wide topography estimated by the two models are sufficiently distinct to allow quantitative disambiguation between the two , but this would require quantitative network modeling ( see subsections Model I: Spread of Epileptogenic Activity and Model 2: Spread of Atrophy via Progressive Degenerative Process under Materials and Methods section ) . The purpose of this paper is to develop network theoretic models of regional atrophy dynamics resulting from each of the above hypotheses , and to statistically determine which model is a better descriptor of the spatial patterning of real TLE atrophy . Both models are based on simple graph theoretic models of influence spread as a Network Diffusion ( ND ) process , enacted on the brain’s structural connectivity network whose nodes correspond to GM regions ( obtained from atlas-based parcellation of T1-weighted MRI ) and edges correspond to WM tracts between them ( obtained from diffusion tensor imaging , DTI , followed by fiber tractography ) . The first model captures the network-centric spread of epileptogenic activity , using a recently developed network model of the spread of functional activity in brain networks [22] . Regional atrophy is treated as a simple consequence of established patterns of epileptic activity . This model only considers the time-averaged propensity of a region of experiencing epileptic activity , hence it does not preclude different patterns of seizure activity at different times in the same individual . The second model captures the spread of atrophy as a progressive degenerative process resulting from deafferentation . We show that this too can be adequately modeled by a very similar ND process , with the critical difference from the first model being that an initial atrophy seed is needed , putatively hippocampus in TLE-MTS , followed by a time-resolved progression of atrophy into distal regions . Epileptogenic activity does not enter this model , and deafferentiation is the primary propagating event . A similar model was employed to capture the spread of neurodegeneration in dementias [23] . Note that the complex neurobiology of excitotoxicty and remote degeneration cannot be tested using these macroscopic graph theoretic models . Our main contribution is to mathematically encode two competing but equally plausible mechanisms of spread , and show that they have differential macroscopic consequences which can be rigorously tested using imaging data alone , without requiring electrophysiology data . It is of course possible and likely that both excitotoxicity and deafferentation occur concomitantly in the same epileptic brain; this study is not designed to separate these two effects in an individual , but to judge , from group data , which of these two mechanisms plays a dominant role in the entire TLE population . We tested the two models using group level atrophy distributions observed in two TLE cohorts: a ) non-lesional temporal lobe epilepsy with mesial temporal sclerosis , and b ) non-lesional temporal lobe epilepsy with no MRI-visible mesial temporal sclerosis . We show that while both models mimic group-level measured atrophy distribution , the ( remote degeneration ) spread-of-atrophy model performs better than the spread-of-activity model in both TLE-no and TLE-MTS groups . The study cohort , like the disease itself , contains considerable inter-subject variability , hence group-level tests will not be valid at the individual level . However , our purpose is not to apply this approach to individuals , but only to test which of two network models is better supported by group level data reflecting the relative involvement of different brain regions in the TLE population as a whole . Other potential etiologies of TLE atrophy unrelated to the network , including pre-existing conditions such as subtle cortical malformation microdysgenesis [24] or genetic variants that could be associated with specific atrophy patterns , are not considered in this study .
The TLE-MTS measured atrophy reflected in Fig 1 ( a ) indicates , as expected , pronounced atrophy in the hippocampus , with less marked atrophy ipsilaterally in pars orbitalis , amygdala , VDC , pallidum , thalamus and inferior temporal gyrus . Contralateral temporal pole , transverse temporal gyrus , entorhinal cortex ( ERC ) and pars opercularis also reveal some atrophy . The measured atrophy of TLE-no , Fig 3 ( a ) , reveals pronounced atrophy broadly , bilaterally distributed across cortical and subcortical regions , particularly in the frontal and temporal lobes . The region with the highest atrophy is the contralateral transverse temporal gyrus , with t-statistics of 3 . 74 . Other regions with high atrophy include the contralateral precentral gyrus ( t-statistics 3 . 29 ) and the postcentral gyrus ( t-statistics 3 . 45 ) . To visualize the possibility of outliers or anomalous distribution of points in both models , we scatter plot the measured neuronal atrophy vs . its estimate for both models and both types of epilepsy . Fig 4 ( a ) and 4 ( b ) give the scatter plots for the case of TLE-MTS Models 1 and 2 respectively . The Model 1 plot reveals more widely scattered points than in the case of Model 2 , where the model is more selective in its estimate of atrophy . This is also consistent with the higher R obtained from Model 2 . Similar observations can be made for the TLE-no case , Fig 4 ( c ) and 4 ( d ) ; while the plots are more scattered than in the TLE-MTS case above , Model 2 still gives a more accurate estimate of the neuronal atrophy than Model 1 . In order to further explore the models’ sensitivity to outliers , Fig B in S1 Text gives the scatter plots of the empirical atrophy vs the logarithm of the estimated atrophy . The resulting R is computed for all four cases . As reflected in the figure , Model 2 is once again superior to Model 1 for both TLE-MTS ( R = 0 . 400 vs R = 0 . 340 , Figs . B ( a ) and B ( b ) ) , and TLE-no ( R = 0 . 270 vs R = 0 . 170 , Figs . B ( c ) and B ( d ) ) . To rule out the possibility that the above results were obtained by chance , we predict a randomly shuffled version of the measured atrophy for both types of epilepsy using both Models 1 and 2 . For each model and each epilepsy type we run 1 , 000 iterations of randomly shuffled versions of the measured atrophy and compute the resulting R . For Model 1 , in each iteration we find R for Φ1 Eq ( 10 ) with the sum evaluated up to K with 2 ≤ K ≤ 86 . For Model 2 , we evaluate Φ2 Eq ( 14 ) over a range of t when the hippocampus is seeded ( case TLE-MTS ) , and when the paracentral sulcus is seeded ( case TLE-no ) . For each iteration , the correlation R is computed in a similar fashion to sections TLE-MTS and TLE-normal above . Fig 5 summarizes the findings . Fig 5 ( a ) indicates the histogram resulting from the randomly shuffled measured atrophy in the case of Model 1 and TLE-MTS . None of the shuffled atrophies achieved a correlation exceeding that obtained from the empirical measured atrophy ( R = 0 . 394 ) . Similarly , measured atrophy randomization in Model 2 gives a histogram where the measured atrophy has the highest R , Fig 5 ( b ) . The TLE-no case does not perform as well . Fig 5 ( c ) gives the histogram obtained in the case of Model 1 . The measured atrophy has a correlation of R = 0 . 213 , with 57 randomly shuffled instances of the atrophy yielding higher R , with a maximum R = 0 . 426 . Nonetheless , only a small percentage of all iterations , 5 . 7% give R > 0 . 213 . Model 2 gives improved results , with only 8 random atrophy patterns having R > 0 . 283 ( 0 . 8% of all iterations ) . The highest correlation obtained is R = 0 . 323 . The fact that all reported R values are at the extreme end of the null distribution conveys a strong suggestion that the network is indeed a relevant modulator or atrophy in TLE . The fact that the reported R statistic of Model 1 , but not of Model 2 , is only moderate in relation to the null histogram further cements our conclusion that Model 2 ( degenerative spread ) is a superior network model than Model 1 ( excitotoxicity spread ) .
The brain graph’s Laplacians eigen-modes should be intuitively understood as sub-networks within which an excitotoxicity-driven process would get “trapped” and reverberate . Network eigen-modes are increasingly appearing in brain science; for instance , they act as attractors for dementia pathology [23] . A description of the neuroscientific meaning of these eigen-modes , as applied to brain activity spread , was given in our previous paper on this topic—see [22] . Our results support a somewhat speculative role of network eigen-modes in epileptic processes , although we note that this study was not designed to explore this aspect in detail . Our theory finds that atrophy ( and activity ) would be predominantly contained within a few small spatially distributed but separate sub-networks of the brain , given by the eigen-modes of the network Laplacian–Eqs ( 10 ) and ( 14 ) . Closer inspection of selected small eigen-modes ( Fig 2 ( a ) and 2 ( b ) ) reveals a compelling picture whereby u2 matches the spatial pattern of TLE-no atrophy , and u5 matches TLE-MTS . Both eigen-modes are dominated by temporal , medial and subcortical structures—precisely the regions that are most frequently associated with TLE . To be sure , other eigen-modes also bear resemblance to atrophy data ( Fig 2 ( c ) and 2 ( d ) ) , but due to their large eigen-value they do not contribute significantly to either model . The marked predilection of epilepsies to occur in medial temporal and subcortical regions is well known , but poorly understood more than 60% of all epilepsies are of the temporal type , and of those 80% have MTS ( statistics from epilepsy . com ) . Why are certain regions especially vulnerable to epileptic seizures , and why are the patterns of atrophy in certain epilepsies so stereotyped ? Given that seizures can in theory arise in any part of the brain ( as they frequently do , in frontal , parietal and other regions ) , that seizures frequently generalize outside of onset zones , and sometimes even recruit the entire brain , the principle of parsimony would dictate that epilepsies should recruit all regions equally . That this is not so suggests that vulnerable structures enjoy a privileged location in the brain network . However , these regions do not stand out in terms of degree , clustering , path length or other conventional network theory metrics . That small network eigen-modes consistently implicate these regions , which are central actors in epileptogenic activity and atrophy , could provide a potential explanation for regional and stereotyped predilection of TLE , and why it happens to be the most common variant of focal epilepsy [25] . Thus , temporal , limbic and subcortical regions are located in a network neighborhood that anchors some of the smallest network eigen-modes , which act as attractors for epileptogenic activity or degenerative processes . This interpretation of small eigen-modes was first put forward in [23] in the context of neurodegenerative disease modeling , and current results suggest a similar , albeit speculative role in epilepsy . Similar to previous morphometric results [5 , 31 , 32] , the spread-of-activity model ( Model 1 ) finds widespread atrophy in the ipsilateral temporal lobe ( fusiform and parahippocampal ) . In addition to the regions outlined in [5] for TLE-MTS , Model 1 finds atrophy in the ipsilateral middle temporal gyrus , lateral orbital frontal gyrus , and precentral and postcentral gyri . The model also finds significant subcortical atrophy , particularly in the hypothalamus , amygdala , and hippocampus . High atrophy is also found in the contralateral thalamus and hippocampus . TLE-MTS atrophy is also widely reported in the frontal lobe [33] where it is proposed that under certain conditions mesiotemporal epileptic activities would propagate through the thalamus to the frontocentral areas . Frontal and parietal regions play a crucial role in the evolution of complex partial seizure [28] , and Model 1 ( but not Model 2 ) captures this , especially in lateral orbitofrontal regions . On the other hand Model 2 reveals atrophy patterns chiefly in the subcortical ipsilateral region , chiefly the hippocampus , amygdala , hypothalamus , and thalamus , all strongly implicated in epileptogenesis [5 , 32] . An additional region captured by Model 2 ( but not Model 1 ) , is ERC ( Fig 1 ( f ) ) . TLE-normal atrophy is known to be widespread bilaterally , with no apparent focus nor sclerosis visible on MRI [5 , 34 , 35] . Both network models give bilateral effects without a clear focus . In this group , the progressive degeneration model is not as strongly supported as for the MTS group , since the highest R from seeding various regions does not show a clear winner ( Fig 3 ( d ) ) . The most plausible seeds are precentral gyrus and frontal pole—neither being an especially prominent source of either seizure activity or atrophy in TLE . The progressive degenerative model with precentral seeding largely agrees with previously published results [5 , 34 , 35] , with significant atrophy found in the ipsilateral inferior temporal gyrus , an epileptogenic focus candidate [5] . Regions with significant estimated atrophy not reported in [5] include the contralateral fusiform and the ipsilateral superior frontal gyrus . The latter node is structurally connected with the ipsilateral putamen where the proposed network diffusion model estimates high atrophy . This study is related to but distinct from conventional analysis of epilepsy networks [6 , 17 , 36–39] or functional connectivity analyses [40–42] . Additionally , the effect of TLE on the structural network topology has been well studied , see for example [43–46] . Here our focus is in developing models of networked spread from first principles , rather than obtaining descriptive network statistics for epilepsy networks . The general class of network dynamics developed here , network diffusion , is well known in the area of signal and image processing [29 , 30] . For example it has been used for image enhancement [29] using weighted graphs . In [30] the authors propose an image smoothing method using heat kernel and weighted undirected network . Bougleux et al consider discrete image smoothing and denoising on weighted graphs using Laplacian operators [47] . Our laboratory was the first to develop diffusive dynamics enacted on networks as a macroscopic model of the evolution of neurodegenerative brain diseases like Alzheimer’s disease and other dementias [23] . However this does not imply any similarity in pathophysiological mechanisms between dementia and epilepsy , two different disease processes . The spread-of-activity model is based on our prior work on modeling the patterns of functional connectivity in the brain [22] , where we hypothesized that on a given structural network , functional influences must travel along connected edges , and satisfy first-order diffusive dynamics which are natural for this kind of influence propagation . The ability of a common mechanistic network model to mimic disparate brain behaviors , from neurodegenerative spread to functional correlations to epilepsy does suggest a striking convergence in the behavior of large scale brain phenomena . Conceptually , the prime limitation of this study is that it assumes no cross-talk between the two alternative models of spread , activity and atrophy—necessitated by the purpose of testing which model “wins” . It is not known whether this is strictly accurate , and several reports suggest evidence of interplay [9 , 19–21] . However , we believe that a statistically sound first step mandates that the two mechanisms be tested and judged separately before potential interplay can be realistically modeled . Additional effects such as developmental abnormalities may come into play . This however is always an issue in any disease with a wide spectrum of causative factors , such as epilepsy . This should not , and does not , reduce the utility of network modeling efforts as long as one can strongly believe that the network is modulating the advance of disease , regardless of etiology . There is no doubt that such is the case with epilepsy . We emphasize that the presented models cannot address disease etiology or pathophysiology; its value lies in showing that the macroscopic effect of network dynamics can largely explain the stereotyped patterns of atrophy in TLE , regardless of individual subjects etiologic factors . Another limitation is that while we model the spread of activity , we do not utilize electrophysiological data to test this , instead relying on regional atrophy patterns . This too is necessitated by the current goal of testing between competing models , but in the future it would be desirable to explore the spatiotemporal dynamics of EEG recordings . For this work the whole brain EEG data needed to achieve network modeling has proven exceedingly difficult to acquire on our patients . Current connectome technology based on diffusion MRI and tractography underestimates long-range connections , and is especially unreliable at estimating inter-hemispheric connections and tracts passing through complicated neighborhoods , for instance frontotemporal connections via the uncinate fasciculus and the connections between the limbic system and orbitofrontal cortex . As a result the model’s estimated atrophy shows bias for ipsilateral hemisphere and underestimates contralateral atrophy . Orbitofrontal cortex is strongly connected to limbic structures and shows significant atrophy in both TLE-MTS and TLE-no cases , but is underestimated by our models due to underweighted uncinate fasciculus connectivity . Another limitation is that our models assume static connections and do not account for change in connections due to alteration in activity or atrophy . Since this study involves measured atrophy , we could only include epilepsy subtypes TLE-MTS and TLE-no which have stereotyped topography of atrophy . A preliminary investigation of cases of more heterogeneous epilepsies gave inconsistent patterns amongst subjects as expected , and made the model correspondingly non-informative . Hence proposed models may not be applied at group level to heterogeneous epilepsies .
This study was approved by UCSF’s IRB and written informed consent was obtained from each subject according to the Declaration of Helsinki . Seventy nine patients suffering from drug resistant TLE were recruited between mid 2005 and end of 2007 from the Pacific Epilepsy Program , California Pacific Medical Center and the Northern California Comprehensive Epilepsy Center , UCSF , where they underwent evaluation for epilepsy surgery . In a brain network each node represents a GM region located on either the neocortex or in deep brain subcortical areas . We define a network 𝒢 = ( 𝒱 , ℰ ) with a set of N nodes given by 𝒱 = { v i ∣ i ∈ 1 , … , N } and a set of edges given by an ordered node pair ℰ = { ( i , j ) ∣ i ∈ 𝒱 , j ∈ 𝒱 } [48] . Between any two nodes i and j there might exist an edge representing a fiber tract whose connectivity weight ci , j ∈ [0 , ∞ ) can be measured from the streamlines of the dMRI tractography , and this defines a connectivity matrix C = { c i , j ∣ ( i , j ) ∈ ℰ } . Although some individual neurons are known to be directional , dMRI does not allow measurement of directionality . Major fiber bundles resolvable by dMRI , especially cortico-cortical pathways are generally bidirectional , having roughly equal number of connections in either direction [49] . We define the connectivity strength or the weighted degree of a node i in this network as the sum of all connection weights: δ i = ∑ j ∣ ( i , j ) ∈ E c i , j . ( 1 ) Table 3 describes the various parameters and variables used in this work . We codify here , from first principles , the hypothesis that epileptogenic activity in the epileptogenic focus spreads outward following known anatomical pathways . What is described here is a simple diffusion process , a model that applies to any spreading quantity . Here , that quantity is brain activity , and we model how it would propagate along the structural network of the brain . For an isolated GM region R1 , the average activation signal over all its neurons x1 ( t ) is proportional to the number of hyperactive neurons per voxel . We assume a simple damped system behavior , given by dx1 ( t ) /dt = −βx1 ( t ) . This damped behavior is consistent with epileptogenic activity , which has a transient nature and dies away exponentially . Damping in such a system may be assumed to arise from gradual loss of synchrony due to dephasing between neurons . Damping behavior of this sort has a long history in brain signal modeling , especially in neural mass approximations of neuronal assemblies [50] , firing rate neural models [51] , and is ultimately governed by membrane time constant of leaky-integrate-and-fire neurons [52] in all these exponential signal decay is the key component . Next consider a pair of cortical regions R1 and R2 , whose connectivity weight is c1 , 2 . The number of hyperactive neurons in R2 is proportional V2 x2 , where V2 is the number of voxels in R2 . Of these , the number of axonal projections from R2 to R1 is proportional to c 1 , 2 1 δ 2 V 2 x 2 , where we divide the connectivity by the weight of R2 , δ2 ( see Eq ( 1 ) , to get a ratio . The proportion of neurons in R1 which experience a hyperactive afferent from R2 , assuming uniform mixing of afferents , is then given by 1 V 1 c 1 , 2 1 δ 2 V 2 x 2 . Our key assumption next is that each afferent hyperactive neuron is able to induce hyperactivity in connecting neurons at a constant rate , modeled as a Poisson process . Therefore , the rate of change in the number of R1 neurons which undergo hyperactivation secondary to enervation by R2 , after accounting for the internal ( damped ) dynamics of R1 , is given by d x 1 ( t ) d t = β ( 1 V 1 c 1 , 2 1 δ 2 V 2 x 2 ( t ) - x 1 ( t ) ) ( 2 ) where we assume identical rate constant β for both the internal and external signals contributing to the dynamics of R1 for simplicity . For multiple afferents into R1 , we modify this to d x i ( t ) d t = β ( 1 V i ∑ j c i , j 1 δ j V j x j ( t ) - x i ( t ) ) . ( 3 ) Assuming a previously proposed relationship between GM volume and degree given by V k ∝ δ k ∀ k ∈ 𝒱 [22] we have d x i ( t ) d t = β ( δ i - 1 2 ∑ j c i , j δ j - 1 2 x j ( t ) - x i ( t ) ) . ( 4 ) Expanding to include the entire network , Eq ( 4 ) can be expressed compactly as: d x ( t ) d t = - β L x ( t ) , ( 5 ) where x ( t ) is an N × 1 vector describes the fraction of hyperactive epileptogenic neurons in all brain regions , and the matrix ℒ is the well-known symmetric and nonnegative definite network Laplacian L = I - Δ - 1 / 2 C Δ - 1 / 2 , ( 6 ) where Δ is the diagonal degree matrix with the node degree δi as the ith diagonal element . A closed form solution to Eq ( 5 ) is given by x ( t ) = exp ( - β L t ) x 0 . ( 7 ) Details of relevant network theory can be found in [48] . This describes the first order dynamics of the spread of epileptogenic neuronal populations along brain networks , starting at an initial configuration x0 . The ND model above is mass conserving , entailing no increase of hyperactivity overall , merely its distribution from focal loci to wider networks . This is appropriate for TLE , where ictal activity in medial temporal regions rarely generalizes systemically or leads to a total loss of consciousness [28] . It is important to note that the time-scale of above equations is related to neural signal transmission , measured in milliseconds . The spread-of-activity model assumes that atrophy in any brain region is the consequence of excitotoxicity induced by lifetime epileptogenic activity in that region . The process of neuronal death secondary to hyperactivity is not fully understood , and several processes have been implicated , including trophic exchanges , remodeling of brain networks [17] , and complex cascade of neurobiological events [18] . At a macroscopic level , however , these complexities may not be germane , and the overall accumulative effect on atrophy may simply be captured by: Φ 1 ∝ ∫ 0 ∞ x ( τ ) d τ . ( 8 ) In this model , the stronger and more frequent the level of hyperactivity experienced by a region , the more atrophy it will suffer . Although a full verification of the network diffusion model of activity spread would require multi-channel ictal and interictal EEG measurements , using the above model relating activity to atrophy we are able to circumvent this , and instead utilize MRI-derived regional atrophy as the end measure of the model . Due to integration to ∞ , atrophy in this model does not explicitly depend on diffusion depth t . Expanding Eq ( 8 ) in terms of Laplacian eigen-modes and exchanging the order of summation and integration: Φ 1 = ∑ i = 1 N u i u i ′ x 0 ∫ 0 ∞ e - β λ i t d t , ( 9 ) and where {λi} are the eigenvalues of the Laplacian matrix ℒ , and {ui} are the corresponding eigen-modes . Evaluating the definite integral above we obtain Φ 1 = 1 β ∑ i = 1 N 1 λ i u i u i ′ x 0 = 1 β L † x 0 , ( 10 ) where we consider only the terms with i > 1 , since the first eigen-mode simply represents uniform activation of the entire brain , which is not relevant in TLE , which rarely displays generalized epileptogenic activity that recruits the entire brain . ℒ † refers to the interesting observation that the above expression evaluates to the pseudo-inverse of ℒ . Further , due to the 1 λ i term , the smallest eigenvalues have the most dominant contribution , thus the summation above is only needed for the first few eigen-modes . In Results section , we evaluate the similarity between measured atrophy patterns and Φ1 over varying number of eigen-components . This model assumes that rather than epileptic activity the process of progressive deafferentation is the propagating event , a process initiated at onset zones by excitotoxicity and other causes , but which thereafter spreads throughout the brain network via remote degeneration . Again , the complex neurobiological cascades leading to frank remote degeneration , involving axonal reaction , inflammation , autophagy , oxidative damage , synaptic dysfunction , loss of trophic support , anterograde and retrograde degeneration , and finally frank neuronal death [19] are not attempted to be modeled here . Instead , we assume that on a macroscopic level , these details are not germane , and that the overall behavior is governed by linear dynamics . Given that degeneration is a result of slow processes which build up over time rather than act instantaneously , it is appropriate to model the local ( to the region ) degenerative dynamics set in motion by a single event entailing loss of y 1 0 neurons per voxel in an isolated region R1 via an impulse response function , which is commonly used in linear systems theory to characterize such non-instantaneous effects [53] . Since the exact shape of this impulse response is not known , we choose one of the simplest but most widely used plausible causal functions , given by the exponential decay function y 1 ( t ) = y 1 0 exp ( − γ t ) , which simply encodes the expectation that a single insult will cause further but non-instantaneous degeneration , persisting in the region with a half-life of 1/γ . This corresponds again to a simple damped system behavior , which may be written as dy1 ( t ) /dt = −γy1 ( t ) . Next consider a pair of cortical regions R1 and R2 , whose connectivity weight is c1 , 2 . In a short time interval δt , let the number of newly deceased neurons in R2 be V2 y2 , where V2 is the number of voxels in R2 . This group of external newly deceased neurons then cause degeneration in R1 , modeled as a Poisson process in close analogy to Model 1 . Thus , accounting for both the internal and externally-induced atrophy dynamics in R1 , we have d y 1 ( t ) d t = γ ( 1 V 1 c 1 , 2 1 δ 2 V 2 y 2 ( t ) - y 1 ( t ) ) ( 11 ) where we assume identical rate constant γ for both the internal and external atrophy dynamics for simplicity . Since these equations are fully analogous to Model 1 , on the entire network we have d y ( t ) d t = - γ L y ( t ) , ( 12 ) where y ( t ) is an N × 1 vector describes the density per voxel of dying neurons in all brain regions . Eq ( 12 ) admits a closed form solution y ( t ) = exp ( − γ ℒ t ) y 0 , giving a time-dependent process starting with initial “seed” map y0 at t = 0 , and ending at a uniform distribution at t = ∞ . Since y ( t ) denotes the number of newly deceased neurons at any instant , the overall atrophy during the degenerative process is given by the time integral Φ 2 ( t ) ∝ ∫ 0 t y ( τ ) d τ , ( 13 ) which has a closed form solution Φ 2 ( t ) = 1 γ ∑ i = 1 N 1 λ i ( 1 - exp ( - γ λ i t ) ) u i u i ′ y 0 , ( 14 ) In contrast to Φ1 , Φ2 ( t ) can be viewed as a function of time , but here t has units of years rather than milliseconds , since the model captures the slow spread of degenerating neurons . Since the true time since onset is not empirically accessible in general , we will estimate t as the instant tcrit when the theoretical pattern Φ2 ( t ) best matches measured atrophy pattern in the subject . Both tcrit and the rate constant γ are a priori inaccessible , and must be empirically determined by data fitting . Seed vector y0 is known in the TLE-MTS case to a high level of confidence , since prominent hippocampal sclerosis indicates a high likelihood that it is indeed the focus location . Thus , we initialize y0 by a unit vector which is zero except for the element corresponding to the hippocampus node which is 1 . Inspection of Eqs ( 10 ) and ( 14 ) reveals that they differ only in the indefinite integral over t; consequently Model 1 is simply Model 2 , evaluated at t = ∞ . Thus , one simple test of which model is better would be to determine whether the fit with real data peaks at an intermediate value of t ( Model 2 wins ) or whether the fit is monotonically increasing in t , peaking at t = ∞ ( Model 1 wins ) . The epilepsy population consisted of 29 TLE-MTS subjects ( mean age 39 . 79±10 . 77 , left TLE/right TLE 15/14 , females/males 17/12 ) . The presence of MTS in TLE-MTS was suggested by hippocampal subfield volumetry using high-resolution T2-weighted images aimed at the hippocampus . The TLE-no cohort consisted of fifty subjects ( mean age 37 . 64±10 . 19 , left TLE/right TLE 20/30 , females/males 27/23 ) . The healthy control group had mean age 35 . 93±9 . 92 and females/males 45/16 . Identification of epileptogenic focus was based on seizure semiology and prolonged ictal and interictal video/EEG/telemetry ( VET ) in all patients . In this work non-lesional epilepsy is defined as TLE with or without MTS but no other pathology detectable on visual inspection , e . g . tumor , dysplasia , vascular or other malformation . All imaging of epileptic subjects was performed on a Bruker MedSpec 4T system controlled by a Siemens Trio TM console and equipped with an eight-channel array coil ( USA Instruments ) . The following sequences were acquired: ( 1 ) For cortical thickness and thalamus measurements a volumetric T1-weighted gradient echo MRI ( MPRAGE ) : TR/TE/TI = 2300/3/950 ms , 1 . 0 × 1 . 0 × 1 . 0mm3 resolution , acquisition time 5 . 17min . ( 2 ) For the measurement of hippocampal subfields , a high-resolution T2-weighted fast spin echo sequence: TR/TE = 3500/19 ms , 0 . 4 × 0 . 4mm2 in plane resolution , 2mm slice thickness , 24 slices acquisition time of 5 . 30min . ( 3 ) For the determination of intracranial volume ( ICV ) , a T2-weighted turbo spin echo sequence: TR/TE = 8390/70 ms , 0 . 9 × 0 . 9 × 3mm3 nominal resolution , 54 slices , acquisition time of 3 . 06min . A cohort of normal subjects were collected jointly by Weill Cornell Medical College and the Brain Trauma Foundation to create the normative connectivity information in the form of tractograms , see [54] for details . Seventy three healthy subjects were used to create the normative connectivity information in the form of tractograms . T1-weighted structural and diffusion-weighted MR images were collected on a 3T GE Signa EXCITE scanner ( GE Healthcare , Waukesha , WI ) . The High Angular Resolution Diffusion Images data were acquired with 55 isotropically distributed diffusion-encoding directions at b = 1000sec/mm^2 and one at b = 0sec/mm^2 , from 72 1 . 8mm thick interleaved slices ( no slice gap ) and 128 × 128 matrix size , zero-filled during reconstruction to 256 × 256 . Proposed and validated in Iturria-Medina et al [55] , the tractography algorithm implemented here incorporates tissue classification probability and orientation distribution information using Bayesian methods . We define measured atrophy as the volumetrics t-statistics s obtained from a healthy group and an epileptic group ( TLE-MTS and TLE-no ) . We assume that the neuronal atrophy resulting from epilepsy is proportional to the cortical/subcortical volumetric change relative to healthy brains . The two-sample t-statistic of the epileptic groups were computed relative to the healthy control group . For two samples x and y of sizes n and m and with variances σ x 2 and σ y 2 , the t-statistic is given by s = x ¯ - y ¯ σ x 2 n + σ y 2 m , where x ‾ and y ‾ are respectively the means of x and y . In this work , x and y are the volumetrics matrices obtained from the healthy group x and the epileptic groups y . The use of the t-statistic between healthy and disease groups’ regional volumes in this manner is a well known and widely used surrogate for measuring atrophy at a regional level , and forms the basis for almost all brain volumetric pipelines , e . g . [26 , 31 , 56] . We apply the network models described by Eqs ( 10 ) , ( 14 ) on the Laplacian ℒ of the 86 nodes mean structural connectivity of a population of healthy brains . In the case of the spread of activity model , Eq ( 10 ) , we bilaterally seed all of the temporal nodes ( vector x0 of Eq ( 10 ) has ones for the elements corresponding to both temporal lobes , and zero elsewhere ) . The atrophy Φ1 is then computed over subsets of the Laplacian eigen-modes ( 1 β ∑ i = 1 K 1 λ i u i u i ′ x 0 , 1 ≤ K ≤ 86 ) until all eigen-modes have been exhausted . At each value of i , R between group atrophy and Φ1 is computed . The Φ1 with the highest R is taken as Model 1’s estimate of neuronal atrophy . Regarding Model 2 , atrophy via remote degeneration , a seed is placed at a given node , then Φ2 ( t ) Eq ( 14 ) is computed over a range of diffusion depth values t . At each value of t the correlation of Φ2 with group atrophy at the given node is computed . The Φ2 with the highest R is then Model 2’s estimated atrophy for the seeded node . The process is repeated for all nodes . The Φ2 yielding the highest R of all nodes is the model’s estimate of the neuronal atrophy . In this work we discard the correlation R obtained in less than three time points ( t < 3 ) diffusion since this does not allow the diffusion to propagate significantly into the Laplacian network . In such cases , we choose instead the highest local maximum . We note however that all of the obtained atrophy estimates result in R vs t curves with unique maxima with t > 2 . The resulting correlation curve generally takes on a bell shape , but occasionally exhibits local maxima or even monotony , in which case we choose the time point corresponding to the highest one . | Medial temporal lobe epilepsy is the most common form of focal epilepsy . In this work we investigate two models describing the dynamics of epilepsy . In the first model the extrahippocampal spread of seizure activity is primarily responsible for the apparent topographic distribution of atrophy . The second hypothesis is that loss of hippocampal neurons leads to remote deafferentation followed by gradual and progressive neuronal loss in connected regions . Impoverishment of hippocampal connections can lead to reduced complexity of remote circuitry . The purpose of this work is to develop network theoretic models of regional atrophy dynamics resulting from each of the above hypotheses , and to statistically determine which model is a better descriptor of the spatial patterning of real TLE atrophy . Both models are based on simple graph theoretic models of influence spread as a network diffusion process , enacted on the brains structural connectivity network . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2015 | Relating Cortical Atrophy in Temporal Lobe Epilepsy with Graph Diffusion-Based Network Models |
Killed oral cholera vaccines ( OCVs ) have been licensed for use in developing countries , but protection conferred by licensed OCVs beyond two years of follow-up has not been demonstrated in randomized , clinical trials . We conducted a cluster-randomized , placebo-controlled trial of a two-dose regimen of a low-cost killed whole cell OCV in residents 1 year of age and older living in 3 , 933 clusters in Kolkata , India . The primary endpoint was culture-proven Vibrio cholerae O1 diarrhea episodes severe enough to require treatment in a health care facility . Of the 66 , 900 fully dosed individuals ( 31 , 932 vaccinees and 34 , 968 placebo recipients ) , 38 vaccinees and 128 placebo-recipients developed cholera during three years of follow-up ( protective efficacy 66%; one-sided 95%CI lower bound = 53% , p<0 . 001 ) . Vaccine protection during the third year of follow-up was 65% ( one-sided 95%CI lower bound = 44% , p<0 . 001 ) . Significant protection was evident in the second year of follow-up in children vaccinated at ages 1–4 years and in the third year in older age groups . The killed whole-cell OCV conferred significant protection that was evident in the second year of follow-up in young children and was sustained for at least three years in older age groups . Continued follow-up will be important to establish the vaccine's duration of protection . ClinicalTrials . gov NCT00289224 .
Cholera is a major global public health problem , causing both epidemic and endemic disease . Although conventional , injectable cholera vaccines have been abandoned as public health tools , modern oral cholera vaccines ( OCVs ) have been found to be safe and effective [1] . A recently revised World Health Organization ( WHO ) position paper expands the potential role of vaccination as a preventive tool against both endemic and epidemic cholera [2] . There are two licensed OCVs currently available: one containing cholera toxin B subunit ( BS ) and killed cholera whole cells ( WC ) , which is licensed in over 50 countries , and the other containing only killed WC , which is licensed in India and Vietnam [1] , [3] . A field trial of BS-WC vaccine in Bangladesh found that a three-dose regimen was safe and conferred high grade ( 85% ) short-term protection against cholera; protection was clearly evident throughout the first two years of follow-up , but markedly declined in the third year [4] . An advantage of the WC-only vaccine is its low cost , now at $1 . 85 per dose to the public sector . We conducted a placebo-controlled , randomized trial to assess the safety and protection conferred by a two-dose regimen of the WC-only vaccine against cholera severe enough to warrant solicitation of medical care . An initial analysis of an ongoing field trial in Kolkata of the WC-only vaccine found a two-dose regimen to be safe and to confer 67% protective efficacy against cholera at two years of follow-up [5] . Here we present results from the third year of follow-up of the Kolkata trial .
Each dose of the killed WC OCV ( Shanchol™ , Shantha Biotechnics ) , contains inactivated Vibrio cholerae 01 cells representing the El Tor and classical biotypes and the Inaba and Ogawa serotypes , as well as serogroup 0139 cells . Vials containing identical–appearing heat-killed Escherichia coli K12 cells were used as placebo . Single-dose vials were labeled with one of four letter codes , two for vaccine and two for placebo . Project staff and study subjects were unaware of the identities of the codes . Participants were randomly assigned , by residential dwelling , to vaccine or placebo groups . Randomization was done before enrollment by an independent statistician ( AD ) , using a random number table . Dwellings were randomized in blocks of 4 , corresponding to the 4 code letters used to label vaccine and placebo , within strata defined by the ward of residence and the number of residents in the dwelling ( six strata ) . Each agent was given as a two-dose regimen with an inter-dose interval of at least 14 days . Enrollment and administration of the pre-assigned agents was performed by dosing teams in vaccination centers serving the population . Codes were kept secretly at Shantha Biotechnics and the International Vaccine Institute by staff who were not involved with the trial . The agents were administered in two rounds in 2006: from July 27 to August 13 and from August 27 to September 10 , 2006 . Surveillance was performed in nine community clinics established for the trial and in two hospitals serving the study population . Study physicians completed structured study forms to obtain pertinent clinical information , and fecal specimens were tested for V . cholerae as previously described , including identification of 01 and 0139 serogroups with agglutination tests . Biotype was ascertained for all 01 isolates , and the biotype of the cholera toxin genetically encoded was identified as previously described [5] , [6] . Confirmation that the subject had indeed visited the treatment site on the date of the visit was assessed through domiciliary visits for all patients whose samples yielded V . cholerae O1 or O139 . A diarrheal visit was defined as having , in the 24 hours before presentation: 3 or more loose or liquid stools; or , at least 1 loose or liquid stool with blood; or , if 1–2 or indeterminate number of loose or liquid stools were reported , the patient must have exhibited at least some evidence of dehydration , using WHO criteria [7] . The onset of a diarrheal visit was the day on which the patient first reported loose or liquid stools . Diarrheal visits for which the date of onset was less than or equal to 7 days from the date of discharge for the previous visit were grouped into the same diarrheal episode . The onset of a diarrheal episode was the onset of the first diarrheal visit of the episode . The primary endpoint , a cholera episode , was defined as a diarrheal episode in which no component visit was described as bloody , in which a fecal specimen yielded V . cholerae O1 , and a domiciliary check confirmed that the subject had indeed visited the treatment center for diarrhea on the recorded date of presentation . Demographic surveillance for migrations and deaths among the study population was maintained during the three years of follow-up , and verbal autopsies were done for identified deaths . The sample size calculation for the trial was previously reported [5] . Prior to the analysis , data were frozen , and a detailed analytic plan was approved by the data and safety monitoring board . The primary analysis was a per-protocol analysis of vaccine protection among subjects who completely ingested two doses of an agent with the assigned treatment code , and included first cholera episodes with onsets between 14 days and 1 , 095 days after receipt of the second dose . A modified intention-to-treat analysis was done for all individuals who received at least one dose of an agent regardless of the amount ingested , and regardless of whether the agent received was as assigned . First cholera episodes that began from 1 to 1 , 095 days after the intake of the first dose were included in this analysis . All analyses were conducted and interpreted prior to unblinding of the codes . Survival analyses were used to calculate vaccine protective efficacy with measurements of the time to the first episode of cholera , censoring the follow-up of individuals who died or migrated out [8] . Kaplan-Meier curves were constructed for descriptive analyses . We also fitted unadjusted and adjusted Cox proportional hazards regression models , after verifying that the proportionality assumptions were fulfilled for all independent variables [9]–[11] . We estimated the hazard ratios by exponentiating the coefficient for the vaccine variable in these models and calculated the vaccine efficacy ( PE ) as : ( 1- hazard ratio ) ×100% . To estimate P values and confidence intervals ( CI ) for the hazard ratio , we used the standard errors for the coefficients . Robust sandwich variance estimates were used to account for the design effect of cluster randomization , allowing inferences for vaccine efficacy at the individual level [12] . Variables used for stratified randomization as well as baseline variables that were found to be significantly associated with time to event at p<0 . 10 in bivariate analyses were candidates as independent variables in the final models assessing vaccine efficacy . To avoid overfitting the models , we used a backward elimination algorithm to select independent variables in addition to the vaccination variable . Vaccine efficacy was evaluated in different subgroups that were defined prior to analyses . Heterogeneity of vaccine protection was assessed in these subgroups by analyzing interaction terms in the models . All P values and confidence intervals ( CI's ) were calculated as one-sided except for assessing heterogeneity of vaccine efficacy in different subgroups , for which stochastic estimates were two-sided . An interim analysis at 2 years of follow-up , using the Haybittle-Peto rule , set the P value for statistical significance for the primary analysis of PE at P< . 01 [13] . Because the three-year analysis was the major objective of the trial , all analyses reported in this paper were evaluated at a threshold of P< . 0 . 05 , with corresponding one-sided 95% CIs . All statistical analyses were performed using SAS version 9 . 1 . While the initial plan for surveillance was only for three years , follow-up is ongoing to assess the duration of protection up to five years post-vaccination . The study protocol was approved by the ethics committee of the National Institute of Cholera and Enteric Diseases , the Health Ministry Screening Committee of India and the International Vaccine Institute Institutional Review Board . Written informed consent was obtained from older residents and from the guardians of residents aged 1 to 17 years of age . Additional written assent was obtained from residents aged 12 to 17 years . An independent data and safety monitoring board reviewed the study protocol , assessed serious adverse events , and approved freezing of data and the analytical plan prior to starting the analysis .
The study was prospectively registered at ClinicalTrials . gov ( NCT00289224 ) . In the per-protocol analysis , there were 1 , 721 clusters and 31 , 932 participants in the vaccine group and 1 , 757 clusters and 34 , 968 participants in the placebo group ( Figure 1 ) . In the intention-to-treat analysis , there were 1 , 727 clusters and 33 , 127 participants in the vaccine group and 1 , 768 clusters and 36 , 202 participants in the placebo group . 4 , 252 and 4 , 661 participants in the vaccine and placebo groups , respectively , died or migrated out of the study area after the second dose . As previously reported , individual-level and cluster-level baseline characteristics were similar for vaccinees and placebo recipients [5] . There were no substantive imbalances in baseline variables among participants in each arm who were excluded or lost to follow-up . All detected cholera episodes were due to V . cholerae 01 , El Tor biotype that genetically encoded classical biotype cholera toxin . As shown in Table 1 , 38 and 128 episodes were detected in per-protocol analysis of the vaccine and placebo groups , respectively ( adjusted cumulative protective efficacy 66% , one-sided 95% CI-lower bound = 53% , p<0 . 001 ) . In the intention-to-treat analysis , there were 49 and 137 cholera episodes in the vaccine and placebo groups ( adjusted cumulative PE = 61% , one-sided 95% CI lower bound = 47% , p<0 . 001 ) . Survival curves for each analysis are presented in Figure 2 . Most of the isolates were Ogawa serotype . In the per-protocol analysis of protective efficacy against Ogawa cholera , there were 34 and 118 episodes , respectively , in the vaccine and placebo groups ( adjusted PE = 68% , one-sided 95% CI lower bound = 54% , p = <0 . 001 ) . Inaba serotype was detected in only 4 and 10 episodes in the vaccine and placebo groups , respectively ( unadjusted PE = 56% , one-sided 95% CI lower bound = −14% , p = . 08 ) . There were no deaths due to cholera identified in the the treatment centers , or due to acute watery diarrhea in verbal autopsies among study participants . Table 1 presents the per protocol analysis by year of follow-up and by age at vaccination . Cumulative three-year vaccine efficacy was highest for children vaccinated at ages 5–14 years ( adjusted PE 88% , one-sided 95% CI lower bound = 71% , P< . 001 ) , intermediate for persons vaccinated at older ages ( 61% , one-sided 95% CI lower bound = 37% , P< . 001 ) , and lowest for children vaccinated at ages 1–4 years ( adjusted PE 43% , one-sided 95% lower bound = 7% , P = . 03 ) , and differed significantly ( P = . 02 , two-sided ) among the three age groups . Protection of all age groups was 65% during the third year of follow-up ( one-sided 95% CI lower boundary = 44% , P< . 001 ) , and showed no evidence of decline over time ( P = . 24 , two-sided , for comparison of PE in years 1 , 2 , and 3 of follow-up ) . Variations in vaccine protection for each age group , by year of follow-up , did not reach statistical significance ( two-tailed P values for comparison of PE in years 1 , 2 , and 3 of follow-up in the 1–4 year , 5–14 year , and ≥15 year age groups were . 11 , . 87 , and . 98 , respectively ) . Vaccine protection was significant during the third year in the 5–14 year and ≥15 year age groups , but was significant only in the second year in follow-up of younger persons .
Our findings demonstrate that a two-dose regimen of the killed , WC OCV conferred protection of 66% protection during the three years following vaccination . Vaccine protection was clearly evident in the third year of follow-up in persons vaccinated at ages five years and older and during the second year in children vaccinated at 1–4 years of age . Due to small numbers of outcomes during the third year , however , further follow-up will be required to assess the duration of protection in the youngest age group . Protection was clearly evident against El Tor Ogawa , and suggestive against El Tor Inaba , though the latter analysis was limited by a small number of outcome events . Of note , all episodes of cholera were due to V . cholerae 01 that manifested the El Tor phenotype but genetically encoded classical biotype cholera toxin , a hybrid strain that now accounts for nearly all cholera cases in many parts of both Africa and Asia and that may be associated with cholera of increased severity [6] , [14] , [15] . An apparently counterintuitive finding was that vaccine protection was lower in the first year of follow-up than in the subsequent two years . However , the most likely explanation for this finding is chance variation , as there were no significant differences in estimates of vaccine protection , either for all age groups combined or for the <5 year , 5–14 year , and ≥15 year age groups individually , during the three years of follow-up . Comparing the results of different vaccines tested in different trials provides less conclusive evidence of their comparative efficacy than head-to-head comparisons of vaccines in the same trial . Nevertheless , it is of interest to contrast the long-term results for the killed WC OCV studied in this trial with those for the killed BS-WC OCV tested in three doses in Bangladesh in the 1980s , the only evaluation of BS-WC with long-term follow-up [4] . In contrast to the trial of killed WC OCV in Kolkata , which demonstrated efficacy during the third year of follow-up , BS-WC vaccine's protection against cholera in Bangladesh was significant only during the first two years of follow-up . Of interest , protection by BS-WC vaccine in the Bangladesh trial was also lowest and of shortest duration in the under-five age group , an observation that has been attributed to the lower level of pre-existing , anti-cholera immunity in this group , owing to less exposure to natural cholera infections in the youngest group . It should be emphasized , however , that long-term protection is only one consideration for the use of an OCV . Enhanced short-term protection may be a distinct advantage when considering the use of a vaccine in self-limited outbreaks of cholera . In this respect , the BS-WC OCV has been shown to confer 85% protection lasting 4–6 months after dosing [16] , Another advantage of BS-WC OCV is its ability to confer cross-protection against LT-producing enterotoxigenic Escherichia coli diarrhea for several months after dosing [17] . The potential of the killed WC OCV tested in this study for use in control of endemic and epidemic cholera is substantial . However , much remains to be done . The study remains blinded and surveillance will continue to assess the duration of protection provided by the vaccine up to five years after dosing . Increasing access to this vaccine is important , not only in India , where it is currently licensed , but also in other cholera-endemic countries . Access should be increased in the near future by WHO prequalification of the vaccine so that it may be purchased by UN agencies for use in other countries for disease control . | New-generation vaccines against cholera are given orally , to stimulate intestinal immunity . An internationally available oral cholera vaccine ( OCV ) consists of killed vibrio whole cells together with the B subunit of cholera toxin , is safe , and protects vaccinated individuals against cholera for two years , but this vaccine has seen limited use due to its high cost . We developed a simpler , inactivated whole-cell only OCV that can be produced inexpensively and might therefore be attractive for use in developing countries , as well as for travelers from industrialized countries . We tested this new OCV in a randomized , controlled field trial that enrolled 69 , 328 individuals aged one year and older living in urban slums of Kolkata , India . At three years of follow-up after receiving at two-dose regimen of this OCV , the vaccinated population experienced 66% protection against all episodes of cholera occurring during the three years , and 65% protection against episodes occurring during the third year . Significant protection was evident in the second year in children vaccinated at ages 1–4 years and in the third year in persons vaccinated at ages of five years and older . Follow-up of the study population will continue for five years to ascertain the duration of vaccine protection . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [
"medicine"
] | 2011 | Efficacy of a Low-Cost, Inactivated Whole-Cell Oral Cholera Vaccine: Results from 3 Years of Follow-Up of a Randomized, Controlled Trial |
The complex interplay between the response regulator ComA , the anti-activator RapF , and the signaling peptide PhrF controls competence development in Bacillus subtilis . More specifically , ComA drives the expression of genetic competence genes , while RapF inhibits the interaction of ComA with its target promoters . The signaling peptide PhrF accumulates at high cell density and upregulates genetic competence by antagonizing the interaction of RapF and ComA . How RapF functions mechanistically to inhibit ComA activity and how PhrF in turn antagonizes the RapF-ComA interaction were unknown . Here we present the X-ray crystal structure of RapF in complex with the ComA DNA binding domain . Along with biochemical and genetic studies , the X-ray crystal structure reveals how RapF mechanistically regulates ComA function . Interestingly , we found that a RapF surface mimics DNA to block ComA binding to its target promoters . Furthermore , RapF is a monomer either alone or in complex with PhrF , and it undergoes a conformational change upon binding to PhrF , which likely causes the dissociation of ComA from the RapF-ComA complex . Finally , we compare the structure of RapF complexed with the ComA DNA binding domain and the structure of RapH complexed with Spo0F . This comparison reveals that RapF and RapH have strikingly similar overall structures , and that they have evolved different , non-overlapping surfaces to interact with diverse cellular targets . To our knowledge , the data presented here reveal the first atomic level insight into the inhibition of response regulator DNA binding by an anti-activator . Compounds that affect the interaction of Rap and Rap-like proteins with their target domains could serve to regulate medically and commercially important phenotypes in numerous Bacillus species , such as sporulation in B . anthracis and sporulation and the production of Cry protein endotoxin in B . thuringiensis .
Two-component signaling systems , consisting of a sensor histidine kinase and a response regulator transcription factor , are the principal mechanism of signal transduction in bacteria [1] . Upon phosphorylation of their receiver ( REC ) domains , response regulators bind target DNA promoters and activate or repress transcription . A number of auxiliary factors have garnered significant attention because they function as anti-activators that inhibit response regulator function without affecting their phosphorylation state . These proteins inhibit the interaction of response regulators with their target promoters [2]–[5] or RNA polymerase [6] , [7] . To our knowledge , the X-ray crystallographic , biochemical , and genetic results presented here show for the first time how an anti-activator structurally inhibits the binding of a response regulator to DNA . The Rap proteins are a family of auxiliary factors that have been most thoroughly studied in Bacillus subtilis , where they regulate two-component and phosphorelay signal transduction . Despite their relatively high sequence similarity , different Rap proteins exert their influence on these pathways via disparate activities [5] . Rap proteins ultimately influence diverse B . subtilis developmental and cellular differentiation processes including sporulation , genetic competence , and biofilm formation , as well as the production of secreted enzymes and the movement of a conjugative transposon [8] , [9] . B . subtilis encodes 11 Rap proteins on its chromosome and another five on plasmids [10]–[12] . Rap proteins were named after the founding members of the family , which were shown to be response regulator aspartate phosphatases [13] . Rap proteins including RapA , RapB , RapE , RapH , and RapJ are phosphatases that dephosphorylate the intermediate response regulator Spo0F , which consists solely of a REC domain [5] , [13]–[15] . In contrast , genetic and biochemical results show that a subset of Rap proteins , including RapC , RapF , and RapH , are anti-activators that bind to the response regulator transcription factor ComA , inhibiting its interaction with DNA promoters without affecting its phosphorylation state ( Figure 1 ) [2] , [3] , [5] . Additional genetic results also suggest that RapD , RapG , and RapK may inhibit ComA as well [16]–[18] . Similarly , B . subtilis RapG inhibits the binding of the response regulator transcription factor DegU to its target promoters [4] . Consistent with the inability of the Rap proteins to dephosphorylate ComA or DegU , it was previously shown that RapC , RapF , and RapH interact stably with the ComA helix-turn-helix ( HTH ) DNA binding domain and not with its REC domain [2] , [5] . Secreted signals called Phr peptides contribute to the complexity of Rap protein signaling . Phr peptides are imported into the cell , where they bind to Rap proteins and antagonize their activity ( Figure 1 ) [19] , [20] . Mature Phr peptides are generated from immature pro-Phr polypeptides [21] , [22] . The genes encoding the pro-Phr polypeptides overlap with the 3′ end of the rap genes , forming rap-phr gene cassettes . Pro-Phr polypeptides are secreted from the cell and subsequently undergo proteolytic maturation . Mature Phr peptides are then imported into the cell , where each peptide inhibits its cognate Rap protein ( e . g . , PhrA inhibits RapA , and PhrC inhibits RapC ) and in some cases a non-cognate Rap protein [23]–[25] . To demonstrate how anti-activators such as the Rap proteins inhibit the binding of response regulators to target DNA promoters , we determined the X-ray crystal structure of a B . subtilis Rap protein , RapF , in complex with the DNA binding domain of the response regulator ComA ( ComAC ) . As expected , comparison of the RapF-ComAC crystal structure with the previously determined structure of RapH-Spo0F [15] revealed that RapF and RapH are structurally similar; however , ComAC and Spo0F bind to RapF and RapH , respectively , at distinct , non-overlapping sites . Furthermore , we show that RapF is monomeric either alone or in complex with PhrF , and that RapF undergoes a conformational change upon binding PhrF that is likely the cause of ComA dissociation from RapF-ComA complexes . Finally , we propose a model that explains the long-standing observation that some Rap proteins dephosphorylate response regulator REC domains while others sequester response regulator HTH DNA binding domains .
To begin to determine how RapF inhibits response regulator binding to DNA without affecting its phosphorylation state , we solved its X-ray crystal structure in complex with ComAC to 2 . 30 Å resolution ( Figure 2A and Table S1 ) . The crystallographic asymmetric unit contains one molecule of RapF bound to one molecule of ComAC . It is worth noting that the RapF-ComAC crystallization conditions are identical to conditions that yield crystals in our initial screens containing RapF and full-length ComA ( ComAFL ) . However , RapF-ComAFL crystals diffracted anisotropically and to very low resolution . Presumably , the ComA N-terminal REC domain , which does not interact with RapF [2] , is responsible for the disorder observed in the RapF-ComAFL crystals . RapF consists of two distinct domains , an N-terminal antiparallel 3-helix bundle ( residues 7–72 ) and a C-terminal tetratricopeptide repeat ( TPR ) domain ( residues 92–381 ) ( Figure 2B ) . The RapF N-terminal 3-helix bundle consists of two similarly sized helices , α1 ( residues 8–23 ) and α2 ( residues 26–42 ) , and a significantly longer third helix , α3 ( residues 47–72 ) . The 3-helix bundle and TPR domain are connected by an elaborate linker region ( residues 73–91 ) , containing a 310 helix ( residues 75–77 ) joined by a short loop ( residues 78–80 ) to helix α4 ( residues 81–91 ) . As discussed in detail below , the N-terminal 3-helix bundle and the linker region form the ComAC binding surface ( Figures 2C , 3A , and 3B ) . The RapF C-terminal TPR domain contains seven helix-turn-helix ( HTH ) folds that assemble into a large superhelix . HTH folds 1–5 and 7 are bona fide TPR folds containing TPR signature sequences ( Figure 2B ) [26] . The sixth HTH fold lacks the TPR signature motif but perpetuates the TPR superhelix . Therefore , we refer to the entire C-terminal region containing seven tandem HTH folds as the TPR domain . The RapF-ComAC X-ray crystal structure reveals an extensive protein-protein interface that buries a total of 1 , 845 . 7 Å2 surface area ( Figures 3A and 4A ) . The structure of ComAC and its interaction with target binding sites within the srfA promoter were previously studied using NMR spectroscopy [27] . The RapF-ComAC crystal structure shows that most of the ComA residues that interact with the DNA phosphate backbone , including Ser181 , Ser184 , Tyr187 , Ser188 , and Thr190 , are buried in the RapF-ComAC interface ( Figures 4B , 4C , and S1 and unpublished data ) . Furthermore , both of the ComA residues shown to interact with the DNA major groove , specifically Arg183 and Tyr187 , are buried in the RapF interface ( Figures 4C , S1 , and S2 ) . In sum , the RapF-ComAC interaction buries six of the seven ComA DNA binding residues . It is also notable that in addition to interacting with ComA DNA binding residues , RapF mediates extensive interactions with ComAC residues not directly involved in DNA binding . As described in detail below , we rigorously tested the functional importance of the crystallographically observed RapF-ComAC interface both in vitro and in vivo . To begin to test the functional significance of the RapF-ComA interactions observed in the RapF-ComAC crystal structure , we generated RapF mutants containing individual alanine substitutions at RapF-ComA interfacial positions ( Figures 3B and S1 ) , and native PAGE was used to examine the binding of the RapF mutants to wild-type full-length ComA ( Figure 3C ) . RapF Asp28 hydrogen bonds to the ComA Arg157 and Thr155 side chains , and RapF Leu67 forms a hydrophobic interaction with ComA Tyr187 ( Figure S1 ) . Consistent with these observations , RapF-D28A and RapF-L67A exhibited a complete loss of ComA binding activity ( Figure 3C ) . Furthermore , RapF P27A forms a hydrophobic interaction with ComA Ile161 and Ser188 , and RapF Gln78 hydrogen bonds with ComA Asn194 and with a water molecule at the RapF-ComA interface ( Figure S1 ) . Compared to the interaction of wild-type RapF with ComA , the interaction of RapF-P27A and RapF-Q78A with ComA was significantly reduced ( Figure 3C ) . In addition to the RapF mutants evaluated above , we analyzed the binding of RapF-F24A to wild-type ComA ( Figure 3C ) . RapF Phe24 is located in the loop connecting RapF helices α1 and α2 ( Figure 3B ) . Its main-chain carbonyl oxygen forms a hydrogen bond with the ComA Ser191 side-chain hydroxyl , and its carbonyl carbon atom mediates hydrophobic interactions with ComA Lys195 ( Figure S1 ) . Native gel analysis showed that the F24A mutation in RapF results in a complete loss of ComA binding ( Figure 3C ) . While a portion of the RapF Phe24 side chain and main chain are buried in the ComA interface , the rest of its side chain is buried in a hydrophobic pocket formed by residues in RapF helices α3 and α4 . We propose that the interaction of the RapF Phe24 side chain with RapF helices α3 and α4 is essential for the RapF-ComA interaction because it ( 1 ) positions the RapF Phe24 main chain carbonyl for interaction with ComA and ( 2 ) mediates the packing of the RapF 3-helix bundle and linker , which both in turn interact with ComA . Also consistent with this hypothesis , Phe24 is conserved in the Rap proteins previously shown to bind ComA directly [2] , [3] , [5] . One RapF mutant , RapF-E71A , exhibited a significant but subtler ComA-binding defect than the other RapF mutants tested ( Figures 3C and unpublished data ) . While RapF Glu71 mediates contacts with ComAC ( Figure S1 ) , it is located at the periphery of the RapF-ComA interface where it is exposed to bulk solvent . Consistent with previous studies of protein-protein interfaces ( see , for example , [28] ) , interfacial residues such as RapF Glu71 that are exposed to bulk solvent routinely have little impact on the free energy of binding . In addition to the RapF mutants described above , we engineered full-length ComA mutants containing single alanine substitutions at the RapF-ComAC interface ( Figures 4A–C and S2 ) . The binding of the ComA mutants was then evaluated using native PAGE ( Figure 4D ) . ComA Arg183 forms a salt bridge with RapF Glu71 ( Figures S1 and S2 ) , and it also interacts intramolecularly with Tyr187 , which is also in the RapF interface . ComA-R183A displayed a nearly complete loss of RapF binding , while numerous other ComA mutants , including ComA-Y187A , ComA-T190A , and ComA-N194A , displayed significantly reduced RapF binding activity compared to wild-type ComA ( Figures 4D and S2 ) . Only one mutant tested , ComA-R157A , appeared to bind RapF with a similar affinity to wild-type ComA ( Figure 4D ) . As described above , ComA Arg157 and ComA Thr155 hydrogen bond to RapF Asp28 ( Figure S1 ) . ComA-T155A was insoluble and therefore could not be studied in vitro , but it is likely that the interaction of ComA Thr155 and RapF Asp28 is primarily responsible for the complete loss-of-function displayed by RapF-D28A ( Figure 3C ) . Finally , we estimate based on the results of the qualitative analysis ( Figures 3C and 4D ) and additional native PAGE binding studies employing a broad concentration range of RapF and a fixed quantity of ComA ( unpublished data ) that the dissociation constant ( Kd ) for RapF-ComA complex formation is in the 1–10 µM range . To confirm that the RapF-ComA interface observed in the crystal structure is important for the regulation of ComA activity in vivo , we overexpressed RapF mutants containing single alanine substitutions in RapF-ComA interfacial positions and evaluated their affects on ComA transcriptional activity in B . subtilis ( Figure 3D ) . More specifically , we measured RapF activity as a function of ComA-driven srfA transcription using the Photinus pyralis luciferase gene fused to the srfA promoter . As previously described , P . pyralis luciferase activity corresponds closely with the rate of its transcription in B . subtilis [15] , [29] . The PsrfA-luc reporter bioassay shows that srfA expression is delayed in proportion to the amount of RapF induced ( Figure S3A ) . However , we questioned why srfA was expressed at all considering the fact that the cells overexpressed RapF , which inhibits ComA-driven srfA expression . We hypothesized that PhrF expressed from its endogenous locus ( Figure S4 ) was antagonizing RapF activity . To confirm that this was the case , we overexpressed a RapF mutant , RapF-D194N , which contains a mutation previously shown to render RapA and RapC immune to the effects of their cognate Phr peptides [3] , [13] . Indeed , overexpressed RapF-D194N completely suppressed PsrfA-luc expression throughout the course of the entire experiment ( unpublished data ) . Moreover , when we added synthetic PhrF peptide to the PsrfA-luc reporter strain overexpressing RapF , PsrfA-luc expression was indistinguishable from the non-induced control ( Figure S3B ) . Together , these results show that overexpressed RapF is antagonized at least to some degree by PhrF expressed from its endogenous locus . Nonetheless , overexpressing RapF significantly delays srfA-luc expression . Consistent with these results and as described below , strains overexpressing RapF proteins containing mutations in the RapF-ComA interface should express srfA-luc at earlier time points than the wild-type RapF control . Mirroring the results of the RapF-ComA binding studies , RapF-D28A and RapF-L67A displayed a loss-of-function , and RapF-P27A and RapF-Q78A displayed intermediate phenotypes ( Figure 3D ) . Also consistent with the in vitro results ( Figures 3C ) , RapF-E71A displays a slight loss of function in vivo ( Figure 3D ) . Western blotting confirmed that the RapF mutants were produced at levels comparable to wild-type RapF ( unpublished data ) . Thus , the results of the RapF-ComA in vitro binding studies and the RapF-ComA luciferase bioassays show that the RapF-ComA interface identified in the RapF-ComAC crystal structure is functionally important in vitro and in vivo . RapF dimerizes around a crystallographic 2-fold symmetry axis in the RapF-ComAC crystals . To begin to determine whether RapF also dimerizes in solution , we analyzed RapF and RapF-ComA complexes by gel filtration ( Figure 5A ) . The results of these studies suggested that RapF alone and RapF in complex with ComA could be forming dimers in solution ( Table 1 ) . However , molecular weight determination using gel filtration becomes less reliable as the shape of the analyzed protein or protein complex deviates from that of a sphere , and RapF has an elongated structure ( Figure 2A ) . In fact , RapF is approximately twice as long as it is wide , and its shape more closely resembles a rod than a sphere . Therefore , to determine unambiguously whether RapF dimerizes in solution , we studied RapF using sedimentation-equilibrium ( SE ) analytical ultracentrifugation ( AUC ) . The theoretical molecular weight of a RapF monomer is 46 . 3 kD , and its molecular weight as determined by SE AUC is 47 kD ( Figure 5B and Table 1 ) . Therefore , we conclude that RapF is monomeric in solution . The RapF-ComAC crystallographic asymmetric unit ( Figure 2A ) contains one molecule of RapF bound to one molecule of ComAC , suggesting that perhaps a monomer of RapF binds to a monomer of ComA in solution . However , ComA alone and ComAC alone homodimerize in solution ( Figure 5A , Table 1 ) [27] , [30] , and ComA dimers bind to DNA regulatory sites composed of three recognition elements [30] . Therefore , we examined whether RapF is binding a ComA monomer or dimer in solution using gel filtration in combination with total amino acid analysis and SE AUC . A mixture of RapF and ComA was passed over a gel filtration column and total amino acid analysis was performed on the RapF-ComA complex fractions as described in Materials and Methods . The results of the total amino acid analysis ( unpublished data ) indicate that the stoichiometry of the RapF-ComA complex is 1∶1 . Finally , SE AUC analysis of the identical RapF-ComA sample indicated the formation of a single species of approximately 74 kD ( Figure 5B ) . This is consistent with a complex composed of one molecule of RapF ( 45 . 7 kD ) and one molecule of ComA ( 26 . 3 kD ) ( Table 1 ) . Together , the results of the SE AUC , gel filtration chromatography combined with total amino acid analysis , and X-ray crystallography suggest that a monomer of RapF binds to a monomer of ComA or ComAC , and that the stoichiometries of the RapF-ComA and RapF-ComAC complexes are 1∶1 . In Figure 5F , ComAC from the RapF-ComAC X-ray crystal structure is aligned with a molecule of ComAC from the dimeric ComAC NMR structure [27] . While the RapF-ComAC structure shows that RapF-bound ComAC undergoes conformational changes near the ComAC dimerization interface , we cannot rule out the possibility that these changes were influenced by lattice contacts involving ComAC . Nonetheless , consistent with the RapF-ComAC crystal structure , we find that RapF binds to a ComA monomer in solution . Furthermore , while RapF does not interact with ComAC dimerization interface residues , which are located in helix α8 , α10 , and the α7–α8 loop ( Figure 5F ) , we hypothesize that the RapF-induced ComAC conformational change is transmitted to ComA α10 through contacts between RapF and ComA residues in the C-terminus of α9 ( residues N194 and K195 ) and the α9–α10 loop ( residues N197 , V198 , and G199 ) ( Figure 5F ) . In fact , RapF Gln78 and RapF Phe24 contact ComA Asn194 and Lys195 , respectively , and RapF-Q78A and RapF-F24A both exhibited severe ComA binding defects ( Figure 3C ) . Despite the numerous contacts between RapF and ComA residues directly adjacent to the ComAC dimerization interface , because RapF does not interact with the surface of the ComAC dimerization interface , we conclude that RapF allosterically inhibits ComAC dimerization . The gel filtration analysis of RapF-ComA and RapF performed above ( Figure 5A ) were repeated in the presence of PhrF ( Figure 5C , 5E and Table 1 ) . Consistent with previous native-PAGE studies [2] , we found that PhrF caused the dissociation of the RapF-ComA complex ( Figure 5E and Table 1 ) . Also consistent with these native-PAGE studies where a faster migrating RapF band was observed in the presence of PhrF , we found that PhrF caused RapF to elute at a significantly higher volume than RapF alone ( Figure 5C , 5E , and Table 1 ) . However , in the previous native PAGE studies it was assumed that RapF was dimeric [2] , and why a faster migrating band appeared in the presence of PhrF was unknown . As discussed above , SE AUC shows that RapF alone is monomeric ( Figure 5B and Table 1 ) . Consistent with this result , the molecular weight of RapF complexed with PhrF is 50 kD as determined by SE AUC ( Figure 5D and Table 1 ) , and there is no change in RapF stoichiometry upon binding to PhrF . Therefore , we conclude that RapF migrates through the gel filtration matrix radically slower in the presence of PhrF than in its absence , and RapF migrates faster in the presence of PhrF when analyzed by native PAGE [2] , because it undergoes a large conformational change upon binding to PhrF .
Response regulators are the primary mechanism for converting sensory perception into transcriptional output in bacteria . How auxiliary factors such as the Rap proteins function structurally to inhibit response regulator binding to DNA promoters was not known . The X-ray crystallographic , biochemical , and genetic results presented here reveal that RapF disrupts response regulator binding to DNA using a two-pronged mechanism . First , RapF buries ComA DNA binding residues in the RapF-ComA interface , sterically blocking the binding of ComA to its DNA recognition elements . Second , RapF binding allosterically weakens the ComAC dimerization interface , inhibiting the formation of transcriptionally active ComA dimers ( Figure 5B ) . In theory , each mechanism alone could effectively inhibit response regulator activity; however , RapF uses both . To our knowledge , the data presented here reveal the first atomic level insight into the inhibition of response regulator DNA binding by an anti-activator . However , the structures or regulatory mechanisms of other anti-activators and anti-repressors that target transcription factors other than response regulators have been described , for example TraM , CarS , TrlR , AppA , and ExsD ( Figure S5 ) [31]–[35] . These proteins repress the activity of their targets by ( 1 ) directly blocking the interaction of DNA binding residues and DNA , ( 2 ) inhibiting their functionally required multimerization , or ( 3 ) causing allosteric rearrangements that preclude their binding to DNA . RapF does not share obvious structural similarities with these anti-activators and anti-repressors ( Figure S5 ) ; however , a comparison of the structural and functional data available for the different systems is informative . The TraR-TraM crystal structure showed that the anti-activator TraM interacts with the TraR N-terminal dimerization domain ( NTD ) in addition to its C-terminal DNA binding domain ( CTD ) and linker region connecting the NTD and CTD ( Figure S5 ) [35] . In contrast to the interactions observed in the RapF-ComAC structure , TraM does not contact the TraM DNA binding residues . Rather , TraM allosterically regulates TraR activity . CarS-CarA interactions were mapped using NMR chemical shift perturbation and site-directed mutagenesis studies [31] . Interestingly , like RapF ( discussed below ) , the anti-repressor CarS mimics DNA to interact with its target's DNA binding domain; however , the RapF and CarS folds are entirely different ( Figure S5 ) . While RapF is entirely alpha helical , CarS resembles an SH3 domain and consists of an antiparallel beta sheet and a 310 helix . How TrlR , AppA , and ExsD function structurally to regulate their targets is not as well understood as it is for the systems discussed above . However , it was shown that TrlR is a truncated form of TraR that regulates TraM by forming inactive TrlR-TraR dimers [32] , and that the flavin binding protein AppA inhibits PpsR repressor activity by reducing a disulphide bond in PpsR and forming inactive AppA-PpsR2 complexes [36] . Finally , how ExsD functions structurally to inhibit ExsA transcriptional activity is unknown , and different studies suggest that ExsD interacts stably with either the ExsA N-terminal oligomerization domain alone [37] or only the intact full-length protein [38] . ComA is a member of the LuxR/FixJ/NarL family of transcriptional regulators whose HTH DNA-binding domains are tetra-helical bundles [39] , [40] . Aligning ComAC of the RapF-ComAC structure with a molecule of NarLC of the NarLC-DNA complex structure ( 1JE8 ) revealed that RapF's ComA-binding surface adopts a conformation reminiscent of a DNA major groove ( Figure 6A and 6B ) [41] , [42] . Additionally , analogous to DNA , at the core of the RapF ComAC-binding surface , there is a large electronegative patch ( Figure 6C ) [43] . Moreover , six of the seven previously identified ComA DNA binding residues are buried in the RapF-ComAC interface . For example , the sidechain of ComA Arg183 forms a salt bridge with the side chain of RapF Glu71 , and the side chain of ComA Thr190 forms a hydrogen bond with the side chain of RapF Glu71 ( Figure S2 ) . These contacts appear to mimic the hydrogen bonds that the ComA residues make with the DNA major groove and phosphate backbone , respectively [27] . Thus , the RapF-ComAC X-ray crystal structure reveals that ComA binds to a RapF surface that , at least in part , mimics DNA . HTH DNA binding domains are ubiquitous in all three superkingdoms of life [39] . In fact , B . subtilis alone encodes 10 proteins containing tetra-helical bundle HTH domains similar to the ComA DNA binding domain [44] . The identity of residues in helix α9 and the α8–α9 loop are primarily responsible for determining LuxR/FixJ/NarL-type HTH DNA recognition element specificity [27] , [41] , [45] , [46] . For example , ComA Tyr187 in helix α9 inserts into the DNA major groove and contacts the phosphate backbone [27] , while no other B . subtilis response regulator encodes tyrosine at the equivalent position ( Figure 6D ) . Similarly , ComA Arg183 and Thr190 insert into the DNA major groove and contact the phosphate backbone , respectively . Only one other B . subtilis response regulator , YdfI , encodes identical residues at these positions ( Figure 6D ) . YdfI is in fact the B . subtilis protein containing a LuxR/FixJ/NarL-type HTH domain most highly homologous to ComAC , yet they bind to different DNA recognition elements [30] , [47] . Interestingly , we found that purified RapF and YdfI did not stably interact ( unpublished data ) . We propose that Rap protein interactions with the HTH DNA binding residues serve to sterically block the binding of response regulators with their DNA recognition elements . Contacts with these DNA binding residues may enable Rap proteins to preferentially recognize their response regulator targets , e . g . ComA or DegU , among the myriad B . subtilis HTH DNA binding domains . Some Bacillus Rap proteins are phosphatases that bind to and dephosphorylate Spo0F , a stand-alone REC domain , while others bind to the HTH DNA binding domain of the response regulator ComA rather than its REC domain [2] , [5] , [15] . Structural alignment of RapF of the RapF-ComAC structure with RapH of the RapH-Spo0F structure ( 3Q15 ) showed that their tertiary structures are essentially identical , and they align with a root mean-square deviation of 1 . 61 Å ( Figure 7A–C ) [15] . Remarkably , this alignment also revealed that ComAC and Spo0F bind to non-overlapping Rap protein surfaces ( Figure 7C ) . ComAC interacts only with the RapF linker region and 3-helix bundle , while Spo0F interacts with the RapH TPR domain and 3-helix bundle at a site distant from the ComAC binding site on the RapF 3-helix bundle . Rap proteins have not been observed to bind simultaneously to both the REC and HTH DNA binding domains of a single response regulator . However , it is possible that an ancestral Rap protein did in fact interact with both domains . The Spo0F ( REC ) and ComAC ( HTH ) binding sites do not overlap , and a Rap protein could theoretically bind to both a REC and HTH domain concurrently ( Figure 7C ) . Alternatively , there may not have been an ancestral Rap protein that could bind a REC and HTH DNA binding domain . In this case , the fact that Rap proteins commonly target one or the other of two domains found in response regulator transcription factors could be a coincidence resulting from independent evolution of Rap protein interactions with their targets . Furthermore , we speculate that Rap proteins such as RapC , RapF , and RapH may have evolved to interact stably with the ComA HTH domain but not its REC domain [2] , [5] because the short linker connecting them precludes their simultaneous interaction with the relevant Rap protein binding sites . Indeed , structural alignment of RapF of the RapF-ComAC structure with RapH of the RapH-Spo0F structure shows that the C-terminus of Spo0F and the N-terminus of ComAC are separated by greater than 49 Å . Rap proteins could interact with the REC and HTH domain of a single response regulator simultaneously if the domains were connected by a long linker as found in the B . subtilis uncharacterized protein YesN , or an additional domain common to the response regulators in the NtrC family [40] , [44] . However , the relatively short linker region connecting the ComA REC and HTH domains would prevent their simultaneous interaction with the identified Rap protein REC [15] and HTH domain binding sites ( Figure 7C ) . Previous work suggests that Phr peptides may bind to the Rap protein TPR domain [3] , [13] , [24] , and it was hypothesized that response regulators may compete with Phr peptides for binding to a common site on the TPR domain [3] . However , the RapF-ComAC X-ray crystal structure shows that the RapF TPR domain does not in fact contact ComAC . RapF-ComAC contacts are mediated only by the RapF 3-helix bundle and linker region . Thus , it appears that PhrF may allosterically inhibit the RapF-ComA interaction , and the dissociation of RapF and ComA presumably results from the PhrF-induced conformational change in RapF ( Figure 5C and 5E ) . The Rap protein conformational changes induced by Phr peptide binding are currently unknown , and it seems likely that crystal structures of Rap proteins in their Phr peptide bound conformations will be required to reveal these conformational changes as well as the mechanistic nature of the Rap-Phr interaction . Furthermore , in regard to Rap protein stoichiometry , it is worth noting that RapH dimerizes around a crystallographic 2-fold symmetry axis similar to RapF , and both RapH and RapK migrate through gel filtration matrix in a manner consistent with that of a dimer ( [15] and unpublished data ) . However , SE AUC analysis of both RapH and RapK ( unpublished data ) show that they are in fact monomeric in solution like RapF . Additional SE AUC studies are required to determine whether other Rap proteins are monomeric similar to RapF , RapH , and RapK , or whether they are dimeric as previously suggested [48] . The results of our X-ray crystallographic studies , SE AUC , and gel filtration combined with total amino acid analysis suggest that a RapF monomer binds to a monomer of ComA or ComAC . However , the reported ComAC homodimerization interface residues [27] are not buried in the RapF-ComA interface ( Figure 5F ) . Thus , it is theoretically possible that a molecule of ComA in a RapF-ComA complex could bind to another molecule of ComA using its ComAC homodimerization surface . Also , a molecule of ComA bound to RapF could bind to another molecule of ComA using its N-terminal receiver domain , which might be accessible because it does not interact stably with RapF . However , even if ComA can in fact homodimerize to some extent when bound to RapF , it is in all likelihood a non-functional ComA dimer . That is , if each molecule of ComA in a ComA dimer is required to interact with DNA at a target promoter [30] , then RapF would disable the ComA dimer when it binds directly to and buries nearly all of the DNA binding residues belonging to one molecule of ComA . Rap proteins are proposed to be the most ancient member of the RNPP protein family [49] . The RNPP proteins are related to the Bacillus Rap , NprR , and PlcR proteins , as well as the Enterococcal protein PrgX . These proteins are widespread in Firmicutes , and a rap-phr cassette was also recently identified outside of the Firmicute phylum encoded on a plasmid in a highly multidrug resistant strain of Salmonella enterica serovar Dublin [50] . Structure-function analysis showed that PlcR and PrgX are transcription factors that contain N-terminal DNA binding domains and C-terminal TPR or TPR-like domains [49] , [51] . There are no structural data and very limited functional data available describing the NprR proteins , which include a number of proteins in Bacillus and Clostridia . Like PlcR and PrgX , NprR proteins are transcription factors; however , sequence analysis suggests that NprR proteins are essentially Rap proteins containing a tetrahelical bundle HTH DNA binding domain fused to their N-terminus [20] . We hypothesize that the NprR structure may closely resemble the structure of RapF-ComAC , except that in the case of NprR the HTH DNA binding domain is covalently attached to the N-terminus of the NprR 3-helix bundle . Similar to RapF-ComAC , DNA binding residues in the NprR HTH DNA binding domain may be buried in an interface with the NprR 3-helix bundle and linker region . Like the Rap proteins , NprR , PlcR , and PrgX activity is regulated by oligopeptides reminiscent of the Phr peptides ( review in [52] ) . Peptide binding to NprR could disrupt the interaction of the NprR 3-helix bundle and its DNA binding domain , triggering NprR transcriptional activity . This mechanism is analogous to the PhrF-driven dissociation of the RapF 3-helix bundle and ComA . In conclusion , we speculate that a protein containing only the Rap protein 3-helix bundle and linker region may be sufficient to inhibit the binding of HTH DNA binding domains to DNA . Engineering this relatively simple scaffold to bind HTH domains other than those belonging to ComA or DegU would create useful in vivo tools for studying bacterial signaling . Furthermore , Rap-Phr and NprR-Phr systems regulate medically and commercially important phenotypes in numerous Bacillus species , e . g . sporulation in B . anthracis [53] and sporulation and the production of Cry protein endotoxin in B . thuringiensis [52] , [54] , respectively . Therefore , compounds that regulate the activity of Rap and Rap-like proteins could serve as commercially important additives that modulate the overproduction of insecticidal endotoxins or as antibacterial drugs .
RapF was overexpressed as an N-terminal fusion to glutathione S-transferase ( GST ) in E . coli strain BL21 grown in LB media supplemented with 100 µg/ml ampicillin . The cultures were first grown at 37°C to an OD600 of 0 . 4 and then transferred to 25°C and induced with 0 . 1 mM isopropyl β-D-thiogalactopyranoside ( IPTG ) at an OD600 of 0 . 6 . After growth for an additional 16 h at 25°C , cultures were harvested by centrifugation at 4 , 000 RPM , lysed in Buffer I ( 20 mM Tris pH 8 . 0 , 150 mM NaCl , 5 mM DTT ) supplemented with 5 mM MgCl2 , 20 µg/ml DNaseI , and 2 mM phenylmethylsulfonyl fluoride ( PMSF ) , and insoluble material pelleted at 22 , 000 RPM for 90 min . Cleared lysates were applied to Glutathione-Uniflow Resin ( Clontech ) , equilibrated in Buffer I . The resin was then washed with Buffer I , resuspended in 4 bed volumes of Buffer I and 3 . 5 µg/ml thrombin , and incubated for 1 . 5 h at 25°C . This procedure resulted in complete cleavage of the N-terminal GST affinity tag as determined by SDS-PAGE . Following thrombin cleavage , RapF ( residues 1–381 ) contained two heterologous residues ( Gly-Ser ) derived from the thrombin cleavage signal . RapF was eluted with Buffer I and diluted 2-fold with Buffer II ( 20 mM Tris pH 8 . 0 , 5 mM DTT ) , passed through a 0 . 22 µm filter , and loaded onto an anion exchange column ( Source 15Q , GE Healthcare ) equilibrated with Buffer II containing 75 mM NaCl . RapF was eluted in a 75–750 mM NaCl linear gradient of Buffer II . RapF-containing fractions were further purified by gel filtration using a Superdex 200 ( GE Healthcare ) 16/70 column equilibrated with Buffer II containing 150 mM NaCl . Again , RapF containing fractions were pooled and concentrated . ComAC protein ( His-ComAC ) was purified as described previously with the following modifications to the protocol [2] . Following Ni-NTA affinity column chromatography , His-ComAC containing fractions were pooled , diluted 4-fold with Buffer III ( 50 mM Tris pH 8 . 0 , 5 mM DTT ) , passed through a 0 . 22 µm filter , and loaded onto an anion exchange column ( Source 15Q; GE Healthcare ) equilibrated with Buffer III containing 50 mM KCl . His-ComAC eluted from the anion exchange column in the flow through and was concentrated by ultrafiltration ( MWCO: 3 kDa ) . For storage , His-ComAC was exchanged into Buffer IV ( 50 mM 4- ( 2-Hydroxyethyl ) -1-piperazinepropanesulfonic acid ( EPPS ) pH 8 . 0 , 200 mM KCl , 1 mM DTT ) using G-25-50 Sephadex ( Sigma ) . RapF-ComAC crystals were produced by the vapor diffusion method at 20°C using a 1∶1 mixture of RapF∶ComAC ( 100 µM RapF and ComAC each in its corresponding storage buffer ) and well solution ( 6% [w/v] PEG 8 , 000 , 240 mM calcium acetate , 80 mM sodium cacodylate pH 6 . 5 , and 20% glycerol ) . Data were collected at a wavelength of 1 . 0750 Å on nitrogen-cooled crystals at NSLS beamline X29A . Diffraction data were processed using HKL2000 [55] . The RapF-ComAC structure was determined by molecular replacement with PHASER [56] using the N-terminal domain ( residues 4–68 ) and C-terminal domain ( residues 77–376 ) of RapH ( 3Q15 ) as search models [15] . ComAC was not included in the search model . The single molecular replacement solution identified using PHASER was refined in REFMAC5 [57] . ARP/wARP was used to generate an initial model [58] , and iterative cycles of building in COOT [59] and refinement in PHENIX [60] were performed to complete the model . The final model lacks only residues 1–5 corresponding to the N-terminus of RapF , and 146–152 and 211–214 corresponding to the N- and C-terminus of ComAC , respectively . The building of a single Mn2+ atom into clear electron density during the final stages of refinement in PHENIX was justified based on its coordination to oxygen ligands , i . e . the carbonyl oxygens of RapF Leu40 and Met43 , and the side chain oxygens in the RapF Glu45 side chain , the metal-oxygen ligand distances , the similarity of the Mn2+ refined B-factor to those of its surrounding atoms , and the absence of calculated Fo-Fc difference density surrounding the Mn2+ ion . Mn2+ was not added to the RapF purification or crystallization buffers and may have been acquired through adventitious buffer contamination . Secondary structure assignments were calculated using PROMOTIF [61] . Ramachandran statistics were calculated in Molprobity [62] . Molecular graphics were produced with PyMOL [63] . The RapF-ComAC structure has excellent geometry , with 98 . 4% , 1 . 6% , and 0% of residues falling within the favored , allowed , and outlier regions of the Ramachandran plot , respectively . 10 µM RapF and either 10 µM ComA or 10 µM His-ComA as indicated were incubated in Buffer A ( 20 mM Tris pH 8 . 0 , 300 mM NaCl , 5 mM MgCl2 , 1 mM DTT , and 5% glycerol ) for 30 min at 4°C ( see Text S1 for additional details regarding the purification of ComA and His-ComA ) . 5× sample buffer was added to each reaction mixture and samples were analyzed by native PAGE using 12% Tris-glycine gels . Gels were run under constant voltage ( 85 V ) for 4 h at 4°C and then stained with Coomassie Brilliant Blue R-250 ( Amresco ) . The disappearance of the lower band ( ComA ) was the primary indicator of complex formation . The degree of top band ( RapF ) shifting corresponded with the disappearance of ComA and provided additional evidence of RapF-ComA complex formation . Gel filtration runs were performed with RapF , His-ComA , and PhrF ( NH2-QRGMI-COOH ) ( LifeTein ) at 20 µM , 40 µM , and 200 µM , respectively . Prior to analysis , the proteins were incubated in Buffer A for 30 min at 4°C and filtered by centrifugation through a 0 . 22 µm cut-off cellulose acetate filter ( Spin-X; Costar ) . 100 µl of each sample was injected onto a Superdex 200 HR10/30 gel filtration column ( Amersham Biosciences ) equilibrated in Buffer A . 0 . 5 ml fractions were collected and analyzed by SDS-PAGE . Gels were stained with Coomassie Brilliant Blue R-250 . RapF-ComA was prepared for total amino acid analysis by injecting a mixture containing 40 µM RapF and 80 µM His-ComA in Buffer A onto a Superdex 200 HR10/30 gel filtration column and collecting fractions spanning 12 . 0 to 13 . 5 ml as shown in Figure 5A . The RapF and ComA bands were separated by SDS-PAGE and excised for total amino acid analysis at the Keck Biotechnology Resource Laboratory ( Yale ) . Analytical ultracentrifugation measurements were carried out on a Beckman XL-A ( Beckman Coulter ) analytical ultracentrifuge equipped with an An-60 Ti rotor ( Beckman Coulter ) at 20°C . 50 µM RapF and 50 µM RapF mixed with 500 µM PhrF were dialyzed overnight against 20 mM Tris pH 8 . 0 and 150 mM NaCl and analyzed at rotor speeds of 13 , 000 ( Figure 5B ) and 16 , 000 rpm ( unpublished data ) . RapF-ComA Superdex 200 HR10/30 gel filtration fractions spanning 12 . 0 to 13 . 5 ml as shown in Figure 5A were dialyzed overnight against Buffer A minus DTT and analyzed at rotor speeds of 9 , 000 rpm ( Figure 5D ) and 16 , 000 rpm ( unpublished data ) . Data were acquired at two wavelengths per rotor speed setting and processed simultaneously with a nonlinear least squares fitting routine [64] . Solvent density and protein partial specific volume were calculated according to solvent and protein composition , respectively [65] . Luciferase assays were performed as previously described with the following modifications [15] . The PsrfA-luc reporter strains ( see Text S1 ) were grown in LB medium to an OD600 of 2 . 0 , centrifuged , and resuspended in fresh Competence Medium ( CM ) [66] to an OD600 of 2 . 0 . The cultures were then diluted 20-fold in fresh CM supplemented with 0 . 25 µM IPTG to induce expression of wild-type or mutant rapF from the Pspank ( hy ) promoter . 200 µl of the induced cultures were dispensed per well in duplicate in a 96-well black plate ( Corning ) . Luciferin was added to each well at a final concentration of 0 . 47 µM . The cultures were then incubated at 37°C under agitation in a PerkinElmer Envision 2104 Multilabel Reader . The plate lids were heated to 38°C to avoid condensation . Relative Luminescence Unit ( RLU ) and OD600 were measured at 1 . 78 min intervals . Atomic coordinates and structure factors for RapF-ComAC have been deposited in the Protein Data Bank under accession code 3ULQ . | Upon phosphorylation , bacterial proteins called response regulators bind to DNA promoters and activate or repress transcription . These response regulators are themselves regulated by anti-activator proteins , which can control response regulator activity without altering their phosphorylation state . We have determined the X-ray crystal structure of the anti-activator RapF complexed with the DNA-binding domain of the response regulator ComA . Our structure-function studies show that RapF disrupts the binding of ComA to DNA using a two-pronged mechanism . First , a RapF surface mimics DNA , and this DNA-like surface binds to nearly all of the ComA DNA-binding residues , thus blocking ComA's interaction with DNA . Second , RapF inhibits ComA dimerization . RapF is also regulated by the PhrF peptide; we find that the RapF-ComA interaction surface is distant from the proposed PhrF binding site . Furthermore , we found that RapF undergoes a conformational change upon binding to PhrF , which likely causes its dissociation from ComA . From these observations , we conclude that PhrF binding to RapF allosterically triggers its dissociation from ComA . Finally , we compared the RapF/ComA DNA-binding domain complex structure with the structure of another response regulator , Spo0F , complexed with the phosphatase RapH . This reveals that while RapF and RapH are structurally similar , they have evolved distinct , non-overlapping surfaces to interact with their different cellular targets . | [
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"development",
"molecular",... | 2011 | Structural Basis of Response Regulator Inhibition by a Bacterial Anti-Activator Protein |
Asian schistosomiasis is a zoonotic parasitic disease infecting up to a million people and threatening tens of millions more . Control of this disease is hindered by the animal reservoirs of the parasite , in particular the water buffalo ( Bubalus bubalis ) , which is responsible for significant levels of human transmission . A transmission-blocking vaccine administered to buffaloes is a realistic option which would aid in the control of schistosomiasis . This will however require a better understanding of the immunobiology of schistosomiasis in naturally exposed buffaloes , particularly the immune response to migrating schistosome larvae , which are the likely targets of an anti-schistosome vaccine . To address this need we investigated the immune response at the major sites of larval migration , the skin and the lungs , in previously exposed and re-challenged water buffaloes . In the skin , a strong allergic-type inflammatory response occurred , characterised by leukocyte and eosinophil infiltration including the formation of granulocytic abscesses . Additionally at the local skin site , interleukin-5 transcript levels were elevated , while interleukin-10 levels decreased . In the skin-draining lymph node ( LN ) a predominant type-2 profile was seen in stimulated cells , while in contrast a type-1 profile was detected in the lung draining LN , and these responses occurred consecutively , reflecting the timing of parasite migration . The intense type-2 immune response at the site of cercarial penetration is significantly different to that seen in naive and permissive animal models such as mice , and suggests a possible mechanism for immunity . Preliminary data also suggest a reduced and delayed immune response occurred in buffaloes given high cercarial challenge doses compared with moderate infections , particularly in the skin . This study offers a deeper understanding into the immunobiology of schistosomiasis in a natural host , which may aid in the future design of more effective vaccines .
Asian schistosomiasis , caused by Schistosoma japonicum , is a parasitic disease endemic in the marsh and lake regions of China , particularly along the Yangtze River basin and mountainous regions [1] , [2] , and parts of the Philippines and Indonesia [3] . Up to 1 million people are infected with 50 million at risk of infection [1] , [4] . Schistosomiasis causes significant morbidity in chronically infected individuals , perpetuating poverty in endemic communities [5] , and is considered to be one of China's major public health priorities [6] . Control of Asian schistosomiasis relies largely on chemotherapeutic treatment with praziquantel ( PZQ ) however this is complicated by the fact that it is a zoonotic disease and infects a wide variety of mammalian hosts [7] , [8] . In particular , the domestic water buffalo , Bubalus bubalis , is considered the most significant animal reservoir of schistosomiasis in China [1] , [9] , [10] where it has been shown to account for up to 75% of human transmission [11] . This results in the need for routine PZQ treatment of livestock in addition to humans for parasite control [1] . However as a complementary strategy , a vaccine could contribute significantly to the elimination of schistosomiasis [1] , [12] , [13] , [14] , [15] , [16] , [17] , [18] . An effective vaccine for buffalo alone would significantly reduce human morbidity as well as improve buffalo health [19] , and for this reason an effort has been made to develop a transmission-blocking veterinary vaccine [17] . Much of our understanding of the immunobiology of schistosomiasis comes from murine models [20] but there is a distinct lack of understanding of the immunological mechanisms during schistosomiasis in natural large animal hosts , including water buffaloes . Moreover the vast majority of previous studies were performed in naive mice , and there is limited information on the response from endemically-exposed hosts , which are the intended recipients of a vaccine . There are several reports of age-related acquired resistance in buffaloes that show that older buffaloes have reduced intensity and prevalence of infection [2] , [9] , [21] , [22] . Also , buffaloes are known to have a natural resistance to S . japonicum infection compared with other hosts , such as cattle , Bos taurus [8] , [23] . While this acquired and natural immunity has been documented , it has not been studied in depth and the mechanism is unknown . Since the migrating schistosomula are recognised as a susceptible target in humans and other animals [14] , [24] , [25] , [26] , it is likely that this stage may be also targeted by immune effector mechanisms in buffaloes . In order to elucidate the immunobiology of schistosomiasis in an endemic host , we investigated the local immune response directed at the migrating schistosome larvae in water buffaloes from an endemic area in China . We characterised the type of immune response at each local site of larval migration , the skin and the lungs , in comparison with the liver where the majority of eggs are trapped . The results of this study may shed light on a mechanism of immunity in a natural host of S . japonicum and inform the design of more effective vaccines .
All experimental animal procedures were performed with written approval from the Ethical Review Board of the Hunan Institute of Parasitic Diseases ( approval no . 110818 ) , and from the Monash University Animal Ethics Committee ( no . 2011-124-FW ) . The handling and care of the animals were performed by trained staff adhering to good animal practice guidelines according to the Animal Ethics Procedures and Guidelines of the People's Republic of China . Eleven mixed-sex ( 8 male and 3 female ) water buffaloes ( B . bubalis ) were selected from a schistosomiasis-endemic area in Hunan Province , aged between 1–1 . 5 years ( Table 1 ) . Animals previously exposed to S . japonicum were selected based on positive faecal egg counts , which required testing over 50 buffaloes from the region . Animals were then moved to S . japonicum-free pasture in a non-endemic region of Hunan Province to rest for 7 weeks , during which time they were treated with praziquantel ( PZQ ) twice to cure the existing schistosomiasis . Because PZQ is only effective on adult worms older than 4 weeks , the second dose was to cure any parasites acquired immediately prior to being moved to the parasite-free pasture . Subsequent to the second PZQ treatment the miracidial hatch test was performed [27] and was negative for all buffaloes , indicating they were no longer excreting viable eggs . Eight buffaloes were chosen randomly for experimental infections , and these were divided into two groups for sacrifice at two time points; either at 5 and 6 days post-infection ( 5–6 DPI ) to study the skin-penetrating cercariae , or 11–12 DPI to focus on the lung-migrating larvae . These particular time points were chosen to study the matured adaptive response in the local draining lymph node ( LN ) , since the peak of an adaptive immune response occurs around 4–5 days in draining LN following antigenic challenge [28] , [29] . The response in the skin-LN was likely to be maximal at 5–6 DPI while 11–12 DPI was chosen to study the response against the lung-stage of infection , as the peak of larvae migration through the buffalo lung is around 7 DPI ( unpublished observations ) . Three buffaloes were left un-infected ( group NI ) and sacrificed at different days throughout the trial ( −1 , 5 and 11 DPI ) . Infection with S . japonicum cercariae was administered percutaneously on the shaved inner thigh , by transferring cercariae with a sterile culture loop onto a glass cover slip , and then resting this on the moistened skin for at least 30 minutes . The S . japonicum cercariae were obtained from Oncomelania hupensis snails collected from the marshlands of the Dongting Lake Region , Hunan Province . Three buffaloes in each challenge group were given a moderate infection of 200–600 cercariae , depending on availability ( Table 1 ) . Matched pairs were given the same cercarial number , and numbered according to this dose . One buffalo at each time point ( #4 ) received a high dose of 2400 cercariae to be used as a source of highly-stimulated samples for a separate study . At post-mortem , lymph nodes ( LN ) draining the skin of the inner thigh ( inguinal LN ) , lungs ( mediastinal LN ) and liver ( portal LN ) were collected and sliced into pieces in a sterile petri dish containing approximately 5 ml of media ( RPMI 1640 Glutamax , supplemented with penicillin ( 100 U/ml ) and streptomycin ( 100 µg/ml ) ( Life Technologies ) ) . These were stored on ice until processed . A sample of skin ( ∼1 cm2 ) was removed from the site of parasite infection , as was a similar piece of lung tissue . Half of each was placed into RNAlater ( Ambion ) , and the other half fixed in 10% formal saline for up to 2 weeks , followed by storage in 70% ethanol until they were processed into paraffin for histological analysis . Lymph node slices were gently teased apart in cold media under sterile conditions . Cells were washed twice in cold media , each time collected by centrifugation at 400× g for 10 min at 4°C . Lymphocytes were counted using a haemocytometer and trypan blue to exclude dead cells , and were resuspended to 1×107 cells/ml in media supplemented with AlbuMAX II ( 2 mg/ml; Life Technologies ) . Lymphocytes were cultured for 48 hours at 37°C with 5% CO2 and stimulated by adding phorbol myristate acetate ( PMA; 10 ng/ml ) and ionomycin ( 1 µg/ml ) . After culturing , the cell supernatants were collected and stored at −20°C until required . Fixed skin samples were paraffin-embedded and 4 µm sections were cut and stained with haematoxylin and eosin ( H&E ) for leukocyte counting , toluidine blue for mast cell differentiation , or stained overnight with Llewellyn's Sirius red for eosinophil identification [30] . Cells were counted in 5–7 different fields-of-view at 400× magnification to determine the numbers of these cell types in the skin . To count the number of leukocytes in the epidermis , 5 sections of 0 . 26 mm in length were enumerated . To determine the thickness of the epidermis and stratum corneum layers , ImageJ [31] was used to calculate the volume of each layer relative to the length of the skin section ( determined by measuring the stratum granulosum ) . For each sample , three sections of skin ( approximately 0 . 8 mm ) were measured . Skin eosinophilic abscesses were avoided for the cell counts since individual cells could not be distinguished . Buffalo skin samples were disrupted using a T10 basic Ultra-Turrax homogeniser ( IKA ) in the presence of Qiazol ( Qiagen ) , and the RNA was subsequently extracted according to Qiagen's protocol . After resuspending the RNA pellet in water , it was purified using the Total RNA Extraction Miniprep System ( Viogene ) and the concentration determined on a NanoDrop spectrophotometer ( Thermo Scientific ) . Total RNA ( 90 ng ) was converted to cDNA using the QuantiTect Reverse Transcription Kit ( Qiagen ) which includes a genomic DNA removal step . To perform the qPCR , primers were used based on B . bubalis sequences [32] , except for galectin-14 which was based on the ovine sequence since the B . bubalis sequence was not available . The sequences of the primers are shown in Table 2 . The qPCR reactions included SYBR Master Mix ( Applied Biosystems ) and the above primers at 0 . 5 µM and 5 µl of 1∶20 diluted cDNA , and were run in triplicate on an Eppendorf Realplex4 Mastercycler for 40 cycles , using the annealing temperatures ( Tm ) shown in Table 2 . Melt curve analysis was done to ensure a single product was amplified . The relative copy number of SYBR green for each gene was calculated from a standard curve of serial dilutions of cDNA , and then the relative expression of the target gene was determined relative to the reference gene copy number . The levels of IFN-γ , IL-4 and IL-10 were quantified in cell culture supernatants by sandwich ELISA . To determine IFN-γ levels , the Bovine IFN-γ ELISA kit ( AbD Serotec ) was used according to the manufacturer's instructions . For IL-4 and IL-10 , monoclonal antibody pairs were used ( AbD Serotec ) . Plates were coated with mouse anti-bovine IL-4 or IL-10 monoclonal antibody ( 2 . 5 µg/ml ) overnight at 4°C . All subsequent steps were done at room temperature , and washing consisted of 5 changes of PBS with 0 . 05% Tween-20 ( PBST ) . After blocking with 0 . 1% bovine serum albumin in PBS for 1 hour , samples were incubated for 1 . 5 hours ( the supernatants or recombinant bovine IL-4/IL-10 for standard curves ) . The supernatant samples were diluted 1∶20 for IL-4 and IFN-γ and 1∶50 for IL-10 measurement . After washing , biotinylated anti-bovine IL-4 or IL-10 was added at 1∶1600 or 1∶1000 , respectively , and incubated for 1 hour and subsequently washed . Finally , horseradish peroxidise-conjugated streptavidin ( Dako ) was added at 1∶1000 for 1 hour , and plates were developed using TMB Substrate ( Life Technologies ) and optical density measured using a spectrophotometer . Supernatant OD readings were compared to the linear part of the standard curve to determine the cytokine amount . This study was limited in the sample sizes that could be included for each group , due to the high cost , the large scale and the logistics involved in using water buffaloes . Therefore many of the differences are described as trends , and statistical results are only reported when significance was achieved at p<0 . 05 . When comparing three groups , a one-way analysis of variance test followed by Tukey's post-hoc test was performed , and when comparing two groups the student's t-test was employed . To measure correlations between two parameters , the Spearman's rank correlation was performed and the rank coefficient ( rs ) was determined . Considering that the two high-dose buffaloes ( #4 ) received up to 12 fold more cercariae than the moderate infections , their data are not included in the means or statistical analyses but are represented separately in the figures .
Buffaloes were infected via the cover-slip method on the inner thigh and at 5 DPI the skin site where cercariae were applied had an obvious raised inflammatory reaction ( Fig . 1A ) . By 11–12 DPI the skin reaction had reduced and was no longer raised . No difference was seen in the lungs between infected and uninfected buffalo , at either time point ( data not shown ) . All buffaloes were selected based on positive S . japonicum faecal egg counts ( see Table 1 ) , and this previous infection history was reflected in the gross morphology of the livers where white nodules were visible ( Fig . 1B ) . All livers had a similar appearance due to these prior infections , which is typical of a patent bovine schistosome infection [33] . Compared with uninfected controls , the skin of infected buffaloes at both time points showed signs of strong inflammation ( Fig . 2 ) , characterised by large numbers of infiltrating leukocytes in infected ( Fig . 2B ) compared with control skin ( Fig . 2A ) . Thickening of the two outer layers , the epidermis and stratum corneum , was observed at 5–6 DPI ( Fig . 2C ) ; although not statistically significant , the thickness of all infected skin samples were above NI values . The epidermis was thickest at 5–6 DPI and then reduced to near NI level at 11–12 DPI , whereas the thickened stratum corneum increased slightly further at 11–12 DPI . Large , eosinophilic abscesses were seen in all skin sections at 5–6 DPI ( Fig . 2D ) , in most cases severely disrupting the architecture as they spanned multiple skin layers; these had mostly cleared up at the later time point with only one of the four buffaloes from 11–12 DPI group ( #1 ) showing abscesses . These abscesses consisted of dense nuclei , likely to be infiltrating leukocytes , with eosinophilic granules staining throughout ( not shown ) . Leukocytes were enumerated in the epidermal ( Fig . 2E ) and dermal layers ( Fig . 2F ) and showed a similar pattern: a large increase in leukocytes initially at 5–6 DPI and a slight reduction at 11–12 DPI . This was significantly higher only in the epidermal layer . A large number of infiltrating eosinophils were seen in infected buffalo skin compared with controls ( Fig . 3A–C ) , and this was predominantly in the dermal layer . Some eosinophils were seen invading discrete sections of infected epidermis ( Fig . 3B ) ; however this was not consistent and seen in very few sections , hence numbers were not determined . Eosinophils were enumerated in the dermal layer ( Fig . 3C ) , showing a dramatic increase from a negligible amount ( 19 cells/mm2 ) in control skin to 710 cells/mm2 of dermis at 5–6 DPI and then dropping slightly to 410 cells/mm2 at 11–12 DPI . When individual data points of eosinophil numbers in infected dermis from buffaloes given the same dose of infecting cercariae were linked , it was observed that for all buffaloes receiving a moderate challenge dose the number of eosinophils was highest at 5–6 DPI , reducing by 11–12 DPI ( Fig . 3D ) . However , for the pair of buffaloes given the high dose ( #4 ) the number of eosinophils was highest at the later time point . Mast cells were observed in the dermis of all skin samples ( Fig . 3E ) , including NI skin , with a consistent increase in mast cell numbers only at 11–12 DPI compared to uninfected skin; despite no statistical significance , all samples at this latest time point had a higher number of mast cells than NI sections , increasing from a mean of 64 to 90 cells/mm2 . The type of immune response occurring in the skin infection site was investigated by measuring the transcript level of IFN-γ ( a typical Th1 cytokine ) , IL-4 ( a Th2 cytokine ) , IL-5 ( a Th2 cytokine which promotes eosinophil proliferation and survival [34] ) , IL-10 and galectin-14 ( a molecule specific to eosinophils [35] ) ( Fig . 4A–E ) . The levels of these transcripts were normalised to a reference gene ( β-actin ) and then calculated relative to the mean of control samples to observe changes resulting from infection . There was a slight non-significant drop in skin IFN-γ following infection , and IL-4 levels remained fairly constant in each group . At both time points post-infection , the level of IL-10 expression was reduced significantly ( p<0 . 001 ) to approximately one-fifth of the control level . IL-5 dramatically increased to 7 . 5 times the level of normal skin at 5–6 DPI and then returned to the NI level at 11–12 DPI , although this was not significant ( p = 0 . 302 ) . The level of galectin-14 increased to approximately 14 times the level of expression in NI skin at 5–6 DPI , reducing to 5 times the level at 11–12 DPI , also not significant ( p = 0 . 491 ) . Furthermore , there was a positive and significant correlation between the number of eosinophils in the skin with the level of galectin-14 ( p = 0 . 011 , rs = 0 . 817; Fig . 4F ) , but not with the level of IL-5 ( p = 0 . 744; data not shown ) . As found with the number of eosinophils in tissues , only the #4 buffaloes given the high dose had an increase in galectin-14 expression at 11–12 DPI compared to 5–6 DPI . Levels of IFN-γ and IL-4 , the prototypical Th1 and Th2 cytokines respectively , and IL-10 , were measured in stimulated cell cultures of LN draining the skin , lung and liver , and the means for the six moderate infection dose animals are shown in Fig . 5 . For the skin-LN , there was an increase in all three cytokine levels at 5–6 DPI , which was reduced at 11–12 DPI . While only IL-4 showed a statistically significant increase at 5–6 DPI above NI levels ( p = 0 . 047 ) , all three buffaloes at 5–6 DPI had higher IFN-γ levels than NI animals and two remained higher at 11–12 DPI . Interestingly , the high-dose buffaloes had higher levels of each of these cytokines at 11–12 DPI than 5–6 DPI in the skin-LN cultures ( Fig . 5 ) , similar to the eosinophil and galectin-14 levels in the skin . For the lung-LN response , only the IFN-γ levels showed a significant increase at the latest time point of infection compared to the NI level ( p = 0 . 036 ) ; there was no change in IL-4 or IL-10 over the infection period . Among the uninfected animals , the lung-LN had a higher level of IL-4 and IL-10 than skin-LN , indicating a difference in basal stimulation of the lungs draining LN compared to those of the skin . The IFN-γ levels were consistent among each LN in NI individuals . Finally , the liver-LN response showed a trend towards an increase for each cytokine at the latest time point , although this was only significantly above NI for IFN-γ ( p = 0 . 011 ) .
This study investigates the local immune response against migrating schistosome larvae in the water buffalo . Despite the evidence of acquired immunity in this natural host , there have been few studies on the buffalo immune response during schistosomiasis [14] , and to our knowledge none exploring the anti-larval response . In the present study , the skin and lungs were examined at two time points after cercarial infection , estimated to reflect a mature immune response in the LN draining those tissues . Overall the findings indicated that these time points did correspond with the timing of larval migration , since we observed consecutive cytokine responses; the response in the skin-LN was highest at 5–6 DPI , with the lung-LN IFN-γ highest at 11–12 DPI . The local immune response against invading cercariae in the skin was the most obvious pathological change evident during the study and was characterised by a strong type-2 inflammatory response . Examination of the skin site showed raised allergic ‘wheal’ type reactions at 5–6 DPI , which had reduced by 11–12 DPI . Histological and qPCR analysis , as well as skin-LN cell stimulations , confirmed a predominant type-2 response against the penetrating larvae at this tissue site . Large numbers of eosinophils were observed infiltrating the dermis even as late as 12 DPI , which was confirmed by transcript levels of the eosinophil-specific galectin-14 . Large eosinophilic abscesses were also observed to disrupt the skin layers in these regions , similar to those described by Incani and McLaren [36] in mouse skin after a secondary infection . They found these abscesses contained dead larvae and discarded tails at 6 hours post-infection and were associated with eosinophils . Although we did not observe trapped larvae , they may have been present at earlier time points than investigated in our study or revealed by more thorough investigation of skin samples . The stratum corneum , which is the principle defensive layer of the skin [37] , showed significant thickening at 5 DPI and remained thickened even at 11–12 DPI , and this may play a protective role from further cercarial penetration . Further examination of the skin at 11–12 DPI indicated that the larvae-associated damage had begun to heal , with several parameters returning to normal levels at this time point . The increase in mast cells at this late time point could be evidence of tissue recovery , since their numbers are known to increase in skin during wound healing [38] . The most marked changes in skin cytokine transcript levels were the increase seen in IL-5 at 5–6 DPI , and the sharp and sustained reduction in IL-10 levels at both time points . In contrast , IFN-γ and IL-4 remained fairly constant . IL-5 is predominantly secreted by Th2 cells and promotes eosinophil proliferation and survival [34] , consistent with the large number of eosinophils observed at this time point . By 11–12 DPI , IL-5 levels had returned to baseline indicating the type-2 response was waning , consistent with a reduction in skin-resident eosinophils . Importantly , IL-5 responses to the vaccine candidate paramyosin have been correlated with immunity for S . japonicum [39] and S . mansoni [40] . In contrast to our findings , IL-10 has been shown to increase after cercarial penetration of naive mouse and human skin [41] , [42] . As a regulatory cytokine , IL-10 limits excessive inflammation by targeting leukocytes , down regulating pro-inflammatory cytokine secretion [43] . In schistosomiasis it has been considered to be a key immunomodulatory strategy of the schistosome parasite to limit the immune response during skin penetration [44] . The lack of IL-10 induction in the buffalo skin could explain the sustained skin inflammation and suggests a significant host response difference to cercarial penetration at the local site . There was an increase in IL-10 in the skin-LN however , which has been seen similarly in other hosts [44] . Additionally in the draining skin-LN there was an increase in IFN-γ and IL-4 following infection , suggesting a mixed type-1 and -2 response . A similar mixed response has been reported following primary infection of mouse skin [41] . However in the present study , the ratio of these two cytokines suggests it was predominantly oriented towards the type-2 response , and this agrees with the allergic-type reactions visible in the skin at the same time . In contrast to the immune events described in the skin , there was no detectable gross or histological change in lung tissue post-infection ( data not shown ) . This could be due to the fact that the relatively small schistosomula disperse throughout the lungs , which are of considerable size in water buffalo . However , the larvae did induce a measurable and significant type-1 response in the lung-draining LN at 11–12 DPI , where the IFN-γ level increased compared with non-infected levels . There was a high IL-10 level in all lung samples including uninfected animals . This could limit the extent of the inflammatory Th1 response and reduce larval killing in the lungs because a strong lung Th1 response is considered the mechanism of immune killing in the murine radiation-attenuated vaccine model [45] . The liver-LN also had more of a type-2 response than uninfected skin-LN , likely due to trapped liver eggs from previous infections which are strong type-2 inducers [20] . Similar to the lung-LN response at 11–12 DPI , the liver-LN had a late IFN-γ response , although this was less pronounced . This slight type-1 response was possibly due to the arrival of immature worms into the liver . The timing of larvae entering the liver is likely to be shortly after leaving the lungs , as occurs in the mouse model [46] , since the transit time between organ vasculature is minimal [47] . One animal at each time point was infected with a significantly higher dose of cercariae in an attempt to generate highly stimulated LN samples for a separate study . Surprisingly , in almost every parameter these high-dose buffaloes consistently had the lowest response in the skin draining lymph nodes compared to the moderate infected buffaloes . In addition , the number of eosinophils and galectin-14 transcripts in the skin , and the cytokine levels in skin-LN cultures , was greater in the high-dose animal at 11–12 DPI than at 5–6 DPI , whereas the inverse was true for each of the buffaloes receiving a moderate challenge infection . While these differences were intriguing , it should be noted that it is based on a single animal in each group and could be due to individual variation , and hence should be repeated with larger numbers to draw firm conclusions . However a possible explanation for these observations is that there is greater down-regulation of the immune response in the skin and draining LN with the higher challenge dose . It is well documented that cercarial secretions have immunomodulatory properties which reduce inflammation and facilitate immune evasion [44] , and the higher number of cercariae would mean more secretions present in the skin . Additionally , while cercariae are generally thought to discard their tails before skin penetration , Wang et al . [48] recently showed that with high-density cercariae infections more tails enter the skin . It is possible that the products derived from these tails may also suppress or delay immune responses in the high-dose buffaloes . Doses of several thousand cercariae have been used in previous buffalo studies [23] , [33] , [49] and in future immunological investigations lower doses may be more appropriate . Overall , the data show that an inflammatory type-2 response was generated following cercarial invasion of water buffalo skin , with a mixed but predominantly type-2 response in the skin draining LN . Then as the larvae migrated to the lungs , a moderate type-1 response was seen in the lung-LN . To our knowledge , this is the first time the immune response to migrating schistosome larvae has been investigated in an endemically-exposed host , shedding light on the immune mechanisms induced during a natural infection . The current paradigm of schistosomiasis holds that the migrating larvae induce a primarily Th1 response , which then becomes a strong Th2 response several weeks later after the worms mature and eggs are deposited in tissues [20] . This has largely been elucidated from primary infections in naive rodent models , where the eggs provide a strong Th2-inducing stimulus [50] . The radiation-attenuated vaccine has also been studied extensively in mice , where immunity is mostly attributed to a protective Th1 response primed in the skin , which is then effected in the lungs [24] , [45] , [51] . In contrast to the Th1-protective mechanism in the mouse , protection in humans from schistosomiasis-endemic communities is linked with type-2 responses and is positively correlated with specific IgE levels [52] , [53] , [54] and Th2 cytokines [39] , [55]; protection is thought to be effected by eosinophils against the incoming larvae using antibody-dependant cellular cytotoxicity [3] , [20] , [56] , [57] , [58] . Cook et al . [59] recently showed that mice given repeated cercarial infections , before egg deposition , have a Th2 phenotype in the skin with eosinophil infiltration indicating the larvae can also skew towards this response in this animal model . A similar allergic inflammatory skin response was seen in rhesus monkeys given repeated immunisations with radiation-attenuated S . japonicum larvae [60] . In this model the skin is the major site of larval attrition during the first 24 h , and larvae were cleared by 3 days [61] . Since buffaloes have a natural and acquired immunity , it is conceivable that the strong inflammatory type-2 response with eosinophil infiltration in the skin region observed here is a mechanism for immunity against penetrating larvae in a similar fashion to that seen in immunised rhesus monkeys . It also reflects what is thought to be the mechanism in human immunity , since human eosinophils have been shown to kill newly-transformed larvae mediated by specific antibody [56] . Unlike in other hosts , IL-10 was not induced in the skin and this suggests the possibility that a local regulatory environment is not induced in buffalo skin , allowing an effective skin response to be induced . Furthermore it is possible that the increased skin thickness of large animal hosts compared to murine skin may delay larval migration and allow more effective immunity to develop at this tissue site . While trapped or killed larvae were not observed in buffalo skin here , dead larvae would most likely be cleared before 5–6 DPI as in immunised rhesus monkeys , and further studies at earlier time points would be required to confirm this . The present study provides a novel insight into the immune response of water buffaloes , important transmission reservoir hosts for schistosomiasis japonica , and indicates that distinct type-1 and -2 responses occur in distinct tissue regions against migrating larvae , with a strong eosinophil-dominated type-2 response in the skin . Since water buffaloes may be able to generate partial immunity to S . japonicum , vaccine formulations replicating the response described in this study may be needed in order to be effective . | Schistosomiasis is caused by a parasitic blood fluke , and in parts of Asia it infects both humans and livestock such as water buffaloes . This makes controlling the disease more difficult , because both humans and livestock must be treated regularly . A vaccine given to buffaloes is likely to reduce human infection rates and improve buffalo health by providing long-lasting protection from re-infection; at present no vaccines are available . Older buffaloes are known to have some immunity to schistosomiasis which is acquired over time; however how this occurs is not understood . In this study we investigated the immune response of buffalo against the schistosome larvae , which are vulnerable to immune attack , and hence are the ideal stage to target for vaccination . We found that the buffalo produces a profound allergic type-2 response as larvae penetrate the skin , with significant cellular infiltrates and abscesses . When the larvae move next to the lungs , a uniquely type-1 response was induced . This skin response is much greater than more susceptible animals such as mice , and may be a mechanism for larval killing in the buffalo . This study offers insight into the immunobiology of an important host for schistosomiasis and may help in designing better vaccines . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [] | 2013 | Local Immune Responses of the Chinese Water Buffalo, Bubalus bubalis, against Schistosoma japonicum Larvae: Crucial Insights for Vaccine Design |
Macrophages are cells with remarkable plasticity . They integrate signals from their microenvironment leading to context-dependent polarization into classically ( M1 ) or alternatively ( M2 ) activated macrophages , representing two extremes of a broad spectrum of divergent phenotypes . Thereby , macrophages deliver protective and pro-regenerative signals towards injured tissue but , depending on the eliciting damage , may also be responsible for the generation and aggravation of tissue injury . Although incompletely understood , there is emerging evidence that macrophage polarization is critical for these antagonistic roles . To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied . A comprehensive literature-based Boolean model was developed to describe the M1 ( LPS-activated ) and M2 ( IL-4/13-activated ) polarization types . The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages . By dynamic modeling of gene expression , the chronology of pathway activation and autocrine signaling was estimated . Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation , indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization .
A large part of macrophage populations originate from monocytes released from the bone marrow that upon injury or inflammation migrate into several tissues of the body such as lung , liver , spleen , lymph node , bone or the central nervous system [1 , 2] . As it harbors the body’s largest pool of sessile tissue macrophages the liver owns a distinguished role within the macrophage system . Lineage tracing experiments [3 , 4] suggest that under homeostatic conditions macrophages recruited from circulating monocytes are of minor importance for maintenance of the population of tissue resident liver macrophages ( also termed as Kupffer cells ) . These cells have janitorial and immune regulatory functions but play a subordinate role for induction and regulation of inflammatory reactions evoked by tissue injury . The latter is mainly adopted by macrophages recruited to the liver from circulating monocytes . They play a critical role for the generation and control of inflammatory reactions that either cause and aggravate tissue injury or mediate processes required for tissue repair [3] , which are also the basis for the unique capability of the liver to regenerate [5] . These diverging functions of macrophages , being either responsible for induction and aggravation of tissue injury or for induction and maintenance of repair processes , are attributed to the fact that macrophages exhibit a remarkable plasticity and can embrace a large spectrum of different activations states and functions . They can be activated by a variety of external stimuli including microbial products , nucleotide derivatives , growth factors , glucocorticoids as well as cytokines and change their physiology in response to these environmental factors [6] . Macrophages are classified into classically activated macrophages ( M1 ) or alternatively activated macrophages ( M2 ) [7] representing the two poles of a continuous spectrum of polarization states [8] . In accordance with experimental guidelines that have been published recently , the M1 polarization of macrophages can be triggered by stimulation with lipopolysaccharide ( LPS ) and/or IFNγ [6] . These cells exhibit microbicidal or tumoricidal activity , and secrete high amounts of pro-inflammatory cytokines and upregulate the surface marker CD69 . Contrariwise , M2 polarization of macrophages can be induced by stimulation with the two interleukins ( IL ) IL-4 and IL-13 . Among others , these cells are characterized by enhanced expression of the mannose receptor ( CD206 ) and by increased arginase activity [1 , 9 , 10] . The toll-like receptor 4 ( TLR4 ) pathway in M1 macrophages that is induced in response to LPS stimulation is quite well characterized [11 , 12] . It primarily leads to activation of the transcription factors NF-κB and interferon regulatory factor 3 ( IRF3 ) [11] which in turn are involved in the regulation of gene expression including that of interferons [13] , pro-inflammatory cytokines such as IL-1β , IL-6 and TNFα [14 , 15] and chemokines [16] . Other key proteins that regulate amount and duration of transcript expression during an inflammatory response towards bacterial or viral infections are the p38 mitogen-activated protein kinase ( p38MAPK ) as well as the p38MAPK-activated protein kinases MK2 and MK3 [15 , 17–22] . For a functional inflammatory response , LPS needs to successively trigger autocrine and paracrine signaling events that are critical for regulation of the initiation , the propagation and , finally , also the resolution of an inflammatory response . A cytokine which is critically involved in the resolution of an inflammatory response and controlled via such autocrine cycles is the anti-inflammatory cytokine IL-10 . Lack of this cytokine or specific deletion of its major effector protein , the transcription factor ( TF ) signal transducer and activator of transcription ( STAT ) 3 in macrophages [15 , 23–25] , results in an overwhelming and deleterious inflammatory response towards pathogens or pathogen stimuli such as LPS . The expression of this cytokine and subsequent activation of STAT3 requires the auto- and/or paracrine activation of the type I interferon receptor ( IFNAR ) via LPS-induced release of type I interferons ( IFN ) such as IFNβ . Activation of IFNAR has been demonstrated to be a prerequisite for LPS-inducible expression of interleukin 10 ( IL-10 ) and subsequent activation of STAT3 [15 , 17 , 26] . The molecular mechanisms involved in the induction of alternatively activated M2 macrophages by IL-4 and IL-13 are less well characterized . Identification of the mannose receptor as a marker protein for alternative activation of macrophages was first proposed by Stein et al . [9] . Apart from this receptor , arginase 1 ( Arg1 ) , the chitinase-like molecules Ym1 and Ym2 , as well as Fizz1 [27] have been identified as genes characterizing the M2 phenotype . M2 macrophages are generally involved in homeostasis , wound healing and tissue repair [28] , but are also implicated in pathological processes like asthma and allergy [29] . The phosphatidylinositol 3-kinase ( PI3K ) /Akt pathway is activated both after stimulation with LPS [30–32] and IL-4/13 [33] . Knockout studies demonstrated an important role of the two isoforms Akt1 and Akt2 in macrophage polarization [34 , 35] . To illustrate the relevance of macrophage polarization , the liver may be a good example as it clearly is central for the control of the systemic acute phase response . The maintenance of a critical balance of pro-survival and pro-apoptotic as well as pro- and anti-inflammatory signals in the liver is essential as dysregulation can have detrimental effects and might lead to organ damage [36] . Risk factors such as alcohol abuse , intake of drugs or toxins , obesity , and chronic HBV/HCV infection can disturb this balance and might develop into chronic hepatitis leading to liver fibrosis and , in the final stage , liver cirrhosis and organ failure [37] . All these conditions involve chronic activation of inflammation and , as a result of ongoing inflammation , impaired wound healing . In this context , the activation of inflammatory pathways in the liver and the release of cytokines from inflammatory cell populations , including macrophages , and their implications on hepatocytes are of particular importance [38 , 39] . For example , the cytokines IL-1β and TNFα not only promote inflammation , but also influence cell death signaling in hepatocytes [40 , 41] . Depending on the eliciting damage , most of the cytokines and pathways involved exhibit dual functions . For instance , NF-κB has been identified as a pathway , which mediates effects important for hepatocyte survival , but has been also identified as a signaling intermediate that propagates liver damage upon ischemia-reperfusion ( I/R ) injury [38] . Likewise , chemokines , such as Cxcl2 have controversial functions in the regulation of liver regeneration . It supports liver regeneration after partial hepatectomy , whereas it impairs recovery from I/R injury [42 , 43] . Similarly , macrophage activation into the various phenotypes mediate heterogeneous effects . Overwhelming M1 activation can induce sepsis , predispose surrounding tissue for neoplastic transformation as well as promote insulin resistance and type 2 diabetes . Whereas excessive M2 activation can induce liver fibrosis during chronic infection and aggravate allergic responses [1] . A systematic understanding and description of the influences leading to activation of macrophages into the various phenotypes as well as the corresponding cytokine pattern is of particular importance for understanding the role of macrophages in health and disease . In this work , we present a large-scale Boolean model that describes the major signaling pathways in M1 ( LPS-activated ) and M2 ( IL-4/13-activated ) primary murine bone marrow derived macrophages ( BMDMs ) as well as a dynamic model of the gene expression module . The initial literature-based network structure is adapted using high-throughput gene expression data from primary murine BMDMs in which , according to recent suggestions , M1 polarization was induced by treatment with LPS whereas M2 polarization was triggered by exposure towards IL-4 and IL-13 . In addition , the dynamics of mRNA expression are investigated in more detail using an ordinary differential equation-based model to disentangle the chronology of direct signaling events and autocrine feedback loops . By estimating the time frame of transcription factor activity , the model structure is further refined , for example IL-10 appears to be upregulated before the transcription factor Stat3 is activated , suggesting that Stat3 may not be essentially required for LPS-inducible expression of IL-10 . Investigation of secreted cytokines and autocrine feedback loops results in the last model refinement , that is the major pro-inflammatory cytokines inducing the acute phase response in hepatocytes , the interleukins IL-1β and IL-6 as well as TNFα [15 , 36] , exert no or minor autocrine effects on macrophages regarding the gene expression profile .
For a first description of macrophage activation , we constructed a comprehensive large-scale Boolean model including relevant signaling pathways activated in macrophages upon induction of M1 polarization by stimulation with LPS or induction of a M2 phenotype in response to combined stimulation with IL-4 and IL-13 ( S1 Fig ) . The model was based on extensive literature research and its first version contained 148 nodes and 176 interactions . Abbreviations of the network nodes are explained in S1 and S2 Tables . The model was implemented using the MATLAB Toolbox CellNetAnalyzer ( CNA ) [50] . It contains three input nodes ( black ) , namely LPS and the interleukins IL-4 and IL-13 . Output of the model is the gene expression pattern that results from the respective stimulation . A number of species ( grey ) have been demonstrated to induce autocrine feedback loops after secretion by macrophages . Among many others , this includes the cytokines IL-1β , IL-6 , IL-10 and TNFα as well as the interferons IFNα , IFNβ , and IFNγ [15 , 54–56] . The following signaling events are reproduced by the Boolean model: ( 1 ) LPS stimulation results in activation and formation of the TLR4 receptor complex containing TLR4 , MD-2 and CD14 as well as LPS and the lipopolysaccharide binding protein ( LBP ) . The activated TLR4 receptor complex can signal via the MyD88-dependent signaling pathway resulting in early activation of NF-κB and the mitogen-activated protein ( MAP ) kinases p38MAPK , JNK , and ERK , as well as the MyD88-independent , TRAM-/TRIF-dependent signaling pathway accounting for late phase NF-κB activation and activation of the transcription factor IRF3 [11 , 12] . Furthermore , the activated TLR4 complex leads to activation of the PI3K/Akt pathway [32] and increased expression of the microRNA miRNA-155 enhancing NF-κB activity [34 , 35] . The transcription factors NF-κB and IRF3 mediate expression of a number of genes associated with the classical M1 phenotype of macrophages . After secretion , some of these proteins induce autocrine signaling: ( 1 . 1 ) IL-1β binds to the IL-1R and its downstream signaling pathway shares similarity with TLR4-mediated signaling . Thus IL-1β also results in activation of NF-κB and the MAP kinases [54] . ( 1 . 2 ) Binding of TNFα to the TNFR1 results in assembly of the receptor complex 1 including TRADD , TRAF2 and Rip1 . This activates the kinase TAK1 leading to activation of NF-κB and the MAP kinases similar to TLR4 signaling [54] . ( 1 . 3 ) IL-6 signals through its receptor consisting of the ligand binding domain gp80 and the signaling subunit gp130 that subsequently gets phosphorylated by the associated Janus kinase 1 ( Jak1 ) leading to recruitment and activation of the TF Stat3 [57] . Activation of STAT3 is inhibited by suppressor of cytokine signaling ( SOCS ) 3 a member of a family of endogenous feedback inhibitors of STAT signaling , which can be induced by STAT family members themselves but also by other pathways , including the p38MAPK/MK2 pathway [58 , 59] . ( 1 . 4 ) The type II interferon IFNγ binds to its receptor IFNGR resulting in activation of the TF Stat1 via Jak1 or Jak2 [56] . ( 1 . 5 ) The type I interferons signal via the IFNAR leading to activation of the Stat transcription factors Stat1 and Stat3 followed by expression of respective target genes [55] . ( 1 . 6 ) One of the target genes upregulated upon activation of IFNAR1 is IL-10 that also acts in an autocrine feedback loop via the IL-10R mediating late and sustained activation of Stat3 that , in contrast to the activation of STAT3 by other cytokines , is resistant to the action of SOCS3 [60] , and important for the resolution phase of the inflammatory response [15] . ( 2 ) Stimulation with IL-4 and IL-13 results in activation of various receptor complexes . Both cytokines share a common receptor subunit , the IL-4Rα chain , which can either engage with the common γ chain ( IL-2Rγ ) upon IL-4 binding or with the IL-13Rα1 chain upon IL-13 binding [33] . IL-13 can also bind to the IL-13Rα2 whose cytoplasmic domains do not contain any Jak/Stat binding sequences [61] and thus is likely to act as a decoy receptor congruently with the phenotype of IL-13Rα2-deficient mice being consistent with enhanced IL-13 responsiveness [62] . IL-4/IL-13 signaling results 1 ( Arg1 ) and mannose receptor type 1 ( Mrc1 ) [27 , 63] . In addition , binding of IL-4 to IL-4Rα leads to activation of the PI3K/Akt pathway [64] resulting in decreased expression of the microRNA miRNA-155 and increased expression of the M2 marker gene Arg1 [34 , 35] . The PI3K pathway and the two isoforms Akt1 and Akt2 differentially influence macrophage polarization [34 , 35] . Arranz et al . [34] demonstrated that Akt1 KO gives rise to an M1 phenotype whereas Akt2 KO induces M2 polarization accompanied by high levels of Arg1 . Furthermore , they showed that downregulation of the microRNA miRNA-155 in Akt2 KO macrophages leads to elevated levels of the CCAAT/enhancer binding protein beta ( C/EBPβ ) and enhanced binding to the Arg1 promotor while Stat6 phosphorylation remains unaffected . In contrast , Akt1 KO leads to elevated levels of miRNA-155 which contributes to NF-κB activity and M1 polarization [35] . However , both isoforms , Akt1 and Akt2 , are concurrently activated in the WT after LPS as well as IL-4/13 stimulation and differentially influence miRNA-155 expression . For correct polarization of macrophages , i . e . induction of the M1 phenotype after LPS stimulation and induction of the M2 phenotype following IL-4/13 stimulation , the introduction of an additional signal into the model was necessary . This coregulating signal is induced by the activated TLR4 receptor complex and acts on the level of Akt activation or downstream signaling . Otherwise , the positive influence of Akt2 on miRNA-155 expression overcomes the inhibitory influence mediated by Akt1 leading to induction of the M1 phenotype even after IL-4/13 stimulation ( S2 Fig ) . Indeed , we observed increased expression of miRNA-155 after 6 h treatment of BMDMs with LPS and decreased expression after 6 h IL-4/IL-13 stimulation ( S3 Fig ) . In order to reproduce the sequential release of various factors by macrophages and following induction of autocrine signaling loops , timescale constants are introduced to the Boolean model [49] . A so called timescale constant τ can be assigned to all logical interactions as a parameter that specifies when a distinct signaling event takes place . A lower timescale constant indicates that the reaction becomes active earlier than an interaction with a higher timescale constant . The model introduced here contains eight timescales τ = {0 , 1 , 2 , 5 , 7 , 10 , 12 , 15} that are used to separate distinct signaling events ( Table 1 ) . Early or direct signaling events can thereby be distinguished from secondary signaling events resulting from autocrine signaling and negative feedback loops when analyzing the Boolean model . All logical equations with their assigned timescale constant are listed in S3 and S4 Tables . The housekeeping node , indicated by green boxes in the model scheme ( S1 Fig ) , represents constitutively expressed genes , such as adaptor proteins and receptors that are already present in the unstimulated state of the cell . Thus , these nodes are activated at the first timescale τ = 0 . The MyD88-dependent signaling events downstream of TLR4 leading to early NF-κB and MAP kinases activation as well as the signaling events downstream of the other receptor complexes are assigned to the timescale τ = 1 . The MyD88-independent signaling pathway downstream of TLR4 accounting for late phase NF-κB activation is triggered at timescale τ = 2 . All transcriptional and translational events take place at the timescale τ = 5 and τ = 7 , respectively . In order to induce the autocrine feedback , the interferons as well as IL-1β , IL-6 , and TNFα first need to be secreted by macrophages . Thus , these proteins occur twice in the model , one being the newly synthesized , intracellular protein at the bottom of the model scheme and one being the secreted protein at the top of the scheme activating the receptor on the cell surface . This secretion step is assigned to the timescale τ = 10 . For synthesis and secretion of IL-10 , first Stat3 needs to be activated by secretion of type I interferons and activation of IFNAR . Hence , IL-10 secretion occurs on a later timescale τ = 12 . All inhibitory reactions are assigned to the last timescale τ = 15 to allow discrimination of feedforward signaling events induced by stimulation of macrophages and negative feedback loops for model analysis . For model validation , primary murine BMDMs are differentiated via macrophage colony stimulating factor ( M-CSF ) stimulation according to experimental guidelines suggested recently for the investigation of macrophage polarization [6] . LPS stimulation was used to induce M1 polarization whereas the M2 phenotype was triggered via stimulation with IL-4 and IL-13 for 0 . 5 , 1 , 2 , 6 or 10 h . Prior to stimulation and after stimulation , macrophages were extensively characterized using cytometric analysis for the expression of different surface markers including CD14 , F4/80 , CD11b , CD68 , CD69 , CD86 and CD206 ( S4 Fig ) . Quantitative , time-resolved data on 48 transcript expression levels were generated using the high-throughput Taqman Fluidigm Technology . Data were analyzed using multivariable regression [48] , normalized to untreated controls and results are displayed as a heat map in Fig 1 . The mean expression values with standard deviation , number of samples and p-values for regulation compared to untreated controls as well as the official full name and gene ID as given in the NCBI gene database and the Applied Biosystems assay number is presented in S1 Dataset . Among the 48 selected genes , we identified those clearly regulated above a threshold in response to either LPS or IL-4/13 stimulation ( S1 Protocol 1 . 2 ) resulting in 16 and 7 genes that are regulated in M1 and M2 macrophages , respectively . In order to compare the Boolean model with time resolved data , the model was analyzed by calculating the logical steady states ( LSS ) [49] for a given input setting and timescale to study the resulting gene expression pattern of M1 vs . M2 macrophages at different time points . For better comparability , the quantitative data on mRNA expression were transformed into balanced ternary values that is 1/-1 if the gene is significantly up/downregulated at least 2-fold and 0 otherwise . Boolean simulation results and experimental data are compared in Fig 2 . All pro-inflammatory chemokines and cytokines that are classified as upregulated from the measurement data of M1 macrophages ( Fig 2A ) are also upregulated in the Boolean model after LPS stimulation ( Fig 2C ) . M2 macrophages , as already mentioned , are less well characterized compared to M1 macrophages . Thus , only four Stat6 target genes that are supposed to be regulated upon stimulation with IL-4/13 are part of the Boolean model ( Fig 2D ) . Generally , M2 macrophages show a diminished response in the expression pattern of the analyzed genes compared to M1 macrophages ( Fig 1 ) , but upregulation of these four genes is confirmed by the experimental data ( Fig 2B ) . However , there are some discrepancies between the measured and simulated expression patterns of M1 and M2 macrophages: ( 1 ) the chemokine Ccl7 is not part of the first model version but was shown to be upregulated in M1 and M2 macrophages ( Fig 2A/2B ) and co-expressed with Ccl2 ( Fig 3A ) . Thus , Ccl7 was included in the model as NF-κB and Stat6 target gene in response to LPS and IL-4/13 stimulation , respectively . ( 2 ) The chemokine Cxcl5 was not upregulated at 0 . 5 h after LPS stimulation in contrast to the other CXC-type chemokines ( Fig 2A ) . Furthermore , Cxcl-5 was not co-expressed with the other CXC-type chemokines such as Cxcl-2 ( Fig 3B ) . Thus , Cxcl-5 as NF-κB target gene was removed from the model . ( 3 ) Similarly , Osm was not significantly regulated after LPS treatment ( Fig 2A ) and not co-expressed with typical NF-κB target genes such as TNFα ( Fig 3C ) and , therefore , was removed from the model . ( 4 ) Also , regulation of the type I interferon IFNα was less clear . It was not ranked as significantly upregulated ( S1 Protocol 1 . 2 ) and was not clearly co-expressed with the other type I interferon IFNβ ( Fig 3D ) as predicted by the Boolean model and thus was also not considered in the model . ( 5 ) Likewise , IFNγ was not significantly expressed; values were close to or even beyond detection limit in most of the measurements . Hence , IFNγ must be excluded from analysis and was removed from the Boolean model . Since IFNγ was not expressed upon LPS and IL-4/13 stimulation , also IFNGR and downstream signaling events were removed . ( 6 ) The Boolean model predicts that IL-10 and Socs3 expression occurs later compared to the pro-inflammatory cytokines at τ = 10 ( Fig 2C ) since its expression requires secretion of type I interferons as well as activation of IFNAR . This is in contrast to the experimental results demonstrating that IL-10 and Socs3 were both similarly upregulated already 0 . 5 h after LPS stimulation comparable to the classical pro-inflammatory cytokines and such as TNFα ( Fig 2A ) . Furthermore , IL-10 and Socs3 were shown to be co-expressed with TNFα ( Fig 3F/3G ) . Since the transcription factor for early upregulation of IL-10 and Socs3 is still unknown but regulation of Socs3 expression via the p38MAPK/MK2 pathways has been shown [59 , 65] , early upregulation of these genes was included in the model in response to MK2 activation . Downregulation is not represented by the Boolean simulation results ( Fig 2C/2D ) . Nevertheless , the dependency matrix which covers the functional relation of all pairs of species in the Boolean model demonstrates an inhibitory influence of Akt1 , which is the Akt isoform that mediates the primary effects in M2 macrophages after IL-4/13 stimulation , on IL-1β and TNFα expression ( Fig 4 , row 15 , column 22–24 ) . The influence of Akt1 on TNFα protein synthesis ( TNFa_syn ) is ambivalent ( Fig 4 , row 15 , column 25 ) , since Akt1 positively influences NF-κB activity and , thus , also favors expression of the phosphatase DUSP1 , deactivation of p38 and inhibition of TNFα protein synthesis via TTP . Only the relevant section of the dependency matrix , that is the PI3K/Akt pathway , is presented for clarity . For a better understanding of the underlying dynamics of the system , particularly to investigate the time course of IL-10 and Socs3 upregulation that is differentially reflected by the Boolean model and the measurement data , the gene expression module is modeled dynamically using a system of ordinary differential equations ( ODEs ) . To reduce complexity , the signaling paths are neglected and only mRNA expression as well as transcription factor activity as input u ( t ) of the system was modeled as explained in [52] . Briefly , information from the Boolean model regarding activated transcription factors and target genes of the respective pathways as well as the chronology of events is translated into five different input functions for the ODE model representing ( u1 ) genes that are directly regulated via NF-κB upon LPS stimulation , ( u2 ) genes that are regulated via NF-κB and secondary by the TFs Stat1 and Stat3 via autocrine feedback loops , ( u3 ) genes , that are solely regulated via Stat TFs in response to LPS treatment , ( u4 ) genes that are Stat6-dependently upregulated in response to IL-4/13 stimulation and ( u5 ) genes that are repressed upon IL-4/13 stimulation . Expression of mRNA is given by d X ( t ) d t = k b + k s * u ( t ) - k d * X ( t ) ( 1 ) where X denotes the relative mRNA concentration , u ( t ) the input function representing transcription factor activity ( TFA ) and kb , ks and kd the basal synthesis , induced synthesis and degradation rate constant , respectively . At t = 0 , the system is at steady state , it is u ( 0 ) = 0 and , thus , scaling reduces the number of parameters by one per gene ( S1 Protocol 2 . 1 ) . If a gene is regulated in response to LPS as well as IL-4/13 stimulation , individual synthesis rates due to regulation by distinct transcription factors but identic degradation rates are assumed . Thus , the overall model contains 23 ODEs and 47 parameters , whereof eight are the time points determining TFA and 39 are synthesis and degradation rate constants . All model equations and parameter values can be found in S1 Protocol . The ODE model was implemented using the MATLAB Toolbox PottersWheel [51] . Accordance of simulation results of the dynamic model and experimental data on transcript expression levels of murine BMDMs is shown in Fig 5 supporting our assumptions regarding transcriptional regulation of inflammatory mediators ( 1 ) – ( 6 ) mentioned above . Besides , one additional modification is implemented: ( 7 ) the IL-1 receptor antagonist ( IL-1rn ) was regulated similar to Ccl5 ( Fig 1 ) , that is both genes are slightly upregulated at 0 . 5 h after LPS stimulation but expression was increased at later time points . In contrast , the initial Boolean model predicted that IL-1rn is a NF-κB target gene such as IL-1β and thus upregulated only early after LPS stimulation . In fact , data analysis revealed that IL-1rn is co-expressed with Ccl-5 ( Fig 3H ) rather than with IL-1β ( Fig 3I ) . Thus , IL-1rn was also included as Stat target gene similar to Ccl-5 in both the Boolean and the ODE model ( u2 ) . Identifiability of parameters was analyzed by investigating the profile likelihood using PottersWheel [53] and 68% confidence intervals were calculated ( S1 Protocol 2 . 3 ) . From the 47 parameters of the ODE model , 33 are identifiable and 14 parameters are practically non-identifiable ( S5 Fig ) . But in most instances , the unidentifiability is caused by the quite low parameter values , for example eight non-identifiable parameters are degradation rate constants of relatively stable mRNAs that are not significantly degraded during the observed time frame . Thus , although there is some uncertainty in parameter values , variations of these parameters only has negligible influence on the model trajectories . Hence , the model is well suited to describe the important dynamics of mRNA regulation of the selected genes in M1 and M2 macrophages . Furthermore , it allows prediction of the time frame of TFA as described in [52] . Upon LPS stimulation , p65 is predicted to be active from 23 min to 46 min and Stat3 shortly afterwards from 47 min to 9 . 9 h . Following IL-4/13 stimulation , Stat6 is predicted to be active from 5 min to 47 min and transcriptional repression is predicted to be mediated from 30 min to 1 . 3 h after treatment . Transcription factor activity is confirmed by immunoblotting ( Figs 6 , 7 and 8 ) . Upregulation of selected mediators secreted by macrophages was also confirmed on the protein level . Murine BMDMs were stimulated with LPS and IL-4/13 for 0 . 5 , 1 , 2 , 6 , 10 and 24 h and protein levels were quantified via Luminex Technology . Protein secretion of differentiated macrophages was also investigated . Macrophages were differentiated for eight days and then 1 x 106 cells were subcultivated in 6 well plates , as usual . Supernatant from those cells was collected after 3 h of cultivation and analyzed via Luminex Technology . Differentiated macrophages constitutively secrete low amounts of the chemokines Ccl2 , Ccl3 , Ccl4 , Cxcl1 , and Cxl2 as well as TNFα ( Fig 9 ) . Significant increase of quantified cytokines and chemokines is detectable after 1 to 6 h after LPS stimulation ( Fig 10A ) . Especially the chemokines are secreted in high concentration after LPS treatment: Ccl3 , Ccl4 , Cxcl1 , and Cxcl2 are secreted after 1 h . Their concentration in the supernatant of cells reach 38 . 9 , 35 . 1 , 14 . 2 and 66 . 4 ng/ml after 24 h stimulation , respectively . The chemokines Ccl2 and Ccl5 increase after 6 h LPS stimulation and reach 3 . 5 and 9 . 4 ng/ml after 24 h . Significant increase of IL-6 is quantified after 2 h stimulation and 182 . 4 ng/ml are detectable after 24 h . TNFα increases after 1 h LPS stimulation and reaches 2 . 5 ng/ml after 24 h , whereas IL-10 reaches 0 . 45 ng/ml . IL-1β is secreted in lower concentration , 43 . 4 pg/ml are detectable after 24 h of LPS treatment . After IL-4/13 stimulation , the secretion of chemokines is only slightly increased compared to the unstimulated macrophages ( Fig 10B ) . Interestingly , the cytokines IL-1β , IL-2 , IL-10 , and TNFα are not detectable in the supernatant of IL-4/13 stimulated macrophages . Secretion of Cxcl2 and IL-6 is transiently increasing . For specific concentrations we refer to S2 Dataset . Further adaptions of the network topology of activated macrophages were performed after analysis of protein data: ( 8 ) Stimulation with LPS leads to IκBα phosphorylation ( serine residues 32 and 36 ) and , subsequently , to the degradation of IκBα ( Fig 7 ) . Furthemore , LPS stimulation induces phosphorylation of p65 on serine 536 , promoting the transactivation by NF-κB ( p65/p50 ) [66 , 67] . This only occurs in the first hour following LPS stimulation , as estimated using the dynamic model of mRNA expression ( Fig 5 ) and confirmed via immunoblotting ( Figs 6A and 7 ) . On the other hand , first significant increase of the cytokines in the supernatant of cells was detectable only after one hour LPS stimulation ( Fig 10A ) . Thus , no effect of these cytokines on NF-κB transactivating activity is visible and , thus , the autocrine feedback loops of IL-1β and TNFα are removed from the Boolean model . ( 9 ) Similarly , when IL-6 is secreted about 2 h after LPS stimulation , Stat3 is already activated ( Figs 6A and 8 ) and Socs3 is highly upregulated on the mRNA ( Figs 1 and 5 ) and also on the protein level ( Fig 8 ) inhibiting Stat phosphorylation at IL-6R and IFNAR . Only IL-10 is able to mediate late and sustained Stat3 activation , since the IL-10R is insensitive to Socs3 [60] . The autocrine feedback loop of IL-6 resulting in Stat3 activation is therefore removed from the model . The combination of modeling and experimental analysis of the inflammatory genes expressed in activated macrophages resulted in the refined version of the Boolean model as presented in Fig 11 . The assumptions mentioned in the previous section ( 1 ) – ( 9 ) are implemented in the refined version of the Boolean model that is now specifically adapted to our experimental system of primary murine BMDMs . Compared to the first literature-based version ( S1 Fig ) , 13 interactions were modified , 33 were deleted and 9 interactions were newly introduced to the Boolean model . Accordingly , 22 species were deleted from the first model , 6 species were added and 12 were modified in the final model version . The final model contains 132 nodes and 152 interactions . All abbreviations and description of network nodes as well as the logic equations are given in S1 and S3 Tables . Modified entries are indicated and the deleted nodes and interactions are listed in S2 and S4 Tables . The model now contains two autocrine feedback loops mediated by secreted IFNβ and IL-10 indicated by grey boxes .
In this study , we present a comprehensive characterization of M1 ( LPS-activated ) and M2 ( IL-4/13-activated ) macrophages and their specific cytokine and chemokines expression profile . We combined current knowledge about macrophages across several cell types and species in the first version of the Boolean model . Based on established procedures [6 , 7 , 44 , 68 , 69] , a protocol for the generation of primary murine BMDMs and their activation into well characterized M1 ( LPS ) and M2 ( IL-4/13 ) -type macrophages was generated . Furthermore , the Boolean model was validated and refined with expression data of cytokines and chemokines on the mRNA and partially also on the protein level . The simulation results of the first Boolean model were compared to mRNA data of BMDMs resulting in elimination of some genes that were not significantly regulated , such as the type II interferon IFNγ whose expression is already controversially reported in literature . Originally , it was assumed that IFNγ production is restricted to activated T cells and natural killer cells , but it has been shown that murine BMDMs secrete large amounts of IFNγ in response to IL-12 and IL-18 stimulation [70] . Another group showed production of IFNγ mRNA and protein in murine macrophages in response to LPS stimulation without detectable amounts of secreted protein [71] . Recently , it has been shown that murine peritoneal macrophages produce moderate amounts of IFNγ in response to LPS stimulation [72] . However , for our system of murine BMDMs , IFNγ was neither detectable on the mRNA nor the protein level in response to stimulation with LPS and IL-4/13 . Also , the type I interferon IFNα was removed since it was not clearly upregulated in murine BMDMs in contrast to IFNβ indicating that IFNβ is one of the principal type I interferons triggering IL-10 dependent sustained activation of STAT3 in response to LPS via an autocrine or paracrine feedback loop . Expression of interferons after TLR4 activation is regulated by two TFs , NF-κB and IRF3 , the first is activated via the early MyD88-dependent pathway and the latter via the delayed MyD88-independent pathway [11] . These two signaling pathways were hence modeled on two separate time scales in the Boolean model . However , no difference in onset of transcription was visible when examining the mRNA data , probably because the measurement time points were too sparse and at the first time point of 0 . 5 h already both TFs were activated . Furthermore , there is evidence that although IRF3 is activated later than NF-κB , both TFs translocate to the nucleus together to activate transcription [73] . Thus , there is no need to include IRF3 and distinct regulation of IFNβ mRNA in the dynamic model of mRNA expression and we focused on NF-κB as major transcription factor directly activated in LPS-stimulated macrophages . After analyzing the time course of mRNA regulation in macrophages in more detail using the ODE model of gene expression , we found that the IL-1 receptor antagonist IL-1rn is not only regulated by NF-κB but also at later time points possibly via the Stat TFs . IL-1rn was already described as a Stat6 target gene [16] . Besides , it was shown that inhibition of NF-κB via adenoviral transfection of IκBα decreases expression of IL-1rn only marginally [74] and that IL-1rn is an IL-10 inducible gene [75] . Also IL-10 and Socs3 turned out to be regulated earlier before its cognate TF Stat3 is activated as shown by the dynamic ODE model and confirmed by immunoblot analysis ( Figs 6A and 8 ) . Regulation of IL-10 expression is diversely reported in literature . Some state that expression of IL-10 is NF-κB independent [74 , 76] . Saraiva et al . showed that a NF-κB p65/RelA site was activated in the IL-10 promotor after stimulation of primary mouse BMDMs through pattern recognition receptors [77] . Also Staples and colleagues demonstrated that IL-10 regulates IL-10 expression itself by an autocrine feedback loop via the TF Stat3 , but the initial IL-10 expression following LPS stimulation is Stat3-independent [78] . So there is a high probability that NF-κB is involved in initial regulation of IL-10 expression , but also the MAP kinases p38MAPK and ERK as well as the p38MAPK downstream effector kinase MAPK activated protein kinase 2 ( MK2 ) were shown to play a role [17 , 79 , 80] . The final modification was removing the autocrine feedback loops of IL-1β , TNFα and IL-6 . Since these cytokines are the major ones inducing the acute phase response in hepatocytes [15 , 36] , it is plausible that they primarily act on surrounding cell types such as hepatocytes and to a negligible amount back on the macrophages . This is supported by the dynamical model of mRNA expression . Although there are some uncertainties in the parameters , the model is well suited to determine the important dynamics of mRNA regulation as well as the time of transcription factor activity which was confirmed by immunoblot analysis ( Figs 6 and 8 ) . Only 14 of 47 parameters are practically non-identifiable . Thereof , eight parameters are mRNA degradation rate constants of IL-1β , IL-1rn , Socs1 and the CCL-type chemokines whose lower bounds are non-identifiable implying that mRNAs are relatively stable and not necessarily degraded over the observed time span of 10 h . Similarly , the lower bounds of the two synthesis rate constants of Ccl3 and Cxcl3 following IL-4/13 stimulation are non-identifiable . Although , by analyzing the measurement data these genes were determined to be downregulated it is also possible that they are non-regulated after stimulation with IL-4/13 . The last four practically non-identifiable parameters are those determining transcription factor activity . More precisely , the lower bounds of the parameters describing the time point of Stat3 activation in LPS-stimulated macrophages and the time point for Stat6 activation as well as the time point for beginning of negative regulation following IL-4/13 stimulation are non-identifiable . This indicates that regulation in response to IL-4/13 is relatively quickly due to the very short signaling paths . For LPS-stimulated macrophages , this indicates that activity of NF-κB and Stat3 is possibly slightly overlapping . Furthermore , the upper bound of the parameter for the time point of Stat3 deactivation is non-identifiable indicating that Stat3 could be active longer than the observed time frame of 10 h after LPS stimulation . In this study , we choose the phenotype of M1 or classically activated macrophages on the one side and alternatively activated or M2 macrophages on the other side for comparative analysis , since these two represent the initially described and most prominent phenotypes [7] . The M1 and M2 terminology originated from the correlating concept of the TH1 and TH2 dichotomy . Later , the concept of M1/M2 macrophage activation was extended as it became clear that activated macrophages are highly heterogeneous [8] . A concept has been evolved describing many shades of macrophage activation with the three main phenotypes , namely classically activated macrophages , wound-healing macrophages , and regulatory macrophages with mixed phenotypes in between , for example tumor-associated macrophages [1] . In a recent proposal , this concept has been refined and a nomenclature has been suggested that is linked to the activation standards [6] . Compared to this classification , the M2 macrophages activated by IL-4 and IL-13 as described in this study would be termed as M ( IL-4/IL-13 ) . As mentioned , classically activated M1 ( LPS ) and wound-healing M2 ( IL-4/IL-13 ) macrophages represent two major phenotypes that are involved in a number of diseases that mainly result from a disturbed balance of macrophage activation with lacking restoration of homeostasis after injury . Thus , systematic characterization of the distinct macrophage phenotypes as well as the corresponding cytokine and chemokine expression patterns as represented by the final , refined version of the Boolean model is particularly important for improving the current understanding of macrophage activation and their roles in health and disease . In this context , the model provided suggests that miRNA-155 represents a critical molecule that determines how activation of Akt by the different stimuli is involved in the determination of the activation type of macrophages . We observed differential regulation of miRNA-155 expression following stimulation with either LPS or IL-4 and IL-13 which is in line with recent concepts demonstrating that the PI3K/Akt pathway plays a critical role in the control of macrophage plasticity with the two Akt isoforms Akt1 and Akt2 being important regulators of M2 and M1 differentiation , respectively [34 , 35] . Among others this may involve Akt1 dependent phosphorylation of the downstream kinase GSK3β at serine 9 , which inhibits GSK3β activity [81] thereby interfering with activation of p65/p50 NF-κB dimers by enhancing formation of the inhibitory p50/p50 NF-κB dimer [82] . Moreover , isolated activation of Akt1 in Akt2 deficient macrophages prevents expression of miRNA-155 resulting in an increased availability of CEBP/β that in turn mediates expression of target genes such as Arginase1 , IL-10 and YM1 . On the other hand isolated activation of Akt2 in Akt1 deficient macrophages leads to an enhanced activation of p65/p50 NF-κB dimers and subsequent upregulation of p65/p50 dependent gene expression [35] . In line with this study , we showed increased expression of miRNA-155 after LPS stimulation which induces M1 polarization and NF-κB-dependent upregulation of inflammatory mediators . Accordingly , stimulation with IL-4 and IL-13 led to decreased levels of miRNA-155 . Notably , although they share with PI3K the same upstream activator , Akt1- and Akt2-driven pathways have distinct regulatory functions with respect to macrophages polarization . This makes it likely that the decision in which direction the polarization is driven is mediated by PI3K-independent co-regulatory signals which are integrated either at the level of the two Akt isoforms Akt1 or Akt2 or at the level of their downstream signaling . Everts et al . [83] recently described a similar role of TBK1 in the context of dendritic cell activation . They identified the kinase TBK1 downstream of the activated TLR4 receptor complex as most important kinase mediating Akt phosphorylation after LPS stimulation . The crosstalk of LPS and PI3K/Akt signaling in the context of macrophage activation needs further investigation .
8–12 weeks old male C57BL/6J mice were used for the generation of BMDMs . All animal applications were reviewed and approved by the appropriate authorities and were performed in accordance with the German animal protection law ( Landesamt für Natur , Umwelt und Verbraucherschutz Nordrhein-Westfalen , Recklinghausen . Animals were handled and housed according to specific pathogen free ( SPF ) conditions in the local breeding facility ( ZETT , Zentrale Einrichtung für Tierforschung und Tierschutzaufgaben , Heinrich-Heine-University Düsseldorf ) . Macrophages derived from bone marrow cultures were obtained according to the method of Meerpohl et al . [44] with some modifications . In brief , both femurs and tibiae of male , 8–12 weeks old C57BL/6J mice were removed and dissected free of adherent tissue . Intact bones were kept in sterile phosphate buffered saline ( PBS; Biochrom , Berlin , Germany ) . Before starting bone marrow preparation , bones were left in 70% [v/v] ethanol for 3–5 minutes , washed with PBS and transferred into washing medium ( DMEM 1000 mg/ml Glucose; Biochrom , Berlin , Germany supplemented with 1% [v/v] Penicillin/Streptomycin; PAN Biotech , Aidenbach , Germany ) . After cutting off the ends of the bones , the bone marrow was flushed out by irrigation with washing medium using syringe and needle ( 23G x 1“ ) . A single cell suspension was obtained by pipetting cells vigorously followed by centrifugation ( 1200 rpm , 4°C , 10 minutes , low acceleration/ deceleration ) . Cells were resuspended in culture medium ( DMEM 1000 mg/ml Glucose , 1% [v/v] Penicillin/Streptomycin , 10% [v/v] fetal calf serum ) without murine macrophage colony stimulating factor ( M-CSF ) . The viable cell counts were performed in a hemocytometer using trypan blue solution . The cell viability was at least about 95–97% and 54 x 106 ± 3 . 8 cells/mouse ( mean ± SEM ) were isolated . Cells were seeded in three vented cell culture flasks ( 75 cm2 ) for overnight incubation at humidified atmosphere ( 37°C , 5% CO2 ) . Adherent cells ( e . g . stromal fibroblasts ) will settle out . The next day , the non-adherent cells were harvested for further cultivation by performing centrifugation . Cells were resuspended in culture medium , supplemented with 10 ng/ml M-CSF ( Peprotech , Rocky Hill , USA ) . Again , trypan blue exclusion was performed and total cell amount was calculated: 37 x 106 ± 2 . 0 cells/mouse ( mean ± SEM ) . Cells were seeded for proliferation and macrophage differentiation in five cell culture dishes with 15 cm diameter and 20 ml medium ( +M-CSF ) each [45] . 10 ml of medium supplemented with M-CSF was added on day 3 , 6 and 7 ( no aspiration of media ) . After 8 days of cultivation , adherent cells were harvested by gentle trypsinisation: Cells were washed twice with prewarmed PBS and treated with 3 ml trypsin/EDTA ( PAN Biotech , Aidenbach , Germany ) for approximately 5–10 minutes . Cells were centrifuged and after evaluation of the total cell yield ( 39 x 106 ± 2 . 7 cells/mouse ( mean ± SEM ) ) , they were adjusted in M-CSF containing culture medium: 3 x 106 cells/3 ml/6 well plate cavity for RNA isolation and 1 x 106 cells/2 ml/6 well plate cavity for supernatant collection . Medium was changed to FCS free culture medium 6 hours before performing the experiments . Cells were stimulated with 50 ng/ml LPS or 25 ng/ml IL-4/ IL-13 , respectively . RNA was isolated or cell supernatant was collected after 0 . 5 , 1 , 2 , 6 and 10 hours of stimulation . Total RNA was isolated using the RNeasy Miniprep Kit ( Qiagen , Hilden ) according to manufacturer’s instructions . Assessment of RNA integrity and quantity was performed by using Agilent Technology ( Agilent 2100 Bioanalyzer , Waldbronn , Germany ) . 600 ng total RNA was reverse transcribed to cDNA with TaqMan Reverse Transcription Reagents ( Applera GmbH , Darmstadt , Germany ) . For qRT-PCR we used the Fluidigm’s BioMark HD high-throughput quantitative chip platform ( Fluidigm Corporation , San Francisco , CA , USA ) with pre-designed gene expression assays from Applied Biosystems according to the manufacturer’s instructions [46] . All TaqMan assays are listed in S1 Dataset . For analysis of miRNA expression cells were grown in 6 well plates and treated with 50 ng/ml LPS , with 25 ng/ml IL-4/IL-13 each or left untreated . Total cellular RNA including miRNA was isolated using the miRNeasy Miniprep Kit from Qiagen ( Hilden , Germany ) according to the manufacturer’s instructions . 100 ng of total RNA was reverse transcribed with miScript II Reverse Transcription Kit ( Qiagen , Germany ) using the HiFlex buffer for transcription of mature and precursor miRNA as well as ncRNA and mRNA . cDNA was diluted 1:5 , and 2 μl of the diluted cDNA was added as template to a final volume of 25 μl including 1 x QuantiTect SYBR Green PCR master mix according to the manufacturer’s instructions ( Qiagen , Germany ) . The following primers were used for real time PCR: mouse miRNA-155 ( sense , 5’-TTA ATG CTA ATT GTG ATA GGG GT-3’; antisense , miScript universal primer ) and the mouse U6 snRNA ( sense , 5’-CGC TTC GGC AGC ACA TAT AC-3’; antisense , 5’-AAA TAT GGA ACG CTT CAC GA-3’ ) . Except the universal primer , which was from Qiagen , Germany , all oligonucleotides were purchased from Eurofins MWG Operon ( Ebersberg , Germany ) . No-template and no-reverse-transcriptase controls were used to control specificity of RT-PCR . Semi-quantitative PCR results were obtained using the ddCT method . Expression values of the miRNA were normalized to the expression levels of the control gene U6 snRNA and referred to untreated controls . Data from four independent experiments are presented as mean ± standard error of the mean ( SEM ) . Antibodies for cytometric analysis were purchased from ebioscience: CD14 ( #12-0141-81 ) , CD11b ( #12-0112-81 ) , F4/80 ( #17-4801-80 ) from BD Pharmingen: CD69 ( # 553237 ) , CD86 ( 558703 ) from Dianova: CD68 ( #MA1-82739 ) and AbD Serotec: CD206 ( #MCA2235FB ) . For exclusion of background fluorescence , cells were stained with the respective isotype control antibodies in parallel: Rat IgG2b K Isotype Control PE , Armenian Hamster IgG Isotype Control PE , Rat IgG2b K Isotype Control APC , Rat IgG2a K Isotype Control FITC ( ebioscience ) . Anti-p65-Ser536 , anti-STAT3-Tyr705 , anti-p38-Thr180/Tyr182 , anti-p38 , anti-IκBα-Ser32/36 , anti-IκBα and anti-STAT6-Tyr641 antibodies used for Western Bot analysis were obtained from Cell Signaling Technology ( Berverly , MA , USA ) . Anti-STAT3 , anti-p65 and anti-STAT6 antibodies were purchased from Santa Cruz Biotechnology ( Santa Cruz , CA , USA ) , the antibody against SOCS3 was obtained from IBL ( Minneapolis , MN , USA ) and against GAPDH from Biodesign ( Saco , ME , USA ) . Cell culture reagents as Dulbecco’s modified Eagle medium ( DMEM ) were purchased from Biochrom ( Berlin , Germany ) , FCS ( Cat . : 10099141 , Lot: 769367 ) was obtained from Invitrogen ( Karlsruhe , Germany ) , Penicillin G/ Streptomycin and Trypsin/ EDTA-Solution were from Cytogen ( Wetzlar , Germany ) . Recombinant murine M-CSF , IL-4 and IL-13 were obtained from Peprotech ( Rocky Hill , NJ , USA ) . LPS from E . coli ( #L3012 ) was purchased from Sigma-Aldrich ( München , Germany ) . The protocol for total protein isolation , Western Immunoblotting and signal detection has been described previously [47] . Equal amounts of protein were subjected to SDS/PAGE . The electrophoretically separated proteins were transferred to polyvinylidene difluoride ( PVDF ) membranes by the semidry Western blotting method . The immunoblots were developed with the enhanced chemiluminescence system ( Amersham Biosciences , MA , USA ) following the manufacturer’s instructions . At the end of the experimental treatment , cell culture supernatant was collected under sterile conditions followed by centrifugation ( 20 minutes , 4°C , 5 . 500 rpm ) . Aliquots were pre-cooled at -20°C and stored in -80°C until quantitative mediator measurement . Analysis of cytokine concentration in supernatants was performed by using Luminex Technology ( Austin , TX , USA ) and the MCYTOMAG-70K Mouse Cytokine/Chemokine Magnetic Bead Panel from Millipore ( Billerica , MA , USA ) according to manufacturer’s instruction . Cell culture media and untreated control samples were used for background normalization . Maturation of macrophages and characterization of macrophage polarization was assessed by using fluorescence-activated cell sorting ( FACS ) and was performed using the FACS Canto II ( BD Biosciences , Heidelberg , Germany ) . Briefly , after differentiation , cells grown in 6 well cavities were harvested by trypsinization ( 3 minutes , 37°C ) . Reaction was stopped by adding 1 ml FACS buffer ( PBS w/o Ca2+/Mg2+ , 2% FCS , 2 mM EDTA , 0 . 1% NaN3 ) . After centrifugation , fixation and permeabilization was performed using the Leucoperm Reagent Kit ( AbD Serotec , Kidlington , UK ) followed by antibody treatment according to manufacturer’s instructions . Each experiment was performed in at least three independent cell preparations using the same experimental conditions . Data analysis was performed with FlowJo Software , version 7 . 6 . 5 ( Ashland , OR , USA ) . Values are expressed as means ± standard deviation , where indicated . Fluidigm data was analyzed using LEMming , a multivariable statistics approach [48] and normalized to untreated controls . Outlier were discarded as explained in S1 Protocol 1 . 1 . Differential expression was assessed using the two-sample Student’s t-tests . All computations were conducted using RStudio Version 0 . 98 . 1091 and MATLAB R2014a . The Boolean model is presented as logical interaction hypergraph ( LIH ) as introduced by Klamt et al . [49] , that is two or more species participate in one interaction . Each species is defined by a logical , binary state variable , that is 0/1 for inactive/active state or absent/present . The model was implemented using the MATLAB Toolbox CellNetAnalyzer ( CNA ) [50] . Logical AND connections are represented by blue dots in the LIH , OR connections by yellow and inhibiting interactions are indicated by red lines . A so called timescale constant τ [49] is assigned to each logical interaction indicating when a reaction becomes active , whereat reactions with lower timescale constants become active earlier than interactions with higher timescale constants . Abbreviations and descriptions of species are given in S1 and S2 Tables , logical equations can be found in S3 and S4 Tables . S1 Model contains all necessary files for starting the Boolean model of macrophage activation with the CellNetAnalyzer . The input/output behavior of the Boolean model is analyzed by calculating the LSS for a given input setting , that is LPS = 1 , IL-4 = 0 , IL-13 = 0 for M1 macrophages and LPS = 0 , IL-4 = 1 , IL-13 = 1 for M2 macrophages . During LSS analysis , the signal is propagated from the fixed inputs along the logical hyperarcs as far as uniquely feasible till the system reaches a LSS , that is the state of each species is consistent with its assigned Boolean function . Assignment of time scales does not modify the manner of LSS calculation rather than altering the analyzed Boolean network by extracting smaller subnetworks . Calculating a LSS for a time scale τ = t computes the LSS for a subnetwork containing all interactions with a time scale constant τ ≤ t whereas interactions with a time scale constant τ > t are not included in the analysis [49] . The gene expression model is based on ordinary differential equations ( ODEs ) and mass action kinetics . It was implemented using the MATLAB Toolbox PottersWheel [51] and comprises 23 species and 47 parameters . The model setup is explained in [52] , a short summary is given in S1 Protocol as well as a list of model equations and parameter values . The ODE system is numerically integrated using the Sundials solver CVODES and parameter values were estimated by least squares fitting . Identifiability of parameters was analyzed by estimating the profile likelihood as explained in [53] . | Macrophages are essential cells of the immune system and indispensable for a defense against bacterial infection . They reside as resting , immune modulatory cells in several tissues of the human body where they continuously sense inputs from their local environment . They react to stimuli such as toxins , injury or bacterial products in a process termed macrophage activation or polarization . For example , the bacterial component lipopolysaccharide induces so-called classical activation of macrophages into the M1 phenotype that secretes a number of inflammatory cytokines and chemokines leading to killing of bacteria and resolution of inflammation . Another prominent phenotype of macrophages is the M2 polarization state that is associated with wound healing and tissue regeneration . Unbalanced activation of macrophages is implicated in a number of diseases . An improved knowledge and extensive characterization of these macrophages as well as the factors determining their phenotypes will improve the understanding of the role of macrophages in disease progression . | [
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"rec... | 2016 | Model-Based Characterization of Inflammatory Gene Expression Patterns of Activated Macrophages |
Enterotoxigenic Escherichia coli ( ETEC ) is a non-invasive enteric pathogen of considerable public health importance , being one of the most common attributable causes of diarrheal illness in infants and young children in developing countries and the most common cause of traveler’s diarrhea . To enhance study-to-study consistency of our experimental challenge model of ETEC in volunteers , and to allow concomitant multi-site trials to evaluate anti-ETEC immunoprophylactic products , hundreds of vials , each containing a standardized inoculum of virulent wild-type ( wt ) ETEC strain H10407 ( serotype O78:H11 expressing colonization factor antigen I and heat-labile and heat-stable enterotoxins ) , were prepared under current Good Manufacturing Practices ( cGMP ) and frozen . Following thawing , the contents of each vial can be used ( diluted as necessary ) to prepare consistent challenge inoculum , even at different study sites . A preliminary human experimental challenge study using this cGMP inoculum was conducted on a research isolation ward and the clinical and cell-mediated immune responses evaluated . Of the 6 healthy adult volunteers challenged 83% ( 5/6 ) developed diarrhea and 50% developed moderate-to-severe diarrhea ( MSD ) . Moderate and severe diarrhea were defined as passage of ≥ 1 liter or ≥ 3 liters of diarrheal stool respectively . We compared the CD4+ T cell responses of volunteers who developed MSD against those who did not and identified significant differences in ETEC-specific cytokine production and gut homing potential . We furthermore demonstrated that increased expression of the gut-homing molecule integrin α4β7 by peripheral T follicular helper cells ( pTfh ) correlated with decreased stool volume and increased ETEC-specific IgA B memory cell ( BM ) development . Collectively , despite small numbers of volunteers , our results indicate a potential role for CD4+ T cells , in particular pTfh , in modulating disease outcome following exposure to wt ETEC in a volunteer experimental challenge model .
Enterotoxigenic Escherichia coli ( ETEC ) is one of the most important pathogens contributing to moderate-to-severe diarrhea ( MSD ) in children in low- and middle-income countries [1] . Moreover , it is also the most common cause of diarrhea among travelers who visit developing countries [2] . In a very recent study , the total estimated ETEC mortality for 2015 was 74 , 100 deaths in all ages with 23 , 600 deaths in children < 5 years of age [3] . Following ingestion of infectious ETEC organisms , adhesion to the epithelium is mediated by colonization factor antigens ( CFAs ) , most of which are fimbria or fibrillae [4 , 5] . Subsequently , watery diarrhea is induced through the action of heat-stable ( ST ) and/or heat-labile ( LT ) enterotoxins . Experimental human challenge studies to investigate the pathogenesis of ETEC diarrhea in humans , study immunity , and evaluate vaccines have been performed at the Center for Vaccine Development ( CVD ) of the University of Maryland School of Medicine since the mid-1970s , as well as at other institutions [4–16] . ETEC strain H10407 , initially isolated in 1971 from the stool of an adult in Bangladesh with severe non-Vibrio cholerae , cholera-like , diarrheal illness , produces human ST ( STh ) , porcine ST ( STp ) , and LTh , and expresses colonization factor antigen I ( CFA/I ) [17] . Although the H10407 strain has been the most commonly used ETEC strain in human challenge studies [18] , prior studies were conducted with freshly harvested organisms . Frozen aliquots of challenge inoculum , which can minimize inter- and intra-site variability with attack rates and severity of illnesses , were successfully used with V . cholerae O1 El Tor Inaba strain N16961 [19] . The availability of these frozen challenge inoculum vials is a precious resource to the scientific community and has been used in efficacy assessments of cholera vaccines [20 , 21] , including a recently completed pivotal efficacy trial [22] . In preparation for testing the efficacy of immunoprophylactic interventions against ETEC , including both vaccine candidates and monoclonal antibodies , H10407 , was fermented under cGMP conditions and identical aliquots were dispensed into several hundred vials that are maintained in GMP frozen storage to provide a source of standardized inocula for future challenge studies . To verify the appropriate challenge inoculum using the frozen material , we conducted a pilot experimental challenge study in volunteers and carefully measured the clinical outcome and immunological responses to experimental ETEC infection . Despite the global health burden posed by this pathogen , there is currently no licensed vaccine available . Vaccine development has been hindered , in part , by lack of known immunological correlates of protection . Human challenge studies provide an ideal opportunity to investigate the immunological responses which are associated with protection from , or resistance to infection . Due to the non-invasive nature of ETEC , antibodies have been proposed as a major contributor to protection . Thus far , however , few studies have investigated the potential contribution of T cells and their role in protection against this organism . Here we describe , for the first time in humans , CD4+ T cell responses pre- and post-challenge and their association with long-term B memory ( BM ) responses . We further demonstrate an association between T cell reactivity and the susceptibility of participants to develop clinical ETEC diarrheal disease and its severity .
The study was approved by the Institutional Review Boards of University of Maryland , Baltimore ( UMB ) , the University of Massachusetts Medical School ( UMMS ) and the Office of Research Protections of the U . S . Army Medical Research and Materiel Command ( since the cGMP production of the challenge inoculum and the clinical trial were supported by the Defense Advanced Research Projects Agency [DARPA] of the U . S . Department of Defense ) . Written informed consent was obtained from healthy adult volunteers 18 to 49 years of age who were screened for the absence of chronic medical conditions , immunodeficiency , and a history of recent foreign travel to an endemic region or prior cholera or ETEC infection ( natural infection or experimental challenge ) , or prior receipt of a cholera or ETEC vaccine . A research cell bank ( RCB ) of ETEC strain H10407 was produced by the Inoculum Preparation Laboratory of the Center for Vaccine Development at the University of Maryland School of Medicine through expansion in animal component-free LB broth ( Remel , Lenex , KS ) . Under cGMP , MassBiologics Manufacturing Facility ( Boston , MA ) produced the challenge inoculum vials starting from the RCB . Briefly , the RCB was inoculated onto CFA agar plates , incubated at 37°C , and the confluent organisms were harvested . The pooled organisms were aliquoted into gasketed screw-cap cryo-vials ( Corning , Tewksbury , MA ) and snap frozen; creating a total of 307 vials , each vial containing ~1010 viable organisms suspended in phosphate buffered saline ( PBS ) and 20% glycerol . The frozen challenge inoculum vials are stored at -80°C under a formal stability monitoring plan . This source of cGMP H10407 challenge inoculum has been registered as an investigational new drug ( BB-IND-15900 ) under the U . S . Food and Drug Administration . After providing informed consent , six eligible volunteers were admitted as inpatients to a Research Isolation Ward two days prior to challenge to acclimate to the ward and to complete medical screening . On the morning of challenge , a single frozen challenge vial was thawed and diluted to the desired concentration ( 108 CFU/mL ) ; the inoculum size was confirmed by quantitative counts . Two grams of NaHCO3 were dissolved in 150 ml of sterile water and each volunteer ( who had been fasting overnight ) drank 120 mL of NaHCO3 buffer solution; 1 min later each participant ingested ~1 x 108 CFU of the ETEC organisms suspended in the remaining 30 mL of NaHCO3 solution . Following ingestion of ETEC organisms , volunteers were closely monitored for illness and every stool was graded as follows: grade 1 , firm; grade 2 , soft; grade 3 , thick liquid; grade 4 , opaque watery; and grade 5 , rice water . All stools ≥ grade 3 were considered loose and were weighed . Any individual who developed loose stools was offered oral rehydration salt solution ( Jianis Brothers ORS , Kansas City , MO ) at a volume of 1 . 5 times the loose stool volume . If unable to ingest ORS or upon their request , intravenous Lactated Ringers solution was used for rehydration . Ciprofloxacin , 500 mg twice daily for 5 days , was administered when a person exceeded 3 liters of cumulative loose stool output or on day 4 post-challenge , whichever occurred first . Individuals were discharged when they were asymptomatic , completed a course of ciprofloxacin therapy , and demonstrated 3 sequential negative stool cultures for ETEC separated by 12 hours each . Diarrhea was defined as the passage of 2 or more loose stools ( grade 3–5 ) over a 48 hour period that equaled or exceeded 200 mL or a single loose stool of 300 mL or greater . Moderate and severe diarrhea were defined as the passage of ≥1 liter and ≥3 liters of diarrheal stool , respectively . Five of the 6 volunteers were Black or African American , 83% were male , and the mean ( median ) age was 32 . 7 ( 32 ) years . Stool specimens were plated directly onto Eosin Methylene Blue agar plates ( Sigma , St . Louis , MO ) , MacConkey agar plates ( Fisher , Pittsburgh , PA ) , and GN enrichment broth ( Fisher , Pittsburgh , PA ) , and incubated overnight at 37°C . For each participant a maximum of 2 stools each day were cultured and a rectal swab was obtained if no stool was passed . Colonies suspicious for E . coli were confirmed by positive agglutination with O78 antisera ( Denka Seiken , Campbell , CA ) on lactose and indole positive colonies . Whole blood was obtained from volunteers pre-challenge ( day -7 ) , and at multiple time-points after challenge ( days 1 , 3 , 7 , 14 , 21 , 28 , 56 , and 180 post-challenge ) . PBMC were isolated from whole blood by Ficoll-Hypaque gradient fractionation and cryopreserved as previously described [23 , 24] . All available time-points from an individual volunteer were thawed simultaneously and rested overnight at 37°C 5% CO2 in complete media ( cRPMI; RPMI 1640 media ( Gibco , Carlsbad , CA ) supplemented with 100 U/mL penicillin ( Sigma ) , 100 μg/mL streptomycin ( Sigma ) , 50 μg/ mL gentamicin ( Gibco ) , 2 mM L-glutamine ( Gibco ) , 10 mM HEPES buffer ( Gibco ) and 10% heat-inactivated fetal bovine serum ( Gemini Bioproducts , West Sacramento , CA ) . Of the 6 volunteers challenged with wild-type ETEC , only 5 were evaluable in immunological studies . Following the overnight rest , PBMC were stimulated for 14–18 hours in the absence ( media control ) or presence of ETEC antigens [whole cell homogenate ( 5 μl/mL; prepared in-house as described below ) , LT-B ( 5 μg/mL; Sigma , St Louis MO ) , double mutant LT ( dmLT ) ( 5 μg/mL; PATH Vaccine Solutions , Seattle WA ) , and purified CFA/I ( 5 μg/mL; prepared at the CVD as previously described [25] ) ] or Staphylococcal enterotoxin B ( SEB—positive control 10 μg/mL; Sigma ) in the presence of Golgi inhibitors ( Golgi Plug and Golgi Stop 0 . 5 μl/mL , BD Biosciences , San Jose CA ) . Anti-CD107a ( LAMP1 ) monoclonal antibody conjugated to 151Eu ( Fluidigm , Sunnyvale , CA ) was added to the culture at 3 μl/mL . Whole cell homogenate was prepared from wt H10407 . Briefly , following overnight incubation at 37°C , bacteria were scraped from the plate and suspended in sterile PBS . A microfluidizer was used to homogenize the bacteria followed by heat inactivation at 56°C for 30 minutes . Following overnight stimulation with ETEC antigens or SEB ( non-specific positive control ) , PBMC were stained for mass cytometric analyses using a panel of 23 metal-conjugated monoclonal antibodies . A table of the monoclonal antibodies used is shown in supplementary materials ( S1 Table ) . Briefly , samples were washed once with staining buffer ( phosphate buffered saline with 2% FBS and 0 . 1% sodium azide ) and then stained for barcoding at 4°C for 30 minutes using anti-CD45-141Pr ( SEB and CFA/I ) , anti-CD45-154Sm ( media and LT-B ) , or anti-CD45-156Gd ( whole cell homogenate and dmLT ) . Following staining with anti-CD45 , sets of 3 samples were combined into a single tube for downstream staining ( media/SEB/whole cell homogenate and LT-B/CFA/I/dmLT ) . Viability staining was performed with cisplatinum ( Sigma; 25 μM ) for 60 seconds . Following cisplatinum staining , samples were Fc-blocked with human immunoglobulin ( Sigma; 3 μg/mL ) for 20 minutes at room temperature followed by surface staining for 30 minutes at 4°C . Fixation and permeabilization were performed according to manufacturer’s recommendations ( eBiosciences , San Diego CA ) followed by intracellular staining for 20 minutes at room temperature . Samples were stained with an Ir191/193 DNA intercalator for cell detection by mass cytometry within 48 hours of sample acquisition and re-suspended in EQ4 normalization beads ( Fluidigm ) . Acquisition was performed using a CyTOF mass cytometer ( Fluidigm , formerly DVS Sciences ) . Data were analyzed with Premium Cytobank . A sample gating strategy for mass cytometric analyses is shown in S1 Fig . Briefly , frozen PBMC were thawed and expanded with B cell-expansion media consisting of 5 μM 2β-ME ( Biorad ) , 1:100 , 000 pokeweed mitogen ( kindly provided by Dr . S . Crotty ) , 6 μg/mL CpG-2006 ( Qiagen/Operon , Huntsville , AL ) , 1:10 , 000 Staphylococcus aureus Cowan ( SAC; Sigma–Aldrich , St . Louis , MO ) in complete RPMI as previously described [26] . Cells were expanded for 5 days ( 1×106 cells/well in 6-well plates ) . The BM ELISpot was performed as follows: Nitrocelulose plates were coated with ETEC antigens [CFA/I ( 5 μg/mL ) , LT-B ( 5 μg/mL ) , ETEC LPS ( 5 μg/mL ) , or dmLT ( 5 μg/mL ) ] , total goat anti-human IgG ( Jackson Immuno Research lab; 5 ug/mL ) , or total goat anti-human IgA ( Jackson Immuno Research lab; 5 ug/mL ) diluted in PBS . Plates were incubated overnight at 4°C and blocked with 10% FBS in RPMI-1640 for 2 hours at 37°C . For ETEC antigens , expanded cells were added at 2×105 cells/well in duplicates and incubated for 6 hours at 37°C , 5% CO2 . For total IgG and IgA , expanded cells were added at 2×104 cells/well in duplicate and serially diluted 2-fold for a total of 7 times . Plates were then washed with PBS-Tween followed by a wash with PBS and incubated with rabbit anti-human biotin conjugated Pan-IgG antibody ( Hybridoma Reagent Laboratory , Baltimore , MD ) or goat anti-human Pan-IgA antibody ( Jackson Immuno Research Labs ) overnight at 4°C followed by HRP-conjugated Avidin D ( Vector Laboratories , Burlingame , CA ) for 1 hour at room temperature . Spots were detected by adding 100 μl/well of 3-Amino-9 etheylcarbazole C ( AEC substrate; Calbiochem , La Jolla , CA ) at room temperature in the dark . The reaction was stopped after 8–10 minutes by washing with distilled water . Spots were counted in an automated ELISpot reader ( Immunospot , Cellular Technology Ltd . , Cleveland , OH ) and analyzed using the Immunospot version 5 . 0 software . Results are expressed as percent of antigen-specific spot forming cells ( SFC ) per total IgA or IgG SFC . Comparisons between Resistant and Susceptible volunteer groups were performed using unpaired t test in GraphPad Prism version 6 . 0 ( Graphpad Software , San Diego , CA ) . Associations between integrin α4β7 expression and stool volume or ETEC-specific IgA BM were determined using linear regression in GraphPad Prism version 6 . 0 . For analyses that involved repeated observations from the same volunteer over time , we used random effects models ( as fit by SAS Proc Mixed ) to account for the correlation between repeated measures from the same person .
Within 18 hours from ingesting the challenge inoculum , volunteers began to produce loose stools . There was a 50% ( 3 of 6 ) attack rate for MSD ( Table 1 ) . Two volunteers achieved the definition of severe diarrhea ( ≥3 liter ) and received early antibiotic intervention . One volunteer achieved moderate diarrhea ( ≥1 liter ) and two volunteers had mild diarrhea and both achieved this on the fourth day post-challenge . Upon initiation of antibiotics , symptoms and shedding of culturable organisms ceased rapidly , within 1–2 days . One volunteer ( number 4016 ) did not experience any diarrhea , but culturable ETEC organisms were detected in his/her stool specimens on the first day post-infection . Additional signs and symptoms associated with challenge are described in S2 Table . For the purpose of examining the cell-mediated immune responses in these participants , we assigned the designation of “Susceptible” to those who experienced MSD . Those who experienced mild ( <1 liter ) or no diarrhea were designated “Resistant” . PBMC from challenged volunteers were stimulated with ETEC antigens followed by mass cytometric analyses to investigate the CMI responses . There were no differences in the % of total CD4+ T cells , T effector memory ( TEM ) , TEM CD45RA+ ( TEMRA ) , T central memory ( TCM ) or T naïve ( TN ) populations between volunteers who developed MSD ( Susceptible ) and those who did not ( Resistant ) ( S2 Fig ) . To identify T cells responding to ETEC antigens , we measured cytokine production by CD4+ T cells stimulated with CFA/I , whole cell homogenate , LT-B , and dmLT . PBMC from Resistant volunteers produced significantly increased levels ( above baseline ) of net TNF-α ( 0 . 2–0 . 4%; p = 0 . 027 unpaired t test ) and IL-2 ( 0 . 1–0 . 2%; p = 0 . 018 unpaired t test ) on day 3 post infection in response to in vitro stimulation with CFA/I while Susceptible volunteers did not show increased cytokine production above baseline when stimulated with CFA/I ( Fig 1 ) . Responses to the other ETEC antigens did not differ significantly between Resistant and Susceptible volunteers with the exception of low levels of whole cell homogenate-specific IL-2 production ( 0 . 05% ) which was significantly higher in Resistant than Susceptible volunteers ( p = 0 . 041 , unpaired t test ) ( S3 Fig ) . Since the strongest responses were noted against CFA/I , we measured the expression of CD154 ( CD40L ) , as well as IFN-γ and IL-17A production following CFA/I stimulation ( S4 Fig ) . While volunteers who were Resistant showed trends towards increases above pre-challenge in net CD154 expression and IFN-γ and IL-17A production on day 3 post-challenge , Susceptible volunteers did not ( S4A , S4B and S4C Fig ) . We further explored the multifunctionality of CFA/I-specific CD4+ T cell responses in the 2 Resistant volunteers , and identified TNF-α single positive cells as well as TNF-α and IL-2 double positive cells ( Fig 2 ) . To determine the CD4+ T cell subsets responsible for CFA/I-specific cytokine production , we calculated the percentage of cytokine response attributable to the 4 memory subsets ( TEM , TEMRA , TCM , and TN ) as well as to pTfh ( Table 2 ) . As expected , the majority of the cytokine production ( 57–85% ) was attributable to the TEM subset . As ETEC is an enteric pathogen , we also investigated the expression of the gut-homing molecule integrin α4β7 by CD4+ T cells . We identified significantly higher percentages of CD4+ T cells expressing integrin α4β7 prior to challenge and at 3 days post-challenge ( p = 0 . 030 and p = 0 . 009 respectively , unpaired t test ) in Resistant volunteers ( Fig 3A ) . In Resistant volunteers , TNF-α and IL-2 were produced by CD4+ T cells with and without gut-homing potential ( Fig 3B ) . Because of the great importance of pTfh in contributing to antigen-specific antibody responses [27] , we then measured the percentages of pTfh in Resistant and Susceptible volunteers prior to and at 3 days post-challenge . Of note , we identified significantly higher percentages of CXCR5+ pTfh in post-challenge specimens of Resistant volunteers compared to Susceptible volunteers ( p = 0 . 006 , unpaired t test ) ( Fig 4A ) . There were also detectable increases in CFA/I-specific TNF-α and IL-2 production by pTfh on day 3 post-challenge ( above baseline ) in Resistant volunteers but not Susceptible volunteers ( Fig 4B & 4C ) . Furthermore , 12–17% of the CFA/I-specific cytokine production by CD4+ T cells was attributable to pTfh which represented 8 . 6–17% of the total CD4+ population ( Table 2 ) . As seen in total CD4+ T cells , there were no significant differences in the increases observed above pre-challenge levels for net production of IFN-γ , IL-17A , or IL-21 or for CD154 or ICOS expression at day 3 post-challenge ( S5 Fig ) . We further explored the homing potential of pTfh and identified significantly higher percentages of pTfh expressing integrin α4β7 or co-expressing CCR6 and CXCR3 ( p = 0 . 014 and p = 0 . 011 respectively , unpaired t test ) in Resistant volunteers on day 3 post challenge ( Fig 5A & 5B ) . Moreover , expression of integrin α4β7 by pTfh was inversely correlated with cumulative stool volume ( Fig 5C; p = 0 . 048 , linear regression ) . We also investigated the association of integrin α4β7 expressing pTfh at early time-points with later IgA BM responses . Volunteers with higher percentages of integrin α4β7 expressing pTfh prior to challenge had more robust CFA/I-specific IgA BM responses on day 21 post challenge ( Fig 5D; p = 0 . 021 , linear regression ) . All evaluable volunteers mounted IgA BM responses against at least one ETEC antigen . We identified higher ETEC LPS-specific BM responses ( as the increase in % of total IgA over baseline ) in Resistant volunteers during days 14–28 post-challenge; however , these differences were not evident at later time-points ( days 56–180 post-challenge ) ( Fig 6A & 6E ) . We did not detect differences in BM responses between Resistant and Susceptible volunteers against any of the other antigens tested ( CFA/I , LT-B , and dmLT ) ( Fig 6B–6D ) We also found that higher integrin α4β7 expression by pTfh following stimulation with whole cell homogenate correlated with net IgA BM responses to LPS antigen on day 14 post-challenge ( p = 0 . 025 , linear regression ) and approached significance on day 28 post-challenge ( p = 0 . 08 , linear regression ) ( S6 Fig ) .
A DARPA grant supported the manufacture of a cGMP standardized inoculum of ETEC strain H10407 to assess the efficacy of candidate anti-ETEC immunoprophylactic products , thereby enhancing study-to-study consistency at the CVD and offering the possibility of concomitant testing at multiple clinical challenge sites . DARPA also supported a small clinical trial to document that the frozen cGMP inoculum was capable of causing typical ETEC diarrhea . This challenge study , in turn , provided an opportunity to measure the humoral and cell-mediated immune responses following challenge . Specifically , to look for reactivity of CD4+ T cells collected pre- and post-challenge , to determine their response following in vitro exposure to ETEC antigens and whether T cell reactivity was associated with some degree of resistance to developing ETEC diarrhea or to manifesting milder illness . Volunteers were recruited from the Baltimore-Washington area . A history of recent foreign travel to an endemic region , prior cholera or ETEC infection ( natural infection or experimental challenge ) , or prior receipt of a cholera or ETEC vaccine were exclusionary criteria . Although the challenged volunteers represent an ETEC-naïve population in terms of travel , U . S . residents are likely repetitively exposed to low doses of either animal or human ETEC through fruits and salad vegetables imported from Mexico and Central American countries where ETEC is endemic , and perhaps from U . S . beef and pork products having low level contamination with animal ETEC [28 , 29] . Thus , non-travelled members of the U . S . population may have had prior exposure to ETEC antigens . At a challenge dose of ~1 x 108 CFU , half of the volunteers challenged with ETEC developed MSD . This dichotomy provided an opportunity to explore differences in immune responses between volunteers who were Resistant and those who were Susceptible . Due to the non-invasive nature of ETEC , it is hypothesized that humoral responses play a major role in protection and much evidence supports this contention . Volunteers pre-treated with immunoglobulin from the milk of cows hyper-immunized with an array of ETEC pathogens that included different serotypes expressing the major CFAs and LT were 100% protected compared to a diarrhea attack rate of 90% in controls when challenged with ETEC strain H10407 . Based on these and similar observations , an antibody preparation from the milk of cows immunized with ETEC is a prophylactic product against ETEC for travelers with evidence of efficacy coming from three separate volunteer challenge studies [30] . In contrast , very limited information is available on the contributions of T cells , which are likely to play a major role in promoting antibody responses . CD4+ T cells perform multiple critical functions within the immune system , including provision of B cell “help” , enhancement and maintenance of CD8+ T cell responses , and regulation/suppression of immune responses to maintain homeostasis [31] . Production of cytokines , i . e . , TNF-α , IL-2 , IFN-γ , and IL-17A , in response to antigenic stimulation is an important function of CD4+ T cells . Although there were no significant differences in the percentages of total CD4+ T cells or CD4+ T cell memory subsets between Resistant and Susceptible volunteers , we identified significantly higher increases above baseline in CFA/I-specific production of TNF-α and IL-2 at day 3 post-challenge by CD4+ T cells in volunteers who did not develop MSD ( Resistant ) following challenge . Furthermore , these responses were multifunctional , a potential indicator of higher quality responses [32 , 33] . TNF-α is a potent pro-inflammatory cytokine and plays an important role in recruiting polymorphonuclear and other immune cells to sites of inflammation , while IL-2 plays a critical role in influencing the proliferation , differentiation , and survival of antigen-specific T cells [34] . Together , these cytokines may play a significant role in a protective immune response against ETEC . Although IFN-γ secretion has been documented following ETEC vaccination [35] , we did not identify a significant difference in IFN-γ production between Resistant and Susceptible volunteers . It is important to note that measurement of IFN-γ secretion by Wenneras et al was by ELISPOT and ELISA which cannot distinguish among the specific cell subset ( s ) producing the cytokine . It is therefore possible that other cell types , besides CD4+ T cells , are responsible for producing IFN-γ in response to CFA/I stimulation . In fact , IFN-γ has been shown to be produced by natural killer cells ( NK ) , macrophages , dendritic cells , and B cells [36] . Cytokine production in response to other ETEC antigens ( whole cell homogenate , LT-B , and dmLT ) was not as pronounced as the responses observed following CFA/I stimulation . It is important to note that the challenge inoculum was grown on CFA agar which may have favored the expression of CFA/I . PBMC from Resistant volunteers demonstrated increases above baseline in production of both TNF-α and IL-2 by CD4+ T cells following stimulation with whole cell homogenate which contains a broad array of ETEC antigens ( likely including CFA/I , LPS , LT and ST , pili , etc ) . The role of CFAs in ETEC pathogenesis has been well documented [37] and CFA/I is a major fimbrial antigen which has been shown to induce IgA antibodies following infection [38] . In the present study , we found CFA/I to be the strongest stimulator of cytokine responses in PBMC from Resistant individuals , indicating that this may be an important antigen for inclusion in future vaccine development . Integrin α4β7 is an important molecule associated with homing of lymphocytes to the gut , the site of initial encounter with ETEC [39] . Importantly , we identified significantly higher levels of integrin α4β7 expression on CD4+ T cells in volunteers who were Resistant , indicating that the ability of CD4+ T cells to home to the sight of pathogen encounter may play a role in protection . We further identified CFA/I-specific cytokine producing CD4+ T cells within both the integrin α4β7 positive and negative populations , suggesting that exposure to wt ETEC elicits specific CD4+ cells capable of homing to the gut as well as to extra-intestinal sites . pTfh are a circulating subset of CD4+ T cells characterized by the expression of CXCR5 and responsible for promoting antibody production by B cells [40] . In fact , the expression of different homing molecules by pTfh has been reported to affect the class of antibodies produced by B cells ( i . e . , CCR6 expressing pTfh cells have been shown to play a role in IgA production ) [40] . Here we describe , for the first time , the expression of integrin α4β7 by pTfh indicating that this subset has the potential to home to the gut . Furthermore , expression levels of integrin α4β7 on pTfh were higher in Resistant volunteers than Susceptible volunteers at day 3 post challenge and higher levels of integrin α4β7 expression by pTfh were associated with lower cumulative stool volumes . Moreover , early expression of integrin α4β7 by pTfh correlated with ETEC-specific IgA BM responses . Interestingly , CFA/I-specific integrin α4β7 expression by pTfh correlated with increased CFA/I-specific IgA BM responses , while whole cell homogenate-specific integrin α4β7 expression by pTfh correlated with increased LPS-specific IgA BM responses . These results support the concept that interaction between antigen-specific pTfh and their B cell counterparts in the gut results in antigen-specific BM responses which may confer long-term protection in humans . While this is the first report of integrin α4β7 expression by pTfh , we have previously reported that oral immunization of volunteers with attenuated oral S . Typhi vaccines or challenge with wild-type S . Typhi , another human-restricted Gram negative enteric bacterial pathogen , elicits S . Typhi-specific TEM and Treg which expressed , or not , the gut homing molecule integrin α4β7 [41–44] . Additionally the gut homing potential of S . Typhi-specific T regulatory cells is associated with increased susceptibility to typhoid disease in a challenge model [42] highlighting the importance of pathogen-specific cells homing to the site of initial antigen encounter . It is , at present , unknown why volunteers with no known previous ETEC exposure ( through natural infection or immunization ) would be Resistant to infection in this challenge model . It is possible that Resistant volunteers have some pre-existing immunity secondary to subclinical exposure to ETEC in the past , or , alternatively , exposure to cross-reactive antigens from related Gram negative organisms such as those in the gut microbiota . It is also possible that volunteers with higher levels of integrin α4β7 expression on circulating CD4+ T cells have a more robust T cell response at the mucosal level resulting in lack of moderate to severe disease . The mechanisms by which volunteers are protected from ETEC challenge may be further elucidated by studies in which volunteers are vaccinated against and then challenged with wt ETEC . An important limitation of the present study is the small sample size . However , we identified several important statistically significant differences between groups of volunteers who were Resistant and Susceptible . Upcoming clinical trials involving challenges with wt ETEC in larger numbers of volunteers will be directed to confirm and extend the novel findings presented herein . Additionally , challenges preceded by immunization will allow us to better define the protective immune responses induced by ETEC vaccine candidate ( s ) . | Enterotoxigenic Escherichia coli ( ETEC ) is an important cause of diarrheal illness in infants and young children in the developing world , as well as in individuals traveling to endemic areas . Due to the lack of suitable animal models for human ETEC infection , we performed a human challenge study in which volunteers ingested wild-type ETEC in a controlled clinical setting . In addition to closely monitoring their clinical status , we studied their ETEC-specific T cell responses prior to and after challenge and studied the presence of associations between CD4+ T cell responses and clinical outcome . We observed differences in the immunological responses of individuals who developed moderate to severe diarrhea following challenge compared to those who did not . These results indicate that T cells may be an important component of the immune response against ETEC . | [
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"analy... | 2017 | Impact of CD4+ T Cell Responses on Clinical Outcome following Oral Administration of Wild-Type Enterotoxigenic Escherichia coli in Humans |
There is growing interest in studying and engineering integral membrane proteins ( MPs ) that play key roles in sensing and regulating cellular response to diverse external signals . A MP must be expressed , correctly inserted and folded in a lipid bilayer , and trafficked to the proper cellular location in order to function . The sequence and structural determinants of these processes are complex and highly constrained . Here we describe a predictive , machine-learning approach that captures this complexity to facilitate successful MP engineering and design . Machine learning on carefully-chosen training sequences made by structure-guided SCHEMA recombination has enabled us to accurately predict the rare sequences in a diverse library of channelrhodopsins ( ChRs ) that express and localize to the plasma membrane of mammalian cells . These light-gated channel proteins of microbial origin are of interest for neuroscience applications , where expression and localization to the plasma membrane is a prerequisite for function . We trained Gaussian process ( GP ) classification and regression models with expression and localization data from 218 ChR chimeras chosen from a 118 , 098-variant library designed by SCHEMA recombination of three parent ChRs . We use these GP models to identify ChRs that express and localize well and show that our models can elucidate sequence and structure elements important for these processes . We also used the predictive models to convert a naturally occurring ChR incapable of mammalian localization into one that localizes well .
As crucial components of regulatory and transport pathways , integral membrane proteins ( MPs ) are important pharmaceutical and engineering targets [1] . To be functional , MPs must be expressed and localized through a series of elaborate sub-cellular processes that include co-translational insertion , rigorous quality control , and multi-step trafficking to arrive at the correct topology in the correct sub-cellular location [2–4] . With such a complex mechanism for production , it is not surprising that MP engineering has been hampered by poor expression , stability , and localization in heterologous systems [5–7] . To overcome these limitations , protein engineers need a tool to predict how changes in sequence affect MP expression and localization . An accurate predictor would enable us to design and produce MP variants that express and localize correctly , a necessary first step in engineering MP function . A useful predictor would be sensitive to subtle changes in sequence that can lead to drastic changes in expression and localization . Our goal here was to develop data-driven models that predict the likelihood of a MP’s expression and plasma membrane localization using the amino acid sequence as the primary input . For this study , we focus on channelrhodopsins ( ChRs ) , light-gated ion channels that assume a seven transmembrane helix topology with a light-sensitive retinal chromophore bound in an internal pocket . This scaffold is conserved in both microbial rhodopsins ( light-driven ion pumps , channels , and light sensors–type I rhodopsins ) and animal rhodopsins ( light-sensing G-protein coupled receptors–type II rhodopsins ) [8] . Found in photosynthetic algae , ChRs function as light sensors in phototaxic and photophobic responses [9 , 10] . On photon absorption , ChRs undergo a multi-step photo-cycle that allows a flux of ions across the membrane and down the electrochemical gradient [11] . When ChRs are expressed transgenically in neurons , their light-dependent activity can stimulate action potentials , allowing cell-specific control over neuronal activity [12 , 13] and extensive applications in neuroscience [14] . The functional limitations of available ChRs have spurred efforts to engineer or discover novel ChRs [11] . The utility of a ChR , however , depends on its ability to express and localize to the plasma membrane in eukaryotic cells of interest , and changes to the amino acid sequence frequently abrogate localization [5] . A predictor for ChRs that express and localize would be of great value as a pre-screen for function . The sequence and structural determinants for membrane localization have been a subject of much scientific investigation [15–17] and have provided some understanding of the MP sequence elements important for localization , such as signal peptide sequence , positive charge at the membrane–cytoplasm interface ( the “positive-inside” rule [18] ) , and increased hydrophobicity in the transmembrane domains . However , these rules are of limited use to a protein engineer: there are too many amino acid sequences that follow these rules but still fail to localize to the plasma membrane ( see Results ) . MP sequence changes that influence expression and localization are highly context-dependent: what eliminates localization in one sequence context has no effect in another , and subtle amino acid changes can have dramatic effects [5 , 16 , 19] . In short , sequence determinants of expression and localization are not captured by simple rules . Accurate atomistic physics-based models relating a sequence to its level of expression and plasma membrane localization currently do not exist , in large measure due to the complexity of the process . Statistical models offer a powerful alternative . Statistical models are useful for predicting the outcomes of complex processes because they do not require prior knowledge of the specific biological mechanisms involved . That being said , statistical models can also be constructed to exploit prior knowledge , such as MP structural information . Statistical models can be trained using empirical data ( in this case expression or localization values ) collected from known sequences . During training , the model infers relationships between input ( sequence ) and output ( expression or localization ) that are then used to predict the properties of unmeasured sequence variants . The process of using empirical data to train and select statistical models is referred to as machine learning . Machine learning has been applied to predicting various protein properties , including solubility [20 , 21] , trafficking to the periplasm [22] , crystallization propensity [23] , and function [24] . Generally , these models are trained using large data sets composed of literature data from varied sources with little to no standardization of the experimental conditions , and trained using many protein classes ( i . e . proteins with various folds and functions ) , because their aim is to identify sequence elements across all proteins that contribute to the property of interest . This generalist approach , however , is not useful for identifying subtle sequence features ( i . e . amino acids or amino acid interactions ) that condition expression and localization for a specific class of related sequences , the ChRs in this case . We focused our model building on ChRs , with training data collected from a range of ChR sequences under standardized conditions . We applied Gaussian process ( GP ) classification and regression [25] to build models that predict ChR expression and localization directly from these data . In our previous work , GP models successfully predicted thermal stability , substrate binding affinity , and kinetics for several soluble enzymes [26] . Here , we asked whether GP modeling could accurately predict mammalian expression and localization for heterologous integral membrane ChRs and how much experimental data would be required . For a statistical model to make accurate predictions on a wide range of ChR sequences , it must be trained with a diverse set of ChR sequences [25] . We chose to generate a training set using chimeras produced by SCHEMA recombination , which was previously demonstrated to be useful for producing large sets ( libraries ) of diverse , functional chimeric sequences from homologous parent proteins [27] . We synthesized and measured expression and localization for only a small subset ( 0 . 18% ) of sequences from the ChR recombination library . Here we use these data to train GP classification and regression models to predict the expression and localization properties of diverse , untested ChR sequences . We first made predictions on sequences within a large library of chimeric ChRs; we then expanded the predictions to sequences outside that set .
The design and characterization of the chimeric ChR sequences used to train our models have been published [5]; we will only briefly describe these results . Two separate , ten-block libraries were designed by recombining three parental ChRs ( CsChrimsonR ( CsChrimR ) [28] , C1C2 [29] , and CheRiff [30] ) with 45–55% amino acid sequence identity and a range of expression , localization , and functional properties ( S1 Fig ) [5] . Each chimeric ChR variant in these libraries is composed of blocks of sequence from the parental ChRs . These libraries were prepared by the SCHEMA algorithm to define sequence blocks for recombination that minimize the library-average disruption of tertiary protein structure [31 , 32] . One library swaps contiguous elements of primary structure ( contiguous library ) , and the second swaps elements that are contiguous in the tertiary structure but not necessarily in the sequence ( non-contiguous library [33] ) . The two libraries have similar , but not identical , element boundaries ( S1A Fig ) and were constructed in order to test whether one design approach was superior to the other ( they gave similar results ) . These designs generate 118 , 098 possible chimeras ( 2 x 310 ) , which we will refer to as the recombination library throughout this paper . Each of these chimeras has a full N-terminal signal peptide from one of the three ChR parents . Two hundred and eighteen chimeras from the recombination library were chosen as a training set , including all the chimeras with single-block swaps ( chimeras consisting of 9 blocks of one parent and a single block from one of the other two parents ) and multi-block-swap chimera sequences designed to maximize mutual information between the training set and the remainder of the chimeric library . Here , the ‘information’ a chimera has to offer is how its sequence , relative to all previously tested sequences , changes ChR expression and localization . By maximizing mutual information , we select chimera sequences that provide the most information about the whole library by reducing the uncertainty ( Shannon entropy ) of prediction for the remainder of the library , as described in [34 , 35] . The 112 single-block-swap chimeras in the training set have an average of 15 mutations from the most closely related parent , while the 103 multi-block-swap chimeras in the training set have an average of 73 mutations from the most closely related parent ( Table 1 ) . While the multi-block-swap chimeras provide the most sequence diversity to learn from , they are the least likely to express and localize given their high mutation levels . The single-block-swap chimeras offer less information to learn from due to their sequence redundancies with other chimeras in the training set , but are more likely to express and localize . Genes for these sequences were synthesized and expressed in human embryonic kidney ( HEK ) cells , and their expression and membrane localization properties were measured ( S1B Fig ) [5] . The expression levels were monitored through a fluorescent protein ( mKate ) fused to the C-termini of the ChRs . Plasma-membrane localization was measured using the SpyTag/SpyCatcher labeling method , which exclusively labels ChR protein that has its N terminus exposed on the extracellular surface of the cell [36] . The training set sequences displayed a wide range of expression and localization properties . While the majority of the training set sequences express , only 33% of the single-block-swap chimeras localize well , and an even smaller fraction ( 12% ) of the multi-block-swap chimeras localize well , emphasizing the importance of having a predictive model for membrane localization . First we explored whether ChR chimera properties could be predicted based on basic biological properties , specifically , signal peptide sequence and hydrophobicity in the transmembrane ( TM ) domains . Each chimera in the library has one of the three parental signal peptides . Although the signal peptide sequence does affect expression and localization ( S2A Fig ) , chimeras with any parental signal peptide can have high or low expression and localization . Thus , the identity of the signal peptide alone is insufficient for accurate predictions of the ChR chimera properties . We then calculated the level of hydrophobicity within the 7-TM domains of each chimera . With very weak correlation between increasing hydrophobicity and measured expression and localization ( S2B Fig ) , hydrophobicity alone is also insufficient for accurate prediction of ChR chimera properties . These models do not accurately account for the observed levels of expression or localization ( S1 Fig ) . Therefore , we need more expressive models to predict expression and localization from the amino acid sequences of these MPs . Our overall strategy for developing predictive machine-learning models is illustrated in Fig 1 . The goal is to use a set of ChR sequences and their expression and localization measurements to train GP regression and classification models that describe how ChR properties depend on sequence and predict the behavior of untested ChRs . GP models infer predictive values from training examples by assuming that similar inputs ( ChR sequence variants ) will have similar outputs ( expression or localization ) . We quantify the relatedness of inputs ( ChR sequence variants ) by comparing both sequence and structure . ChR variants with few differences are considered more similar than ChR variants with many differences . We define the sequence similarity between two chimeras by aligning them and counting the number of positions at which they are identical . For structural comparisons , a residue-residue ‘contact map’ was built for each ChR variant , where two residues are in contact if they have any non-hydrogen atoms within 4 . 5 Å . The maps were generated using a ChR parental sequence alignment and the C1C2 crystal structure , which is the only available ChR structure [29] , with the assumption that ChR chimeras share the overall contact architecture observed in the C1C2 crystal structure . The structural similarity for any two ChRs was quantified by aligning the contact maps and counting the number of identical contacts [26] . Using these metrics , we calculated the sequence and structural similarity between all ChRs in the training set relative to one another ( 218 x 218 ChR comparisons ) . These similarity functions are called kernel functions and specify how the functional properties of pairs of sequences are expected to covary ( they are also known as covariance functions ) . In other words , the kernel is a measure of similarity between sequences , and we can draw conclusions about unobserved chimeras on the basis of their similarity to sampled points [25] . The model has high confidence in predicting the properties of sequences that are similar to previously sampled sequences , and the model is less confident in predicting the properties of sequences that are distant from previously sampled sequences . To build a GP model , we must also specify how the relatedness between sequences will affect the property of interest , in other words how sensitive the ChR properties are to changes in relatedness as defined by the sequence/structure differences between ChRs . This is defined by the form of the kernel used . We tested three different forms of sequence and structure kernels: linear kernels , squared exponential kernels , and Matérn kernels ( see Methods ) . These different forms represent the kinds of functions we expect to observe for the protein’s fitness landscape ( i . e . the mapping of protein sequence to protein function ) . The linear kernel corresponds to a simple landscape where the effects of changes in sequence/structure are additive and there is no epistasis . The two non-linear kernels represent more rugged , complex landscapes where effects may be non-additive . Learning involves optimizing the form of the kernel and its hyperparameters ( parameters that influence the form of kernel ) to enable accurate predictions . The hyperparameters and the form of the kernel were optimized using the Bayesian method of maximizing the marginal likelihood of the resulting model . The marginal likelihood ( i . e . how likely it is to observe the data given the model ) rewards models that fit the training data well while penalizing model complexity to prevent overfitting . Once trained with empirical data , the output of the GP regression model is a predicted mean and variance , or standard deviation , for any given ChR sequence variant . The standard deviation is an indication of how confident the model is in the prediction based on the relatedness of the new input relative to the tested sequences . We used GP models to infer links between ChR properties and ChR sequence and structure from the training data . We first built GP binary classification models . In binary classification , the outputs are class labels i . e . ‘high’ or ‘low’ localization , and the goal is to use the training set data to predict the probability of a sequence falling into one of the two classes ( Fig 1 ) . We also built a GP regression model that makes real-valued predictions , i . e . amount of localized protein , based on the training data ( Fig 1 ) . After training these models , we verify that their predictions generalize to sequences outside of the training set . Once validated , these two models can be used in different ways . A classification model trained from localization data can be used to predict the probability of highly diverse sequences falling into the ‘high’ localization category ( Fig 1 ) . The classification model can only predict if a sequence has ‘high’ vs ‘low’ localization , and it cannot be used to optimize localization . The regression model , on the other hand , can be used to predict sequences with ‘optimal’ properties; for example , a regression model trained from localization data can predict untested sequences that will have very high levels of localization ( Fig 1 ) . The training set data ( S1 Fig ) were used to build a GP classification model that predicted which of the 118 , 098 chimeras in the recombination library would have ‘high’ vs ‘low’ expression , localization , and localization efficiency . The training set includes multi-block swaps chosen to be distant from other sequences in the training set in order to provide information on sequences throughout the recombination library . A sequence was considered ‘high’ if it performed at least as well as the lowest performing parent , and it was considered ‘low’ if it performed worse than the lowest performing parent . Because the lowest performing parent for expression and localization , CheRiff , is produced and localized in sufficient quantities for downstream functional studies , we believe this to be an appropriate threshold for ‘high’ vs ‘low’ performance . For all of the classification models ( Fig 2 and S3 Fig ) , we used kernels based on structural relatedness . For the expression classification model , we found that a linear kernel performed best , i . e . achieved the highest marginal likelihood . This suggests that expression is best approximated by an additive model weighting each of the structural contacts . Localization and localization efficiency required a non-linear kernel for the model to be predictive . This more expressive kernel allows for non-linear relationships and epistasis and also penalizes differing structural contacts more than the linear kernel . This reflects our intuitive understanding that localization is a more demanding property to tune than expression , with stricter requirements and a non-linear underlying fitness landscape . Most of the multi-block-swap sequences from the training set did not localize to the membrane [5] . We nonetheless want to be able to design highly mutated ChRs that localize well because these are most likely to have interesting functional properties . We therefore used the localization classification model to identify multi-block-swap chimeras from the library that had a high predicted probability ( >0 . 4 ) of falling into the ‘high’ localizer category ( Fig 2D ) . From the many multi-block-swap chimeras predicted to have ‘high’ localization , we selected a set of 16 highly diverse chimeras with an average of 69 amino acid mutations from the closest parent and called this the ‘exploration’ set ( S4 Fig ) . We synthesized and tested these chimeras and found that the model had accurately predicted chimeras with good localization ( Fig 2 and Fig 3 ) : 50% of the exploration set show ‘high’ localization compared to only 12% of the multi-block-swap sequences from the original training set , even though they have similar levels of mutation ( Table 1 and S1 Data ) ( chimeras in the exploration set have on average 69 ± 12 amino acid mutations from the closest parent , versus 73 ± 21 for the multi-block-swap chimeras in the training set ) . The classification model provides a four-fold enrichment in the number of chimeras that localize well when compared to randomly-selected chimeras with equivalent levels of mutation . This accuracy is impressive given that the exploration set was designed to be distant from any sequence the model had seen during training . The model’s performance on this exploration set indicates its ability to predict the properties of sequences distant from the training set . The data from the exploration set were then used to better inform our models about highly diverse sequences that localize . To characterize the classification model’s performance , we calculated the area under the receiver operating characteristic ( ROC ) curve ( AUC ) . A poorly performing model would not do better than random chance , resulting in an AUC of 0 . 5 , while a model that perfectly separates the two classes will have an AUC of 1 . 0 . The revised models achieved AUC up to 0 . 87 for “leave-one-out” ( LOO ) cross-validation , indicating that there is a high probability that the classifiers will accurately separate ‘high’ and ‘low’ performing sequences for the properties measured . The AUC is 0 . 83 for localization , 0 . 77 for localization efficiency and 0 . 87 for expression for LOO cross-validation predictions ( S5 Fig ) . To further test the models , we then built a verification set of eleven chimeras , designed using the localization model . This verification set was composed of four chimeras predicted to be highly likely to localize , six chimeras predicted to be very unlikely to localize , and one chimera with a moderate predicted probability of localizing ( S4 Fig ) . The measured localization ( Fig 2E ) and localization efficiency ( S3B Fig ) of the chimeras in the verification set show clear differences , ‘high’ vs ‘low’ , consistent with the model predictions ( Table 1 and S1 Data ) . The verification sets consist exclusively of chimeras with ‘high’ measured expression , which is consistent with the model’s predictions ( Fig 2B ) . The model perfectly classifies the eleven chimeras as either ‘high’ or ‘low’ for each property ( expression , localization , or localization efficiency ) as shown in plots of predicted vs measured properties ( Fig 2B and 2E and S3B Fig ) and by perfect separation in ROC curves i . e . AUC = 1 . 0 ( S5 Fig ) . These models are powerful tools that can confidently predict whether a chimera will have 'high' or 'low' expression ( Fig 2C ) , localization ( Fig 2F ) , and localization efficiency ( S3C Fig ) . Of the 118 , 098 chimeras in the recombination library , 6 , 631 ( 5 . 6% ) are predicted to have a probability > 0 . 5 of 'high' localization , whereas the vast majority of chimeras ( 99% ) are predicted to have a probability > 0 . 5 of 'high' expression . The classification model predicts the probability that a sequence falls into the ‘high’ localizer category , but does not give a quantitative prediction as to how well it localizes . Our next goal was to design chimera sequences with optimal localization . Localization is considered optimal if it is at or above the level of CsChrimR , the best localizing parent , which is more than adequate for in vivo applications using ChR functionality to control neuronal activity [28] . A regression model for ChR plasma membrane localization is required to predict sequences that have optimal levels of localization . We used the localization data from the training and exploration sets to train a GP regression model ( Fig 4A ) . The diversity of sequences in the training data allows the model to generalize well to the remainder of the recombination library . For this regression model , we do not use all of the features from the combined sequence and structure information; instead , we used L1 linear regression to select a subset of these features . The L1 linear regression identifies the sequence and structural features that most strongly influence ChR localization . Using this subset of features instead of all of the features improved the quality of the predictions ( as determined by cross-validation ) . This indicates that not all of the residues and residue-residue contacts have a large influence on localization of ChR . We then used a kernel based on these chosen features ( specific contacts and residues ) for GP regression . The regression model for localization showed strong predictive ability as indicated by the strong correlation between predicted and measured localization for LOO cross-validation ( correlation coefficient , R > 0 . 76 ) ( Fig 4A ) . This was further verified by the strong correlation between predicted and measured values for the previously-discussed verification set ( R > 0 . 9 ) ( Fig 4A ) . These cross-validation results suggest that the regression model can be used to predict chimeras with optimal localization . We used the localization regression model to predict ChR chimeras with optimal localization using the Lower Confidence Bound ( LCB ) algorithm , in which the predicted mean minus the predicted standard deviation ( LB1 ) is maximized [37] . The LCB algorithm maximally exploits the information learned from the training set by finding sequences the model is most certain will be good localizers . The regression model was used to predict the localization level and standard deviation for all chimeras in the library , and from this the LB1 was calculated for all chimeras ( Fig 4B ) . We selected four chimeras whose LB1 predictions for localization were ranked in the top 0 . 1% of the library ( S4 Fig ) . These were constructed and tested ( Fig 3 and S6 Fig and S1 Data ) . Measurements showed that they all localize as well as or better than CsChrimR ( Fig 3 and Fig 4A and Table 1 ) . Cell population distributions of the optimal set show properties similar to the CsChrimR parent , with one chimera showing a clear shift in the peak of the distribution towards higher levels of localization ( S7 Fig ) . These four sequences differ from CsChrimR at 30 to 50 amino acids ( S4 Fig ) . We were interested in how predictive the GP localization models could be with fewer training examples . To assess the predictive ability of the GP models as a function of training set size , we sampled random sets of training sequences from the dataset , trained models on these random sets , then evaluated the model’s performance on a selected test set ( S8 Fig ) . As few as 100 training examples are sufficient for accurate predictions for both the localization regression and classification models . This analysis shows that the models would have been predictive with even fewer training examples than we chose to use . In developing the GP regression model for localization , we used L1-regularized linear regression to identify a limited set of sequence and structural features that strongly influence ChR localization ( Fig 4 ) . These features include both inter-residue contacts and individual residues and offer insight into the structural determinants of ChR localization . To better gauge the relative importance of these features , L2-regularized linear regression was used to calculate the positive and negative feature weights , which are proportional to each feature’s inferred contribution to localization . While not as predictive as the GP regression model because it cannot account for higher-order interactions between features , this linear model has the advantage of being interpretable . When mapped onto the C1C2 structure , these features highlight parts of the ChR sequence and structural contacts that are important for ChR localization to the plasma membrane ( Fig 5 ) . Both beneficial and deleterious features are distributed throughout the protein , with no single feature dictating localization properties ( Fig 5 ) . Clusters of heavily weighted positive contacts suggest that having structurally proximal CsChrimR-residue pairs are important in the N-terminal domain ( NTD ) , between the NTD and TM4 , between TM1 and TM7 , and between TM3 and TM7 . CsChrimR residues at the extracellular side of TM5 also appear to aid localization , although they are weighted less than CheRiff residues in the same area . Beneficial CheRiff contacts and residues are found in the C-terminal domain ( CTD ) , the interface between the CTD and TM5-6 , and in TM1 . C1C2 residues at the extracellular side of TM6 are also positively weighted for localization , as are C1C2 contacts between the CTD and TM3-4 loop . From the negatively weighted contacts , it is clear that total localization is harmed when CheRiff contributes to the NTD or the intracellular half of TM4 and when CsChrimR contributes to the CTD . Interestingly , positive contacts were formed between TM6 from C1C2 and TM7 from CheRiff , but when the contributions were reversed ( TM6 from CheRiff TM7 from C1C2 ) or if CsChrimR contributed TM6 , strong negative weights were observed . Not surprisingly , the sequence and structure of optimal localizers predicted by GP regression ( Fig 4 ) largely agree with the L2 weights ( S9 Fig ) . Using this strategy for model interpretation ( L1 regression for feature selection followed by L2 regression ) , we can also weight the contributions of residues and contacts for ChR expression ( S10 Fig and S11 Fig ) . There is some overlap between the heavily weighted features for ChR expression and the features for localization , which is expected because more protein expressed means more protein available for localization . For example , both expression and localization models seem to prefer the NTD from CsChrimR and the extracellular half of TM6 from C1C2 , and both disfavor the NTD and the intra-cellular half of TM4 from CheRiff . While the heavily-weighted expression features are limited to these isolated sequence regions , localization features are distributed throughout the protein . Moreover , the majority of heavily-weighted features identified for expression are residues rather than contacts . This is in contrast to those weighted features identified for localization , which include heavily-weighted residues and structural contacts . This suggests that sequence is more important in determining expression properties , which is consistent with the largely sequence-dependent mechanisms associated with successful translation and insertion into the ER membrane . In contrast , both sequence and specific structural contacts contribute significantly to whether a ChR will localize to the plasma membrane . Our results demonstrate that the model can ‘learn’ the features that contribute to localization from the data and make accurate predictions on that property . We next tested the ChR localization regression model for its ability to predict plasma-membrane localization for ChR sequences outside the recombination library . For this , we chose a natural ChR variant , CbChR1 , that expresses in HEK cells and neurons but does not localize to the plasma membrane and thus is non-functional [28] . CbChR1 is distant from the three parental sequences , with 60% identity to CsChrimR and 40% identity to CheRiff and C1C2 . We optimized CbChR1 by introducing minor amino acid changes predicted by the localization regression model to be beneficial for membrane localization . To enable measurement of CbChR1 localization with the SpyTag-based labeling method , we substituted the N-terminus of CbChR1 with the CsChrimR N-terminus containing the SpyTag sequence downstream of the signal peptide to make the chimera CsCbChR1 [36] . This block swap did not change the membrane localization properties of CbChR1 ( Fig 6C ) . Using the regression model , we predicted localization levels for all the possible single-block swaps from the three library parents ( CsChrimR , C1C2 and CheRiff ) into CsCbChR1 and selected the four chimeras with the highest Upper Confidence Bound ( UCB ) . These chimeras have between 4 and 21 mutations when compared with CsCbChR1 . Unlike the LCB algorithm , which seeks to find the safest optimal choices , the UCB algorithm balances exploration and exploitation by maximizing the sum of the predicted mean and standard deviation . The selected chimeras were assayed for expression , localization , and localization efficiency ( S1 Data ) . One of the four sequences did not express; the other three chimeras expressed and had higher localization levels than CsCbChR1 ( Fig 6B ) . Two of the three had localization properties similar to the CheRiff parent ( Fig 6B ) . Images of the two best localizing chimeras illustrate the enhancement in localization when compared with CbChR1 and CsCbChR1 ( Fig 6C and S12 Fig ) . This improvement in localization was achieved through single-block swaps from CsChrimR ( 17 and 21 amino acid mutations ) ( Fig 6A ) . These results suggest that this regression model can accurately predict minor sequence changes that will improve the membrane localization of natural ChRs .
The ability to differentiate the functional properties of closely related sequences is extremely powerful for protein design and engineering . This is of particular interest for protein types that have proven to be more recalcitrant to traditional protein design methods , e . g . MPs . We show here that integral membrane protein expression and plasma membrane localization can be predicted for novel , homologous sequences using moderate-throughput data collection and advanced statistical modeling . We have used the models in four ways: 1 ) to accurately predict which diverse , chimeric ChRs are likely to express and localize at least as well as a moderately-performing native ChR; 2 ) to design ChR chimeras with optimized membrane localization that matched or exceeded the performance of a very well-localizing ChR ( CsChrimR ) ; 3 ) to identify the structural interactions ( contacts ) and sequence elements most important for predicting ChR localization; and 4 ) to identify limited sequence changes that transform a native ChR from a non-localizer to a localizer . Whereas 99% of the chimeras in the recombination library are predicted to express in HEK cells , only 5 . 6% are predicted to localize to the membrane at levels equal to or above the lowest parent ( CheRiff ) . This result shows that expression is robust to recombination-based sequence alterations , whereas correct plasma-membrane localization is much more sensitive . The model enables accurate selection of the rare , localization-capable , proteins from the nearly 120 , 000 possible chimeric library variants . In future work we will show that this diverse set of several thousand variants predicted to localize serves as a highly enriched source of functional ChRs with novel properties . Although statistical models generalize poorly as one attempts to make predictions on sequences distant from the sequences used in model training , we show that it is possible to train a model that accurately distinguishes between closely related proteins . The tradeoff between making accurate predictions on subtle sequence changes vs generalized predictions for significantly different sequences is one we made intentionally in order to achieve accurate predictions for an important and interesting class of proteins . Accurate statistical models , like the ones described in this paper , could aid in building more expressive physics-based models . This work details the steps in building machine-learning models and highlights their power in predicting desirable protein properties that arise from the intersection of multiple cellular processes . Combining recombination-based library design with statistical modeling methods , we have scanned a highly functional portion of protein sequence space by training on only 218 sequences . Model development through iterative training , exploration , and verification has yielded a tool that not only predicts optimally performing chimeric proteins , but can also be applied to improve related ChR proteins outside the library . As large-scale gene synthesis and DNA sequencing become more affordable , machine-learning methods such as those described here will become ever more powerful tools for protein engineering offering an alternative to high-throughput assay systems .
Both the GP regression and classification modeling methods applied in this paper are based on work detailed in [26] . Romero et al . applied GP models to predict protein functions and also defined protein distance using a contact map . We have expanded on this previous work . Regression and classification were performed using open-source packages in the SciPy ecosystem [39–41] . Below are specifics of the GP regression and classification methods used in this paper . The hyperparameters and the form of the kernel were optimized using the Bayesian method of maximizing the marginal likelihood of the resulting model . | A protein’s amino acid sequence determines how it will fold , traffic to subcellular locations , and carry out specific functions within the cell . Understanding this process would enable the design of protein sequences capable of useful functions; unfortunately , we cannot predict in detail how sequence encodes function . However , machine-learning models have the potential to infer the complex protein sequence-function relationship by identifying patterns or features that are important for function from sequences with known functions . We used machine learning to learn about and design membrane proteins ( MPs ) . To function , a MP must be expressed , correctly folded in a lipid membrane , and trafficked to the proper cellular location . We built predictive , machine-learning models for this complex process from a set of >200 chimeric MPs and used them to design new sequences with optimal performance on the challenging task of membrane localization . This general approach to understanding and designing MPs could be broadly useful for important pharmaceutical and engineering MP targets . | [
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"an... | 2017 | Machine learning to design integral membrane channelrhodopsins for efficient eukaryotic expression and plasma membrane localization |
Human norovirus ( HNoV ) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after the resolution of symptoms . Elimination of persistent viral reservoirs has the potential to prevent outbreaks . Similar to HNoV , murine norovirus ( MNV ) is spread by persistent shedding in the feces and provides a tractable model to study molecular mechanisms of enteric persistence . Previous studies have identified non-structural protein 1 ( NS1 ) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells ( IECs ) , but its mechanism of action remains unclear . We now find that the function of CR6 NS1 is regulated by apoptotic caspase cleavage . Following induction of apoptosis in infected cells , caspases cleave the precursor NS1/2 protein , and this cleavage is prevented by mutation of caspase target motifs . These mutations profoundly compromise CR6 infection of IECs and persistence in the intestine . Conversely , NS1/2 cleavage is not strictly required for acute replication in extra-intestinal tissues or in cultured myeloid cells , suggesting an IEC-centric role . Intriguingly , we find that caspase cleavage of CR6 NS1/2 reciprocally promotes caspase activity , potentiates cell death , and amplifies spread among cultured IEC monolayers . Together , these data indicate that the function of CR6 NS1 is regulated by apoptotic caspases , and suggest that apoptotic cell death enables epithelial spread and persistent shedding .
Human norovirus ( HNoV ) is the most common cause of epidemic gastroenteritis worldwide , and can be particularly dangerous for infants and the elderly [1 , 2] . Viral shedding often persists asymptomatically for weeks to months after acute infection [3 , 4] , and is a potential source for initiation of outbreaks . Despite recent success in development of in vitro systems to study HNoV infection [5 , 6] , there remains a need for robust small animal models for investigating NoV biology . Murine norovirus ( MNV ) shares genotypic ( ssRNA , positive-sense , ~7 . 5kb genome ) and phenotypic ( fecal-oral transmission , infection of intestinal epithelial cells ( IECs ) , persistent shedding ) features with HNoV . Therefore , studies of MNV have enabled considerable advances in understanding of norovirus biology [7–9] . All noroviruses express a non-structural polyprotein encoded by ORF1 and two structural capsid proteins ( VP1 and VP2 ) encoded by ORFs 2 and 3 , respectively [10–12] ( Fig 1A ) . The non-structural polyprotein is cleaved by the internally-encoded viral protease into six ‘mature’ proteins ( NS1/2 , NS3 , NS4 , NS5 , NS6 , and NS7 ) [12–14] . Unlike related caliciviruses , NS1/2 of NoV remains intact for most of the virus life cycle and may be cleaved by host proteases rather than the viral protease [12 , 13] . NoV non-structural proteins associate with membranes and form the membranous viral replication complex [15–18] . Specific roles of NS5-7 have been defined as VpG cap , protease , and polymerase , respectively . However , the roles of NS1-4 are less well understood [19] . Our prior MNV studies compared acute strain CW3 ( a plaque isolate of MNV-1 ) and persistent strain CR6 to identify viral determinants of pathogenesis and persistence [20–22] . We found that the capsid gene of CW3 ( VP1CW3 ) promotes extra-intestinal spread , whereas the NS1 gene of CR6 ( NS1CR6 ) promotes intestinal replication and persistent fecal shedding . An amino acid substitution at position 94 of CW3 NS1 ( D94E ) alters the conformation of a small structured region and can rescue CW3 persistence , suggesting the importance of this structured NS1 domain in persistence function [20 , 23] . Persistent and non-persistent strains similarly infect macrophages , dendritic cells , T-cells , and B-cells in vitro and in vivo [6 , 24 , 25] , and utilize the same cell-surface receptor , CD300lf [26 , 27] . However , recent studies have shown that tropism for IECs in vivo requires NS1CR6 in addition to CD300lf expression by non-hematopoietic cells [28 , 29] . Furthermore , only a minor lineage of IECs called tuft cells naturally express CD300lf and are susceptible to persistent infection by CR6 [28 , 30] . In contrast to the strain-dependent infection of IECs in vivo , CW3 and CR6 can replicate to similar titer in a cultured IEC cell line ( M2C ) that ectopically expresses CD300lf [29] . Thus , NS1CR6 promotes IEC persistence in vivo via mechanisms other than receptor usage or replicative capacity per se . In addition to viral determinants of MNV infection , critical aspects of the host anti-viral response have been identified . In particular , innate control of MNV replication is determined by the compartmentalized roles of type I ( α/β ) and type III ( λ ) interferon ( IFN ) [31] . Both types of IFN stimulate antiviral gene expression , but the IFN-α/β receptor ( IFNAR ) is expressed on most cell types , whereas the IFN-λ receptor ( IFNLR ) is preferentially expressed on epithelial cells [31] . Correspondingly , IFN-α/β is critical for control of MNV in myeloid cells and prevents pathological dissemination [24 , 32 , 33] , whereas IFN-λ is critical for control of MNV in IECs and prevents persistent shedding [34 , 35] . Deficiency of IFNLR promotes persistent infection , suggesting that NS1CR6 counteracts the IFN-λ response [29] . However , CW3 persistence is not rescued in IFNLR-deficient mice [29 , 35] indicating that other host pathways are also important . In addition to the IFN response , a general host response to many diverse viral genera is initiation of apoptosis . Correspondingly , viruses have evolved ways to hijack apoptotic machinery to their benefit . Apoptosis is a non-inflammatory , highly-regulated disassembly of the cell mediated by a family of cysteine proteases ( caspases ) . Apoptotic cellular contents are packaged and released in membranous apoptotic bodies to be taken up by professional phagocytes and other neighboring cells [36] . Because apoptosis is an innate response to infection , many viruses have developed ways to delay induction of apoptosis such as expression of anti-apoptotic Bcl-2 family homologues [37 , 38] . In contrast , after virus production has reached its peak , many viruses actively promote apoptotic death or utilize ‘apoptotic mimicry’ to facilitate viral dissemination [38 , 39] . Additionally , some viruses rely on apoptotic caspases to cleave viral proteins as an additional level of regulation [40–42] . Thus , depending on its timing in relation to viral replication , apoptosis can be anti-viral or pro-viral and is therefore regulated by a diverse array of viral mechanisms . HNoV and MNV infection both induce apoptosis , as indicated by association with markers of apoptosis in infected intestinal tissue [43–45] . Thus , these viruses have likely also developed mechanisms to regulate apoptosis in a pro-viral manner . Apoptosis has been most extensively studied in MNV-infected myeloid cells and is associated with down-regulation of anti-apoptotic survivin protein and activation of caspases 3 , 7 , and 9 [46 , 47] . Expression of the MNV ORF1 polyprotein is sufficient to induce apoptosis [48] , indicating that one or more non-structural proteins are pro-apoptotic in the absence of viral replication . Additionally , NS1/2 encodes caspase cleavage motifs that are targeted by recombinant caspase 3 in a cell-free assay and separate NS1 from membrane-associated NS2 [12] . Thus , apoptosis during MNV infection may be facilitated by non-structural proteins and host caspases may reciprocally regulate NS1/2 function [20] . Herein , we identify a critical role for NS1/2CR6 cleavage in MNV infection of the intestine and persistence . In vitro , we demonstrate that a minority of total NS1/2 protein is cleaved in a caspase-dependent manner , concurrent with the induction of apoptosis . Despite the relatively low proportion of total NS1/2 that is cleaved , NS1/2CR6 cleavage is absolutely required for early infection in IECs and persistent shedding , independent of IFN-λ . Moreover , the cleavage of NS1/2CR6 promotes apoptotic death of infected myeloid cells and spread among cultured IECs . These data suggest that controlled induction of apoptosis during viral egress promotes spread among IECs and is required for establishment of infection in IECs , the cellular reservoir of persistence .
Previous cell-free studies identified two functional caspase motifs ( DKAD121 and DAMD131 ) within NS1/2 that are cleaved by host caspase 3 [12] ( Fig 1A ) , however , the targeting of these sites during infection and functional consequences of NS1/2 cleavage was unknown . To test their role in persistent infection , we mutated these sites ( D121G and D131G ) within MNV strain , CR6 ( CR6Δcasp , Fig 1B ) . In parallel , we generated a variant of CR6Δcasp with the capsid of the systemic strain CW3 ( CR6Δcasp-VP1CW3 ) to enable determination of a role for NS1/2 cleavage in extra-intestinal tissues ( Fig 1B ) [20 , 49] . Analysis of NS1/2 protein expression following infection of BV2 myeloid cells revealed a similar level of full length NS1/2 production for all strains . However , infection with CW3 , CR6 , or the CR6-VP1CW3 chimera but not NS1/2 cleavage mutant strains resulted in a ~28 kDa NS2 fragment detectable at 18–22 hours post-infection ( Fig 1C ) . The ~18kDa NS1 protein was inconsistently detected in cell lysates for any strain and may reflect the small overall proportion of cleaved NS1/2 or the accumulation of cleaved NS1 in the supernatant reported in a concurrent study [50] . Several fragments larger than 28 kDa ( marked by # ) were also observed , but were not dependent on caspase cleavage sites ( Fig 1C ) and may be generated by other proteases activated during cell death . Overall , these data show that cleavage of NS1/2 occurs during infection , depends on caspase motifs , and is independent of other genetic variation between CW3 and CR6 . To determine the temporal relationship between caspase activity and NS1/2 cleavage , we detected cleaved caspase 3 ( active form ) by western blot . Cleaved caspase 3 was observed at 18–22 hours post-infection for all strains tested , concurrent with NS1/2 cleavage ( Fig 1D ) . To test the requirement of caspase activity for NS1/2 cleavage , we treated infected cells with the pan-caspase inhibitor , ZVAD-fmk . Inhibitor treatment reduced the proportion of cleaved caspase 3 and NS2 in cells infected with CW3 or CR6 but did not reduce the abundance of NS1/2 fragments larger than 28 kDa ( Fig 1D ) . Together , these data demonstrate that NS1/2 is cleaved 18–22 hours post infection with persistent or non-persistent MNV strains , concurrent with induction of apoptosis , in a caspase-dependent manner . In the course of characterizing caspase cleavage , we observed that the timing and magnitude of caspase 3 cleavage differed between viral strains ( Fig 1D ) . To further compare caspase activity triggered by MNV infection , we used densitometry to quantify percentage of cleaved caspase 3 and poly ADP-ribose polymerase ( PARP ) , a known target of apoptotic caspases [51] . We observed three-fold more activated caspase 3 and 10–20% more cleaved PARP in cells 18 hours post-infection with CR6 compared to CR6Δcasp or CW3 ( Fig 2A–2C ) . There were no significant differences in MNV polymerase ( NS7 ) detected at 18–22 hours , but a trend toward more NS7 at 10–14 hours post-infection with strain CR6 compared to CR6Δcasp or CW3 . These data suggested that differences in caspase 3 and PARP cleavage were not due to increased peak replication in CR6-infected cultures , but may be related to earlier differences in replication kinetics ( Fig 2B ) . Together with data in Fig 1 , these data suggest that cleavage of CR6 NS1/2 ( but not CW3 NS1/2 ) potentiates caspase activity . To test whether our panel of MNV strains differed in induction of apoptotic cell death , we quantified surface presentation of phosphatidylserine ( PS , Annexin V staining ) and permeability to the LIVE/DEAD viability dye by flow cytometry following infection of BV2 cells . We found that CR6 infection resulted in two-fold more apoptotic cells ( Alive Annexin V+ ) than CW3 or CR6Δcasp ( Fig 2D–2E ) . In contrast to CR6 , most CR6-VP1CW3 infected cells lost membrane integrity and were stained with the LIVE/DEAD dye in addition to Annexin V ( Fig 2D–2F ) , which is consistent with our recent findings that VP1CW3 drives lytic cell death and inflammatory cytokine production [49] . Even so , cells infected with CR6-VP1CW3 had 30–50% more total Annexin V staining than cells infected with the corresponding mutant strain CR6Δcasp-VP1CW3 , indicating that CR6 NS1/2 cleavage promotes cell death in conjunction with VP1CW3-dependent lysis ( Fig 2F ) . Together , these data suggest that CR6 NS1/2 cleavage by apoptotic caspases reciprocally promotes cleavage of caspase substrates and consequential cell death . The overall abundance of full-length NS1/2 and NS7 on western blots was similar between WT and mutant strains , indicating that viral protein production did not require NS1/2 cleavage ( Figs 1C , 1D and 2A ) . However , many additional steps beyond protein production are required for assembly of infectious virus . So , we compared production of plaque forming units ( PFU ) over time from myeloid cells infected with our panel of MNV strains ( Fig 1B ) to assess whether production of infectious virus was dependent on CR6 NS1/2 cleavage . Mutation of NS1/2 cleavage sites did not significantly alter MNV replication kinetics or peak titers in BV2 cells following infection at a high multiplicity of infection ( MOI , one PFU/cell ) ( Fig 3A ) . Following infection of BV2 cells at a low MOI ( 0 . 01 PFU/cell ) , there were modest differences in early 10–18 hour titer , but all strains reached similar peak titers by 36 hours post-infection ( Fig 3A ) . The similar peak titers are consistent with our ability to obtain high titer viral stocks of NS1/2 cleavage mutants ( generated by low MOI infection of BV2 cells ) and indicate that NS1/2 cleavage is not required for MNV replication in these cells . To test the requirement of NS1/2 cleavage for replication in primary myeloid cells , we generated bone-marrow dendritic cells ( BMDCs ) and infected them with our panel of MNV strains . Similar to BV2 cells , replication of CR6 in BMDCs was minimally impacted by mutation of NS1/2 cleavage sites ( Fig 3B and 3C ) . However , CW3 and CR6-VP1CW3 reached higher peak titer in WT BMDCs compared to other strains tested , suggesting that VP1CW3 promotes BMDC replication . CR6Δcasp-VP1CW3 grew similarly to CR6 but grew to lower peak titers than CR6-VP1CW3 ( Fig 3B ) , suggesting that NS1/2 caspase cleavage may have a VP1CW3-dependent role in BMDCs , possibly related to the increase in lytic cell death observed for CR6-VP1CW3 ( Fig 2D–2F , [49] ) . In parallel with WT BMDCs , we tested the role of STAT1-dependent IFN responses by comparing growth of our MNV strains in Stat1-/- BMDCs . All MNV strains reached higher peak titers in Stat1-/- BMDCs compared to WT BMDCs ( compare Fig 3B and 3C ) , as expected from prior studies of STAT1-dependent immunity to MNV [24] . However , there were no statistically significant differences in growth that could be attributed to NS1/2 cleavage ( Fig 3C ) . Overall , these data indicate that NS1/2 cleavage is not strictly required for replication of MNV in myeloid cells , but may promote BMDC replication in a VP1CW3-dependent manner . Stat1-/- mice exhibit a profound inability to control systemic replication and succumb to infection in a VP1CW3-dependent manner [20–22 , 32 , 33] . To test whether NS1/2 cleavage mutations impair this pathological MNV replication , we orally inoculated Stat1-/- mice with CR6-VP1CW3 or CR6Δcasp-VP1CW3 and monitored animals daily for signs of distress . Infection with CW3 or CR6-VP1CW3 resulted in 100% lethality with similar kinetics ( Fig 4A ) . As expected , CR6-infected controls remained healthy due to lack of VP1CW3-facilitated systemic spread [20] . These in vivo pathogenesis data suggest that NS1/2 cleavage is not required for lethal pathology resulting from uncontrolled , systemic infection of myeloid cells , consistent with no NS1/2 cleavage requirement for in vitro replication in myeloid cells . To directly quantify the effect of NS1/2 cleavage on acute replication and tissue tropism , we inoculated WT mice with CR6 , CR6Δcasp , CR6-VP1CW3 , or CR6Δcasp-VP1CW3 and analyzed tissue and stool titers at three days post-infection ( Fig 4B ) . As expected from our prior studies , CR6 and CR6-VP1CW3 were detected on day three in the mesenteric lymph node ( MLN ) , ileum , and colon . We also detected infectious virus shed in the feces of mice infected with CR6 and CR6-VP1CW3 , consistent with replication in intestinal tissue . In contrast , in mice infected with NS1/2 cleavage mutant strains CR6Δcasp or CR6Δcasp-VP1CW3 , infectious virus was rarely detected in intestinal tissues , or being shed in the feces . However , despite severely compromised replication in ileum ( 10-fold ) and colon ( 1000-fold ) , splenic titers of CR6Δcasp-VP1CW3 were not significantly different from CR6-VP1CW3 ( Fig 4B ) . This indicates that NS1/2 cleavage is not required for systemic spread , consistent with Stat1-/- lethality data ( Fig 4A ) . Overall , these acute titer data demonstrate that caspase cleavage of NS1/2 is specifically required for promoting early infection of intestinal tissue , the eventual site of persistence . Due to limited early detection of NS1/2 cleavage mutants in the intestinal tissues , we predicted that NS1/2 cleavage would be required for intestinal persistence . Indeed , most mice infected with CR6Δcasp or CR6Δcasp-VP1CW3 had undetectable viral genomes in tissues at 21 days post-infection whereas CR6 and CR6-VP1CW3 were present in the ileum , colon , and stool ( Fig 4C ) . Altogether , these findings support a role for CR6 NS1/2 cleavage in establishing early infection of the intestine and subsequent persistence . IFN-λ plays a critical role in protection against persistent MNV infection of the intestine [35] , so we sought to determine whether CR6Δcasp persistence was restored in the absence of IFN-λ signaling . We infected WT or Ifnlr1-/- mice with CR6 or CR6Δcasp and monitored shedding in the stool over time ( Fig 5A and 5B ) , and persistence in the colon at 21 days post-infection ( Fig 5C and 5D ) . Additionally , to determine whether an early barrier to MNV infection could be overcome with increased infectious dose , we included an experimental group inoculated with a 10-fold higher dose of CR6Δcasp ( 1e7 PFU ) . MNV was shed in the stool of all WT mice infected with CR6 beginning on day three and continuing until the experimental endpoint , day 21 . Consistent with the known role of IFN-λ , Ifnlr1-/- mice infected with CR6 shed greater than 10-fold more virus relative to WT mice ( Fig 5A and 5B ) , and CR6 was detected in colon tissue at levels commensurate with fecal shedding ( Fig 5C and 5D ) . In contrast , we were unable to detect shedding or colonic persistence of CR6Δcasp at any time from WT or Ifnlr1-/- mice ( Fig 5A and 5B ) . These data indicate that CR6 NS1/2 cleavage promotes persistent infection via a novel mechanism separate from IFN antagonism . Our preceding data demonstrated that disrupting CR6 NS1/2 cleavage prevented early infection of intestinal tissues and consequent persistent shedding in feces of either WT or Ifnlr1-/- mice ( Figs 4 and 5 ) . Because early infection of IECs is necessary for establishing persistent infection , we hypothesized that CR6 NS1/2 cleavage promoted early IEC tropism . To test this hypothesis , we infected WT or Ifnlr1-/- mice with our panel of MNV strains ( Fig 1B ) and quantified IEC infection at three days post-infection by flow cytometry . We pooled IECs isolated from proximal colon , cecum , and distal small intestine ( devoid of Peyer’s patches ) , and stained intracellularly for MNV NS1/2 ( Fig 6A–6C ) . We rarely detected infected IECs in WT mice at this early timepoint , indicating that acute infection of IECs in our WT mice is below the limit of detection for this assay ( Fig 6B ) . However , IEC infection was increased in Ifnlr1-/- mice relative to WT mice , indicating that IFN-λ limits early establishment of infection in the IEC reservoir ( Fig 6A and 6C ) . CR6 and CR6-VP1CW3 were detected in an average of 40 IECs per million in Ifnlr1-/- mice , but we did not detect significant infection of IECs by NS1/2 cleavage mutants ( Fig 6C ) . In parallel , to determine whether MNV strains that were not detected in IECs had otherwise initiated an infection , we quantified MNV in Peyer’s patches by the more sensitive qPCR method . We detected viral RNA in Peyer’s patches for the majority of CR6-infected mice , ~50% of CR6Δcasp-infected mice , and all mice infected with CR6-VP1CW3 or CR6Δcasp-VP1CW3 ( Fig 6D and 6E ) . The CR6Δcasp-VP1CW3 genomes from Peyer’s patches were examined by sanger sequencing and retained caspase cleavage mutations , which rules out reversion of these mutations as a prerequisite for robust Peyer’s patch infection . The relationship between Peyer’s patch replication for MNV strains in Ifnlr1-/- mice was similar to WT mice ( Fig 6D and 6E ) , consistent with a minimal role of IFN-λ in non-epithelial cells within the Peyer’s patches . These data indicate that the CR6-VP1CW3 strain , which robustly infects Peyer’s patches , does not strictly require NS1/2 cleavage for initial infection of non-epithelial cells therein . Therefore , CR6 NS1/2 cleavage promotes early infection of IECs independent of the ability to initiate replication in Peyer’s patches . In complementary experiments , we infected mice with our panel of MNV strains and detected MNV genomes and EpCAM transcripts in small intestine sections via RNAscope at 48–72 hours post-infection ( Fig 7 ) . We included groups of WT and Ifnar1 x Ifnlr1 double knockout ( DKO ) mice to compare IFN-restricted and unrestricted tropism , respectively . We reproducibly detected CR6 infection of IECs in WT mice at an average of one double positive ( MNV and EpCAM ) cell per cm of tissue length , whereas CR6-VP1CW3 was not consistently detected in IECs of WT mice and NS1/2 cleavage mutants were not detected at all ( Fig 7A and 7B ) . In DKO mice , CR6 and CR6-VP1CW3 were detected with increased frequency relative to WT mice at seven and one DP cells per cm tissue length , respectively ( Fig 7C and 7D ) . In contrast , infection of IECs by NS1/2 cleavage mutants remained below the limit of detection in DKO mice ( Fig 7E ) . Infection of non-epithelial cells was observed inconsistently for all strains in WT mice and did not correlate with IEC infection; in DKO mice , CR6-VP1CW3 and CR6Δcasp-VP1CW3 robustly infected non-epithelial cells , whereas CR6 infection of non-epithelial cells remained inconsistent and CR6Δcasp was not detected ( Fig 7F ) . Taken together , these data demonstrate an IFN-independent role of CR6 NS1/2 cleavage in promoting early infection of IECs , the cellular reservoir of MNV persistence . The infected IECs we observed in vivo on day three may be a result of viral amplification and spread rather than initial infection . To directly test the role of CR6 NS1/2 cleavage on initial infection of IECs , we used a previously characterized CD300lf-transduced IEC cell line ( M2C-CD300lf , hereafter M2C ) , which can be infected by CW3 and CR6 [29] . We infected M2C cells with our panel of persistent and non-persistent strains ( Fig 1B ) and quantified replication over time by plaque assay ( Fig 8A ) . Following infection at an MOI of one , persistent strains ( CR6 and CR6-VP1CW3 ) produced significantly more infectious progeny at 10 hours post-infection compared to non-persistent strains ( CW3 , CR6Δcasp , or CR6Δcasp-VP1CW3 ) . However , between 18–48 hours post-infection all strains reached similar peak titers with at most a two-fold difference between persistent and non-persistent strains ( Fig 8A ) . Likewise , infection of M2C cells at a low MOI ( 0 . 01 PFU/cell ) resulted in higher titers for persistent strains at 10 hours post-infection , but all strains reached similar titers by 18 hours post-infection ( Fig 8B ) . However , between 24–72 hours following low-MOI infection , persistent strains proceeded to replicate robustly and reach similar peak titer as high MOI infection , whereas non-persistent strains replicated more slowly with 10-fold lower titer between 24–72 hours ( Fig 8B ) . These findings differ from our earlier analysis in myeloid cells , where higher peak titers did not correlate with persistence phenotype ( Fig 3 ) , and suggest specific roles of CR6 NS1/2 cleavage in IEC infection . To further characterize infection in IECs , we quantified infected cells by immunofluorescence staining for MNV NS7 following infection at MOI of one . Unexpectedly , only ~10% of M2C cells were NS7-positive between 14 and 24 hours post-infection , suggesting that MNV does not efficiently initiate replication in these cells . Strains CW3 , CR6 , or CR6Δcasp all had a similarly low percentage of initially infected cells at 14 hours post-infection ( Fig 8C and 8D ) . This equal infection suggests that early differences in titer at 10 hours ( Fig 8A and 8B ) are due to different rates of virus production per cell . Although the initial proportion of infected cells was similar between strains , the percentage of CR6-infected cells increased from 10 to 30% by 32 hours whereas the percentage of CW3 or CR6Δcasp infected cells did not significantly increase ( Fig 8C and 8D ) . Furthermore , visual inspection of CR6-infected M2C cell monolayers at 48–72 hours post-infection revealed patches of minimal cellularity whereas CW3 or CR6Δcasp infected monolayers remained intact ( Fig 8E ) . In sum , these data indicate that non-persistent strains are similarly able to initially infect IECs , but spread among IEC monolayers is facilitated by cleavage of CR6 NS1/2 . We next sought to identify the cellular response altered by NS1/2 cleavage that may account for differential spread among IEC monolayers and establishment of IEC infection in vivo . Analyses in myeloid cells indicated that NS1/2 cleavage promoted apoptotic cell death ( Fig 2 ) . To test whether IEC apoptosis was also promoted by NS1/2 cleavage , we infected M2C monolayers with CW3 , CR6 , or CR6Δcasp and characterized cleavage of NS1/2 , caspase 3 , and PARP by western blot . In contrast to the robust caspase 3 cleavage seen in infected BV2 cells , we detected less than 10% cleavage of caspase 3 in M2C lysates from infection with any strain at any timepoint and were infrequently able to detect NS1/2 cleavage at any time post-infection ( Fig 9A ) . This reduced cleavage is consistent with the relatively low frequency of infected M2C cells and loss of cellularity in CR6-infected cultures ( Fig 8C and 8E ) . Additionally , staurosporine treatment of M2C resulted in visible loss of cellularity , but less cleavage of caspase 3 ( 25% vs . 60% ) and PARP ( 10% vs . 95% ) compared to BV2 cells ( Figs 9A–9E and 2A–2E ) . Despite the relatively low amount of caspase cleavage , M2C monolayers infected with CR6 had three-fold more caspase 3 and PARP cleavage than those infected with CW3 or CR6Δcasp ( Fig 9A–9C ) with no significant difference in viral protein production ( NS7 , Fig 9A and 9B ) . To determine if differences in caspase activity of infected M2C cells correlated with a different proportion of apoptotic cells , we quantified Annexin V staining and cell lysis by flow cytometry . At 36 hours post-infection CR6 triggered a three-fold increase in apoptotic cells ( Alive Annexin V+ ) whereas CW3 or CR6Δcasp triggered only a two-fold increase over background ( Fig 9D and 9E ) . Altogether , these data demonstrate that CR6 NS1/2 cleavage promotes apoptotic caspase activity and cell death of myeloid ( BV2 ) and IEC ( M2C ) cell lines . MNV-induced death occurred in a relatively low proportion of M2C cells over an extended time course with strain-dependent loss of cellularity ( Fig 8E ) . To more comprehensively compare strain-dependent differences in this context , we used the incucyte imaging system to quantify confluency , Annexin V staining , and cytotox viability dye staining over time . Control M2C monolayers treated with staurosporine died rapidly with loss of confluency and increasing Annexin V and cytotox staining between 4–12 hours post-treatment ( Fig 10 ) . CR6 infected monolayers also lost confluency over the course of the experiment whereas CW3 or CR6Δcasp infected monolayers remained intact ( Fig 10A ) . Monolayers infected with all MNV strains became positive for Annexin V and cytotox with similar kinetics , beginning at 12 hours post-infection and increased to a maximum at 48 hours ( Fig 9A and 9B , S1–S5 Video ) . However , CR6 infection resulted in more overall Annexin V and cytotox staining over the course of the experiment than CW3 or CR6Δcasp infection , as quantified by area-under-curve ( AUC ) analyses ( Fig 9C ) . Prior to 24 hours post-infection , increased death of CR6-infected M2C cultures cannot be attributed to an increased proportion of infected cells ( Figs 8C and 10A inset ) but may be related to increased early virus production per cell ( Fig 8A and 8B ) . At timepoints beyond 24 hours post-infection , the more effective spread of CR6 among these cultures ( Fig 8C–8E ) amplifies the magnitude of CR6-induced death . Although Annexin V positive M2C cells with intact membranes were detected by flow cytometry ( Fig 9D and 9E ) , these live imaging data suggest that there is a rapid progression from apoptosis to lytic death in virally infected cells . Overall , these analyses of cell death suggest that CR6 NS1/2 cleavage promotes increased apoptotic and lytic death in IECs . To determine whether caspase activity was required for the increased epithelial spread , we infected M2C-CD300lf monolayers with addition of caspase inhibitor ZVAD-fmk or vehicle control ( DMSO ) at 14 hours post-infection and quantified infected cells by immunofluorescence staining for MNV NS7 . As seen in Fig 8 , M2C cells infected with strains CW3 , CR6 , or CR6Δcasp were similarly ~10% NS7-positive at 14 hours post-infection ( Fig 11A ) . The proportion of CW3 or CR6Δcasp NS7-positive cells did not increase over the 32-hour experiment , regardless of ZVAD treatment . However , CR6 NS7-positive cells increased to an average of 25% at 32 hours post-infection , and ZVAD treatment delayed this spread and resulted in significantly fewer NS7-positive cells at 24 hours post-infection ( Fig 11A ) . Furthermore , visual inspection of CR6-infected M2C cell monolayers at 72 hours post-infection revealed a partial restoration of monolayer integrity when CR6-infected cells had been treated with ZVAD compared to when cells had been treated with DMSO control ( Fig 11B and 11C ) . Therefore , caspase activity promotes spread among IEC monolayers . Taken together with our in vivo data , we propose that NS1/2 is a viral sensor of caspase activity whose cleavage promotes execution of cell death programs during viral egress , lateral spread among intestinal epithelial cells , and persistent shedding ( Fig 11D ) .
Herein , we demonstrate that caspase-mediated cleavage of the nonstructural protein , NS1/2 , is critical for persistent MNV infection of the intestine . Importantly , we find that NS1/2 of non-persistent strain CW3 and persistent strain CR6 are similarly cleaved by caspases , suggesting that cleavage is required in conjunction with particular features unique to CR6 NS1 , such as the conformational state of a previously characterized structured domain [23] . Our in vitro and in vivo analyses demonstrate that NS1/2 cleavage is not strictly required for viral replication in myeloid cells but is important for promoting infection in IECs , the cellular reservoir of persistence . Furthermore , our data indicate that CR6 NS1/2 cleavage potentiates apoptosis and lytic death , suggesting that regulation of cell death is a critical aspect of establishing early and consequential persistent infection of IECs . NS1/2 was not previously known to play a role in cell death during MNV infection , but is required for viral replication beginning at the earliest stages of replication complex assembly . Norovirus replication occurs in the cytoplasm on cellular membranes with a variety of organelle markers [15] , and occurs near membranous vesicles [15 , 24] . MNV NS1/2 localizes to the endoplasmic reticulum ( ER ) [16 , 17] and promotes replication via membrane re-organization and through an interaction with the vesicle-associated membrane protein , VAPA [18 , 52] . Similarly , HNoV NS1/2 interacts with VAPA , localizes to vesicles , and disrupts vesicle trafficking when ectopically expressed [18] . Our data indicate that this early and essential role of NS1/2 remains intact when the caspase cleavage sites are mutated , and viral replication occurs normally in myeloid cells ( Fig 2 ) . NS1/2 cleavage only occurs after viral replication has peaked indicating that its role in promoting death , IEC infection , and persistence involves relatively late events in the viral life cycle . The last step of the MNV replication cycle is egress , which occurs concurrently with induction of apoptosis . Therefore , NS1/2 cleavage and associated apoptotic death may be important for regulating release of viral progeny . How noroviruses are released from infected cells is not well understood , but infection of macrophages , DCs , or IECs does result in cell lysis ( [24] , Figs 2 , 9 and 10 ) and non-enveloped viruses generally rely on cell lysis to release newly assembled virions . Apoptotic processes may alter egress by reducing lytic virion release and favoring retention of virions within cellular membranes . Indeed , a number of non-enveloped viruses utilize vesicle-associated egress as a means of evasion from immune detection , and vesicles with exposed phosphatidylserine allow for phagocytic uptake of novel target cells that may not express the viral receptor [53 , 54] . In addition to facilitating phagocytic uptake , membranes with PS can inhibit IFN responses via activation of Tyro3/Axl/Mer ( TAM ) receptor tyrosine kinases on target cells [55] , which would allow more efficient viral replication . Thus , vesicular egress has the potential to dramatically change the outcome of infection via biophysical and immunological mechanisms . Intriguingly , recent studies by the Altan-Bonnet group indicate that MNV can be released from intact cells in PS-positive membranes [56] but the proportion of total virions released in this manner remains unknown . Therefore , it will be important in future work to consider the role of NS1/2 cleavage on regulation of this newly-described membranous egress and the role of viral association with membranes on spread among IECs . The MNV receptor CD300lf is specifically expressed on MNV target cells , including myeloid cells and a specialized IEC subtype , tuft cells [26] . Tuft cells are a rare cell type in the intestinal epithelium and are dispersed among non-tuft , CD300lf-negative IECs [57] . Our analyses often found intestinal villi with multiple , adjacent epithelial cells infected ( Fig 7C ) , raising the possibility of direct MNV spread to neighboring IECs , which could be mediated by cell death or apoptotic egress and regulated by NS1/2 cleavage . Our in vitro studies of infection in IEC monolayers support this hypothesis , and the dispersed nature of CD300lf-expressing tuft cells poses a strong selective pressure for efficient spread across receptor-negative stretches of the intestinal epithelium ( Fig 11 ) . Recent development of in vitro methods for differentiating tuft cells in primary IEC cultures [58] will enable future experiments to characterize spread in an IEC monolayer more representative of the in vivo scenario . Regular turnover of the intestinal epithelium results in a short lifespan for differentiated epithelial cells , which undergo a specialized form of apoptotic cell death ( anoikis ) triggered as the cell detaches from the basal membrane and is shed into the lumen [59] . Specific Bcl-2 family proteins are critical players in anoikis [60 , 61] , and display increased expression as IECs transit up the villus [61] . It is possible cleavage of NS1/2 ( and perhaps NS1 directly ) interferes with Bcl-2 family proteins as a means of altering the kinetics of the highly-regulated pathways that control apoptosis within differentiated IECs . Interestingly , similar to NS1/2 cleavage promoting apoptosis during infection , some Bcl-2 proteins ( e . g . Bid ) are also regulated via cleavage , in which the truncated version is the active , pro-apoptotic form of the protein [62] . An important future direction will be to examine whether NS1/2 interactions with the apoptotic machinery plays a role in maintaining IEC infection . As mentioned above , PS receptor activation associated with apoptotic death could limit host responses . In contrast , virus production that results from cell lysis would promote inflammatory signals to alert the host of an infection . In addition to professional phagocytes , phagocytosis by non-professional phagocytes ( e . g . epithelial cells ) is critical for efficient clearance of apoptotic cells , and for limiting inflammation in tissues with high cellular turnover [63] . Specifically , in a mouse model of colonic injury , phagocytic uptake of apoptotic cells by IECs was sufficient for limiting inflammation in the colon [64] , and phagocytosis mediated by epithelial cells is similarly critical for reducing inflammation in lung tissue [65] and mammary glands [66] . Thus , intestinal infection that promotes apoptotic death and the phagocytic uptake by IECs would limit host detection and provide a non-inflammatory environment that would allow more efficient infection of IECs and benefit MNV persistence . Notably , we have not observed rescue of CR6Δcasp MNV in animals deficient for the IFN-λ receptor , a key mediator of IEC antiviral responses ( Fig 5 ) . However , a concurrent study performed at Washington University has found that CR6Δcasp can be rescued in IFNLR-deficient mice following high dose ( 107 PFU ) inoculation [50] . Additionally , this study by Lee et al . shows that NS1 can be released from infected cells in vitro and immunization with NS1 is protective which strongly suggests an extracellular function [50] . The reason for a difference in IFNLR-dependency between these two studies remains unknown , but may be related to differences in MNV susceptibility between mouse vivaria . Specifically , persistently infected mice at Washington University shed one to two orders of magnitude more MNV genomes compared to mice at OHSU ( Fig 5 in [50] compared to Fig 5 in this study ) , which may overcome the IFNLR-independent barrier to infection we have observed . These studies together raise the possibility that NS1/2 promotes infection of IECs through multiple non-redundant functions . There may be an IFNLR-antagonist function of the cell-free NS1 and a separate function within infected cells to promote apoptosis . Future work to identify molecular interactions with NS1/2 will be required to clarify the relationship between NS1/2 cleavage , IFNLR , NS1 secretion , and cell death . Our discovery of NS1/2 as an “apoptosis sensor” that reciprocally regulates apoptotic death and facilitates epithelial spread ( Fig 11 ) has opened the door to such mechanistic studies and expanded our understanding of the requirements for viral persistence in the intestine .
All mice were maintained in specific-pathogen-free barrier facilities under animal protocols approved by the institutional animal care and use committee at Oregon Health & Science University ( protocol # IP00000228 ) according to standards set forth in the Animal Welfare Act . BV2 cells ( gift from Herbert Virgin IV , Washington University , St . Louis , MO ) , a mouse microglial cell line [67] , were maintained in DMEM ( Life Technologies , ThermoFisher , Carslbad , CA ) with 5% fetal bovine serum ( FBS ) ( VWR Seradigm , Radnor , PA ) , 1X Penicillin/Streptomycin/Glutamine ( P/S/G ) solution , and 10mM HEPES . M2C-CD300lf cells , a murine colonic epithelial cell line transduced with the MNV receptor , CD300lf [29] , were maintained in DMEM with 10% FBS , 1X P/S/G , and 10mM HEPES . For bone-marrow derived dendritic cells ( BMDCs ) , bone marrow was isolated from the long bones of each hind leg of either a WT or Stat1-/- mouse and suspended in RPMI ( Life Technologies ) . Red blood cells were lysed using red blood cell lysis solution ( Sigma , St Louis , MO ) and remaining bone marrow cells were cultured in RPMI with 10% FBS , 1X P/S/G solution , 10mM HEPES , 1X Sodium Pyruvate , 1X non-essential amino acids , and 20ng/mL GM-CSF obtained from supernatants of GM-CSF-secreting cells ( J558L , [68] ) . After seven days of culture ( 37C , 5% CO2 ) , non-adherent cells were collected , infected in suspension with indicated MNV strains ( MOI = 1 ) , and plated without GM-CSF for the indicated time . For indicated experiments , ZVAD-fmk ( Cell Signaling Technology ) was re-suspended in DMSO and added to cells at 70 μM . As a positive control for apoptosis assays , cells were treated with 1uM staurosporine ( SS ) ( CST ) for 3 . 5h , at 37C . CR6Δcasp MNV was generated using site directed mutagenesis as previously described [20] with the following primers: CR6 D121G , 5’- GCCTAAGGAAGATAAAGCCGGTGCGCCCTCCCATGCG and CR6 D131G , 5’-TGCGGAGGACGCCATGGGTGCAAGGGAGCCCATAATTGG . The targeted residue is underlined with the mutation in bold . Correct mutations were verified via sanger sequence analysis . MNV stocks were generated from plasmids , as described [22] . Briefly , plasmids encoding viral genomes of parental CW3 ( Murine norovirus GV/MNV-1/2002/USA , clone CW3 , GenBank accession no . EF014462 . 1 ) , parental CR6 ( Murine norovirus GV/CR6/2005/USA , GenBank accession no . JQ237823 ) , CR6-VP1CW3 [20 , 22] and the two NS1/2Δcasp viral genomes generated herein , were individually transfected into 293T cells ( ATCC #CRL-3216 ) . Forty-eight hours post-transfection , cells were frozen , thawed , vortexed , and spun at 3 , 000xg to remove large debris . Supernatants were expanded by two passages at MOI <0 . 05 in BV2 cells , and p2 virus supernatant was filtered ( 0 . 2 μm ) , concentrated by ultra-centrifugation through a 30% sucrose cushion , and titered via plaque assay . Plaque assays were performed in BV2 cells , similar to previously described methods [20] . Briefly , BV2 cells were grown in 6well plates , infected with serial dilutions of each sample ( 500μl per well , 1h , RT , on a rocking platform ) , after which the inoculum was removed and cells were overlaid with 1% methylcellulose in complete DMEM . At two to three days post-overlay , cells were fixed and stained with 20% EtOH / 0 . 1% crystal violet . M2C-CD300lf cells were grown on coverslips , fixed with 3 . 7% paraformaldehyde/PBS for 15 minutes at room temperature , and permeabilized in PBS with 0 . 1% triton X-100 and 3% normal goat serum for one hour at room temperature . MNV infection was observed by staining with anti-MNV ProPol ( NS6/7 ) ( a gift from Dr . Kim Green , NIH ) followed by anti-Rabbit IgG-Alexa 647 ( Molecular Probes ) . Nuclei were stained with DAPI . Coverslips were mounted on slides with Vectashield ( Vector Laboratories , Burlingame , CA ) , and imaged using a Zeiss ApoTome2 on an Axio Imager , with a Zeiss AxioCam 506 ( Zeiss , Jena , Germany ) . Cells were seeded in a 96-well plate , allowed to form a confluent monolayer ( ~18-24h ) , and subsequently infected with MNV or UV-inactivated MNV ( MOI = 0 . 5 ) . Mock-infected cells , and cells treated with 1μM staurosporine were used for negative and positive controls , respectively . Apoptosis and cell permeabilization were assessed via addition of Incucyte Annexin V Green and Cytox Red reagents ( EssenBioscience , Ann Arbor , MI ) , respectively , following manufacturer protocols . Phase contrast and fluorescent images were collected with a 10x objective using the IncuCyte Dual Dolor ZOOM ( EssenBioscience ) imaging system at 30minute intervals over a 72h time course , maintained at 37°C and 5% CO2 . Images were analyzed using Incucyte 2016A software ( EssenBioscience ) after Top-Hat background subtraction , with a 20μm radius . Stat1-/- ( Stat1tm1Dlv ) mice were originally obtained from the Jackson Laboratories ( stock #012606 ) . Ifnlr1-/- ( generated from Ifnlr1tm1a ( EUCOMM ) Wtsi ) and Ifnar1-/- ( Ifnar1tm1Agt ) were originally obtained from Washington University in St . Louis . Ifnlr1-/- and Ifnar1-/- were bred to generate Ifnlr1-/-/Ifnar1-/- mice ( DKO ) and Ifnlr1+/-/Ifnar1+/- ( Dhet ) . Dhet and DKO crossbreeding yielded littermate mice deficient in neither ( Dhet ) , both ( DKO ) , or a single ( Ifnlr1+/-/Ifnar1-/- or Ifnlr1-/-/Ifnar1+/- ) IFN receptor gene ( s ) ; these littermates were used for experiments to control for variability between litters . All mice ( 7-9weeks old ) were perorally infected with 1e6 PFU in a 25μL volume administered by pipet , with the exception of experiments in Fig 5 where 1e6 or 1e7 PFU ( as indicated in figure ) was administered by oral gavage in a 100μL volume . An equal proportion of males and females were maintained among experimental groups to mitigate results due to any unidentified sex-dependent variables . Same-sex mice , infected with the same MNV strain , were often co-housed for acute infection analyses ( less than three days ) , but singly-housed for extended experiments to prevent continued transmission between animals . Tissue samples , or a single fecal pellet , were collected in 2ml cryovials with 1mm zirconia/silica beads ( Biospec , Bartlesville , OK ) , frozen in a dry ice/EtOH bath and stored at -80C . For plaque assay or RT-qPCR analyses , either 1ml DMEM or 1ml RiboZol ( VWR ) was added , respectively , and tissues were homogenized via bead-beating ( 1min , RT ) prior to continued analysis . RNA from tissues was extracted via RiboZol ( VWR ) protocols , and RNA from cells was extracted with the ZR Viral RNA kit ( Zymoresearch , Irvine , CA ) . DNA contamination was removed with Turbo DNase ( ThermoFisher ) , and 1ug of RNA was used to generate cDNA with the ImpromII reverse transcriptase ( Promega , Madison , WI ) . Quantitative PCR was performed using PerfeCTa qPCR FastMix II ( QuantaBio , Beverly , MA ) , and the following oligo/probes were used for detection: MNV genomic RNA—Forward primer CACGCCACCGATCTGTTCTG , Reverse primer GCGCTGCGCCATCACTC , and Probe TGCGCTTTGGAACAATGG; RPS29—Forward primer GCAAATACGGGCTGAACATG , Reverse primer GTCCAACTTAATG AAGCCTATGTC , and Probe CCTTCGCGTACTGCCGGAAGC . BV2 cells were grown and infected in 12well plates ( 2 e5 cells/well ) . Cells were lifted with trypsin/0 . 25% EDTA ( Gibco , ThermoFisher ) , washed with 1X cold PBS , resuspended in 100 μL NP40 lysis buffer ( Alfo Aesar , Havervill , MA ) with protease inhibitor cocktail ( Sigma , St Louis , MO ) , incubated on ice ( 15min with intermittent mixing ) , cleared via centrifugation ( 1min , 4C , full-speed ) , and diluted 1:1 with 2x SDS loading buffer ( 0 . 125 M Tris [pH 6 . 8] , 4% SDS , 20% glycerol , 0 . 004% bromophenol blue , 10% beta-mercaptoethanol ) . Lysates were run on Novex Tris-Glycine gels ( ThermoFisher ) , transferred to 0 . 2 μm PVDF ( BioRad , Hercules , CA ) , blocked with 5% nonfat dry milk , and probed with the following antibodies ( Ab ) : caspase 3 ( CST ) , PARP ( CST ) , beta-Actin pAb ( Invitrogen ) , MNV NS1/2 ( a gift from Dr . Vernon Ward , U . of Otago , Australia ) , or MNV ProPol ( a gift form Dr . Kim Green , NIH ) . Signal was detected using goat , anti-rabbit IgG-HRP ( Jackson ImmunoResearch ) , developed with ECL substrate ( Pierce , ThermoFisher ) , and imaged with an ImageQuant LAS4000 ( GE Healthcare , Little Chalfont , UK ) . Epithelial fractions were prepared by non-enzymatic striping as previously described [32] . Mice were euthanized and distal small intestine , cecum , and colon ( regions of highest MNV replication ) were isolated . Peyer’s patches were removed from small intestine and intestinal tissues were incubated in stripping buffer ( 10% bovine calf serum , 15 mM HEPES , 5 mM EDTA , 5 mM dithiothreitol [DTT] in PBS ) with shaking for 20 min at 37°C . The dissociated cells were collected as the epithelial fraction , consisting predominantly of IECs and directly used for flow cytometry . BV2 cells were trypsinized and washed once with 1X PBS , stained with the amine-reactive viability dye , Zombie Aqua ( Biolegend , San Diego , CA ) ( 20min , on ice ) , washed once with cold 2% FBS/PBS , washed once with cold Annexin V binding buffer ( CST ) , and stained with Annexin V-FITC ( CST ) ( 15min , RT ) . Cells were immediately analyzed via flow cytometry on an LSR Fortessa ( Becton Dickinson [BD] , Franklin Lakes , NJ ) with FACSDiva software ( BD ) , and data was analyzed using FlowJo software ( FlowJo LLC , Ashland , OR ) . IECs isolated as described above were stained with the Zombie Aqua viability dye ( Biolegend ) , Fc receptors were blocked with purified CD16/CD32 pAb ( clone93; Biolegend ) , and cells were probed for surface markers , EpCAM ( clone G8 . 8; Biolegend ) and CD45 ( clone 30-F11; Biolegend ) . Cells were subsequently fixed with 2% paraformaldehyde/PBS ( 10min , RT ) , and blocked/permeablized in 0 . 2% Triton X/PBS with 2% normal goat serum ( 20min , RT ) . Fixed and permeabilized cells were probed for intracellular viral antigen with 2 distinct antibodies: Rabbit polyclonal anti-NS1/2 ( a gift from Dr . Vernon Ward , U . of Otago , Australia ) and mouse monoclonal anti-NS1 ( clone CM79 , a gift from Dr . Ian Clark , U . of Southampton , UK ) . These unconjugated antibodies were detected with anti-Rabbit Alexa Fluor 647 ( Molecular Probes , Eugene , OR ) , and anti-mouse Dylight 405 ( Jackson ImmunoResearch ) , respectively . Cells were analyzed via flow cytometry , and viable cells that were EpCAM+ , CD45- , NS1/2 pAb+ , and NS1 mAb+ were labeled as infected epithelial cells . Ileum was opened longitudinally , pinned apical side up in wax trays , and fixed in 10% neutral-buffered formalin ( Sigma ) ( 18-24h , RT ) , then moved to 70% EtOH . Tissues were embedded in 2% agar ( ThermoFisher ) , the PP was vertically transected , flipped 90 degrees , and re-embedded in 2% agar . Agar blocks were maintained in 70% EtOH prior to being paraffin-embedded . Tissue sections ( 5μM ) were cut and maintained at RT with desiccant until processed . RNA in situ hybridization was performed using the RNAscope Multiplex Fluorescent v2 kit ( Advanced Cell Diagnostics [ACDBio] , Newark , CA ) per protocol guidelines . Probes were designed by ACDBio for detection of CW3 positive strand RNA ( 471891-C1 ) , CR6 positive strand RNA ( 502631-C1 ) , and EpCAM mRNA ( 418151-C2 ) , and signals were amplified and detected via ACDBio protocols using TSA plus technology ( Perkin Elmer , Waltham , MA ) . Slides were mounted with ProLong Gold antifade reagent ( ThermoFisher ) , and imaged using a Zeiss ApoTome2 on an Axio Imager , with a Zeiss AxioCam 506 ( Zeiss , Jena , Germany ) . Data were analyzed with Prism 7 software ( GraphPad Prism Software , La Jolla , CA ) , with specified tests and significance noted in the figure legends . | Human Norovirus infection is highly contagious and the most common cause of acute gastroenteritis . Norovirus can be persistently shed after resolution of symptoms , perpetuating or initiating new outbreaks . Murine norovirus ( MNV ) is also persistently shed , enabling study of host and viral determinants of norovirus pathogenesis . We previously identified a critical role for MNV non-structural protein 1 ( NS1 ) , in persistence . Herein we find that regulation of NS1 by host apoptotic caspases is required for infection of intestinal epithelial cells , but not for extra-intestinal spread . Additionally , we demonstrate that NS1 reciprocally promotes cell death and spread among epithelial cells . These data identify regulation of NS1 by host proteases and suggest that apoptotic death is a determinant of epithelial spread and persistence . | [
"Abstract",
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... | 2019 | Caspase-mediated cleavage of murine norovirus NS1/2 potentiates apoptosis and is required for persistent infection of intestinal epithelial cells |
Gene conversion is the unidirectional transfer of genetic information between orthologous ( allelic ) or paralogous ( nonallelic ) genomic segments . Though a number of studies have examined nucleotide replacements , little is known about length difference mutations produced by gene conversion . Here , we investigate insertions and deletions produced by nonallelic gene conversion in 338 Drosophila and 10 , 149 primate paralogs . Using a direct phylogenetic approach , we identify 179 insertions and 614 deletions in Drosophila paralogs , and 132 insertions and 455 deletions in primate paralogs . Thus , nonallelic gene conversion is strongly deletion-biased in both lineages , with almost 3 . 5 times as many conversion-induced deletions as insertions . In primates , the deletion bias is considerably stronger for long indels and , in both lineages , the per-site rate of gene conversion is orders of magnitudes higher than that of ordinary mutation . Due to this high rate , deletion-biased nonallelic gene conversion plays a key role in genome size evolution , leading to the cooperative shrinkage and eventual disappearance of selectively neutral paralogs .
All genomes contain similar DNA segments . In diploids , such segments can be classified as orthologs or paralogs . Orthologs , or allelic segments , are paired copies located at the same genomic loci on maternal and paternal chromosomes . In contrast , paralogs , or nonallelic segments , are found at different genomic loci and can have any copy number , in which each copy is derived from an ancestral sequence via gene duplication [1] . The sequences of related DNA segments can diverge via ordinary mutation or converge via gene conversion . Ordinary mutation is generally AT-biased for nucleotide replacements [2]–[4] and deletion-biased for length difference mutations [5] . A number of studies have examined nucleotide replacements produced by allelic and nonallelic gene conversion , some of which have uncovered a GC bias [6]–[9] . Here , we explore length difference mutations produced by nonallelic gene conversion . In contrast to orthologs , paralogs have their own independent long-term phylogenies , making it possible to apply a direct phylogenetic approach to study their coevolution by gene conversion ( Figure 1 ) . For this approach , we utilized multiple alignments of pairs of paralogs in two sister species and an outgroup . First , we ascertained all cases in which , at a particular alignment position , there was an ancestral length difference between the paralogs , i . e . , the difference was present in one sister and in the outgroup . We then examined orthologous positions in the other sister and identified those cases for which there was no length difference between paralogs . Elimination of a length difference was due to an insertion if one paralog acquired an additional nucleotide ( s ) at that position , and was due to a deletion if it lost a nucleotide ( s ) at that position . If the event resulted in the paralogs having identical states at the affected position , it was consistent with gene conversion . A benefit of this approach is that it assumes nothing about the process or biases of ordinary mutation , because an ancestral length difference between paralogs can be caused by either an insertion or a deletion . Moreover , only a small proportion of indels identified using this approach were due to either ordinary mutation or sequencing errors ( see Text S1 ) .
Since our approach required that paralogs be present in the genomes of triplets of closely-related species , we chose to study gene conversion in Drosophila and primate lineages , for which whole-genome sequences of multiple close species are available . For Drosophila , we used D . melanogaster and D . simulans as sister species and D . yakuba as an outgroup , and for primates , we used human and chimpanzee as sisters and orangutan as an outgroup . We obtained 338 ( 199 coding ) and 10 , 149 ( 1 , 740 coding ) pairs of paralogs that are present in all three species of Drosophila and primates , respectively ( Figure 2 ) . Of these , 267 are intra-chromosomal in Drosophila , and 5 , 997 are intra-chromosomal in primates . Our phylogenetic analysis revealed that 101 Drosophila paralogs and 400 primate paralogs underwent gene conversion during the evolutionary timeframes considered . A general prediction of nonallelic gene conversion is that a pair of paralogs should be more similar in the genome of the species in which they underwent conversion than in the genomes of the other sister or outgroup . As expected , 95 paralogs in Drosophila , and 385 paralogs in primates display this trend . Within our set of paralogs , we identified 179 insertions and 614 deletions consistent with gene conversion in Drosophila , and 132 insertions and 455 deletions consistent with gene conversion in primates ( Figure 3a ) . Thus , there were ∼3 . 4 times as many deletions as insertions in both lineages , which was highly significant ( p<0 . 0001 ) . In primates , we found that the deletion bias was substantially larger for long than for short indels ( Figure 3b ) . Exclusion of indels that occurred in coding regions , which were rare ( 45 in Drosophila and 27 in primates ) , did not alter the deletion bias in either lineage , implying that selection on coding paralogs did not affect the overall deletion/insertion ratios observed . One concern of our parsimony-based approach is homoplasy , which would cause us to misidentify two ordinary mutation-induced indels of the same type , one in sister 2 and one in the outgroup , as one conversion-consistent indel in sister 1 ( see Figure 1 ) . However , homoplasy is unlikely in our dataset for several reasons . First , in contrast to nucleotide replacements , identical independent indel mutation events are rare [10] , [11] . Though one group did uncover evidence of homoplastic indels [12] , their analysis compared orthologs in very distantly-related species , the closest sisters being human and mouse , which have an average synonymous substitution rate , or Ks , of ∼0 . 77 [13] . In contrast , the Ks between D . melanogaster ( D . simulans ) and D . yakuba is 0 . 23 ( 0 . 21 ) [14] , and the Ks between human ( chimpanzee ) and orangutan is 0 . 03 ( 0 . 03 ) [15] . Thus , there was much less time for multiple independent indel mutations to occur . Second , paralogs in our dataset do not contain any satellite sequences , which are prone to homoplastic mutations [16]–[19] , and are minimally repetitive in general ( 1 . 69% of Drosophila sequences , and 1 . 51% of primate sequences ) . Third , paralogs containing conversion-consistent indels follow the same genomic distribution as the entire set of paralogs ( Figure S1 ) , making it unlikely that spatial variation in mutation rate led to the observed patterns . Finally , most conversion events occurred between noncoding paralogs , which are less likely to be under selection for similar function , and also are not as limited as coding paralogs in the types of indels ( nucleotide content , size ) that can occur . Even if homoplasy did occur , it would much more likely cause misidentifications of conversion-consistent insertions than deletions , leading to downward biases of deletion/insertion ratios . This is because , for one , homplastic events resembling conversion-consistent deletions require two insertions , which have lower mutation rates than insertions . Additionally , these insertions must be identical in sequence . In the case of single nucleotide insertions , there is a ¼ probability of the second insertion being identical to the first , and this probability rapidly decreases with increasing insertion sequence length . We next estimated the rate of nonallelic gene conversion in Drosophila and primates . For primates , we performed a simple calculation . There are 28 , 701 sites at which there was an ancestral length difference between paralogs . Conversion-consistent indels occurred at 587 of these sites , resulting in ∼0 . 02 indels per site . For Drosophila , a more complex estimate was needed . There were 793 conversion-consistent indels that occurred at 960 possible sites , resulting in 0 . 83 indels per site . Due to this high proportion , it was necessary to correct for multiple conversion events per site . If we assume that gene conversion is a Poisson process , like ordinary mutation , the mean number of events per site is −ln ( 1−0 . 83 ) , or ∼1 . 8 . Because the number of events per site was much smaller in primates than in Drosophila , applying this correction to primate conversion events did not alter the original rate estimate . Strong sequence similarity of paralogs is associated with high gene conversion rate [20] . To study this phenomenon , we computed Spearman correlation coefficients between paralog similarities and the number of gene conversion indels identified . In Drosophila , similarity was indeed positively correlated with gene conversion rate ( ρ = 0 . 35; p = 2 . 3×10−11 ) . However , in primates , there was instead a very weak negative relationship between similarity and gene conversion rate ( ρ = −0 . 04; p = 2 . 45×10−5 ) . Though it is possible that gene conversion rate does not increase with similarity in primates , it is more likely that this result is due to properties of our data . In particular , the primates compared in this study are an order of magnitude more closely related than the Drosophila: the Ks between human and chimpanzee is 0 . 01 [15] , whereas the Ks between D . melanogaster and D . simulans is 0 . 11 [14] . Thus , similarities between primate paralogs tend to be higher and have a narrower range , making it difficult to assess the relationship between similarity and gene conversion rate in primates . Additionally , our data do not reflect “invisible” gene conversion events , or those that occurred between two identical sequences , which are likely to be more prevalent in primates due to the higher similarities of paralogs . The absence of such cases may have also affected our calculation in Drosophila , producing an underestimate of the correlation between sequence similarity and nonallelic gene conversion rate . Physical distance between paralogs is also believed to influence gene conversion rate , with paralogs separated by smaller distances hypothesized to undergo faster gene conversion [21] . To study this relationship , we calculated end-to-end distances between pairs of paralogs located on the same chromosomes and computed Spearman coefficients between these distances and numbers of gene conversion indels detected after correcting for divergence between paralogs . In both lineages , there was a very weak positive correlation ( ρ = 0 . 07 for Drosophila , ρ = 0 . 05 for primates ) , which was statistically significant only in primates ( p = 2 . 79×10−5 ) . These findings agree with those of McGrath et al . [22] , who pointed out that negative correlations between distance and gene conversion rate are likely due to the fact that adjacent paralogs tend to be more similar to each other because of their recent ancestry [23] . Though a higher meiotic recombination rate is associated with elevated rates of allelic gene conversion , the relationship between recombination rate and nonallelic gene conversion is unclear . To investigate the potential relationship between these two parameters , we computed Spearman correlation coefficients between mean recombination rates for pairs of paralogs and numbers of gene conversion indels ascertained . Interestingly , in Drosophila , meiotic recombination rate was negatively correlated with nonallelic gene conversion rate ( ρ = −0 . 22; p = 7 . 38×10−5 ) . In contrast , we did not detect a correlation between these parameters in primates ( ρ = 0 . 005; p = 0 . 63 ) . Next , we calculated the indel mutation rate in Drosophila and primates . To do this , we applied a parsimony-based approach to identify indels produced by ordinary mutation in each lineage . In Drosophila , we observed 202 indels produced by ordinary mutation . Of these indels , 18 were insertions and 184 were deletions , resulting in a deletion bias of ∼10∶1 , which is consistent with previous estimates [24] . The target size for such mutations was half the length of all paralogs , which was 104 , 478 nt . However , as with our conversion analysis , we assumed indel mutations did not occur at the ends of sequences . Subtracting 338 positions , the sequence length along which indels could occur was 104 , 140 nt , resulting in ∼1 . 94×10−3 indel mutations per site in Drosophila . In primates , we observed 1 , 095 indels ( 533 insertions and 562 deletions ) within a total sequence length of 2 , 448 , 263 nt , giving a rate of ∼4 . 47×10−4 indel mutations per site in primates . Comparison of the rates of indels produced by nonallelic gene conversion and ordinary mutation revealed that nonallelic gene conversion is ∼927 . 8 times faster in Drosophila and ∼44 . 7 times faster in primates . This rapid deletion-biased process has a significant effect on genome size evolution . To illustrate this hypothesis , let us consider the life cycle of a length difference mutation within two paralogs . First , ordinary mutation introduces an insertion or deletion in one paralog . Then , deletion-biased gene conversion occurs between the paralogs . If the initial mutation was an insertion , it is removed . Otherwise , the deletion is transmitted to the second paralog , i . e . , fixed within the pair of paralogs . In the absence of selection , this process results in the cooperative shrinkage of these paralogous sequence segments . Cooperative shrinkage of paralogs can be quantified by phylogenetic detection of fixed conversion-induced indels ( Figure 4 ) . To perform this analysis , we ascertained all cases for which , ancestrally , two paralogs had identical lengths at a particular site and , in one sister , they acquired matching indels at that position . This condition implies that , in the ancestral lineage of the sister , ordinary mutation produced an indel in one paralog , and that this indel was later copied to the other paralog , or “fixed” , by gene conversion . In Drosophila , we detected 74 fixed insertions , with a total inserted sequence length of 391 nt , and 176 fixed deletions , with a total deleted sequence length of 1 , 660 nt . In primates , we detected four fixed insertions , with a total inserted sequence length of 4 nt , and 24 fixed deletions , with a total deleted sequence length of 438 nt . Thus , in both lineages , fixed deletions were much longer and more frequent than fixed insertions . Subtracting total insertion lengths from total deletion lengths , we arrived at effective deletion lengths of 1 , 269 nt in Drosophila and 434 nt in primates . The total sequence length of all paralogs was 208 , 956 nt in Drosophila and 4 , 916 , 824 nt in primates . Therefore , the shrinkage rate of paralogs by gene conversion is ∼0 . 11 per Ks unit in Drosophila and ∼0 . 015 per Ks unit in primates . This result implies that , in the absence of selection , these paralogs will exponentially shrink and disappear in ∼138 Ks units , or ∼6 , 210 million years , in Drosophila and ∼1 , 021 Ks units , or ∼612 , 600 million years , in primates .
Whole-genome sequences of Drosophila melanogaster , Drosophila simulans , Drosophila yakuba , Homo sapiens ( human ) , Pan troglodytes ( chimpanzee ) , and Pongo pygmaeus ( orangutan ) were downloaded from the UCSC Genome Bioinformatics site at http://genome . ucsc . edu . We used Mega BLAST [25] ( default parameters ) and Bridges [26] ( KM = 13 , FilterDBase = 20 , FilterQuery = 20 , KS = 12 , CoeffMis = 0 . 01 , CoeffGap = 0 . 05 , FlatGap = 10 , MaxDist = 50 , MinWeight = 100 , CoeffMisPost = 0 . 1 , MaxDistPost = 1000 ) to locate unique pairs of similar sequence segments ( both coding and noncoding ) in the genomes of D . melanogaster and H . sapiens . To avoid short repeats , we required that each sequence in a pair was greater than 100 nt long . After examining the output from these methods , we set a cutoff of 78% sequence identity between pairs of paralogs . If both paralogs were located on the same chromosome , we required that they were separated by greater than 100 nt to avoid sequencing or genome mapping errors . We used the BLASTN [27] ( default parameters ) and Mega BLAST ( default parameters ) algorithms to locate orthologs for each paralog in sister and outgroup species , using conserved synteny of 1 , 000 nt on either side of each sequence to ensure that orthologs were correctly assigned . Orthologs obtained via this method were verified using multiple species sequence alignments downloaded from the UCSC Genome Bioinformatics website ( http://genome . ucsc . edu ) . To identify repetitive regions within paralogs , we ran RepeatMasker ( http://www . repeatmasker . org ) with cross_match ( http://www . phrap . org ) on human paralog sequences with a human-specific repeat library , and on D . melanogaster paralog sequences with a Drosophila-specific repeat library . Pairs of paralogs present in all three species of a lineage were aligned with MUSCLE [28] ( default parameters ) , and alignments , particularly at indel positions , were checked by eye to ensure accuracy . Indels ( both conversion-consistent and ordinary mutations ) were removed from the analysis if they had different lengths or were located at either end of an alignment . We also excluded cases in which , at a particular position where an indel occurred in one sister , all other orthologous sequences were not identical . Meiotic recombination rates were obtained from the Drosophila melanogaster recombination rate calculator [29] for D . melanogaster and from the HapMap website at http://www . hapmap . org [30] for human . Statistical significance was determined with binomial sign tests for deletion biases , and paired t-tests for Spearman correlation coefficients . For each test , we used α = 0 . 05 and reported two-tailed probabilities . | Gene conversion is a process whereby a DNA sequence is copied from one segment of the genome ( donor ) to another ( recipient ) , resulting in the replacement , insertion , or deletion of a DNA sequence in the recipient . This exchange is facilitated by the high sequence similarity of the two segments , which is due to their evolutionary relationship . Here , we study insertions and deletions produced by gene conversion between paralogs , segments related by DNA duplication events . By comparing paralog sequences in multiple species of fruit flies and primates , we find that deletions occur more than three times as frequently as insertions . We also discover that the rate of gene conversion between paralogs is quite high . The deletion bias and high rate of this process causes paralogs to shrink cooperatively and eventually be eliminated from the genome . Because of the abundance of paralogs in animal genomes , this phenomenon can lead to a significant reduction in genome size . Therefore , our finding enhances our understanding of the forces that lead to changes in genome size during evolution . | [
"Abstract",
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] | [
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] | 2012 | A Strong Deletion Bias in Nonallelic Gene Conversion |
Animals employ various types of taste receptors to identify and discriminate between different nutritious food chemicals . These macronutrients are thought to fall into 3 major groups: carbohydrates/sugars , proteins/amino acids , and fats . Here , we report that Drosophila larvae exhibit a novel appetitive feeding behavior towards ribose , ribonucleosides , and RNA . We identified members of the gustatory receptor ( Gr ) subfamily 28 ( Gr28 ) , expressed in both external and internal chemosensory neurons as molecular receptors necessary for cellular and appetitive behavioral responses to ribonucleosides and RNA . Specifically , behavioral preference assays show that larvae are strongly attracted to ribose- or RNA-containing agarose in a Gr28-dependent manner . Moreover , Ca2+ imaging experiments reveal that Gr28a-expressing taste neurons are activated by ribose , RNA and some ribonucleosides and that these responses can be conveyed to Gr43aGAL4 fructose-sensing neurons by expressing single members of the Gr28 gene family . Lastly , we establish a critical role in behavioral fitness for the Gr28 genes by showing that Gr28 mutant larvae exhibit low survival rates when challenged to find ribonucleosides in food . Together , our work identifies a novel taste modality dedicated to the detection of RNA and ribonucleosides , nutrients that are essential for survival during the accelerated growth phase of Drosophila larvae .
Taste discrimination is a common trait of all animals , the most crucial being the ability to distinguish between palatable and mostly nutritional chemicals from aversively perceived , often harmful , and generally bitter-tasting compounds . Calorically nutritious food compounds fall into 3 categories , fats , proteins , and carbohydrates , and their consumption is dependent not only on availability but also on internal physiological states of the animal , such as overall nutrition status , anticipated need for energy expenditure , developmental stage , and reproductive status . To achieve discrimination between different nutrients , different subsets of cells in the taste sensory system express specific receptors for the detection of chemicals belonging to these nutrient groups [1] . Growth of most arthropods , but also some vertebrates , is characterized by a period of rapid body weight gain within a few days . A mouse increases its weight from time of birth ( approximately 1 g ) to the time of weaning ( about 3 weeks; about 10 to 13 g ) by about a factor of 10 . Growth dynamics of many insect larvae are even more dramatic . A freshly hatched Drosophila larva weighs about 9 . 5 μg [2] but grows over a period of only 108 hours to more than 1 . 5 mg at the time of puparium formation [3] , which translates to doubling of weight about every 14 hours . To support this rapid growth , animals are required to consume large amounts of all essential macronutrients , especially carbohydrates , proteins , and fats . In the natural diet of Drosophila , fruits provide the carbohydrates , while fatty acid and proteins are obtained mostly from colonizing microorganisms , such as yeast . Standard Drosophila food used in most laboratories is generally composed of cornmeal , malt extract , and yeast extracts ( see Material and methods ) , which contain all these and other macro- and micronutrients in abundance . In contrast to flies , which express and employ 8 sugar Gr genes for the perception of sweet taste , larvae use a single sugar receptor , Gr43a , for the detection of fructose and sucrose , the most abundant sugars in most fruits [4] . In addition , an Ionotropic Receptor ( IR ) was recently shown to be involved in sensing a limited number of amino acids [5] . Receptors for other macronutrients—including many amino acids , fats , and other compounds potentially critically important for rapid larval growth—have not been identified to date . Here , we report the discovery of a novel taste modality—the taste of ribonucleosides and RNA—and the identification of cognate receptors . We show that larvae have a strong attraction to and feeding preference for ribose , ribonucleosides , and RNA . Feeding on these molecules is mediated by members of the highly conserved taste receptor subfamily Gr28 . Live imaging experiments using the Ca2+ sensor Calcium Modulated Photoactivatable Ratiometric Integrator ( CaMPARI ) show that Gr28a-GAL4-expressing taste neurons respond to ribose , inosine , and uridine , as well as RNA itself , but not to 2-deoxyribose or to adenosine , guanosine , or cytidine . Holidic ( synthetic ) medium ( HM ) lacking inosine and uridine slows larval growth and causes high mortality , and supplementing this medium with RNA rescues both phenotypes . Moreover , when provided with a choice of HM with and without inosine and uridine , wild-type larvae readily select complete HM , leading to fast growth and high survival , while Gr28 mutant larvae fail to do so , resulting in slow growth and low survival . In summary , we have identified the cellular and molecular basis for the taste of ribonucleosides and RNA . We suggest that Drosophila larvae—and possibly other insect larvae—need to feed on RNA precursors to sustain the rapid increase in body weight , which is doubled almost twice a day .
While assessing sugar specificity of Gr43a , we made the surprising observation that larvae are also strongly attracted to arabinose ( Fig 1 ) . When given the choice between agarose containing L-arabinose and agarose alone , larvae showed a strong preference for arabinose ( Fig 1A ) . In flies , arabinose is detected by receptors encoded by the sugar Gr subfamily [6 , 7] , none of which are expressed in larvae [8 , 9] . Instead , larvae sense the main fruit sugars sucrose and fructose through Gr43a , which is expressed in pharyngeal taste neurons , as well as nutrient-sensing neurons in the brain [8] . Other nutritious sugars , such as trehalose and glucose , are sensed postprandially by Gr43a-expressing neurons in the brain , presumably after the conversion of a fraction of these sugars into fructose [8 , 10] . To examine the possibility that arabinose is sensed either through Gr43a or any of the main sugar receptors used by adult flies , the low expression of which might have been missed in previous studies , we examined arabinose preference in Gr43a mutant larvae or larvae lacking all 8 sugar Gr genes ( octuple mutant strain; Fig 1A [11] ) . However , neither Gr43a mutant nor octuple mutant larvae showed any significant loss in arabinose preference in the two-choice preference assay ( Fig 1A ) . L-arabinose is present as a minor component in heteropolysaccharides , such as hemicellulose and pectin [12 , 13] , but it is not a major sugar in fruit and cannot be metabolized by flies [14 , 15] . To assess whether larvae can use arabinose as an energy source , we compared survival rates of second-instar larvae kept on agarose-containing arabinose to larvae kept on plain agarose or nutritious sugar-containing agarose ( Fig 1B ) . Median ( 50% ) survival for larvae on plain agarose was 3 days . Larvae kept on nutritious sugar–containing agarose survived significantly better , with more than 75% of larvae still alive after 3 days , indicating that the consumed sugar provided energy and decreased mortality . In contrast , only about 30% of larvae kept on arabinose-containing agarose survived to the 3-day time point . These observations suggest that arabinose cannot be a nutritionally relevant ligand , and we posit that larvae instead detect a molecule structurally related to arabinose , but one that is nutritious and essential for larval growth . Arabinose is closely related to ribose , the carbohydrate backbone of RNA . We therefore investigated whether ribose and RNA can elicit a similar preference . Indeed , larvae strongly preferred ribose and RNA over plain agarose in the two-choice preference assay , whereas 2-deoxyribose , the sugar moiety of DNA , did not elicit a preference ( Fig 1C ) . Moreover , neither Gr43a nor the sugar Gr genes were required to sense either ribose or RNA , as larvae with respective mutations showed robust preference for both substrates ( Fig 1C ) . We next tested how ribose- or RNA-containing agarose affected larval survival ( Fig 1B ) . In contrast to arabinose , neither of these compounds reduced survival time . However , they did not serve as an efficient energy source either because median survival time was not significantly different from larvae kept on plain agarose ( Fig 1B ) . Taken together , these findings suggest that larvae can sense RNA through a taste or internal chemosensory receptor , presumably recognizing the ribose moiety in the RNA backbone . We next sought to identify the bona fide receptor ( s ) that mediate taste preference for RNA and ribose . Most Gr genes expressed in larvae have not been functionally characterized during that life stage , but expression and function of many of them have been investigated in adults . The vast majority of these genes ( i . e . , encoding bitter taste receptors ) are expressed in bitter taste neurons of taste bristles in the labial palps and legs of the fly , and it has been shown that bitter Gr proteins form multimeric receptor complexes that are activated by a vast array of chemicals perceived as repulsive [16–19] . In addition , Gr21a and Gr63a , which are also expressed in larvae , have noncanonical roles in a small subset of olfactory neurons in the fly , where they function as carbon dioxide receptors [20 , 21] . Because bitter taste receptors and Gr21a/Gr63a are likely to have similar roles in larvae [9] , we focused on the Gr28 gene clade ( Gr28a , Gr28b . a , Gr28b . b , Gr28b . c , Gr28b . d , and Gr28b . e ) , members of which show broad expression in larvae as well as adult flies [22] , whose roles in chemosensation have remained enigmatic . These receptors are also highly conserved across most arthropod orders [23–25] , suggesting important functions that are shared across a range of species . Some of these Gr genes have been implicated in ultraviolet ( UV ) light sensing in larvae [26] , as well as temperature sensing in flies [27] , but no chemical ligands for any of these Gr28 proteins have been identified . To investigate a possible role for proteins encoded by the Gr28 gene family in RNA and ribose sensing , we first examined expression of respective GAL4 lines more closely in larvae , focusing on chemosensory organs and the digestive system , as well as the central nervous system ( CNS ) ( Fig 2 ) . Four of the six Gr28 genes were expressed in small numbers of external ( Gr28a , Gr28b . a , Gr28b . e ) and pharyngeal taste ( Gr28a and Gr28b . d ) neurons . Other sites of expression included neurons in the proventriculus ( Gr28b . a , Gr28b . e ) , cells in the gut ( all but Gr28b . d ) , multidendritic neurons in the larval body wall ( Gr28a , Gr28b . c , Gr28b . d ) , and many neurons in the CNS ( Gr28b . a , Gr28b . b , Gr28b . d , and Gr28b . d ) . Expression of Gr28-GAL4 drivers in the taste system did not overlap with Gr43aGAL4 , which is expressed in a different set of pharyngeal neurons ( S1 Fig ) . We next investigated possible effects on preference for arabinose , ribose , and RNA across a range of concentrations in larvae lacking all Gr28 genes ( ΔGr28; [28] , Fig 3A and S2 ) . Indeed , ΔGr28 mutant larvae completely lost preference for ribose and RNA and showed significantly reduced preference for arabinose , phenotypes that were rescued when reintroducing a genomic construct containing the entire Gr28 locus ( Fig 3A ) . These observations indicate that one or several members of the Gr28 gene cluster mediate the detection of ribose and RNA . To examine whether a single Gr protein can establish ribose sensing , we subjected ΔGr28 mutant larvae expressing each of the Gr28 genes under the control of Gr28a-GAL4 . All Gr28 genes were able to restore at least some preference for ribose ( Fig 3B ) . Notably , Gr28a , Gr28b . a , and Gr28b . e , all of which are expressed in taste neurons ( Fig 2A ) , and Gr28b . b , rescued ribose preference to levels comparable to w1118 controls ( Fig 3B ) . We then asked whether ribose sensing could be conveyed upon other larval taste neurons . We choose the well-characterized sugar-sensing , pharyngeal taste neurons expressing Gr43a , a receptor narrowly tuned to the sugars fructose and sucrose [8] . We examined two-choice preference behavior of ΔGr28 mutant larvae expressing each of the 6 Gr28 genes individually under the control of the Gr43aGAL4 driver ( Fig 3C ) . Indeed , 3 genes—Gr28a , Gr28b . a , and Gr28b . e—endowed such larvae with the ability to sense and preferentially feed on ribose containing agarose in a manner indistinguishable from Gr28+ control larvae , and a fourth gene ( Gr28b . d ) mediated reduced ribose preference . The two other Gr28 genes , Gr28b . b and Gr28b . c , failed to convey any preference , just like Gr28 homozygous mutant larvae ( Fig 3C ) . Of note , neither Gr28b . b nor Gr28b . c is expressed in taste neurons of wild-type larvae ( Fig 2B ) , and while they rescued ribose sensing in Gr28a neurons , they failed to do so in heterologous Gr43a pharyngeal fructose-sensing neurons . One possibility is that Gr28 taste neurons express a cofactor ( i . e . , a chaperone or coreceptor ) absent in Gr43a neurons and that some , but not all , Gr28 proteins are completely dependent on such a factor for taste receptor function . Taken together , our experiments established that larvae possess a taste modality for ribose and RNA and that individual Gr28 proteins are able to mediate ribose and RNA sensing . To establish a role for the Gr28 proteins in ribose detection at the cellular level , we measured responses of terminal organ ( TO ) taste neurons using the fluorescent Ca2+ sensor CaMPARI [29] . In the presence of a high concentration of Ca2+ and simultaneous exposure to blue light , CaMPARI undergoes an irreversible conformational change , leading to a shift in emission properties from green to red fluorescence [29] . This feature allows application of ligand under free-moving conditions of the animal , while neural activation can be analyzed subsequently by quantifying green to red conversion ratios using fluorescent microscopy ( see Material and methods ) . We presented Gr28a-GAL4; UAS-CaMPARI larvae with various ligands while illuminating them with blue light and found that ribose and RNA activated Gr28a-GAL4 neurons , while 2-deoxyribose failed to do so ( Fig 4A ) . Consistent with behavioral assays of ΔGr28 mutant larvae , neurons lacking the Gr28 locus were not activated by either RNA or ribose , phenotypes that were rescued in the presence of a Gr28a transgene expressed in these neurons . To further explore the role of the Gr28 proteins and to assess whether they can mediate ribose sensing to heterologous taste neurons , we co-expressed UAS-CaMPARI with Gr28 genes in fructose-sensing pharyngeal sweet taste neurons using Gr43aGAL4 and measured cellular responses to ribose and RNA ( Fig 4B ) . We chose Gr28a , Gr28b . a , and Gr28b . e because they were competent to mediate ribose preference behaviorally when expressed in these neurons ( Fig 3C ) . Indeed , Gr43aGAL4 fructose-sensing neurons now responded to ribose and RNA in the presence of any of the 3 Gr28 genes . The fructose response in these neurons was similar to that in neurons of control flies ( i . e . , not expressing a Gr28 gene ) , indicating that the function of the endogenous fructose receptor was not impaired . Response to RNA or ribose was lower than responses to fructose , however , suggesting that fructose is more potent in activating Gr43a than ribose or RNA is in activating the Gr28 proteins . Alternatively , activation of Gr28 proteins might be suboptimal in heterologous taste neurons due to the absence Gr28 neuron-specific cofactors or facilitators ( see above ) . Regardless , these experiments , along with the two-choice feeding experiments ( Fig 3C ) , show that expression of a single Gr28 protein can convey ribose- and RNA-sensing properties to a taste neuron normally not responding to these compounds . In contrast to fructose or sucrose , ribose does not act as a major energy source ( Fig 1B ) . Thus , we considered roles of RNA and its precursors as essential nutrients for larval growth . Inosine and uridine are components of HM , a synthetic Drosophila food medium composed of approximately 40 pure chemicals that can sustain larval growth , adult development and fertility [30] . To examine the contribution of these compounds for growth and viability , we examined developmental progression and survival rate of larvae kept on HM , HM lacking inosine and uridine ( HMΔ ) , and modified HM in which RNA was added back to HMΔ ( HMΔ + RNA; Fig 5A; see Material and methods ) . Confirming the findings of Piper and colleagues , HM supported larval growth and adult development with the same survival rate as larvae kept on standard cornmeal food ( SCF ) , albeit at a slightly reduced pace . In contrast , lack of ribonucleosides in the medium ( HMΔ ) sharply increased mortality rate and significantly extended the larval growth phase , while adding back RNA ( HMΔ + RNA ) —but not ribose ( HMΔ + ribose ) —restored both survival and developmental time . Thus , RNA or the RNA precursors uridine and inosine are essential for rapid growth and viability . Given that ribonucleosides contain a ribose moiety , we expected that they are ligands for the Gr28 proteins and could activate TO taste neurons ( Fig 5B ) . To test this , we first performed CaMPARI imaging experiments of wild-type larvae and found that uridine and inosine activated Gr28a-GAL4 neurons , but the other 3 ribonucleosides ( guanosine , cytidine and adenosine ) did not ( Fig 5B ) . Second , we subjected wild-type and ΔGr28 mutant larvae to the two-choice preference assay and found that larvae were attracted to uridine and inosine in a Gr28-dependent manner ( Fig 5C ) . The experiments presented thus far identified a previously unknown taste modality for RNA ( Figs 3 and 4 ) and 2 RNA precursors , inosine and uridine , and established a requirement for these compounds during larval life ( Fig 5 ) . Thus , we sought to determine whether the Gr28 proteins provided a competitive advantage when larvae were presented with a challenging food environment . We devised an assay in which wild-type and Gr28 mutant larvae were required to find nucleosides in HM food during all larval life stages ( Fig 6 ) . Eggs were placed in a feeding arena that consisted of 21 wells , only 9 of which contained complete HM food while the remaining 12 contained HMΔ food ( Fig 6A ) . Control experiments using this setup confirmed a requirement for ribonucleosides ( see above ) , regardless of whether a Gr28 locus was present or not ( Fig 6B , compare solid versus light bars ) . When larvae were provided with the challenging food arena ( HM/HMΔ ) , ΔGr28 mutant larvae showed a large increase in mortality ( red checkered bar ) , while wild-type larvae or ΔGr28 larvae containing the Gr28 genomic rescue construct ( black and green checkered bar ) showed the same high survival rate as larvae kept on HM food ( solid bars ) . Taken together , these findings establish that larvae can discriminate between HM food based on the presence or absence of inosine and uridine and that they use this ability to increase fitness and survival when presented with a challenging food environment .
We have shown that larvae sense and are attracted to ribose-containing compounds , a behavioral feature mediated by the Gr28 protein family , a set of 6 evolutionarily highly conserved Gr proteins [22] . In contrast to sugar receptors and bitter receptors [6 , 7 , 11 , 18 , 19 , 31–34] , the Gr28 proteins appear not to function in combination with other Gr proteins , based on multiple lines of evidence . First , many cells and neurons express only a single member of the Gr28 gene family ( Fig 2 ) , although it remains possible that other Gr genes are co-expressed in some cells . Second , functional rescue experiments indicate that many Gr28 protein can restore preference for RNA and ribose in ΔGr28 mutant larvae when either expressed in Gr28a taste neurons ( all 6 ) or in Gr43a fructose-sensing neurons that do not express any Gr28 gene ( 3 of 6 ) and are therefore also unlikely to express a potential coreceptor that might be present in all Gr28 taste neurons . However , we cannot rule out that Gr43a or another Gr present in these cells is incorporated into a Gr28-based multimeric ribose and RNA receptor complex . At the very least , our data indicate that Gr28 proteins convey specificity for these ligands . We note that a single Gr protein ( Gr28b . d ) conveys temperature sensing in neurons of the fly antenna [35] , although in this case , no chemical ligand for this sensory modality has been identified to date . Surprisingly , while both inosine and uridine are potent ligands for Gr28 , the 3 other ribonucleosides ( guanosine , adenosine , and cytidine ) are not . It will be interesting to determine why some , but not all , of the ribonucleosides can activate the Gr28 proteins . One possibility is that the amino group present in guanosine , adenosine , and cytidine prevents or interferes with binding of these chemicals to the Gr28 proteins . Many Gr28 genes are also expressed in the larval brain and the gut ( Fig 2 ) , and it is possible that these cells contribute to preference of ribose-containing substrates via a postprandial mechanism . Three lines of evidence suggest that peripheral taste is the major driver for sensing these chemicals . First , larvae respond within 2 minutes to ribose and RNA ( S2 Fig ) . Our previous studies on the fructose receptor Gr43a have shown that such rapid decision-making is mediated by peripheral taste neurons , whereas postprandially mediated fructose sensing is a much slower process , requiring about 8 minutes to establish a clear preference [4] . Second , Gr28a-expressing taste neurons are activated by these ligands ( Fig 4A ) , and third , some Gr28 proteins can convey ribose and RNA preference to other taste neurons ( Figs 3C and 4B ) . Together , these observations suggest that Gr28-expressing taste neurons are activated by ribose and ribonucleosides and that this activation leads to the rapidly established preference for these ligands . The discovery that RNA and ribonucleosides are an essential nutrient resource recognized via a distinct taste modality represents a precedent . While detected through the ribose moiety ( Figs 1 , 3 and 4 ) , our data indicate that the ribose serves only as a proxy for the detection of the nucleobase–sugar complex and not as the critical nutrient component per se , e . g . , as a sugar used for energy production . In contrast to RNA , ribose complementation of HMΔ medium did not rescue development time or larval survival ( Fig 5A ) . We propose that RNA or ribonucleosides are sensed by the larval chemosensory system because they are required in large amounts as cellular components with critical roles in gene and protein expression during the accelerated growth phase of larvae . RNA contributes about 4% and 20% of the dry weight of mammalian cells and bacteria , respectively , quantities that are in the same range as fat or polysaccharides ( about 7% in both mammalian cells and bacteria ) . In contrast , DNA contributes only about 1% of the dry weight . Because the larval stage is characterized by unprecedented growth—evidenced by a doubling of body weight almost twice a day over a period of 4 . 5 days—it appears highly beneficial for larvae to be able to sense this abundant cellular constituent as an appetitive taste stimulus . In the natural environment , RNA is likely obtained from microorganisms that colonize decaying fruit . RNAs might be of interest to chemosensory evaluation from another viewpoint . Specifically , microRNAs ( miRNAs ) have recently been implicated in regulating the microbiome in mammals [36] , and double-stranded RNAs ( dsRNAs ) /small interfering RNAs ( siRNAs ) can cross cell membranes of the gut epithelium , a tissue well known for its ability to sense diverse types of chemicals [37] . Thus , it will be interesting to explore potential roles for Gr28 proteins in RNA sensing and transport in the larval gut , where all but 1 of the 6 genes are expressed ( Fig 2 ) . This paper represents the first clear evidence for a chemical compound acting as a ligand for members of this enigmatic Gr protein subfamily . While our study showed a specific role for these receptors in larval feeding on ribose-containing substrates , their function in adult flies remains to be investigated . Using proboscis extension reflex ( PER ) assays , we have found no evidence that adult flies respond to ribose , in either an appetitive paradigm or a feeding suppression paradigm ( S3 Fig ) . This is not surprising given that adults , in contrast to larvae , have a much lower requirement of cell proliferation and growth , which is restricted to the female germline and stem cells in a few organs of the fly . Expression analyses have shown that the Gr28 genes are broadly expressed in all taste organs ( labial palps , tarsi and pharyngeal taste neurons ) , and most of them appear to be expressed in bitter taste neurons [22] . Bitter taste receptors are mutlimeric complexes that are activated by non-nutritious and often toxic chemicals and when activated suppress appetitive taste behavior [16–19] . Thus , in fly taste neurons , Gr28 subunits most likely combine with other Grs to form receptor complexes for such ligands . It will be interesting to see whether these ligands share any structural features with ribose . Gr28 proteins have been reported to have functions in other sensory modalities , such as temperature sensing in flies and light sensing in the larvae . Specifically , Gr28b . d is expressed in 3 neurons of the aristae [22] and was later shown to be important for avoidance of warm temperatures [27] . Gr28b . d conveys thermosensitivity to a number of other cell types , suggesting that this protein acts on its own in the absence of other Gr proteins . Members of the Gr28 protein family were also implicated in light avoidance of larvae , which is mediated by multidendritic neurons in the body wall , where several of the Gr28 genes are expressed [22] ( Fig 2 ) . The Caenorhabditis elegans Gr28 ortholog , lite-1 , is necessary in worms for the avoidance of visible and UV light [38–40] . Two different mechanisms have been suggested for how LITE-1 senses light: one study proposed that light avoidance is an indirect chemosensory response , related to the avoidance of hydrogen peroxide of worms [40] , while another group suggested that LITE-1 directly absorbs light through 2 tryptophan residues [41] . In any case , the Gr28 proteins and its related cousin in C . elegans represent a decidedly atypical type of chemoreceptor that appears to be involved in diverse sensory modalities not associated with taste . Thus , future work will be necessary to identify additional ligands for Gr28 proteins and reveal the many roles of these receptors in physiology and behavior of insects and worms .
Flies were raised on SCF at 25°C on a 12-hour light–dark cycle . SCF in 1 . 5 L of water is composed of 10 . 88 g of agar ( Drosophila agar type II Genesee; 62–103 ) , 78 g of corn meal ( Genesee , 66–101 ) , 165 g malt extract ( Alternative Beverage , MUN-UL ) , 41 . 25 g of yeast extract ( Genesee , 62–106 ) , 4 . 69 g of propionic acid ( VWR , TCP0500-500mL ) , 0 . 075 g chloramphenicol ( Sigma-Aldrich , C0378 ) , and 2 . 11 g of tegosept ( Sigma-Aldrich , PHR1012 ) . Chemicals used for two-choice feeding preference assay and CaMPARI imaging were fructose ( Sigma-Aldrich , F0127 ) , 2-deoxy-d-ribose ( Sigma-Aldrich , 31170 ) , ribose ( Sigma-Aldrich , R7500 ) , arabinose ( Sigma-Aldrich , 10850 ) , t-RNA ( from brewer’s yeast; Sigma-Aldrich , 10109525001 ) , cytidine ( Sigma-Aldrich , C4654 ) , guanosine ( Sigma-Aldrich , G6752 ) , adenosine ( Sigma-Aldrich , A9251 ) , DMSO ( Sigma-Aldrich , D8418 ) , uridine ( Sigma-Aldrich , U3750 ) , and inosine ( Sigma-Aldrich , I4125 ) . Third-instar feeding-stage larvae were collected from food vials by washing them out using water . They were placed along the midline of a feeding arena ( plastic petri plate 60 × 15 mm , Falcon ) containing freshly prepared 1% agarose on one side and 1% agarose mixed with tastant on the other side . They were left feeding , and their location ( agarose versus agarose plus tastant , respectively ) was recorded after 2 , 4 , 8 , and 16 minutes . For simplicity , the preference index ( PREF ) was calculated for the 8-minute time point based on the number of larvae on either half of the plate . Dose-response curves for all sugars were performed using a concentration range from 25 mM to 500 mM ( S2 Fig ) . For preference tests of wild-type and mutant larvae ( Figs 1 , 3 , 4 and 5 ) , a concentration of 100 mM was used for arabinose , ribose , 2-deoxyribose , and inosine , while 50 mM was used for uridine . Stock solutions for all the tastants were prepared in water before mixing in the agarose . For ribose and 2-deoxyribose , the stock solution was treated with charcoal and filtered so as to remove the unrelated odor . “PREF” indicates the number of larvae on agarose plus tastant minus the number of larvae on the agarose only , divided by the total number of larvae . A PREF score of 0 indicates no preference , while a score of +1 ( or −1 ) indicates all larvae preferred ( or avoided ) tastant over agarose alone . Survival time of second-instar larvae kept on a 60 × 15 mm petri plate ( Falcon ) filled with 1% agarose containing various carbohydrates at 100 mM concentration and RNA at 0 . 5 mg/mL . Survival was monitored daily until all larvae died . Dead larvae were removed daily to avoid scavenging . For calcium imaging , we used the slide preparation method as described by Alves and colleagues [42] . A single live larva was placed in 25 μl of distilled water between a cover slip and a perforated slide ( a hole of 0 . 5 cm diameter was made on the slide with a circular drill bit ) . The larva was expressing CaMPARI calcium sensor described by Fosque and colleagues [29] . This preparation was placed on an inverted Nikon A1 confocal microscope and observed with 20× objective . To activate the neurons , a 25 μl solution ( 2× of final concentration ) of the ligand was injected through the hole in the slide . After about 5 seconds , a pulse of photoconversion ( PC ) light of 405 nm with a power of approximately 1 . 8 w/cm2 was delivered to the larvae for 10 seconds . Post activation , the neurons were observed for conversion from green to red emitted wavelengths . The changes were calculated as the ratio of red/green fluorescence ( FRED/FGREEN ) . Data were acquired using the Nikon NIS element acquisition and analysis package . Data are expressed as mean ± SEM . To determine preexposure values , the images were taken without applying ligand or PC light . To determine PC-light–only values , images were taken with applying PC light but without applying ligand . HM was prepared based on diet reported by Piper and colleagues [30] . A detailed list of chemicals is listed in S1 Table . For HMΔ food , inosine and uridine were removed . For supplementation experiments , HMΔ food was complemented by addition of RNA and ribose final concentrations of 0 . 5 mg/mL and 0 . 5 mM , respectively . For survival experiments , HM food and its derivatives were presented in 60 × 15 mm plastic petri plates ( Falcon 5 ml/plate; Fig 5 ) or in 21-well microtiter plates ( 350 μl/well; Fig 6 ) . These plates were then embedded in a 60 × 15 petri plate using 3% agarose . After plates were at room temperature , 40 eggs were placed on each plate , which were sealed with a perforated lid and placed in a humid chamber maintained at 25°C in a 12-hour light–dark cycle . Larvae location was checked once per day , and dead larvae were removed to avoid scavenging . In all figure legends , n indicates the number of experiments , unless otherwise noted . For each experiment , data are presented as mean ± SEM . Statistical analysis was performed using two-tailed nonparametric Mann-Whitney U test to compare 2 different groups of samples . p < 0 . 05 was considered to be statistically significant . Statistical analyses were conducted using Prism 6 . 0 software ( GraphPad Software ) . PER assays were carried out as described in Slone and colleagues [7] with the following modifications . Male and female flies were collected on the day of eclosion and kept on standard corn meal food for 5 to 6 days at 25°C . Prior to performing PER assays , flies were starved for 24 to 26 hours at 25°C in empty vials with a water-saturated cotton ball . Flies were immobilized by cooling briefly on ice and wing-mounted dorsally on a microscope slide using double-sided Scotch tape . Legs were taped to the slide . Mounted flies were allowed to recover for 60 to 90 minutes at room temperature in a humidified chamber . Flies were then allowed to drink water until satiation to ensure that PER responses were nutrient derived . Flies showing no response to water were excluded . Each fly was tested with a given tastant by briefly applying the taste solution to the labellum . Each fly was tested 3 times for each taste solution . A PER response was scored as positive ( 1 ) if the proboscis was fully extended , otherwise it was scored as negative ( 0 ) . PER response scores ( % ) from a single fly were 0% ( 0/3 responses in the 3 applications ) , 33% ( 1/3 ) , 66% ( 2/3 ) , or 100% ( 3/3 ) . Flies were allowed to drink water after each taste application . Taste solutions were delivered with a 20 ml pipette . Stock solutions of sucrose ( Macron , Cat No . 8360–06 ) and ribose ( Sigma , Cat No . R7500 ) were prepared in Millipore Q water and kept at 5°C . Stock solutions were diluted to the final concentration using Millipore Q water prior to each experiment . | Insects that undergo complete metamorphosis grow only during the larval stage of development . In many species , this period is restricted to a few days , during which larvae might increase their weight up to several hundred-fold . Drosophila melanogaster , for example , grow from a tiny first-instar larva of about 10 μg to a wandering third-instar larva weighing about 2 mg over a period of only 4 . 5 days . The main macronutrients known to be critical for this period of rapid growth are amino acids and sugars . In this study , we identify ribonucleosides and RNA as a new , additional type of nutrient necessary for rapid larval growth and survival . We show that larvae harbor taste neurons that express taste receptors necessary for sensing ribonucleosides and RNA . Larvae lacking these taste receptors show high mortality rates when exposed to a complex food environment that requires the location of ribonucleoside-containing food . We hypothesize that the ability to taste RNA evolved as a new taste modality in larvae of insects that go through a rapid growth period because ingestion of ribonucleosides , as opposed to de novo synthesis , provides a survival advantage during a period of extreme growth . | [
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"... | 2018 | The taste of ribonucleosides: Novel macronutrients essential for larval growth are sensed by Drosophila gustatory receptor proteins |
Most West Nile virus ( WNV ) infections are asymptomatic , but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death . Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection . However , other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage . We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen ( HLA ) class II tetramers . Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections . Independent of this , we also observed age related increases in WNV-specific T cell responses . Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells , whose presence was highly correlated with neuroinvasive disease . Moreover , a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects . More globally , subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections . Thus , subjects with neuroinvasive WNV infections exhibited elevated , dysregulated , and atypically polarized responses , suggesting that immune mediated damage may indeed contribute to pathogenic outcomes .
Since its emergence in 1999 , West Nile virus ( WNV ) has become a leading cause of encephalitis in North America . Seasonal outbreaks in various states have led to thousands of documented cases of WNV infection and perhaps millions of undocumented cases , based on serological estimates [1] . Epidemiological data indicate that the majority of WNV infections are essentially asymptomatic [2] . However , a minor yet significant percentage of infections lead to neuroinvasive disease [3] . The symptoms elicited by neuroinvasive WNV infection can include ocular manifestations , muscle weakness , cognitive impairment , tremors , flaccid paralysis , and death , making this virus a significant health concern [4] . While effective vaccines have been developed for other flaviviruses , including yellow fever and Japanese encephalitis , there is currently no approved human vaccine for WNV . As such , a more comprehensive understanding of immune responses elicited by the virus , including the aspects of these responses that accompany favorable and unfavorable outcomes , is a highly desirable goal . Although it is clear that both innate immunity and multiple components of the adaptive response play a role in WNV clearance , direct and indirect evidence indicates that CD4+ T cells play a crucial role in protection against WNV infection . For example , Brien et al . [5] demonstrated that WNV-specific CD4+ T cells are sufficient for protection from WNV in a murine viral challenge model and exhibit direct cytotoxic activity . In particular , CD4+ T cell responses have been shown to be essential for clearance of WNV from the central nervous system ( CNS ) of infected mice [6] . Indirect evidence from human studies also supports the importance of CD4+ T cell responses . For example , WNV infected donors with neurological symptoms were shown to exhibit higher frequencies of CD4+ T cells that expressed Tim-3 , which acts as a negative regulator of Th1 cytokine secretion by T cells [7] . This observation implies that the functional characteristics of WNV-specific T cell responses can differ between subjects with neuroinvasive versus asymptomatic infection and that the quality of T cell responses may influence outcomes . Although T cell responses appear to play an essential role in protecting the host from WNV infection , emerging evidence also suggests that over-exuberant T cell responses may play a role in the pathology of WNV infection . For example , some WNV patients present with autoimmune neuromuscular diseases and demyelinating neuropathies including Guillain-Barre syndrome , myasthenia gravis and stiff-person syndrome , suggesting that immune activation by WNV antigens can lead to immune mediated neurologic damage and/or subsequent cross-recognition of self-antigens [8] . Interestingly , mice that lack perforin and the FAS/FASL pathway were shown to be protected against encephalitis , suggesting that immune mediated damage through both of these pathways may be responsible for neurologic inflammation [9] . More recently , subjects with symptomatic WNV infection were shown to have decreased frequencies of regulatory T cells , implying that regulatory T cells ( Tregs ) may play a role in limiting WNV disease by restricting pathogenic aspects of the immune response [10 , 11] . Cumulatively , these findings suggest that while antiviral T cell immunity is crucial for viral clearance , immune damage mediated by over-exuberant responses may be a component of WNV induced neuropathology . Therefore in this study we sought to characterize WNV-specific T cell responses in subjects who had recovered from laboratory diagnosed WNV infection , including seropositive blood donors with asymptomatic infections and subjects with evidence of neuroinvasive disease recruited through public health organizations . Through the use of HLA class II tetramers , we defined an array of epitopes derived from the structural and non-structural proteins of WNV that are recognized by CD4+ T cells . Utilizing the corresponding tetramers , we performed direct ex vivo analysis of CD4+ T cell responses in subjects with asymptomatic or neuroinvasive infections , examining the number and phenotype of WNV-epitope specific T cells . We further assessed the phenotype of responses in these subjects to correlate the induced T cell response with clinical outcome following WNV infection . This characterization also included assessment of the cytokine profiles of WNV specific T cells and preliminary transcript profiling of WNV-specific responses . Our results demonstrate that the absolute number of WNV specific CD4+ T cells was higher in subjects with neuroinvasive WNV infection , regardless of their HLA restriction or antigen specificity . WNV specific CD4+ T cells from subjects with neuroinvasive infection had increased cytokine production and in these subjects a distinct transcript profile was up regulated in response to WNV-specific stimulation . We then examined the frequency and phenotypic profile of CD4+FoxP3 positive T cells to investigate the possible role of Tregs in restraining WNV-specific responses . Our findings implicate a phenotype of increased immune activation in subjects with neuroinvasive WNV infection that is partially explained by a deficit in immune regulation .
Subjects with prior WNV infection were recruited through the Benaroya Research Institute Infectious Disease Repository and through the Blood Systems Research Institute , with written consent as part of IRB approved studies . Subjects with asymptomatic or mild infections were primarily identified through routine WNV screening of United Blood Services blood donations [10] . Subjects with neuroinvasive infections were identified through Washington and Arizona State and county healthcare organizations . All subjects classified as having neuroinvasive disease had an illness with evidence of an acute infectious process such as fever along with clinical evidence of CNS infection , whereas subjects with asymptomatic or mild infections presented for blood donations with either no symptoms or minimal nonspecific symptoms , such as low grade fever or malaise . The attributes of these subjects are summarized in Table 1 . A total of 24 subjects ( 12 male and 12 female ) with asymptomatic infection were recruited; these subjects had an age range of 23–68 years of age ( median age = 53 , average age = 51 ) and were sampled 59–765 days after diagnosis ( median duration = 486 days , average duration = 397 days ) . A total of 16 subjects ( 8 male and 8 female ) with neuroinvasive infection were recruited; these subjects had an age range of 32–85 years of age ( median age = 61 , average age = 56 ) and were sampled 299–742 days after diagnosis ( median duration = 622 days , average duration = 582 days ) . Comparing the asymptomatic and neuroinvasive subject groups , there was no significant difference in age ( p = 0 . 21 ) but the neuroinvasive cohort had a slightly higher proportion of subjects who were over 65 years of age ( 25% versus 17 . 4% ) . The duration that asymptomatic group was sampled after diagnosis was significantly shorter ( p = 0 . 016 ) than the neuroinvasive group , mainly due to five subjects who were sampled less than 90 days ( earliest sampling was at 59 days ) after diagnosis . WNV , like other arboviruses in the Flaviridae family , contains a single-stranded RNA genome that encodes the capsid ( C ) , envelope ( E ) , premembrane ( prM ) , and membrane ( M ) proteins , as well as seven nonstructural proteins ( NS1 , NS2a , NS2b , NS3 , NS4a , NS4b , and NS5 ) that likely contribute to viral replication [12] . We utilized a tetramer guided approach ( S1 Fig ) to comprehensively identify CD4+ T cells epitopes across the entire WNV proteome , utilizing peripheral blood samples from subjects with documented WNV infection ( Table 1 ) . These epitope defining experiments focused on DRB5*01:01 and nine common HLA-DRB1 alleles that cumulatively provided complete coverage for our cohort of WNV infected subjects . As summarized in Table 2 , this approach allowed us to identify a total of 152 HLA/peptide combinations ( from a library of 451 overlapping peptides that represents the entire WNV proteome ) that were immunogenic in WNV infected subjects . A complete listing of these epitopes is provided in S1 Table . CD4+ T cell epitopes were present in every WNV protein , but only the envelope protein had epitopes that were presented by all of the HLA that we studied ( Table 2 ) . WNV membrane and envelope had the highest epitope densities ( 8 . 0 and 7 . 2 epitopes per 100 amino acids , respectively ) , while NS5 ( the largest protein ) contained the highest overall number of epitopes ( 43 epitopes ) . Capsid , which is the smallest WNV protein , also had a high epitope density ( 6 . 6 epitopes per 100 amino acids ) , containing 7 epitopes despite its modest size . In contrast , NS2a , NS2b and NS4a proteins were less immunogenic , containing only two epitopes each ( Table 2 ) . Cumulatively these results show that although WNV specific CD4+ T cells recognize a broad range of epitopes , the response focuses on a subset of structural and non-structural proteins . To further characterize WNV-specific responses in previously infected subjects , we used phycoerythrin ( PE ) labeled tetramers corresponding to a selected set of epitopes to detect WNV-specific CD4+ T cells directly ex vivo after magnetic enrichment . The patients from our cohort of WNV infected included a total of 26 subjects ( 15 classified as being asymptomatic and 11 classified as having neuroinvasive infections ) who had DRB1*01:01 , DRB1*04:01 , or DRB1*04:04 haplotypes ( Table 1 ) . Tetramers were chosen for these HLA to include at least one envelope epitope and another epitope from at least one non-structural protein ( Env 127–144 , Env 246–263 , and NS1 205–220 for DRB1*01:01; Env 164–180 , Env 356–373 , and NS1 205–220 for DRB1*04:01; Env 246–263 , NS3 91–107 and NS5 159–174 for DRB1*04:04 ) and a total of 23 subjects ( 11 of these were classified as being asymptomatic and 11 of these were classified as having neuroinvasive infections ) were selected for direct ex vivo tetramer analysis . Asymptomatic subject #9 was heterozygous for DRB1*01:01 and DRB1*04:01 , and therefore was analyzed with both sets of tetramers . More comprehensive experiments were not feasible because of the large number of cells and technical demands of direct ex vivo tetramer analysis . As depicted by the representative ex vivo staining result in Fig 1A , we consistently detected a distinct population of CD4+ memory T cells specific for WNV epitopes using these tetramers . In contrast , when tetramers were used to detect WNV-specific CD4+ T cells in subjects with negative WNV serology and no known history of WNV exposure ( Fig 1B ) , these subjects had low numbers of WNV-specific CD4+ T cells that exhibited a naïve ( CD45RA+CCR7+ ) phenotype . In spite of the fact that subjects were sampled 8–27 months after infection ( average time after diagnosis was 18 months ) , WNV-specific T cells were present at high overall numbers in some subjects . Although we were keenly interested in examining differences between subjects with asymptomatic versus neuroinvasive infections , we first characterized the general attributes of WNV specific T cell response in all subjects . As shown in S2 Fig , the number of WNV specific T cells differed for epitopes derived from different antigens ( S2 Fig panel A ) but did not vary significantly based on HLA-DR restriction ( S2 Fig panel B ) . In general , we observed significantly higher numbers of T cells specific for envelope and NS1 epitopes than T cells specific for NS3 and NS5 epitopes ( although NS3 and NS5 were only evaluated in subjects with DRB1*04:04 haplotypes ) . As shown in Fig 1C , expression or lack of CD45RA and CDR7 expression by WNV specific T cells indicated that these cells have a predominantly central memory phenotype ( representative staining results for single markers are shown in S2 Fig panel C ) . However , some subjects also had a significant proportion of WNV specific T cells that exhibited an effector memory phenotype . For WNV specific memory T cells ( CD45RA- cells that were either CCR7+ or CCR7- ) we assessed co-expression of various well-defined combinations of lineage associated surface markers to draw inferences about the functional heterogeneity of WNV specific CD4+ T cell responses . These Th subsets were defined essentially as described by Becattini et al . [13] , including Th1 ( CXCR3+CCR4-CCR6- ) , Th2 ( CCR4+CXCR3-CCR6- ) , Th17 ( CCR6+CCR4+CXCR3- ) , Th1-17 ( CXCR3+CCR4-CCR6+ ) and non-conventional Th1* ( CCR6+CXCR3+CCR4+ ) subsets–plus a newly defined Th1^ ( CXCR3+CCR4+CCR6- ) subset . As shown in Fig 1D , the relative hierarchy for these different Th subsets was: Th1 > Th1^ > Th1-17 > Th1* > Th17 > Th2 . Beyond this , some subjects had a significant proportion of WNV specific T cells that were CD38+ ( S2 Fig panel C ) , which is typically indicative of recent activation when expressed on memory T cells . It seemed possible that WNV specific T cells could be activated and maintained in some subjects by the presence of antigen due to persistent virus . Therefore , we examined blood and urine samples from our cohort , testing for the presence of WNV RNA using transcription-mediated amplification ( TMA ) assays . However , we were unable to detect any WNV RNA in either sample . To follow up on these observations , we analyzed the intracellular cytokine profiles of memory T cells following one round of in vitro expansion , using unbiased culture conditions . As indicated in Table 1 , these experiments utilized samples from 8 subjects with asymptomatic infections ( 4 of which were also sampled for ex vivo tetramer analysis ) and 8 subjects with neuroinvasive infections ( 4 of which were also sampled for ex vivo tetramer analysis ) . Multiple cell lines were isolated from each subject ( using a minimum of three different epitopes per subject ) resulting in a total of 144 WNV specific lines ( 81 from neuroinvasive subjects and 63 from asymptomatic subjects ) . In these experiments , CD4+ T cells were co-stained with tetramer and various anti-cytokine antibodies after in vitro expansion ( representative results are shown in S3 Fig ) . Consistent with the observed ex vivo surface phenotype , interferon γ ( IFN-γ ) was the predominant cytokine produced by WNV specific T cells ( Fig 2A ) . However , smaller percentages of WNV specific T cells produced detectable levels of interleukin 4 ( IL-4 ) , interleukin 10 ( IL-10 ) or interleukin 17 ( IL-17 ) . A minor but notable percentage of WNV specific T cells produced more than one cytokine ( Fig 2B ) . Among these dual production of IFN-γ and IL-4 was the most prevalent combination . To draw inferences about the relationship between the ex vivo surface phenotype of WNV specific T cells and the functional profiles of WNV specific T cell lines , we compared the surface expression of various combinations of CXCR3 , CCR4 , and CCR6 on WNV specific T cells with the intracellular cytokine profiles of WNV specific T cell lines obtained from the same subjects . Regression analysis ( shown in S4 Fig ) indicated a modest correlation between the percentage of T cells with an ex vivo Th2 ( CCR4+CXCR3-CCR6- ) surface phenotype and IL-4 production by WNV specific T cell lines ( p = 0 . 04 ) and a more significant correlation between the percentage of T cells with an ex vivo Th1^ ( CXCR3+CCR4+CCR6- ) surface phenotype and co-production of IFN-γ and IL-4 by WNV specific T cell lines ( p = 0 . 002 ) . Although WNV specific memory T cell responses were detectable in all WNV infected subjects , we observed a wide range of magnitudes and heterogeneous phenotypes for WNV specific T cells . It is well known that elderly subjects have an increased risk of neuroinvasive WNV infection , perhaps due to age related differences in immunity [14] , and the overall age distribution of our study population was diverse–ranging from 23 to 85 years of age . Therefore , we performed multiple regression analyses to investigate the influence of age on the magnitude and phenotype of WNV-specific T cell responses . Interestingly , regression analysis indicated that older subjects generally had significantly higher numbers of WNV-specific T cells ( p = 0 . 009 ) ( Fig 3A ) and tended to have a higher proportion of T cells with a central memory phenotype ( p = 0 . 049 ) ( Fig 3B ) . All other comparisons of surface marker expression , including other naïve and memory subsets ( classified based on surface expression of CD45RA and CCR7 ) and functional subsets ( classified based on surface expression of various combinations of CXCR3 , CCR4 , and CCR6 ) , did not vary significantly versus age . Examining the cytokine profiles of WNV specific T cell lines isolated from various subjects , regression analysis indicated that WNV specific lines isolated from older subjects had a higher proportion of T cells that produced IFN-γ ( p = 0 . 0021 ) ( Fig 3C ) and a higher proportion of T cells that co-produced IFN-γ and IL-4 ( p = 0 . 032 ) ( Fig 3D ) . It seemed possible that other factors such as variations in HLA type or the time since diagnosis could influence T cell number or function . However , additional regression analysis indicated that there was no significant correlation between WNV specific T cell number or function and HLA type or time since diagnosis . Based on a previously observed association between symptomatic WNV infection and reduced Tregs [10] , subjects with neuroinvasive disease could be expected to have stronger WNV-specific CD4+ T cell responses than those with asymptomatic infection . To address this question we directly compared the number of WNV-specific T cells in subjects with asymptomatic infection versus those with neuroinvasive disease . To ensure that this analysis was not confounded by age , we selected groups of subjects ( indicated in Table 1 ) such that the age distributions for the neuroinvasive and asymptomatic groups were statistically indistinguishable ( Kolmogorov-Smirnov test , p = 0 . 639 ) . Consistent with our expectations , subjects with neuroinvasive infection had significantly higher numbers of WNV-specific CD4+ T cells than asymptomatic subjects ( Fig 4A ) , averaging 50±4 cells per million ( mean ±SEM ) and 21±3 cells per million respectively ( p < 0 . 0001 ) . As shown in Fig 4B , neuroinvasive subjects had a lower percentage ( based on CD45RA and CDR7 expression ) of naïve WNV specific T cells ( p < 0 . 05 ) and , correspondingly , a higher percentage of WNV specific TEM cells ( p < 0 . 05 ) . In contrast , the percentages of TCM and TEMRA cells did not differ between the two groups . Comparing the expression of lineage associated surface markers on WNV specific T cells , we observed that subjects with neuroinvasive infections had a significantly lower percentage of Th1-like ( CXCR3+CCR4-CCR6- ) cells ( p < 0 . 0001 ) and a higher percentage of Th1^ ( CXCR3+CCR4+CCR6- ) cells ( p < 0 . 0001 ) ( Fig 4C ) . Other defined combinations of surface markers were not significantly different . Correspondingly , analysis by intracellular cytokine staining indicated that WNV specific T cell lines isolated from subjects with neuroinvasive infections had a significantly higher percentage of WNV specific T cells that were IL-4 positive ( Fig 4D ) . All other cytokines tested were not significantly different . The WNV specific T cell response was ostensibly a Th1 memory response in WNV infected subjects , based on the prevalence of Th1-like ( CXCR3+CCR4-CCR6- ) T cells and production of IFN-γ as the predominant cytokine by WNV specific T cell lines . However , subjects with neuroinvasive infection were distinguished from asymptomatic subjects by their higher overall number of WNV specific T cells , increased prevalence of TEM cells and reduced prevalence of naïve T cells , increased prevalence of TH1^ ( CXCR3+CCR4+CCR6- ) T cells and reduced prevalence of Th1 ( CXCR3+CCR4-CCR6- ) T cells , and increased production of IL-4 . To further clarify the quantitative aspects of WNV specific T cell responses in subjects with neuroinvasive versus asymptomatic infections , we next compared the absolute number of T cells within the various T cell subsets that comprise the WNV specific response . As shown in Fig 5A , subjects with neuroinvasive disease had increased numbers of WNV specific TEM and TCM ( p < 0 . 01 and p < 0 . 0001 respectively ) , whereas both groups had similar numbers of naïve and TEMRA cells . As shown in Fig 5B , subjects with neuroinvasive disease had increased numbers of WNV specific Th1 and Th1^ ( p < 0 . 0001 and p < 0 . 01 respectively ) , but had similar numbers of all other functional subsets . The appearance of CXCR3+CCR4+CCR6- T cells as a distinguishing feature of WNV specific T cell responses is interesting . Although cells that co-express CXCR3 and CCR4 are rarely studied , CD4+ T cells that are CXCR3+CCR4+ comprise approximately 5% of CD45RA- cells in peripheral blood and were previously shown to include cells that simultaneously produce Th1 and Th2 cytokines [15] . More recently Araya et al . [16] reported the induction of T box transcription factor 21 ( Tbet ) and CXCR3 in CCR4+CD25+CD4+ T cells in the context of HTLV-1 infection , leading to induction of IFN-γ secretion by this cell subset . As such , there is precedent to suggest that CXCR3+CCR4+CCR6- T cells represent a population of T cells with an atypically polarized cytokine response . Therefore , we next sought to verify whether subjects with neuroinvasive infections had a significantly higher percentage of WNV specific T cells that co-produced IFN-γ and IL-4 . As summarized in Fig 6 , subjects with neuroinvasive infection had a significantly higher average percentage of WNV specific T cells that produced multiple cytokines than subjects with asymptomatic infections ( 10 . 7% versus 4 . 1% , p < 0 . 0001 ) . The majority of ‘multiple cytokine positive’ WNV specific T cells co-produced IFN-γ and IL-4 ( 8 . 6% and 2 . 4% respectively ) , and this was significantly different between the two groups ( p < 0 . 0001 ) . Smaller fractions co-produced IFN-γ and IL-17 ( 1 . 8% and 1 . 5% respectively ) ; IL-4 and IL-17 ( 0 . 1% for both groups ) ; or IFN-γ , IL-4 and IL-17 ( 0 . 2% and 0 . 1% respectively ) , but none of these combinations were significantly different between the two groups . In summary , subjects with neuroinvasive disease are distinguished from asymptomatic subjects by having increased proportions of WNV specific T cells that are TEM and Th1^ ( CXCR3+CCR4+CCR6- ) , higher overall numbers of WNV specific T cells , and higher numbers of WNV specific T cells that are TCM , TEM , Th1 , and Th1^ . Correspondingly , WNV-specific T cell lines isolated from those with neuroinvasive disease exhibited an atypically polarized functional response that included a significantly higher proportion of cells that co-produced IFN-γ and IL-4 . Our results demonstrated clear differences in the magnitude and functional profile of WNV-specific responses in the two subject groups . We next sought to investigate the transcript profiles elicited by stimulation of PBMC with WNV epitopes in these two groups of subjects . To this end , we activated PBMC from subjects who had asymptomatic or neuroinvasive WNV infection for 20 hours using peptides representing the entire WNV envelope protein , collected RNA , and performed whole genome transcript profiling using RNAseq as described in Materials and Methods . In these experiments the PBMC were stimulated using 20mer peptides because this length of peptide primarily serve to activate a CD4+ T cell response . As shown in Table 3 , 15 transcripts were found to be differentially expressed ( p-values less than 0 . 05 after multiple testing correction ) . Fourteen of those 15 transcripts were differentially up-regulated in subjects with neuroinvasive infections . Among these were multiple genes with known or suspected immunological functions ( BCL6 , CCL19 , CCL3 , and SERPINB7 ) , genes related to cell growth and metabolism ( PPAN , PSAT1 , PYCR1 , and FITM1 ) , genes related to blood coagulation ( F3 , ADTRP , F2RL2 ) and genes from other unrelated pathways ( the matrix metallopeptidase MMP12 , the ELK1 transcription factor inducer C10orf10 , the neuroendocrine related kinase interacting protein MAPK8IP1 , and the cancer-testis antigen CTAG2 ) . Notably , a few of these genes have known functions that would be expected to promote anti-viral immune responses . BCL6 is known to promote T cell survival and plays a role in protecting effector T cells from apoptosis [17] . CCL19 is a CCR7 ligand that regulates CD4+ T cell homing and trafficking and also plays a role in initiating signalling [18] . CCL3 ( also known as MIP1 alpha ) is an inflammatory chemokine that enhances innate immune responses through binding to the CCR1 , CCR4 and CCR5 receptors; among these CCR4 was shown to dictate the inflammatory response to TLR challenge [19] . As shown in Fig 7A , unsupervised clustering analysis revealed two distinct profiles for these differentially expressed genes: a highly up-regulated profile that included 6 of the 7 subjects with neuroinvasive disease and a more weakly up-regulated profile that included 7 of the 9 asymptomatic subjects . Fig 7B depicts a clear segregation of subjects with asymptomatic versus neuroinvasive infections by orthogonal principal components derived from these transcripts . In this analysis , the first two principal components ( PC1 and PC2 ) account for 45 . 2 and 17 . 4 percent of the variance respectively . In total , these results indicate that subjects with neuroinvasive infection have a distinct upregulated transcript profile that includes a trio of immunological transcripts ( BCL6 , CCL19 , and CCL3 ) . This would be expected to promote more robust and inflammatory WNV-specific responses through increased survival , activation , homing , and signaling . Prior studies have shown that subjects with symptomatic WNV infection have decreased frequencies of regulatory T cells ( 10;11 ) . Therefore it is plausible that deficits in regulatory T cell ( Treg ) number or function could contribute to the formation of an atypical and potentially pathological T cell response in neuroinvasive WNV infection . To investigate this possibility , we utilized a multi-color staining panel to compare the number and phenotype of CD4+Foxp3+ Treg in subjects with neuroinvasive versus asymptomatic WNV infection . As shown in Fig 8A , we defined Tregs by gating on CD4+ T cells that were CD25+ and identifying a population that was CD127 low and FoxP3 bright ( all of these cells expressed a higher level of FoxP3 than CD127+ cells ) . Utilizing this gating strategy , we observed no significant difference in the overall percentage of Treg between subjects with neuroinvasive or asymptomatic WNV infection ( Fig 8B ) . Due to sampling limitations , we did not have an adequate number of cells available to directly assess Treg function . To draw inferences about Treg quality , we performed stains to characterize the expression of CLTA-4 and CCR4 on Treg , as both of these markers have been reported to correlate with improved suppressive capacity [20] . We observed that subjects with neuroinvasive WNV infection had a much lower proportion of Tregs that were CTLA-4 positive ( Fig 8C ) , suggesting a possible defect in Treg function . However , the percentage of Tregs that were CCR4+ was not significantly different between subjects with neuroinvasive or asymptomatic WNV infection ( Fig 8D ) .
For reasons that are not fully understood , outcomes for subjects following WNV infection are diverse , raising questions about the mechanisms that influence the severity of disease in infected subjects [21–23] . CD4+ T cell responses have been shown to be sufficient for protection ( independent of B cells and CD8 T cells ) from WNV challenge and crucial for viral clearance from the CNS , but conflicting evidence suggests that either poor viral clearance or immune mediated tissue damage are important causative factors in neuroinvasive disease [5 , 6] . In the present study , we addressed this question by utilizing HLA class II tetramer reagents to directly characterize WNV specific CD4+ T cell responses in subjects with previous WNV infection and compared the immune phenotype in subjects with asymptomatic infections versus neuroinvasive disease . We had previously observed that the yellow fever vaccine elicits T cell responses against every protein in the YFV proteome [24] . Similarly , we observed that CD4+ T cell responses from previously infected subjects with various HLA haplotypes are directed against a wide array of WNV epitopes derived from all of the structural and non-structural proteins . While broad , the epitopes recognized by CD4+ T cells were focused on a subset of viral proteins , including Env , NS1 , NS3 , and NS5 . This observation is in agreement with a previous study in which the majority of positive ELISpot responses in HLA-DR2 , HLA-DR3 , and HLA-DR4 transgenic mice were directed against peptides from Env , NS3 , and NS5 [25] . Utilizing a panel of tetramers , we examined the ex vivo characteristics of the WNV-specific CD4+ T cell response in subjects with DRB1*01:01 , DRB1*04:01 , and DRB1*04:04 haplotypes . In general , T cells that recognized Env and NS1 epitopes occurred at higher numbers than those that recognized NS3 and NS5 epitopes , but WNV specific T cells were primarily TCM ( CD45RA-CCR7+ ) and predominantly Th1-like ( CXCR3+CCR4-CCR6- ) regardless of epitope specificity . Therefore , although the overall number of T cells appears to vary for different WNV antigens , their phenotypic characteristics were similar regardless of epitope specificity . In keeping with their observed surface phenotype , WNV specific T cells mainly produced IFN-γ ( and only lesser quantities of IL-4 , IL-10 , and IL-17 ) , which is consistent with a prior study that observed IFN-γ and IL-2 production by WNV specific T cells following peptide vaccination of mice with CD4+ T cell epitopes [5] . These findings suggest a uniform phenotype for WNV specific T cells that recognize epitopes ( from either structural or non-structural proteins with functional characteristics ) which likely promote effective antiviral immunity . Examining variations in WNV specific T cell responses with respect to age , we observed that older subjects had a higher number of WNV specific T cells , a greater proportion of which were TCM . WNV specific cell lines isolated from older subjects had a higher proportion of T cells that produced IFN-γ and also a higher proportion that co-produced IFN-γ and IL-4 . Such responses against WNV were clearly effective in subjects who cleared the virus and remained essentially asymptomatic . Given that age is an established risk factor for neuroinvasive WNV infection , these observations imply that elevated WNV specific T cell responses may promote pathogenic outcomes rather than protection . We next compared the magnitude and ex vivo characteristics of WNV-specific CD4+ T cell response in subjects with neuroinvasive disease or asymptomatic infection . For this comparison , we selected groups of subjects with similar age distributions , so that this variable would not confound our analysis . We found that , independent of age subjects with neuroinvasive disease had higher numbers of WNV-specific CD4+ T cells . Among these WNV-specific cells , a lower proportion were naïve and a higher proportion were TEM . These differences in proportion coincided with increases in the overall number of TEM and TCM in subjects with neuroinvasive infections . Neuroinvasive disease was significantly associated with an increased proportion and increased number of WNV-specific CD4+ T cells that were CXCR3+CCR4+CCR6- . As described by Kim et al . [15] , CXCR3 and CCR4 expression have been traditionally used designate T cells as being respectively Th1-like or Th2-like , as 88% of Th1 cells in blood are CXCR3+CCR4- , and 90% of Th2 cells are CXCR3-CCR4+ . It has been known for some time that although the majority of memory T cells in the periphery express only one of these markers , approximately 5% are CXCR3+CCR4+ and have the capacity to produce IFN-γ , IL-4 , or both [15] . A more recent study by Araya et al . [16] concluded that HLTV-1 infection induced CXCR3 expression in CCR4+CD25+CD4+ T cells , which then produced IFN-γ through viral upregulation of Tbet . The emergence of these cells was apparently correlated with severity of infection . Another recent report [26] described a case of post-transplant adult T-cell leukemia/lymphoma in whom CXCR3+CCR4+ T cells were observed . As recently described by Becattini et al . [13] , the repertoire of memory CD4+ T cells includes not only cells polarized towards a single fate ( conventional Th1 , Th2 , etc . ) , but also cells whose progeny have acquired multiple fates ( expressing combinations of chemokine receptors and producing measureable levels of the expected cytokines corresponding to these markers ) . That study utilized surface expression of various combinations of CXCR3 , CCR4 , and CCR6 to define the following subsets: Th1 ( CXCR3+CCR4-CCR6- ) , Th2 ( CCR4+CXCR3-CCR6- ) , Th17 ( CCR6+CCR4+CXCR3- ) , Th1-17 ( CXCR3+CCR4-CCR6+ ) and non-conventional Th1* ( CCR6+CXCR3+CCR4+ ) . To these we would add Th1^ ( CXCR3+CCR4+CCR6- ) as a subset that is apparently important in the context of WNV infection . Consistent with coordinate expression of CXCR3 and CCR4 , we observed that WNV-specific T cell lines from subjects with neuroinvasive infection had a higher proportion of WNV-specific CD4+ T cells that co-produced IFN-γ and IL-4 . It has been previously demonstrated that subjects with symptomatic WNV infections have decreased numbers of CD4+ CD25+ Foxp3+ Treg cells and mice depleted of Tregs have increased neurological disease and mortality [10] . Therefore , it could be surmised that neuroinvasive infection is likely to be accompanied by over-exuberant immune responses against the virus that are inadequately restrained . Consistent with this notion , we observed that although subjects with neuroinvasive and asymptomatic WNV infections had similar proportions of CD4+CD25+ CD127lowFoxp3++ cells ( implying similar numbers of Treg ) , Tregs from subjects with neuroinvasive infection had a significantly lower proportion of CTLA-4 expression , which has been shown to be intimately linked to effective suppressive function [20] . Interestingly , Treg ablated mice have also been reported to have significantly higher frequencies of IFN-γ producing CD4+ and CD8+ T cells and an increased proportion of effector T cells that co-produced IFN-γ and TNF-α in response to WNV infection [27] . Therefore , elevated numbers of WNV-specific T cells , many of which co-produce IFN-γ and IL-4 , could be construed to imply an inadequately restrained antigen driven response . Recent functional studies of WNV-specific responses in humans are in conflict about the relationship between the production of various cytokines and different outcomes . Qian et al . [28] report an association between reduced serum IL-4 and severe infection ( implying a correlation between reduced IL-4 and worse outcomes ) , but Garcia et al . [29] report an association between increased pro-inflammatory and antiviral cytokines ( such as IFN-γ , IL-2 , and IL-6 ) and prolonged fatigue following symptomatic clinical WNV disease ( implying a correlation between increased IFN-γ , IL-2 , and IL-6 and worse outcomes ) . Our results are more in agreement with those of Garcia et al . A recent study of CD8+ T cell responses observed that individuals with neuroinvasive WNV infection had WNV-specific CD8+ T cells that were predominately monofunctional , but T cells from subjects with neuroinvasive disease were shown to have significantly increased CD107a , implying greater cytolytic potential [30] . Similarly , a recent report by Gibson et al . [31] observed that reduced polyfunctional CD4 and CD8 T cell responses were associated with persistent circulating CMV DNA in infants , but this study did not measure other cytokines such as IL-4 . Our study provides additional evidence to associate increased functional responses with worse outcomes in the context of viral infection . Persistence of high numbers of WNV-specific T cells many months after initial infection has several possible explanations . Multiple lines of evidence suggest that subjects with neuroinvasive disease might be expected to exhibit deficient immune responses against the virus leading to delayed viral clearance . Elderly and immunosuppressed subjects have been shown to have an increased risk of neuroinvasive WNV infection [14 , 32] . Furthermore , one recent study found that genetic risk variants that are thought to cause impaired resistance to viral infection were associated with symptomatic infection [23] . As such , subjects with neuroinvasive infection may have a higher viral load , leading to higher peak T cell numbers that remain elevated at later times . Indeed , WNV RNA has been shown to persist for three months in blood and others have reported the detection of WNV transcripts at much later times after initial infection [33 , 34] . Therefore , it is possible that WNV-specific T cells could be maintained at higher numbers in some subjects by persistent antigen due to the systemic presence of virus . Alternatively , subjects with neuroinvasive disease may differ from asymptomatic subjects only in the amount of virus that reaches their CNS . Indeed , WNV-specific responses were shown to persist in the CNS of mice for 4–6 weeks post-inoculation , implying that virus may persist for longer times in the CNS [35] . In our phenotyping experiments , approximately one third of WNV-specific memory T cells were CD38 positive , implying recent exposure to antigen . However , levels of CD38 were not significantly different between subjects with neuroinvasive infection and asymptomatic subjects . Also , we found no detectable virus in either the blood or the urine of any subjects from our cohort . Alternatively , it is possible that WNV-specific T cells could be maintained and activated by homologous self-peptides . Indeed , WNV infection has been associated with the development of autoimmune neuromuscular diseases [8] . Interestingly , neurologic symptoms have been reported to arise long after acute infection , raising the possibility that cross-recognition of neural tissue could be a secondary event [36] . Our study does have limitations . For logistical reasons ( mainly related to recruitment ) we were unable to characterize WNV infected subjects at times close to initial infection . Therefore , we were not able to assess parameters such as peak viral titer in our subjects or characterize responses during acute infection . Although we saw clear differences between subjects with neuroinvasive versus asymptomatic infections , it is possible that other compelling differences were present at these earlier time points . In addition , our use of tetramer-based assays allowed detailed and direct interrogation of WNV-specific T cells , but consequently limited this analysis to a few key specificities within the responding T cell population . Therefore , although our data appear to suggest that the functional attributes of the WNV response did not vary for different epitope specificities , we cannot completely rule out that possibility . In addition , although our transcript assays used conditions that were designed to primarily elicit a CD4+ T cell response , these assays were performed on unfractionated PBMC; the resulting profiles could be taken to suggest elevated immune responses that go beyond the CD4+ T cell compartment . This is an important question that exceeds the scope of our current study , but it is likely that future studies may demonstrate important differences in CD8+ T cell or antibody responses that occur in the context of neuroinvasive WNV infection . Finally , our study was inherently limited by its focus on peripheral blood . Although it was only feasible for us access peripheral blood , it is probable that important insights could be gleaned through immune profiling of T cells that have infiltrated neural tissues . Collectively , our results demonstrate important age related differences in WNV-specific immunity and that , independent of this , WNV specific CD4+ T cells occur at higher numbers in subjects with neuroinvasive infection than in asymptomatic subjects , regardless of their HLA restriction or antigen specificity . Neuroinvasive disease was associated with the emergence of a population of WNV-specific TH1^ cells that were CXCR3+CCR4+CCR6- and whose presence correlated with co-production of IFN-γ and IL-4 . Furthermore , WNV-specific responses in subjects with neuroinvasive disease up-regulated distinct transcripts ( including BCL6 , CCL19 , and CCL3 ) that would be expected to promote inflammatory WNV-specific responses through increased survival , activation , homing , and signaling . These results implicate a mechanism of increased immune activation in neuroinvasive disease . However , it remains to be determined whether the increased magnitude of CD4+ T cell responses plays a direct or indirect role in the clinical outcome of WNV infection .
To facilitate experiments with HLA class II tetramers DNA samples from each subject were HLA typed using Dynal Unitray SSP Kits ( Invitrogen ) . Laboratory diagnosis of WNV infection was established by either a positive serum or cerebrospinal fluid ( CSF ) anti-WNV IgM serologic or WNV PCR assay . The subjects were administered a clinical questionnaire either through mail or over the phone . This data was supplemented by medical records obtained from patients’ physicians . Research samples from recruited subjects were tested for WNV RNA using a transcription-mediated amplification assay , the Procleix WNV assay ( Hologic ) as previously described [37] . Libraries of overlapping peptides , generated based on the sequences of each protein in the WNV genome , were obtained through the Biodefense and Emerging Infections Research Resources Repository . These libraries consisted of overlapping peptides , typically 18–20 amino acids long with a 12 amino acid overlap ( peptide length and overlap was adjusted as needed to avoid terminal cysteine residues ) . Peptides were dissolved in DMSO at 20 mg/ml and subsequently diluted as needed . Recombinant HLA-DR proteins were generated as described [38] . Briefly , each HLA-DR was purified from the supernatants of transfected insect cells , biotinylated , and dialyzed into 0 . 1M phosphate buffer . Biotinylated monomer was loaded with 0 . 2 mg/ml of peptide by incubating at 37°C for 72 hours in the presence of 2 . 5 mg/ml n-octyl-β-D-glucopyranoside and 1 mM Pefabloc SC protease inhibitor ( Sigma-Aldrich , St . Louis , MO ) and then conjugated using R-PE streptavidin ( Biosource International , Camarillo , CA ) at a molar ratio of 8 to 1 . The Tetramer guided Epitope Mapping ( TGEM ) procedure was performed as previously described [39] . Briefly , PBMC were isolated by Ficoll underlay and CD4+ T cells purified by negative selection using an isolation kit ( Miltenyi , Auburn , CA ) . Adherent cells from the CD4- fraction were used as antigen presenting cells by incubating 2×106 cells per well ( 200 μL volume ) in 48 well plates for 1 h and washing . Two million CD4+ T cells per well were stimulated with pools of five consecutive peptides . After 14 days , 1×105 cells were then stained with pooled peptide tetramers . Wells which gave positive staining were stained again using each of the corresponding individual tetramers from the positive pool . Direct ex vivo analysis of epitope-specific T cells ex vivo , was accomplished as previously described [24 , 40] . Briefly , PBMC at a concentration of 150 million/ml were treated with dasatinib for 10 min at 37°C followed by staining with 20 μg/ml PE-labeled tetramers at room temperature for 120 min . Cells were then labeled with anti-PE magnetic beads and enriched using a magnetic column according to the manufacturer’s instructions ( Miltenyi Biotec , Auburn , CA ) . For phenotyping studies , magnetically enriched cells were stained with antibodies against each marker of interest , including CD3 V500 ( BD , clone SP34-2 ) , CD4 AF700 ( eBioscience , clone OKT4 ) , CD38 allophycocyanin ( APC ) ( eBioscience , clone HIT2 ) , CD45RA APC-H7 ( BD , clone HI100 ) , CXCR3 PE-Cy5 ( BD , clone 1C6 ) , CCR4 fluorescein ( R&D Systems , clone 205410 ) , CCR6 peridinin chlorophyll protein ( PerCP ) -Cy5 . 5 ( BD , clone 11A9 ) , and CCR7 PE-Cy7 ( BD , clone 3D12 ) . The combination of CD14 Pacific Blue ( Invitrogen , clone TuK4 ) , CD20 Pacific Blue ( Invitrogen , clone H147 ) , and ViViD fixable violet dead cell stain ( Molecular Probes ) was used to exclude monocytes , B cells and dead cells from the analysis . Data acquisition was performed on a 17-color BD LSR II instrument and analyzed using FlowJo software ( Treestar , Ashland , Ore ) . T cell numbers ( expressed as cells per million ) were calculated as previously described [24 , 40] . WNV-specific T cells were expanded by stimulating purified CD4+CD45RA- cells with selected WNV peptides in vitro for 14 days in the presence of autologous adherent cells as antigen presenting cells . These cells were rested for 3 days and then stained with PE-conjugated tetramers for 30 min at 37°C and stimulated with 25 ng/mL PMA and 1 μg/mL ionomycin at 37°C . Brefelden A was then added and the cells incubated for 3–4 hours at 37°C , 5% CO2 . For cytokine staining , surface staining was performed first , followed by fixation/permeabilization as per the manufacturer’s protocol ( eBioscience ) . Cells were then co-stained with antibodies for surface markers and cytokines of interest , including CD3 PE-Cy5 ( BioLegend , clone HIT3a ) , CD4 V500 ( BD , clone RPA-T4 ) , IFN-γ AF700 ( BioLegend , clone 4S . B3 ) , IL-4 fluorescein isothiocyanate ( FITC ) ( eBioscience , clone 8D4-8 ) , IL-10 PE-Cy7 ( Biolegend , clone JE53-9D7 ) , and IL-17 APC-Cy7 ( Biolegend , clone BL168 ) , or alternatively with CD4 PerCP-Cy5 . 5 ( BD , clone RPA-T4 ) and IL-17 APC ( Biolegend , clone BL168 ) . ViViD fixable violet dead cell stain was then added to remove dead cells from the analysis . After 20 min at room temperature , cells were washed and immediately analyzed by flow cytometry . Frozen PBMC from WNV infected subjects were thawed , rested for 30 minutes and then co-stained with antibodies for surface markers and cytokines of interest , including CD4 PerCP ( BD , clone RPA-T4 ) , CD25 PE ( BioLegend , clone BC96 ) , CD127 AF488 ( Biolegend , clone 8019D5 ) , FoxP3 APC ( eBioscience , clone PCH101 ) , CTLA-4 PE-CF594 ( BD , clone BN13 ) and CCR4 BV605 ( Biolegend , clone L29IH4 ) . After 20 min at 4 degrees , cells were washed and immediately analyzed by flow cytometry . To elicit WNV- and influenza-specific transcript profiles ( the latter as a positive control for activation ) , PBMC from WNV infected subjects were activated using peptides spanning the entire WNV envelope protein or the influenza A/new caledonia/20/99 hemagglutinin protein . Briefly , 2 million PBMC per mL were incubated in 14 mL polypropylene round bottom tubes in the presence of peptides at a final concentration of 1 ug/mL ( or a DMSO control ) for 20 hours at 37°C , 5% CO2 . Cells were then washed , pelleted , and resuspended in RNA later according to the manufacturer’s instructions and stored at -80C . Subsequently , cells were thawed , pelleted , re-suspended in RLT buffer and total RNA was isolated using the Qiagen RNeasy kit according to the manufacturer’s instructions . Sequencing libraries were constructed from 100 ng of total RNA using TruSeq RNA Sample Preparation Kits v2 ( Illumina ) . Libraries were clustered onto a flowcell , using a cBOT amplification system with a HiSeq SR v3 Cluster Kit ( Illumina ) . Single-read sequencing was carried out on an Illumina HiScanSQ sequencer , using a HiSeq SBS v3 Kit to generate 50-base reads , with a target of approximately 10 million reads per sample . FASTQs were aligned to a human reference genome to generate gene counts . RNAseq data was analyzed in R version 3 . 0 . 1 using package edgeR [41–46] . Genes that were not expressed in at least 10% of the samples were excluded from analysis . To compare the data across different conditions , the raw counts were normalized as the number of counts per million ( cpm ) mapped for each sample . Next , 1 was added to all normalized data to allow taking ratio , stimulated over unstimulated samples ( FC ) . Responders to the stimulations were identified as those with log2 ( FC ) expression for 2 out of 3 genes selected as response indicator genes: CXCL9 , CXCL10 , and IFNG , ≥ log2 ( 2 ) . After estimating dispersions , a linear model was fit to determine genes responding differently to WNV in symptomatic vs asymptomatic groups . The model was y = μ + β1XS + β2XA:d10 + β3XA:d2 + β4XS:d2 + β5XA:d3 + β6XS:d3 + β7XA:d4 + β8XS:d4 +β9XA:d5 + β10XS:d5 + β11XA:d6 + β12XS:d6 + β13XA:d7 + β14XS:d7 + β15XA:d8 + β16XA:d9 + β17XA:WNV + β18XS:WNV +β19XA:Flu + β20XS:Flu + ε where μ is an estimates for log-expression level for asymptomatic unstimulated sample from asymptomatic donor 1 , and d , S , A , WNV , and Flu are abbreviations for donor , symptomatic , asymptomatic , and peptides derived from WNV and flu , respectively . The genes are considered differentially expressed if absolute estimate of the log2 ( FC ) corresponding to the effect ≥ log2 ( 1 . 5 ) at false discovery rate ( FDR ) adjusted p-value ≤ 0 . 05 . Principal component analysis was performed for the responders with WNV stimulation using the differentially expressed genes in log2 ( FC ) values . Statistical comparisons between symptomatic and asymptomatic subjects were performed with the GraphPad Prism software version 5 . 0a ( GraphPad Software , La Jolla , CA ) using Student’s t test with Welch’s correction for two column comparisons and ANOVA analysis followed by Holm-Sidak's multiple comparisons test for multi-column comparisons . The authors' respective Institutional Review Boards ( the Benaroya Research Institute at Virginia Mason Institutional Review Board and UCSF Committee on Human Research ) approved this study . All adult subjects provided informed written consent . The study did not include any participants who were children . | Most West Nile virus ( WNV ) infections are asymptomatic , but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death . Epidemiologic evidence suggests that neuroinvasive disease is a consequence of poor viral control , as the risk of neurological symptom is highest in immune compromised individuals . However , conflicting evidence suggests that neurologic symptoms may arise because of immune mediated damage . We demonstrate that subjects with neuroinvasive West Nile Virus infections have exaggerated and atypical responses to the virus . Subjects with neuroinvasive infections had higher numbers of WNV-responsive cells and these cells had more potent and diverse functional responses . In particular , we observed that subjects with neuroinvasive infections had a significantly increased population of atypically polarized T cells , whose presence was highly correlated with a globally upregulated transcript profile . Thus , we conclude that immune mediated damage may indeed contribute to neurologic symptoms and pathogenic outcomes in the setting of WNV infection . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2016 | Neuroinvasive West Nile Infection Elicits Elevated and Atypically Polarized T Cell Responses That Promote a Pathogenic Outcome |
We used allometric scaling to explain why the regular replacement of the primary flight feathers requires disproportionately more time for large birds . Primary growth rate scales to mass ( M ) as M0 . 171 , whereas the summed length of the primaries scales almost twice as fast ( M0 . 316 ) . The ratio of length ( mm ) to rate ( mm/day ) , which would be the time needed to replace all the primaries one by one , increases as the 0 . 14 power of mass ( M0 . 316/M0 . 171 = M0 . 145 ) , illustrating why the time required to replace the primaries is so important to life history evolution in large birds . Smaller birds generally replace all their flight feathers annually , but larger birds that fly while renewing their primaries often extend the primary molt over two or more years . Most flying birds exhibit one of three fundamentally different modes of primary replacement , and the size distributions of birds associated with these replacement modes suggest that birds that replace their primaries in a single wave of molt cannot approach the size of the largest flying birds without first transitioning to a more complex mode of primary replacement . Finally , we propose two models that could account for the 1/6 power allometry between feather growth rate and body mass , both based on a length-to-surface relationship that transforms the linear , cylindrical growing region responsible for producing feather tissue into an essentially two-dimensional structure . These allometric relationships offer a general explanation for flight feather replacement requiring disproportionately more time for large birds .
Flight feather molt is a time-demanding activity in the avian annual cycle [1]–[3] . Yet , annual or alternate-year replacement of flight feathers is essential , because physical abrasion and ultraviolet light rapidly degrade even the most sturdy wing quills after two years of use [4] , [5] . Because flight performance declines during molt as new feathers are growing [6]–[8] , most birds do not overlap molt and breeding , and those that do overlap these activities replace few flight feathers at a time , presumably to minimize the energetic and flight-performance costs of molting on reproduction [5] , [9] . Overlap of molt and breeding may be more common in larger species because the time required to rear young , as well as the time required to replace flight feathers , increases with body size [3] , [10] , [11] . Most smaller birds ( i . e . , generally <1 kg ) replace all their flight feathers annually , and a few do so twice a year [12]–[14] . In contrast , many larger birds ( >3 kg ) that depend on flight for feeding during the molt shed only a part of their flight feathers annually [5] , [15] and require two , and sometimes three , years to complete the molt [16] , [17] . For example , no albatross regularly replaces all of its flight feathers in a single bout of molting [18] , [19] , and the largest albatrosses ( Diomedea exulans and D . epomophora ) , whose masses reach 10 kg , avoid reproducing during years following successful breeding because of the competing time and resource demands of reproduction and molt . Although ornithologists have been aware of the protracted molts of large birds for many years , no general argument has been proposed to account for the increased time required for flight feather replacement . To the best of our knowledge , we show for the first time how the allometric scaling of flight feather length and flight feather growth rate with body mass sets an upper limit to complete annual replacement of the primaries at a body mass of about 3 kg . Because feather growth rates do not differ between similarly sized species exhibiting simultaneous versus sequential replacement of the primaries , the resource and energy demands of molting cannot explain why primary growth rate fails to increase with mass as fast as primary length . Rather , we suggest that the architecture of a two-dimensional structure emerging from an essentially one-dimensional follicle constrains the rate of feather growth to slow relative to increasing feather length in larger birds . Finally , these allometric relationships prompt us to ask how the 70-kg raptor , Argentavis magnificens , a flying teratorn from the Miocene of Argentina [20] , [21] , could have organized the replacement of its enormous flight feathers to have had sufficient time also to reproduce .
Primaries are the longest flight feathers of the wing , technically defined as the quills that attach to the bones of the hand . Most extant birds have 9 or 10 functional primaries [5] . We used allometric scaling to explain the basis for time constraints on primary replacement in the life histories of large birds . We have related primary growth rate ( K , from the literature , defined as the daily increase in length of individual primaries ) and both length of the longest primary and summed length of all the primaries ( L , from museum specimens ) to body mass ( M ) across a wide size-range of birds ( masses from [22] ) by the allometric function Y = aMb , where a is a scaling constant and b is the power of the relationship of Y to mass . Primary growth rate scales as M0 . 171 ( Figure 1A ) , close to a value of M0 . 19 found by Hedenstrom [3] using other data and assumptions , whereas the combined lengths of all the primary flight feathers ( as well as the length of the longest primary ) increases with body mass almost twice as fast , as M0 . 316 ( Figure 1A ) . The ratio of length ( mm ) to rate ( mm/day ) , which is the time required to replace all the feathers one at a time ( days ) , and which is also proportional to the time required to grow the longest primary ( Figure 1B ) , increases as the 0 . 14 power of mass ( M0 . 316/M0 . 171 = M0 . 145 ) . This illustrates why molt is so time consuming for large birds . These scaling relationships set upper and lower limits to the time that birds of different size would need to replace their primaries . Figure 1A approximates the upper time required for molt by assuming the primaries are grown one feather at a time , and Figure 1B approximates the lower limit when all primaries are lost and re-grown at the same time . Of course the actual duration of primary molts varies between these extremes by a factor of close to 10 , depending on the number of primaries grown simultaneously . Birds that fly while molting usually grow only two or three primaries on each wing at the same time . For example , rough-winged swallows , Stelgidopteryx serripennis , are 15 . 9-g aerial foragers that replace an average of only 1 . 8 primaries at a time , because they forage on the wing while molting [23] . For a 15 . 9-g bird , the allometric relationships in Figure 1A predict that replacing the nine primaries , one feather at a time , would take 190 days . Adjusting for the number of primaries grown simultaneously reduces this estimate to 105 . 5 days , which closely matches empirical observations [23] . Simultaneous replacement of the flight feathers is characteristic of many water birds ( loons , grebes , waterfowl , many rails , and some alcids ) that can swim and dive to forage and escape predators while flightless [24] . In Figure 2B , the distance between the allometric relationships of the length of the longest primary ( M0 . 313 ) and of primary growth rate ( as in Figure 1A , M0 . 171 ) estimates the time ( M0 . 142 ) that simultaneous replacement of the primaries would render an individual flightless . In most forms of simultaneous primary replacement , secondary flight feathers ( shorter flight feathers , proximal to the primaries ) are replaced at the same time as the primaries , so the full period of flightlessness corresponds to the time required to replace the longest primary—estimated to be 57 days , and observed to be 63 days , for 11 . 8-kg mute swans Cygnus olor . The actual period of flightlessness is somewhat less , because individuals regain flight a few days before the longest primaries are fully grown [25] . For birds that continue to fly while molting , the diverging allometric curves of Figure 1A illustrate how the time required to replace the primaries one by one increases dramatically with body size . Large birds that continue to fly while molting reduce the time spent replacing primaries both by growing several primaries simultaneously and by retaining individual feathers for two or , rarely , even three years [2] , [17] , but they still spend an ever-increasing fraction of the annual cycle replacing flight feathers . Figure 1C illustrates the body size–dependence of the shift from complete to incomplete primary molts . Most individuals of species with masses below 1 kg replace all of their primaries annually , whereas most individuals of species with masses over 3 kg spread the primary molt over two or more years ( Figure 1C ) . The broad size range for this transition reflects special circumstances for many species . For example , numerous small owls have incomplete molts , possibly because the flight feathers of these nocturnal birds suffer little degradation from ultraviolet light [4] and can be used for more than one year . Some very large birds replace all of their flight feathers every year because they overlap molt and breeding [10] , [11] or because they molt for many months . Male wild turkeys ( Meleagris gallopavo ) do not participate in parental care and so they can replace primary feathers for six months each year , beginning well before females [26] . Early theoretical analyses suggested that the size of birds with sustained flapping flight would be limited by the power required for flight , which increases as M7/6 , and the power available for flight , which increases as M3/4 [27] , [28] . If these curves actually crossed at about 15 kg , the theory might explain the size of the largest swans and pelicans with sustained flapping flight . Recent analyses by Chattergee and colleagues [21] , [29] applying helicopter streamtube theory have confirmed this suggestion , finding that the upper limit of sustained powered flight for birds and pterosaurs is about 15 kg . We explored whether flight feather replacement might additionally constrain body size evolution in flying birds by considering size distributions of species using each of the three fundamental modes of primary replacement . Patterns of primary replacement have been described for many birds; further , most birds replace the secondary flight feathers during the primary molt , so secondary replacement does not add to the time spent molting flight feathers [12] , [16] , [30] . Thus , analyses of size constraints based on primary replacement patterns should be general to most birds . We made three predictions . First , the mode of the size distribution for birds with simple molts ( species with a single wave of primary replacement ) should be small and the right tail of this distribution should fail to approach the 15-kg limit for powered flight [27] , [28] . When the primaries are replaced in a single wave , the only way to reduce the time in molt is to grow more feathers simultaneously . However , the resulting large gaps in the primaries would be detrimental to flight [7] , [8] , especially in large species that are heavily wing-loaded [31] . Thus large size and simple primary molts should be incompatible for most species . Second , the size distribution of birds with complex modes of primary replacement should be larger than that for birds with simple primary replacement . Complex molts generate multiple waves of feather replacement ( either by stepwise molts or by dividing the primaries into at least two replacement groups ) . Complex primary molts allow more feathers to be replaced at once , and also reduce the size of gaps in the wing surface because adjacent primary feathers partially overlap each other [32] . Because complex molts reduce time constraints on molting by maximizing the number of feathers growing simultaneously , compared with loss in wing area , the modal size of species with complex molts should exceed that for species with simple molts; further , the right tail of this distribution could extend towards the upper size limit for powered flight of about 15 kg [27] , [28] . Third , species with simultaneous flight feather replacement must be constrained least by the time required to replace their flight feathers ( Figure 1B ) . Hence their size distribution should exhibit the highest mode , and its right tail should extend to 15 kg . This prediction assumes that species that molt simultaneously can meet the energetic and nutritional demands of growing their many flight feathers simultaneously and , thus , reduce the time required to replace all their flight feathers to the time needed to grow their longest primary ( Figure 1B ) . The observation that feather growth rates do not differ between species with simultaneous and sequential replacement of the primaries ( see below ) supports this assumption . Because replacing the flight feathers simultaneously is so time-efficient , simultaneous replacement of the wing quills should also be favored in small aquatic species that can safely undergo a period of flightlessness , possibly giving the body size distribution for simultaneous molters a left skew . Figure 2 presents four sets of size distributions with cartoons illustrating the primary and secondary flight feathers ( Figure 2A ) and the three fundamental modes of primary replacement ( Figure 2B–2D ) . The distribution of log-transformed body masses for all flying birds regardless of molt strategy ( Figure 2A , masses from [22] ) exhibits a relatively small modal size ( ∼13 g ) and a strong right skew ( g1 = 0 . 794 , p<0 . 0001 , n = 9 , 324 ) , which characterizes size distributions for most animals [33] , [34] . Among species with simple molts , log10 ( M ) ( mode = 24 g , Figure 2B ) is also strongly right-skewed ( g1 = 0 . 634 , p<0 . 0001 , n = 4 , 163 ) , but the extreme right tail of this distribution falls short of the upper size limit of contemporary flying birds ( 15 kg ) . Species with complex molts are much larger than those with simple molts ( Mann Whitney p<0 . 0001 ) , having a modal body mass of 133 g and a right tail that reaches the size of the largest flying birds ( Figure 2C ) . The size distribution of species with complex molts is not significantly skewed ( g1 = 0 . 096 , p>0 . 20 , n = 1 , 043 ) , as predicted , presumably because the right tail is constrained by the power requirements for flight [27] , [28] and because the left tail is drawn out by numerous small tropical species that have complex modes of primary replacement to increase breeding frequency [32] . That complex molts permit larger body sizes than simple molts suggests that , if birds must fly while molting , a transition to one of the two complex modes of primary replacement is prerequisite to evolving body sizes that approach 15 kg . Species with simultaneous primary replacement crowd the maximum size of flying birds ( Figure 2D ) and do so even more strongly than those with complex primary molts ( Figure 2C ) . At 750 g , the modal size for species that molt simultaneously significantly exceeds that of species having both simple and complex molts ( Mann-Whitney p<0 . 001 for both simple and complex molts ) . The size distribution associated with simultaneous primary molts is slightly , but not significantly , left skewed ( Figure 2D; g1 = −0 . 201 , p>0 . 10 , n = 344 ) , presumably because the power requirements for flight sets an upper size limit [27] , [28] , constraining skew to the left tail of this distribution . The left skew is generated by small species—such as dippers , small alcids , and small rails—with safe molting sites that permit temporary flightlessness . Simultaneous flight feather molts should be favored in these small species for several reasons . First , simultaneous molts are always complete ( Figure 1C ) , eliminating replacement asymmetries that have fitness costs [35] . Second , no developmental organization is required to maintain symmetry in flight feather replacement during simultaneous molt . Third , simultaneous replacement of the primaries minimizes time conflicts between molt and breeding . Finally , simultaneous flight feather molts may be particularly energy efficient if feathers that do not suffer the strain of use while growing can be grown with less cost [36]; we know of no data addressing this possibility . We found no evidence that primary growth rate during simultaneous molt is reduced by the energy and nutrient demands of growing all of the flight feathers at once . We divided the 43 species with feather growth rates ( Table 1 ) into two groups , those that replace their primaries simultaneously ( n = 15 species in two orders ) and those that fly while molting ( n = 28 species in eight orders ) . We used analysis of covariance ( see Methods ) , with body mass as the covariate to compare feather growth rates between these groups . Remarkably , growth rate did not differ between species with simultaneous primary molt and those that fly while molting ( F = 1 . 0; degrees of freedom = 1 , 40; p = 0 . 32; Figure 3 ) . Because primary growth rates are similar for birds that grow two or three versus ten primaries simultaneously , primary growth rate seems not to be limited by energy or nutrient demands; others have suggested that growth rate might be limited by follicular-level constraints on the rate at which feathers can be generated [1] , [37] , and we explore this below .
Our discovery that the time required to replace the primaries ( either one by one or all simultaneously ) increases disproportionately with body size as M0 . 14 provides a general explanation for much of the variation in primary molt patterns in birds . All birds share the same annual cycle of environmental conditions and seasonal periods available for reproduction , molt , migration , and other activities . The slower feather growth in large species constrains their allocation of time to molt and the degree to which the molt can be completed in a single year . At one extreme , most small temperate species are well known to replace all of their primaries annually , whereas many large species take two or more years to complete their primary molt ( Figure 1C ) . Large species , in which flight feather replacement is typically incomplete , probably grow stronger flight feathers , but we are unaware of data addressing this possibility . Two special adaptations in the molt sequence typify large birds that fly while molting and that often or always have incomplete molts—stepwise primary replacement and division of the primaries into two molt series ( Figure 2C ) . These modes of primary replacement likely evolved to minimize time conflicts between molt and breeding in large birds and to minimize the increase in wing loading that accompanies reduced primary feather area during molt . Large birds that are rendered flightless by simultaneous flight feather molts always replace all of their primaries annually ( Figure 1C ) , and the modal size for species with simultaneous primary molts most closely approaches the size of the largest flying birds ( Figure 2D ) , implying that simultaneous replacement of the primaries permits size increases by dramatically reducing the time required to replace the flight feathers . Finally , primary growth rate is not depressed in species that grow all their flight feathers simultaneously , suggesting that feather growth rate does not depend on the availability of energy or nutrients . We suggest below that feather growth rate is similarly constrained in sequential and simultaneous molt systems by similar architecture of the growing region at the base of the feather . That flight feather growth rate increases less rapidly with respect to body mass than does feather length offers a general explanation for the impact of molting on avian life histories . Large species have long reproductive cycles and molt periods , with the result that individuals often replace fewer flight feathers in a molt that follows successful breeding [38] , [39] . An individual having a succession of such incomplete molts might accumulate so many worn feathers that its success in subsequent breeding attempts might decline , even to the point of skipping a breeding opportunity to clear over-worn flight feathers from the wing [2] . A large investment in breeding in one year often results in reduced adult survival or reduced breeding success in the following year [40]–[43] , but the mechanism underlying this trade-off has been elusive . Accumulated feather wear may well be the culprit , particularly for large species . Even small species with complete molts apparently grow low-quality feathers after heavy investment in breeding [44] , [45] . This suggests that feather quality likely links high breeding investment in one year to low success the following year , even though the long post-breeding period available to small temperate-latitude species seems more than adequate for a complete physiological recovery from investment in reproduction . Feathers simply cannot be repaired ! The time constraint on molting in large birds cannot be overcome by growing more feathers simultaneously because of the size-related scaling of the power required for sustained flight and the maximum power available from the flight muscles [28] . For small birds , maximum power is considerably larger than that required for sustained flight , so small birds can fly with large molt gaps in their wings . For large birds , the difference in the power required for sustained flight and the maximum power available is relatively small , making flight with proportionately similar molt gaps impossible . Thus large birds that fly while molting cannot compensate for the relatively slow growth of their primaries by replacing more primaries simultaneously . The allometric disparity between feather size and feather growth lead us to ask how the 70-kg Argentavis , with a wing span of 7 m and outer primaries that were 1 , 500 mm long [20] , almost four times those of the mute swan , could replace its enormous wing quills frequently enough to maintain good flight performance and reproduce . California and Andean Condors have masses of only 10 kg and 12 . 5 kg , respectively , and California Condors need 2–3 years to replace all of their primaries [17] . Argentavis was simply so huge that it might have overcome time constraints on molting by replacing its enormous flight feathers simultaneously , as do the largest geese and swans . Perhaps it did so every 2–3 years by storing sufficient protein in muscles to shelter in caves or cliffs for a simultaneous replacement of the wing quills , which was estimated to require 74 days ( Figure 1B ) . Although no living raptors replace their primaries simultaneously , the evolutionary transition from sequential to simultaneous replacement of the flight feathers might require few changes in the neurophysiological controls that regulate molt; indeed , some individual flamingos and hole-nesting hornbills have been observed change molt patterns , sometimes molting sequentially and retaining flight , and sometimes molting synchronously and becoming flightless [46] , [47] . Because basal metabolic rate increases with body size at an allometric coefficient of about 0 . 72 , whereas fat loading increases with an allometric coefficient that is greater than 1 . 0 , long fasts are possible for large species [48] . All living penguins fast while replacing their body plumage on land and use protein stored in their breast muscles to build feathers . In the 35-kg Emperor Penguin Aptenodytes fosteri , this fast lasts about 35 days , during which time individuals lose 50% of their body mass [49] . Several fossil penguins , which surely also fasted while molting on land , weighed up to 100 kg [50]–[52] . If penguins can store enough protein and energy to replace their very dense and heavy body plumage while fasting , then our suggestion that Argentavis could have replaced its flight feathers from stored reserves seems plausible . The constraints that feather growth places on molt and other aspects of the annual cycle depend on the positive allometry dictating increasing time required to complete the growth of a single flight feather with respect to increasing body mass . Flight feather growth rate approximates 1/6 power scaling , while flight feather length approximates 1/3 power scaling . Among species of different size that maintain isometric proportions , lengths scale as the 1/3 power of volume . Thus , feather length is dimensionally isometric . Feathers elongate by cell division within a cylinder of collar cells at the base of the growing feather in the follicle , an invagination of the skin [53] . Cell division is followed by cell enlargement , differentiation , and keratinization further along the base of the growing feather and is supplied by blood circulation through the dermal feather pulp within the feather base . The growth zone , within which the barbs of the feather vane also grow , is essentially a linear structure that produces a two-dimensional feather . If the growth zone were to scale in proportion to the length of the grown feather , then the rate of growth would be inversely proportional to the square root ( allometric scaling factor 0 . 5 ) of feather length . We tested this prediction using a log-log regression of feather growth rate on length of the longest primary , and found the predicted allometric coefficient of 0 . 5 ( b = 0 . 50±0 . 05 SD , F = 86 . 7 , degrees of freedom = 1 , 41; p<0 . 0001 ) . Other considerations would include the diameter of the follicle , which clearly increases with feather size , but no comparative data are available . If the length of the cylinder of collar cells had a fixed number of rows of dividing cells regardless of feather length , then the growth rate of the two dimensional feather structure would be related to the one-dimensional circumference of the collar , again leading to an 0 . 5 allometry of growth as a function of size . The length of the growing region of a feather might be constrained by structural considerations , because the base of the feather , which is filled with a soft dermal pulp within a non-keratinized cylinder of dividing and differentiating epidermal cells , is quite weak . It is not unusual for growing feathers to break at this point . Although further measurements of the growing regions of primary feathers will be required to work out the basis for the square-root allometry between growth rate and feather size , the linear-to-surface relationship that transforms the cylindrical growing region into a two-dimensional feather provides a plausible mechanism at this point for understanding variation in patterns of primary feather molt as a function of body size in birds and how molt might set an upper limit to the size of flying birds .
Estimates of primary growth rate ( from repeated measures of growing feathers ) were obtained from the literature for 43 species of birds ( Table 1 ) . For each species , we measured the lengths of the primary flight feathers for one adult male and one adult female using museum specimens , and averaged values for the two sexes in our analyses . For 77 species across a large size range of flying birds , we estimated the fraction of adults that had replaced all their primaries in the previous molt by examining flight feather condition on 20 museum specimens obtained during nonmolting periods . When 20 adults were not available for a species in the collections we examined , fewer specimens were sampled ( Table S1 , numbers of each species examined ) . To compare distributions of avian masses with respect to mode of primary replacement , we used the masses for the birds of the world compiled by Dunning [22] . We included races within species if they differed in mass by 10% or more ( some differed by more than 100% ) . We used references [5] , [15] , [54] , [55] and Rohwer ( data not shown ) to characterize the mode of primary replacement , which , unfortunately , has not been described for several major groups of birds . When mode of primary replacement was assigned using [55] or Rohwer ( data not shown ) , we assumed that all members of a genus followed the molt strategy known for any member of that genus , unless additional data were available or unless body mass variation was too great to safely generalize . To analyze the relationship between the mode of primary replacement and body size , we divided the complexities of primary replacement across birds into three basic modes , Simple , Complex , and Simultaneous [15] . Some cuckoos and kingfishers do not fit these categories and were omitted [5] . In species with Simple primary replacement , molt begins at innermost P1 and proceeds distally until P9 or P10 is replaced . All species in this category feature a single wave of feather replacement , but they often lose adjacent feathers in quick succession , resulting in large gaps in their wings . Complex primary replacement occurs in two ways , one or the other of which usually characterizes large species that maintain the ability to fly while molting . In the first , called stepwise molting , the primaries constitute a single molt series [56] , but several waves of feather replacement progress through the primaries simultaneously in adults [5] , [32] , [37] , [57] . In the second , the primaries are organized into two separately activated and nonoverlapping molt series; this mode of replacement generates two waves of growing primaries if both series are activated during a single episode of molting [5] , [16] , [58] . The third mode of primary replacement is Simultaneous , whereby all primaries ( and , usually , all secondaries ) are lost and re-grown more or less simultaneously , resulting in a 3–6-week period of flightlessness . Allometric relationships between feather length , feather growth rate , and body mass were determined by regression and analysis of covariance of log-transformed values based on type III sums of squares , in which taxonomic orders ( Anseriformes [n = 10] , Passeriformes [16] , Coraciiformes [1] , Procellariiformes [1] , Columbiformes [1] , Falconiformes [4] , Galliformes [3] , Charadriiformes [2] , and Gruiformes [5] ) were entered as a main effect to avoid fortuitous relationships resulting from heterogeneity among taxa . Interactions between taxa and the independent variable were not significant and were dropped from the models . Because the regression slopes of models with taxon as a main effect did not differ from those obtained from simple regressions , we report here the slopes of the simple regressions ( 41 error degrees of freedom in each case ) : longest primary feather versus body mass , b = 0 . 325±0 . 010 , p<0 . 0001 , R2 = 0 . 961; sum of primary lengths versus body mass , b = 0 . 316±0 . 009 , p<0 . 0001 , R2 = 0 . 965; growth rate of primary versus body mass , b = 0 . 171±0 . 017 , p<0 . 0001 , R2 = 0 . 713 . Analyses were carried out with the GLM procedure of the Statistical Analysis System version 9 . 1 ( SAS Institute ) . | The pace of life varies with body size and is generally slower among larger organisms . Larger size creates opportunities but also establishes constraints on time-dependent processes . Flying birds depend on large wing feathers that deteriorate over time and must be replaced through molting . The lengths of flight feathers increase as the 1/3 power of body mass , as one expects for a length-to-volume ratio . However , feather growth rate increases as only the 1/6 power of body mass , possibly because a two-dimensional feather is produced by a one-dimensional growing region . The longer time required to grow a longer feather constrains the way in which birds molt , because partially grown feathers reduce flight efficiency . Small birds quickly replace their flight feathers , often growing several feathers at a time in each wing . Larger species either prolong molt over two or more years , adopt complex patterns of multiple feather replacement to minimize gaps in the flight surface , or , among species that do not rely on flight for feeding , simultaneously molt all their flight feathers . We speculate that the extinct 70-kg raptor , Argentavis magnificens , must have undergone such a simultaneous molt , living off fat reserves for the duration . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [
"evolutionary",
"biology/evolutionary",
"ecology",
"ecology/physiological",
"ecology"
] | 2009 | Allometry of the Duration of Flight Feather Molt in Birds |
Dengue , caused by the four serotypes of dengue virus ( DENV ) , is the most prevalent mosquito-borne viral disease of humans . To examine the incidence and transmission of dengue , the authors performed a prospective community-based cohort study in 5 , 545 children aged 2–14 years in Managua , Nicaragua , between 2004 and 2010 . Children were provided with medical care through study physicians who systematically recorded medical consult data , and yearly blood samples were collected to evaluate DENV infection incidence . The incidence of dengue cases observed was 16 . 1 cases ( range 3 . 4–43 . 5 ) per 1 , 000 person-years ( 95% CI: 14 . 5 , 17 . 8 ) , and a pattern of high dengue case incidence every other year was observed . The incidence of DENV infections was 90 . 2 infections ( range 45 . 2–105 . 3 ) per 1 , 000 person-years ( 95% CI: 86 . 1 , 94 . 5 ) . The majority of DENV infections in young children ( <6 years old ) were primary ( 60% ) and the majority of infections in older children ( ≥9 years of age ) were secondary ( 82% ) , as expected . The incidence rate of second DENV infections ( 121 . 3 per 1 , 000 person-years; 95% CI: 102 . 7 , 143 . 4 ) was significantly higher than the incidence rate of primary DENV infections ( 78 . 8 per 1 , 000 person-years; 95% CI: 73 . 2 , 84 . 9 ) . The rigorous analytic methodology used in this study , including incidence reporting in person-years , allows comparison across studies and across different infectious diseases . This study provides important information for understanding dengue epidemiology and informing dengue vaccine policy .
Dengue is the most common mosquito-borne viral infection worldwide , causing an estimated 40 million cases and 500 , 000 hospitalizations annually [1] . Most infections with the four dengue virus serotypes ( DENV1-4 ) occur in urban and semi-urban areas of tropical and sub-tropical countries , where DENV is transmitted by Aedes aegypti and Ae . albopictus mosquitoes [2] . DENV infection results in a spectrum of disease , ranging from inapparent infection ( 50–90% of infections ) to classic dengue fever , a self-limiting acute illness with headache , retro-orbital pain , myalgia , arthralgia , rash , and at times hemorrhagic manifestations , to life-threatening syndromes characterized by vascular leakage , hemorrhage , and shock [3] . Exposure to one serotype of DENV provides lifelong immunity to that serotype , but does not confer lasting protection to the other three serotypes; in fact , prior infection with a different DENV serotype is the single greatest risk factor for development of severe disease [4] . The earliest reports of dengue in the Americas date back to the 18th century , and dengue caused epidemics in North and South America throughout the 20th century [5] . Improved socioeconomic status , waste and water management , and behavioral factors controlled dengue in the United States , and the Pan American Ae . aegypti eradication campaign in the 1950's and '60's greatly reduced DENV transmission in Latin America [5] . However , when the program ceased , Ae . aegypti mosquitoes and DENV soon returned . In the early 1970's , only DENV-2 was endemic throughout the Americas , with limited reports of DENV-3 activity [6] . In 1977 , DENV-1 was introduced , followed by DENV-4 in 1981 and an Asian DENV-3 genotype in 1994 [7] . Currently , all four serotypes circulate throughout the continent . The first reported dengue epidemic in Nicaragua occurred in 1985 , when >17 , 000 cases and seven deaths were documented , caused by DENV-1 and DENV-2 [8] . Intensive vector control efforts resulted in no dengue outbreaks until the early 1990's , when yearly outbreaks of DENV-1 , DENV-2 and DENV-4 were reported , followed by a large DENV-3 epidemic in 1994–5 [9] , . Since then , all four DENV serotypes have circulated , but unlike Asia where they are hyperendemic , in Nicaragua one serotype typically dominates each season [11] , [12] , [13] , [14] , [15] . In our cohort , epidemics peak during the rainy season ( especially August-January ) , although a low level of cases occur throughout the year [16] . DENV is the only flavivirus known to be circulating in humans in Nicaragua , and since yellow fever is not an endemic disease , the Nicaraguan population does not receive the yellow fever vaccine ( A . Balmaseda , unpublished ) . The Pediatric Dengue Cohort Study , an ongoing prospective cohort study of dengue in children in Managua , Nicaragua , was established in August 2004 to determine the incidence of DENV infection and dengue cases , characterize the clinical spectrum of disease , and study viral and immunological determinants of DENV infection outcome [17] . A report presenting data from the first four years of the study has been published previously [16] . This paper presents results from the first six years of the Pediatric Dengue Cohort Study .
This study was conducted as a collaboration between the Nicaraguan Ministry of Health and the University of California , Berkeley . The study was approved by the Institutional Review Boards ( IRBs ) at the University of California , Berkeley , the Nicaraguan Ministry of Health , and the International Vaccine Initiative . Written consent was obtained from a parent or guardian , or if the guardian was illiterate , the consent form was read aloud in the presence of a witness and the guardian's thumbprint was obtained in lieu of a signature , as approved by the IRBs . Verbal assent was obtained from all children aged six years and older . The Nicaraguan Pediatric Dengue Cohort Study ( PDCS ) is an ongoing prospective cohort study in children two to fourteen years of age in Managua , Nicaragua . A detailed description of the study design , methods , and study population has been published previously [17] . Briefly , recruitment into the study began through house-to-house visits in August 2004 . All children two to nine years old living within the study area were invited to participate . At enrollment , families agreed to bring their children to the study health center , Health Center Sócrates Flores Vivas ( HCSFV ) , for medical care at the first sign of illness . Each year , an annual blood sample was collected in July/August to enable assessment of DENV infection . The initial consent covered three years of study participation; in 2007 , the study was extended for an additional three years; in 2009 , the study was extended for an additional year , and in 2011 , for another three years . During the first three years of the study , children were eligible to participate until they reached the age of 12 . From the fourth year onwards , the protocol was modified to extend eligibility to 14 years of age . The focus of this report is the first six years of the study . The cohort was sized such that even in years of relatively low DENV transmission , a minimum number of symptomatic cases would be identified . Participants are provided with free medical care 24 hours/day , 365 days/year through study physicians at the HCSFV . Children requiring hospitalization are transferred to the study hospital , the National Pediatric Reference Hospital ( Hospital Infantil Manuel de Jesús Rivera ) by study staff . At the HCSFV , children are examined by study physicians , and medical data is recorded systematically on study collection forms . Data is collected on approximately 80 variables including vital signs , temperature , musculoskeletal pain , respiratory symptoms , gastrointestinal symptoms , indicators of dehydration , and rashes and other skin anomalies . Upon medical examination , children are categorized into one of four categories according to their symptoms: suspected dengue case meeting the WHO case definition; undifferentiated febrile illness; febrile illness with defined non-dengue focus; and non-febrile illness or injury . Follow-up time was calculated as the amount of time between enrollment and the end of the reported study period ( June 30 , 2010 ) or withdrawal from the study . For those lost to follow-up , person-years were calculated as the time between enrollment and the last contact with study personnel , plus one-half the time between the last contact and the date recorded as lost to follow-up . Dengue cases were excluded from contributing person-time for one month following illness . Analysis of DENV infections was limited to those participants who completed the year and contributed a blood sample at the beginning and the end of the year . Analysis of second and third DENV infections was further limited to participants who entered into the cohort dengue-naïve . In order to provide conservative estimates of DENV infection incidence , since the exact timing of the DENV infection could not always be ascertained , persons who experienced a DENV infection in a given year contributed person-time for that entire year . A Poisson distribution was used to calculate 95% CIs for the incidence rates . Participant age used in the analysis of dengue case incidence was defined on a weekly basis since their exact age at the time that they were a dengue case can be ascertained . For the DENV infection analyses , participant age was defined as the age of the child when their annual sample was collected . In order to adjust for the sensitivity of the Inhibition ELISA test , each child's study ID was randomly sampled with replacement to match the observed number of children in the dataset , and then all observations from the selected children were used to create a new dataset . The expected number of DENV infections was equal to the number of observed infections in this new dataset divided by the sensitivity of the Inhibition ELISA test . The sensitivity was obtained by determining the number of symptomatic DENV infections reported in this study that were correctly identified as DENV infections by Inhibition ELISA in paired annual samples . The incidence per 1 , 000 person-years was 1 , 000 times the number of DENV infections divided by the number of person-years in the new dataset ( = 1 , 000*infections/ ( sum ( days ) /365 . 25 ) ) . This was performed 1 , 000 times to calculate a mean incidence and confidence intervals . Statistical analyses were performed in STATA , version 12 ( StataCorp LP , College Station , TX ) .
During the first six years of the PDCS ( August 2004 to June 2010 ) , 5 , 545 children participated , contributing 21 , 839 person-years . Yearly participation ranged from 3 , 693 to 3 , 953 children ( Table 1 ) , and mean participation time was 3 . 9 years per child ( range 0 . 02–5 . 9 ) . A total of 1 , 204 ( 21 . 7% ) children were lost to follow-up , 341 ( 6 . 1% ) children completed the time that they had consented to participate in the study and were not re-enrolled into the study , and 340 ( 6 . 1% ) were withdrawn ( Figure 1 ) . Of those lost to follow-up , 844 ( 62 . 2% ) had moved and could not be contacted . Annual loss to follow-up ranged from 3 . 8% to 6 . 9% . Of the 341 children who did not re-enroll in the study , 278 ( 81 . 5% ) did not qualify to re-enroll either because they had reached the maximum age for participation or had moved from the study area . In Years 1–6 of the PDCS , 2 , 601 suspected dengue cases or undifferentiated febrile illnesses were documented , of which 351 ( 13 . 5% ) were laboratory-confirmed as dengue-positive , and 138 ( 39 . 3% ) children with DENV infection were hospitalized . The overall incidence rate of dengue in the cohort was 16 . 1 dengue cases per 1 , 000 person-years ( 95% CI: 14 . 5 , 17 . 8 ) , with yearly symptomatic incidence ranging from 3 . 4 to 43 . 5 dengue cases per 1 , 000 person-years . The highest incidence of dengue was observed in children ≥8 years old ( Table 2 , Figure 2A ) . A majority of cases occurred from August to February , although limited numbers of dengue cases were observed year-round ( Figure 3 ) . In the first year of the study , a majority of symptomatic cases were caused by DENV-1 infection ( 52 . 9% ) ; however , the percentage of cases caused by DENV-2 increased in 2005 through 2008 ( study Years 2–4 ) , reaching 95 . 3% of detected cases in 2007–2008 ( Year 4 ) ( Figure 4 ) . DENV-3 entered the study population in 2008–2009 and was the predominant serotype in 2008–2009 ( 86 . 4% ) and 2009–2010 ( 82 . 4% ) . Two cases of co-infection with two different DENV serotypes were also identified: a DENV-1 and DENV-4 co-infection in 2004–2005 and a DENV-1 and DENV-2 co-infection in 2005–2006 ( Figure 4 ) . A spatio-temporal video of dengue cases in the PDCS from 2004–2010 can be viewed at http://youtu . be/yQOEdsMhoSk . Using the 1997 WHO definitions , of 351 dengue-positive cases , 319 ( 90 . 9% ) had classic dengue fever , 21 ( 6 . 0% ) had DHF , and 11 ( 3 . 1% ) had DSS . The majority of these DHF/DSS cases occurred in two years of the study , with five DHF cases ( 23 . 8% ) and three DSS cases ( 27 . 3% ) in the 2007–2008 dengue season , and 12 DHF cases ( 57 . 1% ) and seven DSS cases ( 63 . 6% ) in the 2009–2010 dengue season . Using the 2009 WHO definitions of dengue severity , 182 cases were classified as dengue with warning signs ( 51 . 6% of dengue cases ) and 53 ( 15 . 0% ) as severe dengue . There was one death due to DENV infection during the six years of the cohort . Analysis of DENV infections was limited to participants who completed each year and contributed a blood sample at the beginning and at the end of the year . In total , 5 , 073 children contributed 19 , 708 person-years with 1 , 778 DENV infections , for an incidence rate of 90 . 2 infections per 1 , 000 person-years ( 95% CI: 86 . 1 , 94 . 5 ) ( Table 3 ) . The incidence of DENV infections by study year ranged from 67 . 0 to 119 . 7 infections per 1 , 000 person-years . Excluding 14-year-old children due to small sample size , among one-year age groups , the highest DENV infection incidence was observed in four-year old children , with an incidence of 100 . 4 infections per 1 , 000 person-years , although the incidence was fairly constant across age groups ( Figure 2B ) . Of the 5 , 073 children who contributed at least one set of paired samples , 3 , 570 ( 70 . 4% ) did not experience a DENV infection during the study , 1 , 250 ( 24 . 6% ) experienced one documented DENV infection , 231 ( 4 . 5% ) experienced two DENV infections , and 22 ( 0 . 4% ) experienced three DENV infections , according to serological analysis . Over the six years , 2 , 779 dengue-naïve children contributed 8 , 881 person-years of time . The incidence of primary DENV infections was 78 . 8 infections per 1 , 000 person-years ( 95% CI: 73 . 1 , 84 . 9 ) ( Table 4 ) . Yearly incidence rates ranged from 45 . 2 to 105 . 3 primary DENV infections per 1 , 000 person-years . Excluding 12- and 13-year-old children due to small sample size , the highest incidence rate of primary DENV infection was observed in nine-year old children ( 109 . 4; 95% CI: 81 . 7 , 146 . 6 ) . Although the highest incidence rates of primary infections were seen in older children , relatively few older children were at risk for a primary infection and therefore the majority of DENV infections in older children were secondary ( Figure 5 ) . The 2 , 813 non-dengue-naïve children contributed 10 , 830 person-years . The incidence of secondary DENV infections was 99 . 5 infections per 1 , 000 person-years ( 95% CI: 93 . 8 , 105 . 7 ) ( Table 4 ) , with annual secondary infection incidence rates ranging from 59 . 6 to 115 . 2 infections per 1 , 000 person-years . The highest incidence of secondary infections was observed in children aged five and under . However , when the percentage of primary and secondary infections was examined by year of age ( Figure 5 ) , the majority of DENV infections in younger children were primary , presumably due to the relatively small percentage of young children at risk for a secondary infection . To examine the potential effects of both waning antibody levels and the use of the Inhibition ELISA assay on our estimate of overall DENV infection incidence , we performed a sensitivity analysis by determining how many symptomatic DENV infections reported in this study were correctly identified as DENV infections by Inhibition ELISA in paired annual samples . This yielded a sensitivity of 79 . 7% , which we then applied to our observed estimate of incidence to arrive at an incidence rate of 112 . 5 DENV infections per 1 , 000 person-years ( 95% CI: 107 . 7 , 117 . 4 ) . Therefore , our observed incidence is a conservative estimate of the true incidence in the cohort . Of the 700 children who entered the cohort dengue-naïve and experienced a primary DENV infection , 138 went on to experience a second DENV infection . The 700 children contributed 1 , 137 person-years of time , yielding an incidence rate of 121 . 3 second DENV infections per 1 , 000 person-years ( 95% CI: 102 . 7 , 143 . 4 ) ( Table 3 ) . Of the 138 children who contributed 188 . 3 person-years of time at risk , 16 experienced third DENV infections , for an incidence rate of 84 . 9 third DENV infections per 1 , 000 person-years ( 95% CI: 52 . 0 , 138 . 7 ) ( Table 3 ) . There were no fourth DENV infections observed during the 8 . 0 person-years of time at risk for a fourth infection . The overall rate of symptomatic cases among DENV infections was 18 . 2 dengue cases per 100 DENV infections ( Table 5 ) . This rate varied dramatically by year; from 4 . 9 cases per 100 infections ( 95% CI: 2 . 7 , 8 . 9 ) in 2006–2007 to 40 . 8 cases per 100 infections ( 95% CI: 34 . 8 , 47 . 8 ) in the 2009–2010 season . The lowest rates of symptomatic cases were seen in the youngest age groups , two- and three-year olds , with rates of 11 . 8 and 10 . 8 cases per 100 infections , respectively . The highest rates were observed in the older age groups , especially in children aged nine and over , with rates of 23 . 6 to 29 . 9 .
To date , this is one of the longest continuous , large-scale , community-based prospective cohort study to characterize dengue cases and DENV infections , and it is currently ongoing . We show that there is a high incidence of DENV transmission in children in Managua , Nicaragua , leading to a substantial incidence of dengue cases , a large proportion of which were hospitalized . Although quite variable year-to-year , on average approximately six times as many DENV infections as cases were documented , illustrating that the number of symptomatic cases substantially underestimates DENV transmission in Nicaragua , consistent with our previous report on this cohort [16] and reports from other regions [16] , [25] , [26] , [27] . Several prospective cohort studies of dengue and DENV infection in Asian and Latin American countries have been reported [16] , [25] , [27] , [28] , [29] , [30] , [31] , [32] , [33] . In Indonesia , during a one-year period of a study in children 4–9 years old , 23 . 2% of children were reported to have experienced a DENV infection [28] . In a seven-month dengue season during a prospective serological study in Thai schoolchildren aged 4–16 years , 5 . 6% of the children experienced a DENV infection , of whom 86% presented as asymptomatic or minimally symptomatic ( defined as absent from school for one day ) [29] . In a three-year prospective cohort study of DENV infections in Thai elementary schoolchildren , 5 . 8% of the children were infected with DENV , with 53% of infections presenting as inapparent DENV infections [25] . In a four-year prospective study of DENV infections in Thai children , 2 . 3% of children experienced a DENV infection per season , and the ratio of inapparent-to-symptomatic infection was 1 . 8∶1 [34] . In Iquitos , Peru , an incidence rate of 20–30 DENV-1 or DENV-2 infections per 1 , 000 person years was observed in a 3 . 5 year-long cohort study of ∼2 , 400 children and adults in years where the population had previously been exposed to DENV-1 or DENV-2; however , a peak rate of 890 infections per 1 , 000 person-years was observed with the introduction of DENV-3 into this population that was completely susceptible to DENV-3 [32] . The overall incidence of DENV infections ( 90 . 2 per 1 , 000 person-years ) observed in this study in Nicaragua was within the range of the estimates reported in studies in Asia , while the incidence of dengue cases ( 16 . 1 per 1 , 000 person-years ) was in general lower than observed in Asia . The incidence rate of second DENV infections in the Nicaraguan PDCS was significantly higher than that of primary infections , presumably due to the fact that children who have already experienced a primary DENV infection and are at risk for second DENV infections live in areas where they are more likely to be exposed to DENV than the overall dengue-naïve study population , who are at risk for primary DENV infections . The rates of second and third DENV infections were not significantly different . In this manuscript , two dengue classification schemes , from the 1997 and the 2009 WHO Guidelines , were used [3] , [24] . This allows for comparison to historical as well as future studies . The percentage of dengue cases qualifying as severe dengue ( 15% ) or dengue with signs of alarm ( 53% ) was higher than those meeting the traditional DHF/DSS classification ( 9% ) , consistent with other reports [35] . Overall , the observed rate of dengue cases among DENV infections was 18 . 2 cases per 100 infections ( range 4 . 8–40 . 8 cases per 100 DENV infections ) , and a clear pattern of alternating seasons of high and low dengue case incidence was observed . This pattern was not due to differences in DENV infection rates , but rather to differences in the rate of dengue cases per DENV infection . As reported in the first four years of this study [16] and contrary to what has been described in Thailand [25] , [26] , [27] , no positive correlation between the incidence of DENV infections and the incidence of dengue cases among DENV infections by year was observed . There were two years in particular where the rate of dengue cases per 100 DENV infections was high , the 2007–2008 dengue season ( 25 . 0 infections per 100 person-years ) and the 2009–2010 dengue season ( 40 . 8 cases per 100 DENV infections ) . An increase in disease severity was also observed in the same two years [15] , [36] . In 2007–2008 , several factors appear to have contributed: waning DENV-1 immunity followed by DENV-2 resulted in more disease severity after a period of approximately two years , plus a clade replacement in DENV-2 led to greater disease severity in DENV-3-immune children infected by the replacing DENV-2 clade , NI-2B [15] . In 2009–2010 , an unusual overlap with influenza pandemic strain H1N1 may have led to the observed atypical dengue presentation , more symptomatic DENV infections , and greater disease severity [36] . In this study , the highest incidence of primary infections was observed in older dengue-naïve children . This seems counter-intuitive , as the majority of DENV infections in older children are secondary ( Figure 5 ) . However , one possible reason for the higher rates of primary infections in older dengue-naïve children is that older children spend more time away from the house and thus have more opportunity to encounter DENV-infected mosquitoes both in the house and in other venues . Interestingly , although the majority of secondary DENV infections are in older children , the highest incidence rates of secondary infections were observed in the youngest children . Two possible explanations for this are first , that older non-dengue-naïve children may have already experienced more than one previous DENV infection and therefore have increased immunity ( type-specific immunity to more serotypes and presumably some level of cross-reactive immunity ) , whereas younger non-dengue-naïve children are more likely to have only experienced a single prior DENV infection and are therefore more likely to be susceptible to the circulating DENV serotype . In fact , some of the older children may have experienced infection with all four DENV serotypes and therefore may not be at risk for infection . However , since many children enter the cohort non-naïve , we are unable to determine who is no longer at risk and therefore should not contribute person-time . Another possible explanation is that there may be a small percentage of the general population that is at greatest risk for DENV infection due to environmental or behavioral risk factors and therefore some children may experience multiple infections at an early age , thereby increasing the rate of secondary infection in some younger children . These findings highlight the importance of analyzing infection incidence rather than only proportions . The strengths of this study include its large size , low loss to follow-up , high participation rates , and year-round follow-up . Many dengue cohort studies only operate during the dengue season , which results in an underestimate of annual incidence of dengue . The rigorous analysis used here , including incidence reporting in person-years , allows comparison across studies and across different infectious diseases . We are currently investigating factors that affect the incidence of symptomatic cases among DENV infections . Another strength of the study is the extensive use of information technologies and intensive quality control procedures since the study's inception [17] . Limitations of the study include ascertainment of cases through enhanced passive surveillance . Thus , some dengue cases may not have been detected due to parents not bringing their child in for healthcare [17] . Another limitation is that serum samples for cohort-wide serological testing were only available from participants once per year; however , due to the longevity of the study , more frequent sampling is not tolerated well by cohort participants . In this study , a ≥4-fold rise in antibody titer in paired annual samples was used to define a DENV infection . It is known that antibody levels begin to wane several months post-infection in dengue cases , but little to no information is available about antibody kinetics in inapparent DENV infections . A third limitation is that our annual sample was collected in July/August of each year , and during several of the study years , dengue transmission began during the sampling period . Thus , it is possible that some individuals may have been sampled very close to a DENV infection . This would result in misclassification of their infection by study year or classification as a DENV infection both in both years ( in those individuals whose antibody levels were still rising at the time of sampling ) when in fact they only experienced one DENV infection . Additionally , we exclude acute dengue cases for one month following infection , which makes incidence estimates conservative as children are likely protected from re-infection for substantially longer . This study relies on Inhibition ELISA to assess inapparent infections . Although no gold standard formally exists for assessing inapparent DENV infection , the plaque reduction neutralization test ( PRNT ) is considered the best method . However , PRNT is extremely labor-intensive , and given the resources available for the study , PRNT or other flow-based neutralization assays [37] , [38] were not a feasible laboratory technique for a cohort of this size . Finally , as this study is limited to children , we are unable to provide information on the burden of dengue in adults in Nicaragua; however , establishing the incidence of dengue in children is particularly important as it is expected that they will be the first target population once there is a licensed dengue vaccine . In summary , we have reported substantial incidence of DENV infections and dengue cases in children in Nicaragua using rigorous analytic methods . We have included several additional years of the cohort study and have presented the data in a form that can be readily used for comparison with incidence rates for other studies or other diseases and extrapolation for burden of disease estimates . Although costly and labor-intensive , cohort studies are the only way to determine the incidence of DENV infection and one of the few ways to capture the true rate of disease , which is greatly underestimated via national passive surveillance systems [39] , [40] . These data are then utilized as a benchmark for establishing expansion factors that are used to estimate the actual disease burden [39] , [41] , [42] and the economic cost of the disease [43] , [44] , [45] , as well as for cost-effectiveness studies of vaccines or other interventions [46] , [47] . Thus , our incidence estimates have significant policy implications for dengue vaccines as they become available . | Dengue is a major public health problem worldwide with 40 million cases annually . We conducted a large-scale prospective cohort study of dengue virus infection in children aged 2–14 years in Managua , Nicaragua . The observed rate of dengue cases was 16 . 1 cases per 1 , 000 persons per year . The observed rate of dengue virus infections was 90 . 2 infections per 1 , 000 persons per year . The observed rate of dengue virus infections is similar to the rates reported in Asian cohort studies , while the rate of dengue cases was lower than that observed in Asian cohort studies . The rate of dengue cases varied more than the rate of dengue virus infections , and a clear pattern of high dengue case incidence every other year was observed . The rigorous analytic methodology used in this study allows comparison of incidence of dengue virus infections and dengue cases across studies and across different infectious diseases . Our estimates of the burden of dengue in Nicaraguan children have significant policy implications for dengue vaccines as they become available . | [
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] | [] | 2013 | The Nicaraguan Pediatric Dengue Cohort Study: Incidence of Inapparent and Symptomatic Dengue Virus Infections, 2004–2010 |
Prediction of drug action in human cells is a major challenge in biomedical research . Additionally , there is strong interest in finding new applications for approved drugs and identifying potential side effects . We present a computational strategy to predict mechanisms , risks and potential new domains of drug treatment on the basis of target profiles acquired through chemical proteomics . Functional protein-protein interaction networks that share one biological function are constructed and their crosstalk with the drug is scored regarding function disruption . We apply this procedure to the target profile of the second-generation BCR-ABL inhibitor bafetinib which is in development for the treatment of imatinib-resistant chronic myeloid leukemia . Beside the well known effect on apoptosis , we propose potential treatment of lung cancer and IGF1R expressing blast crisis .
Biomedical research is changing towards a systems pharmacology view of drug action [1] . In parallel , chemical proteomics ( Figure 1 ) , a postgenomic version of classical drug affinity purifications which use is growing rapidly , has been developed to measure drug target profiles in an unbiased manner [2]–[5] . It usually reveals larger than expected spectra of targets which are causing both therapeutic and adverse effects . Such unbiased target profiles are very valuable entry points to understand which regions of the cell machinery are perturbed by a drug . It is hence desirable to develop new specific algorithms exploiting chemical proteomics profiles . Generally , it is natural that protein interaction networks are involved to characterize drug targets , action on diseases , and potential side effects [6]–[11] . Existing methods are mainly based on the network topology and on an integration of gene expression data and phenotype similarities [12]–[14] . Alternatively , precise modeling of perturbations which change the protein interaction network has the potential to predict new drug targets and to provide a detailed mechanism of action simultaneously [15]–[17] . Beside network approaches , classical gene ontology ( GO ) enrichment analyses of drug targets are commonly used which result in no detailed mechanism but identify different processes and functions of direct involvement [11] , [18] . However , one pivotal aspect is that drug targets can perturb protein interaction networks and biological processes without being directly part of the latter . Therefore , we present a new algorithm which combines direct and peripheral perturbations of functional sub-networks and exploits chemical proteomics drug target profiles . The idea of functional sub-networks is based on the finding that genes associated with the same disease often share protein-protein interactions and gene ontology terms [19] . Our algorithm estimates the drug impact on biological processes and the detailed perturbation effects can be visualized as a network , which facilitates interpretation . Furthermore , we introduce an affinity score to weigh the drug target profile on the basis of interaction strengths . We applied our algorithm to the bafetinib ( NS-187 , INNO-406 ) target profile . Bafetinib is a small molecule tyrosine kinase inhibitor in development for chronic myeloid leukemia ( CML ) [20] . It has been designed to potently and specifically inhibit BCR-ABL and the SRC family kinase ( SFK ) LYN , but no other SFKs , with the purpose of displaying an improved safety profile over multi-kinase and pan-SFK inhibitors , such as dasatinib , while retaining the advantageous dual mechanism of action . We have recently characterized the detailed target profile of bafetinib by chemical proteomics and to interpret the complex dataset obtained is challenging . One of the most popular methods for distinguishing ( and potentially quantifying ) specific drug targets from non-specific background proteins is the competition of soluble drug molecules with the affinity matrix for drug binding proteins ( Figure 1 ) [21]–[23] . Comparison of the protein eluates from a competed and a non-competed drug pulldowns will highlight specific binders , while non-specific binding proteins will not be affected . However , even after correct identification and potentially determination of quantitative interaction parameters for distinct drug-protein pairs , a global or mechanistic understanding of drug effects is but a distant goal requiring some sophisticated experimental and/or theoretical follow-up . Our theoretical effort advances significantly our mechanistic understanding of the effects of bafetinib and provides others with a computational strategy applicable to different drug profiles .
The network is constructed from protein-protein interactions found in the public interaction databases HPRD , MINT , Intact , DIP and BioGRID [24]–[28] . Furthermore , it is supplemented with published interactions of the BCR-ABL core complex which is the primary target of bafetinib in chronic myeloid leukemia ( CML ) [29] . The resulting undirected network contains 11505 proteins and 80363 interactions . The human network of all known protein-protein interactions is associated with its biological processes of gene ontology ( GO ) derived from UniProtKB and Entrez Gene [30]–[32] . All ancestors of the GO tree are assigned in addition to achieve a complete and consistent annotation . In total , the human interaction network consists of 6390 different BP terms . 8939 ( 78% ) nodes of the human interactome are at least associated with one biological process . A uniform functional sub-network is a connected fraction of the interactome , in which all the proteins share the same function , i . e . , one unique GO term . The interactome can contain multiple disjoint functional sub-networks for the same annotation . The recently published drug target profile of the kinase inhibitor bafetinib measured in the cell line K562 is used [33] . Rix et al took three quality criteria into account: ( 1 ) The drug target profile is devoid of proteins in the K562 core proteome . ( 2 ) No frequent hitters are included . ( 3 ) The proteins must be seen in replicates . In addition , splice variants and protein fragments are excluded . The 33 proteins are listed in Table S1 . Bafetinib can impact the uniform functional sub-networks in two ways via its targets ( Figure 2 ) : It is difficult to predict which mode of perturbation has a higher impact . Directly inhibiting a pivotal sub-network member can completely disrupt a function . Nonetheless , biological signaling networks often have multiple alternative routes and protein isoforms to rescue the cell . Drug targets acting at the periphery can modify significantly the function through interaction or modulation of a modification , e . g . , phosphorylation . By this mechanism , the inactivation of different branches and isoforms is possible . Furthermore , functional boundaries are often loosely defined and incompletely annotated . We thus treat both perturbation modes equally and therefore the perturbed functional sub-network ( Figure 2 ) combines the uniform functional sub-networks with all interactions to the peripheral drug targets . This combination could result in joining otherwise disjoint uniform functional sub-networks via a drug target as linker ( see MAPK14 in Figure 3 ) . The direct targets are already members of the uniform functional sub-networks . We define a score snet which predicts how strong functional sub-networks are perturbed by bafetinib . For this purpose different features of the perturbed functional sub-network are combined . The first feature describes how frequent the annotation is present in the sub-network . Peripheral drug targets don't share the functional annotation ( Figure 2 ) , hence they dilute the functional annotation of the sub-networks . To ensure that the function is not underrepresented in the network , a first factor of the score is the ratio of the number nannot , net of nodes which have a specific annotation to the total size nnet of the perturbed functional sub-network . The second feature puts the drug impact in relation to the sub-network size . Generic biological functions result in very big sub-networks , in which the drug targets play overall no important role anymore . Furthermore , the drug should preferentially perturb a function at several different points . Hence , the proportion of the number ndrug , net of drug target nodes to the number nnet of all nodes in the perturbed functional sub-network resembles a good measure . Lastly , the binding affinity at of bafetinib to its targets or the potency of inhibition is important for effective perturbation . In theory , the affinities can be measured in biochemical assays which are not always available . However , we propose hereafter an ad hoc affinity measure derived from chemical proteomics data directly . The impact is summarized in the sum of drug affinities to its targets in the perturbed functional sub-network Tdrug , net divided by the overall affinity of all possible drug targets Tdurg . Combining these factors results in a score for each disrupted functional network: ( 1 ) The last two factors of equation ( 1 ) have an additional role and benefit . Mass spectrometry detection as used in chemical proteomics does not detect direct drug interactors only; it can also detect secondary interactors , i . e . proteins that bind to direct drug interactors . Without prior knowledge , it is difficult to distinguish between direct and indirect interactors but we believe that it is advantageous to use the complete target profile of bafetinib as it embeds the true drug targets into a specific context and increases the crosstalk with annotated nodes of the sub-networks ( the second factor in equation ( 1 ) increases ) . The affinity factor ensures that also true and strong drug targets are part of the sub-network . The affinity of bafetinib to its targets is used to score the impact on sub-networks in equation ( 1 ) . The higher the protein amount in mass spectrometry analysis , the higher the number of different detected peptides covering the protein sequence [34] . Hence , the peptide count pt of each protein is a rough estimate of the amount of pulled-down protein . If soluble bafetinib is supplemented , the soluble drug blocks the binding pocket of its target yielding a reduced amount of pulled-down proteins that are specific drug binders ( Figure 1 ) and thus , their peptide counts pt , comp decrease . This observation is expressed in the first factor of the affinity score . Since in chemical proteomics the drug is always present at a large excess of constant concentration , it is only possible to distinguish the affinities of completely competed proteins by taking the protein amount into account . To down weigh this parameter influence , the logarithm is applied to pt . Thus , the affinity score can be expressed by the following equation: ( 2 ) Due to the reduced complexity of the competed pull-down , it can sporadically happen that pt , comp>pt . This case is seen as no competition and thus the affinity is set to 0 . An empirical p-value is calculated via randomization of the interactome . First , the interaction partners of each node are randomly selected . It is ensured that the degree of each node remains constant . Second , the annotation is randomly assigned to the nodes , while the total number of each term is preserved . The presented algorithm is applied to 500 random instances of the interactome . The empirical p-value is calculated from the fraction of randomized interactomes containing a sub-network with a score equal or better to the tested score divided by the total number of random instances . The highest score of all the random instances smax , rand is 0 . 124 . The presented approach is programmed in the statistical environment R/Bioconductor and available at http://bioinformatics . cemm . oeaw . ac . at/drugDisruptNet [35] , [36] . The provided R package depends on graph , RBGL , snow and GO . db [37]–[39] . Parallelization is done with snow to generate and score random instances [40] . Additionally , the above described data and the results are stored as R data objects . Networks are visualized with Cytoscape [41] . For comparison , classical GO/KEGG/Biocarta enrichment analysis of sets are performed with DAVID [18] .
We present a novel strategy to analyze the mechanisms of action of bafetinib . The target profile is weighted with respect to its drug affinity and its impact on protein interaction networks is scored . Ten perturbed functional sub-networks are scored higher than any sub-network of the 500 randomized interactomes ( smax , rand = 0 . 124 ) , see Table 1 and Figure 3 , 4 and supplementary Figure S1 , S2 . The sub-networks do not necessarily contain all the components of a specific function since several disjoint functional sub-networks can be constructed . Bafetinib is designed to treat BCR-ABL dependent chronic myeloid leukemia ( CML ) . Constitutively active BCR-ABL interferes strongly with apoptosis in malignant cells . We catch this process in our significantly perturbed sub-networks at rank 6 ( Table 1 ) . Furthermore , MAP kinase signaling can also be brought together with pathogenesis and treatment of CML . The top ranked perturbation of “Epidermal growth factor receptor ( EGFR ) signaling pathways” and “Insulin receptor signaling pathway” suggest potential novel domains of treatment for bafetinib and “heart development” indicates a putative side effect . The hit signaling pathways further play important roles in the general perturbed processes of aging , extracelluar structure organization and cell cycle . Finally , phosphorylation is an obvious process to be perturbed by a kinase inhibitor . We discuss pathogenesis , potential new domains of drug treatment and putative side effects of bafetinib in more details . Inactivated apoptosis signaling plays a pivotal role in BCR-ABL dependent CML pathogenesis [42] and is well represented in the significant sub-networks . The perturbed functional sub-network of apoptosis ( Figure 3 ) is disrupted by inhibition of ABL2 , MLTK , LYN and MAPK14 ( p38α ) . These kinases are not annotated themselves as “induction of apoptosis by intracellular signals” but act at the periphery of the uniform functional sub-network . K562 cells express ABL1 , a central node of the network , and its fusion protein BCR-ABL . High amounts of BCR-ABL hide specific ABL1 detection with mass spectrometry . However , western blots proved ABL1 as a competed target of bafetinib in K562 [33] . Hence , the score of perturbation underestimates the impact of bafetinib on apoptosis in CML . The impact of bafetinib on apoptosis in CML is manifested with 5 targeted kinases at the periphery ( Figure 3 ) . The method strongly prefers networks which are attacked by several high affinity drug targets . In theory , a single perturbation might be enough to significantly interfere with a biological function . However , biological signaling networks are often highly redundant thus requiring perturbation at several points in order to observe an effect [43] . Hence , promiscuous drugs like dasatinib are very successful in CML and other cancers and the multi-targeted networks are likely to be of high relevance in drug treatment . Even if we know that the drug has an inhibitory effect on the target kinases , we cannot predict without additional knowledge whether missing phosphorylation has an enhancing or decreasing effect on the biological process . The constitutively active kinase BCR-ABL results in a strong anti-apoptotic phenotype . Inhibition counteracts this behavior [44] . Inhibition of LYN has a similar effect in this context [45] . Contrary to this , MAPK14 inhibition rescues cells from apoptosis [46] . Only through the complex interplay of different signals , the malignant cells die upon treatment as desired . Hence , visualization of the network together with its disturbers strongly aids in interpreting their influence . This is a great advantage compared to simple GO enrichment analysis which does not display the relationship of the proteins to each other . The top ranked perturbed functional sub-network is based on the epidermal growth factor receptor ( EGFR ) signaling pathway ( Figure 4 ) . It is peripherally interacting with six kinases of the drug profile . Three additional kinases are directly interacting with EGFR but also interfering with 7 further proteins of the signaling cascade . Additionally , the crosstalk between the pulled down non-kinase members and the functional network is very high . In total 13 out of 33 EGFR signaling components ( 39% ) are interacting with the drug profile . EGFR is not expressed in hematopoietic cells ( such as K562 ) but this sub-network strongly suggests that bafetinib has the potential to interfere with EGFR signaling for instance in lung cancer cells . Recently , it was shown through the combination of chemical proteomics , phosphoproteomics and functional genomics that dasatinib , a broad-spectrum kinase inhibitor , leads to apoptosis in lung cancer cells via inhibition of SRC , EGFR , FYN and , notably , LYN [47] . Therefore , it is possible that also bafetinib might have a pro-apoptotic effect on these cells as it is also a potent inhibitor of LYN . While expression of dasatinib-insensitive gatekeeper mutants of DDR1 ( or ABL1 ) did not rescue the H292 lung cancer cell line from dasatinib action , the role of DDR1 might be quite different in primary lung cancer cells as several recent reports described this receptor tyrosine kinase to be one of the most highly expressed and phosphorylated kinases in primary lung tumor specimens [48] , [49] . Thus , it is conceivable that bafetinib might exert pro-apoptotic effects on lung cancer cells , and it might do so through simultaneous inhibition of LYN and DDR1 . Second highest is the perturbation of insulin receptor signaling pathway . It was suggested that bafetinib , CGP76030 and nilotinib might overcome imatinib resistance in blast crisis patients which feature BCR-ABL gene amplification [50] . Phase 1 studies could not verify this yet [51] . However , we propose to treat only the subgroup of CML blast crisis patients which expresses IGF1R with bafetinib . The drug targets are strongly interacting with the insulin receptor signaling pathway which maintains survival of hematopoietic cells through IGF1R ( supplementary Figure S1 ) . The IGF1R expression frequency is strongly increased in blast crisis patients ( 73% ) . Inhibition of IGF1R was shown in imatinib-resistant CML to induce apoptosis [52] . IGF1R is not a known direct target of bafetinib but attacking several downstream components simultaneously might show a similar effect as a direct IGF1R inhibition . A potential side effect of several tyrosine kinase inhibitors , like sunitinib and dasatinib , is an increased risk for cardiotoxicity [53] . Observed toxicity in rats can be a result of higher concentration than used in patients [54] . Nevertheless , perturbation of the “heart development” network ( Figure S2 ) indicates some possible risks which should be closely monitored during clinical trials . We validated the robustness of the algorithm by following the rank of the biological process upon leaving-one-out ( supplementary Figure S3 ) . The ranks of the first five sub-networks ( Table 1 ) are generally stable upon loss of a node . High affinity targets are essential to the phenotype which results in increased sensitivity of highly ranked terms to high affinity targets . On the contrary , weaker binders , which are not competed away with free drug , have only a modest effect on the rank . Furthermore , we investigated the effect of hubs on the sub-network ranks , which might exert an influence on the phenotype upon inhibition . It is not clear whether hubs are , in the context of our analysis , highly important or “general signal diluters” . Therefore , we weighted up and then down the affinity of the targets by log10 of their node degree . Multiply by this factor , i . e . increasing hubs importance , the top 6 sub-networks remain unchanged and “cell cycle arrest” even improved its rank by one . The others sub-networks were substituted by “response to insulin stimulus” , “response to peptide hormone stimulus” and “cellular response to hormone stimulus” , which are in line with insulin receptor signaling . Upon down-weighting by division , the top 4 sub-networks still remained unchanged . We conclude for robustness and reasonable independence of local topology . In other words , the function of the sub-network at hand seems to play a strong role in scoring , which is appropriate . To show the general interest of our method we applied the algorithm to data we published recently analyzing lung cancer ( HCC297 ) treatment with dasatinib [47] , another kinase inhibitor . Interestingly , dasatinib is highly promiscuous and pulls down 176 proteins ( 33 kinases ) compared to the 33 proteins of bafetinib . In addition , free compound competition data were not available in this case; we thus exploited IC50s of autophosphorylation , which were available instead ( Table S2A of Ref . [47] ) . The log10 of the IC50s ( in the nM range ) were used to weight the effect of dasatinib on the kinases . Analysis results in 681 significantly hit sub-networks due to the huge kinase profile ( P-value<0 . 002 , Table S2 ) . Even though more than 5% of the human kinases are targeted by dasatinib and , subsequently , many sub-networks are significantly impacted , the top disrupted sub-networks are insightful . For instance , the top 10 ranked biological processes are centered on cell cycle arrest , cell growth and apoptosis . In the highest ranked sub-networks , SRC , LYN and EGFR play a pivotal role , which is absolutely consistent with our experimental data where these three proteins were shown with dasatinib gate-keeper mutants to strongly contribute to cell viability of HCC297 [47] . These results show that the algorithm can provide informative data even in very challenging situations . In comparison to our approach , classical GO enrichment analysis ( p-value<0 . 01 ) of the 33 bafetinib drug targets result in 33 significant biological processes with high redundancy in the GO tree ( supplementary Table S3 ) . Basically , they represent 3 GO terms: cytoskeleton organization ( especially actin filament ) , phosphorylation and regulation of stress-activated protein kinase signaling pathway . Except phosphorylation which is obvious in a target profile of a kinase inhibitor there is no overlap with the perturbed functional sub-networks . The GO term of cytoskeleton organization contains competed and non-competed members of the target profile . The combined attack power of few competed kinases is too low to see perturbation of the large uniform functional sub-network ( 387 members ) which is based on cytoskeleton organization . Enrichment analysis with KEGG and Biocarta pathways ( p-value<0 . 01 ) yielded no hit . In contrast to GO enrichment analysis , the presented method does not as much rely on accurate annotations . Possible missing annotations of drug targets interacting at the periphery with a functional sub-network have only a minor effect on the score . However , we would like to point out that boundaries of pathways and biological processes are very diffuse . Crosstalk between different signaling cascades and metabolic pathways is essential for a living cell . Integrating protein interactions to peripheral drug targets provides a way out of this dilemma and can catch therefore more relevant processes than GO enrichment . Alternatively , augmenting the drug target profile with their direct interactors , results in a set of 831 proteins . GO enrichment analysis ( p-value<0 . 01 ) of this set results in 676 biological processes ( Table S4 ) . Again the first hits are related to general phosphorylation which is obvious for a kinase inhibitor profile . At the ninth rank “regulation of programmed cell death” which is related to perturbed apoptosis is presented with 127 proteins of the augmented set . The highest scored perturbed sub-network of EGFR signaling is only found at position 368 . Even though the disrupted processes are detected with the augmented GO analysis , their ranks are so bad that they would not be considered as relevant . Our approach thus picks the most relevant perturbed functional networks and allows for insights beyond traditional GO enrichment analysis . Competition experiments in chemical proteomics provide an additional layer of security to the drug target profile . Secondary and unspecific binders are difficult to distinguish from true drug targets . They are often similar in the range of peptide counts and other properties . The competition with a soluble drug and our affinity score helps in identifying biological target proteins . Interestingly , unspecific binders influence the perturbation algorithm only marginally since the proteins are dispersed all-over the interactome and have no affinity to a specific uniform functional sub-network . Furthermore , their binding affinity score is 0 . On the contrary , secondary binders of true drug targets increase the crosstalk to the functional sub-network which is attacked by the true target . Hence they can be used advantageously embedding the true targets in a specific context . In conclusion , we identified successfully known mechanisms in CML as well as potential new applications and possible side-effects . We believe that the proposed computational approach can shed light in mechanisms of other drugs including highly promiscuous compounds and when soluble compound competition data are lacking . Hence , we provide an R package at http://bioinformatics . cemm . oeaw . ac . at/drugDisruptNet . | Protein interaction data are accumulating rapidly and , although imperfect and incomplete , they provide a valuable global description of the complex interplay of proteins in a human cell . In parallel , modern proteomics technologies make it possible to measure in an unbiased manner the protein targets of a drug . Such data reveal multiple targets in a view that contrasts with a previously prevalent paradigm that drugs had single – or a very limited number of – targets . In this context of newly available systems level data and more precise and complete information about drug interactions , it is natural to try to determine the global perturbation exerted by a drug on a human cell to identify potential side effects and additional indications . We present a computational method that aims at making such predictions and apply it to bafetinib , a recently developed leukemia drug . We show that meaningful predictions of additional applications to other cancers or resistant cases and likely side effects are obtained that are not straightforward to determine with existing algorithms . Our method has a strong potential to be applicable to other drugs . | [
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"Discussion"
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"oncology/myeloproliferative",
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"hematology/myeloproliferative"... | 2010 | A Computational Approach to Analyze the Mechanism of Action of the Kinase Inhibitor Bafetinib |
The human pathogen Pseudomonas aeruginosa is capable of causing both acute and chronic infections . Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections , while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic , persistent infections . While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted , little is known about the role of dispersion in virulence , the active process by which biofilm bacteria switch back to the planktonic mode of growth . Here , we demonstrate that P . aeruginosa dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells . While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells , the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants . Supernatants of non-dispersing ΔbdlA biofilms were characterized by a lack of many of the degradative activities . Expression of genes contributing to the virulence of P . aeruginosa was nearly 30-fold reduced in biofilm cells relative to planktonic cells . Gene expression analysis indicated dispersed cells , while dispersing from a biofilm and returning to the single cell lifestyle , to be distinct from both biofilm and planktonic cells , with virulence transcript levels being reduced up to 150-fold compared to planktonic cells . In contrast , virulence gene transcript levels were significantly increased in non-dispersing ΔbdlA and ΔdipA biofilms compared to wild-type planktonic cells . Despite this , bdlA and dipA inactivation , resulting in an inability to disperse in vitro , correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models . In contrast , bdlA inactivation rendered P . aeruginosa more persistent upon chronic colonization of the murine lung , overall indicating that dispersion may contribute to both acute and chronic infections .
Pseudomonas aeruginosa is a ubiquitous , Gram-negative opportunistic bacterial pathogen , well known for its remarkable ability to replicate and survive in diverse environments , as well as for causing a variety of acute and chronic human infections . Acute infections are characterized by rapid pathogenic progression , high-level toxin production , and often tissue damage . Chronic infections are characterized by colonization , prolonged persistence , evasion of the host's immune response and tolerance and often resistance to multiple therapeutic agents . The capacity to cause either acute or chronic infections depends to a large extent on the ability of P . aeruginosa to transit from growing planktonically ( free living state ) to surface-attached communities known as biofilm [1] , [2] , [3] , [4] , [5] . While planktonic infections are frequently associated with high virulence and fast growth as observed in sepsis or bacteremia [6] , the development of biofilms is considered to be the root cause of chronic infections [1] , [2] , [3] , [7] , [8] , [9] . Biofilms are composed of microorganisms attached to a solid surface and encased in a hydrated polymeric matrix composed of polysaccharides , protein and DNA . Biofilms form when bacteria adhere to surfaces in moist environments . For example , P . aeruginosa forms what are termed Mode II biofilms [10] within the airways of individuals with cystic fibrosis ( CF ) , with this process playing an important role in CF-associated chronic infections and acting as one of the primary causes of mortality in CF patients [11] , [12] , [13] , [14] . In addition , P . aeruginosa causes a variety of chronic infections in immunocompromised individuals or those suffering from wounds , burns , urinary tract infections , or corneal injury [7] , [15] , [16] , [17] , [18] , [19] . The transition from a planktonic to surface-attached lifestyle is highly regulated . The two-component regulatory systems ( TCS ) including the RetS/GacS/GacA/rsmZ signal transduction pathway , as well as the TCS SagS and BfiSR are required for P . aeruginosa to transition to the surface associated mode of growth and to progress from initial attachment to the formation of mature biofilms [1] , [2] , [3] , [4] , [5] , [20] . Additional regulatory systems required for the formation of mature , three-dimensional biofilms include the TCS BfmSR , MifSR , and SadARS [8] , [21] , [22] . However , these regulatory systems not only regulate the motile-sessile switch but also the growth-mode dependent expression of virulence factors . For example , genome-wide transcriptional profiling suggested that RetS is required for expression of the Type III secretion system ( T3SS ) genes ( psc and pcr operons ) , secreted effectors ExoS , ExoT and ExoY [1] , [23] , [24] , and other virulence factors , and for repression of genes responsible for exopolysaccharide components of the P . aeruginosa biofilm matrix [1] . Conversely , inactivation of retS correlated with hyper-adhesion to mammalian cells but loss of cytotoxicity and attenuated virulence in an acute pneumonia model . In addition to RetS and GacS/GacA/rsmZ , regulation of T3SS gene expression also requires SadARS [8] . Considering the number of regulatory systems affecting T3SS expression , it is not surprising that T3SS-mediated cytotoxicity is considered a key virulence mechanism of P . aeruginosa , allowing for the contact-dependent translocation of pathogenicity factors into eukaryotic host cells , with T3SS inactivation attenuating P . aeruginosa virulence in a murine acute infection model [20] , [25] , [26] , [27] , [28] . Additional known virulence factors produced by P . aeruginosa include hydrogen cyanide , elastase , phenazines , and rhamnolipids . Hydrogen cyanide is believed to be the primary toxic factor secreted by P . aeruginosa that is responsible for killing Caenorhabditis elegans [29] , and has been detected at elevated levels in the infected lungs of patients suffering from CF [30] . Chitinase is expressed at high cell densities and in biofilms and was shown to be able to bind and degrade colloidal chitin [31] , while elastase has been shown to cause localized tissue damage [32] . The redox-active pigments phenazines can generate an oxidative stress on the host and are required for “fast killing” of C . elegans [33] . Rhamnolipids interfere with the internalization of attached particles , reducing the level of phagosome-lysosome fusion of internalized targets within macrophages , and inhibiting the response of alveolar macrophages [34] , [35] . While the clinical relevance of planktonic and biofilm cells in acute and chronic infections , respectively , has been well established , little is known about the contribution of biofilm dispersion to the virulence phenotype of P . aeruginosa and infections . Dispersion is the last yet a very important step in the development of biofilms that allows bacteria to successfully return from the biofilm to the planktonic growth state to spawn novel communities in new locales [36] , [37] . Biofilm dispersion can be induced by exposure to matrix-degrading enzymes and surface protein releasing factors [38] , [39] , [40] . In P . putida and P . fluorescens , the large adhesive outer-membrane protein LapA mediates attachment to surfaces and to matrix components [41] , [42] , [43] . Gjermansen et al . [41] demonstrated that in P . putida , release of LapA from the cell surface results in biofilm dispersal and is mediated through the activity of the periplasmic protease LapG . Additional mechanisms linked to dispersion include cell death , with filamentous phage Pf1-mediated cell lysis serving as an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells [44] . The process of biofilm dispersion in various organisms including P . aeruginosa , P . putida and Schewanella oneidensis is furthermore induced upon sensing a myriad of environmental cues such as variation in oxygen or carbon substrate concentration and sensing the signaling molecule cis-2-decenoic acid [36] , [38] , [45] , [46] , [47] , [48] , [49] , [50] . Oxidative or nitrosative stress , induced upon exposure to exogenous or endogenous nitric oxide ( NO ) , has been linked to biofilm dispersion . A role of oxidative or nitrosative stress was further supported by a P . aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration ( nitrite reductase , ΔnirS ) not dispersing [51] . Biofilm dispersion has also been linked to the modulation of the intracellular signaling molecule cyclic di-GMP ( c-di-GMP ) , high levels of which promote sessile growth , while low levels correlate with planktonic existence [52] , [53] , [54] . Levels of c-di-GMP are enzymatically modulated by diguanylate cyclases ( DCG ) , proteins containing a GGDEF domain , and phosphodiesterases ( PDE ) harboring either an EAL or HD-GYP domain [52] . In P . aeruginosa , dispersion upon exposure to NO and elevated nutrient concentrations has been linked to the reduction of the cellular c-di-GMP levels , requiring the phosphodiesterases DipA and RbdA [46] , [55] , [56] , [57] . In addition , the membrane-bound phosphodiesterase NbdA was found to be specific to the dispersion response following exposure to NO [48] . The chemotaxis transducer protein BdlA ( Biofilm dispersion locus A ) appears to play a central role in the dispersion response by P . aeruginosa biofilms , as inactivation of bdlA impaired dispersion by P . aeruginosa biofilms in response to various nutrients , NO , ammonium chloride , and heavy metals [56] , [57] . However , BdlA does not directly contribute to the observed reduction of cellular c-di-GMP levels in dispersed cells as BdlA lacks domains required for c-di-GMP modulation . Instead , BdlA harbors two sensory Per Arnt Sim ( PAS ) domains and a chemoreceptor domain , TarH . The closest known BdlA homolog is the FAD-binding Aer , the redox potential sensor and aerotaxis transducer in Escherichia coli . Alanine replacement mutagenesis of BdlA-PAS domain residues D14A , N23A , W60A , I109A , W182A , that were previously demonstrated to be essential for aerotaxis in Aer , resulted in impaired dispersion , while alanine replacement mutagenesis of residue G31A resulted in the mutant strain transmitting a constant signal-on bias as it rendered P . aeruginosa biofilms hyper-dispersive [58] . The findings suggested BdlA to likely function as a sensory protein [58] . However , for BdlA to contribute to the dispersion response , it must first be activated via unusual , non-processive proteolytic cleavage at a ClpP-protease-like cleavage site located between the PAS sensory domains PASa and PASb within the BdlA protein [45] . Proteolysis of BdlA was stimulated by increased c-di-GMP levels present in biofilms , and dependent on the protease ClpP , the chaperone ClpD , and BdlA phosphorylation at tyrosine-238 [45] . Once activated , BdlA oligomerizes with the phosphodiesterases DipA and RbdA , thus forming a regulatory network that modulates the intracellular c-di-GMP pool to enable dispersion [57] , [58] . Considering the link between the motile-sessile transition and the switch in P . aeruginosa virulence towards chronic infections [1] , [2] , we addressed herein whether induced dispersion , which enables the return to the planktonic mode of growth , is part of an inherent strategy of P . aeruginosa to initiate or contribute to either acute or chronic infections . To address this question , we analyzed virulence factor production and gene expression in cells exhibiting different growth modes by making use of dispersed cells obtained upon induction of dispersion in response to NO or changes in the nutrient glutamate concentration , as well as in a ΔbdlA mutant strain . This mutant was chosen as it is impaired in nutrient- and NO-induced biofilm dispersion [45] , [56] , [58] while not directly affecting c-di-GMP levels [56] , [58] . This was done as intracellular signaling via c-di-GMP has been demonstrated to be an important contributor to virulence in multiple pathogens [59] , [60] , [61] . To assure , however , that the observed effects on virulence and pathogenicity are not specific to BdlA but instead linked to dispersion , we furthermore made use of mutants inactivated in dipA and rbdA .
Several reports have described the process of dispersion to occur from within microcolonies , with dispersing bacteria observed to be motile , followed by them swimming away from the inner portions of the cell cluster through openings in the cluster and entering the bulk liquid [37] , [47] . The in vitro observations suggested bacteria within microcolonies and enmeshed by extracellular polymeric substance ( EPS ) matrix composed of polysaccharides , extracellular DNA , lipids , and proteins ( reviewed in [62] , [63] , [64] ) to be able to liberate themselves in order to evacuate from biofilms . We , therefore , hypothesized that dispersion may correlate with the release of enzymes that assist in the degradation of the EPS matrix . To do so , we first determined the concentration of proteins present in culture supernatants produced by 1×109 P . aeruginosa PAO1 bacteria grown planktonically , as biofilms , and following dispersion in response to nitric oxide ( NO ) or changes in the nutrient concentration , using the Bradford protein assay . NO was chosen as a dispersion inducing cue to mimic the endogenous production of or exogenous exposure to reactive oxygen intermediates to which P . aeruginosa is frequently exposed , both under anaerobic conditions prevalent in the CF lung environment and during infection [48] , [51] , [57] . Glutamate was used as a dispersion-inducing cue to mimic rapid environmental changes . Analysis of the culture supernatants of P . aeruginosa indicated the presence of supernatant proteins under all growth conditions tested ( Fig . 1A ) . However , the highest amount of proteins was detected in supernatants of dispersed cells followed by supernatants from planktonic cells grown to exponential and stationary phase ( Fig . 1A ) . The lowest concentration was routinely detected in biofilm supernatants ( Fig . 1A ) . Overall , up to 10-times more protein was detected in supernatants of dispersed cells than those of planktonic cells ( Fig . 1A ) . It is of interest to note that no difference in the concentration of supernatant proteins following dispersion in response to NO or changes in the nutrient concentration was noted , indicating protein release to be independent of dispersion inducing conditions ( Fig . 1A ) . As dispersion was induced by NO or glutamate , we also analyzed culture supernatants of planktonic cells exposed to glutamate and NO . No difference in the concentration of protein detected in supernatants of planktonic cells and planktonic cells exposed to glutamate or NO was noted ( Fig . 1A ) . Similar trends were observed upon the analysis of supernatants obtained from the hyper-virulent strain P . aeruginosa PA14 ( Fig . S1 ) . Early infection and colonization have been suggested to require the production of a variety of cytotoxic and degradative proteins [1] . To determine whether proteins detected in the supernatants harbored cytotoxic or degradative activity capable of degrading polymeric tissue components or substances that may contribute to the release of bacterial cells from biofilms , we used agar plate-based assays , focusing on the detection of proteolysis , lipid hydrolysis , hemolysis , and Psl degradation . These degradative activities were chosen as they target components of the EPS matrix or are indicators of cytotoxicity . To better quantitate cytotoxic or degradative activities present in the various supernatants , a total of 10 µg of supernatant protein was used for each assay . Supernatants obtained from biofilms , remaining biofilms following nutrient-induced dispersion , dispersed cells following induction of dispersion in response to NO or changes in the medium glutamate concentration , and planktonic cells grown to exponential and stationary phase were used . In addition , supernatants obtained from planktonic cells exposed to NO and glutamate were used as controls . Supernatants obtained from P . aeruginosa PAO1 displayed proteolytic and Psl degradation activities as well as lipid hydrolysis and hemolysis , regardless of the mode of growth tested ( Fig . 2 ) . The highest activity with respect to proteolysis , lipid hydrolysis , hemolysis , and Psl degradation was detected in supernatants obtained from planktonic cells grown to exponential and stationary phase ( Fig . 2A–B ) . Exposure of planktonic cells to NO or glutamate did not affect the overall activity present in supernatants ( Fig . 2A–B ) . In contrast , with the exception of lipid hydrolysis , biofilms remaining attached following induction of dispersion demonstrated the least activity ( Fig . 2 ) . No significant difference in the cytotoxic and degradative activities was noted in supernatants of dispersed cells obtained following induction of dispersion in response to NO or changes in the nutrient glutamate concentration ( Fig . 2C–F ) . Although supernatants of dispersed cells were characterized by levels approaching 10 times higher protein yield compared to planktonic and biofilm cells ( Fig . 1A ) , the enzyme activity present in supernatants of dispersed cells was intermediate between that of biofilm and planktonic cells ( Fig . 2 ) . Overall , the cytotoxic and degradative activities present in 10 µg of supernatant proteins obtained from dispersed cells were , while elevated , more similar to those observed for biofilm supernatants than planktonic supernatants ( Fig . 2 ) . Similar results were observed when supernatants obtained from P . aeruginosa PA14 were used ( Fig . S2 ) . We furthermore determined DNA hydrolase activity in supernatants . The highest DNA hydrolase activity was found in supernatants of dispersed cells and biofilm cells remaining attached following induction of dispersion ( Fig . S3 ) . Our findings suggested that dispersed cells are distinct from planktonic and biofilm cells with respect to cytotoxic or degradative supernatant activities . To determine whether the ability to disperse correlated with the ability to degrade polymeric substances , we made use of a ΔbdlA mutant . Deletion of bdlA was previously demonstrated to render biofilm bacteria deficient in dispersion triggered by multiple environmental cues including various nutrients , NO , ammonium chloride , and heavy metals [45] , [56] , [58] . Compared to wild type bacteria , ΔbdlA grown planktonically and as biofilms released comparable amounts of proteins into the supernatant ( Fig . 1B ) . Moreover , no difference in cytotoxic and degradative activity was noted for supernatants obtained from ΔbdlA mutant grown planktonically compared to that observed for the wild-type supernatants ( not shown ) . Under biofilm growth conditions , however , very little enzymatic activity was detected in supernatants obtained from ΔbdlA biofilms and biofilms exposed to dispersion-inducing conditions ( referred to as remaining biofilms , although no dispersion event occurred ) compared to the wild type . In fact , no proteolysis and lipid hydrolysis was detected in supernatants from ΔbdlA biofilms and remaining biofilms , and hemolytic and Psl polysaccharide degradative activities were reduced compared to wild-type biofilms and remaining biofilms ( Fig . 2C–F ) . Our findings not only suggested differences in P . aeruginosa PAO1 and ΔbdlA strains with respect to the release of degradative enzymes but , furthermore , that the inability to disperse likely corresponded with significantly reduced release or absence of degradative enzymes compared to wild-type biofilms . To further confirm differences in proteins released by the two strains , proteins present in supernatants obtained from biofilm cells were analyzed by SDS/PAGE , followed by subsequent protein identification by LC-MS/MS . As expected , marked differences in the extracellular proteins of wild-type and ΔbdlA mutant biofilms were noted ( Fig . 3 ) . However , the analysis also revealed the presence of a set of proteins common to the extracellular proteins of both wild type and the ΔbdlA mutant ( Fig . 3 ) . These included the outer membrane porins OprD , OprQ , and OprF as well as the flagellin component , FliC , the aminopeptidase PA2939 , a component of the ABC transporter PA1342 , and the sulfate binding protein precursor PA0283 ( Fig . 3 , Table 1 ) . The latter two proteins were detected in a growth mode-dependent manner with PA1342 and PA0283 being absent in supernatants obtained from planktonic cells but detectable in supernatants obtained from biofilms ( Fig . 3 , Table 1 ) . Differences in the extracellular proteome constituted the proteases elastase ( LasB ) and protease IV ( PA3724 ) . While both were detectable in supernatants of P . aeruginosa PAO1 under planktonic and biofilm growth conditions , the proteins were present at elevated levels in supernatants from P . aeruginosa PAO1 grown planktonically to stationary phase . In contrast , no protein bands of respective apparent mass were detected in culture supernatants of the ΔbdlA mutant ( Fig . 3 , Table 1 ) . The only protease detected in the ΔbdlA mutant was the alkaline metalloproteinase , AprA ( Fig . 3 , Table 1 ) . The finding of the ΔbdlA strain lacking proteases is consistent with the reduced proteolytic activity in this strain ( Fig . 2C ) . No lipases or hemolysins were detected . Additional differences in the extracellular proteome were noted with respect to exotoxin A . Production of exotoxin A inhibits eukaryotic host cell protein synthesis by interacting with elongation factor-2 , resulting in cell death [65] . While exotoxin A was detected in the supernatants of P . aeruginosa PAO1 , its presence was limited to biofilms ( Fig . 3 , see black arrow “F2” , Table 1 ) . In contrast , exotoxin A was detectable in supernatants of the ΔbdlA mutant regardless of growth conditions ( Fig . 3 , Table 1 ) . The difference in protease and exotoxin A abundance in the extracellular proteomes obtained from P . aeruginosa wild-type and ΔbdlA biofilms suggested a contribution of dispersion to the production of degradative and virulence factors . To further explore whether dispersion and the return to the planktonic , free-living mode of growth contributes to a switch in virulence gene expression , qRT-PCR was used to quantitatively determine the transcript levels of genes encoding several known virulence factors . In addition to exotoxin A , expression of the virulence factors hydrogen cyanide ( hcnA ) , chitinase ( chiC ) , elastase ( lasB ) , pyocyanin ( phz operon ) , genes encoding T3SS components ( pcrV and pscL ) , and rhamnolipids ( rhlA ) [66] , [67] was assessed . Considering that dispersed cells are exposed to NO or glutamate , we first determined whether expression of virulence genes is affected by the addition of NO or glutamate by analyzing the transcript abundance of virulence genes of interest in exponential phase planktonic cells and exponential phase planktonic cells exposed to glutamate and NO . No significant difference in transcript levels of the tested virulence genes was noted ( Fig . 4A ) indicating that under the conditions tested , exposure to NO or additional glutamate does not affect the expression of the virulence genes . We therefore compared transcript abundance of virulence genes in biofilms and dispersed cells to untreated exponential phase planktonic cells . Compared to planktonic cells , P . aeruginosa PAO1 grown as biofilms were characterized by significantly reduced expression of chiC , lasB , rhlA , phzB , pscL , and hcnA , but increased pcrV and toxA expression ( Fig . 4B , Table S1 ) . Increased toxA transcript levels in P . aeruginosa PAO1 biofilms are in agreement with the biofilm-specific detection of exotoxin A ( Fig . 3 ) . Dispersed cells were likewise characterized by reduced expression of virulence genes , regardless of whether NO or changes in the glutamate concentration were used to induce dispersion ( Fig . 4B , Table S1 ) . However , all virulence genes tested including pcrV and toxA were significantly reduced in dispersed cells compared to both planktonic or biofilm cells ( Fig . 4B , Table S1 ) . Similar changes in transcript levels were observed for dispersed P . aeruginosa PA14 cells compared to planktonic and biofilm cells ( Fig . S4 ) and suggested that dispersion induced upon exposure to environmental cues to be more than merely a transitional episode of cells leaving the biofilm and adapting to the planktonic mode of growth . Considering the similarity in the trend of virulence gene expression between biofilms and dispersed cells compared to planktonic cells , we hypothesized that dispersion may contribute to virulence gene expression and assessed this hypothesis by testing the dispersion-deficient ΔbdlA mutant strain grown as a biofilm . We anticipated finding transcript levels of virulence genes to be higher in biofilms impaired in dispersion compared to wild-type biofilms . qRT-PCR analysis revealed that transcript levels of chiC , hcnA , lasB , phzB were significantly increased in ΔbdlA biofilms relative to wild-type planktonic cells ( and biofilm cells ) . In contrast , the transcript levels of rhlA and the T3SS genes pscL and pcrV were similar those observed for wild-type planktonic cells ( but significantly increased compared to wild-type biofilm cells , Fig . 4C , Table S1 ) . The only transcript present at similar levels in ΔbdlA biofilm and wild-type biofilm cells was toxA . The finding is in agreement with the supernatant protein analysis ( Figs . 3 , 4C , Table S1 ) . It is of interest to note that similar results were obtained when virulence gene transcript levels of the dispersion-deficient ΔdipA mutant were determined ( Fig . 4C ) . In contrast , multi-copy expression of bdlA , which renders PAO1/pJN-bdlA biofilms hyper-dispersive [45] , [58] , resulted in significantly reduced expression of chiC , lasB , rhlA , phzB , pscL , pcrV , and hcnA compared to planktonic cells . Moreover , with the exception of rhlA and toxA , the transcript levels of chiC , lasB , phzB , pscL , pcrV , and hcnA detected in PAO1/pJN-bdlA biofilms were overall reduced compared to wild type biofilms ( Fig . 4D , Table S1 ) . Instead , the transcript levels of virulence genes in this hyper-dispersive biofilm were overall similar to those observed for dispersed cells ( Fig . 4B-C , Table S1 ) . A similar trend was observed upon overexpression of dipA in biofilms which has previously been demonstrated to also render P . aeruginosa biofilms hyper-dispersive [46] . With the exception of pscl and rhlA , the transcript levels of virulence genes detected in PAO1/pJN-dipA biofilms were significantly different relative to wild type biofilms ( Fig . 4D ) . Our findings indicated non-dispersing and hyper-dispersing biofilms to express virulence genes in a manner distinct from biofilm and planktonic cells , with non-dispersing ΔbdlA and ΔdipA biofilms demonstrating increased expression of virulence genes compared to biofilm and planktonic cells while hyper-dispersing biofilms appeared to express virulence genes at reduced levels compared to planktonic and biofilm cells , with the expression profile overall being more similar to that of dispersed cells . Moreover , our findings further indicate that toxA expression is specific to the biofilm mode of growth . Inactivation of bdlA correlated with increased expression of virulence factors but reduced release of cytotoxic and degradative enzymes relative to wild-type cells . In contrast , overexpression of bdlA resulted in significantly reduced transcript levels of virulence genes . We therefore asked whether bdlA inactivation affected the virulence phenotype of this mutant strain using an A . thaliana virulence model . This alternative nonvertebrate , plant host model was chosen as it has been previously demonstrated to result in the identification of bacterial virulence factors and to correlate with virulence outcomes obtained using vertebrate infection models such as the burned mouse pathogenicity model [68] , [69] . Compared to the wild type , mutants lacking BdlA were less virulent . While more than 58% of Arabidopsis plants were killed by P . aeruginosa PAO1 within 7 days post infection ( Fig . 5A ) with the percent of dead plants rising to 70% following 9 days of infection ( not shown ) , the isogenic ΔbdlA mutant was unable to establish infection within 7 days . Only 12% of all plants were killed 7 days post infection ( Fig . 5A ) . In contrast , overexpression of bdlA , which was found to mimic a hyper-biofilm dispersion phenotype in vitro [45] , [58] , had no additional effect on P . aeruginosa virulence and plant mortality compared to the wild type ( Fig . 5A ) . To ensure that the factors encoding the tested virulence genes chiC , lasB , rhlA , phzB , pscL , hcnA pcrV and toxA contribute to the pathogenicity of P . aeruginosa in the A . thaliana virulence model , mutants inactivated in these virulence factors were tested . Inactivation of exoA , hcnA , chiC , lasB , phz , and rhlA and T3SS-coding genes pcrV and pcsL resulted in significantly reduced plant death compared to the wild type . While more than 55% of Arabidopsis plants were killed by P . aeruginosa PAO1 within 7 days post infection , infections with mutants inactivated in these virulence genes ( with the exception of chiC ) resulted in significantly reduced plant mortality , with less than 20% of all plants being killed over the same period of time ( Fig . 5B ) . The findings confirmed a contribution of the respective virulence factors to P . aeruginosa pathogenicity . Considering , however , that the majority of these factors are up-regulated in ΔbdlA biofilms but reduced in PAO1/pJN-bdlA relative to wild-type biofilms , our findings further suggested that the ability to disperse , rather than the differential expression of a particular set of virulence factors , is contributing to the pathogenicity of P . aeruginosa . To exclude the possibility that the effect on virulence was due to the absence of BdlA rather than linked to the ability to disperse , we made use of recently identified site-directed mutants of BdlA that were unable to restore the ΔbdlA-dispersion phenotype to wild-type levels [58] . These included alanine substitutions of the amino acids D14 , N23 , W60 , I109 , and W182 in BdlA . ΔbdlA mutants complemented with any of these BdlA variants were as avirulent as a ΔbdlA mutant ( Fig . 5A ) . Similarly , complementation of ΔbdlA with a truncated BdlA lacking the N-terminal located PAS domain of BdlA , which was previously shown to not restore the ΔbdlA dispersion phenotype to wild-type levels [58] , resulted in such a bdlA strain remaining avirulent ( Fig . 5A ) . In contrast , alanine substitution in amino acid positions E36 , R75 , and W84 that did restore the biofilm-deficient phenotype of ΔbdlA to wild-type levels [58] , rendered the complemented bdlA strain as virulent as wild-type bacteria ( Fig . 5A ) . Similarly , a ΔbdlA mutant complemented with intact bdlA was used as positive control and found to be as virulent as the wild type ( Fig . 5A ) . To furthermore support the link between dispersion and virulence , we additionally tested the mutant strains ΔdipA and ΔrbdA , that have been previously demonstrated to be impaired in dispersion of P . aeruginosa biofilms in response to NO and glutamate [46] , [55] . Compared to the wild type , mutants lacking DipA and RbdA were significantly less virulent ( Fig . 5C ) . While up to 80% of Arabidopsis plants were killed by the parental P . aeruginosa strain within 7 days post infection ( Fig . 5C ) , the isogenic ΔrbdA and ΔdipA mutants were unable to establish infections . Overall , less than 10% of all plants were killed 7 days post infection ( Fig . 5C ) . Our findings strongly indicate that the process of dispersion is a major contributor to P . aeruginosa pathogenicity , as strains impaired in dispersion are avirulent , while strains capable of the dispersion response demonstrated wild-type virulence levels . To determine whether reduced virulence in a plant model of infection correlates with reduced acute infection and attenuation of virulence in vivo , we next examined the ability of the ΔbdlA mutant , which is impaired in dispersion in vitro , to colonize in an acute infection model , using a murine model of acute pneumonia . Wild-type P . aeruginosa is able to colonize the lungs of an infected mouse and grow about 100-fold in the course of 24 hr ( Fig . 6A–B ) . The isogenic ΔbdlA mutant , in comparison , was unable to establish infection; bacterial load was reduced by 10-fold as compared to infection with the wild-type parental strain ( Fig . 6A–B ) . Additionally , the P . aeruginosa ΔbdlA mutant was unable to establish systemic infections and no bacteria were recovered from the liver or spleen ( data not shown ) . Competitive mixed infection assays have been widely used to assess the fitness of individual P . aeruginosa mutants versus their parental strains during in vivo infection [70] , [71] . Wild-type and ΔbdlA mutant bacteria were used to infect adult CD-1 mice ( in groups of five ) intranasally with 1×107 cells ( 1∶1 ratio ) . Following 16 hr , infected lungs were recovered for bacterial load determinations . Remarkably , the ΔbdlA mutant was only 2% as ( or 45-fold less ) competitive as its parental strain PAO1 ( Fig . 6C ) . To further ensure that the effect on virulence was linked to the ability to disperse , we also examined the ability of the ΔdipA mutant to establish an acute infection . Compared to the parental strain , the isogenic ΔdipA mutant , in comparison , was unable to establish infection , as apparent by the bacterial load being reduced by 80-fold compared to the wild-type strain ( Fig . 6D ) . Moreover , while the bacterial load of wild-type bacteria increased about 20-fold in the course of 24 hr compared to the initial inoculum titer , the ΔdipA mutant load was reduced 4-5-fold ( Fig . 6 ) . The results suggested BdlA and DipA , and thus , likely the process of dispersion to play an important role in virulence of P . aeruginosa during acute infection of mouse airways . To determine the contribution of bdlA and dispersion to the ability of P . aeruginosa to persist at the site of infection in vivo , we next tested this mutant in a murine model of chronic pneumonia . Based on the ΔbdlA dispersion-deficient phenotype in vitro , we expected the P . aeruginosa ΔbdlA mutant to have an advantage in establishing biofilms and/or persistent infection compared to the wild type , due to its reduced virulence phenotype and the reduced number of cells released from the biofilm . We made use of a chronic infection model established by Cash et al . [72] except that we made use of a murine rather than a rat model . The model is based on the intratracheal administration of agarose beads impregnated with P . aeruginosa . This model was chosen because it allows the study of chronic infection , marked by the formation of persistent bacterial biofilm populations at the site of infection that can persist for up to 6 months and mimic biofilm-related infections [70] , [71] , [73] , [74] , [75] . Following 14 days of infection , the bacterial load in the lungs of mice infected with ΔbdlA was significantly higher ( 1 . 5-log increase ) than that of P . aeruginosa PAO1 ( Fig . 7 ) . Moreover , compared to the initial inoculum ( 1 . 2×106 CFU ) , a less than a 100-fold reduction was noted for ΔbdlA while a more than 4000-fold reduction was detected between the initial inoculum and the bacterial burden 14 days post infection for the wild type . To ensure that the bacterial load in the lungs coincided with biofilm formation , we also tested P . aeruginosa overexpressing PA2133 . PA2133 encodes a phosphodiesterase , and PAO1 strain overexpressing the PA2133 was previously shown by Hickman et al . [76] to be significantly impaired in biofilm formation in vitro , even after 72 h of incubation . Reduced bacterial burden correlated with reduced or impaired biofilm formation as indicated by the reduced detection of PAO1/pJN-PA2133 bacterial cells in the lung . Compared to PAO1/pJN-PA2133 , the bacterial burden by P . aeruginosa PAO1 was increased by 1 . 5 logs , and ΔbdlA showed a 4 log increase ( Fig . 7 ) . Decreased burden correlated with a significant reduction in bacterial load compared to the original inoculum . The findings suggested that the bacterial burden at the site of infection depended on the ability to not only form biofilms but also to disperse .
The opportunistic pathogen P . aeruginosa causes a variety of human diseases , ranging from acute and chronic persisting infections , fatal bacteremia in immunocompromised patients and superficial skin infections to chronic colonization of patients with CF and chronic obstructive pulmonary disease . This remarkable adaptability depends to a large extent on its ability to transition from a planktonic ( unattached ) to a biofilm mode of growth and to activate the expression of genes required for viability and growth in a particular environment , while simultaneously repressing genes that are unnecessary or even detrimental for survival in that particular niche . Our findings are in support of previous proteomics and genetic studies of P . aeruginosa [1] , [2] , [3] , [8] , [9] , [77] , indicating that planktonic and biofilm cells differ with respect to virulence gene expression with biofilms displaying a biofilm-specific virulence phenotype , and expressing virulence factors up to 30-fold less compared to planktonic cells . The observed down-regulation of virulence factors following biofilm formation likely serves as an additional adaptation for the bacterial communities to avoid recognition and targeting by the immune system . Accordingly , while releasing similar amounts of protein into the supernatant , biofilm cells were found to produce fewer cytotoxic and degradative proteins than planktonic cells ( Figs . 1–2 ) . Given that early infection and colonization has been suggested to require the production of a variety of cytotoxic and degradative proteins , some of which function after direct translocation into host cells , the observed difference in cytotoxic and degradative activities is in agreement with planktonic cells causing more considerable host cell damage [1] , [78] , [79] . In addition to the established acute-to-chronic virulence switch accompanying the motile-to-sessile mode of growth switch , we demonstrate here that dispersion in response to environmental cues is likely another contributing factor to the virulence phenotype of P . aeruginosa . However , while our findings support dispersion to contribute to virulence , dispersion is not simply a reversion of the motile-to-sessile switch . Instead , our findings suggest dispersion to represent a distinct virulence phenotype . For one , dispersed cells were characterized by significantly reduced transcript levels of virulence genes compared to both planktonic and biofilms cells . Moreover , while up to 10-fold higher protein concentrations were detected in supernatants of dispersed cells , the cytotoxic and degradative activity levels were found to be intermediary to those detected in supernatants of planktonic and biofilm cells . Our findings of the distinct virulence gene expression profile of dispersed cells are in agreement with our previous findings of biofilm cells in the dispersion stage and dispersed cells exhibiting a phenotype that is distinct from both planktonic and biofilm bacteria [36] , [37] , [56] , [80] . P . aeruginosa dispersed cells not only display a virulence phenotype distinct from those of planktonic and biofilm cells , but also likely employ mechanisms enabling them to evade the immune response following the loss of protection associated with biofilm growth . This was apparent in the difference of cytotoxic and degradative enzymes present in supernatants compared to planktonic cells . It is likely that the difference represents a rapid initial response at the level of protein activity to liberate the dispersing cells from the enclosing matrix . While direct evidence of dispersion occurring in the lung environment is lacking , dispersion is widely documented in natural systems and is a basic property of biofilms in general . Moreover , it is likely that ability to degrade the biofilm matrix extends to an ability to degrade the surrounding mucus present in the CF lung , which is chemically similar to known biofilm matrix polymers . Additionally , while P . aeruginosa rarely escapes the lung environment , there are indications that dispersion takes place , as dispersed cells are more susceptible to immune function and antimicrobial treatment compared to biofilm cells [47] , [56] . For example , Donaldson et al . [81] demonstrated that treatment with aerosolized hypertonic saline causes the vasculature to release water from the blood stream , thereby hydrating the thick mucus , correlated with biofilm bacteria being far more susceptible to the antibiotic amiloride compared to bacteria present in untreated mucus [81] . Differences in virulence factor gene expression may likewise indicate a dispersion-specific adaptation to temporarily avoid the host immune response , enabling dispersed cells to release themselves from the biofilm matrix but remain “immune-masked” until they have either reverted to a planktonic phenotype or have reattached . This was supported by our findings of both ΔbdlA and ΔdipA mutants , which are impaired in dispersion in vitro [45] , [46] , [56] , [58] , being attenuated in virulence while demonstrating heightened persistence in vivo ( Figs . 5–7 ) . The contribution of BdlA and DipA to virulence and persistence furthermore indicated that both proteins likely contribute to dispersion not only in vitro but also in vivo . The ΔbdlA and ΔdipA virulence phenotypes further suggested the presence of environmental cues in both the plant and the murine lung environment that contribute to dispersion-inducing conditions . This was apparent as the inability to disperse in vitro affected outcomes in both acute and chronic infection models , with dispersion enhancing the virulence of P . aeruginosa during an acute infection , but interfering with the ability to establish chronic infections . The manner in which BdlA , DipA , and dispersion contribute to persistence is distinct from biofilm formation , as inactivation of bdlA or dipA had the opposite effect to overexpression of the phosphodiesterase PA2133 , which impairs biofilm formation [76] . Our findings thus likely suggest that in addition to biofilm biomass accumulation , dispersion may also be a contributing factor to P . aeruginosa persistence in the murine lung . This was further supported by the finding of a ΔbdlA mutant complemented with BdlA harboring the D14A , N23A , W60A , I109A , W182A substitutions , that were previously demonstrated to result in a null phenotype for dispersion , being attenuated in virulence . Additional support for a role of dispersion in P . aeruginosa pathogenicity was provided by ΔdipA and ΔrbdA mutants , that are impaired in dispersion in vitro [46] , [55] , being attenuated in virulence ( Figs . 5–6 ) . To our knowledge , this is the first report linking dispersion to virulence of P . aeruginosa in vivo . It is of interest to note that the ΔbdlA virulence phenotype is similar to that of a ΔretS mutant in the sense that both promote transitions between the planktonic and the sessile lifestyles . While inactivation of retS promotes the motile-sessile transition [1] , ΔbdlA mutants are impaired in the sessile-motile transition , with inactivation of both retS or bdlA resulting in decreased initial colonization . While the findings suggest that mutations favoring the biofilm lifestyle ( by altering either one of the transitions ) would be selected for in vivo , long-term selection in a host would likely drive populations towards a biofilm phenotype rather than favoring a non-dispersing phenotype . Instead , we expect the dispersion phenotype to be a short-term behavioral change in bacteria and to be associated specifically with acute phase infections . In summary , we demonstrate for the first time that dispersed cells have a unique virulence phenotype , with dispersion and the reversion to the planktonic mode of growth contributing to virulence and persistence of P . aeruginosa in infections . While the ability to disperse in vitro in response to various exogenous cues contributes to virulence of P . aeruginosa in acute infections , dispersion instead reduces the ability of P . aeruginosa to persist at the site of infection . Our observations of dispersion thus reciprocally regulating acute virulence and persistent , chronic infections suggest that dispersion functions as a regulatory switch , mediating the global transition from initial colonization to chronic infections . Thus , our work establishes induced dispersion as not only an integral part of both acute and chronic infections , but also as a potential mechanism of infection control .
The use of animals for this study was reviewed by the IACUC committee at the University of Cincinnati ( UC ) that is a centralized , campus wide animal care and use program under Laboratory Animal Medical Services ( LAMS ) . LAMS is staffed by three full time veterinarians , six veterinary technicians plus animal care staff . One LAMS veterinarian; one veterinary technician and one LAMS husbandry supervisor is on call after hours . LAMS approved the animal care protocol and use protocol/permit/project license . The approved IACUC protocol number is 12-09-06-01 ( “The Molecular Basis of Pseudomonas aeruginosa , Francisella novicida , and Staphylococcus aureus Virulence in Mammalian Hosts” ) . All animals were handled in strict accordance with good animal practice and animal keeping . UC has an Animal Welfare Assurance on file with the NIH-OLAW ( Assurance Number A-3295-01 , expires November 30 , 2015 ) . UC fully complies with the Guide for the Care and Use of Laboratory Animals ( Guide ) , the Public Health Service Policy on the Humane Care and Use of Laboratory Animals ( PHS Policy ) and all U . S . Animal Welfare Act Regulations . P . aeruginosa strain PAO1 and its isogenic mutant strain bdlA were used in this study . P . aeruginosa strain PA14 was used to validate the generality of the findings . All bacterial strains and plasmids used in this study are listed in Table S2 . All planktonic strains were grown in Lennox Broth ( LB , BD Biosciences ) or minimal medium containing glutamate as the sole carbon source [82] in shake flasks at 220 rpm in the absence or presence of 0 . 1–1 . 0% arabinose . Escherichia coli cultures were grown in LB in the absence or presence of 1 mM Isopropyl β-D-1-thiogalactopyranoside ( IPTG ) . Antibiotics for P . aeruginosa were used at the following concentrations: 50–75 µg/mL gentamicin and 200–250 µg/mL carbenicillin . All planktonic cells used in this study were obtained by reinoculating 0 . 5 ml of overnight grown cells into 50 ml minimal medium . To obtain exponential phase planktonic cells , bacteria were allowed to grow to an optical density ( 600 nm ) of 0 . 4 . To obtain stationary phase planktonic cells , bacteria were allowed to grow for 8 hr at which time they reached an optical density of 1 . 2 . Under the conditions tested , onset of stationary phase was noted following 6–7 hours of growth . Planktonic cells growth to exponential and stationary phase were furthermore exposed to an additional 18 mM glutamate or the addition of 500 µM sodium nitroprusside ( SNP , source of NO [51] ) 30 min prior to harvesting the planktonic cells . The timing was chosen to mimic the exposure time of dispersed cells to glutamate and NO . Biofilms were grown in a continuous flow tube reactor system ( 1 m long size 14 silicone tubing , Masterflex , Cole Parmer , Inc . ) at 22°C for up to 5 days to obtain proteins and RNA . For biofilm dispersion assays , biofilms were cultivated in once-through continuous flow tube reactor system composed of size 13 silicone tubing ( Masterflex , Cole Parmer , Inc . ) at 22°C for 5 days . After 5 days of biofilm growth , biofilm dispersion was induced by the sudden addition of glutamate ( 18 mM ) to the growth medium as previously described [56] . Moreover , dispersion was induced by 500 µM sodium nitroprusside ( SNP ) which was used as a source of NO [51] . Dispersion was indicated by an increase in turbidity at 600 nm in the effluent from the silicone tubing . Supernatants were obtained from P . aeruginosa PAO1 grown planktonically ( exponential and stationary phase ) , as biofilms , and following dispersion in response to NO or changes in the nutrient glutamate concentration . While the volume of the supernatants varied depending on the growth conditions tested , all supernatants tested represented supernatants produced by 1×109 cells . Briefly , supernatants of cells grown planktonically to exponential and stationary phase and as biofilms were collected by centrifugation . Similarly , supernatants of dispersed cells and biofilms remaining attached to the surface upon induction of dispersion ( remaining biofilms ) were collected after induction of dispersion of 5 day-old P . aeruginosa biofilms were collected . The resulting supernatant was filter-sterilized , dialyzed , lyophilized , and subsequently resuspended in sterile water . The concentration of proteins present in supernatants was determined using the Bradford assay ( Biorad ) . Cell pellets were collected and sonicated as previously described [37] , and cell debris removed by centrifugation . The protein concentration of the resulting total cell extract was determined using the modified method of Lowry [83] . The resulting protein concentration was then used to calculate the number of cells with 1 ug of total protein being equivalent to 3 . 6*107 CFU [84] . Proteins present in supernatants were visualized by SDS/PAGE analysis using 12% SDS-gels and Coomassie staining . A total of 10 µg of cell extract was loaded onto the gel . Proteins of interest were excised from the gel , tryptic digested , and subsequently identified by LC-MS/MS essentially as previously described using a QStarXL mass spectrometer ( Applied Biosystems ) [85] . Supernatants were tested for the presence of hydrolytic enzymes using agar-plate based biochemical assays . To do so , wells having a 7 mm diameter were punched into the agar . A total of 10 µg of secreted proteins resuspended in a total of 100 µl of water was added to per well . The agar plates were subsequently incubated at 37°C for 18 hr before measurements of the halo surrounding the wells were taken . All measurements were normalized by subtracting the diameter of the well . Lipid hydrolysis was determined using Tributyrin HiVeg Agar Base ( BD Bioscience ) supplemented with 1% tributyrin while proteolysis was determined using milk agar containing 10% skim milk . Hemolysis was determined using trypticase soy agar supplemented with 5% sheep's blood . Psl degradation was determined using 1 . 5% agar containing 1% Psl . Psl polysaccharide was extracted using the rapid Psl protocol [86] . Each agar plate contained the Psl equivalent obtained from a Psl overexpressing P . aeruginosa having an OD of 25 . Psl degradation was visualized following 24–48 hr of incubation using 1% iodine solution ( Grams iodine , Thermo Scientific ) . DNAse activity was determined using 100 µg of supernatant protein and a colorimetric assay as previously described by Sinicropi et al . [87] using DNA-methyl green as a substrate . A total of 0 . 2 mg/ml salmon sperm DNA was used and DNA degradation monitored at 620 nm . Isolation of mRNA and cDNA synthesis was carried out as previously described [84] , [85] , [88] , [89] . qRT-PCR was performed using the Eppendorf Mastercycler ep realplex ( Eppendorf AG , Hamburg , Germany ) and the KAPA SYBR FAST qPCR Kit ( KAPABIOSYSTEMS , Woburn , MA ) , with oligonucleotides listed in Table S3 . mreB was used as a control . The stability of mreB levels were verified by 16S RNA abundance using primers HDA1/HDA2 [90] . Relative transcript quantitation was accomplished using the ep realplex software ( Eppendorf AG ) by first normalizing transcript abundance ( based on Ct value ) to mreB followed by determining transcript abundance ratios . Melting curve analyses were employed to verify specific single product amplification . The role of BdlA in virulence was assessed using the Arabidopsis thaliana infection model which provides a quantitative approach and permits the tracking of bacterial cell proliferation in planta [91] . Following two weeks of growth in 1/2MS ( 2 . 2 g/L Murashige and Skoog basal medium ) , plants were infected with P . aeruginosa PAO1 and its isogenic mutant strains at a final optical density of OD600 = 1 . 0 in 1/2MS and incubated for a period of 12 days at a 25/22°C , 16 hr-light/8 hr-dark cycle . All plants were inspected daily for signs of infection and/or death as evidenced by wilting , discoloration , and necrosis . P . aeruginosa strains PAO1 and ΔbdlA were grown to late stationary phase ( ∼ OD 3 . 0 ) . Cells were harvested , and washed three times with 10 mM MgSO4 . Bacteria were serial diluted in 10 mM MgSO4 , mixed in a 1∶1 ratio , and approximately 1×107 cells were intranasally inoculated into the lungs of four CD-1 mice ( Charles River , Boston , MA ) previously anesthetized with isofluoran . For single infection studies , the mouse lungs were harvested at 24 hr post-infection , homogenized and serially diluted for bacterial burden determination . For competitive index studies , the mouse lungs were harvested at 16 hr post-infection . The bacterial titer is expressed as CFU/g of tissue . A chronic infection model established by Cash et al . [72] was used to test whether the dispersion-deficient mutant strains are able to establish a chronic infection . The infection model by Cash was modified by using a murine model instead of a rat model . For the preparation of the agarose beads , P . aeruginosa wild type and mutant strain ΔbdlA , were grown at 32°C in a low phosphate succinate medium [72] to stationary phase and mixed in a 1/10 ratio with 2% agarose in phosphate-buffered saline ( PBS , pH 7 . 4 ) . The mixture was added to heavy mineral oil equilibrated at 55°C , stirred for 6 min at room temperature , and cooled for 10 min . Free bacteria were removed by washing with 0 . 5% and 0 . 25% deoxycholic acid sodium salt in PBS once and then in PBS alone three times . The beads were passively filtered through sterile 200 µm diameter nylon mesh and then verified for size ( 70- to 150-µm diameter ) and uniformity by microscope examination . An aliquot of beads were homogenized , serially diluted , and plated on LB agar plates to enumerate colony-forming units ( CFU ) . A 100 µl inoculum containing 1×106 CFU of viable P . aeruginosa entrapped in agarose beads was then introduced into the lungs of adult CD-1 mice ( six week old , groups of 10 ) via the trachea with nonsurgical methods by a 21-gauge blunt-end needle to the back of the tongue above the tracheal opening . Successful delivery of the beads to the lungs was manifested by choking of the mouse immediately after instillation followed by rapid breathing , and was confirmed by harvesting lungs three minutes post-inoculation and CFU determination of lung homogenates . Mice were sacrificed by asphyxiation in a precharged CO2 chamber , or by transection of the abdominal aorta after an overdose of pentobarbital ( 2 . 0 ml per kg body weight ) . The bacterial load was expressed as CFU/g of tissue . A Student's t-test was performed for pair-wise comparisons of groups , and multivariant analyses were performed using a 1-Way ANOVA followed by a posteriori test using Sigma Stat software . For the animal studies , Fisher exact test was used for statistical analysis ( http://www . quantitativeskills . com/sisa/statistics/fishrhlp . htm ) . | Pathogenic bacteria , including the human pathogen Pseudomonas aeruginosa , can cause acute and chronic infections . The difference in these infection modes can be explained by how bacteria grow . Acute infections occur when individual bacteria rapidly replicate , produce high levels of virulence factors , and disseminate from the nidus of infection . Chronic infections occur when bacteria adhere to tissue or implanted medical devices and form multi-cellular , matrix-encased aggregates known as biofilms . The acute-to-chronic infection switch occurs when bacteria transition from planktonic to biofilm growth . However , the contribution of dispersion , the process by which bacteria leave a biofilm to return to planktonic growth , remains unclear . Here , we demonstrate that , while having left a biofilm , dispersed cells are distinct from planktonic cells with respect to gene expression , release of matrix-degrading enzymes , and pathogenicity . We found that a mutant impaired in nutrient-induced dispersion , while enhancing chronic infections , is impaired in mounting acute infections in both plant and mouse hosts . Overall , this work establishes that dispersed cells have a unique virulence phenotype , with nutrient-induced dispersion not only serving as an integral part of both acute and chronic infections but also as a potential mechanism of infection control . | [
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"microbiol... | 2014 | BdlA, DipA and Induced Dispersion Contribute to Acute Virulence and Chronic Persistence of Pseudomonas aeruginosa |
During development , certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact . How these neurons regulate pruning activity in the dendrites remains unknown . Here , we identify a coiled-coil protein Spindle-F ( Spn-F ) that is required for dendrite pruning in Drosophila sensory neurons . Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning . Spn-F exhibits a punctate pattern in larval neurons , whereas these Spn-F puncta become redistributed in pupal neurons , a step that is essential for dendrite pruning . The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association , and depends on the function of microtubule motor dynein complex . Spn-F is a key component to link Ik2 kinase to dynein motor complex , and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning . Our findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons .
The precise assembly of neural circuits is crucial for the nervous system to function properly . The developing nervous systems often start with a primitive prototype , characterized by exuberant branches and excessive connections . Thus , further remodeling is required to refine the developing nervous systems to maturity . Neuronal pruning , one such remodeling mechanism , is a highly regulated self-destruct process that eliminates excessive neuronal branches in the absence of cell death . Pruning is widely observed in the nervous systems of both vertebrates and invertebrates [1 , 2] , that not only ensures precise wiring during development , but also allows for adjustment of neuronal connections in response to injury and disease . Various studies have shown that defects in developmental pruning affect the function of the nervous systems in C . elegans [3] and Drosophila [4] . Moreover , a progressive loss of neurites far ahead of cell death is commonly observed in many neurodegenerative disorders [5 , 6] . Thus , any dysregulation of pruning activity even at the level of individual neurons would bring catastrophic consequences to the nervous systems . Although the primary triggers for developmental pruning and pruning that ensues upon neuronal injury and disease are diverse , the downstream machinery that eliminates neuronal processes shared some common features . For example , microtubule disruption is the earliest cellular event observed in all types of pruning [2 , 7 , 8] , and the ubiquitin-proteasome system is required in all circumstances [7–10] . During Drosophila metamorphosis , substantial neuronal remodeling takes place in both the central and peripheral nervous systems [11–14] . Most of the larval peripheral neurons die during metamorphosis , whereas few , including some class IV dendritic arborization ( C4da ) neurons , survive and undergo large-scale dendrite pruning [13 , 14] . Dendrite pruning of the dorsal C4da neuron ddaC starts with severing of the proximal dendrites at 4–6 h APF ( after puparium formation ) [15] . Subsequently these disconnected dendrites become fragmented and eventually eliminated by the surrounding epidermal cells [16] by 16–18 h APF . In contrast to the central brain mushroom body ( MB ) γ neurons where both larval dendrites and axons are pruned during development , the peripheral C4da neurons specifically prune their dendrites keeping the axons intact [14] . The molecular basis for how the pruning activity is confined to the dendrites of C4da neurons remains unknown . We reasoned that molecular differences between dendrites and axons should be considered for such differential pruning activity in C4da neurons . It is known that microtubule polarity is different in the dendrites and axons of neurons [17] , including in the Drosophila sensory neurons [18] . For example , C4da neurons have polarized microtubules in their proximal dendrites predominantly with microtubule minus end pointing away from the cell body , but have an opposite polarity in their axons [18 , 19] . This difference in microtubule polarity is essential for maintaining the proper function and compartmental identities of dendrites and axons , and might be an important determinant for spatially restricting pruning activity in the dendritic compartments of C4da neurons . Based on this assumption , some molecules are required to connect the pruning activity with the distinctive microtubule polarity of the dendrites in C4da neurons during dendrite pruning . Previous studies have shown that dendrite pruning in C4da neurons is initiated by the steroid hormone ecdysone and its heterodimeric receptors , ecdysone receptor B1 ( EcR-B1 ) and Ultraspiricle ( Usp ) [13 , 14] . Through transcriptional regulation of sox14 , ecdysone signaling activates the Sox14 target gene mical , which encodes a cytoskeletal regulator , to regulate dendrite pruning [20] . A few other molecules mediating specific cellular activities have been shown to participate in dendrite pruning of C4da neurons , such as the ubiquitin-proteasome system [14] , caspases [21 , 22] , matrix metalloproteases [14] , microtubule severing proteins [15] and mediators of dendritic calcium transients [23] . Our previous studies identified Ik2 kinase , a homologue of vertebrate IKK- ε ιν Drosophila , that plays an essential role in dendrite pruning of pupal neurons , and further demonstrated that Ik2 is sufficient to induce precocious dendrite severing in larval neurons [15] . To our knowledge , Ik2 is the only known molecule sufficient to induce premature dendrite severing in larvae , reflecting a central role of Ik2 kinase in dendrite pruning . Therefore , in this study we aimed to elucidate the mechanism by which Ik2 kinase signaling is transduced and regulated in Drosophila sensory neurons during dendrite pruning . As Ik2 is essential for dendrite pruning , to elucidate the mechanism of Ik2 kinase signaling , we searched for candidate molecules that mediate Ik2 signals during dendrite pruning . Several lines of evidence suggested that Spn-F , a coil-coiled protein , is a good candidate . Firstly , spn-F mutant flies showed defects in developing oocytes and bristles [24] , similar to the phenotypes observed in ik2 mutants [25] . Secondly , Spn-F physically interacts with Ik2 [26] . It implied that ik2 and spn-F may act in the same pathway during oogenesis and bristle morphogenesis , and raised the possibility that a similar pathway might also be involved in dendrite pruning of C4da neurons . Here , we demonstrate Spn-F playing a key role in linking Ik2 kinase to microtubule motor dynein complex for dendrite pruning . Spn-F acts downstream of Ik2 kinase in the same pathway for dendrite pruning . We show that Spn-F displays a punctate pattern in larval neurons and these Spn-F puncta become dispersed in pupal cells . The redistribution of Spn-F from puncta is essential for dendrite pruning , and depends on the activity of Ik2 kinase and the function of microtubule motor dynein complex . Our data also demonstrate that Spn-F not only links Ik2 to dynein motor complex , but also mediates the formation of Ik2/Spn-F/dynein complex , that is critical for Spn-F punctum disassembly and dendrite pruning .
To examine the role of spn-F in dendrite pruning , we expressed spn-F double-strand RNAs ( dsRNAs ) under the control of class IV-specific ppk-GAL4 [27] to reduce endogenous Spn-F expression . By 18 h APF wild-type neurons have pruned their dendrites ( Fig 1A ) , however the primary dendrites remained connected to the cell body of C4da neurons with spn-F RNAi ( RNA interference ) ( Fig 1B ) . We observed a similar phenotype in spn-F loss-of-function mutants ( spn-F2 , [24] ) ( Fig 1C ) . Dendrite severing was likewise suppressed in neurons of spn-F2/Df mutants ( Fig 1D ) , which carry the spn-F2 allele and a deficiency uncovering the entire spn-F gene locus . To confirm that these pruning defects were due to loss of spn-F from C4da neurons , we expressed the full-length spn-F-GFP directed by ppk-GAL4 in spn-F2 mutants and found that the impaired dendrite pruning was rescued ( Fig 1E and 1F ) . Moreover , we also examined the dendritic morphology of larval C4da neurons of spn-F and ik2 mutants , and found that dendrites develop normally in both mutant neurons ( S1 Fig ) [15] , suggesting that the pruning defects observed in both mutants are not secondary to abnormal dendrite development . Taken together , these results indicated that spn-F is required for dendrite pruning in C4da neurons . Since ik2 and spn-F mutant neurons displayed the same phenotype , we hypothesized that both genes function in the same pathway during dendrite pruning . To test this hypothesis , we performed genetic analyses between ik2 and spn-F . It was known that ik2 over-expression in larval C4da neurons causes cell death [15] . To avoid excessive apoptosis , we employed ppk-GAL4 coupled with its temperature-sensitive inhibitor GAL80ts to achieve spatial and temporal ik2 expression in larval neurons by shifting temperature . Consistent with previous studies [15] , no abnormality was detected in larval neurons under permissive temperature 25°C ( Fig 2A ) . After shifting to non-permissive temperature 29°C , ik2 overexpression not only triggered precocious dendrite severing ( Fig 2B ) , but also caused apoptosis ( Fig 2C ) in wild-type larval neurons . Interestingly , we found that both precocious dendrite severing and apoptosis caused by ik2 overexpression were significantly suppressed in neurons of spn-F mutants ( Fig 2D ) , suggesting that spn-F functions downstream ( or in parallel ) of ik2 in dendrite pruning . Additionally , we found no significant difference between the dendrite pruning defects observed in spn-F2 mutants and that in spn-F2 mutants with ik2 RNAi ( Fig 2E ) , indicating that both ik2 and spn-F act in the same pathway of dendrite pruning . Together , our findings suggested that spn-F acts downstream of ik2 in the same pathway during dendrite pruning . Given that the spn-F-GFP transgene could rescue observed defects in spn-F mutants , we concluded that this transgene could functionally substitute for the endogenous spn-F gene . Therefore , studying the role of Spn-F-GFP in C4da neurons should help us to uncover the molecular function of endogenous Spn-F in dendrite pruning . First , we examined the distribution of Spn-F-GFP in larval and pupal neurons . The Spn-F-GFP proteins displayed a punctate pattern in the soma , dendrites and axons of larval C4da neurons ( Fig 3A and S2A Fig ) . However , those punctate Spn-F-GFP proteins were redistributed in the soma of pupal neurons at 5 h APF ( Fig 3B ) . Since both Ik2 kinase and ecdysone are required for dendrite pruning [15] , we asked whether Ik2 kinase activity and ecdysone signaling regulate the dispersion of Spn-F-GFP puncta in pupal neurons during pruning . To test this possibility , we examined the Spn-F-GFP distribution in mutant pupal neurons with expression of ik2-RNAi , kinase-dead mutant ik2-G250D [28] or dominantly negative ecdysone receptor ( EcR-DN ) , and found that the Spn-F-GFP puncta remained intact in all three mutant neurons at 5 h APF ( Fig 3C–3E ) . These results indicated that there is a negative correlation between Spn-F puncta and dendrite pruning , and both Ik2 kinase and ecdysone signaling are required to redistribute Spn-F from puncta in neurons during dendrite pruning . Moreover , we also found that redistribution of Spn-F from puncta was also observed in the larval C4da neurons with Ik2 , but not with Ik2-G250D , overexpression ( S2F–S2I Fig ) . To gain mechanistic insight into the redistribution of Spn-F from puncta , we performed live-cell imaging to monitor Spn-F-GFP distribution in the same C4da neurons from larvae to pupae ( S1 and S2 Movies ) . The live imaging and signal profiling of Spn-F-GFP showed high punctate and low dispersed signals in the cytosol of larval neurons ( S2B and S2E Fig ) . We quantified the numbers and the sizes of Spn-F puncta in single live ddaC neurons , and found both numbers and sizes of Spn-F puncta decrease along the pupation time ( Fig 3F ) . In contrast , the dispersed Spn-F-GFP signals in the cytosol of pupal neurons increased as the punctate signals decreased ( S2C–S2E Fig ) . We quantified the averaged fluorescent intensity of dispersed cytosolic Spn-F-GFP signals in the soma , and measured the fold changes of these signals in the same neurons at various time points . In the cytosol of wild-type C4da neurons , the dispersed cytosolic GFP signals started to increase soon after pupation ( Fig 3G ) , peaked at 1 h APF , and decreased to a steady state after 2 h APF . We noticed that these dispersed cytosolic signals measured in pupal neurons were all higher than those in larval cells ( Fig 3G ) . Furthermore , this prompt increase of dispersed Spn-F-GFP signals in wild-type neurons was not observed in the mutant neurons with ik2-RNAi , ik2-G250D , or EcR-DN expression ( Fig 3G ) , indicating that Ik2 kinase and ecdysone signaling promote the increase of dispersed Spn-F in early pupal neurons . Next , we assessed Ik2 kinase activities in C4da neurons by staining with antibodies against phosphorylated Ik2 ( P-Ik2 ) on serine 175 , whose phosphorylation is essential for Ik2 activation [29] . Consistent with the observation of maximum dispersed Spn-F-GFP signals in the cell body of pupal neurons at 1 h APF ( Fig 3G ) , strong P-Ik2 signals were detected in C4da neurons at the same stage ( Fig 3I and 3L ) , whereas no specific signals were found in larval cells ( Fig 3H and 3L ) or in mutant neurons expressing Ik2-G250D ( Fig 3J and 3L ) . These results indicated that Ik2 kinase activity is upregulated in early pupal neurons , but downregulated in larval neurons . Since transiently increasing Ik2 expression caused precocious dendrite severing of larval neurons ( Fig 2B ) [15] , we concluded that elevation of Ik2 activity in pupal neurons initiates dendrite severing , and suppression of Ik2 activity in larval neurons prevents premature severing . We also examined P-Ik2 signals in mutant neurons with disrupted ecdysone signaling and failed to detect activated Ik2 signals ( Fig 3K and 3L ) , indicating that Ik2 kinase acts downstream of ecdysone signaling in dendrite pruning . Taken together , these data demonstrate that Ik2 kinase is activated promptly in early pupal neurons and regulates Spn-F distribution in C4da neurons during dendrite pruning . The inverse correlation between Spn-F puncta and dendrite pruning suggests that redistribution of Spn-F from puncta in C4da neurons might be a critical event for dendrite pruning . If this is true , a mutant Spn-F that remains punctate and disperse-resistant in pupal neurons should have pruning defects . To generate such a mutant Spn-F , understanding the regulatory mechanism of Spn-F distribution is prerequisite . The detection of a band shifting of Spn-F-GFP , which is only expressed in C4da neurons , specifically in the larval lysates with Ik2 expression , but not with kinase dead Ik2-G250D expression or without Ik2 expression ( S3 Fig ) , indicated that Spn-F could be phosphorylated by Ik2 kinase in C4da neurons . Since our study revealed that Ik2 kinase activity regulates Spn-F distribution , and Dubin-Bar et al . also shown that Ik2 phosphorylates Spn-F in kinase assays [26] , we further characterized how the phosphorylation of Spn-F by Ik2 kinase regulates Spn-F distribution and dendrite pruning . First , to identify the residues of Spn-F phosphorylated by Ik2 kinase , we carried out mass spectrometry analyses . Given that Ik2-dependent redistribution of Spn-F-GFP in neurons could also be observed in Drosophila S2 cells , we reasoned that the mechanism underlying the reduction of Spn-F puncta is conserved in both cell types . Thus , we purified 1 ) Spn-F proteins from S2 cells with Ik2 expression referred to as the group of high Ik2 activity , and 2 ) Spn-F from S2 cells alone or 3 ) S2 cells with kinase-dead Ik2-K41A expression [30] referred as the group of low kinase activity for mass spectrometry analyses . The results revealed five serine residues ( S53 , S85 , S264 , S270 and S349 ) of Spn-F that are phosphorylated by Ik2 kinase specifically ( S1 and S2 Tables and Fig 4A ) , and three other serine residues ( S172 , S202 and S325 ) showing increased level of phosphorylation in the group of high Ik2 activity ( Fig 4A and S4 Fig ) . To determine the function of phospho-Spn-F , we substituted all eight serine residues with alanine to generate the phospho-deficient Spn-F-8A , and with aspartic acid to make the phospho-mimetic Spn-F-8D ( Fig 4A ) . We observed evident gel mobility shift of Spn-F in the cell lysates of S2 cells co-expressing Ik2 , but not Ik2-G250D ( Fig 4B ) . In contrast , we detected no mobility shift of Spn-F-8A in any case ( Fig 4B ) . This confirmed that the eight serine residues identified by mass spectrometry are the main sites of Spn-F phosphorylated by Ik2 kinase . Both Spn-F-8A and Spn-F-8D retained the interaction with Ik2 as the control Spn-F did ( S5 Fig ) , consistent with previous reports that Ik2-dependent phosphorylation of Spn-F does not affect the interaction between Ik2 and Spn-F [26] . The presence of Spn-F puncta in cells suggested that Spn-F might form oligomers through self-association . To test this possibility , we performed co-immunoprecipitation ( co-IP ) in S2 cells and found that Spn-F proteins could interact with themselves ( Fig 4J ) . Moreover , we observed reduced self-association of Spn-F in the S2 cells with Ik2 co-expression , but not in either the control S2 cells alone or the S2 cells with Ik2-G250D expression ( Fig 4J ) . These results indicated that the phosphorylation of Spn-F by Ik2 kinase decreases Spn-F self-association . As expected , the self-association of Spn-F-8D , but not Spn-F-8A , was strongly reduced in both the control and the Ik2-expressing cells ( Fig 4J ) . These data suggested a mechanism that Ik2 phosphorylates Spn-F to decrease Spn-F self-association and promotes the redistribution of Spn-F from punctate to dispersed in cytosol . Next , we examined the distribution of Spn-F-8A-GFP and -8D-GFP proteins in larval and pupal neurons respectively . The puncta formed by SpnF-8A-GFP were comparable to those formed by Spn-F-GFP in larval neurons ( Fig 4C and 4D ) . However , the puncta formed by SpnF-8D-GFP were generally smaller than those by Spn-F-GFP in larval cells ( Fig 4E ) . Moreover , the dispersed GFP signals in the cytosol of larval neurons with SpnF-8D-GFP expression were higher than those with Spn-F-GFP expression ( Fig 4E ) . Notably , SpnF-8A-GFP puncta were resistant to disperse in neurons at 5 h APF ( Fig 4G ) , while both Spn-F-GFP and SpnF-8D-GFP puncta were redistributed ( Fig 4F and 4H ) . These findings confirmed that phosphorylation of Spn-F by Ik2 kinase promotes the dissociation of Spn-F oligomers and facilitates Spn-F redistribution in early pupal neurons . Finally , to determine the role of Spn-F redistribution in dendrite pruning , we examined whether SpnF-8A , whose puncta are resistant to redistribution in pupal neurons , could rescue dendrite-pruning phenotypes in spn-F mutant neurons . Compared to the pruning defects rescued by wild-type Spn-F , we found Spn-F-8A is incapable of fully rescue the pruning defects in spn-F mutants ( Fig 4I , p<0 . 005 ) . This result confirmed that Spn-F redistribution in pupal C4da neurons is required for dendrite pruning . Moreover , SpnF-8D rescued the pruning phenotypes in neurons of spn-F mutants as efficiently as wild-type Spn-F did ( Fig 4I ) . However , SpnF-8D cannot rescue the pruning defects in mutant neurons with ik2-G250D overexpression ( Fig 4K ) , suggesting that other unidentified factors are required for dendrite pruning . Together , these results demonstrated that phosphorylation of Spn-F by Ik2 kinase , Spn-F redistribution and unidentified factors are critical for dendrite pruning in C4da neurons . Since Spn-F redistribution from puncta is critical for dendrite pruning in C4da neurons , to further study the mechanism underlying Ik2-dependent Spn-F redistribution , we searched for other candidates that involve in Spn-F redistribution during dendrite pruning . Given that Spn-F interacts with a subunit of cytoplasmic dynein complex [24] , Cut up ( Ctp ) , the Drosophila homologue of dynein light chain 1 [31 , 32] , and dynein is a microtubule-based motor protein , we questioned whether cytoplasmic dynein is involved in dendrite pruning . To address this question , we began to examine the roles of microtubules and cytoplasmic dyneins in Ik2-dependent redistribution of Spn-F in S2 cells with disrupted microtubule cytoskeletons or impaired dynein function . The redistribution of Spn-F from punctate to dispersed in the cytosol of S2 cells is also Ik2-dependent ( Fig 5A and 5B ) . We found that Ik2-dependent Spn-F redistribution was suppressed in cells treated with microtubule-disrupting chemical , colchicine ( Fig 5C ) . The similar observation was also obtained from S2 cells treated with cytoplasmic dynein specific inhibitor , ciliobrevin D [33] ( Fig 5D ) . Notably , Spn-F puncta formed normally in S2 cells treated with either colchicine or ciliobrevin D ( S6 Fig ) , suggesting that the intact microtubule cytoskeletons and functional dynein complexes are not required for the formation of Spn-F puncta . These results indicated that Ik2-dependent redistribution of Spn-F requires both functional dynein and intact microtubule cytoskeletons , and further suggested that Spn-F redistribution is caused by the movement of cytoplasmic dynein on microtubules in cells . Next , to verify whether the function of cytoplasmic dynein complexes is required to redistribute Spn-F in neurons for dendrite pruning , we examined the distribution of Spn-F-GFP in mutant C4da neurons with impaired dynein function . Considering the interaction between Spn-F and Ctp proteins , we first examined the Spn-F-GFP distribution in ctp mutant neurons . Since ctp is an essential gene for embryonic development [32] , we used a hypomorphic ctp mutant allele ctpG0153 , which had few mutant larvae developing to pupae , for analyses . The Spn-F-GFP puncta formed normally in larval neurons of ctpG0153 mutants ( Fig 5F , compared to Fig 5E ) , but failed to disperse in mutant pupal neurons at 5 h APF ( Fig 5I , compared to Fig 5H ) . We next examined the role of dynein heavy chain ( Dhc ) , which encodes the only motor subunit in dynein complex , in the redistribution of Spn-F in C4da neurons . Among seven Dhc genes in fly genomes [34] , Dhc64C ( thereafter referred to as Dhc ) is the only one exhibiting ubiquitous expression , including the nervous systems , throughout development [35] . We employed Dhc RNAi lines to knockdown endogenous Dhc . The thick axons found in larval neurons with Dhc dsRNAs expression ( Fig 5G’ , compared to Fig 5E’ ) were consistent with reported phenotypes in mutant neurons with impaired dynein function [36] , and validated the knockdown efficiency . The Spn-F-GFP puncta formed normally in larval neurons of Dhc-RNAi mutants ( Fig 5G ) , but remained intact in mutant neurons at 5 h APF ( Fig 5J ) . These findings demonstrated the requirement of functional dynein complex to redistribute Spn-F in C4da neurons during dendrite pruning . Finally , to verify the roles of dynein complexes in dendrite pruning , we examined whether mutant C4da neurons with impaired dynein function show pruning defects . At 16 h APF , the primary dendrites remained connected to the soma of C4da neurons in Dhc RNAi mutants ( Fig 5L , compared to Fig 5K ) , suggesting a critical role of dynein motor in dendrite pruning . We also verified the dendrite pruning defects in neurons of trans-heterozygous Dhc mutants with hypomorphic alleles , Dhc6-6 and Dhc6-10 ( Fig 5Q ) [37] . In order to confirm that the phenotypes observed in hypomorphic Dhc mutants were not due to other unidentified mutations on the same chromosome , we examined C4da neurons of Dhc/Df mutants ( deficiency uncovers the entire Dhc64C gene ) . The defective dendrite pruning was evident in C4da neurons of Dhc/Df mutants; 61% ( n = 90 ) of ddaC neurons in Dhc6-6/Df mutants ( Fig 5M and 5Q ) and 20% ( n = 100 ) of cells in Dhc6-10/Df mutants ( Fig 5Q ) showed phenotypes at 16 h APF . Since dynein has been shown to play an essential role in larval dendrite development [36] , to verify the pruning defects in Dhc mutants were not secondary to the early dendrite development defects , we examined the dendritic morphology of C4da neurons in Dhc mutant larvae , and observed normal dendrites of most larval C4da neurons in Dhc mutants ( S7 Fig ) . These findings demonstrated that Dhc plays a critical role in dendrite pruning of C4da neurons . Furthermore , at 16 h APF , about 22 . 5% of C4da neurons ( n = 80 ) in hypomorphic ctpG0153 mutants exhibited severe dendrite clearance phenotypes , which is characterized by long disconnected dendrites surrounding the soma ( Fig 5N ) . We hypothesized that ctpG0153 mutant neurons might have delayed dendrite severing , which resulted in deferred clearance . To test this hypothesis , we examined the dendrites of C4da neurons in wild-type and ctpG0153 mutant pupae at 12 h APF . All dendrites of wild-type neurons ( n = 38 ) were severed at 12 h APF ( Fig 5O ) ; however , about 26 . 8% of neurons ( n = 56 ) in ctpG0153 mutants still retained their primary dendrites attached to the soma at the same stage ( Fig 5P ) . These results indicated that dendrite severing was delayed in hypomorphic ctp mutant neurons , and confirmed that ctp plays a role in dendrite pruning . Finally , we asked whether SpnF-8D could rescue the dendrite pruning defects observed in Dhc mutants . We found no significant difference between the pruning defects of C4da neurons in Dhc6-6/Df mutants and that in Dhc mutants with SpnF-8D overexpression at 16 h APF ( Fig 5R ) , indicating that SpnF-8D cannot rescue the dendrite pruning defects in Dhc mutants . Taken together , our data demonstrates that cytoplasmic dynein motor complex is required for Spn-F redistribution in C4da neurons and for dendrite pruning . Since Spn-F interacts with both Ik2 and Ctp [24 , 26] , we hypothesized that Ik2 , Spn-F and Ctp might form ternary complexes , which could be transported by cytoplasmic dynein . To test this hypothesis , we performed co-IP and antibody staining in S2 cells . The formation of Ik2/Spn-F/Ctp ternary complexes could be detected by co-IP only from the lysates of S2 cells with Spn-F co-expression , but not from that without Spn-F co-expression ( Fig 6A ) , suggesting the central role of Spn-F in the ternary complex formation . As Ik2 kinase causes Spn-F redistribution , the even distribution of all three molecules observed in S2 cells ( S8 Fig ) did not provide convincing evidence to support the colocalization . To visualize the colocalization of these three molecules in S2 cells , we used Ik2-G250D for antibody staining based on the following reasons . First , the dependence of Spn-F to form ternary complex was also observed between Ik2-G250D and Ctp proteins ( Fig 6A ) . Second , since Ik2 kinase activity does not affect Ik2/Spn-F interaction [26] , kinase-dead Ik2-G250D still possesses normal interaction with Spn-F and remains as puncta in S2 cells . The immunofluorescence staining ( Fig 6B and 6C ) and quantification ( Fig 6D ) showed that the colocalization of Ik2-G250D , Spn-F and Ctp molecules were observed only in S2 cells with Spn-F co-expression , but not in cells without Spn-F co-expression , supporting the formation of Ik2/Spn-F/Ctp ternary complex in cells . To test whether Spn-F phosphorylation by Ik2 kinase may affect the formation of Ik2/Spn-F/Ctp complexes , we performed co-IP and colocalization experiments with SpnF-8A and SpnF-8D in S2 cells . Our results showed that SpnF-8A and SpnF-8D have similar abilities as wild-type Spn-F in mediating the formation of Ik2/Spn-F/Ctp complexes in S2 cells ( S9 Fig ) , suggesting that Spn-F phosphorylation have no or minor effects on Ik2/Spn-F/Ctp complex formation . These data demonstrated that Spn-F acts as a central mediator to link Ik2 kinase to dynein motor complex via Spn-F/Ctp interaction in cells . As Ik2 , Spn-F and Ctp are crucial for dendrite pruning , and Spn-F plays a central role in the formation of Ik2/Spn-F/Ctp ternary complex in cells , we asked whether this ternary complex is required for the Spn-F redistribution and for dendrite pruning in C4da neurons . To approach this question , we set out to map the Ik2- and Ctp-interacting domains of Spn-F , and determined the role of Ik2/Spn-F/Ctp ternary complex in neurons for dendrite pruning . Spn-F protein contains three coiled-coil domains [38] , namely CC1 , CC2 and CC3 , and an Spn-F conserved domain ( SCD ) at its C terminus ( Fig 4A ) , which is evolutionarily conserved among different insect species ( S10A Fig ) . To identify the Ik2- and Ctp-interacting domains of Spn-F , we generated a series of Spn-F deletion mutants ( S10B Fig ) and examined their interactions with Ik2 and Ctp in S2 cells by co-IP . The results showed that removal of Spn-F CC3 domain completely abolished the interaction between Ik2 and Spn-F ( Fig 7A ) , but remained weak interaction between Spn-F and Ctp ( Fig 7B ) , indicating that both Ik2/Spn-F and Spn-F/Ctp interactions are disrupted in SpnF-ΔCC3 . Moreover , the co-IP experiments revealed normal interaction between Ik2 and SpnF-ΔSCD ( Fig 7A ) , but weak interaction between SpnF-ΔSCD and Ctp ( Fig 7B ) , demonstrating that SpnF-ΔSCD keeps a normal interaction with Ik2 , but a defective interaction with Ctp . Both the SpnF-ΔCC3 and -ΔSCD puncta formed normally in larval neurons ( Fig 7D and 7E ) , comparable to the wild-type Spn-F puncta ( Fig 7C ) ; however , both Spn-F deletion mutants remained punctate in neurons at 5 h APF ( Fig 7G and 7H , compared to Fig 7F ) , indicating that both Spn-F CC3 and SCD domains are crucial for Spn-F redistribution in pupal neurons . Finally , to determine the role of Ik2/Spn-F/Ctp ternary complex in C4da neurons for dendrite pruning , we performed rescue experiments with SpnF-ΔCC3-GFP and -ΔSCD-GFP in spn-F mutant neurons . SpnF-ΔCC3-GFP failed to rescue the dendrite pruning defects of ddaC neurons in spn-F mutants at 16 h APF ( Fig 7I ) , demonstrating that the Spn-F CC3 domain , which mediates both Ik2/Spn-F and Spn-F/Ctp interactions , is essential for dendrite pruning in C4da neurons . In contrast to SpnF-ΔCC3-GFP , SpnF-ΔSCD-GFP only partially rescued the dendrite pruning defects in spn-F mutants at 16 h APF ( Fig 7I ) , suggesting the role of Spn-F/Ctp interaction in facilitating dendrite pruning . Collectively , our data demonstrated that the Ik2/Spn-F/Ctp complex is critical for Spn-F redistribution and for dendrite pruning in C4da neurons .
In addition to apoptosis , neurons have a second self-destruct program in their axons for axonal pruning during development and in response to neuronal injury and disorders [39] . Here , we propose a third self-destruct program , which is mediated by Ik2 kinase activity in Drosophila sensory neurons , specific for dendrite pruning . Ik2 is essential for dendrite severing in pupal C4da neurons [15] , and currently is the only known molecule sufficient to cause precocious dendrite severing in larval cells [15] , indicating that Ik2 activation must be regulated temporally . For temporal regulation , ecdysone signaling plays a key role in dendrite pruning [13 , 14] . Our studies showing that no Ik2 activation was detected in pupal C4da neurons with impaired ecdysone signaling and thus placed Ik2 kinase downstream of ecdysone signaling . Microarray studies have identified ik2 as one of the ecdysone/EcR up-regulated genes in brain MB γ neurons during axon pruning [40] . This suggests one possible mechanism where ecdysone/EcR regulates Ik2 activation through increasing ik2 expression in C4da neurons . Although Ik2 kinase activity is crucial for oogenesis and bristle morphogenesis [25 , 29] , the activation mechanisms of Ik2 kinase in both processes remain unknown . Since pruning activity is considered as a self-destruct program , how to regulate this activity spatially in subcellular compartments within individual neurons is an intriguing issue to investigate . In this study , we identified Spn-F and cytoplasmic dynein complex as critical regulators of Ik2-mediated dendrite pruning activity in C4da neurons . It is known that endogenous Spn-F exhibits a punctate pattern in nurse cells [24] , consistent with our observation of punctate Spn-F-GFP in larval C4da neurons . The formation of Spn-F puncta in cells is through self-association , and does not depend on the integrity of microtubule network or the function of cytoplasmic dynein ( S6 Fig ) . Since Ik2 could form oligomers in cells [29] , the interaction between Ik2 and Spn-F might also play a role in Spn-F puncta formation . Indeed , we observed that SpnF-ΔCC3-GFP has normal interaction with either SpnF-ΔCC3 or full-length Spn-F ( S11 Fig ) , but formed fewer puncta than the wild type Spn-F-GFP did in larval neurons ( Fig 7D ) . Therefore , the Spn-F puncta formation could be attributed not only to Spn-F self-association , but also to Ik2/Spn-F interaction and Ik2 oligomerization . In larval C4da neurons , Ik2 kinase is inactive and associates with Spn-F as puncta in the cytosol . After puparium formation , Ik2 kinase becomes activated promptly and phosphorylates Spn-F in C4da neurons . This Ik2-dependent phosphorylation on Spn-F decreases Spn-F self-association , and subsequently the numbers and sizes of Spn-F puncta were reduced ( Fig 3F ) . One may question that protein degradation might contribute to decrease the numbers and sizes of Spn-F puncta in C4da neurons during dendrite pruning . It was known that Ik2 promotes caspase inhibitor DIAP1 degradation via proteasomes during the development of sensory organ precursors [28]; therefore , Ik2 might promote Spn-F degradation in C4da neurons during dendrite pruning . However , Ik2 overexpression does not alter the protein level of Spn-F in either S2 or germline cells [26] . Thus , protein degradation by proteasomes is unlikely the mechanism leading to decreased Spn-F puncta after Ik2 activation . Since P-Ik2 signals were indistinguishable between wild-type and Dhc64C RNAi neurons ( S12 Fig ) , it is reasonable to presume that both Ik2 activation and Spn-F phosphorylation occur normally in dynein mutant neurons . We also found no significant differences between the pruning defects of C4da neurons in spn-F mutants and that in spn-F mutants with Dhc-RNAi ( S12D Fig ) , and between the pruning phenotypes observed in Dhc mutants and that in Dhc mutants with ik2-RNAi ( S12E Fig ) . These findings further support that Ik2 , Spn-F and dynein complex function together in the same pathway in dendrite pruning of C4da neurons . However , our finding of Spn-F puncta in mutant pupal neurons with impaired dynein function indicated that dynein is required for Spn-F redistribution after Ik2 activation . Furthermore , Spn-F remains punctate in S2 cells with Ik2 overexpression even after microtubule depolymerization and inhibition of dynein function , suggesting that dynein might redistribute Ik2/Spn-F complexes via transporting complexes toward the minus ends of microtubules in C4da neurons during dendrite pruning . Based on our results in this study and studies in germline cells that more Spn-F puncta accumulated in nurse cells with colchicine treatment and with Dhc mutation [24] , we favored the mechanism of protein redistribution for Spn-F punctum reduction in dendrite pruning of C4da neurons . It has been shown that during Drosophila bristle elongation , directional transport of activated Ik2 and of Spn-F to the bristle tips , where the microtubule minus ends are concentrated [41] , requires the function of cytoplasmic dynein , and Spn-F acts as an adaptor to link Ik2 to dynein complexes [42] . These are similar to our findings that both Ik2 activation and dynein complex are essential for Spn-F redistribution , and Spn-F plays a central role in the formation of Ik2/Spn-F/dynein complex , which is crucial for Spn-F redistribution and for dendrite pruning in C4da neurons . However , the studies in bristle elongation indicating that spn-F acts upstream of ik2 [42] disagree with our finding that ik2 acts upstream of spn-F in dendrite pruning . The discrepancy between the epistasis analyses of ik2 and spn-F in these two different processes might be due to different cell-type specific factors in these two types of cells that affect the morphological readouts in genetic studies . Moreover , we demonstrated that Ik2-dependent phosphorylation of Spn-F decreases Spn-F self-association , promotes Spn-F redistribution , and finally leads to dendrite pruning in C4da neurons . The activated Ik2 signals are found accumulated at the microtubule minus ends in cells with polarized microtubule distribution , such as oocytes , follicle cells and bristles [29 , 43] . This is consistent with our conclusion that dynein transports activated Ik2 toward microtubule minus ends in C4da neurons . Since Drosophila sensory neurons have polarized microtubules in their proximal dendrites predominantly with microtubule minus end pointing away from the cell body [18] , our studies revealed a possible mechanism that Spn-F and minus-end directed motor dynein complex confine Ik2-dependent pruning activity to the somatodendritic compartments of C4da neurons . During Drosophila bristle elongation , the accumulation of endogenous Spn-F observed at the bristle tip , where the microtubule minus ends are enriched [41] , led us to examine Spn-F-GFP signals along the dendrites of C4da neurons during dendrite pruning . However , we did not observe enriched Spn-F-GFP signals in the proximal dendrites , where dendrite severing is expected to occur , by live imaging during pruning . Previous studies [15] showed that microtubules are first disassembled in the proximal dendrites of C4da neurons during dendrite severing , and this local disassembly of microtubules was suppressed in ik2 mutant neurons . Since our genetic studies indicated that both ik2 and spn-F acts in the same pathway of dendrite pruning , we tested whether local microtubule disassembly happens normally in spn-F mutants . We found that local breakage of microtubules in the proximal dendrites of C4da neurons was also suppressed in spn-F RNAi mutants ( S13 Fig ) , suggesting that Spn-F , like Ik2 , plays a role in dendrite severing that involves local microtubule disassembly . However , the molecular mechanisms by which activated Ik2 and Spn-F lead to dendrite severing in the proximal dendrites of C4da neurons will be an important question for future studies . It is known that there was no decrease in cell death in wing discs with ik2 knockdown and in ik2 mutant embryos [28] , indicating that the primary function of Ik2 is not involved in apoptotic pathway during development . However , ectopic Ik2 activation by overexpression leads to cell death in fly compound eyes [28] and in C4da neurons [15] , suggesting that excessive Ik2 kinase signaling could trigger a crosstalk with signaling molecules in apoptotic pathway and result in apoptosis . It is known that Ik2 kinase regulates the nonapoptotic function of caspase through promoting DIAP1 degradation [28] . In a similar manner , the confinement of activated Ik2 kinase in the dendritic compartments might restrict the detected caspase activity in the degenerating dendrites after separating from the soma of C4da neurons during dendrite pruning [21] . Therefore , this raises a possibility that de-regulation of pruning activity in neurons may trigger a crosstalk with molecules in apoptotic pathway and lead to undesired cell death during neuronal injury and disorders . Recently , a caspase cascade , including caspase 3 and 6 , was identified in mice to play a role in developmental axon pruning and in sensory axon pruning after trophic factor withdrawal [44 , 45] . Moreover , activated caspase 6 was detected in human patient brains of Alzheimer and Huntington diseases long before cell death [46] , highlighting a critical role in regulating caspase activity in both diseases . Understanding the regulatory mechanisms that confine pruning activity into proper subcellular compartments of the neuron might provide molecular insights into the pathogenesis of neural disorders .
spn-F2 [24] , Dhc6-6 , Dhc6-10 [37] , ppk-eGFP [47] , ppk-GAL4 [27] , UAS-ik2 ( UAS-DmIKK ε ) , and UAS-ik2-G250D [28] have been described previously . Df ( 3R ) tll-e , Df ( 3L ) BSC436 , tub-GAL80ts , and UAS-EcR-F645A were obtained from Bloomington Stock Center . ctpG0153 was obtained from Drosophila Genetic Resource Center ( DGRC ) . RNAi lines were obtained from Vienna Drosophila RNAi Center ( VDRC ) , and Fly Stocks of National Institute of Genetics ( NIG-FLY ) . Flies were raised at 25°C . For the experiments with tub-GAL80ts , flies were transferred and kept at 29°C for 40–42 h before analysis and imaging . Transgenic flies were generated by standard P-element mediated transformation . Our previous study with ddaC neurons labeled with ppk-eGFP or ppk-GAL4::UAS-mCD8GFP indicated normal severing occurs around 4–6 h APF , and complete debris clearance around 16–18 APF [15] . Therefore , we analyzed the pruning phenotype of ddaC neurons at or after 16 h APF to exclude individual temporal differences . At time of analysis , ddaC neurons having any single dendrite branch with continuous GFP signals extended from the center of soma more than 100 μm was considered pruning defective . The spn-F full-length and deletion DNA fragments were generated by using PCR to amplify the DNA fragments from the cDNA clone LD01470 ( Drosophila Genomics Resource Center ) as the template . The spnF-8A and spnF-8D DNA fragments were generated by PCR-based site-directed mutagenesis . All spn-F DNA fragments were inserted together with EGFP coding sequence or the coding sequences containing HA , myc , or FLAG tags into pUAST vector to make various UAS-spn-F constructs . To generate UAS-ctp-FLAG construct , a DNA fragment encoding Ctp protein was amplified by PCR from the genomic DNA of w1118 flies and inserted together with FLAG coding sequence into pUAST vector . The DNA sequences of all spn-F and ctp inserted fragments were verified by DNA sequencing . Drosophila Schneider cells ( S2 cells ) were cultured in Shields and Sang M3 insect medium ( Sigma ) supplemented with 10% FCS and antibiotics at 25°C . Plasmids were transfected using Effectene ( QIAGEN ) . To disrupt microtubule cytoskeletons , S2 cells were treated with 10 μM cholchicine . To suppress dynein function , S2 cells were treated with 10 μM ciliobrevin D . Cells were transfected with tubulin-GAL4 to drive various UAS constructs: UAS-ik2-HA , UAS-ik2-FLAG , UAS-ik2-G250D-HA , UAS-spn-F-myc , UAS-spn-F-FLAG , UAS-spnF-8A-myc , UAS-spnF-8D-myc , UAS-spnF-N-myc , UAS-spnF-C-myc , UAS-spnF-ΔCC1-myc , UAS-spnF-ΔCC2-myc , UAS-spnF-ΔCC3-myc , UAS-spnF-ΔSCD-myc , UAS-GFP-myc , and UAS-ctp-FLAG . Cells were lysed in lysis buffer ( 20 mM Tris pH 7 . 0 , 150 mM NaCl , 2 mM EDTA , 1% TritonX-100 , 1 mM DTT and protease inhibitors ( Roche ) ) . The cell lysates were incubated with anti-FLAG M2 agarose beads ( Sigma ) on ice and washed thoroughly with lysis buffer . Proteins were eluted and detected using immunoblot assays with rat antibody to HA ( Roche ) , rabbit antibody to Myc ( Santa Cruz Biotechnology ) , mouse antibody to FLAG ( Sigma ) , and appropriate HRP-conjugated secondary antibodies ( Jackson Immuno Research ) . Primary antibodies used in this study were rabbit anti-P-Ik2 antibodies ( 1:50 , a gift of Dr . S . Hayashi ) [29] , rat anti-HA 3F10 ( 1:500 , Roche ) , mouse anti-FLAG ( 1:500 , Sigma ) . The secondary antibodies conjugated with DyLight dyes ( Jackson Immuno Research ) were used at 1:500 dilution . The larvae and pupae were dissected in cold PBS , fixed with 4% formaldehyde for 20 min at room temperature , and stained with proper primary antibodies and subsequent secondary antibodies . For antibody staining in S2 cells , transfected S2 cells were transferred to coverslips coated with Concanavalin A ( Sigma ) , fixed with 4% formaldehyde , and stained with proper primary antibodies and appropriate secondary antibodies . For live imaging , pupae were placed on a double-sided tape ( 3M ) with dorsal side up , and pupal cases were removed by forceps . The pupae were overlaid with distilled water and coverslip for imaging using laser scanning confocal microscope . Images of fluorescent signals were acquired in live animals or fixed samples on confocal microscopes of Bio-Rad MRC-600 , Leica TCS SP5 , or Zeiss LSM 710 . The z-stack images were collected and the maximum intensity projection was used for further analysis . The images were processed using ImageJ ( National Institutes of Health ) , and brightness and contrast were adjusted by Photoshop ( Adobe ) . For timelapse imaging of Spn-F-GFP distribution in ddaC neurons from larvae to early pupae , larvae and pupae were placed with dorsal side up , overlaid with halocarbon oil and coverslip , and imaged with Zeiss LSM 510 laser scanning confocal microscope . To analyze the cytosolic fluorescence of Spn-F-GFP in neurons , we first drew a line across the center of nucleus of cells , and applied the profile analysis module of the Zen software ( Zeiss ) to reveal the signal intensity along the line ( an example in S1B and S1E Fig ) . The peaks of fluorescence intensity were considered as Spn-F-GFP puncta , and the rest of cytosolic fluorescence , which excludes the peaks , was considered as the dispersed cytosolic Spn-F-GFP in cells ( an example as a region marked by an asterisk in S1E Fig ) . The cumulative fluorescence intensities of dispersed cytosolic Spn-F-GFP ( excluding the signals of any Spn-F-GFP puncta ) were calculated and divided by the pixels numbers to obtain the average dispersed fluorescence intensity in the cytosol of each neuron . The average cytosolic dispersed fluorescence intensity of larval neurons was considered as one to calculate the fold changes of average cytosolic dispersed signals in the same neuron at various time points after pupation . Statistical analysis was conducted and graphs were generated in GraphPad Prism . Spn-F proteins were isolated by anti-FLAG M2 beads ( Sigma ) from S2 cells transfected with tubulin-GAL4 and UAS-spn-F-FLAG alone , or together with either UAS-ik2 or UAS-ik2-K41A . The bound proteins were eluted with FLAG peptide ( Sigma ) , separated by SDS-PAGE , and stained with Coomassie brilliant blue R-250 ( Sigma ) . The Spn-F-FLAG protein bands were manually excised from the gel and performed in-gel digestion with modified trypsin ( Promega ) . NanoLC-nanoESI-MS/MS analysis was performed on a nanoAcquity system ( Waters ) connected to the Orbitrap Elite hybrid mass spectrometer ( Thermo Electron ) equipped with a PicoView nanospray interface ( New Objective ) ( Core Facilities for Protein Structural Analysis at the Institute of Biological Chemistry , Academia Sinica , Taiwan ) . The tryptic peptide mixtures were loaded onto a 75-μm x 250-mm nanoACQUITY UPLC BEH130 column packed with C18 resin ( Waters , Milford USA ) and separated at a flow rate of 300 nl/min using a gradient from 5% to 80% acetonitrile in 0 . 1% formic acid . The effluent from the HPLC column was directly electrosprayed into the mass spectrometer . The LTQ Orbitrap Velos instrument was operated in data-dependent mode to automatically switch between full scan MS and MS/MS acquisition . All data generated were searched against the NCBI database by the Mascot daemon software . Search criteria used were set as following: NCBI database; trypsin digestion; up to two missed cleavages allowed; variable modification set as carbamidomethyl ( Cys ) , oxidation ( Met ) , phosphorylation ( Ser , Thr ) and phosphorylation ( Tyr ) ; the mass accuracy is set as 10 ppm for precursor ion; peptide charge state set as 2+ , 3+ and 4+ . Phosphorylation sites and peptide sequence assignments contained in Mascot search results were validated by manual confirmation from raw MS/MS data . | In Drosophila , the nervous systems undergo extensive neuronal remodeling during metamorphosis , as many larval neurons die and adult neurons are generated while some larval neurons survive and prune their branches . Pruning that removes specific parts of neuronal branches without causing cell death is a self-destruct process , thus requiring precise regulation to prevent undesired damage to the nervous systems . Certain Drosophila sensory neurons that undergo dendrite pruning , specifically eliminating the dendrites but leaving the axons intact , provide us an opportunity to study the mechanism of how pruning activity is regulated in the dendrites . We reasoned that the distinctive microtubule polarity in dendrites and axons might be involved and factors are required to regulate the pruning activity in the dendrites through their interaction with microtubules . Here , we identified Spindle-F that mediates Ik2-dependent pruning activity in the dendrites by linking Ik2 to the microtubule motor dynein complex . We showed that elevation of Ik2 activity during dendrite pruning promotes Ik2/Spindle-F/dynein complex moving along the microtubules . We also showed that the formation and redistribution of Ik2/Spindle-F/dynein complex are essential for dendrite pruning . Our study reveals a connection between the polarized microtubules of dendrites and the pruning activity through Spindle-F for dendrite pruning . | [
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] | [] | 2015 | Spindle-F Is the Central Mediator of Ik2 Kinase-Dependent Dendrite Pruning in Drosophila Sensory Neurons |
Malignant tumors are caused by uncontrolled proliferation of transformed mutant cells that have lost the ability to maintain tissue integrity . Although a number of causative genetic backgrounds for tumor development have been discovered , the initial steps mutant cells take to escape tissue integrity and trigger tumorigenesis remain elusive . Here , we show through analysis of conserved neoplastic tumor-suppressor genes ( nTSGs ) in Drosophila wing imaginal disc epithelia that tumor initiation depends on tissue-intrinsic local cytoarchitectures , causing tumors to consistently originate in a specific region of the tissue . In this “tumor hotspot” where cells constitute a network of robust structures on their basal side , nTSG-deficient cells delaminate from the apical side of the epithelium and begin tumorigenic overgrowth by exploiting endogenous Janus kinase/signal transducer and activator of transcription ( JAK/STAT ) signaling activity . Conversely , in other regions , the “tumor coldspot” nTSG-deficient cells are extruded toward the basal side and undergo apoptosis . When the direction of delamination is reversed through suppression of RhoGEF2 , an activator of the Rho family small GTPases , and JAK/STAT is activated ectopically in these coldspot nTSG-deficient cells , tumorigenesis is induced . These data indicate that two independent processes , apical delamination and JAK/STAT activation , are concurrently required for the initiation of nTSG-deficient-induced tumorigenesis . Given the conservation of the epithelial cytoarchitecture , tumorigenesis may be generally initiated from tumor hotspots by a similar mechanism .
In epithelial tissues , cells communicate with their neighbors and receive information from the surrounding environment through signaling networks , adhesion molecules , and junctional molecules in order to form complex organs and maintain their integrity and morphology . This robust , self-organizing system , however , is progressively disrupted during tumor development . In tumorigenesis , transformed mutant cells evolve into a malignant neoplasm through a multistep process whereby the transformed cells acquire traits that enable them to become tumorigenic and ultimately malignant [1] . Although many genes have been identified as involved in different steps of cancer cell progression , little is known about the beginning of tumorigenesis , in which only small subsets of mutant pro-tumor cells deviate from the robustly organized microenvironment to evolve into aggressive tumors . Studies of a group of Drosophila tumor-suppressor genes , lethal giant larvae ( lgl ) , discs large ( dlg ) , and scribble ( scrib ) , highlighted the critical relationship between loss of epithelial organization and tumor development . These genes play key roles in regulation of apical-basal cell polarity and cell proliferation in epithelial tissues [2] . In developing imaginal discs , epithelial tissues that are homozygous mutant for any of these three genes cause cells within the normally monolayered epithelia to lose structure and polarity , fail to differentiate , and overproliferate , thus becoming multilayered amorphous masses that fuse with adjacent tissues [2] . Similarly , loss or alteration in expression of these genes in mammals has been shown to be involved in development of malignant tumors [3 , 4] . The neoplastic phenotypes exhibited by the mutant tissues have led to the classification of these three genes as conserved neoplastic tumor-suppressor genes ( nTSGs ) [2 , 3] . However , when nTSG mutant fly cells are generated in otherwise wild-type epithelia using the FLP/FRT-mediated mitotic recombination technique , they undergo cell-competition-dependent apoptosis and are eliminated from the epithelial tissue [5–8] . This cell-competition-dependent elimination of nTSG-depleted cells has recently been confirmed in mammalian cells [9] , suggesting cell competition is an evolutionarily conserved tissue-homeostatic mechanism that ensures elimination of pro-tumor cells and epithelial integration . Here , we show that nTSG-deficient pro-tumor cells evade competitive pressure to take a first step toward evolving into aggressive tumors when they are located in a specific region within an epithelium and how this tissue-intrinsic local microenvironment has a decisive role for the life-or-death fate of pro-tumor cells .
Through close examination of nTSG-deficient cells in Drosophila wing imaginal discs , we found RNA interference ( RNAi ) -induced silencing of a single nTSG caused tumorigenic overgrowth in specific areas of the disc ( Fig 1A and 1B ) . At the same time , many of the nTSG-knockdown cells adjacent to wild-type normal cells underwent cell-competition-induced apoptosis ( Fig 1C , S1A and S1B Fig ) , as has been previously shown [6–8] . When lgl was knocked down by ptc-Gal4-driven lgl-RNAi along the anterior-posterior ( AP ) boundary of developing wing imaginal discs , dysplastic overgrowth was induced in the dorsal hinge region where the epithelial sheet is folded ( Fig 1B , 1C and 1D ) . In contrast , the ptc-Gal4-induced misexpression of a constitutively active form of Yorkie ( YkiM123 ) , a transcriptional co-activator of the Hippo tumor-suppressor pathway , did not cause dysplastic overgrowth and instead showed hyperplastic overgrowth throughout the entire region of misexpression ( S1C Fig ) . This clear phenotypic difference between lgl knockdown and Yki over-activation can be explained by their functions in two different tumor suppressor pathways; nTSGs are involved in epithelial organization through maintenance of apical-basal polarity [3] and mitotic spindle orientation [10–12] , while the Hippo hyperplastic tumor suppressor pathway is involved in regulation of cell proliferation and apoptosis [13] . However , the difference between the phenotypes induced by lgl knockdown in the dorsal hinge and other regions in the same epithelial tissue was unexpected . We therefore hypothesized that specific regions in the imaginal epithelial tissue are intrinsically susceptible to tumorigenic stimuli . To test the hypothesis of site-specific tumorigenesis , we generated random clones of nTSG-knockdown cells using the heat-shock-activated flip-out Gal4 system [14] . The dysplasia induced by lgl or scrib knockdown became clear in the hinge region 4 d after heat-shock-induced RNAi expression , and dysplastic tumor growths in the region were clearly observed 7 d after RNAi induction ( Fig 1E–1H ) . These tumors showed strongly up-regulated expression of both phosphorylated c-Jun N-terminal kinase ( pJNK ) and matrix metalloproteinase-1 ( MMP1 ) ( S2A and S2B Fig ) , indicating an invasive cellular behavior [15] . In the hinge area , the occurrence ratio of nTSG-knockdown-induced dysplastic cell mass ( each cell mass was counted as a single event of tumorigenesis ) was the highest in the dorsal hinge ( Fig 1I ) . In the wing pouch region , however , these nTSG-knockdown cells did not show dysplastic tumor growth ( 0% , n = 85 ) . Apoptosis was detected in some nTSG-knockdown cells in both the wing pouch and hinge regions of the mosaic wing disc 3 d after RNAi induction ( Fig 1J and 1K ) . These apoptotic cells , detected by cleaved Dcp-1 staining , were mostly at the boundary between the nTSG-knockdown cells and wild-type cells ( 81 . 7% , n = 278 apoptotic lgl-knockdown cells ) , suggesting cell competition is involved in eliminating nTSG-knockdown cells , as described previously [6–8] . Despite cell-competition-induced apoptosis , surviving nTSG-knockdown cells showed dysplastic tumor growth in the hinge region but not the pouch area . These results suggest the tumorigenic potential of pro-tumor cells with apical-basal polarity defects is dependent on their local environment in the epithelial tissue . We therefore refer to the wing pouch region where pro-tumor cells do not show dysplastic tumor growth as a “tumor coldspot , ” whereas the hinge region where pro-tumor cells induce tumorigenesis is referred to as a “tumor hotspot . ” Tumorigenesis is normally associated with activation of growth-promoting signaling pathways [16] , so we suspected the tumor hotspots in the wing disc might be related to local activation of these pathways . The Janus kinase/signal transducer and activator of transcription ( JAK/STAT ) pathway has been shown previously to be endogenously active in the hinge region of developing wing imaginal discs [17] , and its dysregulation has been implicated in diverse types of human cancers [18] and in several tumor models in Drosophila [19 , 20] . Its secreted cytokine-like ligand Unpaired ( Upd ) [21] , a Drosophila homolog of mammalian Interleukin-6 ( IL-6 ) , is endogenously expressed in dorsal , anterior lateral , and ventral posterior compartments of the hinge area [22] . The expression pattern of 10xSTAT92E-GFP , a JAK/STAT activity reporter , in wild-type tissues confirmed the endogenous activity of this pathway in these same tumor hotspots , particularly in the dorsal hinge region where its endogenous activity appears to be the highest ( Fig 2A ) . The dorsal hinge has three epithelial folds: proximal , medial , and distal folds . The endogenous activity displayed by the JAK/STAT pathway is high in the medial fold , weak in the proximal fold , and barely detectable in the distal fold ( Fig 2A and 2B ) [23] . If tumorigenesis depends on JAK/STAT activity in the tumor hotspot , we predicted it would occur most obviously in the medial fold , where JAK/STAT activity is the highest . Indeed , the dysplastic tumor growth induced by lgl- or scrib-knockdown clones was mostly observed in the medial fold ( Fig 2C and 2D ) . This site-specific tumorigenesis was further examined by suppressing nTSGs using different Gal4 drivers ( act-Gal4 , sd-Gal4 ) , each of which has a broad expression pattern , including both the wing pouch and hinge regions in the wing disc ( Fig 2E , S3A and S3B Fig ) . We found that the dysplastic tumor growth induced by these different drivers occurred most frequently in the medial fold of the dorsal hinge ( 71 . 19% , n = 59 tumors in dorsal hinge ) , with strong up-regulation of 10xSTAT-GFP in the tumorous tissue ( Fig 2E ) . Conversely , no dysplastic tumor growth was detected in the wing pouch area where JAK/STAT signaling is inactive ( 0% , n = 59 ) , and the nTSG-knockdown cells in this tumor coldspot did not show up-regulation of 10xSTAT-GFP ( Fig 2E and S3C Fig ) . These findings suggest a strong correlation between endogenous JAK/STAT activation and tumorigenesis induced by nTSG knockdown in the wing imaginal disc . To determine whether JAK/STAT activation is required for tumor growth , we suppressed STAT92E , the Drosophila homolog of mammalian STAT3 and STAT5 [24 , 25] , in lgl-RNAi-expressing mosaic discs by co-expression of STAT92E-RNAi . The mosaic wing disc bearing clones expressing STAT92E-RNAi alone did not show any obvious phenotype ( Fig 3A ) . Depletion of STAT92E in lgl-knockdown cells , however , blocked the dysplastic tumor growth ( Fig 3C ) , as has been shown in scrib and dlg mutant cells [26] , indicating STAT activation is necessary for nTSG-knockdown-induced tumorigenesis . Next , we asked whether the activation of the JAK/STAT pathway is sufficient for the dysplastic tumor growth of nTSG-knockdown cells . To address this question , we ectopically activated JAK/STAT signaling in lgl-RNAi-expressing mosaic discs by co-expression of a constitutively active form of STAT92E ( STAT92EΔNΔC ) [27] . First , we confirmed that ectopic expression of STAT92EΔNΔC without lgl- or scrib-knockdown showed no dysplastic tumor growth in wing discs ( Fig 3B ) . However , over-activation of STAT in lgl- or scrib-knockdown cells in tumor hotspots , including the distal fold of the dorsal hinge , dramatically enhanced the tumor size ( Fig 3D–3F ) when compared with nTSG-knockdown-induced tumors without STAT over-activation from the same time point post-clone induction ( Figs 1F and 3F ) . Three days after heat-shock induction , clones with co-expression of lgl- ( or scrib- ) RNAi and STAT92EΔNΔC showed clear dysplasia in the hinge regions ( Fig 3D and 3E ) . This dysplasia developed into large tumor masses a day later ( Fig 3F ) . In contrast , nTSG-knockdown cells without STAT over-activation only started to display dysplasia 4 d after clone induction ( Fig 1F ) . To determine whether ectopic activation of STAT can transform a tumor coldspot into a hotspot , STAT92EΔNΔC was co-expressed with lgl- ( or scrib- ) RNAi in the pouch region . Still , tumors were not found in this coldspot , where apoptotic cell death was prevalent ( Fig 3G ) . These results indicate that JAK/STAT activity is required yet not sufficient to induce tumorigenesis of nTSG-knockdown cells when they reside in tumor coldspots . Therefore , we hypothesized that additional factor ( s ) are responsible for the difference in tumorigenesis between coldspots and hotspots . One key difference between coldspots and hotspots is the morphology of columnar epithelial cells composing the pseudostratified monolayer , such that cells in the wing pouch ( coldspot ) have a long and straight shape along their apical-basal axis , whereas cells in the valley-folded hinge regions ( hotspot ) are shorter ( Fig 4A ) . Therefore , we examined subcellular localization patterns of various proteins that function in cytoarchitectural construction of epithelial cells , which might serve to characterize the differences between morphologies . Measurement of proteins involved in the actin cytoskeleton ( F-actin ) , components of the cell cortex ( α-Spectrin and FasIII ) , apical complexes ( aPKC ) , focal adhesion complexes ( βPS-Integrin ) , adherens junctions ( E-cadherin and Armadillo ) , and septate junctions ( Dlg and Lgl ) showed no significant differences in subcellular localization between the coldspot and hotspot ( Fig 4A , 4B , and 4G ) . Interestingly , however , we found that intrinsic subcellular distribution of cortical microtubules ( MTs ) , a cytoskeletal component crucial for the maintenance and modification of epithelial cell structures [28] , showed distinct pattern differences between the coldspot and hotspot ( Fig 4A and 4C–4F ) . MTs were localized primarily at the apical side of epithelial cells in the coldspot ( Fig 4E ) . By contrast , the hotspot area lacked this apical enrichment of MTs . Instead , MTs appeared enriched at the basal side ( Fig 4F ) . Furthermore , in transmission electron micrographs ( TEM ) , we found other striking differences in cytoarchitecture between the hotspot and coldspot . The basement membrane , a sheet-like form of extracellular matrix ( ECM ) underlying epithelial tissues , is composed of approximately ten thin laminae in the imaginal epithelia . These ECM laminae were loosely organized in the coldspot , whereas they were tightly aligned in the hotspot ( Fig 5A , 5B , 5D and 5E ) . Another remarkable difference was also identified in the basal side of the epithelial layer; in the valley-folded hotspot regions , cellular membranes displayed a complicated set of bends at the basal side , whereas in the coldspot they appeared straight along the apical-basal axis ( Fig 5A and 5D ) . In addition , the TEM images revealed many circular-shaped double membrane structures at the basal side of the hotspot , but very few in the coldspot ( Fig 5A and 5D ) . These circular membrane structures , which appeared similar to cross-sections of filopodial protrusions , varied in size ( 50–500 nm in radius ) and had microtubules passing inside ( Fig 5E ) . Indeed , when single cells were marked by randomly expressed membrane-GFP , laterally-elongated filopodial protrusions ( 5–10 μm ) were consistently detected at the basal side of cells in the hotspot ( Fig 5F ) . The web of protrusions appeared intricately intertwined among neighboring cells . In contrast , only shorter filopodial protrusions ( 1–5μm ) were found at the basal side of coldspot cells ( Fig 5C ) . Taken together , these observations suggest that the basal side of the hotspot contains robustly organized membrane and ECM structures , which may prevent delamination of pro-tumor cells from its surface . The aforementioned morphological observations led us to examine the early-phase behavior of pro-tumor cells in different regions of the wing disc through three-dimensional confocal imaging . In the wing pouch , the lgl-RNAi- or scrib-RNAi-expressing cells underwent apoptosis ( Fig 1J and 1K ) and were extruded from the basal side of the epithelial layer 2 d after clone induction ( Fig 6A–6C ) . Apoptotic nTSG-knockdown cells were similarly observed in the basal side of the folded hinge hotspot region , yet remained within the epithelial layer ( Fig 6A , 6B and 6D ) . These results suggest that apoptotic pro-tumor cells , found in both coldspot and hotspot regions , tend to be extruded toward the basal side of epithelial sheets , likely by myosin-II-dependent pulling forces generated by apoptotic cells , as has been previously shown [29] . Consistent with this observation , sporadic wing-disc cells misexpressing a pro-apoptotic gene , Reaper , were also extruded toward the basal side in both coldspots and hotspots ( S4 Fig ) . Interestingly , we found some surviving nTSG-knockdown cells in the hotspot delaminated from the apical side of the epithelial layer ( Fig 6F ) . In contrast , no apical delamination of the nTSG-knockdown cells was detected in the coldspot ( Fig 6E and 6H ) , consistent with the previous report that mitotic spindle misorientation caused by nTSG knockdown induces live-cell delamination from the basal side in the wing pouch . These basally delaminated cells then undergo apoptosis [10] . In the hotspot , by contrast , apically-delaminated cells appeared to have proliferated 5 d after clone induction , forming an amorphous mass of cells at the apical side ( Fig 6G–6I ) . Indeed , the dysplastic tumor growth induced by nTSG knockdown always occurred at the apical side of the epithelial layer ( 100% , n = 127 ) . As has been described above , the tumorigenic overgrowth was detected most frequently in the medial fold where JAK/STAT activity was high ( Fig 2D ) , suggesting apically delaminating pro-tumor cells are exposed to endogenous JAK/STAT signaling activity that is specifically activated in the medial fold for their survival and growth . Similar to reports in mammalian epithelial cells [30 , 31] , Drosophila Upd is secreted from the apical surface of epithelial cells to transduce the signal to the neighboring cells , where it binds the receptor Domeless , which is also localized on the apical membrane [32] . Therefore , apical delamination in the valley-folded hotspot where the JAK/STAT ligand abundantly accumulates favors the survival and proliferation of the pro-tumor cells . If this hypothesis is true , we predicted that nTSG-knockdown cells delaminated apically would not undergo tumorigenesis in the coldspot where JAK/STAT signaling is not active . The guanine nucleotide exchange factor RhoGEF2 , an activator of the Rho family small GTPases [33 , 34] has been implicated in apical constriction and the cell-shape change of epithelial cells during gastrulation [35 , 36] , actomyosin contraction during blastoderm cellularization [37 , 38] , and segmental groove formation during embryogenesis [39] in Drosophila . Furthermore , knockdown of p115 RhoGEF , a mammalian homolog of RhoGEF2 , has been shown to disrupt the apically directed extrusion of apoptotic cells in cultured epithelial monolayers from human bronchia [40] . Therefore , we examined whether a defect in RhoGEF2 activity could disturb the delamination direction of nTSG-knockdown cells in the wing imaginal disc . Using the en-Gal4 driver to knockdown RhoGEF2 in the posterior compartment of a wing disc ( Fig 7A–7C ) , we found that both apical enrichment of F-actin and basal enrichment of βPS-integrin were decreased in the pouch cells ( Fig 7D and 7E ) . Unexpectedly , RhoGEF2 knockdown also reversed the subcellular localization of cortical MTs in the coldspot: MTs became enriched at the basal side , opposite to their normal apical localization in the same region ( Fig 7F ) . Moreover , the thickness of basement membranes visualized with a functional GFP-tagged Collagen IV ( Vkg-GFP ) was strongly reduced by RhoGEF2 knockdown in the coldspot ( Fig 7G and 7H ) where ECM laminae were loosely organized ( Fig 5A and 5B ) . These observations suggest that RhoGEF2 knockdown induces hotspot-like cytoarchitectures in the coldspot area . To determine whether RhoGEF2 knockdown has an effect on the delamination direction of nTSG-deficient cells in the disc , we co-knocked down RhoGEF2 in scrib-RNAi- or lgl-RNAi-expressing mosaic discs . Consequently , loss of RhoGEF2 resulted in apical delamination of both scrib- and lgl-knockdown cells in the coldspot ( Fig 8A and 8C ) . Because nTSG-knockdown alone resulted in only basal delamination in this same region ( Figs 6H and 8A ) , we conclude that depletion of RhoGEF2 can disturb the delamination direction of nTSG-deficient cells . However , these apically delaminated scrib-knockdown cells did not show tumorigenic overgrowth in the lumen of coldspots 4 d after clone induction ( Fig 8C ) . Since endogenous JAK/STAT signaling is required for tumorigenesis in the hotspot ( Figs 2 and 3C ) , we asked whether ectopic activation of JAK/STAT signaling can induce tumorigenic overgrowth of nTSG-deficient cells delaminated apically at the coldspot . To this end , we misexpressed STAT92EΔNΔC together with scrib-RNAi and RhoGEF2-RNAi and found that scrib-knockdown cells with RhoGEF2 suppression and STAT ectopic-activation showed apical delamination and tumor growth in the lumen of coldspots 4 d after clone induction ( Fig 8B and 8D ) . These tumors showed strongly up-regulated expression of MMP1 ( Fig 8E ) , which was not detected in scrib- and RhoGEF2-co-knockdown cells located in the coldspot ( Fig 8D ) . These results indicate that both apical delamination and JAK/STAT activity are required for nTSG depletion-induced tumorigenesis in this Drosophila epithelial tissue . Ectopic activation of STAT by STAT92EΔNΔC alone did not change the epithelial cytoarchitecture such as MT subcellular distribution in epithelial cells ( S5A Fig ) . It did not change the extrusion direction of scrib-knockdown cells in the coldspot either ( S5B Fig ) . On the other hand , RhoGEF2 knockdown did not cause a change of JAK/STAT signaling activity in the disc ( S5C Fig ) . These data suggest that the effects of each modification on nTSG-knockdown pro-tumor cells are discrete and that collaboration of these two independent processes ( apical delamination and JAK/STAT activation ) is required for the initiation of nTSG-depletion-induced tumorigenesis . Considering that ectopic activation of STAT alone in basally extruded nTSG-knockdown cells did not induce tumorigenesis in the coldspot , apical delamination provides the pro-tumor cells with a crucial survival advantage . Apoptosis of pro-tumor cells , such as nTSG mutant cells , is known to be induced by Eiger , the Drosophila homolog of mammalian tumor necrosis factor ( TNF ) -α , which is produced by circulating hemocytes recruited to tumor tissues [41] . Because hemocytes directly associate with cells along the basal side of the epithelial layer [42] , apical delamination could have prevented the pro-tumor cells from receiving the death signal . Our data describe how two different causal factors are closely involved in initiation of nTSG defect-induced tumorigenesis in tumor hotspots: apical delamination of surviving pro-tumor cells and high levels of endogenous JAK/STAT activity . The different sensitivity to tumorigenic growth between the distal pouch and proximal hinge regions in Drosophila wing imaginal discs has been reported in some previous studies [8 , 43 , 44] . However , the tumors induced by lgl mutant clones in those studies were created under specific experimental conditions in which the competitive pressure was removed or attenuated by conferring oncogenic activity on lgl mutant clones via overexpression of oncogenic RasV12 or Yki , or lowering competitiveness of the surrounding cells via the Minute technique . In contrast , the site-specific tumorigenesis phenotype reported here was induced by knockdown of an nTSG alone , revealing an important role played by the endogenous local microenvironment on determining where tumors can grow . The tumor hotspots display a network of robust basal structures , including a web of intertwining filopodia and tightly laminated basement membranes , which could force pro-tumor cells to delaminate from the apical side and enter the lumen formed by the disc epithelium and peripodial membrane . In the disc lumen where JAK/STAT activity is high , such as the medial fold , tumorigenesis takes place in this niche-like environment . The cells in the medial fold of the dorsal hinge region also show intrinsic resistance to ionizing radiation ( IR ) - and drug-induced apoptosis and help tissue regeneration , further suggesting the intrinsic tumor susceptibility of this subpopulation [45] . A recent in vitro study using organotypic human mammary acini has shown apical translocation of single cells from the epithelial layer allows the oncogenic mutant cells to undergo luminal outgrowth [46] . In addition , chronic inflammation is linked to all stages of tumor development . It is well known that activation of STAT family members ( particularly STAT3 ) , which are closely linked to inflammatory processes in multiple tissues , is implicated in tumorigenesis , promoting proliferative and survival signaling [47] . In pathological histology , it is also known that tumors frequently arise in transitional zones where two different types of epithelial tissue meet , resulting in the appearance of a distinct abrupt transition , which can be found in numerous locations within various tissues [48] . For example , tumors in the anal canal were found to arise primarily in a transitional zone between stratified squamous epithelium of anal skin and mucosal epithelium of the large intestine [49] . Intriguingly , even in wild-type mice , the epithelium of the transitional zone in the anus intrinsically shows many features reminiscent of hyperproliferative epidermis including aberrant expression of differentiation markers , enhanced Ras-MAPK signaling , and locally increased inflammation [49 , 50] . Future studies will determine if the transitional zones in mammalian tissues have the tumor-hotspot-specific cytoarchitectures that we found in the Drosophila imaginal discs . Besides the transitional zones , however , it is also likely that tumor-hotspot microenvironments are easily formed by small alterations in epithelial cytoarchitectures . Given the widely conserved cytoarchitectures of metazoan epithelia , disruption of tissue organization initiated from intrinsic tumor hotspots could explain a general mechanism of tumorigenesis .
Drosophila stocks were maintained by standard methods at 25°C . For generation of mosaic UAS-transgene overexpression clones in imaginal discs , first instar larvae ( 48 h after egg deposition ) were heat-shocked for 15–60 min at 37°C . To control the timing of UAS-transgene overexpression with the temperature-sensitive Gal80 ( Gal80ts ) in imaginal discs , fly larvae were kept in 18°C until they reached second instar stage and were then transferred to 29°C . The following fly strains were used: ptc-Gal4 ( Bloomington #2017 ) , sd-Gal4 ( Bloomington #8609 ) , en-Gal4 ( Bloomington #30564 ) , act-Gal4 ( Bloomington #4414 ) , UAS-RhoGEF2-RNAi ( Bloomington #34643 ) , UAS-rpr ( Bloomington #5823 ) , UAS-lgl-RNAi ( VDRC #51247 ) , UAS-scrib-RNAi ( VDRC #105412 ) , UAS-STAT92E-RNAi ( VDRC #106980 ) , UAS-YkiM123 [51] , UAS-3HA-STAT92EΔNΔC [27] , 10xSTAT-GFP [22] , and vkg-GFP ( G00454 ) . All genotypes of flies used in each experiment are described below . Fig 1: Fig 2: Fig 3: Fig 4: Fig 5: Fig 6: Fig 7: Fig 8: S1 Fig: S2 Fig: S3 Fig: S4 Fig: S5 Fig: Immunofluorescent stainings of imaginal discs were performed according to standard procedures for confocal microscopy as described previously [52] . The following antibodies were used: rabbit anti-aPKC ( 1:1000 , Santa Cruz Biotechnology ) , rabbit anti-cleaved Caspase-3 ( 1:100 , Cell Signaling ) , rabbit anti-Dcp-1 ( 1:100 , Cell Signaling ) , mouse anti-armadillo N2 7A1 ( 1:40 , Developmental Studies Hybridoma Bank [DSHB] ) , rat anti-DE-Cadherin DCAD2 ( 1:30 , DSHB ) , mouse anti-Dlg 4F3 ( 1:40 , DSHB ) , mouse anti-Fasciclin III 7G10 ( 1:15 , DSHB ) , mouse anti-Integrin βPS CF . 6G11 ( 1:100 , DSHB ) , mouse anti-MMP1 ( 1:1:1 mixture of 3B8 , 3A6 and 5H7 were diluted 1:10 , DSHB ) , mouse anti-α-Spectrin 3A9 ( 1:50 , DSHB ) , mouse anti-α-Tubulin AA4 . 4 ( 1:100 , DSHB ) , rabbit anti-Lgl ( 1:100; a gift from J . A . Knoblich , IMBA , Vienna ) [53] , rabbit anti-phospho JNK pTPpY ( 1:100 , Promega ) , and rabbit anti-Laminin-γ ( 1:100 , abcam ) . Alexa Fluor 488 , 546 , and 633 ( 1:400 , Molecular Probes ) were used for secondary antibodies . F-actin was stained by Alexa Fluor 546 Phalloidin ( 1:50 , Molecular Probes ) . Images were captured on a Zeiss LSM 510 confocal microscope or on an Olympus FV1200 confocal microscope . 3-D reconstructions of confocal z-stack images were rendered with ImageJ 3-D Viewer , an ImageJ plugin ( B . Schmid , 2007 ) . Signal intensities were plotted with Interactive 3-D Surface Plot , an ImageJ plugin ( K . U . Barthel , 2004 ) . The sample preparations were performed according to standard procedures for TEM as described previously [54] . Dissected imaginal discs were fixed in a solution of 2 . 5% glutaraldehyde and 2% paraformaldehyde buffered with 0 . 1 M sodium cacodylate to pH7 . 4 . They were post-fixed with 1% osmium tetroxide in the same buffer , stained en bloc with 0 . 5% uranyl acetate in distilled water , dehydrated in ethanol , and embedded in Epon . Ultra-thin sections ( 70 nm ) were stained with 2% uranyl acetate solution and Reynold’s lead citrate solution . Images were obtained with a VELETA CCD Camera ( Olympus Soft Imaging Solutions ) mounted on a JEM 1010 transmission electron microscope ( JEOL ) . Tumorigenesis was induced by random expression of nTSG-RNAi using the heat-shock activated flip-out Gal4-UAS system , and the occurrence ratio of tumorigenesis was counted 4 to 7 d after clone induction . Each occurrence of tumorigenesis includes a dysplastic cell mass deviating from the disc-proper epithelial layer with a diameter larger than four cells . The percentages of tumor growths were quantified from more than three independent experiments for each genotype . The numerical data used in Fig 8A and 8B are included in S1 Data . Two-tailed unpaired t tests assuming equal variances were performed for all statistical analyses . p-Value < 0 . 001 was considered statistically significant for all analyses . | Transformed mutant cells ( pro-tumor cells ) can evolve through a multistep process in which they become tumorigenic and invasive . Many genes that are involved in the different steps towards cancer development have been identified; however , how certain mutant cells destroy normal tissue organization and undergo uncontrolled proliferation during the initial stages of this process remains largely unclear . Using the epithelial tissue of the wing imaginal discs of the fruit fly ( Drosophila melanogaster ) larvae as a model system , we have analyzed these initial stages of inducing tumors by depletion of a neoplastic tumor suppressor gene ( nTSG ) . We discovered that these tumors always originate from specific regions of the epithelial tissue of the wing disc . We show that in other regions that we dubbed “tumor coldspots” and that lack specific cellular structures , pro-tumor cells are eliminated from the epithelial tissue by the surrounding cells . However , in “tumor hotspots , ” cells constitute specific structures in their basal side , and we found that pro-tumor cells successfully avoid potential elimination and deviate from the apical side of the tissue , initiating tumorous overgrowth . Our findings reveal the molecular and cellular mechanisms underlying the initial steps of tumorigenesis at tumor hotspots in the Drosophila imaginal wing discs . | [
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"dro... | 2016 | Epithelial Tumors Originate in Tumor Hotspots, a Tissue-Intrinsic Microenvironment |
Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway . Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role , but the evidence for this is not robust . We aimed to carry out a whole-genome loss of heterozygosity analysis , followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas . An initial panel of 9 sessile serrated adenomas ( SSA ) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity . This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples . Methylation analysis of genes of interest was carried out using methylation specific PCR ( verified by pyrosequencing ) and immunohistochemistry used to correlate loss of expression of genes of interest . All experiments used adenoma samples and normal tissue samples as control . SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15 . 1–4p15 . 31 , which was not found in the sole TSA , adenomas , or normal tissues . Genes of interest in this region were PDCH7 and SLIT2 , and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2 . Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development , and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson “two hit” hypothesis .
Serrated adenomas are a type of colorectal polyp [1] that possess features of both hyperplastic and adenomatous polyps . The classical feature of serrated adenomas is saw-toothed infolding of dysplastic colonic epithelium as described by Longacre et al ( 2 ) . The classification of serrated adenomas into distinct types has presented a challenge . Snover et al [2] suggested subdivision into sessile serrated adenoma ( SSA ) , traditional serrated adenoma ( TSA ) , mixed serrated polyp ( MSA ) and sessile serrated polyp ( SSP ) , with the latter two subtypes being reserved for difficult to classify or atypical mixed polyps with serrated morphology . Rex et al [3] suggested polyps be subdivided into SSA/P ( with or without cytological dysplasia ) , TSA and stated that these two lesions were the predominant pre-malignant lesions in the serrated pathway . They also stated that dysplasia observed in SSA/P represented the progression of a SSA towards malignancy . The differences between SSAs and TSAs were examined by Torlakovic et al [4] who described SSAs as flat , sessile lesions with crypts that grow parallel to the muscularis mucosae and highly variable and irregular Ki67 expression , whereas TSAs are pedunculated lesions with similar crypts to SSA but low Ki67 expression and a highly eosinophillic cytoplasm . SSA are thought to form a unique subset of colorectal polyps with a distinct molecular pathogenic pathway , known as the “serrated pathway” . The hypothesised molecular pathway in the development and progression of SSA is thought to be different from the conventional adenoma-carcinoma pathway . The most important molecular event known to be highly frequent in the pathogenesis of SSAs is an activating mutation in BRAF ( 1 ) . Multiple studies have found BRAF mutations within SSAs at frequencies of 62–100% [5]–[8] . However , BRAF mutations are not specific to serrated tumours , also being found in colonic adenomas and cancers without a serrated morphology [9] . Also , BRAF mutations have been found in TSA with dysplasia at a high frequency [10] . Consequently BRAF mutations in isolation cannot be said to cause sessile serrated morphology . It has been proposed that a hyperplastic polyp ( HP ) is initiated by a BRAF mutation . The HP then acquires the CpG island methylator ( CIMP ) phenotype , leading to silencing of genes such as IGFBP7 and MLH1 . This is accompanied or followed by progression to a SSA . Methylation of the MLH1 promoter is also hypothesised to be responsible in part for the acquisition of microsatellite instability and for progression to microsatellite-unstable cancer . However , a set of BRAF-mutant , CIMP-high , microsatellite stable cancers also exists [1] and the origins of this type of tumour are currently unknown . Since the serrated adenoma molecular pathway is not fully elucidated , and is unknown whether a common pathway exists to give the serrated phenotype , which then leads to the formation of SSA or TSA , we used genome-wide SNP typing to screen for regions of copy number change and loss of heterozygosity in a set of sessile serrated adenomas . On the basis of our results , we investigated candidate genes in regions of loss of heterozygosity for promoter methylation and changes in expression .
Of the 42 total serrated adenomas , 20/42 were from male patients , and 22/42 from female patients . The median age of patients was 69 years ( range 34–87 years ) . In terms of location , 8/42 were located in the caecum , 2/42 in the ascending colon , 11/42 in the sigmoid colon and 21/42 in the rectum . Of the forty two serrated adenomas , 1/42 was a mixed traditional serrated & sessile serrated adenoma , 1/42 was a mixed sessile serrated/tubovillous adenoma ( reported predominantly as a sessile serrated adenoma ) , 3/42 were filiform TSA [3] and the remaining 37/42 were sessile serrated adenomas . In terms of levels of dysplasia , the mixed SSA/Adenoma was mildly dysplastic , and of the 37 SSA , 19 had mild dysplasia and 18 had no dysplasia . We divided the SA's into two sets , an initial discovery set consisting of 10 SA's ( 9 SSA , 1 TSA ) and a second verification set consisting of 32 SA's ( 2 TSA , 1 mixed SSA , 29 SSA ) . In the classical adenoma samples , 6/10 were from female patients and 4/10 from male patients , with a median age of 58 years ( range 45–73 years ) . They were located in the sigmoid colon ( 2/10 ) , Caecum ( 3/10 ) and Descending colon ( 2/10 ) and rectum ( 3/10 ) . In the classical adenoma tissue microarray there were 100 samples , of which 45/100 were from male patients and 55/100 from female patients with a median age of 58 years ( range 30–85 years ) . In terms of histology , 55/100 were mildly dysplastic , 40/100 moderately dysplastic and 5/100 severely dysplastic . Of the 100 polyps on the array , 22/100 were located in the rectum , 25/100 in the sigmoid , 10/100 in the descending colon , 21/100 in the transverse colon , 10/100 in the ascending colon and 12/100 in the caecum . The initial ten serrated adenoma samples ( 9 SSA , 1 TSA ) from the discovery set were successfully genotyped on the Goldengate array . Of the 9 SSA , 4 had dysplasia and 5 did not . The average genotyping call rate was 0 . 993 . Samples were assessed for copy number changes and LoH events using the log R ratio and the B allele frequency in Illumina GenomeStudio software . There were no large-scale chromosomal gains or losses , except for one sample where there was copy-neutral LoH for the majority of the p-arm of chromosome 6 ( Figure 1 ) . There was only one recurrent region of small-scale copy number change ( Figure 2 ) , a single-copy deletion at chromosome 4p15 . 1–4p15 . 31 ( Chr4:19 , 837 , 583–33 , 138 , 129 ) . Nine out of ten samples ( all SSA samples , with and without dysplasia ) demonstrated this region of copy neutral LoH and this result was confirmed by MIP array analysis in the single SSA sample for which the assay was successful . In total 2 SSA samples were analysed via MIP array with 1 sample failing due to insufficient sample quality . Due to cost constraints the whole genome genotyping was not repeated on the subsequent 32 samples of the verification set . The sole SA in the discovery set which GoldenGate array analysis did not demonstrate LoH at 4p15 . 1–15 . 31 was the SA classified as a TSA . In order to validate the SNP array data , microsatellite LOH analysis was performed on the 9 initial SSA samples ( SA A - I ) in the discovery set ( specifically excluding the single TSA sample ) . The results of this are shown in Table 1 , as well as the measured heterozygosity at these loci , the genetic and cytogenetic position and BP length of the microsatellite . Although there was variable performance of markers and hence LOH frequency , presumably related to chance differences in allelic amplification from FFPE-derived DNA , LOH in the region was confirmed . Although there was no normal tissue we noted that all SSA samples appeared to be homozygous . We could not distinguish this between acquired and constitutional homozygosity however the initial samples matched with the SNP arrays so we assume this represented acquired homozygosity . In the sessile serrated adenoma samples of the discovery set , AFM112XD4 was found to show LOH in all 9 samples except sample C . Being the most informative marker in the region , AFM112XD4 was used to test LOH in the second verification set of 32 SAs . Within this group , of the 29 SSA's , 26/29 had LOH ( 89 . 7% ) . The mixed SSA also demonstrated LOH , whereas the 2 TSA's did not demonstrate LoH , giving a total of twenty-seven of 32 ( 87% ) samples showing LOH . This was significantly different to the observed rate of heterozygosity in the blood control samples ( 78% heterozygosity , p<0 . 001 , Z = −6 . 930 , Rank sum ) . This also reassured us that the observed homozygosity was highly likely to be acquired rather than constitutional . None of the 10 classical adenoma samples had LoH at this marker . In this region of LoH there are 7 genes: SLIT2 ( ENSG00000145147 ) , GPR125 ( ENSG00000152990 ) , PACRGL ( ENSG00000163138 ) , DHX15 ( ENSG00000109606 ) , SOD3 ( ENSG00000109610 ) , RBPJ ( ENSG00000168214 ) and PCDH7 ( ENSG00000169851 ) . Of these , we prioritised two genes – SLIT2 and PCDH7 – as candidate LOH targets , based on their functions . SLIT2 is a negative regulatory component of the DCC/netrin pathway ( 14 ) which leads to malignant transformation in colorectal epithelial cells by Hakai-mediated E-cadherin degradation [11] , [12] , and PCDH7 encodes a protocadherin , the C-isoform of which interacts with ATM in double strand break repair ( 15 ) . Methylation specific PCR of the PCDH7 and SLIT2 promoter regions was carried out in the verification group only . No further examination of other gene specific promoter regions in this area ( i . e . GPR125 , PACRGL , DHX15 , SOD3 or RBPJ ) was carried out as a visual examination of the ENCODE DNA methylation tracks in the UCSC Genome Browser did not suggest any long range methylation in this region . For PCDH7 , 12/32 ( 37 . 5% ) SAs had methylation ( 12/29 SSAs , 0/1 mixed SSA/TSA , 0/2 TSA ) , whereas none of the classical adenoma ( 0/10 ) samples and 1/5 ( 20% ) of the normal mucosa samples were methylated ( Figure 2 ) . For SLIT2 , 31/32 ( 96 . 9% ) SAs had methylation ( 29/29 SSA's , 1/1 mixed TSA/SSA , and 0/2 TSAs ) . There was no methylation in the adenoma ( 0/10 ) or normal mucosa samples ( 0/5 ) ( Figure 3 ) . No methylation analysis of additional normal mucosa samples was carried out at this point as the frequency of methylation observed in SLIT2/PCDH7 was so low it was thought unlikely to add any additional information . The MSP result was verified in a limited subset of samples using pyrosequencing . Five CpG dinucleotides were interrogated in the pyrosequencing PCR , based on the coverage of the MSP , and abnormal methylation was defined as a methylation percentage greater than two times the standard deviation of the average methylation in normal mucosa . It was found that in SSAs , there was consistent and informative promoter methylation at the 2nd , 3rd and 4th CpG's within the MSP region ( Table 2 ) . There was no hypermethylation in a caecal polyp , nor in normal mucosa . As part of a separate experiment conducted in the laboratory , it was found that there was hypermethylation in SLIT2 within a panel of cell lines ( Table 2 ) . Using a separate pyrosequencing primer for SLIT2 ( PM00018634 ) , the SSA were checked for long range methylation in the serrated adenomas further down the CpG island . There was no methylation seen ( Table 2 ) . In analysis of BRAF mutation status , 30/42 ( 73 . 1% ) of analysed serrated adenomas were BRAF mutant , with 29/41 being V600E and 1/41 being V600L , in line with previous studies . Of the three TSA within the SA group , two had V600E mutations and the other was wild type and in the SSA group , 27/41 had BRAF mutations . To study the relationship between methylation and expression in serrated adenomas , IHC for PCDH7 and SLIT2 was carried out on the 32 SAs within the verification group . For PCDH7 expression , 23/32 ( 72 . 0% ) SA samples ( 23 SSA only ) scored either a median expression level of 1 or 0 ( minimal/no expression ) , and 9/32 ( 28 . 1% ) had normal expression ( 6 SSA , 1 mixed TSA/SSA , 2 TSA ) , very similar to the control normal mucosa . For SLIT2 , 31/32 ( 97% ) SA samples ( 29/29 SSA , 1 mixed TSA/SSA and 1 TSA ) had a median expression score of 1 or 0 ( minimal/no expression ) , and 1/32 ( 3% ) had normal expression ( 1 TSA ) . Inter-observer variation for PCDH7 expression was κ = 0 . 70 ( SE = 0 . 12 , Z = 5 . 60 , p<0 . 001 ) and for SLIT2 expression κ = 0 . 74 ( SE = 0 . 16 , Z = 4 . 53 , p<0 . 001 ) . Of the SLIT2 samples , there was only 1 sample where there was disagreement between examiners , with examiner one scoring it as 2/3 and examiner two as 3/3 . After discussion , the score for this sample was set at 3/3 . In Figure 4a ( 5× magnification ) & 4b ( 5× , 10× , 20× magnification ) , normal expression of SLIT2 can be seen in both the conventional adenoma and normal tissue , whereas there is complete loss of expression in the serrated adenoma . A particularly interesting result was found in a polyp with mixed adenomatous/serrated architecture , as demonstrated in Figure 5 . In this image , there was very reduced expression of SLIT2 within the crypts with serrated morphology but normal expression of SLIT2 within the crypts with “classical” adenomatous morphology . There was a poor correlation between PCDH7 promoter methylation and expression ( Table 3 ) , in SA , normal and classical adenoma tissues ( n = 47 ) suggesting that there may be no relationship between the two ( Fisher's exact test , p = 0 . 478 ) . By contrast , there was a clear positive association between SLIT2 methylation and loss of expression ( Fisher's exact test , p = 4 . 97×10−9 , Table 3 ) . Similarly , in comparing PCDH7 expression vs . LOH at AFM112XD4 ( Table 4 ) , in samples where there was loss of PCDH7 expression there was significantly higher rates of LOH at AFM112XD4 ( Fishers exact p = 0 . 001 ) . In comparing SLIT2 expression vs . LOH at AFM112XD4 ( Table 4 ) , in samples where there was loss of SLIT2 expression there was significantly higher rates of LOH at AFM112XD4 ( Fishers exact p = 5 . 16×10-9 ) . For the classical adenomas on the tissue microarray , 83/85 ( 97 . 6% ) demonstrated strong expression of PCDH7 , and 2/85 ( 2 . 4% ) loss of expression . For SLIT2 , 4/83 ( 4 . 8% ) had a median expression score of either 1 or 0 ( minimal/no expression ) and 79/83 ( 95 . 2% ) had normal expression . As part of a separate independent study [13] conducted in our laboratory into whole genome methylation patterns comparing paired colorectal tumour and normal mucosa a gene set enrichment analysis ( GSEA ) was carried out comparing tumour vs . normal mucosa . On leading edge analysis ( an analysis designed to ascertain the most frequently differentially methylated genes within a gene set ) of the GSEA , SLIT2 was the most highly ranked gene in the set . This suggests SLIT2 may be implicated as a tumour suppressor in colorectal carcinogenesis .
A consistent and specific region of LOH occurs in both sessile serrated adenomas ( irrespective of presence/absence of dysplasia ) , located around chromosome 4p15 . 1–4p15 . 31 ( Chr 4:18 , 500 , 000–34 , 000 , 000 ) . Of two excellent candidate genes within this region , SLIT2 shows very frequent promoter methylation and low protein expression . The other gene , PCDH7 , is not infrequently methylated , but shows no convincing evidence of reduced protein expression , and the association between methylation and expression is weak . It is conceivable that long range methylation may be occurring within this region , causing promoter methylation in a number of genes . However , there is no concordance between SLIT2 & PCDH7 methylation and so we thought that this was unlikely . A potential weakness of our study is the use of methylation specific PCR in analysing promoter methylation . As discussed in the methods , we felt that MSP provided a reasonable balance between cost and sensitivity , and we took all possible methods to reduce false positives . We verified the MSP using bisulfite pyrosequencing in a limited sample set , finding informative methylation within the SLIT2 region studied . We were also reassured by the lack of methylation observed in both normal mucosa and classical adenoma samples , and the fact there was no long range methylation of SLIT2 outside the identified region . Alterations in the chromosome 4p15 region have been identified in a variety of cancers . Shivapurkar et al [14] demonstrated a LOH rate of 47% in 4p15 using microsatellite markers in a panel of colorectal cancers . In a separate study [15] , they also demonstrated LoH in this region in 57% of breast cancers studied . This finding was also replicated [16] in a study of malignant mesotheliomas and small cell lung cancer ( >50% ) . However , these findings may have been by aneuploidy that is commonly found in these types of cancer , whereas all our SAs were near-diploid as assessed by SNP arrays . SLIT2 has been postulated for several years to act as a tumour suppressor gene in several cancers [17] . On the basis that SLIT2 expression is regulated by methylation , the findings of our experiment agree with those of Dallol et al [18] . They examined the role of SLIT2 as a tumour suppressor in a panel of 32 colorectal cancers , finding that 23 ( 72% ) were methylated in this region . Furthermore , after treatment of 6 colorectal cancer cell lines with the demethylation agent 5-aza-2-deoxycytidine ( 5-aza-C ) , they demonstrated re-expression of SLIT2 . In order to ascertain whether SLIT2 acted as a tumour suppressor , Dallol et al exposed colorectal cancer cell lines to ectopic SLIT2 , finding that it could suppress colony formation . The mechanism by which SLIT2 exerts pro-apoptotic and growth-suppressive effects is unclear . Prasad et al [19] suggested that SLIT2 regulates the β-catenin and PI3K signalling pathways and enhances β-catenin/E-cadherin-mediated cell adhesion . The pro-apoptotic role of SLIT2 may be caused by interaction with the DCC pathway , as SLIT2 interacts with ROBO [20] which in turn interacts with DCC ( 20 ) , with apoptosis occurring via the caspase pathway [21] . A recent study [22] has also identified low frequency SLIT2 methylation as a marker of high risk dysplasia in ulcerative colitis cancers . This study is the first to demonstrate SLIT2 LOH , promoter methylation and down-regulation consistently occurring in a pre-malignant lesion ( the sessile serrated adenoma ) , and may explain partially the alternate pathway by which sessile serrated adenomas progress towards carcinoma . We did observe a single TSA where there was loss of SLIT2 expression without LOH or SLIT2 promoter methylation . This could be because of somatic mutation within SLIT2 , although this was not examined due to the excessive cost of sequencing SLIT2 . Whilst further studies are required to show a causal relationship between loss of SLIT2 expression and the pathogenesis of SAs , in principle , the two hits we have found at SLIT2 – methylation and LOH – could promote tumorigenesis by a revised version of the Knudson two hit hypothesis [23] , in which LOH renders an epimutation homozygous .
Forty-two formalin-fixed , paraffin-embedded ( FFPE ) serrated adenomas were obtained from Mayday University Hospital , Gloucester NHS Hospitals Foundation Trust and St George's Hospital , London NHS Trust . All patients had had colonoscopy for screening purposes and none reported a family history of colorectal cancer . Representative haematoxylin and eosin-stained sections were reviewed independently by two consultant histopathologists to confirm serrated architecture and the presence/absence of dysplasia . 100 “classical” adenomas from patients undergoing screening colonoscopy , but without a family history of cancer , were examined using a tissue microarray . Normal tissue samples were obtained from 10 unrelated patients . All mucosa samples sample from patients undergoing screening colonoscopy for a change in bowel habit who did not have a family history of cancer , no polyps on colonoscopy and on biopsy were found to have completely normal histological appearances , without any mucosal inflammation being present . Ethical approval was obtained from Wandsworth Ethics Committee ( 07/H0806/109 ) and Oxford Research Ethics Committee ( 05/Q1605/66 ) . 40 µM tissue scrolls were cut and DNA extraction was performed using proteinase K digestion at 55°C . DNA was diluted to a concentration of 50 ng/µl . Genome-wide SNP genotyping was carried out using the Illumina Goldengate HumanLinkage system ( Panel V ) on 10 serrated adenoma samples of which 9/10 were sessile serrated adenomas and 1/10 was a traditional serrated adenoma ( TSA ) . This was included to see if the SSA and TSA demonstrated similar characteristics . Briefly , 1 µg of genomic DNA was amplified as per the manufacturer's instructions and hybridised to the Sentrix BeadArray overnight . The chip was then washed and stained , and scanned immediately using an Illumina BeadArray reader . GenomeStudio software which was used to identify regions with loss of heterozygosity ( LOH ) and/or copy number changes in unpaired sample mode DNA . In order to verify the Illumina array results , 100 ng DNA from two sessile serrated adenoma samples were analysed using the Affymetrix OncoScan FFPE Express service utilising Affymetrix whole genome molecular inversion probe ( MIP ) SNP arrays . Data were analysed using GoldenHelix SNP & Variation Suite 7 utilising the copy number analysis function . Microsatellites ( D4S230 , D4S418 , D4S914 , D4S616 , D4S1593 , D4S2289 and AFM112XD4 ) were chosen to cover the region of interest comprising chromosome bands 4p15 . 1–4p15 . 31 ( Chr4:18 , 500 , 000–35 , 500 , 000 , Genome Build 36 ) . PCR products from the SAs ( Text S1 ) underwent capillary electrophoresis fragment analysis on an Applied Biosystems 3730xl analyser . In the absence of paired normal DNA , we exploited the fact that the SA samples contained a small amount of contaminating normal cells and hence did not show complete LOH . On this basis , we were able to analyse unpaired samples of normal colonic mucosa with the same genotypes as the tumours for scoring LOH . In order to verify the levels of heterozygosity in our study against previously observed population norms for microsatellite markers , we also carried out microsatellite PCR for all analysed microsatellites on 96 blood derived DNA controls from the CORGI study ( primers and conditions available in Text S1 ) . Data were analysed using Applied Biosystems GeneMapper 4 . 0 software . Samples were called as having LOH if the peak area ratio of the second allele compared to the first was less than 0 . 5 or greater than 2 . 0 . For methylation-specific PCR ( MS-PCR ) & pyrosequencing , 1 µg genomic DNA was bisulphite converted using the Zymo DNA Methylation Gold bisulphite conversion kit and eluted into a 20 µL volume , which was then diluted to give a final concentration of 20 ng/µl . MS-PCR primers ( conditions in Text S1 ) were designed using MethPrimer to span the CpG Island of the PCDH7 promoter and the SLIT2 promoter region [24] ( primers and conditions available in Text S1 ) . After MS-PCR , SA sample reaction products were run on a 2% agarose gel alongside those from unpaired normal colonic mucosa DNA , 100% methylated DNA and 100% unmethylated DNA as controls . Samples were classed as methylated if a band was visualised in the methylation-specific reaction and unmethylated if no band was present in the methylated reaction but was present in the unmethylated reaction . If there was a band in both samples , the sample was said to be methylated as this could represent either hemi-methylation or heterogeneity for methylation within the sample . If there were no bands in either sample , the sample was said to have failed . It was thought that MSP would provide an adequate method due to use of highly sensitive and specific PCR reagents ( Qiagen Multiplex PCR kit ) , the low number of cycles used in the PCR reaction ( 35 cycles ) and previous studies documenting the reasonable sensitivity and specificity of MSP [25]–[27] . However , to verify the results , bisulfite pyrosequencing primers ( primer sequences available in Text S1 ) were designed to sequence the region of interest using Pyromark Assay Design v2 . 0 ( Qiagen ) . A off-the-shelf primer set for the SLIT2 CpG island ( PM00018634 ) , available from Qiagen , was also used to analyse long range methylation of SLIT2 . A bisulfite pyrosequencing PCR was set up with standard conditions ( available in Text S1 ) and carried out on a limited subset of samples . Products were analysed using a PyroMark Q96 instrument . To analyse BRAF mutation status , 20 ng of SA DNA was used in a standard sequencing PCR reaction for the region surrounding BRAF codon 600 , and sequenced using an ABI BigDye sequencing reaction ( primers & conditions available in Text S1 ) . 4 µM serrated adenoma sections were cut from FFPE blocks and mounted on poly-L-lysine glass slides for use in immunohistochemistry . Immunohistochemistry for PCDH7 was performed using an indirect method against the primary antibody ( Sigma Prestige rabbit anti-PCDH7 ( ID HPA011866 ) , 1∶100 dilution or Sigma Prestige rabbit anti-SLIT2 ( ID HPA023088 , 1∶35 dilution ) for 1 hr . A positive control of normal colonic mucosa obtained from a normal screening colonoscopy of an individual without colorectal tumours or a family history of colorectal cancer was used , and the negative control was an SA which followed all staining steps except application of the primary antibody . Additional comparison was made with 100 classical colonic adenomas was made using an in-house tissue microarray and with 5 normal colonic tissue samples . PCDH7 and SLIT2 IHC slides were read by two independent assessors ( see Acknowledgements ) . From normal tissue in this study , expression of both PCDH7 and SLIT2 was found to be primarily cytoplasmic , as previously observed by the Human Protein Atlas project . IHC scoring was graded from 0–3 ( 0 = no expression , 1 = mild expression , 2 = moderate expression , 3 = heavy expression ) as compared to the normal control ( heavy expression ) and the median of the two observers was calculated . If there was any disagreement , the higher expression value was used , as this was conservative , based on an assumption that loss of expression would be functionally important . Assessment of inter-observer variation was estimated using Cohen's Kappa ( κ ) . As part of a separate independent experiment [13] , paired colorectal tumour and normal mucosa was also analysed for whole genome methylation patterns on the Illumina HumanMethylation27 platform and subjected to analysis via Gene Set Enrichment Analysis ( GSEA ) . Statistical analyses were carried out using STATA 11 . 1 ( StataCORP , Texas ) . For the SLIT2 and PCDH7 protein expression , variables were dichotomised into strong/moderate expression ( 2 or 3 ) , or no/weak expression ( 0 or 1 ) . For all comparisons , we used the χ2 test , or Fisher's exact test if any cell within the table had a count of less than five . | Polyps in the bowel are the precursors of development of bowel cancer . Much is known about the biology and pathology of these polyps . These polyps have classically been divided into two main types: adenomas and hyperplastic polyps . The former has pre-malignant potential , whereas it is thought the second does not . Within the last 10 years , a third type of polyp has been discovered , called the “serrated adenoma , ” that has features of both adenomas and hyperplastic polyps . However , they seem to have very different characteristics , and we are not certain of the distinct molecular event that leads to their development . In this study we investigate the genetic differences that set apart serrated adenomas from other types of polyps . We report on a gene change within these polyps that seems to be associated with their development . The gene change is highly unusual as it consists of the loss of one copy of the gene as well as methylation of the other gene . This methylation switches off the remaining copy of the gene and stops its associated protein from being expressed in the polyp . To our knowledge , this is the first time this phenomenon has been seen . | [
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] | 2013 | Loss of Expression and Promoter Methylation of SLIT2 Are Associated with Sessile Serrated Adenoma Formation |
The relevance of Cryptosporidium infections for the burden of childhood diarrhoea in endemic settings has been shown in recent years . This study describes Cryptosporidium subtypes among symptomatic and asymptomatic children in rural Ghana to analyse subtype-specific demographic , geographical , seasonal and clinical differences in order to inform appropriate control measures in endemic areas . Stool samples were collected from 2232 children below 14 years of age presenting with and without gastrointestinal symptoms at the Agogo Presbyterian Hospital in the rural Ashanti region of Ghana between May 2007 and September 2008 . Samples were screened for Cryptosporidium spp . by PCR and isolates were classified into subtypes based on sequence differences in the gp60 gene . Subtype specific frequencies for age , sex , location and season have been determined and associations with disease symptoms have been analysed within a case-control study . Cryptosporidium infections were diagnosed in 116 of 2232 ( 5 . 2% ) stool samples . Subtyping of 88 isolates revealed IIcA5G3 ( n = 26 , 29 . 6% ) , IbA13G3 ( n = 17 , 19 . 3% ) and IaA21R3 ( n = 12 , 13 . 6% ) as the three most frequent subtypes of the two species C . hominis and C . parvum , known to be transmitted anthroponotically . Infections peak at early rainy season with 67 . 9% and 50 . 0% of infections during the months April , May and June for 2007 and 2008 respectively . C . hominis infection was mainly associated with diarrhoea ( odds ratio [OR] = 2 . 4; 95% confidence interval [CI]: 1 . 2–4 . 9 ) whereas C . parvum infection was associated with both diarrhoea ( OR = 2 . 6; CI: 1 . 2–5 . 8 ) and vomiting ( OR = 3 . 1; 95% CI: 1 . 5–6 . 1 ) . Cryptosporidiosis is characterized by seasonal anthroponotic transmission of strains typically found in Sub-Saharan Africa . The infection mainly affects young infants , with vomiting and diarrhoea being one of the leading symptoms in C . parvum infection . Combining molecular typing and clinical data provides valuable information for physicians and is able to track sources of infections .
The parasitic protozoa Cryptosporidium spp . attracts attention with large epidemics in industrialized countries while being undiagnosed and neglected in many developing countries [1 , 2] . Cryptosporidiosis is an opportunistic infection in immune-compromised patients , which may results in severe and life-threatening diarrhoea [3–5] . In immunocompetent hosts the disease is usually self-limited , however it has been shown to induce weight loss , growth stunting , sustained impact on child development and increased case fatality [2 , 6–8] . In recent years a number of studies revealed the high proportion of Cryptosporidium spp . on the burden of diarrhoeal disease in endemic settings [1 , 2 , 8–12] . The Global Enteric Multicenter Study ( GEMS ) calculated the second highest attributable fraction on diarrhoeal disease for Cryptosporidium spp . in children less than 12 months [8] . Only in the last decade , increasing species-specific PCR-based diagnosis and sequencing-based subtyping methods ( e . g . sequencing of the gp60 gene ) untangled routes of transmission of Cryptosporidium infections [13–16] . While zoonotic and anthroponotic transmission in industrialized countries varies by regions , human-to-human transmission of Cryptosporidium hominis and anthroponotic subtypes of Cryptosporidium parvum seem to be the dominant infection route in sub-Sahara Africa [9 , 13 , 17–23] However most studies from Sub-Sahara Africa were not conducted in rural areas , but in tertiary care hospitals or in metropolitan areas , where zoonotic transmission might be low . Previous molecular-typing showed species and subtype specific reservoirs , as well as differences , in terms of diarrhoea manifestation , growth faltering and seasonality [12 , 13 , 24–26] . Cryptosporidiosis has been recognised as an important cause for childhood diarrhoea in Ghana [5 , 10] . Nevertheless , no further molecular studies have been performed in Ghana or its neighbouring countries . Molecular typing data on Cryptosporidium spp . for West Africa is up to now limited to publications from Nigeria [18 , 27 , 28] . This study aims to describe Cryptosporidium subtypes among symptomatic and asymptomatic children in rural Ghana and to analyse subtype specific routes of transmission , demographic characteristics as well as clinical differences . The combination of molecular typing results and clinical data will provide valuable information on reservoirs , endemic strains and pathogenicity .
This study was performed in the context of a case-control study on the aetiology of diarrhoea in rural Ghana . Study site was the children’s Outpatients Department ( OPD ) of the Agogo Presbyterian Hospital ( APH ) , a district hospital with 250 beds in the Asante Akim North municipality . This municipality has an estimated population of 142 , 400 inhabitants , spread over an area of 1 , 160 square kilometres . The region has a tropical climate and is mainly covered by secondary rain forest and cultivated land [29] . Children below 14 years of age visiting the hospital’s OPD between May 2007 and September 2008 with complaints of gastrointestinal symptoms ( diarrhoea and/or vomiting ) were included into the study and served as study cases . Diarrhoea was defined as at least three loose stools within the last 24 hours . Vomiting had to occur within the last 24 hours prior to the hospital visit . During the same study period , children of the same age visiting the OPD without any gastrointestinal symptoms , defined as absence of diarrhoea , vomiting or acute malnourishment were recruited as study controls . Each study participant had to provide one stool sample on the day of the hospital visit . All participants were informed about the study’s purpose and procedures . Written informed consent was obtained from the parents or the guardian on behalf of the study children prior to study enrolment . Non-participation had no effect on the medical treatment provided . The Committee on Human Research , Publications and Ethics , School of Medical Science , Kwame Nkrumah University of Science and Technology , Kumasi , Ghana , approved the study design and the informed consent procedure . Immediately after stool collection , the sample was frozen at −20°C . DNA was extracted from the frozen stool samples using the QIAamp DNA Stool-Kit ( Qiagen , Hilden , Germany ) . Extracted DNA was transported to Germany and subsequently a real-time polymerase chain reaction ( RT-PCR ) test was applied to identify C . hominis/C . parvum DNA as previously described [30] . The PCR targets the 18S small subunit ribosomal RNA gene and amplifies a 139 bp DNA fragment . As controls for DNA extraction and/or PCR inhibition each sample was contaminated with traces of phocine Herpesvirus-1 ( PhHV-1 ) prior to faecal DNA extraction . RT-PCR for PhHV-1 was performed as previously described [31] . All PCR-confirmed Cryptosporidium spp . DNA samples were subject to amplification of a 850-bp fragment of the gp60 gene using a nested PCR with the outer primers AL3531 and AL3535 and inner primers AL3532 and AL3534 as described before [20 , 32] . Amplicons were sent for purification and bidirectional sequencing to Eurofins ( Hamburg , Germany ) . Forward and reverse sequences were assembled using Seqscape Software v3 . 0 ( Applied Biosystems , USA ) and subsequently aligned using the ClustalW program running within the BioEdit software package , version 7 . 0 . 9 . 0 . Classification on species level was conducted by an alignment to reference sequences retrieved from the NCBI GenBank . The assignment to subtypes was performed according to the number of trinucleotide repeats ( TCA , TCG or TCT ) coding for the amino acid serine as described elsewhere [17] . The subtype IIcA5G3 was subdivided according to differences in the 3’ region of the gp60 gene [17] . To further support the subtype classification , a neighbour-joining tree was built using the Kimura two-parameter model in MEGA 5 [33] . The reliability of all phylogenetic groupings was determined through a bootstrap resampling analysis ( 1 , 000 replicates ) . Six reference sequences of Cryptosporidium spp . subtypes from the NCBI GenBank database were included in the alignment as controls . All sequences were uploaded to NCBI GenBank ( Accession numbers KM538975—KM539062 ) . Categorical variables were described as frequencies with corresponding percentages . Continuous variables were described using their mean and SD or the median and interquartile range ( IQR ) , respective their distribution . To describe gastrointestinal symptoms in children infected with C . hominis or C . parvum case-control studies for both species were conducted ( S1 Dataset ) . In these analyses exposed children were infected with either C . hominis or C . parvum , un-exposed children were free of Cryptosporidium spp infection . Study outcomes were presence or absence of diarrhoea or vomiting , respectively . Age-adjusted odds ratios ( OR ) , along with the 95% confidence intervals ( CI ) were calculated using the Mantel-Haenszel method . Missing values were excluded from the analysis , thus the denominators for some comparisons differ The discriminatory power ( D ) was calculated as the average probability that the typing system differentiates two unrelated strains [34] . A typing method , which categorizes all different strains to different subtypes , gives a value of 1 . 0 , while a value of 0 . 0 results from different strains being grouped into one single subtype . All data analyses were performed with Stata 12 ( StataCorp LP , College Station , USA ) .
Between May 2007 and October 2008 stool samples from 2 , 322 children were collected . Median age was 33 months ( IQR 14–74 ) and 1 , 239 ( 53 . 4% ) were male . In 116 ( 5 . 0% ) samples Cryptosporidium spp . were detected by PCR . Compared to the whole study group , the median age of Cryptosporidium spp . infected children was lower ( median = 14 months; IQR 8–24 ) . One hundred one ( 87 . 1% ) and 87 ( 75 . 0% ) patients were below the age of three and two years , respectively ( Fig . 1 ) . The majority of the cases were male ( n = 66; 56 . 9% ) . The mean age for male subjects ( 0 . 85; SD 0 . 90 ) was lower than for females ( 1 . 74; SD 2 . 36 ) . Patients were not equally distributed over the study period . In 2007 , 10 . 5 patients/month were observed , while only 3 . 2 patients/month occurred in 2008 . The proportion of infected patients peaked in the months May to July and April to July in 2007 and 2008 respectively , ranging between 5 . 3% ( 5/94 ) in May 2008 to 12 . 9% ( 8/62 ) in July 2008 ( Fig . 2 ) . For a subset of 88 ( 75 . 9% ) out of the 116 Cryptosporidium patients/isolates the gp60 gene was successfully amplified . Alignments to reference strains classified 51 ( 58 . 0% ) strains as C . hominis and 37 ( 42 . 1% ) as C . parvum . Four different C . hominis subtype families ( Ia , Ib , Id , Ie ) and two C . parvum subtype families ( IIc , IIe ) were identified . Further sub-classification led to 16 different subtypes of which the three most frequently observed types were IIcA5G3a ( n = 26 , 29 . 6% ) , IbA13G3 ( n = 17 , 19 . 3% ) and IaA21R3 ( n = 12 , 13 . 6% ) ( Table 1 ) . Ia was the genetically most diverse subtype family , consisting of eight different subtypes , while the other C . hominis subtype families comprise only one single subtype ( IbA13G3 , IdA15 , IeAA11G3T3 ) . The subtype families IIc and IIe contained two subtypes each ( IIcA5G3a , IIcA5G3b and IIeA10G1 , IIeA10G2 , respectively ) . This heterogeneity resulted in a discriminatory power of 0 . 848 for subtyping of the gp60 gene ( Fig . 3 ) . The analysis of demographic patient data for the 88 sequenced strains showed that 23 ( 62 . 2% ) of the C . parvum infected children were males , while the sex distribution for C . hominis infections was relatively equal ( n = 27; 52 . 9% ) ( Table 1 ) . Particularly , the C . parvum subtype family IIc ( n = 20; 66 . 7% vs . n = 10; 33 . 3% ) and the C . hominis subtype family Ia ( n = 16; 64 . 0% vs . n = 9; 36 . 0% ) dominated in male patients . The median age was similar for C . hominis ( 13 months; IQR 8–18 ) and C . parvum ( 12 months; IQR 7–24 ) . 90 . 2% ( 46/51 ) of C . hominis and 89 . 2% ( 33/37 ) of C . parvum infections occurred below the age of three . No age related differences were seen on subtype level . The patients originated from 17 communities . 72 . 4% ( 63/87 ) of cases came from the three largest communities , which are Agogo ( 42 cases ) , Hwidiem ( 10 cases ) and Konongo ( 11 cases ) . In the other 14 communities cases were observed sporadically . When looking at the distribution of subtypes over time for each community separately , not more than three cases of the same strain per month were detected . No particular cluster of specific species or subtype families could be observed over time during or outside of the transmission peaks ( Fig . 2 ) Clinical information on the presence of diarrhoea and vomiting was available for 109 ( 94 . 0% ) Cryptosporidiosis patients . Of those 73 ( 67 . 0% ) and 50 ( 45 . 9% ) patients had diarrhoea and vomiting , respectively . Both symptoms were reported from 41 ( 37 . 6% ) patients . In the 2 , 206 ( 95% ) children without Cryptosporidium spp . infection 929 ( 42 . 1% ) and 785 ( 35 . 6% ) had diarrhoea or vomiting , respectively . In this group 399 ( 18 . 1% ) children showed both symptoms . This corresponds to 7 . 3% and 2 . 7% of Cryptosporidium spp . infections in children with and without diarrhoea symptoms and to 6 . 0% and 4 . 0% in children with and without vomiting , respectively . The age-adjusted case-control study showed associations for C . hominis and diarrhoea ( OR = 2 . 4; 95% CI: 1 . 2–4 . 9 ) , whereas for C . parvum associations with vomiting ( OR = 3 . 1; 95% CI: 1 . 5–6 . 1 ) and diarrhoea ( OR = 2 . 6; 95% CI: 1 . 2–5 . 8 ) were shown ( Table 2 ) .
The absence of any subtype typically associated with zoonotic transmission of C . parvum/hominis , strongly suggests a predominantly anthroponotic transmission among children living in the rural Ashanti region of Ghana , despite close contact to livestock . Transmission is endemic with peaks in the rainy season , but without clonal clusters . As for other diarrhoeal diseases , public health control programmes need to primarily focus on hygienic conditions , particularly among young infants below the age of two years . Physicians should be aware that apart from diarrhoea , vomiting is a frequent symptom , especially in C . parvum infections . The finding of rare subtypes such as IbA13G3 and the IIe subtype family , previously mainly isolated from Sub-Saharan Africa , hints to the existence of regional endemic strains . | Cryptosporidium spp . are a frequent cause of diarrhoea worldwide . While both animal-to-human and human-to-human transmission has been reported from industrialized countries , human-to-human transmission clearly prevails in Sub-Saharan Africa . However , data on the distribution of zoonotic and human subtypes is limited for rural African regions , where children are in very close contact to animals . We conducted a case-control study with 2232 stool samples from symptomatic and asymptomatic children living in the rural Ashanti region of Ghana . The combination of molecular typing results and clinical data helped to untangle transmission routes and to analyze the association of clinical symptoms with specific Cryptosporidium subtypes . Our study results demonstrate seasonal transmission with no clusters of specific subtypes . All subtypes detected have so far been only encountered from human specimens , strongly suggesting a predominantly human-human transmission among children living in the rural Ghana , despite close contact to livestock . Therefore , public health control programmes need to primarily focus on hygienic conditions among young infants below the age of two years . Of interest for practicing physicians , vomiting is a frequent symptom , especially in C . parvum infections . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [] | 2015 | Molecular Characterization of Cryptosporidium spp. among Children in Rural Ghana |
Introduced in the 1950s , ethidium bromide ( EB ) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial . EB has long been known to cause loss of the mitochondrial genome , named kinetoplast DNA ( kDNA ) , a giant network of interlocked minicircles and maxicircles . However , the existence of viable parasites lacking kDNA ( dyskinetoplastic ) led many to think that kDNA loss could not be the mechanism of killing . When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome , we investigated the effect of EB on kDNA and its replication . We here report some remarkable effects of EB . Using EM and other techniques , we found that binding of EB to network minicircles is low , probably because of their association with proteins that prevent helix unwinding . In contrast , covalently-closed minicircles that had been released from the network for replication bind EB extensively , causing them , after isolation , to become highly supertwisted and to develop regions of left-handed Z-DNA ( without EB , these circles are fully relaxed ) . In vivo , EB causes helix distortion of free minicircles , preventing replication initiation and resulting in kDNA loss and cell death . Unexpectedly , EB also kills dyskinetoplastic trypanosomes , lacking kDNA , by inhibiting nuclear replication . Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA , we conclude that minicircle replication initiation is likely EB's most vulnerable target , but the effect on nuclear replication may also contribute to cell killing .
Trypanosoma brucei , the African trypanosome , is a protozoan parasite causing human sleeping sickness and the disease nagana in cattle . For both humans and livestock , there is a compelling need for less toxic and more effective drugs . One drug , ethidium bromide ( EB , known in the veterinary world as homidium; see structure in Fig . S1 ) was synthesized as a trypanocide over a half-century ago by chemists at Boots Pure Drug Co . , Ltd , in Nottingham , U . K . [1] . EB is an intercalating agent [2] widely used as a fluorescent stain for DNA in electrophoresis gels although many scientists are concerned about its mutagenicity . Given its potential dangers , many would be shocked to learn that EB is still used for treating cattle that provide beef and milk for human populations [3] . EB has long been known to promote loss of the trypanosome's mitochondrial genome , the giant DNA network named kinetoplast DNA ( kDNA ) . kDNA is an amazing structure , constituting ∼5% of the cell's total DNA , that consists of several thousand minicircles ( each 1 kb ) and a few dozen maxicircles ( each 23 kb ) . These circles are interlocked together in one huge planar network that has a topology like that of medieval chain mail ( reviewed in [4] , [5] ) . The kDNA network in vivo is condensed into a compact disk-shaped structure residing in the mitochondrial matrix . Maxicircles , like conventional mitochondrial DNAs , encode rRNAs and a few mitochondrial proteins ( e . g . , subunits of respiratory complexes ) . However , to form a functional mRNA with an open reading frame , most maxicircle transcripts are edited by insertion or deletion of uridylates at specific sites . Minicircles encode guide RNAs that are editing templates ( reviewed by [6] ) . The unusual characteristics of kDNA and its replication pathway ( discussed below ) , coupled with the lack of DNA networks in mammalian cells , would suggest that kDNA and its replication proteins should be attractive targets for selective chemotherapy . But this possibility was ignored for many years because of the existence of bloodstream form ( BSF ) trypanosomes lacking kDNA . These dyskinetoplastic ( Dk ) BSFs appear spontaneously or have been induced by treatment with DNA binding agents such as acriflavin or EB [7] , [8] , [9] . Their existence suggested that kDNA is not essential for viability of BSFs and therefore would not be a drug target . Acriflavin causes loss of kDNA in other trypanosomatids , such as Leishmania tarentolae , but in this case the cell dies because maxicircles encode essential mitochondrial proteins [10] . However , a recent discovery changed our thinking on kDNA as a target for chemotherapy . A report that RNAi knockdown of a mitochondrial RNA ligase involved in editing is lethal to BSFs indicated that editing , and therefore kDNA , are indeed needed for viability [11] . Subsequent studies revealed that an essential maxicircle gene product in BSFs is the A6 subunit of the membrane-embedded Fo component of the F1 Fo ATP synthase [12] . In procyclic trypanosomes this enzyme makes ATP , but in BSFs it couples the reverse reaction , ATP hydrolysis , to generation of a mitochondrial membrane potential required for viability [12] , [13] . If editing of A6 is inactivated in either lifecycle stage , then the ATP synthase cannot function and the parasite dies . The reason that some Dk cell lines survive is that they acquire a compensating mutation in a nuclear gene ( encoding the gamma subunit of the F1 portion of the ATP synthase ) that rescues Dk cells and permits their survival [12] , [14] , [15] . Since the compensating mutation occurs at low frequency , kDNA and proteins involved in its replication or gene expression should be valid drug targets in BSFs . Since EB-mediated kDNA loss could be due to a block in network replication , we will briefly discuss this pathway ( reviewed in [4] , [5] ) , focusing first on minicircles . In the first step , a topoisomerase ( topo ) II releases monomeric covalently-closed minicircles from the network into the kinetoflagellar zone ( KFZ ) , a region of the mitochondrial matrix between the kDNA disk and membrane near the flagellar basal body . Proteins in the KFZ initiate and propagate unidirectional theta-type replication . Free minicircle progeny probably segregate in the KFZ and then migrate to the antipodal sites , two protein assemblies flanking the kDNA disk and positioned ∼180° apart . Here RNA primers are removed , and most but not all gaps and nicks ( we will refer to these discontinuities as gaps ) are repaired . The newly replicated minicircles , still containing at least one gap , are then attached to the network periphery by the mitochondrial topoisomerase II ( TbTopoIImt ) positioned in the antipodal sites . Following completion of replication , when the minicircle copy number has doubled , the network splits in two and minicircle gaps are repaired . The progeny kinetoplasts then segregate into the daughter cells during cytokinesis . Much less is known about maxicircle replication . They also replicate unidirectionally as theta structures , but unlike minicircles , they remain linked to the network during replication ( see [16] , [17] , [18] for information on maxicircle replication ) . In this paper we report new and unexpected effects of EB on trypanosomes that reveal , for the first time , how this drug kills these parasites .
We tested EB concentrations ranging from 0 . 01 µg/ml to 10 µg/ml on growth of BSF T . brucei 427 . Although 0 . 02 µg/ml EB stopped growth in 3 days , in most experiments we used 2 µg/ml ( 5 µM ) EB to compare our results with previous data [19] . This concentration arrested growth and killed the cells ( Fig . 1A ) . We next used fluorescence microscopy of DAPI-stained cells to examine EB's effect on the kinetoplast ( Figs . 1B , C ) . It was not surprising that EB initially caused production of small kinetoplasts , and by 72 h , more than 75% of cells had no detectable kDNA ( we refer to these cells as Dk even though we did not confirm that they are completely devoid of kDNA ) . Examples of cells with normal kinetoplasts , small kinetoplasts , and none at all ( Dk ) are shown in Fig . 1C . We then isolated and DAPI-stained networks from EB-treated cells and measured their surface areas ( Fig . 1D ) . Networks from untreated cells averaged ∼5 . 5 µm2 ( in agreement with previous measurements [20] , [21] ) , but after a 3 day EB treatment had shrunk to an average area of ∼2 . 1 µm2 ( Fig . 1D ) . Because kDNA isolation involves centrifugation , there may have been selective loss of the smallest networks; thus the average area may be smaller than indicated . To prove that kinetoplast shrinking is due to loss of minicircles and maxicircles , we digested total DNA with Hind III/Xba I . After electrophoresis , we probed a Southern blot for maxicircle and minicircle fragments ( Fig . 1E ) . Taking into account the loading control , more than half of the minicircles and maxicircles were lost by 72 h . Covalently-closed circular DNAs isolated from prokaryotic or eukaryotic cells are always negatively supertwisted , with one exception . Remarkably , covalently-closed minicircles in a kDNA network [22] or free replication intermediates [23] are fully relaxed in vivo . However , we expected that minicircles isolated from EB-treated cells , either in a network or free , would become negatively supertwisted . This prediction was based on the equation , Lk = Tw + Wr , that relates linking number ( Lk ) to twist ( Tw ) and writhe ( Wr ) [24] . Since minicircles in vivo are relaxed ( Wr = 0 ) , then Lk = Tw , and for a 1 kb minicircle Tw is ∼96 ( assuming 10 . 4 bp/helical turn ) . EB in the mitochondrial matrix should intercalate into minicircles , thereby reducing their helical twist . Since EB binding does not change Lk , the decrease in twist is compensated by an increase in writhe , or positive supertwisting . A mitochondrial topoisomerase could then remove the positive supertwists , reducing Lk . EB , however , would be still bound to the DNA and would be removed only when DNA is isolated . After EB removal , the twist would revert to its normal level of ∼96 , but since Lk had been reduced in vivo , the writhe would decrease , producing negative supertwisting . To test the effect of 2 µg/ml EB on free minicircle replication intermediates , we fractionated total DNA from treated cells on an agarose-EB gel that resolves relaxed covalently-closed minicircles ( CC; the replication precursors ) from gapped minicircles ( G , the replication products ) . A Southern blot ( Fig . 2A ) revealed a profound increase in a new minicircle species , designated fraction E , that migrated near covalently-closed minicircles . Fraction E forms a smear that broadened between 3 and 72 h , and at later time points its level declined . There was also an increase in multimeric minicircles , and at least one species , probably an interlocked dimer ( marked * ) , formed a smear like that of fraction E . As observed previously [19] , EB caused a rise in linearized minicircles ( maximum at 1 h , data not shown ) that then leveled off . Gapped free minicircles rose during the first 3 h followed by a small decline . We then set out to identify fraction E , first using a 2-D gel to compare DNA from untreated cells with that from cells treated 6 h with 2 µg/ml EB . The first dimension conditions were the same as those used for the gel in Fig . 2A; the second dimension was run in 30 mM NaOH . This gel system is effective in resolving free minicircle species [25] , [26] . We then probed a Southern blot with 32P-oligonucleotides of comparable specific radioactivity and complimentary to either the L-strand ( Fig . 2B , left panel ) or H-strand ( Fig . 2B , right panel ) . From untreated cells , we observed the well-documented resolution of covalently-closed minicircles ( CC ) and gapped minicircles ( G ) . These two species hybridized equally to both probes because their strands are equimolar . Most importantly , this experiment showed that fraction E consists of minicircles in the 1-kb range that also have roughly equimolar amounts of L and H strands . In our second approach , we treated fraction E ( purified by sucrose gradient sedimentation [25] ) with E . coli topo I , an enzyme that relaxes negative but not positive supertwists [27] . Electrophoresis on an agarose-EB gel ( Fig . 2C ) demonstrated that the fraction E smear collapsed into a band that migrated with covalently-closed relaxed monomers . Finally , EM of Fraction E provided the most compelling evidence on its structure ( Fig . 2D ) , proving that Fraction E is a family of supertwisted free minicircles . The topo I experiment ( Fig . 2C ) showed that the supertwisting is negative . In addition to supertwisting , DNA can compensate for severe underwinding by forming single-stranded regions and regions of left-handed helix [28] . In fact , we previously detected Z-DNA in highly supertwisted free minicircles from cells undergoing RNAi of p38 , a protein involved in replication initiation [25] . To determine whether a region of Z-DNA forms in free minicircles from EB-treated cells , we immunoprecipitated total DNA with anti-Z DNA antibody , centrifuged the immune-complexes , and fractionated the DNA from the supernatant and pellet on an agarose-EB gel . A Southern blot ( Fig . 2E ) showed that minicircles from untreated cells or following 6 h of EB treatment had no fraction E in the pellet , indicating that these minicircles contain no Z-DNA . However , after 16 or 24 h of EB exposure , we observed part of fraction E ( with the slowest electrophoretic mobility ) in the pellet , demonstrating that some fraction E minicircles contain regions of Z-DNA . We conclude that fraction E minicircles have a broad distribution of linking numbers and the most severely underwound have sequences that flip into Z-DNA . T . brucei has 3 known mitochondrial topoisomerases , TbTopoIImt [29] , TbTopoIAmt [30] , and TbTopoIB ( the latter is functional in both nucleus and mitochondrion [31] ) . In RNAi experiments in Fig . S2 , we showed that knockdown of TbTopoIImt , but not TbTopoIAmt or TbTopoIB , prevented reduction of the linking number of EB-bound free minicircles and thereby inhibited production of Fraction E . Thus , TbTopoIImt is responsible for supertwisting free minicircles isolated from EB-treated cells . Experiments already presented provide strong evidence that EB blocks free minicircle replication . The dramatic rise in covalently-closed monomeric free minicircles in the form of Fraction E ( Fig . 2A ) indicates that minicircle release from the network occurs in the presence of EB . In addition , their accumulation indicates that the subsequent step , initiation of replication , is inhibited . We therefore assessed replication directly by measuring incorporation of bromodeoxyuridine ( BrdU ) , a thymidine analog , into free minicircles . We incubated log phase cells ( ±2 µg/ml EB for 6 h ) with 50 µM BrdU for the last 40 min . We then isolated total DNA , and fractionated the minicircle species first on a 1-dimensional agarose-EB gel ( Fig . 3A , left panel ) . A Southern blot confirmed that minicircles were more abundant after EB treatment because of the large increase in Fraction E and multimers . To assess BrdU incorporation , we ran the same amount of DNA on a 2-dimensional gel ( like that in Fig . 2B ) . We used this gel to evaluate whether any BrdU-labeled minicircle species accumulated in the presence of EB . Probing a blot with anti-BrdU antibody revealed BrdU incorporation into gapped minicircles in the absence of EB but little or none in any minicircle species during the EB treatment ( Fig . 3B ) . This experiment provided even stronger evidence that EB inhibits initiation of minicircle replication . Based on our findings with free minicircles , we predicted that minicircles in networks isolated from EB-treated cells would also be negatively supertwisted and we used EM to test this possibility ( for this experiment it was essential to extract EB from the DNA prior to EM ) . We were astonished that most minicircles in networks isolated from cells exposed to 2 µg/ml EB for up to 24 h remained relaxed . Fig . 4B shows a network from a cell treated for 24 h ( compare with untreated network in Fig . 4A ) . Although it is impossible to evaluate many minicircles in the network interior , most on the periphery are relaxed . Only a few ( some marked by arrows ) appear highly twisted . Fig . S3 shows many more examples of networks from cells treated with EB for 6 h , 15 h , and 24 h . By 24 h , most networks were smaller and more loosely packed ( Fig . S3 , networks J , K , and L ) . In these it was easier to evaluate minicircles in the network interior , and most were relaxed . However , one example ( Fig . S3 , network F ) has a substantial region of the network periphery in which most minicircles are twisted . From the data presented so far , EB treatment of trypanosomes causes severe underwinding of free minicircles but , despite our inability to assess minicircles in the crowded interiors of many networks , it does not have this effect on many and probably most network minicircles . This is remarkable because both DNAs reside in the same cellular compartment , the mitochondrial matrix . One possibility is that the EB caused nicking of network minicircles , thereby preventing supertwisting . To test for nicking , we studied the same networks used in the previous section , but we spread them for EM in the presence of a high concentration of EB ( 100 µg/ml ) . If the network minicircles are nicked , they could not supertwist under these conditions . However , as shown by the example in Fig . 4C ( others are in Fig . S3 , networks H and M ) , we observed extensive supertwisting of minicircles in most networks . However , some , such as network I in Fig . S3 , contained relaxed minicircles . These networks must contain mostly gapped minicircles , and they will be further evaluated below . These data prove that most networks from EB-treated cells contain minicircles that are covalently-closed and that lack of supertwisting is not due to nicking . A second possibility is that network minicircles bind EB , but there is no topoisomerase available to reduce the minicircle Lk . We showed in Fig . S2 that there is considerable active TbTopoIImt present . Furthermore , we recently reported that TbTopoIImt is present throughout the kDNA disk where it mends holes made by minicircle release during replication [20] . Thus , this possibility is unlikely . We addressed a third possibility , that network minicircles bind little or no EB , by fluorescence microscopy of EB-treated live cells . We found that brief EB treatment ( 2 µg/ml added to culture medium ) caused distinct staining of the kinetoplast in nearly all cells , but it is possible that some of this staining could be due to free minicircles . Unfortunately , the high motility of trypanosomes precluded capture of adequate images of live cells . Even when the cells were restrained by adherence to poly-lysine coated slides and other methods , they quickly died and lost their staining . Before this happened their localized movement prevented imaging . Instead , Fig . S4 shows images of 16 fluorescent kinetoplasts in formaldehyde-fixed cells . Our conclusion is that kDNA in vivo probably binds some EB , but it could be far less than that observed with free minicircles . A likely reason for low EB binding could be that the condensed network is associated in vivo with basic proteins [32] , some of which may stabilize the network in its disk-shaped configuration [33] . Such proteins could prevent unwinding of the minicircle helix and therefore would block EB binding . One candidate protein for this role is p19 ( GeneDB Accession Number Tb11 . 02 . 2420 ) , recently discovered in our laboratory and shown to localize throughout the kDNA disk ( unpublished studies of J . Wang , Z . Zhao , B . Liu , P . T . Englund , and R . E . Jensen ) . Recombinant p19 condenses isolated networks in vitro ( not shown ) to a size comparable to that of the kinetoplast in situ . In fluorometric experiments , we observed that network-bound recombinant p19 inhibited EB binding as measured by the marked reduction in EB fluorescence ( Fig . 5 , compare middle panel with left panel ) . In a similar assay , p19 could displace EB from a network ( Fig . 5 , right panel ) . Thus , the most likely explanation for the absence of supertwisting in many if not most network minicircles is that EB binds poorly to networks in vivo . Perhaps those minicircles that do become supertwisted are devoid of proteins , and it is EB binding to these , along with free minicircles , that contributes to the kinetoplast fluorescence observed in Fig . S4 . As mentioned above , some networks from EB-treated cells , examined by EM in the presence of 100 µg/ml EB , had many minicircles that did not twist , suggesting that these minicircles were gapped ( an example is network I in Fig . S3 ) . We further investigated these networks by in vitro labeling the 3′-OH groups at gaps with terminal deoxynucleotidyl transferase ( TdT ) and fluorescein-dUTP [34] . In networks from cells untreated with EB , we detected the well-characterized polar labeling of partly replicated networks [34] , [35] , [36]; labeling is polar because gapped minicircles are attached to the network adjacent to the antipodal sites . From these cells , never exposed to EB ( Fig . 6A ) , 28% of networks were stained by TdT ( TdT-positive ) . This value changed little during 24 h of EB treatment ( see time course in Fig . 6B ) . However , EB dramatically changed the labeling pattern , causing an increase in uniformly labeled networks and a steady decline in polar labeling . The latter suggested that reattachment of newly-replicated minicircles to the network periphery must be strongly reduced by EB , as expected from the fact that EB blocks minicircle replication . In the Discussion we will comment further on these data . If EB kills trypanosomes by blocking initiation of replication of free minicircles , then it would be logical to predict that Dk trypanosomes would be resistant to EB killing . We therefore studied the effect of EB on Dk 164 , a BSF trypanosome lacking kDNA [8] , [37] . Our cytotoxicity assay , using a range of EB concentrations , involved colorimetric measurement of acid phosphatase activity released by lysing living cells after 24 h in wells of a 96-well plate [38] . To our great surprise , we found that EB efficiently killed Dk parasites , and its EC50 , 0 . 3 µg/ml , was slightly lower that of the wild type 427 strain , 0 . 65 µg/ml ( Fig . 7A ) . Thus , EB must have an efficient killing mechanism unrelated to kDNA . A likely possibility for the killing of Dk trypanosomes was that EB also targets nuclear DNA replication . We therefore incubated 427 BSFs with BrdU in the presence and absence of EB for 6 h . Incorporation of this thymidine analog into nuclear DNA increased with time in untreated cells and was inhibited substantially with 2 µg/ml EB ( Fig . 7B ) . At first glance , the data in Fig . 7A suggested that a killing mechanism targeting nuclear replication may be as important , or possibly more important , that that targeting kDNA . However , we then considered the possibility that our 24 h cytotoxicity assay may not reveal the effects of EB-mediated kDNA loss in wild type 427 cells . The reason is that the killing would not occur until essential maxicircle gene products were depleted , which , depending on their half-lives , may take a few days . Thus , the 427 cells might contain a time bomb that would not explode until after termination of the 24 h cytotoxicity assay . To evaluate this possibility , we conducted a new cytotoxicity assay using 427 trypanosomes ( Fig . 7C ) . In this assay , we collected cells not only for evaluation of the effect of EB on growth , but also for measurement of kinetoplast size by DAPI staining . In addition , we measured nuclear replication by adding BrdU to the wells for the last 2 h of the 24 h assay . As shown in Fig . 7C , inhibition of nuclear replication occurred at the same EB concentration as the growth effect; thus , inhibiting nuclear replication kills with little delay . In contrast , kDNA loss occurs at a >10-fold lower EB concentration and this loss will ultimately lead to cell death but not during the 24 h cytotoxicity assay . To further evaluate this possibility , we chose an EB concentration , 0 . 02 µg/ml , that , based on the graphs in Fig . 7C , should eventually kill 427 trypanosomes by targeting kDNA and not kill DK-164 cells which would require a higher EB concentration to inhibit nuclear replication . As predicted , there was little effect of EB on Dk-164 cells ( Fig . 7D ) . Also , as expected , there was little effect on 427 cells during the first 24 h ( the duration of the cytotoxicity assay ) , but survivors were reduced by 75% at day 2 and few cells were alive at day 3 . We also noted in our initial studies mentioned in the first paragraph of Results that 0 . 02 µg/ml EB killed trypanosomes within 3 days . Most studies in this paper on the effect of EB were conducted at 2 µg/ml . Now it was essential to evaluate further the effects of 0 . 02 µg/ml EB on minicircle and nuclear DNA replication . For minicircles we conducted an analysis of free minicircles to determine whether covalently-closed free minicircles ( including Fraction E ) accumulate ( because they are unable to initiate replication ) . In a control experiment , similar to that in Fig . 2A , we found that 6 h treatment with 2 µg/ml EB caused a 36-fold increase in covalently-closed minicircles ( Fig . 7E ) . Reducing the EB concentration 100-fold to 0 . 02 µg/ml , increased the covalently-closed minicircles 15-fold , still a very potent inhibition of replication . As with 2 µg/ml , the maximum effect of 0 . 02 µg/ml was at 6 h and at both concentrations there was a large increase in minicircle oligomers . It was not surprising that 0 . 02 µg/ml EB had a smaller effect in reducing the free minicircle linking number; thus its fraction E smear is more compact . To measure the effect of 0 . 02 µg/ml EB on nuclear replication , we treated a culture for 0 , 6 , 12 , and 24 h . At each time point we incubated a sample with [3H]thymidine for 2 h and then measured acid-insoluble radioactivity ( Fig . 7F ) . Since kDNA constitutes only about 5% of the total DNA , nearly all of the incorporation is in nuclear DNA . These results show that 0 . 02 µg/ml EB has no effect on nuclear replication at 6 h and a small effect thereafter . All the experiments in Fig . 7 provide strong evidence that the most sensitive mechanism by which EB kills trypanosomes is to block minicircle replication initiation . However , inhibiting nuclear replication also contributes to the trypanocidal activity .
Our goal in these studies was to determine whether EB kills trypanosomes by its action on kDNA and , if it does , to determine the killing mechanism . One remarkable and unexpected effect of EB treatment was that it caused profound supertwisting of free minicircles ( Fig . 2 ) , whereas many if not most network minicircles , in the same cellular compartment , remained relaxed ( Figs . 4 , S3 ) . The most likely explanation is that the EB binding to the network , although detectable by fluorescence microscopy ( Fig . S4 ) , occurs at a much lower level than it does to free minicircles . The large reduction in EB binding to network minicircles is most likely explained by the fact that basic proteins like p19 condense the network in vivo and by preventing helix unwinding , inhibit EB binding ( Fig . 5 ) . The conclusions from this experiment are limited because they are based on only one protein , p19 , whose function is unknown , and because the kDNA disk in vivo is likely bound and stabilized by multiple proteins [32] . However , the p19 experiments clearly demonstrate that such proteins can reduce EB binding in vivo ( Fig . 5 ) . Polyamines also bind DNA and prevent EB binding [39] , but proteins seem more likely to be compartmentalized , binding to networks and not free minicircles . Our EM data provides some clues into the replication status of maxicircles . Gel electrophoresis of topo II-decatenated networks [40] from cells treated with 2 µg/ml EB for 6 to 24 h revealed that 20–50% of maxicircles were covalently-closed ( data not shown ) . Furthermore , in 121 EM images of networks ( from cells treated similarly with EB and then observed by EM after EB removal ) , we observed no supertwisted maxicircles . Thus , the covalently-closed network-bound maxicircles , like most network minicircles , must bind little if any EB in vivo . We speculate that the lack of EB binding to maxicircles , as in minicircles , is due to bound protein . If EB does not bind to the maxicircles , it is possible that it has no effect on maxicircle replication . Further studies are needed to address this issue . Our most significant finding was that the most effective mechanism by which EB kills trypanosomes is by inhibition of initiation of free minicircle replication . We initially based this conclusion on the dramatic accumulation of covalently-closed free minicircles , the substrates for replication ( Figs . 2A ( using 2 µg/ml EB ) and 7E ( using 0 . 02 µg/ml EB ) ) . Especially in the case of 2 µg/ml EB , the high level of EB that bound these molecules in vivo not only resulted in the development of extensive negative supertwisting after isolation but also , by distorting their helix in vivo , must have prevented assembly of replication proteins at the origin . The inhibition of BrdU incorporation into gapped minicircles confirmed the replication block ( Fig . 3 ) . Since newly replicated free minicircles attach to the network poles adjacent to the antipodal sites , the EB-mediated decline in networks with polar TdT labeling led to the same conclusion ( Fig . 6 ) . But why does the number of networks uniformly-labeled with TdT increase during EB treatment ? The likely reason is that a partially-replicated network contains gapped minicircles ( labeled by TdT ) in the two polar regions and covalently-closed minicircles ( not yet replicated and unlabeled by TdT ) in the central region [34] , [35] . In the presence of EB , few if any minicircles attach to the network poles , preventing enlargement of the polar regions . However , Fig . 2A shows that covalently-closed minicircle release continues , and these must derive from the central region of the network . Once all are released , the two polar regions , containing gapped minicircles , then fuse . Thus the network appears , after TdT labeling , to be uniformly-labeled and small in size . As the network shrinks , minicircle release may become less precise , resulting in release of multimers ( Fig . 2A ) . We previously found that EB poisons mitochondrial TbTopoIImt ( but not nuclear topo II ) and that cell lysis by SDS-proteinase K results in linearization of ∼2% of the minicircles [19] . This inhibition of TbTopoIImt is unlikely responsible for cell death because only a small fraction of the TbTopoIImt is trapped as a cleavable complex ( for comparison , a more powerful topo II poison , etoposide , causes linearization of 12% [41] ) . Furthermore , the fact that TbTopoIImt is responsible for the EB-mediated reduction in free minicircle linking number ( Fig . S2 ) proves that much of this enzyme is catalytically active in the presence of EB . Since EB blocks initiation of replication of free minicircles , we were initially surprised , and troubled , to find that Dk cells were also killed by EB . However , further study showed that the EB-susceptibility of Dk cells could be explained by inhibition of nuclear DNA replication ( Fig . 7B ) . Furthermore , the effect of EB on kinetoplast size occurred at a >10-fold lower concentration , indicating that kDNA replication is the primary target for EB killing of wild type trypanosomes . Our realization that killing of wild type cells by low EB concentrations does not occur during our 24 h cytotoxicity assays fully clarified this mechanism . The delay in killing must be due to the time needed for the cell to run out of its essential maxicircle gene product , the A6 subunit of the ATP synthase [12] . When we analyzed free minicircles from cells treated 6 h with 0 . 02 µg/ml EB , we confirmed that this low concentration also inhibits free minicircle replication ( Fig . 7E ) . Although our cytotoxicity assays suggest that EB acts primarily on kDNA , this drug has at least some effect on nuclear DNA replication . The effect is modest at low concentrations ( 0 . 02 µg/ml EB; see Figs . 7C and 7F ) , but inhibition of nuclear replication becomes significant at higher concentrations ( 2 µg/ml EB; see Fig . 7C and compare Fig . 7B with 7F ) . Therefore , the contribution of inhibition of nuclear DNA replication to trypanosome killing depends on the level of EB in the treated animal . The blood concentration of ethidium in cattle injected intra-muscularly with the standard dose of 1 mg/kg is 0 . 070–0 . 268 µg/ml at 15 min and ∼0 . 075 µg/ml after 24 h [42] , [43] , [44] . These concentrations are for the free base form of ethidium . At the highest concentration detected , 0 . 268 µg/ml ethidium base ( equivalent to 0 . 34 µg/ml EB ) , which is transient , inhibition of nuclear DNA replication is only about 40% of maximum effectiveness ( see Fig . 7C ) . The 24 h level , ∼0 . 075 µg/ml ( equivalent to 0 . 095 µg/ml EB ) , is about 5 times higher than the 0 . 02 µg/ml EB used in our experiments , suggesting that inhibition of nuclear DNA replication could contribute to killing . One problem with comparing drug activity in vitro to that in animals is that many drugs bind tightly to serum proteins , thereby reducing markedly in animals the concentration of free active drug [42] . We could find no reports on the extent of EB binding to serum proteins , but if high this could substantially reduce the levels of free EB in animals . One reason why EB may target minicircle replication so effectively is that the intra-mitochondrial EB concentration could be higher than the extracellular concentration , due to a membrane potential-driven transport of ethidium into the mitochondrial matrix [43] . We think it unlikely , but cannot rule out the possibility , that non-specific effects of EB , such as binding to RNA or membranes , could contribute to the killing of trypanosomes . Another anti-trypanosomal drug still used for treating African cattle is isometamidium chloride [3] , an EB derivative ( see structure in Fig . S1 ) . In a previous study of whether isometamidium could target kDNA , the effect of the drug on wild type and Dk trypanosomes was investigated using in vitro assays [44] . As we found with EB , those authors reported a slightly lower EC50 for the Dk cells and concluded that an effect on the kinetoplast could not be the primary target of isometamidium . As with EB , it is possible that an effect on maxicircle gene products was not manifested during their cytotoxicity assays even though theirs were longer than ours . Thus , like EB , the primary target of isometamidium could be minicircle replication . In conclusion , these experiments prove decisively that low concentrations of EB kill trypanosomes by blocking initiation of minicircle replication . However , it is likely that curing of trypanosome infections depends in part on the effect of EB on nuclear replication . Although it has been shown previously that kDNA is essential for viability of BSF parasite [11] , our studies emphasize that enzymes involved in kDNA replication could be valid drug targets for chemotherapy for BSF trypanosomes , not only for those causing bovine trypanosomiasis but also for the parasites causing human sleeping sickness .
All experiments except those involving RNAi or the dyskinetoplastic Dk 164 strain ( gift from Ken Stuart , Seattle Biomedical Research Institute ) were conducted on BSF T . brucei wild-type 427 strain ( gift from George Cross , Rockefeller University ) , cultured as described [45] , [46] . For RNAi we used procyclic T . brucei 29–13 [47] ( also from George Cross ) cultured in SDM-79 medium [48] containing 15% fetal bovine serum . Ethidium bromide ( Bio-Rad , 10 mg/ml ) was kept at room temperature in the dark and added to a final concentration of 2 µg/ml unless otherwise indicated . Total DNA , ( purified as described [49] ) from 7×106 EB-treated ( 6 h , 2 µg/ml EB ) cells was centrifuged on a 33 ml 5–20% sucrose gradient ( Beckman SW28 rotor , 25 , 000 rpm , 20 h , 4°C ) [24] . Aliquots ( 10 µl ) of 1 ml fractions were electrophoresed as in Fig . 2A and a blot probed for minicircles . Fraction E was detected in tubes 9 to 15 and the pool was dialyzed overnight against 25 mM Tris-HCl , pH 8 . 0 , 50 mM NaCl , 10 mM MgCl2 ( final volume , 9 ml ) . It was used for E . coli topo 1 treatment ( Fig . 2C ) and EM ( Fig . 2D ) . Networks were isolated as described [50] . Fraction E was prepared for EM using the cytochrome c drop spreading method [51] . After shadowing , samples were examined in an FEI ( Hillsboro , OR ) Tecnai 12 TEM and images recorded using a Gatan Inc . ( Pleasanton , CA ) Ultrascan400 CCD camera . Isolated kDNA networks were spread on grids using the formamide method [52] . Since many experiments required removal of EB from networks prior to EM , we extracted 6 times with an equal volume of water-saturated n-butanol . However , we observed identical results if we did not extract; in that case EB must have been removed by formamide or by ethanol-washing of the grids . After rotary shadowing , networks were photographed using a Hitachi 7600 transmission electron microscope and a DVC 1412M-FW digital camera with AMT Image Capture Engine Software . EM images were uniformly adjusted for brightness and contrast using Adobe Photoshop . Undigested total DNA was electrophoresed to analyze free minicircles , and HindIII/XbaI digests were electrophoresed to analyze for total minicircles and maxicircles [25] . Both were run in a 1 . 5% agarose gel in 1X Tris-borate-EDTA ( TBE ) buffer containing 0 . 5 µg/ml EB ( 18 h , 70 V ) [41] . DNA was detected by probing Southern blots , and the hexose transporter gene was the load control . For 2-D electrophoresis [25] , the first dimension used conditions just described and the second was run in 30 mM NaOH . After electrophoresis , DNA was transferred to GeneScreen plus membrane ( Perkin Elmer ) and probed with strand-specific 5′-[32P]oligonucleotides [25] . Untreated and 6 h EB-treated cells ( 50 ml , 0 . 7×106 cells/ml ) were pulse-labeled for 40 min with 50 µM bromodeoxyuridine ( BrdU ) and 50 µM 2′-deoxycytidine [53] . The latter was added to overcome potential allosteric inhibition of ribonucletotide reductase by high concentrations of BrdU . Total DNA was isolated , electrophoresed on either a one-or-two dimensional gel followed by transfer to a nitrocellulose membrane ( Schleicher and Schuell ) . Membranes were blocked , probed with anti-BrdU antibody , and BrdU level was detected by chemiluminescence [25] . | Trypanosoma brucei is a protozoan parasite that causes cattle disease and human sleeping sickness in Africa . Trypanosomes are primitive eukaryotes with atypical biological features . One well-studied example is their mitochondrial genome , known as kinetoplast DNA or kDNA . kDNA , resembling medieval chain mail , is a giant network of interlocked DNA rings known as minicircles and maxicircles . Here we study ethidium bromide , a drug used for over 50 years for treating cattle infected with trypanosomes . EB's killing mechanism has been elusive , and many thought it could not involve kDNA . We now report that EB kills these parasites by blocking the initiation of minicircle replication , and , at higher drug concentration , it also blocks nuclear replication . | [
"Abstract",
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] | [
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"biology/dna",
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] | 2010 | The Killing of African Trypanosomes by Ethidium Bromide |
Hepatitis C virus ( HCV ) is a significant pathogen , infecting some 170 million people worldwide . Persistent virus infection often leads to cirrhosis and liver cancer . In the infected cell many RNA directed processes must occur to maintain and spread infection . Viral genomic RNA is constantly replicating , serving as template for translation , and being packaged into new virus particles; processes that cannot occur simultaneously . Little is known about the regulation of these events . The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years , but the details have remained enigmatic . NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented . NS5A domain III is not required for RNA replication , and the function of this region in the HCV lifecycle is unknown . We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication . This deletion disrupts virus production at an early stage of assembly , as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells . Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype . This serine residue lies within a casein kinase II consensus motif , and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus . We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production . A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity . These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV . The ability to uncouple virus production from RNA replication , as described herein , may be useful in understanding HCV assembly and may be therapeutically important .
Hepatitis C virus ( HCV ) chronically infects nearly 3% of the population of the planet [1] . Persistent virus replication in these individuals often progresses to chronic liver disease , including cirrhosis and hepatocellular carcinoma . Since the discovery of HCV as the causative agent of non-A , non-B hepatitis in 1989 [2] , considerable progress has been made in therapeutics , but current anti-virals are still ineffective for the majority of patients . One of the major obstacles to developing new anti-viral strategies is the nebulous nature of many aspects of the HCV lifecycle . One particularly vague area of HCV biology is that of the regulation of the transit of RNAs from active replication to virion biogenesis . HCV is a member of the Flaviviridae family of enveloped , single strand positive sense RNA viruses [3] . The 9 . 6 kb viral genome contains a single open reading frame encoding a polyprotein that is cleaved co- and post-translationally to yield ten viral proteins [4] , [5] . These include the structural proteins ( Core , E1 and E2 ) and the non-structural proteins ( p7 , NS2 , NS3 , NS4A , NS4B , NS5A , and NS5B ) . HCV RNA replication occurs in association with ER-like cellular membranes and requires several viral non-structural ( NS ) proteins including; NS3 , NS4A , NS4B , NS5A , and NS5B , as well as host cell factors [6] . The site of virion assembly is unknown , but recent data has proposed the recruitment of HCV RNA and non-structural proteins by the HCV core protein from the replicase to lipid droplets as an early event in virion assembly [7] . Viral genomes that lack core , or contain mutations in NS5A domain I that block lipid droplet binding , prevent the production of infectious virions [7] . The regulatory events that control these events are not known , but it is clear that productive virus assembly requires the NS5A protein . The complexity of intracellular events associated with HCV infection is staggering , with RNA involved in active RNA replication on positive and negative strand templates , serving as message for translation of viral proteins , and serving as a substrate for progeny virus assembly . These events presumably take place in functionally , and perhaps physically , distinct sub-cellular compartments and require different viral and host factors . There is little information of what regulates these processes to avoid functional conflicts , but the HCV NS5A protein is an attractive candidate for at least some of these roles . NS5A is an absolutely required phosphoprotein component of the replicase , but little is known about its function . NS5A is a zinc metalloprotein organized into three discreet domains ( Figure 1A ) [8] . The crystal structure of domain I suggests the functional form of NS5A is a dimer , with a large putative RNA binding groove located at the interface of the monomers [9] . The interaction of NS5A with RNA is thought to be critical for the function of this protein in RNA replication [10] . The region of NS5A required for association with lipid droplets , a prerequisite of virion production , is located in domain I [7] . No structural information has yet been obtained for domains II or III . These regions are genetically variable among HCV isolates and can tolerate insertions and deletions without disrupting RNA replication [11]–[15] . Recent genetic mapping has shown many residues in domain II are essential for RNA replication , but domain III is dispensable [15] . Despite having no role in RNA replication , regions of sequence conservation exist in domain III , suggesting a function in the HCV lifecycle . NS5A localizes to the site of RNA synthesis via an N-terminal amphipathic α-helix membrane anchor [16]–[18] . NS5A , unlike the other NS proteins , can be trans-complemented , suggesting it may have functions outside of the viral replicase [19] . The interaction of NS5A with numerous host-signaling pathways has been described , suggesting this protein may modify the host cell environment to a state favorable for the virus , events that may require an extra-replicase form of NS5A [20] . In recent years , the phosphorylation of NS5A has become a major focus area , as this modification may modulate the activity of NS5A in viral and cellular processes . NS5A is phosphorylated on multiple serine and threonine residues and exists in hypophosphorylated ( 56 kDa ) and hyperphosphorylated ( 58 kDa ) forms [21] . The identification of which of the many potential phosphoacceptor sites in NS5A are relevant to these forms is not known . Nonetheless , evidence has accumulated suggesting that hypophosphorylation primarily targets residues in domains II and III , whereas hyperphosphorylation sites tend to cluster in and around domain I ( reviewed in [22] . Cell culture adaptive mutations that increase RNA replication tend to decrease hyperphosphorylation , suggesting NS5A phosphorylation may regulate replication [23] . Compounds selected to decrease the hyperphosphorylation of NS5A increase RNA replication , and these compounds have been shown to function as potent and specific casein kinase 1α ( CKIα ) inhibitors [24] . CKIα requires pre-phosphorylation of residues near sites of phosphorylation in NS5A for productive modification , suggesting that other kinases also play a role in NS5A phosphorylation [25] . This effect on replication may be due to the differential association of phosphoforms with the host hVAP-A protein , an essential replicase component [26] , [27] . A host of other kinases have been shown to target NS5A in heterologous expression/screening systems , including AKT , p70s6K , MEK , MKK1 , and CKII [22] . Perhaps the best-characterized NS5A kinase activity , after that of CKIα , is casein kinase II ( CKII ) , with a number of biochemical experiments implicating casein kinase II ( CKII ) as an NS5A directed kinase [28]–[30] . In vitro phosphorylation of purified NS5A by CKII incorporates 5 moles of phosphate per mole of protein , suggesting at least five sites exist in NS5A [29] . Low-resolution mapping studies have implicated residues in domain III as the predominant sites of CKII phosphorylation ( Kim et al . , 1999 ) . Further experiments have indicated CKII is likely involved in basal phosphorylation of NS5A , as hyperphosphorylated NS5A is not produced by CKII treatment in vitro [25] . The mutation of a number of potential CKII acceptors does not alter RNA replication [11] . Nonetheless , a body of evidence exists implicating CKII as an NS5A directed kinase . Recent advances in HCV biology have allowed the complete recapitulation of the virus lifecycle in cell culture , allowing the assessment of the function of NS5A phosphorylation in virion production [31]–[34] . In this study we identify a deletion within domain III of NS5A that disrupts the production of infectious HCV virions without altering the replication of HCV RNA . Mapping studies suggest that a single serine residue in a consensus CKII phosphorylation motif is important for the observed phenotype , and phosphomimetic mutants at this position suggest this residue represents a phosphorylation dependent switch controlling virion production . CKII chemical inhibition and siRNA silencing impair infectious virus release for viruses with a serine residue at this position , whereas those with the aspartic acid substitution are insensitive to these treatments . Furthermore , the over expression of CKII increases virus production for serine but not aspartic acid bearing viruses . These results implicate NS5A as a CKII phosphorylation dependent regulator of particle assembly .
Our previous mapping experiments in the context of the subgenomic replicon system , and the work of others , suggested that domain III of NS5A was not required for RNA replication [11]–[15] . We hypothesized that this region might have a function in virus particle production , at the level of RNA release from the replicase for virion production . This hypothesis is supported , at least in part , by the recent demonstration that the localization of NS5A to lipid droplets is an essential prerequisite for virion production [7] . We generated a panel of deletions within the domain III region of NS5A of the cell culture infectious genotype 2a J6/JFH-1 virus clone to assess the importance of domain III in virion production , including deletion A ( amino acids 432–442 ) and deletion B ( amino acids 443–457 ) ( Figure 1A ) . The preliminary analysis of these two deletions in RNA replication and infectious virus production by an immunohistochemical ( IHC ) assay is shown in Figure 1B . In this assay in vitro transcripts of various HCV genomic RNAs are generated and electroporated into the highly permissive human hepatoma cell line , Huh-7 . 5 [35] . Following a 48 hour incubation , the supernatants are harvested , filtered , and used to infect naïve cells . Following an additional 48 hours , cells are fixed and IHC staining for the presence of the NS5A is performed . Wild type virus ( Figure 1B , J6/JFH-1 ) , is capable of replication and production of infectious virus , as evident by the presence of NS5A staining in cells in both the electroporated and infected cells . A viral RNA with a lethal lesion in the HCV RNA polymerase ( J6/JFH-1 pol- ) coding sequence is incapable of replication and infection . Deletion A generated results very similar to the wild type virus , with replication and infection occurring at levels comparable between these two constructs . Deletion B , although showing RNA replication levels similar to that seen for the wild type J6/JFH-1 construct , did not generate any detectable infectious virus production ( Figure 1B , last column of images ) , indicating deletion B could disrupt infectious virus production , while not altering RNA replication . A more quantitative approach than NS5A IHC was used to better characterize the deletion B virus . For these experiments , HCV RNA transcripts corresponding to wild type J6/JFH-1 , J6/JFH-1 pol- , and J6/JFH-1 deletion B were generated and electroporated into Huh-7 . 5 cells . The cells were plated and following a four hour recovery time , total cellular RNA was harvested in a time course experiment spanning 48 hours . HCV RNA was detected in these samples by quantitative real-time reverse transcription PCR using HCV specific probes and primers . Human GAPDH mRNA was also measured for purposes of data normalization . Figure 1C shows the RNA replication kinetics for J6/JFH-1 and J6/JFH-1 deletion B are identical , indicating deletion B does not affect HCV RNA replication . As lateral spread of virus infection in the J6/JFH-1 electroporated cells , and not in the J6/JFH-1 deletion B cells , could possibly skew results in this type of experiment , we further analyzed the effect of deletion B on RNA replication in the context of the non-infectious HCV 2a replicon , pSGR-JFH-1 . Figure 1D shows that RNA replication was not altered for deletion B bearing RNAs in the context of non-infectious HCV replicons . From these data it is clear than deletion B does not alter HCV RNA replication . Turning to the production of infectious virus , we electroporated J6/JFH-1 , J6/JFH-1 pol- , and J6/JFH-1 deletion B RNAs into cells , harvested supernatants after 48 hours , and titered the infectious virus in these supernatants by a limiting dilution assay . Viral titers are shown in Figure 1 E in units of 50% tissue culture infectious units per milliliter ( TCID50/ml ) . J6/JFH-1 yielded a TCID50/ml titer of 5 . 5×105 and the lethal pol- lesion prevented production of infectious particles . J6/JFH-1 deletion B , despite having RNA replication levels indistinguishable from J6/JFH-1 , failed to produce any detectable infectious virus in this assay . To determine if deletion B simply delayed the kinetics of virus production , we incubated electroporated cells for six days post RNA delivery and titered the output of infectious virus from these cultures . We saw no infectious virus production from cells electroporated with deletion B RNA from these six day cultures ( Figure 1E ) , indicating deletion B did not simply induce a kinetic delay in virion production . Furthermore , we have kept deletion B RNA in the context of a neomycin selectable , bicistronic full-length viral genome ( FLneo J6/JFH-1 , as described in [31] for more than 5 months with no detectable infectious virus output from these cultures ( data not shown ) . Only a small fraction of the total HCV non-structural proteins present within a cell are actively involved in the process of viral RNA replication [36] . One can easily imagine a mutant that destabilizes NS5A sufficiently so as to eliminate the non-replicative form of the protein , yet retaining enough trace NS5A protein to allow for productive RNA replication . If the non-replicative excess of non-structural proteins are somehow involved in particle assembly , a simple defect in protein stability could lead to a phenotype similar to what we observe with deletion B . To investigate this possibility we performed Western blots to assay the level of NS5A expression in cells electroporated with replication competent wild-type and deletion B full-length viral genomes . Figure 1C shows the results of this analysis . No obvious defect in the levels of NS5A were apparent , but , interestingly , the phosphoform distribution was altered . Wild-type NS5A was present as an even mixture of hyper- and hypophosphorylated forms , whereas deletion B was present in predominantly the hypophosphorylated form , with a small but detectable amount of hyperphosphorylated protein . This observation , combined with the removal of a number of serine residues in the deletion B NS5A protein relative to the wild type sequence suggested that our mutation disrupted one or more phosphorylation sites . The lack of detectable infectious virus observed for the J6/JFH-1 deletion B construct raised the question as to where in the HCV life cycle the defect in infectivity release might be located . HCV , by analogy to related viruses , most likely buds virions into the ER or an ER-like compartment , and virions are exported from the cell using the host secretory machinery . It has been shown that significant amounts of infectious HCV virions are present inside the infected cell , and can be liberated by simple repetitive freeze-thaw lysis of the cell [37] . To determine if J6/JFH-1 deletion B generates infectious particles that fail to exit the cell , we performed electroporations of this RNA and the parental J6/JFH-1 RNA , incubated the cells for 48 hours , and lysed the cells by freeze thaw after extensive pre-lysis washing to remove virus released by normal exocytosis . Cell lysates were then titered by limiting dilution on naïve Huh-7 . 5 cells . J6/JFH-1 pol- , as expected , produced no infectious virus in these experiments ( Figure 1F ) . J6/JFH1 liberated approximately 104 infectious units after freeze thaw . Deletion B failed to release any infectious virus in this experiment , suggesting the defect observed for deletion B was not at the step of virus release from the cell , but at an earlier step in virion biogenesis . We next monitored HCV RNA release into the supernatant of infected cells to determine if these cells release RNA and/or HCV particles that are somehow defective in initiation of infection . Figure 1G presents the results of this analysis , with HCV RNA present in cell supernatants quantified by real-time reverse transcriptase PCR using HCV specific primers and probe and presented as relative RNA release to the level of RNA released by a J6/JFH-1 pol- RNA . As expected , wild type J6/JFH-1 infected cells release RNA into the cell culture supernatant , consistent with the release of packaged infectious particles . Deletion B bearing J6/JFH-1 released approximately 525 fold less RNA than wild type J6/JFH-1 . RNA release for J6/JFH-1 deletion B was comparable to the non-infectious H77/JFH-1 chimeric virus construct [31] and less than that observed for a subgenomic RNA , pSGR-JFH-1 [38] . These data suggest that the J6/JFH-1 deletion B either does not assemble virions at all or it may release non-infectious particles lacking the HCV genome . To address these possibilities we next looked at HCV core protein , a component of the HCV virion , in the supernatants of J6/JFH-1 and J6/JFH-1 deletion B electroporated cells using the commercially available Ortho Diagnostics HCV core ELISA assay . The use of this assay in HCV cell culture infection experiments as a means to measure virion release is well documented [39] , [40] . Table 1 summarizes core protein released at 24 hours post RNA electroporation . J6/JFH-1 pol- RNA electroporated cells released 21 . 33 femptomoles/L of core protein , establishing the baseline for a non-replicating viral genome . J6/JFH-1 electroporated cells , in contrast , released 2225 femptomoles/L of HCV core , indicating a robust release of core protein . The J6/JFH-1 deletion B electroporated cells released 58 femptomoles/L , considerably less core than the parental J6/JFH-1 electroporated cells . Cell lysates of J6/JFH-1 and J6/JFH-1 deletion B contained comparable amounts of core protein when analyzed by this ELISA assay , indicating no defect in core protein production existed ( data not shown ) . Collectively , these data suggest the defect in J6/JFH-1 deletion B is at an early step of virion production . No viral RNA , capsid protein , or infectious virus are released from cells replicating deletion B RNA , and infectious virions are not merely trapped within the secretory pathway for this mutant . As the preliminary characterization of J6/JFH-1deletion B suggested this region of NS5A was important for the production of infectious HCV virions , we decided to map what residues in deletion B were responsible for this phenotype . We therefore generated three smaller deletions , designated B1 ( amino acids 443–447 ) , deletion B-2 ( amino acids 448–452 ) and deletion B-3 ( amino acids 453–457 ) , in the context of the wild type J6/JFH-1 virus ( Figure 2A ) . Each of these deletions was capable of robust HCV RNA replication ( Figure 2B , top row of images ) . Deletions B-1 , B-2 , and B-3 were also capable of producing infectious virus , although deletion B-3 produced considerably less infectious virus than deletion B-1 and B-2 ( Figure 2B , bottom row of images ) . However , none of these deletions was capable of reproducing the complete loss of infectivity release observed with the original deletion B mutant . From these mutants it was clear that none of the residues in deletion B-1 or B2 were essential for virus production . Residues in deletion B-3 clearly have some impact of infectivity release , but not to the extent of deletion B . As deletions B-1 , B-2 , and B-3 completely span the parental deletion B region , we were puzzled by our inability to map the region of deletion B involved in the disruption of infectivity release . Upon closer examination of the deletions we generated we noticed that generating deletion B-3 positioned an upstream serine residue at position 452 ( residue highlighted in red in Figure 2A ) in a position analogous to serine 457 that was removed in deletion B-3 . This data hinted that having a serine in this position might be important for the production of infectious virus , and also suggested that residues upstream from that serine ( deletion B-3 region ) might have some impact of infectivity release , as evidenced by deletion B-3's reduced infectivity release . We therefore generated two additional deletions , deletion B-4 ( amino acids 457–460 , sequence SEED ) and deletion B-5 ( amino acids 458–460 , sequence EED ) , to evaluate the importance residues near this serine ( Figure 2C ) . Both deletions B-4 and B-5 were capable of efficient replication upon electroporation of these RNAs into cells ( Figure 2D ) . Deletion B-4 , removing serine 457 and three downstream residues , failed to generate infectious virus in a manner similar to that observed for the parental deletion B ( Figure 1B ) . Deletion B-4 , retaining serine 457 and deleting only the three downstream residues , produced a robust level of infectivity . Collectively , these studies implicate serine 457 as a key residue in the production of infection virus , although the involvement of other residues in this region ( specifically in deletion B-3 ) may affect the efficiency of this process . We have subsequently generated deletions of serine 457 alone , and this also blocks infectivity release ( data not shown ) . We next analyzed the stability and phosphorylation state of our NS5A deletions by Western blotting lysates prepared from cells electroporated with the constructs shown in Figure 2 , panel B and D . Figure 2 , panels E and F show the results of this analysis . No obvious defects in NS5A stability were evident for any of the mutant NS5A proteins . Interestingly , we observed less hyperphosphorylated NS5A with deletion B-3 and B-4 , deletions which reduced infectivity release . This is reminiscent of our observations with the parental deletion B , again suggesting these mutations alter hyperphosphorylation of NS5A . As our mapping studies suggested that serine 457 of NS5A was an important residue for the production of infectious HCV , and the phosphorylation of NS5A by a number of cellular kinases is well documented in the literature , we explored the possibility that serine 457 was a phosphoacceptor site . The web based NetPhosK server strongly predicted serine 457 as a site of CKII phosphorylation , with serine 457 present in an ideal motif for CKII phosphorylation ( SEED sequence ) [41] . We proceeded with the hypothesis that serine 457 is a phosphoacceptor site , and that this site is a target of CKII . We therefore generated mutations at position 457 in the context of the infectious J6/JFH-1 viral genome , including an alanine substitution ( AEED ) that is incapable of phosphorylation at position 457 , an aspartic acid ( DEED ) that should mimic phosphorylation at this site , and a serine to serine codon change ( SEEDcc ) that should be capable of phosphorylation and eliminate any chances that our previous mapping work merely disrupted an RNA packaging signal or other RNA element required for infectious virus production . Figure 3A shows the analysis of these mutants using the IHC NS5A detection assay for replication and infection . The substitution of alanine for the serine at position 457 did not alter RNA replication , but blocked the production of infectious virus and the subsequent infection of naïve cells , although a few infected cells were visible in this experiment . The aspartic acid mutation ( DEED ) and the serine codon change mutation ( SEEDcc ) had not obvious effect on RNA replication or infectivity release in this assay . Using the more quantitative real-time PCR based assay , we generated transient RNA replication curves for all of our mutations at position 457 and the wild type and deletion B bearing J6/JFH-1 RNAs ( Figure 3B ) . J6/JFH-1 , J6/JFH-1 deletion B , J6/JFH-1 AEED , and J6/JFH-1 SEEDcc RNAs had no measurable defect in RNA accumulation levels or the kinetics of RNA accumulation . The J6/JFH-1 DEED mutant showed a small but reproducible ½ log reduction in RNA levels in this assay . We next looked at the release of infectious virus for these mutant RNAs by titering released virus in the supernatants by limiting dilution ( Figure 3C ) . J6/JFH-1 released approximately 5 . 5×105 TCID50/ml , with the lethal polymerase lesion ( J6/JFH-1 pol- ) releasing no infectious virions . J6/JFH-1 deletion B , as shown in previous experiments , failed to release any detectable infectivity . The J6/JFH-1 AEED mutant virus released infectious virus at very low levels ( about 60–80 TCID50/ml ) , in good agreement with the observation of a few NS5A positive cells in the lower infection panel in Figure 3A , indicating that substitution of serine 457 to alanine inhibited infectivity release by about five orders of magnitude . The low level of infectivity release suggested these infected cells might be harboring revertants . We expanded these serine 457 to alanine infected cultures , extracted RNA , and performed RT-PCR based sequencing on the bulk population . This analysis revealed that the input alanine mutation was retained in the infectious virus produced by this mutant , indicating the requirement of serine 457 may not be absolute , or that other changes elsewhere in the HCV genome may compensate for this mutation ( data not shown ) . We sequenced the entire HCV coding sequence and were unable to identify any additional sequence changes , furthering the idea that a low level of particle production may occur even in the absence of serine 457 . The J6/JFH-1 DEED released virus at about 3×105 TCID50/ml , indicating aspartic acid was an acceptable substitution at position 457 . J6/JFH-1 SEEDcc produced a virus titer similar to that of wild type J6/JFH-1 , indicating changes in the HCV RNA sequence at this position were not responsible for the observed reduction in infectious virus production . Analysis of the stability and phosphorylation state of these mutants ( Figure 3D ) revealed no obvious defect in stability and a loss of some hyperphosphorylated NS5A in the AEED mutant . The reduction of NS5A hyperphosphorylation seems to be common to all of our mutants that are of reduced infectivity release . At first glance it would seem likely that serine 457 is a phosphoacceptor site , and the alteration or deletion of this residue leads to reduced levels of hyperphosphorylated NS5A . However , the situation is more complex , as the DEED mutant also eliminates this serine residue , yet has levels of hyperphosphorylation comparable to wild type NS5A or the many virus release competent mutations . The data suggests that serine 457 phosphorylation is a prerequisite to modification of other phosphoacceptors involved in generating hyperphosphorylated NS5A . To determine if CKII phosphorylation of serine 457 was important for the process of virion biogenesis in vivo we investigated the use of the specific CKII inhibitor 2-dimethylamino-4 , 5 , 6 , 7-tetrabromo-1H-benzimidazole ( DMAT ) on infectious virus release . DMAT is a cell-permeable ATP-competitive inhibitor that displays a 1 , 300 fold selectivity for CKII over CKI and has been shown to minimally affect a large number of other protein kinase activities [42] . Huh-7 . 5 cells were electroporated with transcripts of the infectious J6/JFH-1 RNA and DMAT was added to cell culture media four hours after RNA delivery . Following three days of further incubation , supernatants of these cultures were collected and used to infect naïve Huh-7 . 5 cells . Figure 4A shows IHC staining of the NS5A protein in these infected cells for two doses ( 0 . 5 µM and 2 µM ) of DMAT and a DMSO only vehicle control . Robust infections of naïve cells is evident with the transfer of supernatants from the DMSO only treated cells , and a dose dependent reduction in infectious virion content is seen in infections using supernatants from DMAT treated cells . A more quantitative analysis of the effects of DMAT treatment on infectious virus production is presented in Figure 4B . For this experiment , cells were electroporated with J6/JFH-1 RNA transcripts , plated and incubated for four hours , and doses of DMAT were added to the cells . Following three days of incubation , cell supernatants were harvested for tittering on naïve cells . As observed in Figure 1A , a dose dependent inhibition of infectious virus release was observed with DMAT treatment , with no effect observed for DMSO vehicle only treatment . Infectious virus titers were plotted as the percent of infectivity release relative to the DMSO only treated cells versus the dose of DMAT . The EC50 ( effective concentration 50% ) of DMAT inhibition of infectivity release was calculated at 180 nM , in good agreement with the published IC50 ( inhibitory concentration 50% ) of DMAT of 140 nM as assayed on purified rat liver CKII [42] . Although these data suggested CKII activity was important for the production of infectious virions , it did not provide information about the phosphorylation of serine 457 of NS5A in this process . We therefore assayed the aspartic acid mutation at position 457 of NS5A in these experiments , based on the concept that aspartic acid should mimic phosphorylation of position 457 and make NS5A insensitive to CKII , and therefore , DMAT inhibition . The aspartic acid mutation at position 457 ( Figure 4B , DEED ) was insensitive to DMAT inhibition of infectivity release , with an EC50 value ( 5 . 5 µM , Figure 4B red line ) close to the LD50 ( lethal dose 50% ) of DMAT on Huh-7 . 5 cells ( 6 . 5 µM , Figure 4B dashed line ) , suggesting that the inhibition of infectivity release for the aspartic acid mutation was largely due to cytotoxic effects of drug treatment . The roughly 30-fold difference in EC50 values of DMAT on wild type NS5A versus the aspartic acid mutation implicates serine 457 phosphorylation as an important event in infectious virus production and suggests that other possible CKII sites in NS5A have little role in this process . The replication of wild type or DEED HCV RNA , as determined by monitoring levels of genomic RNA in cells by real-time PCR , was not affected by treatment with DMAT up to the level where DMAT toxicity was observed ( Figure 4C ) . Treatment of cells with an unrelated nucleotide analog kinase inhibitor , 2-aminopurine , had no effect on infectious virus release when used either to treat cells prior to infection or when maintained during the entire course of the experiment ( figure 4D ) . As the NS2 protein is known to be a key determinant in the production of infectious virus , and also documented to contain a CKII site that can regulate the stability of NS2 [43] , we treated cells infected with the JC1 HCV virus [44] with the DMAT inhibitor and monitored the stability of NS2 over the course of our previous measurements of infectivity ( Figure 4E ) . The use of the JC1 virus , which alters the location of the junction between J6 and JFH-1 genomes in the region of NS2 was necessary , as we only have antibodies that recognize the JC1 form of NS2 , and not that present in J6/JFH-1 . In any case , the putative CKII site in NS2 is not different in these different viral constructs . We observed no difference in NS2 stability from 0 . 125 to 2 µM DMAT , a slight reduction in NS2 levels at 4 µM , and a nearly complete loss of NS2 at 6 µM . These data , combined with our observed EC50 for DMAT on infectious virus production for wild type NS5A bearing virus ( 180 nM ) , suggest that alterations in NS2 levels are not a significant factor in our experiments . Although the chemical inhibition of infectious virion production by the specific CKII inhibitor DMAT suggested CKII phosphorylates NS5A , the possibility existed that other kinases were inhibited by DMAT and were responsible for infectious virus production . To address this point we chose to specifically silence CKII gene expression . CKII exists as a tetramer composed of 2 β regulatory subunits and two α kinase subunits [45] . Two isoforms of CKII α subunits exist , designated α and α′ . These α/α′ isoforms can assemble with 2 β subunits in a homotypic ( all α or all α′ subunits in the CKII holoenzyme ) or heterotypic manner ( α and α′ in the CKII holoenzyme ) . As it was unclear which subunit might be involved in NS5A phosphorylation , we chose to silence both α and α′ expression individually as well as α′ and α simultaneously . siRNAs ( CKII α , α′ , a mix of α and α′ , or an irrelevant siRNA ) were delivered to cells via electroporation and the cells were plated and incubated for 24 , 48 and 72 hours prior to harvesting protein lysates and total cellular RNA to determine the efficacy of silencing . Figure 5A shows Western blotting of cells treated with the various siRNAs using CKII α and α′ specific anti-sera . In all samples , a decrease in the amount of the targeting CKII alpha subunit ( s ) was evident within 24 hours , with little or no CKII α or α′detectable by 72 hours . To further confirm our silencing of the CKII kinase subunits we performed real-time reverse transcription PCR using primers and probes targeting CKII α and α′ mRNA ( Figure 5C ) . We were able to reduce CKII α mRNA levels by about 60% relative to the level of an untreated control , and CKII α′ by approximately 65% . Cells treated with both CKII α and α′ RNAs ( Figure 5C , Mix ) showed similar silencing to cells treated with a single siRNA , although silencing was somewhat more efficient in cells receiving the mixture of both CKII siRNAs . Measurement of cell viability at 72 hours indicated that our siRNA treatments had little negative affect on the cells , with all cells at least 95% or better than the viability of untreated cells ( Figure 5B ) . We therefore chose to conduct all future experiments 72 hours post siRNA delivery . Following siRNA electroporation and a 72 hour incubation , cells were infected with J6/JFH-1 or DEED virus at a multiplicity of infection of five . After five hours , the virus inoculum was removed and the cells washed extensively . After 48 hours of infection , supernatants were harvested and titered for the presence of infectious virus . J6/JFH-1 virus production was inhibited by silencing of either CKII α or α′ , with reductions in virus yield of about 80% relative to an irrelevant siRNA control ( Figure 5D ) . Silencing of CKII α and α′ together impaired wild type virus production by more that 98% . It should be noted that although silencing of CKII α or α′ individually did not significantly alter HCV RNA replication in these experiments , but silencing both CKII α and α′ together led to a one log reduction in HCV RNA levels ( Figure 5D ) . This lower RNA replication may explain the observed increase in infectious virus inhibition in samples where both CKII α and α′ were silenced . The DEED mutation bearing virus infected cells were not inhibited in virus release when CKII α or α′ were silenced individually or in combination , in contrast to what was observed in J6/JFH-1 wild-type infections ( Figure 5E ) . The DEED infections also had about a one log drop in HCV RNA in cells in which CKII α and α′ were both silenced ( Figure 5D ) , but this did not alter virus production for these cells ( Figure 5E ) . This , perhaps , not surprising as virus constructs such as JC1 are capable of increasing infectivity release without altering the levels of RNA replication , suggesting the relationship between replicating RNA and virion packaged RNA is not stoichiometric [44] . The siRNA data overall is similar to our use of the CKII inhibitor , DMAT , and collectively these experiments highlight the importance of serine 457 phosphorylation by CKII in the production of infectious virus . Although chemical and genetic inhibition of CKII activity has suggested the importance of this kinase in the production of infectious virus for HCV , it remains a possibility that these treatments altered some other aspect of the host cell physiology . We therefore chose to over express the α and α′ subunits of CKII from a CMV promoter based plasmid and assay the effects of this on infectious virus release . The expression of CKII α has been shown to increase the production of the regulatory β subunit in cells , making delivery of exogenous CKII β not necessary [46] . We transiently transfected cells with pCMVSport6 CKII α , pCMVSport6 CKII α′ , or a pCMVSport6 empty vector control , and following a 24 hour incubation to allow for expression of CKII α subunits , cells were infected with J6/JFH-1 virus . A Western blot image showing the level of CKII alpha subunit relative to a β-actin loading control are shown in Figure 5F . After 48 hours , cell culture supernatants were titered for the presence of infectious virus on naïve Huh-7 . 5 cells . Table 2 presents the results of this experiment , with values listed as fold increase in infectious virus release relative to a non-plasmid transfected cell control . The presence of the pCMVSport6 control vector had no affect on the production of infectious virus , but the expression of CKII α from this vector led to a 3 . 44 fold increase in infectious J6/JFH-1 virus yield . Similarly , CKII α′ led to a 2 . 95 fold increase in virus titers . We also conducted a parallel experiment in which similarly transfected cells were infected with a J6/JFH-1 DEED mutant virus . The DEED mutant virus showed no increase in virus production with the over expression of CKII α ( 0 . 83 fold change ) or CKII α′ ( 0 . 94 fold change ) ( Table 2 ) . These data highlight the importance of CKII to infectious virus production , and in the case of the DEED mutant virus , the importance of a single residue of the NS5A protein to this process . Although our genetic and chemical manipulations of NS5A suggest serine 457 is phosphorylated by CKII , we have not shown direct modification of this residue by this kinase . Indeed , only two sites of NS5A phosphorylation have been mapped despite intense efforts in this regard over the past 15 years [47] , [48] . This is , at least in part , due to the large number of potential acceptor sites in NS5A and the unusual repetitive amino acid usage of potential acceptor sites in several regions of NS5A . Our efforts in attempting to identify serine 457 as a phosphoacceptor from NS5A isolated from infected cells by immunoprecipitation and mass spectrometry have not been successful due to poor coverage of the domain III region of NS5A in our spectra ( data not shown ) . We therefore decided to analyze the deletion B region as a potential CKII substrate using in vitro kinase reactions on GST fusion protein substrates . We generated two GST fusion proteins for use as in vitro substrates for CKII phosphorylation . The first contained amino acids 449 to 465 ( GSGSWSTCSEEDDTTVV , in single letter amino acid designation ) from the JFH-1 NS5A protein fused to the C-terminus of GST . The second fusion protein was identical with the exception of the substitution of serine 457 with an alanine residue ( GSGSWSTCAEEDDTTVV ) . Following incubation with recombinant human CKII in the presence of γ-33P ATP , we captured the proteins on glutathione agarose beads and determined radiolabel incorporation by liquid scintillation counting . This data is presented in Figure 6 . Proteins were first analyzed in the absence of added CKII to assess the level of phosphate incorporation that might be present from co-purifying kinase activities . All of the proteins showed similar levels of incorporation in these reactions , indicating that the purified proteins were essentially equivalent in regard to potential kinase contamination . The fusion protein containing serine 457 ( designated SEED in Figure 6 ) incorporated about 3 times more radiolabel than GST with no fusion in the presence of recombinant CKII , indicating residues in the fusion sequence were modified by CKII . The fusion protein in which serine 457 was changed to an alanine residue , but was otherwise identical to the SEED fusion protein , did not show this increased label incorporation relative to the GST control , suggesting that the modification of the SEED fusion protein involved serine 457 . These data confirm that serine 457 is a valid CKII target , at least in these in vitro reactions . We have attempted to detect the effects of CKII α over-expression and the DMAT inhibitor on NS5A phosphorylation in the context of an HCV cell culture infection . Unfortunately , we observe only a very small effect on NS5A hyperphosphorylation with CKII α over-expression ( Figure 6B ) , and only a very moderate decrease in hyperphosphorylation ( Figure 6C ) with these treatments . These data are far less dramatic that our previous genetic manipulations , suggesting more advanced phosphopeptide mapping will be required to provide a definitive answer on whether NS5A phosphorylation is altered by these treatments . Recent research has implicated the lipid droplet as an important organelle in the production of HCV virions [7] . The localization of HCV core to lipid droplets , and the subsequence recruitment of viral RNA and non-structural proteins are prerequisites of virion production . A mutation in the domain I region of NS5A that disrupts lipid droplet association was shown to prevent the production of virions in this work . The localization of our deletion B bearing NS5A to lipid droplets was therefore of significant interest . Using a lipid droplet specific fluorescent dye and NS5A indirect immunofluorescence microscopy we compared the localization of wild type and deletion B NS5A proteins in the context of cells electroporated with replication competent full-length viral genomes . Figure 7 shows the results of this analysis , with panels showing lipid droplet and NS5A staining , as well as merged images to examine co-localization . Panels B and C show close up views of this analysis for wild type and deletion B bearing genomes , respectively . In the case of both mutant and wild type NS5A proteins , we see localization of NS5A to lipid droplets . We were unable to observe any difference in the localization of NS5A in any of our efforts in this regard , suggesting that deletion B is functionally quite different than the NS5A mutant that blocks infectivity release by disrupting localization to lipid droplets [7] .
The idea of NS5A as a regulator of events in the HCV lifecycle via phosphorylation by cellular kinases is not a new concept . Indeed , this idea has been prevalent in the HCV community since the discovery that NS5A exists in multiple phosphorylation states [21] . Considerable progress has been made in deciphering the role of CKIα phosphorylation of NS5A in RNA replication [22] . The functional significance of CKII phosphorylation of NS5A , however , has long been an enigma . Our data provides the first evidence for a function of these events , not in RNA replication , but in the process of virion assembly . We have shown that CKII likely phosphorylates serine 457 of genotype 2a NS5A , and this modification is required for infectious virus production . Generating an NS5A that is incapable of serving as a phosphoacceptor ( alanine mutation at position 457 ) does not alter RNA replication , but blocks the production of infectious virus . Generating an aspartic acid mutation at position 457 lowers RNA replication slightly but does not affect infectivity release . This lack of direct coupling of replication levels and infectivity release is in agreement with that observed for the JC1 chimeric virus [44] , in which infectivity is increased ten fold with no change in RNA replication levels . Our first interpretation of this data was that phosphorylation of serine 457 , mimicked by the aspartic acid mutation , destabilized the replicase leading to release of NS5A and viral RNA from the replicase for assembly events . This idea is roughly equivalent to the models of phosphorylation as a regulator of the replicase first proposed by Evans , et al . [26] , [44]The case is clearly not this simple for our mutants , as we see NS5A participating in later events in assembly , such as association with lipid droplets even when serine 457 ( and surrounding residues ) are deleted . In this regard , our data is quite different than the recently described NS5A mutants in domain I that block infectious virus production by hindering the association of NS5A with lipid droplets [7] . These mutations are severely impaired in RNA replication , yet release what appear to be non-infectious virus particles containing both HCV core and viral RNA . In contrast , our deletions and single amino acid serine 457 to alanine mutant in domain III does not alter HCV RNA replication , but blocks the production of infectious virus , and does not release large amounts of viral RNA and HCV core . We see no defect in the association of NS5A with lipid droplets with our deletion B mutant , further highlighting the difference between domain I and domain III mutants in NS5A assembly activities . Collectively , these data suggest our mutants affect a different step in the HCV assembly pathway than the domain I mutations [7] . Our lack of understanding of the mechanisms of HCV assembly make it difficult to speculate exactly what step in assembly our mutations are affecting . We are clearly manipulating an early step in the assembly process , as we do not see virus particles inside the cell secretory pathway , suggesting , in conjunction with the lack of RNA and core release , that particles are simply not generated . The data suggests that we are affecting a later stage in assembly than the domain I mutants , as we see clear evidence of lipid droplet association of NS5A in the context of our mutant . What this later step of assembly might be is unknown . Perhaps it involves NS5A making productive interactions with the structural proteins at the lipid droplet , NS5A moving and releasing viral RNA , NS5A functioning to package viral RNA , or NS5A directly being packaged into virions as a minor component of the infectious virion . Unfortunately , our understanding of the properties of NS5A is still in its infancy , making directly addressing these issues difficult at the present time . Although our genetic manipulations of NS5A suggest serine 457 is phosphorylated by CKII , and we can show phosphorylation of fusion protein substrates corresponding to this region of NS5A , we have not shown direct modification of this residue by this kinase in the context of an authentic virus infection . Indeed , only two sites of NS5A phosphorylation have been mapped to date , and not in the context of authentic RNA replication , despite intense efforts in this regard over the past 15 years [47] , [48] . This is , at least in part , due to the large number of potential acceptor sites in NS5A and the unusual repetitive amino acid usage of potential acceptor sites in several regions of NS5A . Our efforts in attempting to identify serine 457 as a phosphoacceptor from NS5A isolated from infected cells by immunoprecipitation and mass spectrometry have not been successful due to poor coverage of the domain III region of NS5A in our spectra ( data not shown ) . Based on the lack of evidence in the literature of documented NS5A phosphorylation sites , and our lack of understanding of what the differences in phosphorylation that generate the different phosphoforms of NS5A , it will likely be some time before the complete complement of NS5A phosphorylation sites are known . Even understanding whether this site is a component of the hypo or hyperphosphorylated form of NS5A is not a simple task . Serine 457 lies in a region of NS5A domain III that is believed to primarily contain sites of hypophosphorylation based on alanine scanning mutagenesis of potential acceptor sites in domain III , although this particular residue position has never been evaluated by mutagenesis in the context of the 2a NS5A sequence [11] . Our Western blot data suggests serine 457 may either be a hyperphosphorylation site , or a residue that when phosphorylated , promotes hyperphosphorylation of NS5A . The later seems more likely , as aspartic acid mutations at serine 457 have normal levels of hyperphosphorylation , despite being incompetent as an acceptor site . The work of Miyanari , et al . shows that the lipid droplet associated form of NS5A , a requirement for virus assembly , is enriched for hyperphosphorylated NS5A relative to what is seen in viruses lacking the core protein , suggesting hyperphosphorylation might be important in assembly . This is , at least in broad terms , supported by the observation that hyperphosphorylated NS5A destabilizes the replicase . Nonetheless , in vitro phosphorylation reactions of NS5A with recombinant CKII only generate the hypophosphorylated form of NS5A , even in conditions of excess CKII [25] . At least for CKI phosphorylation of NS5A in vitro , several acceptor sites require activation by pre-phosphorylation of upstream sites before they are modified , suggesting that in vitro reactions probably do not fully represent what occurs in vivo [25] . Although we have shown serine 457 is an important residue in infectious virus production , it may be that this residue merely ‘activates’ other phosphoacceptor sites , as has been observed for CKIα phopshorylation . This concept fits well with our observations of altered levels of hyperphosphorylation with our mutants . Although the cell culture infectious genotype 2a strain clearly possesses an insertion relative to other HCV genotypes ( Figure 1A ) , it is unlikely that this insertion is directly related to the ability to generate infectious virus . Clearly , isolates of genotype 1a are infectious in cell culture , but lack this insertion [34] . Indeed , we can delete this region without disrupting infectious virus production . The conservation of a CKII site around residue 457 seems to be a more likely candidate for an important conserved feature , with other defects preventing the production of infectious virus in cell culture for other genotypes . In this regard , it is worth noting that the web based NetPhosK 1 . 0 server [41] can predict CKII sites in all 35 HCV genotype reference sequences in the Los Alamos HCV database [49] that involve direct serine 457 modification . A recent paper looking at the sequence of patient isolated HCV RNA for genotypes 1a , 1b and 3 identified a number of potential CKII sites in NS5A , including sites very close to the location of serine 457 in genotype 2a , however the majority of the sites predicted have residues corresponding to serine 457 as sites of modification by another enzyme that generate a CKII recognition site just upstream of this position [50] . This analysis was based on the site motif described by Pinna [51] , in which most CK2 sites typically have a serine or threonine , followed by an acidic residue at the n+3 position . This work did not take into account , as mentioned by Pinna , that whenever the negatively charged determinant is absent at position n+3 , it is invariably present at position n+1 and vice-versa , leading to some variability in CK2 sites [51] . The majority of sites we predicted as conserved across all HCV reference strain had met these rules of the more conventional n+3 , but many were of the less common n+1 variety . We also were able to predict the sites using an upstream CK2 site with serine 457 serving as an activator when phosphorylated , as described by Dal Pero and colleagues [50] . Unfortunately , it will likely be some time before the intricacies of NS5A phosphorylation , RNA replication , and now virion assembly , will be fully understood , thereby addressing the question of what type of CK2 sites are utilized in this region of NS5A . The lack of solid information on the regulation of CKII further complicates our understanding of when CKII might be active on NS5A and at what location in the infected cell ( see [52] for review ) . The requirement for phosphorylation of a viral non-structural viral replication protein by a host cell kinase for the production of infection virus particles , although novel for HCV , has been described previously for other viruses . Bluetongue virus ( BTV ) is perhaps the closest example of this phenomenon to what we observe in the case of HCV , despite the great evolutionary distance between these viruses . The BTV NS2 protein is phosphorylated by CKII , and this phosphorylation is required for the assembly of the site of replication virion assembly , termed viral inclusion bodies [53] . As is the case for HCV , the precise mechanisms of this phenomenon are not fully understood in BTV biology . Viruses more closely related to HCV , such as the flaviviruses Dengue ( DENV ) and West Nile virus ( WNV ) , require a functional c-Src ( or c-Src like ) kinase activity for the assembly and maturation of infectious virus particles at the site of RNA replication [54] , [55] . The mechanism of these events is also unknown , but the relatively close relationship of DENV and WNV to HCV suggest c-Src and c-Src like activities and CKII may perform similar regulatory functions in switching between replication and assembly . It seems that the regulation of switching between replication and assembly by phosphorylation , or controlling later stages of assembly and mturation may be a common , but understudied mechanism in virology . In summary , we have identified serine 457 of the HCV NS5A protein as a key determinant for the production of infectious virions and presented data showing this site is likely a target for CKII phosphorylation . Phosphorylation of this residue appears to be an important regulatory step in infectious virus production . Via genetic and chemical manipulations we have shown that HCV RNA replication can be uncoupled from infectious virus production without altering HCV RNA replication efficiency . It remains to be demonstrated whether these observations are valid in the context of an infected liver , but at least in cell culture these events appear to be a critical step in the virus lifecycle . Nonetheless , these new genetic and chemical tools , when combined with recently identified mutants in NS5A that alter localization to the site of virion assembly , will be of significant value in further dissection of the mechanisms of HCV RNA replication and virion biogenesis .
The construction of the J6/JFH-1 , J6/JFH-1 pol- , H77/JFH-1 , and pSGR-JFH-1 replicon have been described previously [31] . We generated these clones using a J6/JFH-1 chimeric virus backbone generated in house by oligo based gene synthesis . Deletions and mutations in NS5A were generated using the QuikChange mutagenesis system following the manufacturer's instructions . All constructs , subclones , and mutants were confirmed by DNA sequence analysis . The pCMVSport6 vectors encoding the human CKII α and α′ cDNA sequences were obtained from Dr . Michael Conkright . Huh-7 . 5 ( human hepatoma ) cells were obtained from an original seed stock of this clone and cultured as described previously [35] . The anti-human CKII α ( A300-197A ) and CKII α′ ( A300-199A ) purified rabbit polyclonal antibodies were obtained Bethyl Labs . The anti human β-actin mouse monoclonal loading control antibody ( A-5441 ) was obtained from Sigma Aldrich . The anti-NS5A 9E10 antibody has been described previously [31] . Secondary antibody horseradish peroxidase conjugates were obtained from Pierce Biotechnology . Viral RNAs were generated and purified as described previously [31] . For electroporation , subconfluent monolayer cultures of Huh-7 . 5 cells were trypsinized , washed twice in ice cold RNase free phosphate buffered saline ( Cambrex , Bio-Whitaker ) , and resuspended at a concentration of 1 . 25×107 cells/ml in ice cold RNase free PBS . A 400 µl aliquot of cells was then mixed with 1 µg of HCV viral RNA and electroporated using a BTX ECM830 square wavelength electroporator in a 2 mm gap cuvette . Electroporation conditions were 5 pulses of 920 volts for 99 microseconds with 1 . 1 second intervals . Following a ten minute recovery time , cells were removed form the cuvette and diluted into 13 . 6 milliliters of DMEM +10% FBS . Monitoring virus replication and infectivity by IHC was performed as described previously [31] , with minor modifications . Briefly , Huh-7 . 5 cells electroporated with HCV RNA were plated and incubated for 48 hours . Cell supernatants were then harvested and filtered through a 0 . 2 micron filter and stored for infection of naïve cells . The cell monolayers were then washed twice in PBS and fixed in methanol . For infection studies , naïve cells were inoculated with the filtered supernatant . Following a six hour incubation , the inoculum was replaced with fresh DMEM +10% FBS and cells were incubated for a total of 48 hours . Cells were then washed and fixed in methanol . Fixed cells were then probed for NS5A expression using the 9E10 monoclonal antibody and procedure described previously [31] . The limiting dilution virus titering assay was performed as described previously [31] . For measuring HCV RNA levels in cell lysates following various experimental treatments , cells were washed twice in PBS and RNAs were then purified using the Qiagen RNeasy kit using the manufacturer's instructions . For measuring HCV RNA levels in cell culture supernatants , electroporated cells were plated for 24 hours , washed 3 times in PBS , trypsinized/split 1∶2 , and plated for an additional 48 hours . Supernatants were then harvested and RNA extracted using the Qiagen Qia Amp UltraSens Virus kit using the manufacturer's instructions . Extracted , purified RNAs were quantified by UV absorbance and 100 nanograms of total RNA was used for real time reverse transriptase PCR . All real time PCR experiments were performed using a Roche lightcycler 480 using HCV specific primers and probe as described previously [39] . Values are normalized using the human GAPDH endogenous control primer set ( Applied Biosystems , # 4326317E ) . Huh-7 . 5 cells were electroporated with the appropriate HCV RNAs as described herein . 5×105 electroporated cells were plated in a 35 mm dish in DMEM +10% FBS and incubated at 37°C for 2 days . Culture media was then removed , the cells were washed twice in PBS and trypsinzed . The cells were then collected by centrifugation at 5 , 000× g for 5 minutes and resuspended in 2 ml of DMEM +10% FBS and lysed by four rounds of freezing and thawing . Cell lysates were then centrifuged at 5 , 000× g for 5 minutes , filtered through a 0 . 2 micron filter and buffered with the addition of HEPES pH 7 . 4 to a final concentration of 20 mM . 2-dimethylamino-4 , 5 , 6 , 7-tetrabromo-1H-benzimidazole ( DMAT ) was purchased from EMD Biosciences and dissolved in sterile dimethyl suffixed ( DMSO ) . Following the electroporation of J6/JFH-1 or J6/JFH1 DEED mutant RNA , cells were plated and incubated at 37°C for four hours . Cell culture media was then replaced with media supplemented with DMAT in DMSO or DMSO only . Cells were then incubated for 48 hours . Cell supernatants were then collected , filtered through a 0 . 2 micron filter , and titered for the presence of infectious virions . Cell lysates were collected for determination of HCV RNA levels . Cell viability was determined using the Celltiter-Glo assay on DMAT treated or control DMSO only treated cells . For experiments using the control compound , 2-aminopurine ( 2-AP ) , cells were treated with 2-AP for one hour prior to infection , washed extensively , and infected with a high titer stock of J6/JFH-1 virus . In some experiments , 2-AP was left in the culture medium at 1 mM ( maximum concentration without toxicity ) for the course of the experiment . Output virus was harvested and titered by limiting dilution . To determine the effects of DMAT inhibition of NS2 stability , cells electroporated with JC1 viral RNA were treated with DMAT for 48 hours , lysed and probed for NS2 using the 6H6 monoclonal antibody . β-actin was used as a loading control for these experiments . Pre-annealed , validated Silencer siRNA mixtures targeting human CKII α ( siRNA # 1337 ) and α′ ( siRNA # 183 ) and an irrelevant siRNA ( siRNA # 4611 ) were purchased from Ambion . Electroporation of siRNAs was performed as described previously [56] , with minor modifications . One nanomole of RNA duplexes was electroporated with 2 . 5×106 Huh-7 . 5 cells in 0 . 4 ml of RNase free PBS . Electroporation conditions were the same as used for HCV genomic RNA delivery . Cells were infected 72 hours after electroporation with a multiplicity of 5 for 6 hours , rinsed with media , then maintained for 2 days at 37°C . Cell supernatants and lysates were then harvested for assessment of HCV replication ( infectious particles released and intracellular RNA level ) and cell viability ( Celltiter-Glo assay , Promega ) . Real-time reverse transcriptase PCR analysis of CKII gene expression was performed using Applied Biosystems pre-designed Taqman assays and total cellular RNA prepared as described for HCV specific real time PCR analysis . CKII α was assayed using assay number Hs00601957_m1 . CKII α′ was assayed using assay Hs00176505_m1 . Values were normalized for RNA level using the expression of human GAPDH ( Applied Biosystems , # 4326317E ) . Huh-7 . 5 cells were transfected with pCMV-Sport6 vectors expressing CKII α , α′ , or an empty vector control using Fugene 6 transfection reagent . Following a 24 hour incubation , cells were infected with J6/JFH-1 virus for 4 hours , washed , and incubated a further 48 hours , at which point supernatants were collected for virus titering . The levels of CKII α and α′ in these experiments were demonstrated by Western blotting of cell lysates using CKII specific anti-sera as described above . β-actin was used as a loading control in these experiments . For core protein release assays , Huh-7 . 5 cells were electroporated with J6/JFH-1 , J6/JFH-1 deletion B , or J6/JFH-1 pol- RNA . Supernatants were harvested at 24 hours post transfection and HCV core protein was measured using the Ortho Diagnostics Trak-C HCV core ELISA kit following the manufacturer's instructions . To generate GST fusion proteins of the deletion B region of NS5A , complementary 5′ phosphorylated oligos were generated coding for amino acid 449–465 of the JFH-1 NS5A protein with either a serine or an alanine at position 457 . The oligos we designed such that when annealed they generated appropiate ends for direct cloning into a pGEX6p1 vector previously digested with BamHI and EcoRI . The resulting pGEX6p1 vectors encoded deletion B fused to the C-terminus of GST . After DNA sequence analysis to confirm the clones were correct , we transferred these vectors to E . coli strain BL21 and induced the production of the fusion protein with IPTG . Briefly , 1 liter LB broth cultures of BL21 cells with the appropriate fusion protein encoding vector were grow at 37°C until an OD600 of 0 . 6 was reached . Cells were then induced by the addition of 1 mM β-2-D-isopropyl-thio-galactoside and grown for an additional 4 hours at 37°C . Cells were then pelleted , resuspended in 20 ml of cold buffer A ( 25 mM Tris-HCl pH 8 . 0 , 25 mM NaCl , and 20% glycerol ) and lysed by three passes through an Avestin air emulsifier . Lysates were clarified by centrifugation at 25 , 000× g for 20 minutes at 4°C , and the soluble fractions were loaded onto a 5 ml HiTrap GST FF column equilibrated in buffer A . After extensive washing of the column to remove unbound material , the fusion proteins were eluted with buffer A supplemented with 10 mM reduced glutathione . The reduced glutathione was then removed by desalting the proteins into buffer A using a HiPrep 26/10 desalting column . The purity of proteins was assessed by SDS-PAGE and concentrations were determined by Bio-Rad protein assay . For in vitro phosphorylation experiments 25 µl reactions were prepared containing 2 µM of each fusion protein , 100 µM unlabeled rATP , 5 µCi of γ-32P-rATP , in 1× CKII kinase reaction buffer ( New England Biolabs ) . Phosphorylation was initiated by the addition of 500 units of recombinant human CKII ( New England Biolabs ) , and reactions were subsequently incubated at 30°C for 30 minutes . After incubation , 100 µl of PBS was added to the reaction , followed by 50 µl of a 50% slurry of glutathione 4B agarose ( GE Healthcare ) equilibrated in PBS . Proteins were allowed to bind to the agarose beads for 10 minutes at 4°C , the centrifuged at top speed in a microfuge , and the unincorporated label ( supernatant ) was removed . This pelleting and washing step was repeated 4 times . After washing , pellets were resuspended in 100 µl of PBS and transferred to a scintillation vial with 10 ml of aqueous scintillation cocktail and counted using a Beckman liquid scintillation counter . To visualize lipid droplets and NS5A , cells were electroporated with 1 µg of the appropriate HC RNA , plated in glass slide micro-chambers , incubated 48 hours , fixed by a 20 minute incubation in 4% paraformaldehyde and probed with 5 µg/ml BODIPY 493/503 ( Invitrogen/Molecular Probes ) for lipid droplet staining and a 1∶15 , 000 dilution of the 9E10 monoclonal anti-NS5A antibody and an Alexa 568 secondary antibody ( Invitrogen/Molecular Probes ) . Slides were then mounted in Vectashield ( Vector Labs ) soft mounting medium and visualized . | Hepatitis C virus ( HCV ) is a life-threatening infection afflicting some 170 million people worldwide , and current antiviral therapies are only marginally effective in treating these patients . Clearly , more effective anti-viral drugs for HCV are needed . Of paramount importance to this process is understanding the detailed mechanisms HCV uses to infect cells , replicate the viral genome , assemble progeny virus , and exit the cell . Using genetic mapping , we have identified a single amino acid residue of the HCV NS5A protein that is phosphorylated by host cell kinase , and this modification regulates the production of new infectious virus particles . This modification of NS5A results in the release of some of the viral genome from replicative events , thereby making this material available for progeny virus particle production . We have identified genetic and chemical methods to modulate this event , resulting in our ability to control the production of infectious virus particles in the laboratory . The ability to separate the replication of the virus genetic material and the assembly of new viruses allows us a valuable tool to monitor how this process occurs and , potentially , a novel target for the development of much needed anti-viral drugs . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"infectious",
"diseases/viral",
"infections"
] | 2008 | Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein |
Recent genome-wide association studies ( GWAS ) have identified multiple new loci which appear to alter coronary artery disease ( CAD ) risk via arterial wall-specific mechanisms . One of the annotated genes encodes LMOD1 ( Leiomodin 1 ) , a member of the actin filament nucleator family that is highly enriched in smooth muscle-containing tissues such as the artery wall . However , it is still unknown whether LMOD1 is the causal gene at this locus and also how the associated variants alter LMOD1 expression/function and CAD risk . Using epigenomic profiling we recently identified a non-coding regulatory variant , rs34091558 , which is in tight linkage disequilibrium ( LD ) with the lead CAD GWAS variant , rs2820315 . Herein we demonstrate through expression quantitative trait loci ( eQTL ) and statistical fine-mapping in GTEx , STARNET , and human coronary artery smooth muscle cell ( HCASMC ) datasets , rs34091558 is the top regulatory variant for LMOD1 in vascular tissues . Position weight matrix ( PWM ) analyses identify the protective allele rs34091558-TA to form a conserved Forkhead box O3 ( FOXO3 ) binding motif , which is disrupted by the risk allele rs34091558-A . FOXO3 chromatin immunoprecipitation and reporter assays show reduced FOXO3 binding and LMOD1 transcriptional activity by the risk allele , consistent with effects of FOXO3 downregulation on LMOD1 . LMOD1 knockdown results in increased proliferation and migration and decreased cell contraction in HCASMC , and immunostaining in atherosclerotic lesions in the SMC lineage tracing reporter mouse support a key role for LMOD1 in maintaining the differentiated SMC phenotype . These results provide compelling functional evidence that genetic variation is associated with dysregulated LMOD1 expression/function in SMCs , together contributing to the heritable risk for CAD .
Coronary artery disease ( CAD ) is the leading cause of mortality worldwide , and poses considerable public health burden despite current treatment and lifestyle interventions . Both genetic and environmental factors contribute to CAD susceptibility , however early detection and prevention requires a deeper understanding of the primary causes of the disease process itself . Recent meta-analyses of genome-wide association studies ( GWAS ) have identified 161 genetic loci associated with CAD at genome-wide significance[1–6] . Importantly , around 60% of these loci have been associated independently of classical risk factors[7] , and given previous functional evidence of candidate causal genes at these loci[8–11] , suggests an involvement of pathways intrinsic to the vessel wall . This is supported by the recent CAD GWAS meta-analysis , which combined targeted genotyped , multi-ethnic individuals with individuals from the CARDIoGRAMplusC4D consortium , to identify 15 new loci , within or near genes having arterial wall-specific functions[4] . Interestingly , one of these new loci ( lead SNP rs2820315 ) is found to reside in a smooth muscle cell ( SMC ) -restricted gene annotated as Leiomodin 1 ( LMOD1 ) [4] . LMOD1 is a member of the LMOD family of proteins , known to bear close resemblance to actin capping proteins called Tropomodulins ( TMODs ) . However , unlike TMODs , only LMODs possess an extended C terminus containing an additional WH2 domain ( ~17aa actin monomer motif ) [12 , 13] . While LMOD2 and LMOD3 are found to be primarily restricted to skeletal and cardiac muscles , we and others have previously demonstrated LMOD1 is highly enriched in smooth muscle containing tissues , including main arteries and visceral organs of the gastrointestinal system[14–17] . Furthermore , of the three LMODs , only LMOD1 is identified as a specific downstream target of the SMC master regulator serum response factor ( SRF ) -Myocardin ( MYOCD ) complex[15] . More recently , a homozygous nonsense mutation in LMOD1 has been linked to a rare congenital visceral myopathy called megacystis microcolon intestinal hypoperistalsis syndrome ( MMIHS ) [18] . Detailed analyses performed using Lmod1 deficient mice , revealed this phenotype to be a consequence of disrupted actin filament assembly , cytoskeletal arrangement and impaired SMC contractility[18] . Interestingly , this defect is reportedly analogous to the thin-filament disorganization and nemaline myopathy observed in mice and humans with Lmod3 deficiency[19] . In addition , our recent epigenomic profiling study of human coronary artery smooth muscle cells ( HCASMC ) has led to the identification of a novel CAD associated regulatory variant , rs34091558 , in the LMOD1 locus[20] . Although we determined this variant resides in a region of accessible chromatin defined by the Assay for Transpose Accessible Chromatin ( ATAC-seq ) and is marked by the H3K27ac active enhancer histone modification , the underlying mechanism for this association remains unknown . Herein , we provide confirmatory evidence that rs2820315 , near LMOD1 , is a CAD-specific GWAS association , which co-localized with the expression quantitative trait locus ( eQTL ) association in both GTEx and STARNET artery tissues , and that rs34091558 ( in tight LD with rs2820315 ) is a likely causal variant at this locus . We investigate the genetic regulatory mechanism using a combination of in silico , in vitro and in vivo approaches in primary HCASMC and human and animal disease models , which highlight a role for LMOD1 in maintaining the differentiated SMC phenotype . Collectively these findings are expected to improve our understanding of the ‘hidden risk’ for CAD , and further close the gap between disease-associated regulatory variation and relevant vascular wall phenotypes .
The LMOD1 locus was previously associated with CAD ( lead SNP rs2820315; P = 7 . 70E-10 ) in a meta-analysis ( 88 , 192 CAD cases and 162 , 544 controls ) of de novo genotyped multi-ethnic cohorts combined with previous genotyped CARDIoGRAMplusC4D cohorts in a study that identified 15 loci related to arterial wall mechanisms[4] . To follow up on these findings , we mapped the lead risk variant , rs2820315 , in the latest CARDIOGRAMplusC4D and UK Biobank meta-analysis for CAD ( 10 , 801 CAD cases inclusive of angina and 137 , 371 controls ) [5] and identified rs2820315 associated at sub genome-wide significance ( P = 2 . 09E-06 , Fig 1A ) , consistent with previously published reports[4] . Interestingly , rs2820315 was not associated with low-density lipoprotein ( LDL ) cholesterol ( Fig 1B ) in large-scale studies such as the Global Lipids GWAS Consortium[21] . Similarly , we did not detect a significant association for rs2820315 with other CAD risk factors such as fasting glucose , fasting insulin , systolic blood pressure and low vWF in previous GWAS datasets from MAGIC[22] , in the International Consortium for Blood Pressure ( ICBP ) [23] , and CHARGE[24] ( S1 Fig ) . These results extend previous findings implicating the LMOD1 locus in the ‘hidden’ heritable risk for CAD , potentially through regulation of vessel wall processes . The majority of published GWAS results report the nearest gene ( s ) to the lead SNP , however recent findings from the multi-tissue Genotype-Tissue Expression ( GTEx ) project[25] demonstrate that this is correct for only 40% of GWAS signals ( 21 traits tested ) [26] . Therefore , we evaluated eQTL results in GTEx to identify potential causal genes at the LMOD1 locus . Using such an approach , we identified a significant eQTL association of rs34091558 with LMOD1 ( P = 5 . 0E-09; r2 = 0 . 94 with lead GWAS SNP rs2820315 in Europeans ) in tibial artery tissue ( Fig 1C ) , but not in liver tissue ( Fig 1D ) . We also identified significant eQTLs for variants associated with LMOD1 in SMC containing tissues in GTEx , such as lung ( S2A and S2B Fig ) and esophagus mucosa ( S2C and S2D Fig ) . Importantly , we only observed GWAS overlapping eQTLs in artery GTEx tissues for variants associated with LMOD1 ( P = 5 . 0E-09 ) , and not nearby genes: NAV1 , MIR1231 , IPO9-AS1 , IPO9 , SHISA4 , RP11-307B6 . 3 ( Fig 1C , S1 Table ) . Thus , we queried the relative expression of all genes at the LMOD1 locus ( Fig 2A , upper panel ) in atherosclerosis prone tissues such as human coronary artery in the GTEx dataset . We observed abundant LMOD1 expression in this tissue type , however the expression of neighboring genes was barely detectable ( Fig 2A , lower panel ) . A similar expression pattern was observed in other atherosclerosis prone and SMC-enriched tissues such as aorta , and tibial artery in GTEx ( S3A Fig ) . As expected little or no LMOD1 expression was detected in liver ( S3A Fig ) . Similarly , we evaluated relative gene expression in the STARNET datasets and observed highest expression of LMOD1 in normal mammary artery ( MAM ) and atherosclerotic aortic root ( AOR ) compared to liver ( S3B Fig ) . We then ranked candidate gene expression in all GTEx tissues ( normalized by transcripts per million ) and observed LMOD1 is highly ranked in both artery and SMC-enriched tissues than compared to neighboring genes SHISA4 , IPO9 and NAV1 ( Figs 2B and S4 ) . These results were consistent with the direction of the rs2820315-T and rs34091558-T risk alleles associated with lower LMOD1 expression in tibial arteries ( P = 1 . 2E-07 and 5 . 0E-09 , respectively ) in the GTEx ( Fig 2C and 2D ) . Despite the high expression of LMOD1 in coronary artery tissue , eQTLs were undetectable , potentially due to the lower sample number and greater tissue heterogeneity in coronary arteries relative to tibial arteries ( www . gtexportal . org ) . Using summary-based mendelian randomization ( SMR ) colocalization analysis of CARDIoGRAMplusC4D GWAS and multi-tissue GTEx and STARNET cis-eQTL summary statistics[27] , we observed LMOD1 to be the top candidate gene at this locus ( PSMR = 7 . 25E-05 , PHET = 0 . 137 ) . Taken together , these data strongly implicate LMOD1 as a causal gene at this new CAD locus . To further validate cis-acting effects on LMOD1 gene expression , we measured allelic expression imbalance ( AEI ) in cultured HCASMCs using the coding SNP , rs2820312 , as a proxy ( r2 = 0 . 83 with lead SNP rs2820315; S5A Fig ) and detected an approximate 0 . 5–0 . 8 fold imbalance of the reference allele ( G ) over the variant allele ( A ) in 11 out of 12 heterozygous samples ( P = 5 . 9E-0 . 4 ) using TaqMan based quantification of cDNA/gDNA ( Figs 2E and S5B ) . We extended our findings by performing pyrosequencing based allelic quantification and observed a similar trend ( S5C Fig ) . Together these data are consistent with the direction of the eQTL results reported above ( Fig 2C ) and suggest that LMOD1 levels are decreased through a cis-regulatory mechanism in subjects carrying the risk variant . To investigate the regulatory landscape at the LMOD1 locus and prioritize potential causal variants responsible for attenuated LMOD1 expression , we leveraged the Roadmap Epigenomics dataset comprising 98 different primary cells and tissues . Having previously identified rs34091558 ( in tight LD with the lead SNP rs2820315 ) , to reside in a region of open chromatin and H3K27ac marked active enhancer in HCASMCs[20] , we thus focused on the H3K27ac mark across several CAD related , SMC-enriched tissues , as well as other primary tissues . Consistent with the GTEx RNA-seq tissue distribution , we observed rs34091558 to reside in an H3K27ac marked region in aorta and SMC-enriched gastrointestinal tissues ( S6A Fig ) . Interestingly , this region also displayed high specificity in HCASMCs when compared to other ENCODE cell datasets ( S6B Fig ) . We also analyzed the 15 chromatin state ChromHMM Roadmap datasets , and found that rs34091558 preferentially resides in active enhancer states in these SMC-enriched tissues , consistent with activation rather than repression of gene expression ( S7 Fig ) . To further test whether chromatin annotations are sufficient to delineate causal variants at this locus we implemented probabilistic fine-mapping approaches such as the Probabilistic Annotation INTegratOR ( PAINTOR ) [28] and Probabilistic Identification of Causal SNPs ( PICS ) [29] algorithms , based on HCASMC functional annotations , haplotype structure and summary GWAS results . As expected , the lead SNP rs2820315 was the top ranked variant using the LD-based PICS , but rs34091558 had the highest posterior probability using PAINTOR ( S2 and S3 Tables ) . We augmented these results using an exhaustive shotgun stochastic search algorithm ( FINEMAP ) to identify causal configurations from GWAS and eQTL variants[30] , and again observed rs34091558 with the highest posterior probability ( S4 Table ) . This is consistent with our findings on rs34091558 being the only CAD risk variant that overlapped a conserved sequence and multiple functional annotations in HCASMC ( S5 Table ) . This is also consistent with conditional analysis results of rs34091558 on the latest CAD GWAS association results at the LMOD1 locus ( S8 Fig ) . While it remains possible that multiple causal variants may exist under specific regulatory contexts , these fine-mapping results strongly indicate that rs34091558 is a top candidate causal variant at this locus . Given these findings we then pursued the potential molecular mechanism ( s ) linking the rs34019558 risk variant ( single nucleotide deletion ) to reduced LMOD1 expression . To this end , we scanned the sequence containing rs34091558-TA and rs34091558-T for putative transcription factor binding sites ( TFBS ) using a combination of in silico prediction tools , including JASPAR[31] , MATCH[32] , HOCOMOCO[33] and HaploReg[34] . Interestingly , we identified that the ancestral protective allele ( rs34091558-TA ) is predicted to bind a mammalian conserved family of transcription factors annotated as Forkhead box ( FOX ) , while the derived risk allele ( rs34091558-T ) is predicted to significantly disrupt this binding ( JASPAR PWM scores 0 . 91 > 0 . 67 ) ( Fig 3A and S6 Table ) . RNA-seq analysis of all candidate FOX transcription factors in GTEx datasets , identified FOXO3 as highly expressed in coronary , tibial , and aorta artery tissues in comparison to other FOX family members ( P < 2 . 2E-16 ) ( Figs 3B and S9 ) and in a manner similar to that of LMOD1 , thus revealing FOXO3 as a potential regulator of LMOD1 . In order to evaluate the impact of rs34091558 on FOXO3 protein binding and activity , we utilized enhancer luciferase reporter constructs containing either the protective or risk allele [20] , driven by a minimal promoter , and thereafter tested the response of each construct to an expression plasmid encoding human FOXO3 in cultured A7r5 SMCs . As predicted , we detected significant basal allelic effects with the ancestral protective allele ( rs34091558-TA ) having greater enhancer activity than the risk allele , rs34091558-T ( Fig 3C , P = 6 . 9E-05 ) . These results provide independent confirmation of our previous findings [20] . Importantly , we observed even more pronounced allelic differences in reporter activity upon FOXO3 overexpression ( Fig 3C , P = 2 . 9E-05 ) . Given that FOXO3 strongly activated the protective allele but not the risk allele , suggests that FOXO3 mediated trans-activation is partly responsible for these allelic effects . Given the known limitations of episomal based reporter assays , we then performed FOXO3 chromatin immunoprecipitation ( ChIP ) in cultured HCASMCs to investigate the impact of rs34091558 in the native chromatin context . Using such an approach , we first demonstrated significant enrichment of FOXO3 protein at the rs34091558 enhancer region ( P = 3 . 25E-06 ) ( Fig 3D ) , following which we measured the allele-specific effects of rs34091558 on FOXO3 binding using both homozygous and heterozygous HCASMC donors at rs34091558 . In agreement with the reporter assays and eQTL direction of effect above , we observed the ancestral protective allele , rs34092558-TA , to preferentially bind FOXO3 protein relative to the derived risk allele rs34091558-T ( P = 3 . 0E-13 ) ( Fig 3E ) . These results suggest that carriers of the risk allele have reduced LMOD1 expression in part due to the inhibition of FOXO3 protein binding at rs34091558 . To assess whether FOXO3 specifically regulates endogenous LMOD1 expression , we first transfected HCASMCs with a small interfering RNA ( siRNA ) to FOXO3 and then measured LMOD1 gene expression after 24 hours . In addition to observing reduced FOXO3 expression ( S10A and S10C Fig ) , cells transfected with siFOXO3 also had reduced LMOD1 mRNA expression ( Fig 3F ) . Extending these findings , we overexpressed FOXO3 in A7r5 SMCs ( S10B Fig ) , and observed a consistent increase in LMOD1 expression in these cells ( Fig 3G ) . To begin exploring the relevant upstream signals responsible for FOXO3 regulation of LMOD1 , we treated HCASMC with various growth factors and cytokines known to influence SMC differentiation . Silencing of transforming growth factor beta ( TGFB1 ) , a known pro-differentiation cytokine did not alter LMOD1 expression in HCASMC ( S11 Fig ) . However , treatment with platelet derived growth factor BB ( PDGF-BB ) an established mitogenic anti-differentiation growth factor[35] , significantly downregulated LMOD1 mRNA levels via qRT-PCR ( P = 0 . 0054 ) , consistent with previously shown HCASMC RNA-seq results [20 , 36] ( Fig 4A and 4B ) . These confirmatory results support that LMOD1 expression is regulated by PDGF-BB signaling and preferentially associated with the differentiated phenotype in HCASMC . We extend these findings by Western blotting , which revealed attenuated LMOD1 protein levels but increased phosphorylated FOXO3 levels in response to PDGF-BB ( Fig 4C ) . Increased phosphorylated FOXO3 downstream of PDGF-BB signaling has been shown to limit the transcriptionally active FOXO3 protein[37] . Previous findings have demonstrated significant differential expression of LMOD1 in human atherosclerotic carotid artery plaque microarray data ( n = 127 ) from the Biobank of Karolinska Endarectomies ( BiKE ) study[36] . Using this dataset we identified a strong correlation of LMOD1 and FOXO3 transcripts ( Fig 4D ) , which suggests that LMOD1 and FOXO3 may be co-regulated during atherosclerosis in vivo . Having confirmed RNAi mediated LMOD1 knockdown at the mRNA and protein levels ( Fig 5A ) we next performed a series of functional assays to interrogate the effects of reduced LMOD1 expression on SMC differentiation . Compared to control siRNA , LMOD1 siRNA transfected cells displayed a significantly higher proliferation rate as measured using trypan blue cell viability/proliferation assays ( P = 0 . 024 ) ( Fig 5B ) and CellTiter96 ( Promega ) cell proliferation assays ( P = 0 . 013 ) ( Fig 5C ) . Also , LMOD1 deficient HCASMCs were associated with increased migration in Boyden chamber assays ( P = 1 . 4E-04 ) ( Fig 5D ) . In keeping with these findings , cell contraction assays revealed that loss of LMOD1 significantly repressed HCASMC contraction on collagen gel substrate , which was maintained for 18 hours ( Fig 5E–5G ) . This effect on contraction is consistent with previous results in intestinal SMCs[38] . These data suggest that LMOD1 is essential to preserving the contractile phenotype observed in differentiated SMCs and its loss potentiates SMCs to adopt a de-differentiated phenotype often associated with CAD . SMCs have been shown to play critical roles in both the expansion of the intima ( largely comprised of proliferative SMCs ) in early stages of atherosclerosis and stabilizing the developing lesion in later stages through the formation of a thick fibrous cap ( comprised of differentiated SMCs ) [39] . While previous reports demonstrated differential staining of LMOD1 in carotid atherosclerosis [36] , it was still not clear what type of SMCs are implicated . This is important given that SMCs have heterogeneous responses during atherosclerosis and may elicit detrimental or protective effects depending on the nature of their phenotypic transitions to a de-differentiated state [40 , 41] . To validate our in vitro findings and determine the potential role of SMC-specific LMOD1 in atherosclerosis , using a previously authenticated LMOD1 antibody [42] , we tracked LMOD1 expression in atherosclerotic lesions in the brachiocephalic artery of the SMC-specific lineage tracing mouse Myh11-Cre-ERT2 ROSA26-STOPFlox tdTomato Apoe-/- [40] after 18 weeks of high-fat diet and observed that LMOD1 co-localizes with tdTomato-positive cells of SMC-origin in both the media and fibrous cap outside the lesion ( Fig 6A–6C ) . In contrast , LMOD1 was largely absent from tdTomato-positive cells within the lesion , which mostly consist of de-differentiated/proliferative SMCs[40] . We observed similar results in independent mouse lesions and noted that LMOD1 was highly expressed along the thin fibrous cap and throughout the media ( Fig 6D–6F ) , suggesting that LMOD1 , as a differentiation SMC marker gene , may participate in plaque stability . This is consistent with previous immunohistochemistry results showing a loss of LMOD1-expressing cells in carotid atherosclerotic lesions [36] , and refines the localization of LMOD1 in specific SMC-derived populations during disease . These data also suggest that LMOD1 downregulation may represent a critical step in SMC phenotypic transitions during atherosclerosis , while maintained LMOD1 expression in differentiated SMC may be beneficial . Taken together our working model ( Fig 7 ) demonstrates that individuals carrying the protective allele , rs34091558-TA have elevated LMOD1 expression levels due to increased FOXO3 binding , promoting SMCs to adopt a differentiated phenotype , a hallmark of stable atherosclerotic lesions thought to reduce CAD risk . In contrast , individuals carrying the risk allele , rs34091558-A have reduced LMOD1 levels due to the inhibition of FOXO3 binding , resulting in SMCs adopting a proliferative phenotype , a hallmark of unstable lesions and increased disease risk .
In this study we performed a comprehensive functional analysis of a SMC-restricted gene associated with CAD . By combining large-scale regulatory annotations and fine-mapping with mechanistic perturbations and functional assays in primary HCASMCs , we confirm previous studies and reveal new insights into the causal CAD mechanisms operating in the vessel wall . In particular , this integrative approach identified a critical role for the rs34091558 deletion risk variant in regulating FOXO3 mediated transcription of LMOD1 . This regulation of LMOD1 was strongly antagonized by the well-established mitogenic PDGF-BB signaling pathway , consistent with previous reports and provides a physiological link in coronary SMCs . Further , by employing loss-of-function assays in HCASMC and immunostaining of atherosclerotic lesions in vivo , we identified a role for LMOD1 in controlling the differentiated SMC phenotype and potentially regulating plaque stability . These findings are expected to shed light on the understudied heritable risk associated with CAD and may inform the development of more precise therapeutic interventions in future investigations . To date , 161 loci have been identified for CAD from large-scale genome wide association studies , with the majority of these associations being independent of traditional risk factors[3 , 5 , 6] . Previous studies have focused on systems based approaches to categorize and prioritize these loci into regulatory networks and functional pathways for future follow up[43–45] . Fifteen loci , within or near genes having arterial wall-specific functions were recently reported[4] . Of these , 5 loci are found to reside near genes preferentially expressed in SMCs ( LMOD1 , SERPINH1 , DDX59–CAMSAP2 , TNS1 , PECAM1 ) . SERPINH1 ( rs590121 lead SNP ) is predicted to increase risk of hemorrhage; DDX59-CAMSAP2 ( rs1867624 ) may be involved abnormal vascular development; PECAM1 ( rs1867624 ) may regulate vascular inflammation and endothelial dysfunction; and LMOD1 ( rs2820315 ) is implicated in SMC differentiation[4] . In our previous study we also discovered regulatory variants at seven candidate CAD loci , including 9p21 . 3 , SMAD3 , PDGFD , IL6R , BMP1 , CCDC97/TGFB1 and LMOD1 , that associated with enhancer epigenetic marks in HCASMC[20] . However , LMOD1 is the only gene that appears to have a SMC-restricted expression pattern as a SRF/MYOCD target gene . Given that LMOD1 is a member of the LMOD family of actin filament nucleators[12] , it may potentially play a critical role in regulating smooth muscle actin filament nucleation and function . This is supported by the recent identification of a recessive mutation in a premature stop codon of LMOD1 as a cause of a rare congenital visceral myopathy , MMIHS[18] , which usually arises due to autosomal dominant mutations in the smooth muscle enriched actin gamma 2 ( ACTG2 ) gene . The identification of common regulatory variation in LMOD1 associated with CAD supports the notion that a genetic dosage effect exists for Mendelian and complex diseases involving smooth muscle . Here , we confirm that the lead associated variant at the LMOD1 locus , rs2820315 , is also the lead SNP in multiple CAD cohorts including the most recent UKBiobank cohort comprising ~500 , 000 individuals . We also show that this variant is an eQTL for LMOD1 , with the risk allele associated with lower LMOD1 expression levels , in SMC tissues including tibial artery from the GTEx dataset . Fine-mapping and expression analyses demonstrated that LMOD1 is the most likely causal gene at this locus , which is enriched in vascular tissues , and allelic imbalance assays in HCASMC suggest a cis-regulatory mechanism of LMOD1 . Pinpointing causal variants at a given locus is often challenged by the strong LD between the lead risk variant and hundreds of candidate regulatory variants[46] . Many of these variants reside in regions of accessible chromatin , and are occupied by transcription factors or histone modification . Using ATAC-seq and ChIP-seq molecular profiling in HCASMC we previously identified a top candidate regulatory variant at this locus , rs34091558 ( r2 = 0 . 94 with rs2820315 ) , that resides in a region of open chromatin , TF binding , and enhancer activity . In fact , this was the only such variant identified at the LMOD1 locus ( S4 Table ) . Here , we provide more definitive statistical fine-mapping evidence supporting its causality and identify this variant to disrupt binding of the FOXO transcription factor family , particularly FOXO3 , given its endogenous expression in HCASMC and vascular tissues . It remains possible that other variants ( despite lack of complete functional annotation ) contribute to the regulation of LMOD1 . This is in agreement with a ‘multiple enhancer variant’ model for complex diseases[47] , in which multiple risk alleles in the same haplotype could contribute regulatory effects on LMOD1 through cooperative or independent mechanisms . Also , it is important to note that this proposed mechanism probably relates to European and Asian populations where the LD is generally high ( r2~0 . 9 ) but not in African populations , where due to higher recombination rates these two SNPs ( rs34091558 and rs2920315 ) are not in LD . The mechanistic link of FOXO3-LMOD1 is intriguing given that FOXO3 has been associated with body mass index and adiposity[48 , 49] and may explain the strong eQTL association of rs2820315 with LMOD1 in adipose tissues . Interestingly FOXO3 was found to be the most highly expressed of the FOXO family members , particularly in artery tissues . Previous evidence suggests that FOXO transcription factors can regulate tissue-specific expression either directly or by heterodimerizing with cell-specific cofactors[50] . Here , we also identified an enrichment of FOXO3 motifs in HCASMC-specific open chromatin peaks compared to other tissues ( S12 Fig ) , supporting a potential tissue-specific binding preference in SMCs . Previous studies have observed altered FOXO3 expression and phosphorylation with PDGF-BB and IGF mediated signaling in dermal fibroblasts[37] . We demonstrate here that LMOD1 protein levels are significantly down-regulated with PDGF-BB stimulation , while phosphorylated FOXO3 ( P-FOXO3 ) levels are upregulated . This supports a potential mechanism by which PDGF-BB mediated P-FOXO3 sequesters available nuclear FOXO3 for binding to this enhancer , thus resulting in reduced LMOD1 . FOXO3 binding sites were also identified at the promoter of LMOD1 , thus it remains possible that mechanisms independent of this enhancer could partially contribute to the PDGF-BB mediated effects . Our functional assays in HCASMC with LMOD1 gene silencing support a role for this gene in regulating the differentiated SMC phenotype , consistent with previous reports in intestinal SMCs[18] . LMOD1 downregulation resulted in increased SMC proliferation , viability , migration , and decreased contraction . Further , using an established SMC lineage tracing mouse bred on the Apoe-null background with 16 weeks of high-fat diet challenge , we localized LMOD1 to the forming fibrous cap in the brachiocephalic artery . Given the direction of the human genetic association studies and prior expression analyses in carotid arteries[36] , these results suggest that LMOD1 may serve a protective role in the setting of atherosclerosis , and potentially stabilize the developing lesion . Supporting this ‘atheroprotective SMC’ hypothesis , LMOD1 was also localized to the medial layer of the lesion . While LMOD1 appears to selectively mark differentiated SMCs in vivo , the functional effects of LMOD1 deficiency should also be validated in vivo using various models of atherosclerosis progression . Future studies using single-cell transcriptomic profiling in lineage-traced SMCs during atherosclerosis may further define the causal functional role of LMOD1 as a key regulator of SMC transformation during the disease process .
Association between lead variant , rs2820315 , and candidate regulatory variant , rs34091558 , with LMOD1 gene expression was determined by querying the public eQTL database from the Genotype-Tissue Expression ( GTEx ) project and the Stockholm-Tartu Atherosclerosis Reverse Engineering Task ( STARNET ) [51] project , as previously described[20] . Briefly , both rsIDs and candidate genes in the entire locus were queried in all tissues in GTEx and STARNET and results represented as -log10p-values across a ~1Mb region in LocusZoom . Note that rs34091558 is misannotated in GTEx as rs397789675 and can be found based on its chromosome:position ID , chr1:201886769 ( hg19 ) . Alternatively , dosage plots of normalized gene expression levels relative to variant genotype are shown for both GTEx and STARNET eQTLs . For comparing the tissue distribution of candidate gene expression levels , reads per kilobase per million mapped reads ( RPKM ) or transcripts per million ( TPM ) values were extracted from the GTEx databases . Similarly , EDAseq normalized reads were extracted from the STARNET database ( atherosclerotic aortic root , internal mammary artery , and liver ) , as previously described [20] . Conditional analysis of rs34091558 or rs2820315 SNP association with CAD was performed using the Genome-wide Complex Trait Analysis ( GCTA ) software package , GCTA-COJO[52] . Briefly , summary statistics data from the latest CARDIoGRAMplusC4D and UK Biobank meta-analysis was downloaded from the consortium website . A 1Mb region flanking lead SNP rs2820315 was extracted using subset function and data was reformatted in R according to the input file requirements . Either rs34091558 or rs2820315 was added to snp file cond . snplist and cojo was run using the default parameters: gcta64—bfile test—cojo-file test . ma—cojo-cond cond . snplist—out test . Tissue-specific regulatory mapping of LMOD1 variants was assessed using genome-wide epigenomic data in the Roadmap Epigenomics[53] and Encyclopedia of DNA Elements ( ENCODE ) [54] projects . Cardiovascular tissues , smooth muscle enriched tissues and non-smooth muscle tissues were selected and queries were performed using the rsID for regulatory variants in the locus and ChIP-seq data for the active enhancer histone modification H3K27ac . Chromatin states from the 15-state ‘core’ model using Roadmap histone modification ChIP-seq data were queried using ChromHMM[55] and visualized in the Roadmap Epigenomics browser . HCASMC epigenomic profiles generated from custom ATAC-seq and ChIP-seq ( H3K27ac , H3K4me1 , and H3K4me3 ) were queried for each variant as previously described[20] . Statistical fine-mapping of the LMOD1 locus was first conducted using the Probabilistic Identification of Causal SNPs ( PICS ) [56] . Given the observed pattern of the association at the LMOD1 locus , using CARDIoGRAMplusC4D and UKBiobank meta-analysis summary statistics and variant coverage using Phase 3 1000 Genomes Project data , 10 , 000 permutations were simulated with the association of the lead SNP rs2820315 fixed and all possible associations obtained from the LD SNPs . Based on these statistical fluctuations the probabilities of LD SNPs being the causal variant were then estimated using Bayes’ theorem . The test makes the assumption that the prior probability of each SNP to be a causal variant or the lead SNP rs2820315 is equal . Also , probabilities were calculated for all SNPs in LD at r2 >0 . 5 with the lead SNP . Fine-mapping analysis was also conducted using the Probabilistic Annotator INTegratOR ( PAINTOR v3 . 0 ) [28] implemented on the command line . Briefly , summary statistics from the latest CARDIoGRAMplusC4D and UK Biobank meta-analysis were integrated with LD data by generating a matrix with pairwise Pearson correlations for each SNP , and a matrix containing binary formatted HCASMC functional annotation data ( ATAC-seq and ChIP-seq for H3K27ac , H3K4me1 , H3K4me3 ) for SNPs overlapping a given annotation . Functional annotations were used as prior probabilities and learned from the data via Empirical Bayes to prioritize causal variants ( without explicit assumptions on maximum number of causal variants , j ) . Candidate causal variants were estimated by posterior probabilities and observed association Z-scores using a standard multivariate normal assumption model , where Cj is the causal vector , λj is the non-centrality parameter , and Σj is the LD matrix of pairwise correlation coefficients: P ( Cj;λj ) =N ( Zj;Σj ( λj∘Cj ) , Σj ) Colocalization fine-mapping of candidate target genes in the LMOD1 locus was performed using Summary-based Mendelian Randomization ( SMR ) [27] by integrating the latest CARDIoGRAMplusC4D and UK Biobank GWAS summary statistics with GTEx and STARNET eQTL summary data . We tested the pleiotropic effect of all cis-eQTLs associated with both gene expression at the LMOD1 locus and CAD phenotype . We considered all cis-eQTL associations at P < 5E-05 and GWAS associations at P < 0 . 05 . We used genetic data from individuals of European ancestry from 1000 Genomes Phase 3 as the reference panel . We also performed the HEterogeneity In Dependent Instruments ( HEIDI ) test to determine whether the GWAS and eQTL associations are likely driven by the same variant ( not significant for HEIDI test ) . Therefore we considered GWAS target genes at a threshold of PSMR < 0 . 05 and PHEIDI > 0 . 05 . Statistical fine-mapping of variants in the LMOD1 locus was performed using both GWAS and relevant eQTL summary statistics in FINEMAP v1 . 0 , an exhaustive Shotgun Stochastic Search ( SSS ) algorithm implemented on the command line[30] . Like other fine-mapping tools , PICS , PAINTOR , and CAVIARBF , FINEMAP uses Bayes’ theorem to estimate a posterior probability of causal configurations . The most computationally efficient method to compute the unnormalized posterior using the Bayes factor ( BF ) was used , where γ is the maximum likelihood estimate ( MLE ) of the causal SNP effects , y is the mean-centered values for a quantitative trait , X is the SNP genotype matrix , m is the maximum number of causal configurations , k is the number of causal configurations , and pk is the prior probability for each causal configuration: p2* ( y , X ) = ( mk ) -1pk×BF ( γ:NULL ) The shotgun stochastic search of FINEMAP allowed more causal configurations to be considered , without the need to fix the number of causal SNPs . Commercially available HCASMCs purchased from Lonza or Cell Applications were cultured in SMC basal medium ( SmBM ) supplemented with SmGM-2 SingleQuots kit as previously described[20] . SMC integrity was established by reports of positive smooth muscle α actin staining and negative von Willebrand ( factor VIII ) performed by the suppliers . Rat aortic SMCs ( A7r5 ) were maintained in DMEM containing high glucose and 10% fetal bovine serum ( Thermo Fisher Scientific ) without antibiotics or antimycotics as previously described[15] . For PDGF-BB stimulation experiments , HCASMCs were seeded at ~70% confluency in 6 well dishes . Once attached , cells were serum starved for 24 hours and thereafter treated with either vehicle or PDGF-BB ( 20ng/ml ) . Following a 24-hour incubation , RNA or protein was isolated from cells . Genomic DNA and RNA were prepared from individual HCASMC donor lots determined to be either heterozygous or homozygous genotypes at proxy SNP rs2820312 ( determined by whole genome sequencing ) . Genomic DNA and total RNA were isolated using the Qiagen DNeasy Blood and Tissue kit and the Qiagen miRNeasy Mini kit , respectively , according to the manufacturer’s instructions . Total cDNA was prepared from 1 μg RNA using the High Capacity cDNA Reverse Transcription kit ( Applied Biosystems ) . Each qPCR reaction contained 20ng Genomic DNA or 20ng cDNA , 40x rs2820312 TaqMan SNP genotyping probe ( Applied Biosystems ) , and 2x Universal Master Mix ( Applied Biosystems ) . The PCR cycling conditions were as follows: an initial denaturation at 95 °C for 10 min , followed by 50 cycles of 95 °C for 10 sec and 60 °C for 45 sec . AEI was determined using the TaqMan SNP genotyping probe for rs2820312 ( S7 Table ) and expressed as the normalized allelic ratio of cDNA/gDNA . Calibration of the SNP genotyping assay was determined by mixing 20 ng of gDNA or cDNA , homozygous for each allele at the following ratios: 4:1 , 2: 1 1:1 , 1:2 , 1: 4 . The Log2 ratio of the VIC/FAM intensity from gDNA at cycle 50 was then plotted against the Log ratio of the two alleles to generate a linear regression standard curve . The Log ratio of VIC/FAM intensity of cDNA was fitted to the standard curve for the correction . These values were then normalized to the ratio of gDNA for each allele to obtain the normalized allelic ratio . Pyrosequencing assays were performed by the Protein And Nucleic acid ( PAN ) facility ( Stanford ) as previously described[57] with assays designed using PyroMark Assay Design software ( Qiagen ) . 20 ng gDNA or cDNA was amplified using forward and reverse pyrosequencing primers under the following conditions: 94°C 4 min , ( 94°C 30 sec , 60°C 30 sec , 68°C 45 sec ) ×35 , 68°C 6 min and PCR products were verified by gel electrophoresis . Pyrosequencing reaction was performed on PCR reactions using PyroMark Q24 ( Qiagen ) according to manufacturer’s instructions . Allelic quantitation was obtained automatically from the mean allele frequencies derived from the peak heights using PyroMark Q24 software . Total RNA was isolated from cell lines using the miRNeasy mini kit ( Qiagen ) as per the manufacturer’s instructions and subsequently quantified using a nanodrop ( Thermo Fisher Scientific ) . cDNA was generated using the iScript cDNA synthesis kit ( Bio-Rad ) or High Capacity cDNA Reverse Transcription Kit ( Thermo Fisher Scientific ) . Differences in mRNA expression were examined and quantified using TaqMan based real time PCR ( Applied Biosystem ViiA™7 ) . Data generated was normalized to GAPDH , converted to relative quantity values using the 2-ddCt method and expressed as the relative fold change . Detailed information of the probes used to assess expression of target genes are listed in S7 Table . HCASMC were seeded at ~70% confluency in 6-well dishes containing medium supplemented with serum and growth factors . Once attached , cells were serum starved ( treated with medium containing only 0 . 2% serum ) overnight and transfected the following day with either 10nM negative control ( siCtrl , Catalog # 4390846 ) or siRNA to FOXO3 ( siFOXO3 , Catalog # s2843 ) using Lipofectamine RNAiMAX ( Life Technologies ) as per the manufacturer’s instructions . 24 h post transfection , RNA was isolated from these cells for measuring mRNA expression . In the case of overexpression studies , cDNA generated from HCASMC derived RNA and enzyme-clamped primers ( S7 Table ) were used to PCR amplify a 2021 bp fragment encompassing the human FOXO3 coding sequence . The resulting PCR product was cloned into the EcoRV-HindIII site of the pCMV-Tag2B plasmid ( Agilent ) . The authenticity of the plasmid generated was confirmed via Sanger sequencing . Confluent A7r5 cells were transfected with either 1ug pCMVTag2B or FOXO3-pCMV-Tag2B using Lipofectamine 2000 ( Life Technologies ) as per the manufacturer’s instructions . 4–6 hours post transfection , cells were re-fed with fresh growth medium and total RNA was isolated after 48 hours . LMOD1 enhancer reporters were constructed by first identifying the putative enhancer sequence based on HCASMC ATAC-seq and H3K27ac flanked region at chr1: 201886639–201886794 ( hg19 ) . 160 nt single stranded oligonucleotide sequences ( S7 Table ) containing either the protective rs34091558-TA allele or the risk rs34091558-T allele and cloning site were annealed and the resulting dsDNA oligos were subcloned into the pLuc-MCS ( Agilent ) luciferase reporter vector driven by a minimal promoter . All reporter constructs were validated by Sanger sequencing . A7r5 SMCs were plated at ~70% confluency in 24 well-dishes and co-transfected with either luciferase reporter , Renilla luciferase internal control plasmid , and human FOXO3 or empty vector expression plasmids on the following day using Lipofectamine 3000 , as per the manufacturer’s instructions ( Thermo Fisher Scientific ) . Cells were lysed 48 h later in passive lysis buffer and analyzed on a luminometer ( Molecular Devices ) using the Dual-Luciferase Reporter Assay System ( Promega ) as per the manufacturer’s instructions . All transfections were performed in quadruplicate and were repeated at least three times . Results are presented as the fold change of the ratio of firefly to Renilla luciferase relative light units ( RLU ) with the control condition ( pLuc + Empty vector ) set to 1 . 0 . HCASMC and other tissue-specific open chromatin peaks were selected using the most stringent criteria , i . e . the peak can only appear in the selected tissue . This yielded 24372 specific peaks for brain , 7332 for HCASMC , 6388 for lung fibroblasts , 11249 for heart . Density plots were constructed using ChIP-Cor Analysis Module Feature Correlation Tool v1 . 5 . 3 . Input range was defined as +/- 10000 bp from the centered reference features . Window width was set to 500 bp and counts cut-off value was set to 999999 . Normalization was global , i . e . histogram entries were normalized by the total number of reference and target feature counts and the window width . The output graph represents target feature abundance as fold change relative to a genome average . FOXO3 PWM matrix was taken from JASPAR CORE vertebrates 2018 database . PWMScan—Genome-wide position weight matrix ( PWM ) scanner was used to scan GRCh37/h19 version of the human genome using the following parameters: p-value cut-off 0 . 0001 , background base composition 0 . 29 , 0 . 21 , 0 . 21 , 0 . 29 , search strand—both , non-overlapping matches—on . To confirm that enrichment of FOXO3 PWM near HCASMC-specific open chromatin peaks does not occur due to the correlation in base compositions of FOXO3 PWM and HCASMC specific open chromatin regions , we repeated the analysis using randomized FOXO3 PWM . Using such defined parameters , we obtained 712104 FOXO3 PWM binding site genome-wide and after randomizing the FOXO3 matrix we obtained 740737 FOXO3-randomized sites and performed correlation with ChIP-Cor Analysis Module as described above . ChIP assays were performed as previously described[58] . Briefly , 2x107 HCASMCs were crossed linked using 1% formaldehyde and quenched with glycine . Nuclear lysates were prepared using cold hypotonic buffer , followed by lysis in 1X RIPA buffer ( Millipore ) . Chromatin nuclear lysates were then sheared to fragments of 100–500 bp using a Bioruptor Pico sonicator ( Diagenode ) at constant duty cycle , output control 3–4 , 20 sec pulses for 16 rounds . Equal amounts of anti-rabbit IgG ( Dako ) , FOXO3 ( Novus Biologicals , Cat # NBP2-16521 ) , and JUNB ( Santa Cruz , Cat# sc-73 ) antibody were added to sheared chromatin to immunoprecipitate TF–DNA complexes and incubated overnight at 4°C . Dynabeads Protein G ( Life Technologies ) were used to capture the antibody–TF–DNA complexes and followed by washing with ice-cold RIPA buffer and PBS and eluted twice in 1X TE buffer containing 1% SDS or 0 . 67% SDS for 10 min each at 65 °C . The crosslinked Protein–DNA complex were reversed overnight at 65 °C and ChIP DNA was recovered using Qiagen PCR Purification kits according to the manufacturer’s instructions . For ChIP-qPCR experiments , primers spanning the FOXO3 binding site within the LMOD1 enhancer and EGFR control regions were used . qPCRs were performed using SYBR green chemistry ( Applied Biosystems ) . Primer information is listed in S7 Table . In the case of allele-specific assays , DNA from ChIP experiments and a customized TaqMan probe were used for performing qPCR . Homozygous cell line for the risk allele was used as a positive control and the mean ratio of major allele/minor allele ( FAM/VIC ) at 50 cycles were normalized to input DNA ratios . Total protein was isolated from cultured cell lines using 1X RIPA buffer ( Millipore ) supplemented with 1X Halt Protease Inhibitor Single-Use Cocktail ( Thermo Fisher Scientific ) as previously described[59] . Protein concentration for all samples was determined using Pierce BCA Protein Assay Kit ( Thermo Fisher Scientific ) . Equal amounts of protein samples were loaded and separated on precast gels ( Bio-Rad ) and thereafter transferred onto PVDF membranes ( Bio-Rad ) . Following a 1-hr incubation of these membranes in 5% skim milk solution prepared in 1X TBST , membranes were probed with commercially available antibodies recognizing LMOD1 ( Proteintech , 1:1000 ) , p-FOXO3 ( Cell Signaling , 1:500 ) and alpha-tubulin ( TUBA , Cell Signaling , 1:1000 ) overnight at 4°C . Membranes were subsequently rinsed with TBST and incubated with appropriately matching HRP conjugated goat anti rabbit ( 1:3000 ) secondary antibody for 1 hour at room temperature . Upon completion , membranes were briefly incubated with Pierce ECL Western Blotting Substrate ( Thermo Fisher Scientific ) after which protein expression was detected and captured using the Li-Cor odyssey imaging system . Cell viability was determined by implementing previously described Trypan Blue assays[60] . In short , equal number of transfected SMCs were plated in a 6-well dish and incubated overnight . Once attached , cells were serum starved for 48 hours and thereafter incubated with complete growth medium . 24 hours later cells were trypsinized , and briefly incubated with Trypan Blue , following which cells were manually counted using a hemocytometer . Alternatively , cell viability was also assessed by conducting a CellTiter96 Cell Proliferation Assay/MTT Assay ( Promega ) according to the manufacturer’s instructions . Briefly , transfected serum starved cells treated with complete growth medium for 24 hours , were incubated with 15 μl /well of Dye Solution for 4 hours at 37°C and thereafter with Solubilization Solution/Stop mix for 1 hour . Fluorescence was recorded at 570/650 nm using a plate reader ( SpectraMax 190 , Molecular Devices ) . SMC migration was assessed by performing standard Boyden chamber assays , as previously described[61] . Briefly , transfected SMCs were serum starved for 48 hours following which they were trypsinized and counted such that equal number of cells were distributed onto the upper chamber of 8 . 0 μm transwell migration chambers ( BD Biosciences ) . Following a 24-hour incubation , cells that migrated into the lower chamber were fixed in cold methanol , stained with hematoxylin , imaged using a Leica inverted microscope and manually counted in a blinded manner . 6x105 HCASMCs were transfected with either scrambled siRNA or siLMOD1 . Following 24 hours of transfection , cells were trypsinized and used for a collagen based cell contraction assay according to manufacturer’s instruction ( Cell Biolabs , San Diego , CA ) . Briefly , a mixture of cold Collagen Gel Working Solution ( collagen solution: PBS: Neutralization Solution = 28: 7: 1 ) and 0 . 1 ml 2x105 cell suspension was incubated in a 24-well dish at 37°C for optimum polymerization . Following an hour of incubation , cell culture medium was added atop of each collagen lattice and cells were incubated in a 37°C , 5% CO2 chamber . After 48 hours , cell contraction was initiated by gently releasing collagen gels from the sides of the culture dishes with a sterile spatula and changes in collagen gel size were measured by a ruler at different time points: 0 hr , 1 hr , 3 hr , 6 hr , and 18 hr . Animal protocols were approved by the Stanford University Administrative Panel on Laboratory Animal Care . ROSA26-STOPfloxtdT+/+ , Myh11-CreERT2 and Apoe-/- and were used in this study . To begin , we crossed ROSA26-STOPfloxtdT+/+ with Myh11-CreERT2 to generate ROSA26-STOPfloxtdT+/+; Myh11-CreERT2 mice . Since the Myh11-CreERT2 transgene is located on the Y chromosome , male ROSA26-STOPfloxtdT+/+; Myh11-CreERT2 were bred to female Apoe-/- to generate a cohort of ROSA26-STOPfloxtdT+/+; Myh11-CreERT2; Apoe-/- mice . In order to activate the Cre-recombinase , 5–6 week old male ROSA26-STOPfloxtdT+/+; Myh11-CreERT2; Apoe-/- mice were administered tamoxifen daily through intraperitoneal injections for 5 consecutive days . Upon completion , experimental mice were fed a high-fat ( Western-type ) diet , containing 21% anhydrous milk fat and 0 . 15% cholesterol ( Dyets ) for 18 weeks following which the brachiocephalic arteries from mice were harvested , rinsed with PBS , fixed with 4% paraformaldehyde and embedded in OCT ( Sakura ) and sectioned at 7 μm thickness using a cryotome ( CM1950 , Leica ) . For detecting LMOD1 expression within the atherosclerotic lesion , brachiocephalic artery tissue sections were stained with a primary antibody specific to LMOD1 ( Proteintech , 1:400 ) as previously described[18] . Briefly , tissue sections were rehydrated with PBS , permeabilized with 0 . 1% Triton X-100 and thereafter incubated in serum free protein blocking buffer ( DAKO ) for 1 hour . Upon completion , tissue sections were incubated overnight at 4°C with LMOD1 antibody , briefly rinsed with PBS after which they were incubated in goat anti-rabbit secondary antibody ( A21245 , Thermo Fisher Scientific ) for 1 hour and ultimately mounted with medium containing DAPI ( Sigma , F6057 ) . Images were captured with a Leica SP8 inverted confocal microscope . Human atherosclerotic carotid artery lesions were obtained from patients undergoing endarterectomy surgery for stable ( asymptomatic ) ( n = 40 ) or unstable ( symptomatic ) ( n = 87 ) carotid stenosis , as part of the Biobank of Karolinska Endarterectomies ( BiKE ) [62] . Normal control arterial samples ( n = 10 ) were obtained from the iliac and radial arteries from healthy organ donors without any history of cardiovascular disease . Briefly , tissue was snap frozen in liquid nitrogen before pulverizing to a fine powder using a pre-chilled mortar and pestle , then resuspended in Qiazol lysis reagent ( Qiagen ) and homogenized with a rotor stator tissue homogenizer . Total RNA was extracted as described above using the miRNeasy Mini Kit ( Qiagen ) and RNA quality assessed using a Bioanalyzer 2100 ( Agilent ) . Global gene expression profiles were analyzed by Affymetrix HG-U133 plus 2 . 0 Genechip microarrays from 127 patient derived plaque samples and 10 donor control samples . Robust multi-array average ( RMA ) normalization was performed and processed gene expression data presented in Log2 scale . These processed microarray data are available under Gene Expression Omnibus ( GEO ) accession number GSE21545 . The raw data is also available through a formal application process by contacting the study authors directly . Gene expression and genotyping data from the STARNET database[20] , were obtained from atherosclerotic aortic root ( AOR ) , internal mammary artery ( MAM ) and liver from up to 600 CAD patients ( determined eligible for the study and consented by the ethical committees of the Karolinska Institutet and Tartu University ) that were obtained during coronary artery bypass graft open-heart surgeries . GenomeWideSNP_6 arrays ( Affymetrix ) were used for genotyping gDNA . Total RNA was isolated from the atherosclerotic arterial wall or internal mammary artery . Gene expression levels were determined using a standard RNA-seq library preparation and sequencing protocol ( Illumina HiSeq 2500 ) , followed by normalization of raw read counts to adjust for library size and batch effects . Briefly , samples with <1 million reads were removed , and genes with <1 counts per million in more than 50% of the samples were also removed . EDAseq was then used to normalize the library size and GC % content , and outliers were removed based on a gender-expression test , covariates were adjusted with linear regression , outliers were again removed and last rank quantile normalization was performed . ‘Normalized Expression Counts’ represent counts before rank quantile normalization . Adjusted read counts were subsequently log2-transformed , and the association between genotype and expression was tested using a linear model . eQTLs were called using the MatrixEQTL R package running a linear model and returning all calls with a maximum P value of 0 . 05 . A follow-up conditional analysis was done to test the P ( max SNP | SNP ) as well as P ( SNP | max SNP ) for each gene . This was calculated by regressing the genotype of SNP_1 onto the residuals of SNP_2 regressed onto the gene expression , where SNP_2 is the SNP being conditioned on ( for example , P ( SNP_1 | SNP_2 ) ) . All samples reported in this study were obtained with approval of the Institutional Review Board at the respective institutions under written informed consent . Samples collected in the STARNET study were obtained from patients determined eligible for the study and consented by the ethical committees of the Karolinska Institutet and Tartu University . All atherosclerotic carotid plaque and donor control samples collected from the Biobank of Karolinska Endarterectomies ( BiKE ) were obtained with informed consent from patients , organ donors or their guardians . The BiKE study is approved by the Ethical Committee of Northern Stockholm ( approval numbers 02/147 and 2009/295-31/2 ) . Experiments were performed using at least three independent preparations and individual treatments/conditions performed in triplicate or quadruplicate unless otherwise noted . Sample sizes for individual experiments were determined based on power calculations to detect small effects in cultured cells/tissues . All data are presented as mean + standard deviation of replicates using RStudio for statistical analysis . In experiments requiring comparisons across two groups , standard unpaired t-tests with Welch’s correction were implemented for determining significance using RStudio . In the case of more than 2 groups , a one-way analysis of variance ( ANOVA ) followed by Bonferroni’s post-hoc test for individual significance was performed using RStudio . CARDIoGRAMplusC4D Consortium ( http://cardiogramplusc4d . org/ Genome-wide Complex Trait Analysis ( http://cnsgenomics . com/software/gcta/ Genotype Tissue Expression Project ( https://www . gtexportal . org/home/ ) Roadmap Epigenomics Consortium ( http://www . roadmapepigenomics . org ) Encyclopedia of DNA Elements Project ( https://www . encodeproject . org ) ChromHMM ( http://compbio . mit . edu/ChromHMM/ ) HaploReg ( https://pubs . broadinstitute . org/mammals/haploreg/haploreg . php ) JASPAR ( http://jaspar . genereg . net ) TRANSFAC–MATCH ( http://gene-regulation . com/pub/programs . html ) HOCOMOCO ( http://hocomoco11 . autosome . ru ) ChIP-Cor ( http://ccg . vital-it . ch/chipseq/chip_cor . php ) PWMScan ( http://ccg . vital-it . ch/pwmtools/pwmscan . php ) MatrixEQTL ( https://cran . r-project . org/web/packages/MatrixEQTL/index . html ) LocusZoom ( http://locuszoom . org ) FINEMAP ( http://www . christianbenner . com/# ) PAINTOR ( https://github . com/gkichaev/PAINTOR_V3 . 0 ) PICS ( https://pubs . broadinstitute . org/pubs/finemapping/ ) | It is now estimated that nearly ~50% of one’s lifetime risk for coronary artery disease is genetically predetermined . While genetic studies have led to the identification of several novel disease risk factors , the mechanisms driving many of these associations are yet to be fully identified . Here , we take advantage of a recent finding identifying genetic variation in a smooth muscle cell restricted gene called Leiomodin1 , and investigate the causal gene and mechanism by which this risk factor predisposes individuals to coronary artery disease . By implementing several complementary approaches , we extend previous findings demonstrating that this genetic variant is associated with attenuated expression of Leiomodin 1 . Interestingly , we demonstrate that this association is attributed to altered binding of a transcription factor Forkhead Box 3 . More importantly , we show that reduced Leiomodin 1 results in phenotypic modulation of smooth muscle cells , a process related to diseases such as atherosclerosis . Interestingly , Leiomodin 1 levels were found to be reduced in smooth muscle cells within atherosclerotic lesions relative to those around the fibrous cap . These studies provide new insights into the heritable mechanisms of coronary artery disease with Leiomodin 1 as a potential therapeutic target . | [
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"small... | 2018 | Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus |
Eggs deposited in the liver of the mammalian host by the blood fluke parasite , Schistosoma mansoni , normally drive a T-helper-2 ( Th2 ) -mediated granulomatous response in immune-competent mice . By contrast , in mice deprived of T-cells and incapable of producing granulomata , egg-secreted proteins ( ESP ) induce acute hepatic injury and death . Previous work has shown that one such ESP , the T2 ribonuclease known as omega-1 , is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage . Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase ( ALT ) activity as a marker of heptocyte injury , we demonstrated that S . mansoni eggs , egg-secreted proteins ( ESP ) , soluble-egg antigen ( SEA ) , and omega-1 are directly hepatotoxic and in a dose-dependent manner . Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33% , respectively . Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction . Proteomics of that fraction identified the presence of IPSE/alpha-1 ( IL-4 inducing principle from S . mansoni eggs ) , a known activator of basophils and inducer of Th2-type responses . Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro . Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter's toxicity and diminished the total toxicity of ESP and SEA by 32 and 35% , respectively . Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively . We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S . mansoni eggs .
Schistosomiasis is a chronic parasitic disease that affects more than 200 million people worldwide [1] . The central pathological characteristic during chronic infection is a granulomatous reaction around trapped parasite eggs in the host's liver , bladder , or intestine [2] . Granulomatous inflammation in the liver may result in fibrosis , scarring , portal hypertension , and in the worst cases hemorrhaging and death [3] . Schistosoma mansoni infection in mice is the most common experimental model employed . Approximately five-to-six weeks post-infection , parasite eggs deposited by adult worms induce a T-helper-2 ( Th2 ) -type-polarized immune response [4] . A number of the immunodominant molecules in eggs have been described [5] , [6] , [7] , [8] , in addition to the two molecules central to this report ( see below ) . The ability of S . mansoni eggs to induce Th2-type differentiation during infection is underscored by the observation that eggs alone , or soluble egg antigen ( SEA ) released by the eggs through pores in the shell , are sufficient to drive Th2 polarization in naïve uninfected mice [9] , [10] , [11] . Research from the late 1960s and 1970s has documented that mice lacking T-cells due to genetic loss or surgical removal of the thymus [12] , [13] , [14] , or administration of specific immunosuppressives [15] , [16] , [17] , do not develop a typical granulomatous response to trapped parasite eggs . In these T-cell depleted mice , infection was associated with extensive hepatic parenchymal damage suggestive of a cytotoxic egg product ( s ) diffusing into hepatic tissue [18] . Histopathology of livers from schistosome-infected immunocompromised mice displayed microvesicular hepatocyte steatosis [18] , [19] , [20] , nuclear degeneration , and hepatocyte apoptosis [21] . Coincident with hepatocyte injury , there is an increase in liver cell transaminases in the plasma [19] . Immunosuppressed mice also have higher mortality once egg deposition in the liver begins [17] , [18] , [19] , [22] . In immunologically intact mice , circumoval granulomata , and antibody responses to released S . mansoni egg components , likely protect against hepatocyte damage . Also , hepatotoxicity is prevented in infected T cell-deprived mice by transfer of serum from intact mice immunized with S . mansoni eggs or egg homogenate , whilst antisera against other lifecycle stages do not prolong survival [19] . Egg-induced hepatotoxicity appears to be specific to S . mansoni; it is not observed during infection of T-cell deprived mice with S . haematobium or S . bovis [23] . Finally , transfer of sera from S . japonicum-infected mice failed to alleviate hepatotoxicity induced by S . mansoni eggs in T-cell deprived mice [24] . Research in the 1980s identified several proteins in S . mansoni egg antigen preparations based on their electrophoretic mobility and their recognition by sera from mice with chronic infection [18] , [25] . Two of these proteins , termed omega-1 and alpha-1 , were isolated from S . mansoni egg homogenates ( SmAE ) by cation exchange chromatography in a single salt-eluted fraction that was termed cationic egg fraction 6 ( CEF6 ) [26] . Omega-1 is a 31 kDa monomeric glycoprotein [26] released from S . mansoni eggs [27] that has previously been reported to be hepatotoxic [18] . Monospecific antisera against omega-1 , which is a highly immunoreactive egg antigen , were protective against hepatocyte damage in T-cell deprived mice [18] , [23] . Immunochemical characterization of omega-1 using sera from humans and mice infected with different schistosome species suggested that the antigen is specific to S . mansoni [26] . Omega-1 is a functional T2 ribonuclease ( RNase ) [28] . Cytotoxic RNases ( which includes T2 family RNase members ) have been found in a wide range of species from bacteria to mammals . A range of biological roles has been suggested , including serving as extra or intracellular cytotoxins and modulating host immune responses [29] . Omega-1 was also reported to drive Th2 polarization in human monocyte-derived dendritic cells ( DCs ) , whereas SEA depleted of omega-1 loses this ability [30] . Omega-1 directly affects both DC morphology and the ability of these antigen-presenting cells to interact physically with CD4 T-lymphocytes [31] . Furthermore , when injected into IL-4 dual reporter mice , omega-1 is a potent inducer of the Th2 response in vivo [30] . A second major protein in S . mansoni egg homogenates , originally termed alpha-1 [25] , was recently reported to be identical to IPSE ( IL-4 inducing principle from S . mansoni eggs ) [32] . IPSE/alpa-1 is a glycoprotein [33] that has been crystallized [34] , occurs naturally as a dimer ( 33–35 kDa ) , and is enriched in the sub-shell area of S . mansoni eggs from where it is secreted into the surrounding tissue [32] . It is not found in the miracidium residing within the egg [35] . Various proteomics analyses have identified IPSE/alpha-1 as an abundant protein in egg secretions [5] , [36] , [37] , [38] . IPSE/alpha-1 binds immunoglobulin and activates naïve basophils , leading to histamine release and facilitating the production of Th2-type cytokines [32] . In vivo , IPSE/alpha-1 induces interleukin ( IL ) -4 secretion from murine basophils in an IgE-dependent but antigen-independent manner [39] . Most recently , IPSE/alpha-1 has been shown to contain a functional C-terminal , monopartite , nuclear localization sequence that binds DNA such that it may alter gene expression in the host cell [40] . We employed a primary hepatocyte in vitro culture system to identify and measure direct toxicity of S . mansoni eggs and their derived fractions , including pure proteins . Hepatotoxicity of omega-1 was confirmed , and IPSE/alpha-1 was identified as a novel hepatotoxin . Both proteins together account for more than half of the egg-derived toxicity measured .
These studies were performed in accordance with the recommendations by the University of California San Francisco Institutional Animal Care and Use Committee . The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of California San Francisco ( Permit Number: AN080237-03 ) . All surgery was performed under sodium pentobarbital anesthesia , and all efforts were made to minimize suffering . The protocol followed these guidelines in the study: All U . S . Federal Policy and Guidelines governing the use of laboratory animals , Public Health Service Policy on Humane Care and Use of Laboratory Animals , Guide for the Care and Use of Laboratory Animals , National Academy Press , USDA Animal Welfare Act and Regulations , and NIH Office of Laboratory Animal Welfare Guidelines . S . mansoni eggs were isolated from the livers of female golden hamsters six weeks following infection with 500 cercariae , as previously described [41] . Approximately 0 . 5 million eggs were washed twice in serum-free RPMI-1640 supplemented with 100 mg/ml streptomycin . Eggs were then resuspended in 2 ml RPMI-1640 , and 500 µl aliquots were placed in 12-well culture plates ( Costar ) . Cultures were checked daily by microscopy to ensure sterility . Medium was harvested after 72 h , and centrifuged for 10 min at 200×g to remove eggs . ESP , usually containing approximately 0 . 5 mg/ml protein by Bradford assay [42] , was stored at −80°C . After collection of ESP , egg viability was confirmed by hatching of miracidia; normally >85% of the eggs hatched . Hatching of eggs during the collection period was <1% . SEA was prepared , as described previously [43] . Purified natural omega-1 [30] and recombinant ( r ) IPSE/alpha-1 proteins [32] , and anti-omega-1 ( 140-3E11 ) and anti-IPSE/alpha-1 ( 74-1G2 ) monoclonal antibodies [32] were kindly supplied by Drs . Gabriele Schramm and Helmut Haas of the Research Center Borstel , Germany . Experiments to deplete ESP and SEA ( each 10 µg/ml ) of omega-1 and IPSE/alpha-1 involved incubation for 1 h with 5 µg/ml of the respective monoclonal antibodies bound to Protein G Sepharose ( GE healthcare Biosciences Pittsburgh , PA ) . Protein G Sepharose was then removed by centrifugation for 30 min at 100×g . ESP ( 2 mg ) were added to 2 ml 30 mM Tris-HCl , pH 8 . 0 , and centrifuged at 5000×g for 10 min at 4 C . The supernatant was loaded onto an Hr 5/5 Mono Q column ( GE Healthcare ) , and equilibrated with the same buffer and elute by a 0 to 1 M linear NaCl gradient in six column volumes of Tris-NaCl buffer . Flow-through and eluted fractions ( 1 ml ) were stored at −80°C prior to testing for toxicity with cultured primary hepatocytes ( applied volume 100 µl of each fraction to 2×105 hepatocytes/0 . 5 ml ) . Hepatocytes were isolated from C57/BL6 mice by in situ perfusion of liver with collagenase , as described previously [44] . The portal vein was severed to permit outflow followed by cannulation of the inferior vena cava with a 22-guage catheter . The liver was then flushed with a calcium-chelating buffer ( liver perfusion medium ) for 3 to 5 min , followed by perfusion with collagenase ( liver digest medium ) for an additional 6–8 min . At the end of the digestion , the liver was removed to a sterile dish and minced thoroughly with a scissors . This crude liver cell isolate was suspended in 25 ml of Dulbecco's modified Eagle's medium/Ham's F-12 , filtered through sterile gauze , centrifuged at 70×g for 2 min , and resuspended in Dulbecco's modified Eagle's medium/Ham's F-12 . After an additional round of centrifugation and resuspension , hepatocytes were isolated by centrifugation using a 50% Percoll gradient . Hepatocytes were cultured at a density of 2×105/0 . 5 ml per well in a 12-well culture plate ( Costar ) , previously coated with a 5 mm layer of matrigel ( BD Bioscience ) [45] , which is a tumor biomatrix prepared from the Engelbroth-Holm-Swarm mouse sarcoma [46] . Hepatocytes were allowed to attach for 1 hour at 37°C . Culture plates were gently swirled and the medium containing unattached cells and debris was aspirated . Cultures were then incubated for 72 h in a final volume of 0 . 5 ml RPMI medium containing 50 , 100 , or 200 eggs , unfractionated ESP ( 10 µg/ml ) or 100 µl of chromatography fractions . Supernatants were collected and stored at −20°C and analyzed for alanine transaminase ( ALT ) , a serum marker of hepatoxicity [47] ) using a Beckman Chemical Analyzer in the Clinical Chemistry Laboratory of the San Francisco Veterans Affairs Medical Center ( VAMC ) . ESP was fractionated by SDS-PAGE then silver stained [48] , [49] , and 40 evenly spaced protein bands were sliced out of the gel ( Fig S1 ) . The gel slices were then diced into small cubes , reduced and alkylated with dithiothreitol and iodoacetamide , and in-gel digested with trypsin [50] , [51] . The resulting peptides were extracted and analyzed by on-line liquid chromatography/mass spectrometry , using an Eksigent nanoflow pump and a Famos autosampler , which were coupled to quadrupole-orthogonal-acceleration-time-of-flight hybrid mass spectrometer ( QStar XL or Pulsar , Applied Biosystems ) . Peptides were fractionated on a reversed-phase column ( C18 , 0 . 75×150 mm ) , and a “5–50% B gradient-in-gradient” was developed in 35 min at a 350-nl/min flow-rate . Solvent A was 0 . 1% formic acid in water and solvent B was 0 . 1% formic acid in acetonitrile . Data were acquired in information-dependent acquisition mode: 1 sec MS surveys were followed by 3 sec CID experiments on computer-selected multiply charged precursor ions . Peak lists were generated using Analyst 2 . 0 software ( Applied Biosystems ) with the Mascot script 1 . 6b20 ( Matrix Science ) . Database searches were performed using ProteinProspector v . 5 . 1 . 7 ( http://prospector2 . ucsf . edu ) [52] . Searches were performed first on the SwissProt databank ( December 16 , 2008 , 405 , 506 entries ) to evaluate sample purity , followed by searching in the S . mansoni database SchistoDB v . 4 . 0 ( www . schistodb . net; 13 , 174 entries downloaded July 2009 ) . Batch-Tag settings were selected for samples prepared with trypsin allowing a maximum of one missed cleavage and no non-specific cleavages . Peptide modifications searched for included carbamidomethyl ( Cys ) as the only fixed modification , and up to two variable modifications from among the following: oxidation ( Met ) , acetyl ( N-term ) , oxidized acetyl ( N-term ) , pyroglutamate ( Gln ) and Met-loss ( N-term ) . The mass accuracy considered was 200 ppm , and 300 ppm for the precursor and fragment ions , respectively . The following acceptance scores for database matches were required: a minimum protein score of 22 , a minimum peptide score of 15 , and a maximum expectation value of 0 . 02 required for both peptide and protein identification . These criteria resulted in an approximate 2% false determination rate . Protein identifications are reported with a minimum of two peptide matches per protein . For the analysis of ESP anion exchange fraction #11 , the maximum expectation value was changed to 0 . 05 , and no decoy proteins were identified using these acceptance criteria .
To measure hepatotoxicity caused by parasite eggs ESP , and their chromatographic fractions , we employed an in vitro system involving murine primary hepatocytes cultured on matrigel . ALT was employed as a hepatoxicity biomarker . With parasite eggs , measurements were taken 24 , 48 and 72 h . No alteration in ALT levels was seen at 24 or 48 h ( not shown ) ; however , by 72 h , ALT had increased markedly and in a dose-dependent manner ( Figure 1A ) . Dose-dependent hepatocellular injury elicited by ESP was also measured after 72 h ( Figure 1B ) . To aid identification of those ESP constituents responsible for hepatotoxicity , ESP was fractionated by Mono Q anion exchange chromatography . The flow-through ( component not bound to the column ) , and each of the eluted fractions ( 100 µL ) , was co-incubated with cultured hepatocytes . At 72 h , fraction #11 induced an approximate two-fold greater release of ALT relative to the other eluted fractions and flow-through , such that it was the equivalent of 10 µg/ml unfractionated ESP ( Figure 2 ) . SDS-PAGE and tryptic digestion followed by mass spectrometry of ESP and hepatotoxic fraction #11 identified 99 and nine proteins , respectively ( Tables 1 and 2 ) . Previously , total proteomic analysis of ESP identified 188 proteins [36] , and many are common between this and the present dataset ( Table 1 ) . Among the nine proteins identified in fraction #11 were metabolic enzymes involved in glucose metabolism , glycogen storage , in addition to chaperones . IPSE/alpha-1 was also identified; and , given its potent immunomodulatory properties [32] , [34] , was of immediate interest in discovering of whether or not it was hepatotoxic . Omega-1 is an egg-secreted glycoprotein with RNase activity and in vivo-demonstrated hepatotoxicity [18] , [28] . To measure direct toxicity to primary cultured hepatocytes , purified native omega-1 was co-incubated with primary hepatocytes . At 72 h , a dose-dependent release of ALT was measured ( Figure 3A ) . Likewise , Aspergillus oryzae T2 RNase ( 25 U/µl ) ( Invitrogen , # 18031-013 Carlsbad , CA ) was toxic . Importantly , pre-incubation of pure omega-1 with a monoclonal anti-omega-1 antibody bound to Protein G Sepharose abolished cytotoxicity ( Figure 3B ) . Depleting ESP and SEA with the same antibody decreased toxicity by 47 and 33% , respectively . All reductions in hepatoxicity were statistically significant ( Figure 3B ) . IPSE/alpha-1 was an abundant protein in the hepatotoxic fraction #11 from anion exchange chromatography ( Table 2 ) . Recombinant IPSE/alpha-1 was added to hepatocyte cultures , and a dose-dependent toxicity was measured at 72 h ALT levels that was significantly elevated relative to negative controls ( Figure 4A ) . Similar to that found for omega-1 , specific neutralization of rIPSE/alpha-1 with an anti-rIPSE/alpha-1 monoclonal antibody abolished activity and decreased the cytotoxicity of ESP and SEA by 32 and 35% , respectively ( Figure 4B ) . All reductions in hepatoxicity were statistically significant . To measure the combined contributions of omega-1 and IPSE/alpha-1 to the hepatotoxicity of ESP and SEA in vitro , both egg-derived preparations were depleted of both omega-1 and IPSE/alpha-1 with specific monoclonal antibodies prior to incubation with hepatocytes . The combination of both antibodies diminished hepatotoxicity of ESP and SEA by 60 and 58% , respectively ( Figure 5 ) .
The pathogenesis of hepatic schistosomiasis is due to the host's granulomatous response to eggs deposited in the liver [2] . The initial cellular granuloma is characterized by the presence of activated macrophages , lymphocytes , and eosinophils , as reviewed in both Agnew and Pearce [23] , [53] . Over time , granulomata become fibrotic , and their accumulation in periportal areas , as is the case in chronic S . mansoni infection , can lead to portal hypertension , hemorrhaging , and death [3] . Ironically , in the absence of a granulomatous response , experimental hepatic schistosomiasis in mice leads to a more acute and lethal disease [17] , [18] , [19] , [20] , [22] , [54] . The understanding from such observations is that schistosome eggs release hepatotoxins , toxins that are normally prevented from diffusing by circumoval granulomata . To date , the only hepatotoxin characterized in S . mansoni eggs is omega-1 [18] which is RNaseT2 [28] , that also induces a Th2 response [30] . We established an in vitro primary hepatocyte culture system using ALT as the metric for cell injury to identify egg components with direct hepatotoxicity . We first confirmed the toxicity of S . mansoni eggs and their derivatives , ESP and SEA , and then showed that pure native omega-1 is hepatotoxic in vitro , consistent with previous in vivo observations . Based on the present system , omega-1 is a major toxin released by S . mansoni eggs , as depletion of ESP or SEA with a specific monoclonal antibody decreased ALT levels by 47 and 33% , respectively . To search for additional hepatotoxins , we combined anion exchange chromatography of ESP with proteomics . A single hepatotoxic fraction ( #11 ) was identified which contained a short list of nine proteins . IPSE/alpha-1 stood out as a molecule of interest given its potent immunomodulatory properties [39] , [55] . Subsequent characterization of pure rIPSE/alpha-1 demonstrated that the molecule is indeed directly hepatotoxic . The finding was confirmed using a specific monoclonal antibody that essentially neutralized IPSE/alpha-1 toxicity while decreasing the cell injury produced by both ESP and SEA by approximately one-third . Further depletion of ESP and SEA with a combination of monoclonal antibodies targeting both omega-1 and IPSE/alpha-1 indicated that approximately 60% of the toxicity of the egg-derived material is due to these two proteins . This leaves room for additional hepatotoxins to be identified , perhaps by different chemical and physical separation approaches . We also note that although both omega-1 and IPSE/alpha-1 were identified in the total ESP proteome , only IPSE/alpha-1 was subsequently found in the single hepatotoxic fraction #11 . This suggests that omega-1 was below the mass spectrometry detection limits used to identify proteins . Recently , IPSE/alpha-1 was reported to be internalized by Chinese hamster ovary cells ( CHO ) and primary monocyte-derived dendritic cells , but not by peripheral blood basophils [40]; and in each case without apparent toxicity . This suggests that host cell-specific factors determine how cells interact with and respond to IPSE/alpha-1 . Such factors might explain why IPSE/alpha-1 is directly toxic to hepatocytes . Studies to understand the mechanism of hepatoxicity induced by IPSE/alpha-1 , and other hepatotoxins such as omega-1 , can now be undertaken with the present in vitro system . Ribonuclease activity is often associated with cytotoxicity , and Steinfelder et al noted that omega-1 was initially characterized as a hepatotoxic agent from S . mansoni [28] . Nevertheless , the Th2-promoting activity of omega-1 cannot be explained by a cytotoxic effect , as the molecule failed to induce a detectable reduction in dendritic cell viability . The exact mechanism ( s ) by which the ribonuclease activity of omega-1 may promote Th2 responses is currently under investigation [31] . The results presented here underscore the paradox of the granulomatous response in hepatic schistosomiasis . Though detrimental to the host in the longer term due to its contribution to disease sequelae such as portal hypertension , it nevertheless protects against more acute hepatocyte injury resulting from toxins released by the schistosome egg . | The flatworm disease , schistosomiasis , is a major public health problem in sub-Saharan Africa , South America and East Asia . A hallmark of infection with Schistosoma mansoni is the immune response to parasite eggs trapped in the liver and other organs . This response involves an infiltration of cells that surround the parasite egg forming a “granuloma . ” In mice deprived of T-cells , this granulomatous response is lacking , and toxic products released by eggs quickly cause liver damage and death . Thus the granulomata protect the host from toxic egg products . Only one hepatotoxic molecule , omega-1 , has been described to date . We set out to identify other S . mansoni egg hepatotoxins using liver cells grown in culture . We first showed that live eggs , their secretions , and pure omega-1 are toxic . Using a physical separation technique to prepare fractions from whole egg secretions , we identified the presence of IPSE/alpha-1 , a protein that is known to strongly influence the immune system . We showed that IPSE/alpha-1 is also hepatotoxic , and that toxicity of both omega-1 and IPSE/alpha-1 can be prevented by first mixing the proteins with specific neutralizing antibodies . Both proteins constitute the majority of hepatotoxicity released by eggs . | [
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Two recently published papers describe nuclear DNA sequences that were obtained from the same Neanderthal fossil . Our reanalyses of the data from these studies show that they are not consistent with each other and point to serious problems with the data quality in one of the studies , possibly due to modern human DNA contaminants and/or a high rate of sequencing errors .
The simultaneous publication of two studies with Neanderthal nuclear DNA sequences [1 , 2] was a technological breakthrough that held promise for answering a longstanding question in human evolution: Did “archaic” groups of humans , such as Neanderthals , make any substantial contribution to the extant human gene pool ? The conclusions of the two studies , however , were puzzling and possibly contradictory . Noonan and colleagues [1] estimated an older divergence time ( i . e . , time to the most recent common ancestor ) between human and Neanderthal sequences ( ∼706 , 000 y ago ) , and a 0% contribution of Neanderthal DNA ( 95% confidence interval [CI]: 0%–20 % ) to the modern European gene pool . In contrast , the Green et al . [2] study found a much more recent divergence time and made two striking observations that were highly suggestive of a substantial amount of admixture between Neanderthals and modern humans . Specifically , Green et al . [2] reported a human–Neanderthal DNA sequence divergence time of ∼516 , 000 y ( 95% CI: 465–569 thousand y ) and a human–human DNA sequence divergence time of ∼459 , 000 y ( 95% CI: 419–498 thousand y ) . These two CIs overlap substantially , suggesting that the Neanderthal–human divergence might be within the realm of modern human genetic variation . Indeed , the level of divergence between the Neanderthal sequence ( which is primarily noncoding ) and the human reference sequence is roughly the same as the average sequence divergence between modern human ethnic groups in noncoding regions of the genome ( [3]; J . D . W . and Michael F . Hammer , unpublished results ) . The second surprising observation from the Green et al . [2] study was that the Neanderthal sequence carries the derived allele for roughly 30% of human SNPs in the public domain [4 , 5] . For HapMap SNPs [4] alone , the Neanderthal sequence has the derived allele 33% of the time ( 95% CI: 29%–36 % ) while the human reference sequence has the derived allele 38% of the time ( 95% CI: 34%–42 % ) . Once again , the overlapping CIs suggest either that humans and Neanderthals diverged very recently—much more recently than is consistent with the fossil record—or that there was a substantial amount of admixture between the two groups . In contrast to the Green et al . study , the comparable percentage from the Noonan et al . [1] data is only 3% ( 95% CI: 0%–9% ) . Noonan et al . [1] and Green et al . [2] used different techniques for sequencing Neanderthal nuclear DNA and estimating parameter values . However , since both studies used genetic material from the same sample , they should come to similar conclusions , even if the particular regions sequenced do not overlap ( taking into account the inherent uncertainty in parameter estimation ) . To examine the potential discrepancies more carefully , we reanalyzed both the Noonan et al . and Green et al . datasets using a uniform set of methods . We estimated the average human–Neanderthal DNA sequence divergence time , the modern European–Neanderthal population split time , and the Neanderthal contribution to modern European ancestry using methodology very similar to what was used by Noonan et al . [1] . This method is based on a simple population model of isolation followed by instantaneous admixture ( see Materials and Methods ) , and utilizes information from each base pair about whether the human reference sequence and/or the Neanderthal sequence have the derived ( or ancestral ) allele to estimate parameters . See Noonan et al . [1] for further details .
As in both Noonan et al . [1] and Green et al . [2] , we only use human-specific mutations to calculate sequence divergence times . Neanderthal-specific mutations are excluded because the vast majority of these ( ∼90% ) are thought to be caused by post-mortem DNA damage [1 , 2] . We estimate sequence divergence times of 706 thousand y ago [Kya] ( 95% CI: 466–1 , 028 Kya ) for the Noonan et al . [1] data and 560 , 000 Kya ( 95% CI: 509–615 Kya ) for the Green et al . [2] data . For comparison , the estimates from the original studies were 706 Kya ( 95% CI: 468–1 , 015 Kya ) and 516 Kya ( 95% CI: 465–569 Kya ) , respectively . The slight discrepancies between our estimates and the Green et al . [2] and Noonan et al . [1] ones arise due to minor methodological differences in sequence divergence time estimates between the two original studies and to slight differences in the way that CIs were calculated ( see Materials and Methods ) . Our estimates of population split times differ markedly between the two datasets . We estimate a modern European-Neanderthal population split time of 35 Kya ( 95% CI: 33–51 Kya ) for the Green et al . data and a split time of 325 Kya ( 95% CI: 135–557 Kya ) for the Noonan et al . data . Figure 1B shows that these two estimates are clearly not consistent with each other . The original Noonan et al . study used slightly different methodology to estimate an even older split time ( 440 Kya; 95% CI: 170–620 Kya ) , while the Green et al . study did not estimate population split times from their data . Note that for the model used here the population split time is always much more recent than the sequence divergence time ( see Materials and Methods ) . The estimates of the Neanderthal contribution to modern European ancestry are also dramatically discordant between the two datasets . When we fix the modern European–Neanderthal split time at 325 Kya ( as estimated from the Noonan et al . data ) , the Neanderthal admixture estimates are 94% ( 95% CI: 81%–100 % ) for the Green et al . data and 0% ( 95% CI: 0%–39 % ) for the Noonan et al . data . These admixture estimates are dependent on the assumed modern European–Neanderthal population split time . Only if we arbitrarily fix a very recent split time of 60 Kya or less are the admixture estimates from the two datasets compatible with each other ( unpublished data ) . However , this very recent split time is not consistent with the estimated modern European–Neanderthal population split time from the Noonan et al . data [1] ( Figure 1B ) .
Given the large discrepancies in the parameter estimates from the two studies , it is clear that the conclusions reached by at least one of the studies are incorrect . We examine the two possibilities in greater detail below . If the estimates from the much larger Green et al . [2] dataset are accurate , then the discrepancies in Figures 1B and 1C either arose by chance ( i . e . , due to the uncertainty in the estimates from the much smaller Noonan et al . dataset ) or due to some unknown bias or problem with the Noonan et al data . Under this scenario , either the modern European–Neanderthal split time is very recent ( i . e . , ≤60 Kya ) or the Neanderthal admixture proportion is extremely high ( >>50% ) . Paleoanthropological evidence suggests that Neanderthals formed a distinct group of fossils at least 250 Kya [6 , 7] , so the more recent modern European–Neanderthal split time is highly unlikely . In addition , most of the available evidence from paleoanthropology suggests that the Neanderthal contribution to the modern gene pool is limited [7] , while previous Neanderthal mtDNA studies concluded that the Neanderthal contribution could be no more than 25% [8 , 9] . Furthermore , preliminary analyses of additional nuclear Neanderthal sequences suggest a much older human–Neanderthal sequence divergence time than was found by Green et al . [10] . So , based on studies from the same [9 , 10] and results from other laboratories [6–8] , it seems extremely improbable that the Green et al . estimates are accurate . In contrast , if the Noonan et al . [1] estimates were correct , then no additional assumptions would be needed to understand the Neanderthal nuclear DNA sequence data in the context of previous human evolutionary studies . This leads to consideration of three possible issues that may have compromised data quality in the Green et al . [2] study: contamination with modern human DNA , widespread difficulties in aligning Neanderthal DNA fragments , and abnormally high DNA sequencing error rates . Although Green et al . [2] found little evidence of modern human mtDNA contamination , it is not clear whether this observation generalizes to the autosomal data under study . To examine this in greater detail , we divided the Green et al . sequence data into three groups: short ( ≥30 bp and ≤50 bp ) fragments , medium-sized ( >50 bp and ≤100 bp ) fragments , and large ( >100 bp ) fragments ( see Materials and Methods ) . We then estimated the human–Neanderthal sequence divergence time for each of these groups . The likelihood of the data as a function of the divergence time is shown in Figure 2 . While the short fragments have an estimated divergence time similar to what was found in the Noonan et al . [1] study , the large fragments are much more similar on average to modern human DNA . In fact , the large fragments have an estimated human–Neanderthal sequence divergence time that is less than the estimated divergence time between two Hausa ( West African ) sequences ( see Materials and Methods ) . If true , this would indicate greater similarity between human and Neanderthal than between two extant members of the Hausa population . This pattern thus raises the concern that some of the longer sequence fragments are actually modern human contaminants . Modern human contamination would be expected to be size biased , since actual Neanderthal DNA would tend to be degraded into short fragments [1 , 11] . We note that the observation of a length dependence of the results makes alignment issues alone [10] unlikely to be a sufficient explanation , since we would expect that longer fragments would be easier to align and thus the data from longer fragments should be more accurate . We further note that we did not find a similar signal of potential contamination in the Noonan et al . data ( unpublished data ) . We also tabulated the percentage of HapMap SNPs for which the Neanderthal sequence contains the derived allele for each of the three groups of Green et al . data ( see Table 1 ) , and refer to this percentage as Nd . For comparison , we also estimated ( from simulations ) the expected value of Nd as a function of the European–Neanderthal population split time and the Neanderthal admixture proportion ( see Figure 3 ) . The expected value of Nd increases as the European–Neanderthal population split time decreases and/or the Neanderthal admixture proportion increases , though for reasonable parameter values ( i . e . , a population split time ≥150 Kya and a Neanderthal admixture proportion <25% ) Nd is always <25% . In contrast , Nd = 32 . 9 for the Green et al . data , which is inconsistent with the true value of Nd being ≤25% ( p < 10−4 ) . Moreover , Nd shows a clear trend of higher values for longer fragments , consistent with the hypothesis of widespread contamination with larger-sized modern human DNA fragments . The expected value of Nd for modern human contaminants is 37 . 0 , so the Green et al . data look in some ways more like modern human DNA than they do like Neanderthal DNA . If we use Nd to estimate very roughly the proportion of the Green et al . [2] data that are actually modern human contaminants , this leads to a contamination rate estimate of 73% ( 95% CI: 51%–97%; see Materials and Methods ) . Alternatively , a likelihood-based estimate of the proportion of modern human contaminants yields an estimate of 78% ( 95% CI: 70%–88%; see Materials and Methods ) . These estimates are very approximate , and given the uncertainty in the actual Nd values for Neanderthal and modern human DNA , the data are also consistent with lower ( but still substantial ) levels of contamination . On the other hand , if contamination were prevalent in the Green et al . [2] data , then due to the large number of apparent mutations that appear in Neanderthal DNA due to post-mortem DNA damage [1 , 2] the Neanderthal-specific sequence divergence should be smaller for the Green et al . data than it is for the Noonan et al . data . Instead , we find that the Neanderthal-specific divergences are almost the same for the two studies . It is not clear how to interpret this observation . It is clear that the two studies are inconsistent with each other , and given the weight of evidence from many previous studies , the more parsimonious explanation is still that something is wrong with the Green et al . [2] data . Although we have highlighted strong circumstantial evidence of modern human DNA contamination in the Green et al . data , there are likely other important problems or biases affecting data quality in one or both studies . One possibility is that due to subtle differences in laboratory protocols , there is a higher sequencing error rate in the Green et al . data than in the Noonan et al . data . ( Sequencing errors would look the same as Neanderthal-specific mutations in our analyses . ) There is some indirect evidence of this—post-mortem DNA damage often causes the deamination of cytosine to uracil [12] , resulting in apparent C→T or G→A mutations . These particular mutations make up a significantly larger fraction of the Neanderthal-specific mutations from the Noonan et al . data than they do for the Green et al . data ( χ2 , 1 degree of freedom; X2 = 21 . 2 , p << 10−4 ) , suggesting that some other process ( besides post-mortem damage to Neanderthal DNA ) is leading to the “Neanderthal-specific mutations” in the Green et al . data . In conclusion , the sequencing of Neanderthal nuclear DNA is truly a remarkable technical achievement . However , because contamination with modern human DNA and sequencing error rates are continuing concerns , it will be important to carefully evaluate published and future data before arriving at any firm conclusions about human evolution .
Neanderthal sequence data was obtained from the supplementary online material in Noonan et al . [1] and Green et al . [2] . The mapping coordinates of the Green et al . [2] alignments of the Neanderthal , reference human , and chimpanzee sequences are based on National Center for Biotechnology Information ( NCBI ) build 36 of the human genome . We removed 5 bp of sequence from the ends of each read , and analyzed fragments that contained at least 30 bp of autosomal sequence . When multiple sequence fragments overlapped with each other , we analyzed the largest one and threw out the other ( s ) . For the Noonan et al . [1] study , all of the relevant information was directly available from their Supplementary Methods . For the Green et al . [2] study , we did not have access to their post-filtered dataset for comparison . Using the above criteria , we started with 891 , 406 total bases from 13 , 955 unique fragments . Of these , we removed 115 , 963 bases with missing data or a poor-quality score , 24 , 698 bases found on the sex chromosomes , and 51 bases where the human , Neanderthal , and chimpanzee sequences all had different bases , leaving 750 , 694 bp for our analyses . Green et al . [2] do not specify the total number of base pairs used in their analyses , but they do mention that 736 , 941 base pairs are identical across human , Neanderthal , and chimpanzee . Our filtering criteria yielded 735 , 878 such base pairs , within 0 . 2% of what was analyzed in Green et al . [2] . For all biallelic HapMap SNPs ( available in March 2007 ) that overlap with the Neanderthal autosomal sequences , we used a human–chimpanzee alignment to determine which allele was derived or ancestral . This alignment is available at http://hgdownload . cse . ucsc . edu/goldenPath/hg18/vsPanTro1/ , and uses NCBI build 36 of the human genome and the PanTro1 build of the chimpanzee genome . HapMap SNPs without an orthologous chimpanzee reference allele were excluded , as were SNPs that did not segregate in the Utah residents with ancestry from northern and western Europe ( CEU ) population . CIs for the proportion of HapMap SNPs where the Neanderthal ( or human reference ) sequence had the derived allele were estimated from 5 , 000 bootstrap simulations . When comparing Neanderthal–modern human divergence with human–human divergence , we ignored Neanderthal-specific mutations , since most of them are due to post-mortem DNA damage [1 , 2] . Using the divergence on the human-specific branch to estimate sequence divergence times is expected to be unbiased . For comparison , π for two Hausa individuals is roughly 0 . 11% [3] , and is roughly 0 . 12% for two individuals from other African populations ( JDW , unpublished results ) . These π values are for noncoding DNA and should be compared with twice the divergences displayed in Table 1 . Over 90% of the Neanderthal base pairs from the original studies map to noncoding ( putatively nonfunctional ) areas of the human genome . We follow the model outlined in Noonan et al . [1] for the CEU population . A schematic of the model is shown in Figure 4 ( see also Figures S3 and S6 from Noonan et al . ) . The parameters estimated are TMRCA , the average human–Neanderthal DNA sequence divergence time; Ts , the modern European–Neanderthal population split time; and p , the Neanderthal contribution to modern European ancestry . From Figure 4 , it is clear that TMRCA is always substantially larger than Ts . Estimates of Ts assume that p = 0 , while estimates of p assume that Ts = 325 Kya . See Noonan et al . [1] for further details . Our analyses are similar to those described in Noonan et al . [1] , but with several minor differences . For HapMap SNPs we categorize them into ten different frequency bins when calculating P ( Ascert|freq ) and when tabulating ascertained SNPs by frequency ( see page 10 of Supplementary Online Material from [1] ) . These bins consist of frequencies ( 0 . 0 , 0 . 1] , ( 0 . 1 , 0 . 2] , . . . ( 0 . 9 , 1 . 0 ) . Summaries of the actual data analyzed are provided in Tables 2 and 3 . The parameter values and demographic models used were the same as in [1] , except we took a mutation rate of 2 . 5 × 10−8/bp . For estimating split times , we ran simulations for split times in 5 , 000-y increments ( for times less than or equal to 50 Kya ) or 25 , 000-y increments ( for split times greater than 50 Kya ) . For estimating Neanderthal admixture , we fixed the population divergence time at 325 Kya and ran simulations in increments of 0 . 02 ( i . e . , 0 , 0 . 02 , 0 . 04 , etc . ) for the admixture proportion . Results are based on 50 million replicates for each set of parameter values . Approximate 95% CIs were estimated assuming the composite likelihood was a true likelihood ( as well as the standard asymptotic assumptions for maximum likelihood ) . Since each fragment is so small , assuming each site is independent is a reasonable approximation . We assume that the true Neanderthal admixture rate is 0 ( as estimated from the Noonan et al . data ) , that the true modern European–Neanderthal population split time is 325 Kya ( as estimated from the Noonan et al . data ) , and that the Green et al . data consist of a simple mixture of true Neanderthal DNA ( with Nd = 21 . 8 , cf . Table 1 , Green small fragments ) and modern human DNA ( with Nd = 37 . 0 ) . We estimate contamination rates in two ways . First , we find the proportion of Neanderthal versus modern human DNA that gives an expected Nd value equal to 32 . 9 ( the actual value in the Green et al . data ) . Second , we find what proportion of Neanderthal versus modern human DNA maximizes the ( composite ) likelihood described above . We note that the likelihood of the data for a model with 78% contamination is modestly higher ( 1 . 06 log-likelihood units ) than the likelihood of the data for a model with no contamination and 94% admixture ( maximum-likelihood in Figure 1B ) . | One of the enduring questions in human evolution is the relationship of fossil groups , such as Neanderthals , with people alive today . Were Neanderthals direct ancestors of contemporary humans or an evolutionary side branch that eventually died out ? Two recent papers describing the sequencing of Neanderthal nuclear DNA from fossil bone held promise for finally answering this question . However , the two studies came to very different conclusions regarding the ancestral role of Neanderthals . In this paper , we reanalyzed the data from the two original studies . We found that the two studies are inconsistent with each other , which implies that the data from at least one of the studies is probably incorrect . The likely culprit is contamination with modern human DNA , which we believe compromised the findings of one of the original Neanderthal DNA studies . | [
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] | 2007 | Inconsistencies in Neanderthal Genomic DNA Sequences |
Urinary tract infections ( UTI ) are extremely common and can be highly recurrent , with 1–2% of women suffering from six or more recurrent episodes per year . The high incidence of recurrent UTI , including recurrent infections caused by the same bacterial strain that caused the first infection , suggests that at least some women do not mount a protective adaptive immune response to UTI . Here we observed in a mouse model of cystitis ( bladder infection ) that infection with two different clinical uropathogenic Escherichia coli ( UPEC ) isolates , UTI89 or CFT073 , resulted in different kinetics of bacterial clearance and different susceptibility to same-strain recurrent infection . UTI89 and CFT073 both caused infections that persisted for at least two weeks in similar proportions of mice , but whereas UTI89 infections could persist indefinitely , CFT073 infections began to clear two weeks after inoculation and were uniformly cleared within eight weeks . Mice with a history of CFT073 cystitis lasting four weeks were protected against recurrent CFT073 infection after antibiotic therapy , but were not protected against challenge with UTI89 . In contrast , mice with a history of UTI89 cystitis lasting four weeks were highly susceptible to challenge infection with either strain after antibiotic treatment . We found that depletion of CD4+ and CD8+ T cell subsets impaired the ability of the host to clear CFT073 infections and rendered mice with a history of CFT073 cystitis lasting four weeks susceptible to recurrent CFT073 cystitis upon challenge . Our findings demonstrate the complex interplay between the broad genetic diversity of UPEC and the host innate and adaptive immune responses during UTI . A better understanding of these host-pathogen interactions is urgently needed for effective drug and vaccine development in the era of increasing antibiotic resistance .
Urinary tract infections ( UTI ) are among the most common infections: more than half of women , as well as some men , will have at least one UTI in their lifetime [1] , mostly caused by uropathogenic E . coli ( UPEC ) [2 , 3] . In humans , the adaptive immune response to UTI , and particularly to cystitis ( bladder infection ) , is not well understood . Placebo studies show that in some women an acute UTI spontaneously resolves , whereas other women develop symptomatic or asymptomatic colonization lasting weeks [4–8] . UTI can also be highly recurrent: after receiving appropriate antibiotic therapy , 20–30% of women with an acute UTI will have a recurrent episode ( rUTI ) within six months [1] , despite the fact that about half of rUTI episodes are caused by the same UPEC strain that caused the initial infection [9] . These findings suggest that individuals with UTI frequently fail to mount an adaptive immune response that protects against subsequent infection , even with the same strain . Although bacterial factors and host responses that impact innate immune responses to UTI have been characterized [10 , 11] , less is known about host and/or microbial factors that impact the host’s ability to mount a protective adaptive immune response . Studies of experimental infection in different strains of inbred mice have illustrated the complexity of the role of adaptive immunity in determining UTI outcomes . In C57BL/6 mice , UPEC infection is inherently self-limiting , independent of infectious dose , and adaptive immune responses can partially protect from recurrent infection [12 , 13] . For example , the adoptive transfer of either cellular or humoral immunity after experimental UPEC bladder infection partially protects naive C57BL/6 mice against infection with an isogenic UPEC strain [12] . Other strains of mice have been shown to be susceptible to same-strain recurrences caused by the well-studied clinical cystitis isolate UTI89 [14 , 15] . For example , C3H/HeN mice can develop chronic and severe same-strain recurrent infections with UTI89 in an infectious dose-dependent manner [14] , indicating that adaptive immunity towards UTI89 is insufficient to prevent these outcomes . However , vaccination of C3H mice with bacterial antigens can strongly protect against both acute and recurrent UTI [15 , 16] , demonstrating that a protective adaptive response to UTI can be experimentally induced in these mice . Our previous studies in C3H/HeN mice ( summarized in Fig 1 ) have revealed that within 24 hours post infection ( hpi ) with UTI89 , a cyclooxygenase-2-dependent acute host-pathogen checkpoint determines the outcome of the infection [14 , 17] . The hallmarks of “checkpoint activation” are high urine bacterial titers ( bacteriuria ) , pyuria ( neutrophil accumulation in urine ) , elevated serum levels of pro-inflammatory cytokines , and severe bladder inflammation with bladder epithelial wounding . Elements of this checkpoint are predictive of recurrent UTI in women , demonstrating clinical relevance of the model [17] . Mice that do not activate the checkpoint spontaneously resolve the infection within four weeks , and have bladder titers <104 colony-forming units ( CFU ) at time of sacrifice [14] . In contrast , mice that activate the checkpoint develop chronic cystitis , which we define as persistent , high-titer bacteriuria ( >104 CFU/ml urine for four weeks ) coupled with high bladder bacterial burden ( >104 CFU/bladder ) and bladder inflammation at sacrifice four weeks post infection ( wpi ) . Strikingly , we have found that those mice that develop chronic cystitis lasting at least four weeks remain infected apparently indefinitely ( six months or longer ) [14] , indicating that , once established , chronic UTI89 bladder infections cannot be cleared by adaptive immunity . Antibiotics can clear the chronic infection , but we found that a “molecular imprint” remains on the bladder mucosa in the form of significant bladder mucosal remodeling [15 , 17] that modulates host response to challenge infection . Thus , mice with a history of chronic UTI89 cystitis are highly susceptible ( or “sensitized” ) to checkpoint activation and severe recurrent chronic cystitis upon challenge infection with either UTI89 or other clinical uropathogenic strains [15 , 17] . In this study , we used these well-characterized mouse models of chronic and recurrent UTI in C3H/HeN mice ( Fig 1 ) to probe the roles of bacterial diversity and adaptive immunity in UTI by comparing and contrasting the disease outcomes of two clinical UPEC isolates: the cystitis isolate UTI89 [18] and the pyelonephritis/urosepsis isolate CFT073 [19] . These isolates belong to the B2 clade of E . coli , the clade in which uropathogenic strains are most commonly found , but differ in their serotypes and their carriage of various mobile genetic elements , such as plasmids and pathogenicity islands that are known to harbor virulence genes [20–22] . We found that adaptive immunity was able to restrict chronic and same-strain recurrent cystitis caused by CFT073 , but this restriction did not translate to cross-protection against UTI89 challenge infection . A better understanding of the complexities of the adaptive immune response to UTI could ultimately allow the development of new therapeutics , and inform our understanding of adaptive immunity in the context of chronic bacterial infections in general .
First we assessed differences in host outcomes 28 days after infection with 108 CFU of either UTI89 or CFT073 ( Fig 1C and 1D ) . We found that CFT073 caused chronic cystitis lasting four weeks , but at a significantly lower rate than UTI89 did ( 30% incidence for CFT073 vs . approximately 50% incidence with UTI89; Fig 2A ) , consistent with a previous study [21] . Whether infected with UTI89 or CFT073 , mice with chronic cystitis at 28 dpi had significantly higher bladder bacterial burdens ( Fig 2B ) and bladder edema ( Fig 2C ) than mice that spontaneously resolved their infection , in accordance with our definition of chronic cystitis . Kidney titers were also higher in mice with chronic cystitis than those that resolved their infection ( Fig 2D ) . In a blinded analysis of scanning electron micrographs of chronically infected bladders at 28 dpi , there were no obvious differences between chronic UTI89 and chronic CFT073 cystitis , with both infections causing epithelial disruption , immune cell infiltration , and abundant extracellular rod-shaped bacteria ( Fig 2E ) . However , compared to chronic UTI89 cystitis , chronic CFT073 cystitis at 28 dpi trended toward reduced: bladder titers ( Fig 2B ) , bladder edema ( Fig 2C ) , and kidney titers ( Fig 2D ) , suggesting that chronic CFT073 cystitis at this time point may be less robust than chronic UTI89 cystitis . During chronic cystitis , CFT073 urine bacterial titers dropped significantly between 1 and 28 days post infection ( dpi ) , though they remained above our predefined cutoff of 104 CFU ( Fig 2F ) . As well , in the course of this study we repeatedly observed that a subset of mice resolved their CFT073 infection between 21 and 28 days ( i . e . persistent bacteriuria >104 CFU until 21 dpi , but urine and/or bladder titers <104 at 28 dpi ) , which we dubbed “late resolution . ” Late resolution was not observed with UTI89 infection . Extending our studies past 28 days of infection revealed that every CFT073-infected mouse cleared bacteriuria within eight weeks ( Fig 2G; note that four of 18 mice had “late resolution” between weeks three and four ) . In contrast , urine titers remained persistently high for 14 weeks post infection in the UTI89-inoculated mice that had developed chronic cystitis ( Fig 2G ) . Analysis of a late-resolving CFT073-inoculated bladder by SEM revealed a calm surface at 28 dpi , with an intact epithelium having no immune cells or visible bacteria ( Fig 2H ) . A critical distinction between UTI89 and CFT073 is each strain’s allele of the mannose-binding fimH adhesin , which tips the type 1 pilus and mediates bladder colonization [23] . UTI89 has the FimH variant A62/V163 , whereas CFT073 has the FimH variant S62/A163 , which has a lower in vitro affinity for mannose and reduced virulence in the mouse bladder [21] . We found that a strain of CFT073 that expresses UTI89’s higher mannose affinity fimH allele , CFT073ΔfimH::FimHUTI89 , had a significantly greater incidence of chronic cystitis at 28 dpi than wild-type CFT073 did ( Fig 2A ) , in line with previous findings [21] . Strikingly , this strain was able to persist longer in the bladder than wild-type CFT073 , but nevertheless all but one mouse eventually cleared the infection over the course of 14 weeks ( Fig 2G ) . These observations indicate that while the high mannose affinity variant FimH enhances and prolongs the colonization of CFT073ΔfimH::FimHUTI89 in the mouse urinary tract relative to the wild type strain , other factors unique to CFT073 account for its propensity for being cleared from the host . As well , in the course of this work we became aware that the CFT073 strain used has a frameshift mutation in rpoS [24] . However , a CFT073 strain with a wildtype rpoS allele still had “late resolution” between days 21 and 28 , and was cleared from all mice within eight weeks ( S1 Fig ) , indicating that the rpoS frameshift mutation does not explain CFT073 clearance relative to UTI89 . We have previously shown that chronic UTI89 cystitis in C3H/HeN mice is predicted by an immune checkpoint at 24 hours post infection ( Fig 1B ) , and that chronic bladder inflammation lasting four weeks results in a bladder imprint that persists after antibiotic therapy ( Fig 1E ) . At 24 hpi , mice infected with CFT073 or UTI89 did not significantly differ in overall urine bacterial titers , pyuria , or pro-inflammatory serum cytokines . However , when mice were grouped by disease outcome , regardless of whether infected with UTI89 or CFT073 those that ultimately would develop chronic cystitis lasting four weeks had activation of the immune checkpoint , indicated by: higher urine bacterial burdens ( Fig 3A ) , though here just a trend with UTI89; greater pyuria ( Fig 3B ) ; and increased serum levels of G-CSF and the IL-8 analogue KC , relative to the mice that would resolve their infection ( Fig 3C and 3D ) . As well , there were trends toward higher serum IL-5 and IL-6 levels in mice that would develop chronic CFT073 cystitis ( Fig 3E and 3F ) , but , unlike with UTI89-infected mice , the differences were not statistically significant . Thus , while we saw some minor differences between UPEC strains , checkpoint activation predicts the development of chronic cystitis lasting four weeks for both UTI89 and CFT073 in a similar manner . Scanning electron microscopy ( SEM ) analysis of bladders 28 days after antibiotic therapy showed that mice with a history of chronic CFT073 infection , referred to herein as CFT073HC mice , had significantly smaller urothelial cells ( average cell surface area approximately one eighth the size of Adult Naive cells ) ( Fig 4A and 4B ) , similar to the previously observed reduced size of urothelial cells from mice with a history of chronic UTI89 ( UTI89HC ) infection [15] . This change in bladder cell morphology is long-lasting in UTI89HC mice: SEM analysis performed six months after antibiotics showed that UTI89HC urothelial cells were still about one fourth the size of age-matched naive controls ( S2A and S2B Fig ) . Immunofluorescence microscopy of bladder sections revealed that while CFT073HC mice expressed the terminal differentiation marker uroplakin IIIa ( Fig 4C ) , expression of the terminal differentiation marker keratin 20 was weak and patchy , in line with previous observations for UTI89HC mice [15] ( Fig 4D ) . Weak and patchy keratin 20 staining was also found six months after initial infection in UTI89HC mice ( S2C Fig ) , suggesting that these infection-induced changes to the host may be permanent . Finally , histological examination of bladder sections from CFT073HC mice 28 days after antibiotic therapy revealed the presence of lymphoid follicles ( S3 Fig ) , which were previously found to develop between two and four weeks post infection in UTI89HC mice [14] . Thus , chronic CFT073 infection results in a constellation of bladder remodeling phenotypes similar to those seen after chronic UTI89 infection , suggesting that bladder remodeling is a broad consequence of chronic E . coli infection . C3H/HeN mice become resistant to UPEC UTI with age [14 , 15 , 25] , but UTI89HC mice have enhanced susceptibility to UTI relative to age-matched ( so-called “Adult” ) Naive mice , a phenomenon we have termed “sensitization” to rUTI [14] ( Fig 1G ) . To assess whether CFT073HC mice were also sensitized to rUTI , mice with an initial chronic or mock infection were given antibiotics at four wpi and then challenged four weeks later with 107 CFU of an isogenic strain with a different antibiotic resistance marker . Acute and chronic rUTI outcomes were then assessed . Previously , we found that UTI89HC mice displayed a colonization resistance phenotype within the first 12 hours after challenge infection: UTI89 was less able to invade and replicate within remodeled bladder epithelial cells relative to naive ones , possibly due to their smaller cell size , resulting in significantly lower CFUs in UTI89HC mice relative to age-matched Adult Naive mice [15] . We found that CFT073HC mice also displayed a colonization resistance phenotype , with significantly reduced CFUs six hours after CFT073 infection in both UTI89HC and CFT073HC mice compared to age-matched Adult Naive mice ( Fig 5A ) . Kidney titers were not statistically significantly different at this time point ( Fig 5A ) . Despite early colonization resistance , UTI89 is nevertheless able to cause severe UTI at 24 hpi in about half of UTI89HC mice , as indicated by high urine titers ( median titer ~106 CFU/ml ) ( Fig 5B ) that precede the development of recurrent chronic cystitis over the 28 day challenge ( Fig 5C ) . In contrast , CFT073 is not able to cause severe UTI in CFT073HC mice , with median urine titer ~104 CFU/ml urine at 24 hpi ( Fig 5B ) and zero incidence of chronic cystitis during the 28 day challenge ( Fig 5C ) . Notably , CFT073HC mice were only protected from same-strain challenge: UTI89 titers were high in the urine at 24 hpi ( median titer ~106 CFU/ml ) ( Fig 5B ) and caused recurrent chronic cystitis in 50% of CFT073HC mice ( Fig 5C and S4 Fig ) . Likewise , UTI89HC mice were susceptible to recurrent CFT073 cystitis ( Fig 5B and 5C and S4 Fig ) , in line with previous observations [15] , and remained susceptible to CFT073 for at least six months after antibiotic therapy ( Fig 5D ) . Thus , whereas a history of UTI89 chronic cystitis predisposes to rUTI caused by either UTI89 or CFT073 , a history of chronic CFT073 cystitis predisposes to rUTI caused by UTI89 , but not caused by CFT073 . The observations that chronic CFT073 infection resolves over time ( Fig 2 ) and confers protection against CFT073 challenge ( Fig 5 ) suggest that chronic CFT073 cystitis may elicit an adaptive immune response that clears the infection and protects against challenge . Thus we questioned whether components of the adaptive immune system played a role in this model . We assessed bacterial-specific IgG levels in the serum and found that both CFT073 and UTI89 elicited antibody responses , though anti-CFT073 IgG levels were somewhat higher than anti-UTI89 IgG levels ( S5 Fig ) . We also tested whether T cell depletion would affect chronic and/or recurrent cystitis outcomes . For other chronic Gram-negative bacterial infections , depletion of both CD4+ and CD8+ T cells resulted in significantly worse infection phenotypes than single depletions did [26–29] . Hence , we tested the effects of both single subset depletions and combined depletions , compared to an equivalent dose of isotype ( 0 . 5 or 1 mg , respectively ) , during an initial UPEC infection . Depletion of lymphoid subsets and effects on myeloid subsets was determined four weeks after the initial infection , and at the time of the challenge infection , by flow cytometry of spleen single cell suspensions ( S6 Fig ) . Depletion of CD4+ and CD8+ T cell subsets did not affect the incidence of chronic cystitis caused by UTI89 ( Fig 6A ) . For CFT073 infection , the incidence of chronic cystitis at 28 dpi was higher in the single depletion groups ( 67% for CD4-depleted and 62% for CD8-depleted , compared to 46% in isotype-treated mice , S7A and S7B Fig ) , but the difference was not statistically significant , and similar proportions of mice cleared the infection after seven days ( S7C Fig ) . Furthermore , mice depleted of single T cell subsets were still protected from same-strain rUTI ( S7D and S7E Fig ) . In the combined CD4+ and CD8+ T cell depleted group , the rate of chronic CFT073 cystitis was 61% , vs . 40% in the isotype-treated group ( Fig 6A ) , and CFT073 urine titers at 28 dpi were significantly elevated in the combined T cell-depleted mice relative to isotype-treated controls ( Fig 6B ) . Notably , significantly fewer mice depleted of both CD4+ and CD8+ T cell subsets resolved the CFT073 infection after 7 dpi , relative to isotype-treated mice ( Fig 6C ) . Accordingly , combined CD4+ and CD8+ T cell depletion also prevented the so-called “late resolver” phenotype ( wherein some mice resolve their CFT073 infection between days 21 and 28 post infection; see Fig 2G ) . In the T cell-depleted group , n = 12 mice resolved the CFT073 infection: nine resolved within the first week and none between days 21 and 28 . In contrast , of the n = 21 isotype-treated mice that resolved the CFT073 infection , ten resolved between days 21 and 28 ( Fig 6D and 6E ) . Based on the above results , we tested whether the abrogation of CD4+ and CD8+ T cell responses during the initial CFT073 infection would render mice susceptible to recurrent CFT073 cystitis . Four weeks after the initial infection and concomitant T cell depletion , mice were given antibiotics and then challenged four weeks later with the same strain as their initial infection , without additional T cell depletions . UTI89HC mice were susceptible to developing recurrent UTI89 cystitis , whether they were initially treated with anti-CD4/8 or isotype ( Fig 6F ) . In contrast , T cell depletion had a striking effect on CFT073 challenge of CFT073HC mice . No mice ( out of n = 8 ) in the isotype-treated group developed recurrent chronic CFT073 cystitis , consistent with our observations in untreated mice ( see Fig 5C ) ; one mouse had persistent bacteriuria but had a bladder titer <104 CFU . However , four of 11 mice that received anti-CD4/8 during their initial chronic infection developed recurrent chronic CFT073 cystitis lasting four weeks ( Fig 6F–6H ) and one mouse was a “late resolver , ” similar to what is seen during CFT073 infection of juvenile naive mice ( see Figs 2G and 6D ) . Bladder and kidney bacterial burdens did not differ between isotype vs . anti-CD4/8 treatments in UTI89HC mice after UTI89 challenge , but in CFT073HC mice challenged with CFT073 , titers at sacrifice were higher in those that received anti-CD4/8 than those that received isotype control ( Fig 6I ) . As well , bladder edema four weeks post challenge with CFT073 was significantly greater in CFT073HC mice that received anti-CD4/8 than in those that received isotype control ( Fig 6J ) . In sum , these experiments show that depletion of both CD4+ and CD8+ T cell subsets during an initial CFT073 infection renders C3H/HeN mice susceptible to chronic and same-strain recurrent cystitis , whereas T cell depletion during a UTI89 infection does not impact chronic or same-strain recurrent cystitis .
Some women who suffer from an acute UTI will become afflicted by highly recurrent UTI , but most women do not experience this fate . It may be that adaptive immunity protects at least some women from rUTI , but relatively little is known about adaptive immune responses to cystitis . We showed that the clinical urosepsis isolate CFT073 can cause severe acute and chronic UTI in C3H/HeN mice . In line with our previous findings for the clinical UTI isolates UTI89 and J96 [14] , chronic CFT073 cystitis developed after the triggering of an acute host-pathogen checkpoint , and chronic infection lasting four weeks resulted in bladder mucosal remodeling . Thus , we suggest that checkpoint activation is broadly predictive of chronic UPEC UTI , and that bladder mucosal remodeling is a general consequence of chronic UPEC infection and/or infection-associated inflammation . Unlike UTI89 , however , CFT073 cystitis did not last indefinitely; all mice cleared CFT073 infection within eight weeks , and after an initial chronic infection lasting four weeks , mice were protected from CFT073 challenge , but susceptible to UTI89 challenge , demonstrating strain-specific protection . We found that the combined depletion of CD4+ and CD8+ T cell subsets during the initial CFT073 infection increased the incidence of chronic cystitis and rendered mice susceptible to recurrent chronic CFT073 cystitis . T cell depletion did not significantly impact chronic or recurrent cystitis caused by UTI89 , in line with a previous study of recurrent UTI in C57BL/6 mice [13] . Thus , this study provides evidence for a role of T cells and adaptive immunity in restricting chronic and same-strain recurrent UTI caused by some , but not all , UPEC strains . Placebo-controlled studies have demonstrated two general outcomes of uncomplicated cystitis: while many women can resolve their infections without the aid of antibiotics , others remain bacteriuric ( with or without symptoms ) for weeks [30] , suggesting an ineffective or absent adaptive response . As well , we note that spontaneous resolution of infection in placebo-treated women has been reported to occur both early in infection ( within seven days ) and later on ( between four to seven weeks ) [6] . Much is known about innate immune responses to UTI [10 , 11] , but fewer studies have focused on the role of adaptive immunity . In our study we recapitulated the two general cystitis outcomes seen in women by using inbred C3H/HeN mice , which can spontaneously resolve bladder infections or develop chronic cystitis lasting two or more weeks . A role for adaptive immunity was elucidated via depletion of T cells , which skewed the balance of infection outcomes in a bacterial strain-specific manner . In T cell-depleted mice , the incidence of chronic CFT073 cystitis was increased due to the elimination of the “late resolvers” ( mice that resolved a chronic infection after 21 days ) , while chronic UTI89 cystitis was not affected because late resolution does not occur after infection with this strain . Our finding of strain-specific adaptive immunity echoes the recent discovery that different clinical isolates of the Gram-positive pathogens Staphylococcus aureus and Streptococcus pyogenes vary substantially in their ability to elicit adaptive immune responses in vitro depending on the content of their accessory ( non-core ) genome [31] . It has previously been shown that adaptive responses can protect against same-strain rUTI in C57BL/6 mice or mutants on the C57BL/6 background [12 , 13 , 32] . The work presented herein elucidates the complexity of this response by showing bacterial strain-dependent variability . Studying multiple UPEC strains is critical because ongoing genome sequencing has revealed remarkable genetic diversity in E . coli , as the pan-genome remains “open” and continues to expand beyond more than 16 , 000 genes , resulting in the extremely variable accessory genome , typically comprised of secretory systems , metabolic pathways , and virulence gene networks [33] . A large-scale genomic study of 43 E . coli isolates from 14 women suffering from recurrent UTI [34] revealed that the isolates were phylogenetically diverse , as they represented five major E . coli clades and only about 60% of each strain’s genome was found to be shared among all of the strains . Upon infection in mice , significant heterogeneity in bladder titers was observed with these genetically diverse strains . Notably , some strains from clades that are not considered traditionally uropathogenic ( such as B1 ) could robustly colonize the mouse bladder , while conversely , some strains from the B2 clade ( typically considered uropathogenic ) were poor colonizers of mice . Further , no single genetic signature predicted a strain’s ability to colonize the mouse bladder . This study indicated that a complex interplay between UPEC genetics and host susceptibility was necessary for UTI to develop , and that there may be multiple pathogenic pathways in UPEC that can lead to infection [34] . Based on our work here , we argue that depending on the host and the infecting strain , adaptive immunity may or may not be capable of restricting chronic and same-strain recurrent UTI . Further , even if an adaptive response restricts same-strain rUTI , it may not broadly protect against challenge with diverse UPEC strains . The present study adds further proof of the complexity of UTI in the form of host adaptive immune responses that vary with the infecting strain . By comparing and contrasting different clinical UPEC isolates , we begin to recapitulate the great genetic and phenotypic diversity of uropathogens–and thus , bacterial antigens–to which women are exposed . During chronic cystitis the host is likely to have significant exposure to the highly immunogenic UPEC outer membrane components that comprise the classical bacterial serotype: the O antigen polysaccharide of LPS; the K capsule; and the H antigen , flagellin . The components of the E . coli serotype , defined by their ability to generate an immune response in a diagnostic setting , are numerous: as of 2003 , 170 O antigens , 103 K antigens , and 56 H antigens had been identified [35] . Host immune responses acting against certain serotype components may present a selective pressure that drives the high diversity in serotype components . Although UTI89 and CFT073 are genetically relatively similar and both belong to the B2 clade [34] , they have different serotypes: UTI89 is O18:K1:H7 while CFT073 is O6:K2:H1 [20] . Interestingly , a previous study found that experimental UTI with an E . coli O6:K13:H1 strain elicited a T cell response in rats [36] . One or more components of the UTI89 serotype may be able to subvert adaptive immunity to facilitate long-term persistence and same-strain recurrence . Depletion of both CD4+ and CD8+ T cell subsets during the initial infection was necessary to prevent “late resolution” of chronic CFT073 cystitis and render the host susceptible to recurrent chronic CFT073 cystitis , suggesting that one subset may compensate for the loss of the other . A similar paradigm was observed for infection with the Gram-negative intracellular pathogen Francisella tularensis , wherein studies using T cell depletions or T cell knockout mice have demonstrated that while mice that have lost either CD4+ or CD8+ T cells are able to clear an initial or challenge infection , the loss of both CD4+ and CD8+ T cell subsets impairs bacterial clearance and protective memory responses [29 , 37 , 38] . CD4+ or CD8+ T cells could control F . tularensis growth in macrophages in vitro in part through the production of IFN-γ [39] , and in vivo both subsets produced IFN-γ [38]; interestingly , IFN-γ knockout mice on the C57BL/6 background were previously shown to be more susceptible to UTI than immunocompetent mice [40] . In our study of chronic and recurrent UTI , the mechanism ( s ) of T cell-mediated protection are as yet unknown . CD4+ T cells could play a variety of protective roles , including: i ) directly stimulating neutrophil recruitment via IL-17 production; ii ) activating macrophages and CD8+ T cells via IFN-γ; iii ) supporting B cell affinity maturation and isotype switching; and/or iv ) themselves becoming memory T cells [41] . CD8+ T cells could be directly cytotoxic against infected urothelial cells , and/or could be responding to cross-presentation of antigens by dendritic cells or other cell types; previously , macrophages were found to sequester UTI89 from dendritic cells , limiting adaptive responses to same-strain recurrent UTI in C57BL/6 mice [13] . We found that splenic monocytes and neutrophils , but not macrophages , were elevated in T cell-depleted mice at 28 dpi , which might suggest an expanded role for monocytes and neutrophils in host response in the absence of T cells . Further studies are needed to determine the mechanisms of T cell-mediated protection against specific UPEC strains , as well as to assess the role of B cells and antibodies in this UTI model . A previous study in C57BL/6 mice found that concurrent kidney and bladder infections led to a strong pathogen-specific serum IgG response , compared to bladder-restricted infections [42] . Here we observed that pathogen-specific serum IgG levels were higher in CFT073HC mice than UTI89HC mice . These data suggest that CFT073 is better able to induce an antibody response , though the exact reason for this difference is unclear at this point . Finally , γδ T cells were previously shown to play a role in protection against UTI in C57BL/6 mice [43]; our depletion antibodies are not expected to target γδ T cells , so their potential role in this model of chronic and recurrent UTI remains unexplored . Although UTI are among the most common bacterial infections , most cases are uncomplicated and readily treated by antibiotics . However , our ability to treat these infections is threatened by the global rise of multidrug-resistant “superbugs”; indeed , recently the first United States case of E . coli resistant to the last line drug colistin was reported in a UTI patient [44] . It has become increasingly clear that rUTI result from complex interactions among host risk factors ( both intrinsic , like genetic polymorphisms affecting UTI susceptibility , and extrinsic , i . e . behavioral risk factors such as sexual activity ) [45] and genetically diverse uropathogens possessing an enormous variety of bacterial virulence factors [34] . Our study emphasizes the additional complexity of host immune responses to specific bacterial strains . A major question going forward will be what bacterial factor ( s ) govern the development or suppression of adaptive immunity in susceptible hosts . Many experimental UTI vaccines–including attenuated or inactivated whole bacteria as well as specific bacterial components–have elicited protection against UTI in animal models [46] . However , translating these preclinical studies to a successful and widely used clinical UTI vaccine has proven difficult . Our study demonstrates that the development of a broad spectrum vaccine will need to take into account the genetically diverse uropathogens that women encounter in the community , and emphasizes the importance of incorporating multiple bacterial strains and the effects of previous exposures in animal models of host-pathogen interactions and outcomes .
All mouse experiments were conducted according to the National Institutes of Health guidelines for the housing and care of laboratory animals . All experiments were performed in accordance with institutional regulations after review and approval by the Institutional Animal Care and Use Committee ( IACUC ) at Washington University School of Medicine in St . Louis , MO ( animal protocol number 20150226 ) . The uropathogenic E . coli isolates used in this study were the human cystitis isolate UTI89 [18] and the antibiotic-resistant derivatives UTI89 attHK022::COMGFP ( kanamycin-resistant ) and UTI89 attl::PSSH10-1 ( spectinomycin-resistant ) [47]; the human urosepsis isolate CFT073 , wild-type and ΔrpoS [19] , and the antibiotic-resistant derivatives CFT073ΔrpoS HK::Cm ( chloramphenicol-resistant ) , CFT073ΔrpoS HK::Kan ( kanamycin-resistant ) , and CFT073ΔrpoS ΔfimH::FimHUTI89 ( kanamycin-resistant ) [21] . Strains were cultured statically in lysogeny broth ( LB ) at 37°C for two to three overnight passages to induce type 1 pilus expression . In experiments where mice had an initial infection and subsequent challenge infection , the challenge strain was marked with different antibiotic resistance from the initial strain . C3H/HeN mice were obtained from Envigo Research Model Services , Inc . ( formerly Harlan ) . All mice were female and 7–8 weeks old ( “Juvenile” ) at the time of the first infection ( or mock-infection in the case of the Adult Naive group ) . Bacterial inocula were prepared as previously described [14] and 107 or 108 CFU of bacteria were inoculated into the bladders of C3H/HeN mice by transurethral catheterization under isoflurane anesthesia as previously described [48] . For infections in Juvenile Naive mice , 108 CFU was always used , since C3H/HeN mice develop chronic cystitis in an infectious dose-dependent manner . For challenge infections , 107 CFU ( of a differently antibiotic-marked strain than the initial infection ) was used , to facilitate comparisons with prior studies [14 , 15 , 17] . To monitor infection outcomes , urine , bladder and kidney bacterial burdens were determined as previously described [14] . Mice were humanely euthanized by isoflurane inhalation followed by cervical dislocation . Chronic cystitis was defined as persistent high-titer bacteriuria ( >104 CFU/ml urine ) at every urine collection time point ( 1 , 3 , 7 , 10 , 14 , 21 , and 28 days post infection , and weekly thereafter if experiments continued ) . A cutoff of 104 CFU/ml persistent bacteriuria is highly specific and sensitive for the detection of chronic cystitis [14] . In addition , chronic cystitis was also indicated by a high bladder bacterial burden ( >104 CFU/bladder ) and a visibly enlarged , inflamed bladder ( indicating inflammation and edema ) at time of sacrifice ( ≥28dpi ) . Resolution of cystitis was defined as urine bacterial titer dropping below 104 CFU/ml urine during at least one time point , and/or bladder bacterial burden <104 CFU at time of sacrifice . For long-term “challenge” experiments , beginning four weeks after the initial infection , all mice ( including mock-infected controls , and regardless of infection outcome ) were treated with trimethoprim and sulfamethoxazole in the drinking water ( 54 and 270 μg/ml water , respectively ) for ten days . Urine samples were collected weekly after the initiation of antibiotics to confirm sterile urines and any mice with treatment failure were excluded from subsequent analysis . Four weeks or six months after the initiation of antibiotics to clear initial infection , mice were challenged with 107 CFU of bacteria . The same criteria were used to define chronic cystitis during the challenge infection as in the initial infection ( persistent bacteriuria >104 CFU/ml urine; bladder burden >104 CFU; enlarged bladder ) , with the exception that urine titers could be <104 CFU/ml on the first day post challenge , as we have found that checkpoint activation in mice with a history of chronic cystitis may take longer ( up to 72 hours ) than in Juvenile Naive mice ( 24 hours ) . Bladders were aseptically harvested and fixed overnight in methacarn ( 60% methanol , 30% chloroform , 10% glacial acetic acid ) , bisected to give two halves per bladder , paraffin-embedded and sectioned . For histopathological examination , slides were stained with hematoxylin and eosin and imaged with a Zeiss Axio Scan Z . 1 brightfield slide scanner . Immunofluorescence experiments were conducted as previously described [15] . Primary antibodies used were uroplakin IIIa ( mouse monoclonal , 10R-U103a , Fitzgerald ) , Trp63 ( rabbit polyclonal , GTX102425 , GeneTex ) , E-cadherin ( goat polyclonal IgG , AF748 , R&D Systems ) , cytokeratin 5 ( chicken polyclonal , 905901 , BioLegend ) and cytokeratin 20 ( mouse monoclonal , M7019 , DAKO ) . Samples were mounted in ProLong Gold Antifade Mountant with DAPI ( ThermoFisher Scientific ) and fluorescence was visualized on a ZEISS Axioskop Observer . Z1 microscope . Two microscopy sample preparation protocols were used during the course of this work . For urothelial cell size determination six months after antibiotics , bladders were aseptically harvested , bisected , splayed in dissecting trays , and fixed in EM fixative as previously described [15] . The fixative comprised 2% paraformaldehyde and 2% glutaraldehyde in 0 . 1M sodium phosphate buffer , pH 7 . 4 . Samples were prepared by critical point drying . Briefly , samples were post-fixed in 1% osmium tetroxide , dehydrated in increasing concentrations of ethanol , then dehydrated at 31 . 1°C and 1072 PSI for 16 minutes in a critical point dryer . Samples were mounted on carbon tape-coated stubs and sputter-coated with gold/palladium under argon . After these experiments , subsequent SEM samples were processed with a different protocol , which we verified gave the same results as prior studies . Thus , for all other experiments , the EM fix comprised 2% paraformaldehyde , 2 . 5% glutaraldehyde in 0 . 15M sodium cacodylate buffer with 2 mM CaCl2 at pH 7 . 4 , and after post-fixing in 1% osmium tetroxide and critical point drying , samples were sputter-coated with iridium . For urothelial cell size determination after UTI89 vs . CFT073 infection , bladders were aseptically harvested , bisected , splayed in dissecting trays , and fixed in EM fixative before further processing . For the bladders obtained during chronic UTI89 or CFT073 cystitis at 28 dpi , the “balloon method” was used , wherein bladders were instilled with fixative prior to harvest , as previously described [49] . All bladders were imaged on a Zeiss Crossbeam 540 FIB-SEM or a Zeiss MERLIN FE-SEM . For urothelial cell size determinations , ImageJ 1 . 47v ( National Institutes of Health , USA ) was used to calculate epithelial cell surface area in five 500x magnification fields per bladder half ( ten per mouse ) . Bacteria were enumerated in urine or bladder samples by serial dilution as previously described [14] . Urine sedimentation to determine neutrophil influx was performed as previously described [14] . Briefly , 80 μl of a 1:10 dilution of urine was centrifuged onto poly-L-lysine-coated glass slides and stained with a Hema 3 kit ( Fisher Scientific ) . Slides were examined by light microscopy and the average number of polymorphonuclear leukocytes ( PMN ) per high-powered field ( hpf; 400x magnification ) was calculated from counting five fields . A semi-quantitative scoring system was used: 0 , less than 1 PMN/hpf; 1 , 1–5 PMN/hpf; 2 , 6–10 PMN/hpf; 3 , 11–20 PMN/ hpf; and 4 , >20 PMN/hpf . Bladder edema was assessed by pre-weighing empty bladder homogenization tubes , weighing the bladder-containing tubes , and calculating the difference . Serum cytokines were assessed using the Luminex-based multiplex cytometric bead array platform ( Bioplex , Bio-Rad , Hercules , CA ) using beads individually purchased for IL-5 , IL-6 , KC , and G-CSF . The assay was performed according to the manufacturer’s instructions , except using 10-fold less standard and half the amount of coupled beads and detection antibodies indicated in the protocol , as described in [50] . Individual samples were run in duplicate and the mean value was used for plotting and statistical analysis . Blood was obtained from convalescent mice ( four weeks after the initiation of antibiotics ) via submandibular puncture using a 5 mm lancet ( Goldenrod ) and collected in a BD Microtainer serum separator tube ( BD Biosciences ) . UTI89 and CFT073 were grown as described above and 1 ml of OD600 = 1 bacteria was lysed by boiling at 100°C for 5 minutes . Lysed bacteria were diluted tenfold in PBS and applied to high-affinity Immulon 2 HB 96 well plates ( Thermo Fisher ) overnight at 4°C . Plates were washed with PBS containing 0 . 05% Tween 20 and a dilution series of serum was applied for 90 minutes at room temperature . After washing , a 1:10 , 000 dilution of HRP-conjugated goat anti-mouse IgG ( Invitrogen ) was applied for 90 minutes at room temperature . After a final wash step , the TMB substrate reagent set ( BD Biosciences ) was used to develop the reaction . Absorbance was measured on a VersaMax microplate reader ( Molecular Devices ) . Depletion antibodies were obtained from BioXCell: α-mouse CD4 ( GK1 . 5 , BP0003-1 ) , α-mouse CD8a ( 2 . 43 , BP0061 ) , and rat IgG2b isotype control ( LTF-2 , BP0090 ) . Mice were given intraperitoneal injections one day prior to infection and at days 7 , 14 , and 21 post infection . For depletions of both CD4+ and CD8+ T cell subsets , mice were treated with 500 μg α-CD4 and 500 μg α-CD8 , or 1 mg isotype control , in a 500 μl volume of pH 7 . 0 dilution buffer ( BioXCell ) . For single subset depletions , mice received 500 μg α-CD4 or 500 μg α-CD8 , or 500 μg isotype control , in a 250 μl volume of pH 7 . 0 dilution buffer ( BioXCell ) . T cell depletions were performed only during the initial infection , not the challenge infection . Spleens were dissected and placed in RPMI ( Gibco ) on ice until spleens were removed from all animals . Each sample was dissociated by placing the spleen between two frosted glass slides and rubbing the slides together . The spleens were placed in ACK buffer ( Gibco ) for 5 minutes at room temperature to lyse red blood cells . Ten mls of RPMI was added to each tube and tubes were inverted to mix , then centrifuged at 1300 rpm at 4°C for 5 minutes . The supernatant was discarded and the pellet was resuspended in 3 mls RPMI and pushed through a 70 μm cell strainer ( Fisher ) . The cells were washed twice in 1X dPBS and centrifuged at 500 X g . The pelleted cells were resuspended in Fc block ( BD Biosciences ) and incubated in 1 μl of live/dead blue fluorescent reactive dye ( Thermo Fisher/Invitrogen , L34962 ) per 106 cells for 20 minutes at 4°C , then washed in 1X dPBS . Cells were stained with surface markers , all from Biolegend , in two separate panels: Panel 1: CD45 ( 30-F11 ) , CD8a ( 53–6 . 7 ) , CD3 ( 17A2 ) , CD19 ( GD5 ) , and CD4 ( GK1 . 5 ) ; Panel 2: CD45 ( 30-F11 ) , CD11b ( M1/70 ) , Ly6G ( 1A8 ) , F4/80 ( BM8 ) , and Ly6C ( HK1 . 4 ) . Sample data was acquired on the LSR II flow cytometer ( BD Biosciences ) recording 10 , 000 events . The data was analyzed using FlowJo software ( version 10 . 0 ) . The relative proportion of cellular infiltrates was expressed as a percentage of live cells . Our experience with the C3H/HeN chronic and recurrent cystitis model shows that when assessing bimodal responses , five mice per replicate ( two replicates minimum ) is the minimum number necessary to overcome any biological and/or technical variability and provide reliable and interpretable results within an experiment; a statistical analysis was not used to determine the number of mice per experiment . Investigators were not blinded to the infection history of the animals , except for Fig 2E . Statistics were performed in GraphPad Prism v7 . 01 . A two-tailed Fisher’s exact test was used to test for significant differences in the incidence of chronic cystitis and kinetics of infection resolution . For bacterial titers , pyuria , cytokine expression levels , and surface area differences in superficial cells , comparisons of three or more groups were performed with the Kruskal-Wallis test followed by Dunn’s multiple test correction . Pairwise comparisons were performed with a two-tailed Mann-Whitney U test . P < 0 . 05 was considered statistically significant . | Many patients suffer from highly recurrent urinary tract infections ( UTI ) caused by Escherichia coli , which are genetically diverse bacteria . Recurrent episodes are often caused by the same E . coli strain that caused the first infection , suggesting that some patients may not develop a protective immune response . We found that two E . coli UTI strains behaved differently in a mouse model of UTI . One strain , UTI89 , could infect the bladder indefinitely , whereas strain CFT073 was always cleared within eight weeks . After mice had a CFT073 infection and antibiotic treatment , they were protected from a CFT073 challenge infection , but were susceptible to a UTI89 challenge infection . In contrast , mice with a UTI89 infection and antibiotics were susceptible to recurrent UTI when challenged with either strain . We found that depleting T cells , immune cells important for developing protection against infection , prevented mice from clearing their CFT073 infections and made them susceptible to recurrent CFT073 UTI . Thus , while infection with one E . coli strain could trigger a protective immune response , another strain sidestepped this response . Our findings shed new light on immune responses to UTI and may be important for drug and vaccine development . | [
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"organisms",... | 2018 | Host restriction of Escherichia coli recurrent urinary tract infection occurs in a bacterial strain-specific manner |
Hox genes encode homeodomain-containing proteins that control embryonic development in multiple contexts . Up to 30 Hox genes , distributed among all four clusters , are expressed during mammalian kidney morphogenesis , but functional redundancy between them has made a detailed functional account difficult to achieve . We have investigated the role of the HoxD cluster through comparative molecular embryological analysis of a set of mouse strains carrying targeted genomic rearrangements such as deletions , duplications , and inversions . This analysis allowed us to uncover and genetically dissect the complex role of the HoxD cluster . Regulation of metanephric mesenchyme-ureteric bud interactions and maintenance of structural integrity of tubular epithelia are differentially controlled by some Hoxd genes during renal development , consistent with their specific expression profiles . We also provide evidence for a kidney-specific form of colinearity that underlies the differential expression of two distinct sets of genes located on both sides and overlapping at the Hoxd9 locus . These insights further our knowledge of the genetic control of kidney morphogenesis and may contribute to understanding certain congenital kidney malformations , including polycystic kidney disease and renal hypoplasia .
Mammalian genomes contain 39 genes related to the Drosophila homeotic genes , which encode transcription factors necessary for proper development along the major body axis . In the mouse , these Hox genes are organized in four separate chromosomal clusters and are expressed along the body axis in a spatio-temporal manner that corresponds to their physical positions along the clusters [1 , 2] . Because these four clusters were generated by large-scale duplications , Hox genes are classified into thirteen groups of paralogy , based on both sequence similarities and respective positions along the clusters . These genes have long been recognized as important regulators of developmental processes , in particular due to their function in specifying regional identities along the anterior to posterior body axis [3] . In the course of tetrapod evolution , Hox gene functions were co-opted in a number of developmental processes including the patterning of the limb bud and the formation of several organ systems , where they positively or negatively regulate the expression of genes involved in cell differentiation , adhesion , proliferation and apoptosis [4] . The intricate overlapping expression patterns and structural homology of these gene products underlie many instances of pleiotropy and functional redundancy , phenomena that have substantially complicated their functional analysis . Further untangling of this complex genetic system is expected to yield novel insight into important developmental mechanisms . For example , in the developing mammalian urogenital system , simultaneous expression of about thirty Hox genes has been reported , and some traces of colinear regulation became apparent [5–7] . The early molecular events underlying kidney morphogenesis are functionally well described , and are initiated by reciprocal epithelial-mesenchymal signaling interactions between the epithelium of the ureteric bud ( UB ) and the metanephric mesenchyme ( MM ) [8] . In mouse , the UB forms during embryonic day 10 ( E10 ) as an epithelial protrusion in the more posterior part of the Wolffian duct . The nearby mesenchyme ( future MM ) condenses and signals to induce branching morphogenesis of the UB , whereas signals emanating from the UB in turn promote MM survival and activate nephrogenesis . The induced MM expands , becomes a polarized epithelium through the intriguing process of mesenchyme to epithelium transition and forms vesicles that elongate and differentiate to form the nephrons . Molecular genetic analyses in mice have revealed a complex network of regulatory interactions that control kidney organogenesis , involving transcription factors , cell adhesion proteins and secreted molecules [8] . In this context , few studies have addressed the function of Hox genes during kidney morphogenesis , except for paralogy group 11; while single mutations in any of the Hox11 group gene results in normal nephrogenesis , double or triple mutations lead to either absent , or rudimentary kidneys , with a severely reduced number of nephrons [9 , 10] . Gain of function approaches have suggested a richer variety of HOX function in kidneys , since the over-expression of Hoxb8 and Hoxb7 caused kidney hypoplasia and renal duplication , respectively [11 , 12] , whereas the ectopic expression of Hoxd13 induced renal agenesis [13] . In order to assess the total functional contribution of Hoxd genes to kidney development , we performed detailed comparative phenotypic analysis of a set of mouse strains carrying a variety of targeted genomic rearrangements within the HoxD cluster . We show that the most striking kidney defect at birth is a failure of UB branching morphogenesis , leading to kidney hypoplasia associated with early postnatal death . This defect correlates with a gain of expression of the posterior Hoxd12 gene in developing kidneys . Interestingly however , defects reminiscent of polycystic kidney disease ( PKD ) were also observed , yet exclusively in mutant mice carrying a deletion of more anterior Hoxd genes like Hoxd9 and Hoxd8 . Finally , the distinct functions of Hoxd genes in kidneys correlate well with their specific expression in the MM or UB , respectively . In this context , we use some additional mutant strains to provide evidence for the presence of putative global enhancer and silencer elements , which define and coordinate the expression pattern of Hoxd genes in developing kidneys .
As several Hoxd genes are expressed during kidney development , multiple modes of functional redundancy or compensatory mechanisms may remain easily hidden in single gene inactivation experiments [7 , 10] . We thus addressed this question by using more drastic genetic conditions and first compared renal development in a variety of mouse strains carrying targeted deletions of multiple Hoxd genes in cis ( Figure 1A ) . The shortest of these deletions removed the DNA interval including from Hoxd10 to Hoxd11 , and is referred to as Del ( 10–11 ) , whereas the largest deletion Del ( 1–13 ) removed the entire HoxD cluster , from Hoxd1 to Hoxd13 included ( Figure 1A ) . Other deletions Del ( 10–13 ) , Del ( 4–13 ) and Del ( 4–11 ) removed the corresponding intermediate sets of Hoxd genes . Importantly , while both Del ( 10–11 ) and Del ( 10–13 ) deletions removed Hoxd genes expressed in the MM only , other deficiencies involved heterogenous sets expressed either in the epithelium , or in the mesenchyme [7] . While homozygous pups derived from all mutant strains were obtained at birth following a Mendelian proportion , all homozygous Del ( 4–11 ) newborns ( 100% ) and between 40 to 50% of the Del ( 4–13 ) and Del ( 1–13 ) homozygous pups died shortly after birth . When compared to wild-type ( WT ) littermates at birth , Del ( 10–13 ) , Del ( 4–13 ) or Del ( 1–13 ) homozygous mutants displayed grossly normal kidneys , whereas Del ( 10–11 ) and Del ( 4–11 ) animals showed slight or strong reduction in the size of their kidneys , respectively ( Figure 1B ) . As expected from such severe kidney hypoplasia , histological analysis of Del ( 4–11 ) homozygous kidneys revealed a strong reduction of UB branching reflected in a reduced number of collecting tubules in the medulla region , when compared to control and other mutant strains ( Figure 2A , top ) . In addition , UB branching was slightly decreased in Del ( 10–11 ) kidneys . Because the severe hypoplasia found in Del ( 4–11 ) kidneys was absent from both Del ( 4–13 ) and Del ( 1–13 ) homozygous mice , we concluded that a mis-regulation of the remaining Hoxd12 and/or Hoxd13 genes was likely the cause of this postnatal lethality . The abnormal reduction in the number of UB branches in Del ( 10–11 ) and Del ( 4–11 ) newborn kidneys was further documented by using immuno-histochemical detection of CADHERIN type 1 ( CDH1 ) , a marker specific for developing UB and epithelial tubules ( Figure 2A , bottom ) . As appropriate branching of the UB is essential to set the number of nephrons , we next determined the volume and glomerular density of WT and mutant kidneys by quantitative morphometry . In both Del ( 10–11 ) and Del ( 4–11 ) mice , cortical and medullar volumes were reduced when compared to other strains ( Figure 2B , left ) . Furthermore , the density of glomeruli was significantly reduced in Del ( 4–11 ) kidneys ( Figure 2B , right ) , further confirming a role for Hoxd genes in the control of UB growth . Histological examination also revealed the presence of tubular dilatations in both the medulla and the cortex of Del ( 4–11 ) , Del ( 4–13 ) and Del ( 1–13 ) homozygous newborn kidneys ( Figure 2A , top ) . These defects were observed in heterozygous mutants too , although clearly less frequently . Higher magnifications revealed microcystic changes in these tubules , characterized by abnormally thin epithelial cells and widened lumina , resembling renal cysts observed in PKD ( Figure 3 , top ) . Most cysts originated from the UB lineage , as shown by staining with the collecting-duct specific lectin Dolichos bifluoris agglutinin ( DBA; Figure 3 , middle ) . In addition , CDH1 immuno-staining , an indicator of cystic kidneys in mice [14] , was lost in the plasma membrane of cystic tubules from both Del ( 4–11 ) and Del ( 4–13 ) homozygous kidneys , suggesting a defect in epithelial cell polarity ( Figure 3 , bottom ) . Finally , escaper Del ( 1–13 ) homozygous mice , surviving birth for at least one month , developed different degrees of well-visible unilateral or bilateral polycystic kidneys ( Figure S1 ) . Altogether , the abnormal kidney architecture observed in the different HoxD deletions suggested important roles for these genes during renal morphogenesis . On the one hand , the hypoplastic phenotype found in both Del ( 10–11 ) and Del ( 4–11 ) mice involved a mis-regulation of the remaining posterior Hoxd12 and Hoxd13 genes , reminiscent of gains of expression as previously seen during the development of the limbs [15] . On the other hand , the microcystic defects were likely induced by the loss of either Hoxd4 , Hoxd8 or Hoxd9 , or combinations thereof , as suggested by their occurrence in Del ( 1–13 ) , Del ( 4–13 ) and Del ( 4–11 ) , but not in Del ( 10–11 ) or Del ( 10–13 ) homozygous animals . Kidney morphogenesis depends upon a delicate balance between cell proliferation and apoptosis . We thus assessed whether the growth defects observed in our HoxD deleted mice could derive from modifications in the rates of either one of these processes . Immuno-detection of phosphorylated histone H3 ( P-H3 ) , a nuclear protein present in mitotic cells only , showed distribution of proliferating cells in the different compartments of developing kidneys at birth , with relatively higher levels in the cortex ( Figure 4A , top ) . Quantitative analysis of P-H3-positive cells revealed no significant difference between WT kidneys and age-matched Del ( 10–11 ) , Del ( 10–13 ) , Del ( 4–11 ) or Del ( 4–13 ) mutants ( Figure 4A , bottom left ) . In control newborn kidneys , TUNEL-positive , apoptotic cells were barely detected , either in the nephrogenic zone , or in the medullary papilla ( Figure 4A , top ) . In contrast , apoptosis was increased five to six fold in both Del ( 4–11 ) and Del ( 4–13 ) kidneys ( Figure 4A , bottom right ) . In these cases , apoptotic cells were particularly numerous in the cortical interstitium as well as in the microcystic tubules previously identified in these two HoxD deletions ( Figure 4B ) . No significant changes were observed either in Del ( 10–11 ) , or in Del ( 10–13 ) mutants ( Figure 4A ) , suggesting a critical involvement of the Hoxd4-Hoxd9 genomic interval . Therefore , the precise balance between proliferation and apoptosis was clearly disturbed in both Del ( 4–11 ) and Del ( 4–13 ) cystic kidneys , with a robust increase in tubular epithelial cell apoptosis , similar to what happens in human and animal models of PKD [16] . To explore the molecular mechanisms underlying the UB branching defects , we analyzed the expression pattern of those Hoxd genes flanking the internal Del ( 10–11 ) and Del ( 4–11 ) deletions , using both quantitative RT-PCR ( qPCR ) and in situ hybridization ( ISH ) , to detect potential deletion-induced mis-regulations [15] . By using qPCR and kidneys from embryonic day E13 . 5 fetuses to postnatal day 0 ( P0 ) , we observed a strong gain of expression of Hoxd12 in Del ( 4–11 ) kidneys , and a reproducible increase was also noticed in Del ( 10–11 ) mice ( Figure 5A ) . Del ( 10–13 ) and Del ( 4–13 ) kidneys were used as negative controls for the specificity of Hoxd12 gene amplification . No ( or only very slight ) difference was observed in the expression levels of Hoxd3 under the same circumstances ( Figure 5A ) . We next analyzed the distribution of Hoxd12 mRNA by ISH in WT , Del ( 10–11 ) and Del ( 4–11 ) kidneys at E15 . 5 and P0 , using Del ( 4–13 ) as a negative control ( Figure 5B ) . In WT kidneys , Hoxd12 was expressed rather diffusely within the MM at E13 . 5 , whereas it was detected mainly in the central MM surrounding the main trunk of the UB at birth , with lower expression in MM cells at the periphery of the kidney . In addition to the expression in the MM , Del ( 10–11 ) kidneys also showed Hoxd12 mRNA in some UB branches as well as UB tips , at the junction with the distal segments of the S-shaped body ( Figure 5B , bottom ) . Finally , Hoxd12 was strongly mis-expressed in the UB branches and tips of Del ( 4–11 ) kidneys at both E13 . 5 and P0 . Hoxd13 expression was undetectable at all these developmental stages , both in the various mutants as well as in WT controls ( [7] and data not shown ) . These results demonstrated that , as a result of the intervening deletions , the Hoxd12 transcription unit became progressively mis-expressed and shifted its expression from MM to UB , presumably due to its closer proximity to the more anterior ( 3' ) part of the cluster . Importantly , these changes in the quantitative level and tissue distribution of Hoxd12 expression correlated well with the UB branching defects observed in both Del ( 10–11 ) and Del ( 4–11 ) mutant kidneys . We investigated the expression of various genes known as regulators of kidney development , in order to identify potential genetic targets of Hoxd gene products . We started with the hypoplasia phenotype , common to both Del ( 10–11 ) and Del ( 4–11 ) mutant mice . The hypoplasia observed in Del ( 4–11 ) mice was similar to the effect of Hoxa11/Hoxd11 double loss of function [9] , suggesting that the up-regulation of Hoxd12 expression , in this allele ( Figure 5 ) , negatively affects the function of group 11 proteins . A similar mechanism has been proposed for explaining complete kidney agenesis or severe hypoplasia in the TgH[d9/lac] transgenic configuration , wherein the insertion of a transgene near Hoxd13 up-regulated this latter gene in developing metanephric kidneys [13] . To investigate this hypothesis , we analyzed by qPCR the expression of several genes reported to be strongly mis-regulated in Hoxa11/Hoxd11 double mutant kidneys [17] . However , both Del ( 4–11 ) and Del ( 4–13 ) homozygous kidneys exhibited normal expression of all genes assayed in this respect ( Figure S2 ) . In contrast , similar analyses carried out on the TgH[d9/lac] transgenic mice revealed a significant increase of both Gata6 and Tgfβ2 expression , two genes described as target of Hoxa11/Hoxd11 [17] , together with a massive gain of expression of Hoxd13 in the MM and subsequent kidney hypoplasia ( Figure S3 ) . Because the gain of Hoxd13 expression in the MM of TgH[d9/lac] transgenic kidneys involved modifications in MM-specific genes ( Gata6 and Tgfβ2 ) , we investigated whether the gain of posterior Hoxd gene expression in the UB of Del ( 4–11 ) mice could affect the expression of UB-specific genes , instead of the previously tested MM candidates . Little ( if any ) difference was scored amongst the various HoxD strains , upon analyses of the expression levels of a battery of genes known to regulate UB branching ( Figure 6A and S4A ) . Interestingly however , a severe reduction of embryonic and postnatal integrin-α3 ( Itgα3 ) gene expression was observed in Del ( 4–11 ) hypoplastic kidneys , as compared to their WT and Del ( 4–13 ) counterparts ( Figure 6A ) . A slight down-regulation of Itgα3 was already present in the Del ( 10–11 ) kidney , whereas a normal expression level was found in the TgH[d9/lac] transgenic strain ( data not shown ) . The Itgα3 gene was of particular interest in this context , since its functional inactivation leads to mice with similar defects in branching morphogenesis and microcystic phenotypes than those observed in our Del ( 4–11 ) mutants [18] . Altered expression of Itgα3 in Del ( 4–11 ) kidneys was further confirmed at the protein level by using immuno-fluorescence . ITGα3 is present in both the UB and glomerular podocytes , along the glomerular basement membrane ( GBM ) of WT kidneys ( Figure 6B and reference [19] ) . Strikingly , ITGα3 immuno-staining was virtually lost in Del ( 4–11 ) mutant UB branches and tips , when compared to WT and Del ( 4–13 ) controls ( Figure 6B , top and middle ) . In addition , the GBM was disorganized in a number of Del ( 4–11 ) mutant kidneys , as shown by reduced capillary branching ( Figure 6B , bottom ) . These glomerular anomalies are consistent with an essential role of ITGα3 in the maturation of the GBM [20] , and may result in the loss of normal glomerular filtration barrier in Del ( 4–11 ) kidneys . We then focused on the polycystic kidney phenotype , common to both Del ( 4–11 ) and Del ( 4–13 ) mutant mice . Because these cystic kidneys displayed increased apoptotic activity ( Figure 4 ) , we used qPCR to determine the expression levels of those apoptotic genes known to trigger a cystogenic pathway in mouse dysplastic kidneys ( Figure 7A and S4B ) [21] . The RNA content of c-myc and p53 , two important pro-apoptotic gene markers of PKD [16] , were both increased in Del ( 4–11 ) and Del ( 4–13 ) kidneys , when compared to WT controls ( Figure 7A ) . Expectedly , C-MYC was detected in a nuclear pattern in epithelial cystic tubules of both Del ( 4–11 ) and Del ( 4–13 ) kidneys ( Figure 7B ) . In addition , we scored a strong up-regulation of growth and differentiation factor-5 ( Gdf5 ) in these two mutant kidneys , coincident with the observed cystic dilatations of epithelial tubules . Gdf5 mRNA and protein expression levels were elevated in Del ( 4–11 ) and Del ( 4–13 ) kidneys , in both non-cystic and cystic tubules , as revealed by ISH and immuno-fluorescence , respectively ( Figure 7B , top ) . Members of the TGFβ super-family trigger cellular responses through the SMAD protein pathway , which transduces the signal from the cell surface to the nucleus to activate specific gene transcription [22] . We thus analyzed the expression of phosphorylated SMAD 1-5-8 proteins ( P-SMADs ) in cystic kidneys , as a cellular reporter of TGFβ pathway activation . Although P-SMADs were expressed at low levels , in a cytoplasmic pattern in epithelial tubules from control tissue , they were robustly up-regulated in both the cytoplasms and nuclei of microcystic tubules from Del ( 4–11 ) and Del ( 4–13 ) kidneys ( Figure 7B ) , suggesting an increased activity of the BMP/GDF pathways in these tubules . The polycystic kidney phenotype was observed only in those HoxD cluster deletions which remove both anterior and posterior Hoxd genes , such as Del ( 4–11 ) , Del ( 4–13 ) and Del ( 1–13 ) . In contrast , this phenotype never developed in those strains where only posterior genes had been affected , such as Del ( 10–11 ) and Del ( 10–13 ) , suggesting that this alteration was caused by ( a ) gene ( s ) lying within the Hoxd9 to Hoxd4 interval ( Figure 2 ) . To further clarify the importance of these genes in the formation of microcysts , we used two novel mutant strains , carrying either a deletion or a duplication of the region located between Hoxd9 and the intergenic ‘region i' ( Del ( i-9 ) and Dup ( i-9 ) ; Figure 8A ) , i . e . deleting or duplicating in cis both Hoxd9 and Hoxd8 . Histological examination of Del ( i-9 ) homozygous kidneys revealed the presence of epithelial microcysts , as well as an increased tubular Gdf5 expression ( Figure 8B ) . In this allele , the expression of neighboring posterior and anterior Hoxd genes was not importantly modified , strongly arguing against any potential global regulatory re-allocations that may have triggered a cystic phenotype ( Figure S5 and data not shown ) . As mentioned earlier , microcystic tubules were also observed in Del ( 1–13 ) heterozygous kidneys , with a penetrance of about 50 percent ( 3 out of 6 mice ) , consistent with the loss of function explanation involving Hoxd8 and Hoxd9 . Interestingly , these renal defects were never observed in six compound mutant mice generated on the same genetic background , which were trans-heterozygous for Del ( 1–13 ) and Dup ( i-9 ) , suggesting a rescue from the duplicated allele ( Figure 8C , top ) . In these Del ( 1–13 ) /Dup ( i-9 ) animals , the two copies of Hoxd9 and Hoxd8 , coming from the duplicated DNA segment were likely expressed at similar levels , since expression was about two-fold higher than in Del ( 1–13 ) heterozygous kidneys ( Figure 8D ) . However , while Hoxd9 expression level was similar in the MM , it was clearly higher in the UB , suggesting that the second Hoxd9 gene copy was mostly expressed in the UB ( Figure 8C , bottom ) . By using whole mount in situ hybridization , Hoxd8 was detected only in the UB of WT and Del ( 1–13 ) kidneys , with relatively higher expression in Del ( 1–13 ) /Dup ( i-9 ) kidneys ( data not shown ) . Accordingly , the expression of Gdf5 and c-myc also decreased in Del ( 1–13 ) /Dup ( i-9 ) kidneys , reaching levels observed in WT conditions ( Figure 8D ) . These results suggest a role for Hoxd9 and Hoxd8 ( and perhaps for ‘region i' ) in the regulation of tubular epithelia in postnatal kidneys . In order to determine the influence of Hoxd genes' clustered organization on their transcriptional regulation in MM and UB kidney cell types , we used a collection of mouse strains containing the same reporter transgene together with progressively more extended deletions of the HoxD cluster ( Figure 9A , top ) . In these strains of mice , a Hoxd11/lacZ fusion gene effectively replaced the deleted regions and thus allowed for histochemical detection of β-galactosidase activity in kidneys . In this way , the transcriptional behavior of the same reporter gene could be assessed when located at various positions within the gene cluster . As expected from the absence of Hoxd13 expression in kidneys , reporter activity was not detected when the fusion gene was positioned at the posterior end of the HoxD complex flanked by Hoxd13 on its 3' side ( TgH[d11/lac]; Figure 9A , bottom ) . However , the proximity of progressively more 3' parts of the HoxD cluster induced accompanying progressively more anterior shifts of reporter gene expression along the trunk ( data not shown ) , including a shift from MM to UB . While Del ( 12–13 ) and Del ( 11–13 ) strains showed strong β-galactosidase activity in the MM , only UB but no MM cells were stained when the deletions brought the transgene closer to the 3' ( telomeric ) part of the cluster ( Figure 9A , bottom ) . These expression patterns were suggestive of the existence of two distinct enhancer sequences , one controlling expression of ‘posterior' genes in the MM , the other triggering expression of more ‘anterior' Hoxd genes in the UB . Because reporter transgene activity in the UB persisted even when the HoxD cluster was entirely deleted , a putative UB enhancer ought to localize outside the cluster itself . On the other hand , reporter gene activity in the mesenchyme was scored in the Del ( 11–13 ) strain , whereas absent in Del ( i-13 ) animals , suggesting that a mesenchyme enhancer activity may reside within the cluster , between Hoxd10 and ‘region i' . However , in mice lacking either halves of this region ( i . e . Del ( i-9 ) or Del ( 10–11 ) ) , expression of the remaining posterior Hoxd genes in the MM was still clearly detected ( Figure 5B and S5 ) . From these observations , we conclude that the absence of reporter activity in the MM of Del ( i-13 ) mice is not due to the deletion of the corresponding enhancer sequence . Consequently , this putative regulatory sequence must lie either in the most anterior part of the cluster ( within the Hoxd4 to Hoxd1 region ) or , like for the UB regulation , outside the cluster itself . Because enhancers controlling Hoxd gene expression in limbs are functionally separated and distributed on either sides of the cluster [23] , we further tried to map these putative ‘kidney enhancers' . To this aim , we used mice carrying a large inversion that splits the HoxD cluster into two parts , between Hoxd11 and Hoxd10 [23] , separated by a 3 Mb large DNA fragment ( Figure 9B , top ) . In such mice , the HoxD cluster is split into two sub-clusters with independent regulations [23] . In WT newborn kidneys , the expression of both Hoxd11 and Hoxd10 was detected in the MM only ( Figure 9B , left ) . Interestingly , after separating the complex , mutant kidneys completely lost expression of both Hoxd11 and Hoxd12 , whereas expression of Hoxd10 in the MM remains unchanged . Quantitative analyses by using qPCR confirmed the abrogation of Hoxd12 and Hoxd11 expression in this allele , whereas Hoxd10 or other UB-specific genes , such as Hoxd8 , exhibited slightly higher than normal expression levels , respectively ( Figure 9B , right ) . Altogether , these results strongly suggest that the putative global enhancer sequences controlling the expression of Hoxd genes in both MM and UB are located either within the most telomeric part of the HoxD cluster , or further outside in 3' .
A high proportion of Hox genes are expressed in both human and mouse kidneys [6 , 7] and several reports have pointed to an important role for these genes during renal development . Functional studies in mice have illustrated the functional overlap that exists between paralogous genes as well as between genes that are adjacent to each other in the same cluster . While a single mutation in any gene of paralogous group 11 results in normal nephrogenesis , double or triple mutations lead to either absent , or rudimentary kidneys , indicating a critical role for paralogous group genes in UB branching morphogenesis [9 , 10] . Furthermore , the combined inactivation of Hoxa10 and Hoxd10 altered both the position and the size of developing kidneys [24] , and some renal size reductions were also scored in Hoxd9/Hoxd10 double mutant mice [25] . Here , we show that TgH[d9/lac] and Del ( 4–11 ) mutant mice have a reduced branching morphogenesis . In the former case , we explain this by the strong gain of Hoxd13 expression in developing kidney mesenchyme , which negatively affects the function of group 11 paralogs , through a process known as ‘posterior prevalence' , whereby the product of posterior genes can override the function of more anterior products [26] . Ectopic Hoxd13 expression led to a failure of the mutant MM to properly induce UB branching , similar to the phenotype of combined group 11 loss of functions . In the latter case , the gain of function involves Hoxd12 in the UB , which leads to branching inhibition as well as podocyte GBM defects , coherent with the down-regulation of the cell adhesion molecule ITGα3 . Although experimental evidence supports a link between posterior Hoxd genes and Itgα3 in developing kidneys , the demonstration of a direct interaction between these components , so far hampered by the lack of fully specific anti-HOX antibodies , remains to be established . Firstly , Itgα3 deficient newborn mice exhibit UB branching and GBM defects similar to those reported here , due to impaired epithelial cytoskeleton organization [19] . Secondly , α3 subunit-containing integrins bind with high affinity to isoforms of laminin-10 and −11 , which are the predominant integrin ligands in both the UB and mature glomeruli [19] . Interestingly , targeted disruption of these laminins leads to hypoplastic kidneys [18] . Also , accumulating evidence suggests the existence of functional links between these two groups of genes in a variety of tissues and contexts [27] . In mouse kidney cell lines , for instance , Itgα8 and Iap are targets of Hoxa11 [28] , whereas the ITGα3 gene is regulated by HOXD3 and HOXD10 in some human cancers [29 , 30] . Finally , Hox genes may regulate the progression of human kidney diseases , including glomerulonephritis , diabetic nephropathy and PKD , where integrins have been reported to play an important role [19] . PKD , one of the most frequent human genetic disorders , constitutes a major cause of end stage renal failure worldwide [21] . In human and rodent models of PKD , pathological features include increased apoptosis , as well as dedifferentiation , adhesion and ciliary abnormalities of epithelial cells [16] . Cystic kidneys were previously observed in a fraction of Hoxd11 null mice containing a chimeric Hoxa11 allele with the Hoxa4 homeobox [31] . In this work , we show that the combined deletion of Hox gene functions ( Hoxd9 and Hoxd8 ) leads to the development of polycystic kidneys . The comparative analysis of renal development and gene expression in the various HoxD deleted strains also revealed a significant increase of the cystic phenotype in Del ( 4–13 ) and Del ( 1–13 ) mice , as compared to Del ( 4–11 ) and Del ( i-9 ) , suggesting some participation of Hoxd12 in the regulation of the mesenchymal-epithelial transition in developing tubules . In previous studies , this loss of function phenotype may have been compensated for by functional redundancy , as illustrated either for the group 11 Hox genes [10] , or between non paralogous genes expressed in similar kidney compartments [7] . Of note , mice lacking either all group 9 or all group 8 genes die soon after birth for unknown reasons [32 , 33] , which may illustrate a protective role of Hoxd9 and Hoxd8 against the development of PKD . Also , loss of these genes was reported to have occurred in different human cancers affecting kidneys , the thyroid and breast tissues [34 , 35] , even though a direct causal relationship was not established . In addition , a detailed analysis of the set of genes mis-regulated in a murine model of PKD revealed no difference ( or a slight decrease ) for Hoxd9 and Hoxd8 expression , whereas the neighboring Hoxd10 , Hoxd4 and Hoxd3 were all increased [36] . These observations support the hypothesis that Hoxd9 and Hoxd8 genes might be necessary to maintain a given differentiated phenotype in epithelial cells . In mice , PKD can be induced by mutations in a variety of genes , including several members of the BMP/GDF family of proteins such as BMP4 and BMP7 [37] . Here , we report the up-regulation of another member of the family , GDF5 , in Hoxd mutant polycystic kidneys . Like BMPs , GDF proteins trigger cellular responses through their binding to type-I receptors ( preferentially ALK3 and ALK6 ) and subsequent activation of the SMAD pathway ( Smad 1-5-8 ) , which relays the signal from the cell surface to the nucleus to activate specific gene transcription [22] . Interestingly , both ALK3 and ALK6 receptors are highly expressed in developing kidneys [37] and transgenic mice over-expressing a constitutively active form of ALK3 in the UB lineage develop PKD [38] . In our different HoxD deletions that induce PKD , down-regulation of CDH1 and up-regulation of P-SMADs and C-MYC expression levels were observed , similar to what was scored in cystic tubules from ALK3 transgenic mice . We consider this as an indication that the activation of the BMP/GDF pathway , in our mutants , may be triggered by this receptor . In this respect , it is noteworthy that the primary function of ALK3 seems to be associated with apoptosis , at least in developing limbs [39] . The distinct functions observed at the genetic level for some ‘posterior' and ‘anterior' Hoxd genes in kidneys correlate well with their specific expression in either the MM or the UB , respectively ( [7] and this work ) . The data obtained using our collection of mutant strains suggest that the distribution of Hoxd genes between these two renal compartments is under the control of global enhancers , resembling in this respect the regulation of the HoxD cluster in the developing limbs and intestinal hernia [23] . In developing kidneys , a putative mesenchyme enhancer may control the expression of Hoxd12 to Hoxd9 , whereas a ureteric bud enhancer may control gene expression from Hoxd9 to Hoxd1 , Hoxd9 being thus the only gene responsive to both enhancers ( Figure 9C ) . Because these expression specificities concern two different cell types , it is likely that the regulation of Hoxd gene expression in kidneys involves two distinct enhancer sequences . While the data shown here suggest that both of these sequences unexpectedly reside at the telomeric end ( or side ) of the cluster , their precise nature and location will have to await a more extensive transgenic approach of the suspected genomic region . As it stands , we cannot rule out the possibility that a single global enhancer , controlling expression in both MM and UB , may be at work , involving various cell-type or tissue-specific co-factors . Regardless of the number of enhancers involved , the underlying mechanism ( s ) must be complex to account for the quasi exclusion of the two expression specificities , with the exception of Hoxd9 . The analysis of yet a few more genetic configurations will shed some light on this issue , for example whether part of these expression differences are due to the presence of silencer or boundary elements , as reported in similar instances [40] .
Genomic rearrangements within the HoxD cluster , including deletions ( Del ) or duplications ( Dup ) , were all produced in vivo through targeted meiotic recombination [41] , except for the Del ( 11–13 ) , Del ( 4–13 ) and Del ( 1–13 ) lines , which were engineered by loxP/Cre mediated site-specific recombination in ES cells [42–44] . These three lines were previously referred to as Del3 , Del7 and Del9 , respectively . The Del ( 10–13 ) , Del ( i-9 ) and Dup ( i-9 ) alleles were previously described [45] , whereas Del ( 12–13 ) , Del ( 10–11 ) , Del ( 4–11 ) , and Del ( i-13 ) are novel alleles of the HoxD cluster . The Del ( 12–13 ) , Del ( 11–13 ) , Del ( i-13 ) , Del ( 4–13 ) and Del ( 1–13 ) alleles also contained the Hoxd11/lacZ fusion gene , allowing for histochemical detection of β-galactosidase activity by X-gal staining [46] . Targeted insertions of the Hoxd11/lacZ and Hoxd9/lacZ transgenes to the posterior end of the HoxD cluster ( TgH[d11/lac] and TgH[d9/lac] strains , respectively ) were reported previously [46 , 47] . The split of the HoxD cluster , between Hoxd11 and Hoxd10 ( Split ( 10–11 ) , was produced using the sequential targeted recombination-induced genomic ( STRING ) approach [23] . For histological analyses , newborn kidneys were fixed overnight at 4°C with 4% PFA before alcohol dehydration and paraffin embedding . The whole kidneys were sectioned at 5 μm and stained with hematoxylin and eosin according to standard protocols . Cortical and medullary volumes , as well as glomerular density ( number of glomeruli counted per kidney volume ) were determined in serial sections throughout entire kidneys according to well established morphometric procedures [48] , using the ImageJ free software . For each mouse strain , four kidneys obtained from separate newborn mice were investigated . The independent Student's t-test was used for all statistical analyses . Embryonic or newborn kidneys were fixed in 4% PFA and cryoprotected in 20% sucrose before embedding in OCT compound . In situ hybridization was performed on 10 μm cryosections by using digoxygenin-labelled riboprobes . Sections were hybridized overnight at 65°C with 2 μg/ml of riboprobe in the following hybridization buffer: 50% formamide , 10% dextran sulfate , 1x Denhardt's solution , 1 mg/ml yeast RNA , 200 mM NaCl , 1 . 1 mM Tris-base , 8 . 9 mM Tris-HCl , 5 mM Na2HPO4 , 5 mM NaH2PO4 and 5 mM EDTA . Two washes of 30 min at 65°C were done in 50% formamide , 1x SSC ( 0 . 15 M NaCl plus 0 . 015 M sodium citrate ) and 0 . 1% Tween 20 , followed by two washes at RT in TBS-T ( 137 mM NaCl , 27 mM KCl , 25 mM Tris-base , 0 . 1% Tween 20 , pH 7 . 5 ) . Sections were then incubated for one hour in a blocking buffer consisting of 20% goat serum and 2% blocking reagent ( Roche ) in TBS-T , and then left overnight at 4°C in an alkaline phosphatase-conjugated anti-digoxigenin antibody ( Roche ) at a dilution of 1:2'000 in the blocking buffer . Five washes of 20 minutes were done in TBS-T , and color development was performed in NTMT ( 100 mM NaCl , 100 mM Tris , 50 mM MgCl2 , 0 . 1% Tween 20 , pH 9 . 5 ) containing both nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate ( Roche ) . The mouse antisense RNA probes for Hoxd genes were described previously [45] . The Gdf5 probe was amplified by PCR , as reported [49] . Immuno-fluorescent staining on kidney cryosections was carried out as follows: fixation for 10 minutes with 4% PFA; epitope retrieval for 20 minutes at 95°C in 0 . 01 M citrate buffer ( pH 6 . 0 ) ; blockage for one hour with normal goat serum; incubation overnight at 4°C with the primary antibody: anti-CDH1 ( 1:100 , Cell Signaling ) , anti-ITGα3 ( 1:400 , Chemicon ) , anti-phospho-SMADs ( 1:50 , Cell Signaling ) or anti-C-MYC ( 1:100 , Santa Cruz Biotechnology ) ; incubation with the Alexa Fluor-conjugated secondary antibody ( Alexa Fluor-488 or −568 , Invitrogen ) for one hour . For the detection of GDF5 , immuno-fluorescence was performed using a monoclonal anti-GDF5 antibody ( 20 μg/ml , R&D Systems ) and the MOM immuno-detection kit as described by the manufacturer ( Vector Laboratories ) . To assess cell proliferation and apoptosis in kidneys , a double staining was performed with a combination of immuno-staining for anti-phospho-Histone-H3 ( Ser10 , Upstate ) and TUNEL assay ( TMR red , Roche ) according to the respective manufacturer's instructions . Slides were subsequently counterstained with DAPI ( 4' , 6'-diamidino-2-phenylindole ) for microscopic observation . Lectin staining was performed using fluorescein isothiocyanate-coupled Dolichos biflorus agglutinin ( DBA , Vector Laboratories ) as described previously [9] . Single-stranded cDNA templates were generated from total kidney RNAs using random hexamers and Superscript II reagents ( Invitrogen ) . For each mouse strain , at least three kidneys from separate embryos or newborn mice were investigated . Quantitative RT-PCR was carried out on an iCycler thermal cycler ( Bio-Rad ) using SYBR green . PCR efficiencies and dissociation curves of primer pairs were used to ensure specificity of the amplicons . Tbp , Gapdh and Tubβ4 genes were used as internal controls for normalization . Primer sequences used for PCR amplification are listed in Table S1 . New primer pairs were designed using Primer Express 2 . 0 software ( Applied biosystems ) . | Hox genes encode proteins that control embryonic development along the head-to-tail axis and in multiple organs . Here , we show that several members of this gene family are necessary for the normal development of the mammalian kidneys . These genes are clustered in one site on the chromosome and their respective positions within the group determine which component of the kidneys they will contribute to . Using a large collection of engineered mutations in this system , we show that these genes are required both for the growth of the kidneys and for their proper organization , such that mutations in some genes reduce the size of the organs , whereas mutations in others induce polycystic kidneys . Our set of genetic rearrangements also allowed us to localize the position of regulatory sequences , which control the expression of these genes during kidney development . | [
"Abstract",
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] | 2007 | Distinct Roles and Regulations for Hoxd Genes in Metanephric Kidney Development |
Balancing selection provides a plausible explanation for the maintenance of deleterious alleles at moderate frequency in livestock , including lethal recessives exhibiting heterozygous advantage in carriers . In the current study , a leg weakness syndrome causing mortality of piglets in a commercial line showed monogenic recessive inheritance , and a region on chromosome 15 associated with the syndrome was identified by homozygosity mapping . Whole genome resequencing of cases and controls identified a mutation causing a premature stop codon within exon 3 of the porcine Myostatin ( MSTN ) gene , similar to those causing a double-muscling phenotype observed in several mammalian species . The MSTN mutation was in Hardy-Weinberg equilibrium in the population at birth , but significantly distorted amongst animals still in the herd at 110 kg , due to an absence of homozygous mutant genotypes . In heterozygous form , the MSTN mutation was associated with a major increase in muscle depth and decrease in fat depth , suggesting that the deleterious allele was maintained at moderate frequency due to heterozygous advantage ( allele frequency , q = 0 . 22 ) . Knockout of the porcine MSTN by gene editing has previously been linked to problems of low piglet survival and lameness . This MSTN mutation is an example of putative balancing selection in livestock , providing a plausible explanation for the lack of disrupting MSTN mutations in pigs despite many generations of selection for lean growth .
Leg weakness is a heterogeneous condition causing lameness in pigs , and has negative impacts on both animal welfare and productivity [1 , 2] . Significant heritability estimates have been reported for leg weakness traits [reviewed in 3] , with moderate to high estimates in certain pig breeds , e . g . h2 = 0 . 45 in Landrace [4] . Several quantitative trait loci ( QTL ) have been identified for these traits , albeit they are generally not consistent across studies and breeds [5–8] , which may be partly due to the heterogeneity of this condition . Interestingly , significant genetic correlations between leg weakness and other production traits ( such as growth and muscle depth ) have been detected [4] . Further , in a divergent selection experiment in Duroc lines , selection for high leg weakness was associated with a significant increase in muscle length and weight [9] . Taken together , these results suggest a degree of antagonistic genetic relationship between leg weakness and muscle growth traits in pigs , potentially explaining increases in the syndrome observed with intense selection for lean growth in recent decades . Deleterious alleles can be maintained at relatively high frequency in commercial livestock populations due to heterozygous advantage for traits under selection [10] . Examples of such balancing selection in cattle include a frame-shift mutation in the mannose receptor C type 2 gene ( MRC2 ) responsible for crooked tail syndrome and also associated with increased muscle mass in Belgian Blue [11] , and a large deletion with antagonistic effects on fertility and milk production traits in Nordic Red breeds [12] . In pigs , balancing selection at c . C1843T mutation in the ryanodine receptor 1 ( RYR1 ) gene [13 , 14] is likely to have caused an increase in incidence of porcine stress syndrome ( also known as malignant hyperthermia ) in the 1970s and 1980s , due to the association of the causative missense mutation with reduced backfat–a trait under selection . The ryanodine receptor 1 protein RYR1 acts as a calcium release channel in skeletal muscle . The c . C1843T mutation interferes with the proper function of the calcium release channel rendering homozygotes susceptible to stress induced malignant hyperthermia and death . Involuntary muscle contraction by the leaky channel may contribute to reduced fat levels in carriers and homozygotes [15] . More recently , a study showed an unique example of allelic pleiotropy in which one allele ( deletion ) is responsible for both increased growth and late foetal mortality by affecting two different genes [16] . We investigated genetic parameters and mode of inheritance for a leg weakness syndrome causing piglet mortality in a commercial Large White terminal sire line historically marketed by JSR Genetics under the product name “Yorker” . A monogenic recessive inheritance was observed , and homozygosity mapping was used to identify a genomic region on Sus scrofa chromosome ( SSC ) 15 associated with the trait . A mutation causing a premature stop codon in exon 3 of the Myostatin ( MSTN ) gene ( similar to mutations causing the ‘double-muscling’ phenotype in cattle [17] was the outstanding functional candidate in the region . Comparison of MSTN genotype frequencies at birth and 110 kg supported this hypothesis , and carriers were shown to have significantly higher muscle mass and reduced fat depth than wild type homozygotes . Therefore , we propose that the MSTN mutant allele is highly deleterious in this population in homozygous form , but was maintained at moderate frequency due to heterozygous advantage .
The overall prevalence of leg weakness in the commercial cohort was 6 . 3% ( Table 1 ) . When only affected litters were considered , the mean proportion of affected piglets was 23% ± 0 . 7 . This within-litter prevalence is consistent with the expectation under the hypothesis of a single recessive locus ( i . e . 25% ) . Complex Bayesian segregation analysis [18] suggested that almost all the variation was explained by a single locus with almost no environmental variation . The estimate of the additive effect was 0 . 50 ± 0 . 001 and dominance effect was -0 . 50 ± 0 . 001 , which is in precise agreement with a recessive locus model . Estimates of heritability for the leg weakness syndrome ( analysed as a binary trait on the underlying liability scale ) was high ( 0 . 57 ± 0 . 10 in the sire and dam model ) with low ( 0 . 17 ± 0 . 02 and 0 . 11 ± 0 . 02 ) but significant effects observed for permanent environmental effects due to the dam and litter , respectively ( Table 2 , S1 Table ) . Homozygosity mapping was used to map the underlying recessive variant , and the longest shared homozygous segment was a region of ~ 8 . 3 Mbp on SSC15 . This unique region , contained 55 informative single nucleotide polymorphisms ( SNPs ) on the Illumina PorcineSNP60 SNP chip [19] . These 55 SNPs were homozygous in affected animals , but contained SNPs that were heterozygous or homozygous for the alternative allele in the unaffected animals , albeit there was one unaffected animal also sharing the homozygous segment . The segment started with SNP ALGA0110636 ( rs81338938 ) at position 86 , 745 , 668 in the current Sscrofa11 . 1 reference genome assembly ( Genbank assembly accession GCA_000003025 . 6 ) and finished with SNP H3GA0044732 ( rs80936849 ) at position 95 , 062 , 143 ( Fig 1A and 1B ) . The MSTN gene was located within this homozygous segment , from position 94 , 620 , 269–94 , 628 , 630 . The 8 . 3 Mbp segment was assumed to represent an identical-by-descent ( IBD ) region likely to contain the underlying causative mutation , and became the focus of further analyses to discover and characterise this mutation . The SNP genotype used in the homozygosity mapping at SSC15 is given in S3 Table . To identify candidates for the causative variant , whole genome sequence data from ten cases , six presumed heterozygous carrier dams , and 22 controls were analysed . A total of 40 SNPs identified within the homozygous segment fitted the pattern of a potential causative variant assuming a recessive mode of inheritance . Functional annotation of these SNPs revealed that 19 were intergenic , 19 were intronic , 1 was in a pseudogene , and 1 caused a premature stop codon . There were also 10 InDels identified , 3 of which were intergenic and 7 of which were intronic ( S4 Table ) . The outstanding functional candidate was a mutation in the third exon of the MSTN locus that results in the replacement of a codon for glutamic acid with a stop codon in exon 3 at position 274 ( c . 820G>T; p . E274* ) ( Fig 2A ) . The mutation is located in a region that is highly conserved across multiple species , and is predicted to result in truncation of the protein ( Fig 2B ) . Functional annotation of all other variants detected in the IBD region did not reveal any other obvious causative candidates . This stop gain mutation was not present on the Ensembl variation database , accessed 25th July 2018 . The whole genome sequencing raw reads are available in the NCBI , BioProject accession PRJNA506339 . The MSTN c . 820G>T mutation showed no statistically significant deviation from Hardy Weinberg equilibrium ( HWE ) in the 486 piglets sampled at birth ( q = 0 . 22 , α = 0 . 019 , χ2 = 0 . 18 , P > 0 . 05 ) . Random mating of the dioecious population would be expected to result in a value of α that is slightly negative [20] , and the value observed does not differ significantly from this value . However , the mutation deviated significantly from HWE at 40 kg ( q = 0 . 17 , α = - 0 . 180 , χ2 = 12 . 2 , P > 0 . 001 ) and at 110 kg ( q = 0 . 17 , α = - 0 . 210 , χ2 = 11 . 45 , P > 0 . 001 ) . This was due to the loss of homozygous mutant piglets , with all but one dying ( or being euthanized ) shortly after birth , and the remaining piglet being euthanized due to poor health before it reached 110 kg . For detailed information , see S2 Table . The large change in α in a negative direction is quantitative evidence of the selective disappearance of homozygote genotypes , as opposed to disappearance as a result of selection against the allele itself . There were no significant changes in the relative genotype frequencies ( GG , GT ) over the total period or any sub-period from birth to the end of the test , confirming all changes in q and a are due to the selective loss of homozygotes , and that any other mortality or culling was at random with respect to MSTN genotype . The association of the porcine MSTN c . 820G>T mutation with performance traits was assessed on 384 pigs which had completed a commercial performance test . Given the loss of the homozygous mutant animals the effect of the MSTN c . 820G>T mutation was only estimated by the difference between the heterozygotes and the wild type pigs . The genotype means and differences are shown in Table 3 , with the most notable of these being a major increase in muscle depth and a reduction in fat depth in the carriers ( p < 0 . 001 ) , with no evidence of a difference in live weight at 110 kg . Approximately 31% of the genetic variation in muscle depth and 18% of the genetic variation in fat depth was explained by this single variant ( Table 3 ) . The heterozygous animals had on average 5 mm increased muscle depth , and 1 . 7 mm decreased backfat depth when compared with wild type homozygous animals . Histological comparison of biceps femoris muscle between a piglet carrying the TT genotype ( putative double MSTN knockout ) and a heterozygous piglet revealed had significantly larger myofibre sizes in the homozygote ( 13 . 4 μm2 vs 11 . 5 μm2 , P < 0 . 001 ) , suggesting comparative myofibre hypertrophy . However , there was no difference in myofibre number between the two animals , which does not suggest hyperplasia ( S1 Fig ) .
A piglet leg weakness syndrome identified in a commercial line of Large White pigs showed moderate to high heritability , consistent with the upper range of estimates reported in the literature [2 , 21 , 22] . The within-litter incidence of the syndrome and the complex Bayesian segregation analysis both pointed to a monogenic recessive condition . Homozygosity mapping revealed a 8 . 3 Mbp segment on SSC15 as a putative IBD region likely to contain the causative variant . A single control animal appeared to also carry the putative IBD segment in this region , although it is possible that this animal was an affected animal misclassified as a control . The leg weakness syndrome is not lethal per se , but was debilitating under farm conditions and affected animals were typically crushed or failed to suckle effectively and were therefore typically euthanized . The outstanding functional candidate variant identified by whole genome sequencing of cases and controls caused a premature stop codon within exon 3 of the MSTN gene ( Fig 2 ) , and this variant was in HWE at birth but significantly distorted by 110 kg due to an absence of homozygous mutant animals . Knockout of MSTN by gene editing in previous studies has been associated with poor health and mortality of knockout piglets [23–26] , including observations of a piglet leg weakness syndrome with an inability to stand or walk [24] , strikingly similar to the syndrome described in the current study . MSTN is a member of the transforming growth factor beta ( TGF-β ) superfamily , which is highly conserved across species , and is typically expressed in developing and mature skeletal muscle as a key regulator of muscle growth [27] . The MSTN gene has been a gene of interest to animal breeders for over twenty years since the discovery of loss-of-function mutations in the cattle MSTN gene , which cause muscle hypertrophy leading to double muscling phenotypes [17 , 27 , 28] . Interestingly , these loss-of-function mutations causing double muscling are frequently due to premature stop mutations in the highly conserved exon 3 of MSTN [28] , ( Fig 2B ) . Further , in the case of Marchigiana beef cattle , the MSTN variant causing double muscling results is due to replacement of Glutamic Acid with a stop codon ( the same change observed in the current study ) in the equivalent position in the protein as observed in the current study [29] . This observation provides additional indirect evidence that the porcine MSTN mutation described herein is likely to cause the increase in muscle depth and decrease in fat depth associated with carrier animals . A histological analysis of muscle showed larger myofibre sizes in a homozygous TT ( putative double MSTN knockout ) than a heterozygous piglet suggesting comparative myofibre hypertrophy . A similar phenotype was observed in MSTN gene knockout piglets from a genome editing study [24] . However , unlike in another study of MSTN knockout piglets [25] , where a greater number of myofibre nuclei were observed in mutants compared with the WT group , the density of myofibres in our study was comparable between the two animals which does not suggest hyperplasia ( S1 Fig ) . In livestock breeding schemes , MSTN-inactivating mutations have been retained due to selection for increased lean growth associated with meat production , particularly for double-muscled cattle . However , despite many generations of selection for lean growth in pigs no such mutations have been reported previously . Associations between polymorphisms within the porcine MSTN gene and production traits have been shown in small-scale studies [30–32] . Further , a genome-wide association study in a sire line Large White population ( related to the animals in the current study ) detected SNPs on SSC15 significantly associated with rib fat between 81 . 1 and 87 . 8 Mbp [33] . While these SNPs are between 7 and 13 Mbp closer to the centromere than MSTN , two of these SNPs overlap with the region on homozygosity indicative of an IBD region in the current study . Therefore , it is plausible that this association may be due to linkage disequilibrium ( LD ) with the MSTN variant , or that other variants impacting performance traits exist on SSC15 . The selection index applied in this line explicitly benefited animals with positive muscle depth and negative fat depth estimated breeding values , with analysis of the index showing that heterozygotes had a slight but significant advantage ( p<0 . 05 ) . Therefore , it is likely that the MSTN c . 820G>T mutation was maintained at moderate frequency in this line despite its deleterious impact on piglet mortality due to heterozygous advantage for muscle and fat traits . Similar examples of balancing selection have observed to explain the maintenance of deleterious alleles at moderate frequency in commercial cattle [10 , 12] and pig [16] populations . The results described herein have major implications for the targeted ablation of MSTN via gene editing to increase lean growth in pigs , and provide a plausible explanation of why MSTN loss-of-function mutations have not been previously reported in pigs despite decades of selection for lean growth .
All samples were collected on a commercial nucleus farm as part of standard husbandry and management procedures in the nucleus herd , which complied with conventional UK red tractor farm assurance standards ( https://assurance . redtractor . org . uk/ ) where sick or injured livestock that do not respond to treatment are promptly and humanely euthanized by a trained and competent stockperson . A piglet leg weakness syndrome was characterised in a Large White terminal sire line , historically marketed by JSR Genetics under the product name “Yorker” , reared in a nucleus herd , under standard conditions but with additional data recording . Leg weakness trait observations were collected on 19 , 006 piglets from 2007 to 2010 , during which time a high incidence of the syndrome was detected . In addition , DNA was sampled on a further 119 piglets in 2011 and 486 piglets from the same population in 2012 , of these 384 also had weight and carcass phenotypes available . The cohort born in 2007 to 2010 will be referred to as the commercial cohort and those in 2011 and 2012 as the survey cohort . Pedigree was available for all animals and spanned 9 generations . Details of the data and pedigree structure are presented in Table 1 . The leg weakness in the phenotyped animals was visually classified as normal or affected ( 0 / 1 , respectively ) . The leg defect is characterised by the piglet not being able to straighten its legs to stand , this being most apparent for the front legs , and being slow to suckle . Detailed post mortems were conducted on two affected individuals but the results were ineffective in providing additional diagnostic aids . The Online Mendelian Inheritance in Animals database ( OMIA: http://omia . org/OMIA000585/9823/ ) was searched for previous reports of leg weakness in pigs but the syndrome observed here did not appear . These problems frequently resulted in death from either starvation or being crushed by the sow . Where the outcome resulted in poor welfare the piglets were euthanized in accordance with the Ethics Statement . Additional farrowing data were collected on the females in the commercial cohort including numbers born alive , dead or mummified , parity and year of birth . Body weights , ultrasound muscle and fat depths were obtained for individuals in the survey cohort that were retained in the herd until commercial slaughter age ( when average weight is 110 kg ) . The ultrasonic measurements taken were the average depth of the m . longissimus dorsi and overlaying subcutaneous fat layer across the last four ribs . Initial studies to establish the genetic basis of the leg weakness syndrome were undertaken by fitting linear mixed models to the commercial cohort . The binary record of the syndrome was modelled on the observed 0 / 1 scale and the full model fitted was: y=Xβ+Z1u+Z2v+Z3w+e ( 1 ) where y is the vector of leg weakness phenotypes; β , a vector of fixed effects for month of observation ( 43 df ) , sex ( 1 df ) , parity ( 7 df ) , numbers in litter born dead ( 1 df ) or alive ( 1 df ) , with design matrix X; u , additive polygenic effects , assumed to be distributed MVN ( 0 , Aσ2a ) with design matrix Z1; v , random litter effects assumed to be distributed MVN ( 0 , Iσ2v ) , with design matrix Z2; w , maternal environment effects across litters assumed to be distributed MVN ( 0 , Iσ2w ) with design matrix Z3; and e residuals assumed to be distributed MVN ( 0 , Iσ2e ) . Variations in this model were fitted replacing the individual polygenic effects with sire and/or dam effects , assumed to be distributed MVN ( 0 , Aσ2s ) and MVN ( 0 , Aσ2d ) respectively . All models were fitted using the ASREML software [34] . Likelihood ratio tests were used to assess the random effects . In addition , an analogous threshold mode with an underlying continuous liability was fitted with a logit link function and sire and dam effects associated with the pedigree , but not with individual polygenic effects , following recommendation of Gilmour et al . [34] . For the full model , the phenotypic variance was calculated as σ2p = σ2u + σ2v+ σ2w + σ2e . Where sire and dam models were used , σ2u was replaced by σ2s + σ2d . Heritability ( h2 ) was calculated as σ2u/σ2p or 2 ( σ2s + σ2d ) /σ2p depending on the model . The proportion of variance explained by the litter and maternal environmental effects were estimated as σ2v/σ2p and σ2w/σ2p , respectively . Heritabilities on the observed scale ( 0/1 ) was transformed to an underlying liability scale following Dempster and Lerner [35] using the observed prevalence of the syndrome in the commercial population which was 6 . 3% Inspection of the data suggested that the syndrome may be due to a single gene with the predisposing deleterious allele showing a recessive mode of inheritance , and this hypothesis was tested using chi square tests and segregation analyses . An initial test of a monogenic recessive mode of inheritance was carried out by pooling all affected litters , estimating the probability of being affected conditional on being born in an affected litter , and using chi-squared to test the null hypothesis that the probability of being affected was 0 . 25 . A weakness of this approach is that some litters by chance will have no affected offspring , so a more complex segregation model was fitted . This model included all known phenotypes and pedigree data , and assumed a monogenic inheritance with environmental variation fitted by Gibbs sampling [18] . Ten affected animals from different litters and 10 unaffected full-sib controls from the commercial cohort were genotyped using the Illumina PorcineSNP60 SNP chip [19] . Only those SNPs that mapped to known positions on autosomal chromosomes and were not fixed nor completely heterozygous were retained . This left 38 , 570 segregating autosomal SNPs for the use in homozygosity mapping . Homozygous regions were assessed by alignment with the Sscrofa11 . 1 reference genome assembly sequence ( Genbank assembly accession GCA_000003025 . 6 ) . The genomes of the ten cases used for homozygosity mapping and six separate dams with affected offspring , assumed heterozygotes , were whole genome shotgun sequenced on an Illumina HiSeq 2500 platform as 125 bp paired-end reads . The dams were individually sequenced with a 10x genome coverage . The piglets were barcoded and individually sequenced at 3x coverage to achieve 30x coverage for the pool . The full sequencing output resulted in ~1 . 3 billion paired-end reads with an average of 48 million paired-end reads/sample for the piglets and 157 million paired-end reads/sample for the dams . Quality filtering and removal of residual adaptor sequences was conducted on read pairs using Trimmomatic v . 0 . 32 [36] . Only reads where both pairs had a length greater than 32 bp post-filtering were retained , leaving a total of ~1 . 2 bn paired-end reads . Whole genome resequencing was followed by alignment to the Sscrofa11 . 1 assembly; using the Burrows-Wheeler Aligner with default parameters [37] . The average alignment rate of properly paired reads was of 92% . PCR duplicates were marked using Picard Tools ( http://broadinstitute . github . io/picard ) . Variant calling was performed using the Genome Analysis Toolkit ( GATK ) HaplotypeCaller after read recalibration [38] . The parameter setting for the hard filters that were applied to the raw genotypes were: QualByDepth < 2 . 0 , FisherStrand > 60 . 0 , RMSMappingQuality < 40 . 0 , MappingQualitySumTest < -12 . 5 , ReadPosRankSumTest < -8 . 0 . Candidate loci were identified from the sequences of the 16 animals from this study plus 22 additional Sus scrofa control sequences obtained from a public database [39] , comprising 7 domesticated breeds ( Duroc , Hampshire , Jiangquhai , Landrace , Large White , Meishan and Pietrain ) and wild boar . The following criteria were used to identify candidate SNPs: It is worth noting that the limited sequencing depth means that both alleles will not be detected for all bases in all individuals . This limitation is particularly relevant to the reliable detection of heterozygous SNPs . A ‘kompetitive allele specific PCR’ ( KASP ) assay was designed by LGC Genomics ( Teddington , UK ) to enable genotyping of the mutation in the MSTN stop codon in large numbers of animals . The survey cohort of 486 piglets sampled at birth were genotyped by LGC . Of these , 265 remained as candidates for the final selection at 110 kg with a complete record of their performance test , together with another 119 pigs phenotyped at slaughter age ( total n = 374 ) . In both the 486 piglets and the subsequent subsets surviving to 40 and 110 kg , the frequency of the MSTN mutation ( q ) was calculated by counting , and the departure from Hardy Weinberg equilibrium the genotypes was estimated as α = 1-Hobs/Hexp [20] where Hobs is the observed heterozygosity and Hexp is the expected heterozygosity calculated as 2q ( 1-q ) . The significance of departure from true random mating genotype frequencies ( α = 0 ) was tested using a chi-squared test . Associations between the MSTN c . 820G>T locus and variation in performance traits in commercial testing conditions were examined in the survey cohort ( n = 384 ) . The performance test was started at a target weights of 40 kg , at an average age of 85 days , and continued for 54 ( s . d . 12 ) days . The performance tests were performed over two distinct periods . The traits available were live weights and ages at the start and the end of the test , ultrasonic muscle and fat depths measured at the end of the test , days from birth to 40 kg and days from 40 to 110 kg . Univariate mixed models were fitted to these data in ASReml-R4 using the following model: y=1μ+X1β+X2b+Zu+e ( 2 ) where y is the vector of phenotypes; μ , a fitted mean , and 1 , a vector of 1’s; β , a vector of fixed nuisance effects with design matrix X1; b , a scalar fixed effect for the effect of SNP genotype with design matrix X2 , this has only 1 df due to the absence of homozygotes completing the test; u , additive polygenic effects assumed to be distributed MVN ( 0 , Aσ2a ) , with design matrix Z; and e , residuals assumed to be distributed MVN ( 0 , Iσ2e ) . For all traits , the sex of the piglet ( 1 df ) , parity of dam ( 4 df ) and period of testing ( 1 df ) were fitted as nuisance factors , together with cubic smoothing splines for the start date of the test fitted separately within each period [40] . The age at the time of measurement was fitted as covariate ( 1 df ) for all traits other than days to 40 kg and days from 40 to 110 kg . The significance of fixed effects was assessed using Wald tests [ASReml Manual] . Samples of biceps femoris were taken from one myostatin homozygous knockout piglet ( TT ) and one heterozygous piglet ( TG ) , and fixed in 10% neutral buffered formalin . After 24h of fixation , the tissues were processed to paraffin embedded blocks , and 4 μm sections were taken and stained with haematoxylin & eosin . One Tiff image was taken from each animal in an area where myofibres had been sectioned transversally using a Zeiss Axiocam 105 colour camera using the same light settings , and measurements were calibrated using a microscope graticle . For image analysis , images were loaded in FIJI ( https://fiji . sc/ ) , and for each image blood vessels were manually removed . The green channel was chosen , and the threshold adjusted to identify myofibres . This was followed by two binary functions ( ‘fill holes’ and ‘watershed’ consecutively ) , and the resulting mask was measured using the particle analyser , filtering particles below 5μm2 in size . The myofibre size was compared between the two samples using a Mann-Whitney test to assess hypertrophy . For assessment of hyperplasia , the total number of myofibres was expressed as the number of myofibres per 100 μm2 . | Lameness is an important problem in livestock production for both animal welfare and economic reasons . A severe piglet lameness syndrome was observed in a commercial pig population . The incidence of the condition was low ( 6 . 3% ) , but was higher in affected families ( ~25% ) , which suggested a genetic basis and a recessive mode of inheritance . We discovered a region on Chromosome 15 where cases shared the same alleles that were different to healthy piglets . In this region , we discovered a mutation that causes a premature stop codon in the myostatin gene . Myostatin causes ‘double-muscle’ phenotype in several mammalian species . Piglets with two copies of this mutant allele suffer the lameness syndrome and do not survive post 40 kg live weight . However , those that carry a single copy have higher muscle depth and lower fat depth compared to wild type . We suggest that despite the negative consequences of the mutant allele in homozygous form , the mutation was maintained in the herd due to positive selection for this allele in heterozygous form . This is an interesting example of so-called ‘balancing selection’ and may explain why naturally occurring myostatin mutations have not previously been reported in pigs despite centuries of selection for lean growth . | [
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"loci",
"agricul... | 2019 | Balancing selection at a premature stop mutation in the myostatin gene underlies a recessive leg weakness syndrome in pigs |
The γ-proteobacterial plant pathogen Pseudomonas syringae pv . tomato DC3000 uses the type III secretion system to inject ca . 28 Avr/Hop effector proteins into plants , which enables the bacterium to grow from low inoculum levels to produce bacterial speck symptoms in tomato , Arabidopsis thaliana , and ( when lacking hopQ1-1 ) Nicotiana benthamiana . The effectors are collectively essential but individually dispensable for the ability of the bacteria to defeat defenses , grow , and produce symptoms in plants . Eighteen of the effector genes are clustered in six genomic islands/islets . Combinatorial deletions involving these clusters and two of the remaining effector genes revealed a redundancy-based structure in the effector repertoire , such that some deletions diminished growth in N . benthamiana only in combination with other deletions . Much of the ability of DC3000 to grow in N . benthamiana was found to be due to five effectors in two redundant-effector groups ( REGs ) , which appear to separately target two high-level processes in plant defense: perception of external pathogen signals ( AvrPto and AvrPtoB ) and deployment of antimicrobial factors ( AvrE , HopM1 , HopR1 ) . Further support for the membership of HopR1 in the same REG as AvrE was gained through bioinformatic analysis , revealing the existence of an AvrE/DspA/E/HopR effector superfamily , which has representatives in virtually all groups of proteobacterial plant pathogens that deploy type III effectors .
Many bacterial pathogens of plants and animals disarm and remodel host cells by injecting large repertoires of effectors via the type III secretion system ( T3SS ) [1] , [2] . In some cases , the repertoires of individual strains appear to function as robust systems that can tolerate loss of individual effectors with little or no reduction in virulence because of high-level functional overlap among the effectors [3] . The potential extent of such redundancy is highlighted by recent reports that enterohemorrhagic Escherichia coli 0157:H7 may inject 39 effectors into animal cells via the T3SS and that fungal and oomycete pathogens , using alternative protein translocation pathways , may deliver hundreds of effectors into plant cells [4]–[7] . Unraveling functional redundancy among effectors can potentiate the study of individual effectors through the design of mutants with more easily assayed phenotypes , and elucidation of functional overlaps should help us understand how the various effectors in a repertoire may function as a system in hosts . Pseudomonas syringae pv . tomato DC3000 , which causes bacterial speck of tomato , is an excellent model for investigating the possible operation of type III effector repertoires as systems . The DC3000 genome has been fully sequenced [8]; the DC3000 T3SS , which is encoded by hrp/hrc ( hypersensitive response and pathogenicity or conserved ) genes , is being intensively investigated [9]–[12]; multiple approaches have been used to firmly establish the effector repertoire [13]–[15]; the molecular function of several of these effectors in plants has been determined [16]–[18]; DC3000 can infect the experimentally tractable plants Arabidopsis thaliana and Nicotiana benthamiana [19] , [20]; and this strain has become the pathogen of choice for plant biologists probing the plant defense systems thought to be targeted by the effectors . DC3000 appears to actively deploy 28 effectors and several other proteins associated with extracellular functions of the T3SS [14] , [21] . The genome also harbors 12 effector pseudogenes and seven effector genes that appear only weakly expressed [14] , [21] , [22] . The majority of the active effector genes occur within six clusters in the DC3000 genome [23] . By deleting various effector gene clusters and individual genes , we have shown that effector HopQ1-1 ( Hop denotes Hrp outer protein ) acts as an avirulence determinant in N . benthamiana , that deleting hopQ1-1 enables DC3000 to cause bacterial speck disease in N . benthamiana ( which is otherwise a nonhost ) , and that deleting four of the clusters ( encoding 12 effectors ) strongly reduces symptom production in N . benthamiana but surprisingly does not significantly reduce bacterial growth [20] . According to the current model for P . syringae-plant interactions [24] , the primary function of the effectors is to suppress PAMP ( pathogen-associated molecular pattern ) -triggered immunity ( PTI ) , which is elicited by common bacterial factors like flagellin , LPS , peptidoglycan , and elongation factor Tu . PAMPs are perceived by pattern recognition receptors ( receptor-like kinases ) at the surface of plant cells . A second layer of defense involves detection inside plant cells of injected effectors or their activity by resistance ( R ) proteins , which results in effector-triggered immunity ( ETI ) . Pathogens may evade ETI by mutating the betraying effector gene or by deploying another effector that suppresses ETI . This model predicts a coevolutionary process that would generate the observed amplification of effector genes in pathogens and of R genes in plants , and the model also predicts interplay among effectors in redundantly targeting PTI and suppressing ETI . Three observations support the prediction of interplay among effectors in a repertoire . In the first example , loss of virPphA ( hopAB1 ) from P . syringae pv . phaseolicola 1449B resulted in avirulence in bean , the normal host , because of failure to suppress ETI triggered by another effector in the 1449B repertoire [25] . The other two examples of effector interplay involve work with DC3000 in host tomato and provide a foundation for the work presented here . Although mutations in individual P . syringae effector genes typically produce little or no loss in growth in planta , an ΔavrEΔhopM1 double mutant and an ΔavrPtoΔavrPtoB double mutant were found to be significantly reduced in growth in tomato [26] , [27] . The avrPto and avrPtoB genes are unlinked in the DC3000 genome , but the observation that these two effectors make redundant contributions to virulence is consistent with the observation that they target the FLS2/BAK1 pattern recognition receptor complex important in PAMP detection [28] , [29] . The avrE and hopM1 genes are located in DC3000 effector cluster VI [23] , which is known as the conserved effector locus ( CEL ) [30] . The CEL forms part of the tripartite Hrp pathogenicity island of P . syringae , and avrE , hopM1 , and hopAA1-1 are present in this location in diverse P . syringae strains [13] . We had previously shown that deletion of the CEL region encompassing avrE , hopM1 , and hopAA1-1 strongly reduces DC3000 virulence [30] . AvrE and HopM1 appear to play a major role in promoting cell death in both host and nonhost plants . This ability is not attributable to avirulence activity , as indicated by experiments involving host tomato and nonhost tobacco ( Nicotiana tabacum ) challenged with DC3000 mutants lacking both effectors or with Agrobacterium-mediated transient expression of AvrE in these plants [26] , [31] . Importantly , the ΔCEL mutant fails to suppress PTI-associated callose deposition in Arabidopsis , and either avrE or hopM1 can redundantly restore callose suppression and bacterial growth to the ΔCEL mutant [32] . Here , we extend our analysis of DC3000 effector gene cluster polymutants by deleting cluster I and the CEL to produce a strain lacking all 18 of the clustered effector genes . We explore the basis for the failure of the resulting mutant to grow well in N . benthamiana , and in so doing we identify effector gene clusters that functionally overlap with the CEL . This analysis leads to HopR1 in cluster IV , which we show to be a member of an effector superfamily that also contains the AvrE family and which also contributes to the suppression of PTI-associated callose deposition in N . benthamiana leaves inoculated with DC3000 . We then explore the effects of deleting the flagellin PAMP-encoding fliC gene on the growth of DC3000 effector polymutants . Our observations lead to the proposal that AvrE/HopR1/HopM1 and AvrPto/AvrPtoB form redundant-effector groups ( REGs ) that make major contributions to DC3000 growth in N . benthamiana by targeting different steps in PTI .
As shown in Figure 1A , 18 of the DC3000 active effector genes occur in 6 clusters ( with the duplicated hopAM1-1 and hopAM1-2 genes counted as just hopAM1-1 in this tally ) . We used pK18mobsacB as before to construct unmarked deletions involving cluster I and the CEL in various mutants lacking other effector genes ( Figure 1B ) [20] . We then analyzed the ability of these mutants to grow and produce symptoms in N . benthamiana leaves when inoculated by syringe infiltration or dipping and to grow in tomato leaves when inoculated by syringe infiltration ( Figure 1 C–1G ) . With the exception of the ΔhrcQB-hrcU ( T3SS− ) strain , all mutants carried deletions affecting hopQ1-1 or the entire cluster IV , which is necessary to avoid HopQ1-1-mediated avirulence in N . benthamiana . The ΔCEL mutant showed a significant reduction in growth in dip-inoculated N . benthamiana , and was strongly reduced in growth in tomato , as was previously observed with ΔhopAA1-1ΔhopM1ΔavrE and ΔhopM1ΔavrE CEL mutants [26] , [30] . Deletion of cluster I in a ΔIIΔIVΔIXΔX background produced no virulence reduction in any of our tests . However , a strong reduction in growth in planta and a complete loss of symptom production was observed when the CEL was deleted from the ΔIΔIIΔIVΔIXΔX background to produce mutant CUCPB5500 , which lacks all 18 of the clustered effector genes . A CUCPB5500 derivative was confirmed to perform as well as DC3000 in growth on Hrp-inducing minimal medium and mannitol-glutamate MG medium and in translocation of AvrPto-Cya , expressed from its native promoter in pCPP5702 [11] , into N . benthamiana when inoculated at 1×108 or 1×107 CFU/ml ( Figure S1 ) . Thus , the failure of ΔIΔIIΔIVΔCELΔIXΔX DC3000 to grow well in N . benthamiana can be attributed to a subset of the 18 missing effectors that is important in plant interactions . Several aspects of these observations are notable . First , dipping leaves in inoculum is considered a more natural method of inoculation than infiltration with a blunt syringe and a better test for subtle reductions in virulence . Nonetheless , we saw only minor differences in the relative symptoms in N . benthamiana leaves produced by our panel of mutants using the two inoculation methods ( Figure 1C and 1E ) , and the relative growth in the differently inoculated leaves was essentially identical ( Figure 1D and 1F ) . Given the convenience and higher reproducibility of the syringe infiltration assays , we used this method in all subsequent experiments . Second , the CEL was more important for growth in tomato than in N . benthamiana . However , the strong reduction in growth in N . benthamiana resulting from deleting the CEL from the ΔIΔIIΔIVΔIXΔX mutant revealed the underlying importance of the CEL and suggested that one or more effectors encoded outside of the CEL acts redundantly with CEL effectors . Third , results of virulence and growth assays of the ΔIΔIIΔIVΔCELΔIXΔX mutant suggest that the remaining 10 effectors were not sufficient to promote more than minimally significant growth of the mutant above the T3SS− mutant in all plant tests . To identify any effector gene clusters that function redundantly with the CEL we introduced the ΔCEL mutation into the ΔhopQ1-1 mutant and the lineage of mutants that were used to construct the ΔIΔIIΔIVΔCELΔIXΔX strain . The mutants were inoculated into N . benthamiana leaves and assayed for growth 6 days later . As shown in Figure 2A the ΔIVΔCEL mutant was significantly impaired in growth relative to the ΔhopQ1-1ΔCEL mutant . Additional deletions of clusters X , II , and I had no significant effect , but deletion of cluster IX from a ΔIIΔIVΔCELΔX mutant resulted in a further reduction in growth . Interestingly , all mutants with the ΔCELΔIX mutation no longer elicited chlorosis in N . benthamiana leaves ( Figure 2B ) . A slight reduction in growth of the ΔIV mutant relative to the ΔhopQ1-1 mutant was also observed ( Figure 2C ) , however , the reduction was not significant in this experiment and was absent or insignificant in five of eight repeated experiments; in contrast , in all experiments , the ΔIVΔCEL mutant was significantly impaired in growth relative to the ΔhopQ1-1ΔCEL mutant ( data not shown ) . This suggests that the ΔIVΔCEL mutant growth impairment does not result from an additive effect of mutations involving cluster IV and the CEL . The cluster IX deletion was introduced when the lineage already contained multiple effector gene cluster deletions . Therefore , to uncover underlying functional redundancies of other clusters with cluster IX , we constructed pairs of deletion polymutants lacking hopQ1-1 or cluster IV along with additional clusters , with one member of each pair also lacking cluster IX . This revealed that deleting cluster IX had little impact on the growth of the ΔhopQ1-1 , ΔCEL , or ΔIVΔCEL mutants ( Figure 2C ) . However , the ΔIΔIIΔIVΔCELΔIXΔX mutant was more strongly reduced in growth than the ΔIVΔCELΔIX mutant , which suggested that cluster II or cluster X was acting redundantly with cluster IX . We constructed another series of deletions in which every strain carried the ΔIVΔCEL mutations plus additional deletions involving cluster IX and candidate redundant clusters . Deletion of cluster II from the ΔCELΔIVΔIX mutant significantly reduced bacterial growth , and no further reduction was observed upon further deletion of clusters I and X ( Figure 2D ) . Thus , deletion of the CEL is sufficient to reduce bacterial growth in N . benthamiana , but the stronger growth reductions observed with polymutants reveal that a cluster IV effector other than HopQ1-1 functions redundantly with the CEL , and in a ΔIVΔCEL background further potential redundancies with clusters II and IX are uncovered . The experiments above suggested that an effector in cluster IV was functionally redundant with CEL effectors . Cluster IV contains hopD1 , hopQ1-1 , and hopR1 . We cloned hopD1 and hopR1 and the CEL effector genes hopM1 and avrE ( and their respective chaperone genes , shcM and shcE ) under the direction of the avrPto promoter in the broad-host-range vector pBBR derivative pCPP5372 [12] . These constructs were then expressed in the ΔIVΔCEL mutant . The resulting strains were inoculated into N . benthamiana leaves and assayed for growth 6 days later . Growth of the ΔIVΔCEL mutant was substantially less than that of the ΔIV mutant , and as expected , the positive control clones shcM-hopM1 and shcE-avrE significantly enhanced growth ( Figure 3A ) . Growth was not enhanced by expression of hopD1 , but it was by expression of hopR1 . Thus , HopR1 has functional redundancy with CEL effectors . The ΔhopQ1-1ΔCEL mutant is much more strongly reduced in growth in tomato than in N . benthamiana ( Figure 1 ) . One possible explanation for this is that HopR1 makes less of a contribution to bacterial growth in tomato than in N . benthamiana , thus reducing functional redundancy with CEL effectors . To test this hypothesis , we inoculated tomato leaves with the ΔIVΔCEL strain expressing hopD1 , hopR1 , shcM-hopM1 , or shcE-avrE . As expected , HopD1 did not enhance growth , whereas HopM1 and AvrE did so strongly ( Figure 3B ) . HopR1 produced a slight increase in growth that was not significantly different from the ΔIVΔCEL parent strain in the context of the higher variability in bacterial growth in tomato leaves . Although we cannot conclude that HopR1 makes absolutely no contribution to bacterial growth in tomato , it appears that the contribution of HopR1 , relative to HopM1 and AvrE , is significantly less in tomato than in N . benthamiana . BLAST-P with HopR1 from DC3000 generates hits to putative HopR1 homologs in Xanthomonas campestris pv . campestris and Ralstonia solanacearum ( see http://www . ncbi . nlm . nih . gov/sutils/blink . cgi ? pid=28868103 ) . It also reveals a low quality hit ( E-value = 0 . 042 ) to AvrE from Pseudomonas cichorii . We subsequently found that RSp1281 , the HopR1 homolog from R . solanacearum GMI1000 , generates BLAST-P hits with lower scores to AvrE from P . viridiflava and P . syringae ( see http://www . ncbi . nlm . nih . gov/sutils/blink . cgi ? pid=17549500 ) . To investigate the relationship between AvrE and HopR1 in more detail , we performed a PSI BLAST analysis using the DC3000 HopR1 and stringent inclusion criteria ( E-value<1 . 0E-10 ) . Hits to AvrE occurred in the second iteration and hits to E . amylovora DspA/E occurred in the third iteration . A selection of third iteration PSI BLAST hits was made to maximize strain and species diversity and then used to generate an alignment and neighbor-joining tree using MUSCLE [33] , which was subsequently displayed using NJplot ( Figure 4 ) . This analysis revealed that HopR1 is a member of a superfamily that is comprised of AvrE , DspA/E , and HopR families . BLAST-P analysis also revealed a large number of hits at the C-terminus of the DC3000 HopR1 to metazoan cytoskeleton-interacting proteins , such as rootletin and myosin . This C-terminal domain only aligns for HopR1 homologs from DC3000 , P . syringae pv . phaseolicola 1448a , and X . campestris pv . campestris . Also , analysis of the C-terminus of the X . campestris pv . campestris HopR1 homolog yielded a hit in CDD search to the PFAM02463 SMC_N terminal domain ( cluster ID 410621 ) . Alternatively , a more detailed analysis of the BLAST alignments suggests that the regions of similarity at the C-termini may be more generic , attributable to the presence of coiled-coil domains . Such domains are found in most of the BLAST-P hits to HopR as well as to SMC_N domains , and they have very regular primary structures . To check for the presence of coiled-coil domains , members of the AvrE/DspA/E/HopR superfamily were analyzed using the coiled-coil predictor COILS [34] . The three HopR homologs with the extended C-terminus had coiled-coil domains in that region , which were strongly predicted by COILS . Additionally , most members of the family that were analyzed had a predicted coiled-coil domain near the middle of the protein ( in the vicinity of residue 1000 ) . P . syringae T3SS-deficient mutants and DC3000 ΔCEL mutants are reduced in their ability to suppress the formation of callose deposits , which are associated with papillae that form as appositions inside plant cell walls and which provide a useful assay for cell wall-based PTI defenses [32] , [35] , [36] . Because the expression of avrE in trans has been shown to restore suppression of callose formation to a DC3000 ΔCEL mutant in Arabidopsis [32] , we were curious to see if AvrE and HopR1 could function redundantly in the same assay in N . benthamiana . We accordingly inoculated N . benthamiana leaves with the ΔIVΔCEL mutant and with the mutant expressing shcE-avrE , hopD1 , or hopR1 under the direction of the avrPto promoter in the broad-host-range vector pBBR derivative pCPP5372 and after 12 h assayed for the formation of aniline blue-stained deposits by epifluorescence microscopy . The ΔIVΔCEL mutant elicited the formation of ca . 100-fold more callose deposits than was observed with the buffer mock inoculation ( Figure 5 ) . The ΔIVΔCEL mutant expressing hopD1 in trans elicited at least as many callose deposits , whereas the mutant expressing either avrE or hopR1 expressed substantially fewer . The complementation assays presented in Figures 5 and 3 , respectively , suggest that expression of hopD1 in the ΔIVΔCEL mutant may elicit stronger defenses and interfere with growth in N . benthamiana , whereas expression of hopR1 suppresses defenses and promotes growth in N . benthamiana . The work described above revealed that among the 18 effector genes occurring in clusters in the DC3000 genome , those in the CEL and hopR1 in cluster IV make the largest contribution to the growth of ΔhopQ1-1 DC3000 in N . benthamiana . Among the effector genes outside of the clusters , avrPto and avrPtoB are likely to be particularly important because an ΔavrPtoΔavrPtoB mutant had been shown to be significantly reduced in virulence in tomato [27] . To foster analysis of the ΔavrPtoΔavrPtoB mutant in N . benthamiana and comparison with effector gene cluster polymutants , we introduced the ΔhopQ1-1 mutation into DC3000ΔavrPtoΔavrPtoB [27] . The ΔhopQ1-1 , ΔhopQ1-1ΔavrPtoΔavrPtoB , and ΔIVΔCEL mutants were inoculated into N . benthamiana leaves and assayed for growth 6 days later . The ΔhopQ1-1ΔavrPtoΔavrPtoB and ΔIVΔCEL mutants were significantly reduced in growth relative to the ΔhopQ1-1 strain ( Figure 6 ) . As previously shown with the ΔavrPtoΔavrPtoB mutant in tomato [27] , growth of the ΔhopQ1-1ΔavrPtoΔavrPtoB was partially restored by complementation with avrPtoB ( Figure S2 ) . The ΔhopQ1-1ΔavrPtoΔavrPtoB and ΔIVΔCEL mutants were reduced in growth to a similar degree in N . benthamiana ( Figure 6 ) . The primary function of the P . syringae effector repertoire is thought to be the suppression of PTI , and it appears that flagellin is a particularly important PAMP in P . syringae-plant interactions [37] , [38] . To explore the relative role of the CEL-HopR1 and AvrPto/AvrPtoB effector groups in defeating flagellin-triggered immunity , we deleted the fliC flagellin gene from the three strains analyzed above in Figure 6 . Loss of flagellin had no significant effect on the growth of the ΔhopQ1-1 and ΔIVΔCEL strains , but it significantly enhanced the growth of the ΔhopQ1-1ΔavrPtoΔavrPtoB strain .
A consideration of the effector repertoires of other strains and the P . syringae effector pan-genome is useful in interpreting our results . P . syringae strains are host-specific and are assigned to more than 50 pathovars based largely on host specificity in the field . The effector repertoires have been established for fully sequenced strains in three pathovars – P . syringae pv . tomato DC3000 ( bacterial speck of tomato , Arabidopsis and N . benthamiana ) , P . syringae pv . phaseolicola 1448A ( halo blight of bean ) , and P . syringae pv . syringae B728a ( brown spot of bean and N . benthamiana ) – and recently for draft-sequenced P . syringae pv . tomato T1 ( bacterial speck of tomato ) [13] , [14] , [21] , [39] , [40] . In general , there are only a few effectors that appear to be universally deployed: AvrE , HopI1 , AvrPtoB ( or other members of the HopAB family ) , and HopAF1 ( HopM1 and HopAA1 also appear universal but are truncated in many strains ) [13] . Beyond these universal effectors , the repertoires are remarkably diverse , which is consistent with the observation that effector genes are typically associated with mobile genetic elements and horizontally acquired islands in the genome , and there is no evidence that the effector genes in any island ( other than the CEL ) are clustered by function [23] . Importantly , there appears to be no pattern among the variable effectors in the sequenced strains that would predict host range [13] , [20] , [39] , and well-studied variable effectors such as AvrPto appear to have the same PTI-suppressive activity in diverse plants . Similarly , deleting DC3000 effector cluster IX ( carrying 4 variable effector genes ) results in reduced symptom formation in Arabidopsis , tomato , and N . benthamiana [20] , which is consistent with the partially reduced virulence of hopAO1 mutants that was previously observed [41]–[43] . Interestingly , the DC3000 hopAO1 mutants showed a significant reduction in growth per se in tomato and Arabidopsis , which we did not observe with ΔIX mutants in N . benthamiana except in combination with other effector gene cluster deletions . It will be interesting to see in the future if this possible difference in the importance of HopAO1 results from differences in N . benthamiana targets of HopAO1 or functional redundancy with other effectors that work better in N . benthamiana . In this regard , it is noteworthy that N . benthamiana is unusually susceptible to a wide range of plant viruses and other pathogens , although importantly , not all P . syringae pathovars are virulent on N . benthamiana [20] , [44] . It is also important to note here that deletion of individual effector genes from P . syringae pv . syringae B728a resulted in increased virulence indicative of weak avirulence activity , in a differential manner , in the two susceptible plants tested: bean and N . benthamiana [39] . This finding further supports the widespread importance of effector interactions with the R-protein surveillance system , which we consider to be fundamentally more polymorphic than the virulence targets of the effectors . However , there is clearly some polymorphism in the PTI defense system , as exemplified by the lack of a functional EFR ( elongation factor-Tu receptor ) in N . benthamiana [45] . Similarly , our observation that HopR1 has a stronger capacity to promote growth in N . benthamiana than in tomato would be consistent with polymorphism between the PTI defenses of these plants . Because the distribution of effectors in the sequenced strains shows no relationship with host range , we favor the hypothesis that most effectors are “generalists” with targets that may sometimes be polymorphic , rather than “specialists” with targets unique to different plant groups and therefore essential for the inclusion of that plant group in the pathogen's host range . In summary , these observations suggest that any effector in the super-repertoire of the P . syringae pan-genome ( which currently comprises 42 effector families: http://pseudomonas-syringae . org ) could promote virulence in many ( if not all ) plants , barring detection by an R protein . If the many effectors in the pan-genome are functionally interchangeable in a wide range of potential hosts , what selection pressures , if any , constrain the composition of effector repertoires other than evasion of R-protein surveillance ? We suggest that two types of relationships among effectors might underlie the canonical observation that effectors are collectively essential but individually dispensable . First , the majority of effectors may act independently to defeat PTI through a massively redundant “death by a thousand cuts” strategy . Second , much of the redundancy may be organized around REGs , which attack host defenses in some coordinated way . The key to differentiating between these models is to determine the extent of interplay among effectors . Effector interplay can be detected through both gain-of-function and loss-of-function experiments . The former have been particularly useful in identifying potential interplay involving defined pathogenic processes , such as ETI suppression . For example , nonpathogenic P . fluorescens expressing a cloned P . syringae T3SS and HopA1 ( an effector with avirulence activity in N . tabacum ) was used in a survey for DC3000 effectors that could suppress the HopA1-triggered hypersensitive response when expressed in trans [46] . A recent , powerful example of the gain-of-function approach involves the six Salmonella effectors that manipulate the host cytoskeleton to promote pathogen internalization into non-phagocytic intestinal cells [47] . Augmented expression of effector pairs in interacting bacteria and transfected cultured cells revealed both synergistic and antagonistic relationships among these effectors in promoting internalization . Faced with 28 effectors and the resulting vast number of potential gain-of-function combinations , we chose to identify interplaying effectors by constructing combinatorial deletions and assaying for reduced bacterial growth in planta . By exploiting the clustering of effector genes we could eliminate 18 effectors with just six genetic manipulations , and the anticipated internal redundancy within the CEL promised to further simplify the problem . Importantly , any virulence phenotypes resulting from deletions could be cleanly attributed to the natural function of the relevant effectors ( expressed natively and in the context of the remaining repertoire and other virulence factors ) , which contrasts with experiments involving heterologous expression . By assaying for bacterial growth , we assessed the overall parasitic ability of mutants independently of any disease sub-processes , such as lesion formation , chlorosis , or defense suppression , which can be uncoupled from each other and from growth in complex ways . N . benthamiana is ideal for growth assays because of its large and easily inoculated leaves , the reproducibility of the data , and the possibility of rapidly testing the role of candidate host factors using virus-induced gene silencing . Importantly , our approach of constructing multi-effector deletions , beginning with our previously reported CEL deletion , produces DC3000 derivatives with easily assayed phenotypes for functional analysis of individual effectors through complementation in near-native backgrounds [30] , [32] . Recognition of the AvrE/DspA/E/HopR superfamily was one useful outcome of our work . AvrE was the first effector to be characterized in DC3000 [48] , but the AvrE/DspA/E/HopR superfamily and the relationship between HopR1 and AvrE in DC3000 had not been fully recognized before . HopR1 ( formerly HolPtoR ) had previously been observed to be a homolog of the R . solanacearum GMI1000 RSp1281 and X . campestris . pv . campestris ATCC33913 Xcc1089 proteins [3] , [49] , and RSp1281 orthologs recently were noted in several sequenced plant pathogens [50] . The PSI-BLAST and MUSCLE analyses performed here define the AvrE/DspA/E/HopR superfamily and suggest that it is present in virtually all groups of T3SS-dependent plant pathogens with the notable exception of some xanthomonads that carry the AvrBs3 effector family , which is a highly amplified and important effector family that behaves like a REG in these strains [51] . Proteins in the AvrE/DspA/E/HopR superfamily are large ( ca . 2 , 000 amino acids ) , and regions of similarity are found across the length of the proteins , although the predicted C-terminal coiled-coil domain in the DC3000 HopR1 and Xcc1089 proteins is lacking from RSp1281 and AvrE . There is evidence for at least one member of each family in the AvrE/DspA/E/HopR superfamily contributing to virulence . For example , in the HopR family , the X . campestris pv . campestris 8004 ortholog was identified in a transposon screen for mutants with reduced virulence [52] . DspA/E has been shown to be essential for the virulence of Erwinia amylovora [53] , and it is partially interchangeable with the DC3000 AvrE [54] . AvrE was the one effector identified in a transposon screen for DC3000 mutants with reduced virulence in a sensitive Arabidopsis dip inoculation assay [55] . It is also noteworthy that the RSp1281 , hopR1 , and avrE genes are considered to be ancient constituents of their respective genomes , which further points to the importance of this superfamily in plant pathogenesis [49] , [50] . The pattern of growth defects attending our combinatorial deletions does not support the hypothesis that the DC3000 effector repertoire defeats N . benthamiana through a “death by a thousand cuts” strategy . Notably , deletions involving a majority of the clustered effectors had no impact on growth . Instead , the deletion phenotypes revealed a functional architecture in the effector repertoire that is structured around REGs , whose members are self-identified by phenotype . For example , HopR1 makes a significant contribution to growth only in the context of the CEL deletion , and was thus determined to be in the same REG ( and only later did we recognize the sequence similarity between AvrE and HopR1 ) . The further finding that deletions involving AvrE/HopR1/HopM1 and AvrPto/AvrPtoB produced equivalently strong reductions in bacterial growth in N . benthamiana suggests a model for DC3000 pathogenesis involving these two REGs targeting different high-level processes in PTI ( Figure 7 ) . According to this model , AvrPto and AvrPtoB functionally overlap in blocking PAMP perception and signal transduction . Therefore , removing both of these effectors is necessary to fully unblock PAMP signal processing for robust PTI development in infected plants . This part of the model is supported by ( i ) recent biochemical evidence that AvrPto and AvrPtoB target the FLS2/BAK1 complex needed for flagellin perception [28] , [29] , ( ii ) our observation that deleting fliC compensates for the loss of these two effectors , and ( iii ) the observation that flagellin may be a particularly important PAMP in bacterial interactions with N . benthamiana because this plant lacks the EFR pattern recognition receptor kinase needed for recognition of another important bacterial PAMP , elongation factor Tu [45] . AvrE , HopR1 , and HopM1 are proposed to overlap in blocking a different high-level process in PTI: deployment of antimicrobial factors , including cell wall defenses associated with callose formation . This hypothesis is supported by ( i ) the sequence similarity of AvrE and HopR1 , ( ii ) multiple observations of functional redundancy between AvrE and HopM1 [26] , [32] ( and now HopR1 ) , ( iii ) the evidence that HopM1 blocks the vesicle trafficking that is likely important for deploying antimicrobial factors [56] , ( iv ) multiple observations that proteins in the DspA/E , AvrE , and HopM1 families can promote cell death in compatible host cells [26] , [31] , [57] , [58] , which is consistent with their disruption of a common cellular process , such as vesicle trafficking , and contrasts with the lack of such an effect by AvrPto and AvrPtoB in compatible hosts , ( v ) the failure of the fliC deletion to compensate for the loss of the AvrE/HopR1/HopM1 REG , and ( vi ) the ability of HopR1 to function similarly to AvrE in suppressing callose formation . Also , it is noteworthy that a knockout of the AtMIN7 target of HopM1 only partially restores growth of ΔCEL DC3000 in Arabidopsis , whereas treatment of Arabidopsis with Brefeldin A , an inhibitor of vesicle trafficking , restores growth to wild-type levels [56] . Although this effect was attributed to additional activities of HopM1 , we find attractive the alternative hypothesis that the inhibition of vesicle trafficking by the CEL results from AvrE as well as HopM1 . What selection pressures drive the formation of a REG and expansion of its membership ? Recent advances support the following scenario for the evolution of the AvrPto/AvrPtoB REG [29] , [59]: plants countered the PTI-suppressive kinase-inhibitory activity of these effectors by evolving kinase decoys guarded by R proteins , and the resulting ETI drove pathogen evolution for the acquisition and substitution of alternative effectors in the same REG . The coevolutionary dynamics of the AvrE/HopR1/HopM1 REG are less clear . These are all considered ancient effectors in P . syringae [49] , but intriguingly , no cognate R protein or case of gene-for-gene , race-specific resistance against these effectors has been reported . For example , avrE was originally cloned from P . syringae pv . tomato PT23 on the basis of its ability to confer avirulence to P . syringae pv . glycinea race 4 in soybean [60] , and it was later found to confer such avirulence to all cultivars tested [61] . It is possible that ancient effectors like AvrE are indeed under R-protein surveillance , but this is obscured by ETI suppressors . However , our combinatorial deletion analysis has not supported this hypothesis . In contrast , the observation that AvrE , DspA/E , WtsE , and HopM1 can promote cell death in compatible hosts raises the possibility that the ETI system may detect general perturbations in the process ( most likely vesicle trafficking ) that is targeted by this REG in a manner that is independent of the specific structure and mechanistic details of the individual members of the group . While the existence of multiple effectors within each REG would confer robustness to pathogens that attack a limited set of vulnerable processes in plants , our data suggest a high-level redundancy among these targets that provides robustness to the defense system . That is , it appears that no single REG is sufficient to confer maximal pathogen growth . As depicted in Figure 7 , PAMP perception and antimicrobial deployment are serial processes , and each process has a secondary “backup” pathway , which may be targeted by other effectors . Thus , the strong reduction in bacterial growth observed with the ΔavrPtoΔavrPtoB mutant indicates that these effectors indeed target the major process in PAMP perception in N . benthamiana . But there apparently exists a backup pathway for PAMP perception . Otherwise the ΔIVΔCEL mutant and other effector gene cluster mutants that we constructed would all grow to wild-type levels because AvrPto/AvrPtoB would always block the first step in the PTI process . That is , AvrPto/AvrPtoB are necessary for robust growth in N . benthamiana ( as seen with the ΔavrPtoΔavrPtoB mutant ) , but they are not sufficient for growth ( as seen with the ΔIΔIIΔIVΔCELΔIXΔX mutant ) . The converse argument can be made for redundancy in the high-level process of deploying antimicrobial factors , which is strongly but incompletely blocked by the AvrE/HopR1/HopM1 REG . We postulate that the apparent redundancy in high-level PTI processes requires P . syringae to deploy multiple REGs to adequately suppress PTI . It is also worth noting here that REGs may differ in their internal redundancy and relative importance in different plants . Thus , the AvrE/HopR1/HopM1 REG appears to have less internal redundancy in tomato than in N . benthamiana because of the limited efficacy of HopR1 in tomato , and in contrast AvrPto/AvrPtoB appear to be more important in N . benthamiana than in tomato ( Figures 1 and 6 ) [27] . Quantitative differences in the defense systems of potential hosts may be another factor promoting the expansion and diversity of P . syringae effector repertoires . It is possible that combinatorial deletions involving the eight remaining effector genes outside of the clusters will reveal additional REGs in DC3000 . The widespread effectors HopE1 , HopI1 , and HopAF1 are of particular interest . HopAI1 , although apparently not actively deployed by DC3000 , is a phosphothreonine lyase that inactivates PTI-associated mitogen-activated protein kinases ( MAPKs ) and therefore represents a good candidate for another P . syringae effector targeting PAMP perception and signaling [62] , [63] . It is also possible that the minimal set of DC3000 effectors needed to defeat plants will include REG representatives plus other effectors that act more globally to remodel plant metabolism , such as HopU1 and HopT1-1 , which ADP-ribosylate RNA-binding proteins and suppress the microRNA pathway , respectively [18] , [64] . Additional complexities include the apparent multi-domain structure of many effectors and potential membership in more than one REG . A comprehensive picture of the molecular activities of effectors inside plant cells is beginning to develop [1] , [65] . Our combinatorial deletion analysis complements these studies and provides resources and testable hypotheses for further analysis of effector repertoires . For example , DC3000 REG polymutants could be used to screen effectors in the P . syringae pan-genome or in fungi and oomycetes ( appropriately modified for delivery by the T3SS [6] , [66] ) for their high-level PTI targets , as indicated by REG-specific rescue of bacterial growth . Indeed , there is partial precedence for this in the ability of the Hyaloperonospora parasitica effector ATR13 to restore bacterial growth and callose suppression in Arabidopsis to a DC3000 ΔCEL mutant [66] . Although ATR13 also enhances the virulence of wild-type DC3000 in Arabidopsis , it is possible that an analysis of the relative ability of this effector to restore growth in planta to the ΔavrPtoΔavrPtoB and ΔIVΔCEL mutants could reveal a clear difference indicative of the primary high-level target . Hopefully , approaches such as this , which exploit DC3000 and its well studied effector repertoire , will accelerate elucidation of the effector repertoires produced by diverse pathogens and eventually guide the deployment of more durable R genes for protecting crops against multiple pathogens .
2E . coli and DC3000 strains and plasmids used in this study are listed in Table 1 . Plasmid maintenance and manipulations were typically conducted with either E . coli DH5α or TOP10 . Invitrogen Gateway maintenance and manipulations were conducted in E . coli DB3 . 1 . E . coli S17-1 and SM10 were used for conjugations . DC3000 strains were routinely grown in King's B ( KB ) medium at 25 or 30°C [67] . E . coli strains were routinely grown on LB media at 37°C . Growth of DC3000 and derivatives was also compared on two defined media: mannitol-glutamate medium ( MG ) and Hrp-minimal medium ( HMM ) [68] , [69] . Antibiotics and additives were used at the following final concentrations ( µg/ml ) unless otherwise noted: ampicillin ( Ap ) , 100; kanamycin ( Km ) , 50; gentamicin ( Gm ) , 5; spectinomycin ( Sp ) , 50; rifampicin ( Rf ) , 50; tetracycline ( Tc ) , 20; 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside ( X-gal ) , 40 . DNA manipulations and PCR were conducted according to standard protocols [70] . Plasmid DNA was purified using the QIAprep Spin Miniprep Kit from Qiagen ( Valencia , CA ) . Genomic DNA was prepped using the Wizard Genomic DNA Purification Kit from Promega ( Madison , WI ) . DNA gel extraction and DNA enzyme reaction cleanups were conducted using the Gel DNA Recovery Kit and Clean-up and Concentrator Kit from Zymo Research ( Orange , CA ) . DNA restriction and modification enzymes were from New England Biolabs ( Ipswich , MA ) and were used according to the manufacturer's recommendations . Invitrogen ( Carlsbad , CA ) Gateway recombination was conducted with LR clonase I or LR clonase II from Invitrogen as recommended by the manufacturer . PCR was routinely conducted using Takara Ex Taq Polymerase Premix from Takara Mirus Bio ( Otsu , Shiga , Japan ) . PCR primers were obtained from IDT ( http://www . idtdna . com ) . Primers and primer sequences are listed in Table 2 . DNA sequencing was conducted at the Cornell University Biotechnology Resource Center using an Applied BioSystems 3730xl DNA Analyzer . Sequences were analyzed using the Vector NTI software package from Invitrogen . DC3000 strain deletions were made with pK18mobsacB constructs essentially as described previously [11] . DC3000 strains were conjugated on sterile nitrocellulose squares on LM medium with E . coli S17-1 or E . coli SM10 carrying pK18mobsacB deletion constructs . DC3000 merodiploid transconjugates were selected with KB Rf Ap Km augmented with additional antibiotics when needed . Merodiploids were spread on KB Rf with 10% sucrose to counter-select the integration . For marked deletions , sucrose counter-selection plates were augmented with the appropriate antibiotics to select for the mutant population . SucR colonies were screened by PCR . pCPP5610 was created by PCR amplification of 1 . 5 and 1 . 0-kb flanks to hopU1 and hopF2 with P2308/P2309 and P2310/P2311 respectively . The PCR fragments were digested with XbaI and ligated with T4 ligase . The 2 . 5 kb ligation product was gel purified , digested with BamHI and HinDIII and cloned into BamHI and HindIII digested pK18mobsacB . pCPP5610 was used to delete cluster I from CUCPB5452 to create CUCPB5459 . The deletion was confirmed by PCR with P2312/P2313 . pCPP5734 was created by PCR amplification of 0 . 9 and 1 . 1-kb flanks to shcN and avrE with P2370/P2371 and P2372/P2373 respectively . The PCR fragments were digested with EcoRI and ligated with T4 ligase . The 2 . 0-kb ligation product was gel purified , digested with XbaI and cloned into XbaI digested pK18mobsacB . pCPP5734 was used to delete cluster VI/CEL from CUCPB5460 , CUCPB5459 , CUCPB5440 , CUCPB5442 , CUCPB5450 , CUCPB5452 and CUCPB5451 to create CUCPB5501 , CUCPB5500 , CUCPB5515 , CUCPB5516 , CUCPB5517 , CUCPB5518 and CUCPB5539 respectively . The deletions were confirmed by PCR with P2373/P2501 . pCPP5917 was created by sub-cloning the cluster IX deletion construct from pCPP5397 [11] with EcoRI into EcoRI digested pK18mobsacB . pCPP5917 was transformed into E . coli SM10 and used to delete cluster IX from CUCPB5460 , CUCPB5501 , CUCPB5515 and CUCPB5516 to create CUCPB5529 , CUCPB5530 , CUCPB5531 , CUCPB5538 respectively . Merodiploid transconjugants were selected with KB Rf Ap Km Sp . The FRTSpR cassette was removed from CUCPB5529 , CUCPB5530 and CUCPB5531 by transformation and curing of the unstable FLP expression vector pCPP5264 [11] . The deletions were confirmed by PCR with P1967/P1968 . pCPP5729 was created by PCR amplification of the FRTGmR cassette with P2259/P2260 from pCPP5209 [11] . The cassette was digested with XbaI and sub-cloned into pCPP5608 [20] so it could be used to make mutants in KmR strains by screening for GmR and GmS . Cloning with XbaI destroys the FRT sites . pCPP5729 was used to delete hopQ1-1 from DC3000 ΔavrPtoΔavrPtoB and CUCPB5467 to make CUCPB5484 and CUCPB5489 respectively . The deletions were confirmed with by PCR with P2299/P2300 . pCPP5615 was created by PCR amplification of 1 . 1 and 1 . 0-kb flanks to fliC with P2245/P2261 and P2248/P2262 respectively and PCR amplification of the FRTGmR cassette from pCPP5209 [11] with P2253/P2260 . The three PCR products were digested with AvrII and XmaI and ligated with T4 ligase . The 3 . 4-kb product was gel purified and TOPO cloned into pCR2 . 1-TOPO . The deletion construct was then sub-cloned by SalI and SphI digestion into SalI/SphI digested pK18mobsacB . pCPP5615 was used to delete fliC from DC3000 , CUCPB5489 and CUCPB5515 to create CUCPB5467 , CUCPB5514 and CUCPB5541 respectively . The FRTGmR cassette was removed from CUCPB5467and CUCPB5514 by transformation and curing of the unstable FLP expression vector pCPP5264 [11] . The deletions were confirmed by PCR with P2326/P2327 and loss of motility on a KB swim plate with 0 . 2% agar . hopR1 and shcM hopM1 were PCR amplified with P2502/P2505 and P1932/P1931 respectively and TOPO cloned into pENTR/SD/D-TOPO ( Invitrogen ) to create pCPP5951 and pCPP5233 respectively . shcE with its stop codon was PCR amplified with P2506/P2507 and TOPO cloned into pENTR/D/SD-TOPO . avrE with its native SD but without its stop codon was PCR amplified with P2508/P2509 digested with AscI and cloned in-frame into the AscI digested pENTR/D/SD-shcE to create pCPP5912 . All effector and chaperone clones were confirmed by DNA sequencing . pBBR PavrPto constructs for complementation were made by LR recombination between pCPP5372 [12] and entry vectors carrying hopR1 , shcM-hopM1 , shcE-avrE , hopD1 , or avrPtoB [71] . Plasmids were introduced into DC3000 derivatives by electroporation [72] . Virulence assays were conducted essentially as described previously [20] . N . benthamiana and tomato ( Solanum lycopersicum cv . Moneymaker ) were grown under greenhouse conditions until 4–5 weeks post-germination . Primary streaks of DC3000 strains were made from isolated colonies onto KB plates and grown overnight at room temperature . The plates were then spread with 100 µl sterile KB and incubated overnight at room temperature to produce even bacterial lawns . Cells were scraped from plates with a sterile loop and suspended in 10 mM MgCl2 100 mM sucrose to a final OD600 of 0 . 3 ( 3×108 CFU/ml ) . Bacterial suspensions were diluted to 3×104 CFU/ml in 10 mM MgCl2 for syringe infiltration or 3×105 CFU/ml in dH2O and 0 . 02% Silwet for dip inoculation . The bacterial concentrations of the suspensions were verified by plate count . Tomato and N . benthamiana syringe infiltrations were conducted by infiltrating expanded leaves using a dissecting needle and a blunt syringe . N . benthamiana dip inoculations were conducted by inverting whole plants into bacterial suspensions and gently agitating for 30 sec . The inoculated plants were incubated with a 12 h light cycle at 20–25°C with medium humidity for tomato and high humidity for N . benthamiana . Bacterial populations were assessed at three days post-inoculation for tomato and six days post-inoculation for N . benthamiana . Three 0 . 8-cm leaf discs were harvested with a cork borer from each infiltration area . The discs were ground with a sterile mortar and pestle into 0 . 3 ml 10 mM MgCl2 100 mM sucrose , diluted and plated to determine CFU cm−2 . The data for each figure presented are obtained from a single experiment with a single inoculum preparation , and each experiment was repeated as indicated . Six-week post-germination N . benthamiana leaves were infiltrated with a bacterial suspension with an OD600 of 0 . 5 . Leaf disks were collected 12 hr after infiltration , cleared with 95% ethanol , and stained for callose with 0 . 1% aniline blue in 150 mM K2HPO4 ( pH 9 . 5 ) as previously described [36] . The stained leaf disks were observed by epifluorescence microscopy under ultraviolet light . The numbers of callose deposits per 1 cm2 microscopic field were counted in randomly coded samples from eight leaf disks using images captured with an Olympus digital camera and its software ( DP2-BSW ) . The callose deposit counts were then matched with the corresponding treatment codes for determination of the mean and standard deviation . DC3000 HopR1 was used in a PSI BLAST with the inclusion parameter set to 1 . 0E−10 . By the second iteration members of the AvrE clade and by the third iteration members of the DspA/E clade were identified . A selection of third iteration PSI BLAST hits were made to maximize strain and species diversity and were exported to the MUSCLE web server [33] . An alignment was generated with MUSCLE using default parameters ( Figure S3 ) , and a second iteration neighbor-joining tree was generated . The tree was displayed using NJplot [73] . hopK1 ( AAO53599 ) , hopY1 ( AF458403 ) , hopU1 ( AAO54045 ) , shcF ( AY321312 ) , hopF2 ( AAO54046 ) , hopH1 ( AAO54130 ) , hopC1 ( AAO54131 ) , hopD1 ( AAO54410 ) , hopQ1-1 ( AF458396 ) , hopR1 ( AF458397 ) , hopAM1-1 ( AAO54553 ) , shcN ( AE016853 ) , hopN1 ( AAO54892 ) , hopAA1-1 ( AAO54894 ) , shcM ( AE016853 ) , hopM1 ( AAO54897 ) , shcE ( AE016853 ) , avrE ( AF232004 ) , hopB1 ( AF232004 ) , hopAF1 ( AAO55088 ) , avrPtoB ( AY074795 ) , avrPto ( AAO57459 ) , hopE1 ( AY208297 ) , hopAA1-2 ( AAO58156 ) , shcV ( AE016853 ) , hopV1 ( AAO58158 ) , hopAO1 ( AAO58160 ) , hopG1 ( AY208296 ) , hopI1 ( AAO58206 ) , hopA1 ( AAO58779 ) , hopAM1-2 ( AAO59032 ) , hopX1 ( AAO59038 ) , shcO1 ( NC_004633 ) , hopO1-1 ( AF458392 ) , hopT1-1 ( AF458399 ) , RSp1281 ( AL646053 ) , Xcc1089 ( NC_003902 ) . | Pseudomonas syringae is a Gram-negative plant pathogen that defeats plant defenses through effector proteins that are injected into plant cells via the type III secretion system . P . syringae strains are assigned to pathovars based largely on their host of origin . P . syringae pv . tomato DC3000 causes bacterial speck of tomato and has become a model for studying bacterium–plant interactions because it also attacks the experimentally amenable plants Arabidopsis thaliana and ( if one effector acting as an avirulence determinant is removed ) Nicotiana benthamiana . Genome sequence–enabled studies have revealed that strains in different pathovars harbor large ( 15–30 ) effector repertoires , which are surprisingly diverse and show no obvious correlation with host range . In search of rules governing the composition of effector repertoires in individual strains , we constructed combinatorial deletions involving 20 of the 28 effectors deployed by D3000 . The pattern of growth defects resulting from these mutations suggests an architecture in the effector repertoire involving redundant targeting of a few vulnerable plant defense processes and compensatory redundancies in these defenses . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"microbiology/plant-biotic",
"interactions",
"microbiology/innate",
"immunity",
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] | 2009 | Deletions in the Repertoire of Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Genes Reveal Functional Overlap among Effectors |
Telomeres shorten with each cell division and telomere dysfunction is a recognized hallmark of aging . Tissue proliferation is expected to dictate the rate at which telomeres shorten . We set out to test whether proliferative tissues age faster than non-proliferative due to telomere shortening during zebrafish aging . We performed a prospective study linking telomere length to tissue pathology and disease . Contrary to expectations , we show that telomeres shorten to critical lengths only in specific tissues and independently of their proliferation rate . Short telomeres accumulate in the gut but not in other highly proliferative tissues such as the blood and gonads . Notably , the muscle , a low proliferative tissue , accumulates short telomeres and DNA damage at the same rate as the gut . Together , our work shows that telomere shortening and DNA damage in key tissues triggers not only local dysfunction but also anticipates the onset of age-associated diseases in other tissues , including cancer .
Telomeres are structures composed of DNA repeats and a specific set of proteins ( the shelterin complex ) that protect the ends of eukaryotic chromosomes . Telomeres have two main functions: 1 ) to prevent recognition of chromosome-ends as deleterious DNA double strand breaks ( DSBs ) and 2 ) to ensure complete DNA replication by recruitment of telomerase , a reverse transcriptase that that synthesizes new telomeric DNA repeats [1 , 2] . Due to the “end-replication problem” , telomeres shorten with each round of cell division in cells that do not express telomerase [3 , 4] . On average , human cells lose 50–100 bp from their extremities in every round of division [5–7] . Telomerase expression is restricted in most human somatic cells [8] . Consequently , as observed in population studies , telomeres shorten significantly during human aging [9 , 10] . The rate of telomere decline is most pronounced in early life , from birth to puberty , slowing down in adulthood [9 , 11] . Telomere erosion is a strong barrier to cell proliferation and thus thought to constitute a tumor suppression mechanism [12] . However , by limiting the function of stem cell reserves necessary for tissue renewal , telomere shortening has also been proposed to be a strong promoter of aging-associated degenerative phenotypes [10 , 13 , 14] . Indeed , short telomeres are a frequent feature of diseases that anticipate facets of aging , such as Dyskeratosis Congenita , DC [15–17] . DC patients harbor mutations in several components involved in telomere maintenance such as DKC1 ( dyskerin ) , a component of the active telomerase enzyme complex [18] , hTR ( telomerase RNA ) , hTERT ( telomerase reverse transcriptase ) and in shelterin component TINF2 [19–22] . Mutations in DKC1 associate specifically with an X-linked form of DC [23] . Short telomeres as cause of age-related disease is further supported by other syndromes , such as Idiopathic Pulmonary Fibrosis ( IPF ) and Hoyeraal-Hreiderasson [19 , 24] . These “telomeropathies” exhibit a pattern of genetic anticipation , in which later generations suffer from increased disease severity [15 , 25 , 26] . These diseases are characterized by dysfunction of proliferative tissues , namely bone marrow , lung and liver , ultimately leading to multi-organ failure . Dysfunction and disease severity can be predicted by the extent of short telomere accumulation in different tissues [27 , 28] . Apart from these symptoms , DC patients show severe mucocutaneous alterations and an increased susceptibility for developing cancer [15] . A direct association between telomere shortening and tissue specific dysfunction in vertebrates has been clearly demonstrated in late-generation telomerase knockout mice ( both catalytic enzyme , mTERT-/- and RNA subunit , mTR-/- ) and , more recently , in telomerase deficient zebrafish [29–33] . Telomerase plays a pivotal role in high-turnover mouse and zebrafish tissues [29 , 30 , 32] . In G6 mTR-/- mice , intestinal epithelial cells and male germ cells have reduced proliferative capacity and increased apoptosis , resulting in tissue atrophy and infertility [32] . In low proliferative tissues , such as heart and liver , G4 mTERT-/- mice incur metabolic failure by repressing master regulators of mitochondrial biogenesis , via upregulation of p53 [34] . In zebrafish , first generation telomerase mutants show a tissue and time dependent dysfunction that associates with short telomeres and diminished proliferative rates [30] . Telomere shortening can also disrupt tissue integrity in a non-cell autonomous manner , by inducing cell senescence . Senescent cells accumulate with age in a variety of mammalian tissues , such as skin and eye [35] . These cells secrete a specific set of molecules , including inflammatory cytokines and chemokines , growth and survival factors , which are referred to as the “Senescence-Associated Secretory Phenotype” , SASP [36] . SASP creates a microenvironment within and between tissues which compromises homeostasis and promotes features of tumor malignancy , including invasiveness [36 , 37] . Further evidence supports extratelomeric functions for telomerase ( which are independent of telomere elongation ) and that may influence aging phenotypes . Telomerase stimulates ( stem ) cell proliferation by acting as a cofactor of the beta-catenin transcriptional complex [38] , and promotes tumor development by potentially activating Myc and Wnt signaling [39 , 40] . Additional non-canonical roles of telomerase involve its interaction with the RNA component of mitochondrial RNA processing endoribonuclease ( RMRP ) , which produces double stranded RNAs that can be processed into siRNAs [41] . Finally , telomerase can be targeted to the mitochondria affecting its function , superoxide production and apoptosis [42 , 43] . While short telomeres may underlie part of the cascade of tissue homeostasis decline with time , aging mammalian species are affected by many other factors , such as mitochondrial dysfunction , epigenetic alterations and impaired proteostasis [44–46] . It remains to be defined in which tissues telomeres shorten to critical levels with natural aging , triggering DNA Damage Responses ( DDR ) and , ultimately , organ dysfunction . Identifying the tissues where short telomeres dictate phenotypes of aging and how these tissues then influence the organism as a whole is crucial for understanding the impact of telomere length on aging . Zebrafish has emerged as an important vertebrate model to study both telomere biology and aging [29 , 30 , 47–50] . First generation telomerase mutant zebrafish age and die prematurely [29 , 30] , reminiscent of human telomere shortening syndromes . Yet , a detailed analysis of organ dysfunction and respective telomere shortening dynamics throughout lifetime in wild type animals remains largely unexplored . We conducted a comparative prospective study of telomere dynamics , DNA Damage Response ( DDR ) and aging-related dysfunction and disease in different tissues in both WT ( from 3 to 42 months ) and tert-/- zebrafish ( from 3 to 12 months , when at least 50% of tert-/- mutants are dead ) . This allowed us to define the choreography ( timings and kinetics ) of age-related lesions and how these are conserved between WT and tert-/- . We discovered that tert-/- mutants anticipate the tissue-specific dysfunctional events observed during normal zebrafish aging , including tumorigenesis .
Human population studies revealed variation in telomere length between different tissues , irrespective of their proliferation rates [51] . In birds , variation in length was also found between different somatic tissues [52] . However , population averages are unable to discriminate if telomere shortening precedes tissue dysfunction in aging . Zebrafish has telomeres of human like length , ca . 5–16 kb [30 , 47 , 53] and absence of telomerase limits its lifespan [29 , 30] . To test if different zebrafish tissues have different telomere lengths within an individual , we dissected juvenile zebrafish and performed Telomere Restriction Fragment ( TRF ) [54] analysis of high proliferative ( gut , testis ) and low proliferative tissues ( muscle ) . We observed that , whereas at larval stages telomeres are more homogeneous and longer ( larvae , “L” , Fig 1Aa ) , different tissues have acquired different telomere lengths by the time of sexual maturity , at 3 months ( Fig 1Ab ) . We observed most WT tissues have a mixture of long and short populations of TRFs and tert-/- mutants predominantly have the latter ( Fig 1Ab ) . The longer telomere population is of the same size observed in blood and thus likely to reflect circulating cells in all tissue samples . We therefore decided to use the median Telomere Length ( mTL ) as a conservative measurement of telomeres in different cell populations/tissues . Three-week-old zebrafish larvae had long telomeres ( ca . 12 Kb ) that declined with growth and development ( Fig 1Aa ) . Sexually mature males had long mTL in the testis ( ca . 9 . 9 Kb ) followed by the muscle ( ca . 9 . 4 Kb ) while the shortest telomeres measured were in the gut ( ca . 8 . 4 Kb , Fig 1Aa ) . Strikingly , the relative telomere length difference between highly proliferative tissues testis and gut are globally maintained in tert-/- mutant tissues ( tert-/- larvae had telomeres of ca . 11 . 6 Kb which declined to ca . 8 . 3 Kb in the testis and gut ca . 7 . 8 Kb; Fig 1Aa ) . This suggests that , like in humans , where telomerase activity is restricted in most tissues , telomere length and set points are established during zebrafish development . In order to determine the dynamics of telomere shortening with aging in different tissues , we performed TRF analysis at different ages in the fin , a less proliferative tissue , which we previously reported to have shorter telomeres , and in the kidney marrow ( the hematopoietic organ in zebrafish ) , a proliferative tissue with long telomeres [30] . We measured telomeres in four individuals and observed significant shortening of WT fin telomere length with age at the population level ( Fig 1Ba ) . We detected a linear decline on mTL from 3 to 24 months of age , ranging ca . 8 . 6 Kb to 7 . 3 Kb ( Fig 1Ba ) . From 24 months of age onwards , we did not detect further telomere shortening ( Fig 1Ba ) , suggestive of an exhaustion of proliferation potential in later life stages . By taking advantage of the well-studied regenerative capacity of zebrafish fin , we were able to calculate the telomere shortening rate for two zebrafish individuals for their first 24 months of life , by taking advantage of the well-studied regenerative capacity of zebrafish fin . We estimated a decline of 45 bp/month and 90 bp/month respectively . Since telomerase expression was reported to increase during fin regeneration [47] , we performed this experiment by sampling telomere length in the different fins for each individual at different ages . Contrary to any other tissue analyzed , WT kidney marrow telomeres dramatically elongated from larvae to fully developed adult stages ( S1 Fig ) and shortened with age after 18 months ( Fig 1Bb ) . At the population level , by averaging different individuals’ telomere median length ( N = 3–6 for each time point from 3 months onwards ) , no appreciable shortening could be detected in the first 18 months of life , after which telomeres declined ( Fig 1Bb ) . This is suggestive of higher telomerase activity and , consequently , lesser telomere shortening in kidney marrow than in the fin . Telomeres shortened significantly with age in the fin of WT , matching the length of 12-month old tert-/- mutants ( N = 3 for each time point ) by 18–24 months ( Fig 1Ba ) . In contrast , WT telomeres of the kidney marrow never reached the length of tert-/- mutants ( N = 3 for each time point ) during their lifetimes ( Fig 1Bb ) . These data show that telomere median length , as measured by TRF , correlates directly with zebrafish age up to 18–24 months albeit in a tissue specific manner . In addition , mTL shortening reaches a limit bellow which we are unable to detect further decrease . As previously seen by others [9 , 11] , this is likely due to a selection process that eliminates cells with extremely short telomeres thus creating a virtual barrier to telomere decrease . We have previously shown that tert-/- mutants have shorter telomeres than WT siblings already by 3 months ( Fig 1A and [30] ) . As consequence , tert-/- mutants develop premature cell proliferation defects and DDR which culminate in early tissue degeneration [30] . Thus , in our study , we used tert-/- mTL as proxy for the presence of critically short telomeres that cause tissue dysfunction in WT . To probe for accumulation of short telomeres , we determined the variation in telomere length in WT proliferative tissues gut and testis and less proliferative tissue muscle , over time ( N = 3–4 per time point for gut and muscle; N = 5–6 per time point for testis; Fig 2 ) . While WT kidney marrow mTL shortened within a range of ca . 16–11Kb ( Fig 1Bb ) , other tissues’ mTL shortened to lower ranges of 11–6Kb ( Fig 2 , densitometries shown for one individual per age in S2 Fig ) . For the time points tested after sexual maturity , mTL ranged between ca . 8 . 0–6 . 5 Kb in the gut , ca . 10 . 2–7 . 8 Kb in the muscle and ca . 11 . 1–9 . 3 Kb in the testis ( Fig 2A–2C ) . We further detected significant mTL shortening in the gut and muscle of WT at 24 months ( Fig 2A and 2B ) . Similar to what was observed in the fin , gut and muscle telomeres declined significantly in a linear fashion between 3 and 24 months in WT , stabilizing in the last time point of 36 months ( Fig 2A and 2B ) . Because the muscle is a low proliferative tissue , telomere shortening with age is likely to rely on factors that are independent of local cell replication . Apart from cell division , reactive oxygen species ( ROS ) are prime candidates for generating damage at telomeres and , consequently , promoting telomere shortening [55] . We tested if the levels of ROS increased differently with aging in tissues with distinct turnovers , by measuring total ROS in the gut and muscle of 6 and 36 month-old WT zebrafish , using the cell-permeant 5- chloromethyl-2’ , 7’-dichlorodihydrofluorescein diacetate ( CM-H2DCFDA ) . We found that the muscle accumulated significantly ( ca . 12x ) higher levels of ROS with aging ( p = 0 . 04 , S3 Fig ) . In contrast , we could not find significant differences in ROS levels between the guts of 6 and 36 month-old zebrafish ( S3 Fig ) . tert-/- mutants had shorter mTL already by 3 months of age in all tissues and we could not detect further significant shortening by the age of 12 months ( N = 3–4 per time point for gut and muscle , N = 4–6 per time point for testis; Fig 2A–2C ) . When comparing WT and tert-/- , we found that telomeres in both gut and muscle of old WT reached the length of 12 month old tert-/- ( 6 . 8 Kb for gut and 8 . 5 Kb for muscle; Fig 2A and 2B ) . We estimated that short telomeres in WT reached the level of tert-/- mutants around the age of 20 months ( indicated by blue arrow in Fig 2A and 2B ) . In contrast , WT telomeres did not shorten appreciably with age in the testis ( indicated by blue arrow in Fig 2C ) . In summary , telomeres shorten in old WT zebrafish to the level of tert-/- mutants the fin , gut and muscle , but not in testis and kidney marrow , regardless of their distinct proliferation rates . One of the hallmarks of aging is the general accumulation of DNA damage [56] . This leads to robust activation of DDR pathways , which accumulate preferentially at short or damaged telomeres [57 , 58] . This process is thought to underlie the establishment of cellular senescence . In order to determine if short telomeres precede activation of persistent DDR in tissues , we measured the appearance of nuclear foci of phosphorylated H2AX at Ser139 ( γ-H2AX ) over time . Presence of short telomeres in tert-/- tissues resulted in the accumulation of DDR markers . From 3 to 12 months , DDR markers increased significantly from 0 . 5 to 11 . 4% in the gut ( p = 0 . 0077 , Fig 3Ab , 3Ad and 3Ea; S4 Fig ) , 1 . 4 to 10 . 5% in the kidney marrow ( p = 0 . 0096 , Fig 3Bb , 3Bd and 3Ed; S4 Fig ) and 0 . 5 to 5 . 0% in the testis ( p = 0 . 0065 , Fig 3Cb , 3Cd and 3Eg; S4 Fig ) and showed a slight tendency to increase in the muscle ( p = 0 . 35 , Fig 3Db , 3Dd and 3Ej; S4 Fig ) . During this period , telomere length was always shorter in tert-/- tissues when compared with WT lengths , ranging ca . 7 . 4–6 . 8 Kb in the gut , ca . 7 . 3–7 . 7 Kb in the kidney marrow , ca . 7 . 4–7 . 8 Kb in testis and ca . 8 . 2–8 . 5 Kb in muscle ( Figs 1 and 2 ) . In order to investigate whether short telomeres dictated the onset of tissue dysfunction in WT tissues , we asked if changes in molecular markers related to DDR , cell proliferation and apoptosis were correlated with mTL decline ( Fig 3E ) . In gut and muscle of old WT , shorter mTL directly correlated with the increase in DDR markers , which reached tert-/- levels ( p = 0 . 021 and p = 0 . 044 , Fig 3Ea and 3Ej ) . More specifically , the percentage of WT cells bearing five or more γH2AX foci increased from 2% to 18% in the gut ( p<0 . 0001 ) and from 0 to 9% in muscle ( p = 0 . 0007 ) , between the ages of 3 and 36 months , as mTL declined from 8 to 6 . 9Kb and from 10 . 2 to 7 . 8Kb respectively ( Fig 3Aa–3Ae , 3Da–3De , 3Ea and 3Ej ) . However , an increase in DDR with age , from 3 to 36 months , was also detected in the testis and kidney marrow ( of 12% , p = 0 . 0027 , and 28% , p = 0 . 0003 , respectively ) of WT zebrafish , even though these tissues failed to significantly shorten their telomeres to tert-/- levels ( Fig 3Ba–3Be and 3Ca–3Ce , 3Ed and 3Eg ) . Thus , DDR activation in cells of naturally aged zebrafish strongly correlates with telomere shortening specifically in the gut and muscle . However , some highly proliferative tissues , such as testis , exhibit marks of DDR independently of critically short telomeres , as set by tert-/- mTL . To determine what is the contribution of short-telomere induced damage to the increase in DDR in aging , we performed telomere- Fluorescence In Situ Hybridization ( FISH ) in γH2AX immuno-stained cells isolated from WT and tert-/- gut ( at 3 , 12 and 36 months for WT and at 3 months for tert-/- , N = 3 per age/genotype ) . The number of three of more telomere induced foci ( TIF ) increased significantly from 3 to 36 months in WT gut ( p = 0 . 0001 , arbitrary line indicates 3 or more TIFs , S5A and S5B Fig ) . We also found a significant accumulation in TIFs in 3 month-old tert-/- gut , when comparing with age-matched 3-month old WT controls ( p = 0 . 0001 , arbitrary line indicates 3 or more TIFs , S5A and S5B Fig ) . Our data demonstrates that the presence of dysfunctional telomeres is an important contributor to the age-associated increase in DDR in the zebrafish gut . We next asked whether the observed decline in mTL could anticipate other cellular phenotypes of aging . We looked for changes in rates of cell proliferation and apoptosis emerging with time and correlated them with mTL decline . Indeed , using PCNA immunostaining as readout for cell division , we observed decreased proliferation in the gut and testis of tert-/- mutants already by 3 months of age and by 12 months in the kidney marrow ( Fig 3A–3C ) . Accordingly , the percentage of proliferating cells dropped in the gut of older WT individuals ( 3 month vs . 36 months ) from 38% to 10% ( p<0 . 0001 , Fig 3Af–3Aj and S4 Fig ) and this strongly correlated with telomere shortening ( p = 0 . 015 , Fig 3Eb ) . Testis and kidney marrow also showed a marked decrease in cell division with aging . We observed a decline of 19% ( p = 0 . 0005 ) and 12% ( p< 0 . 0001 ) of PCNA stained cells between 3 months and 36 months , in testis and kidney marrow respectively ( Fig 3Bf–3Bj , 3Cf–3Cj and S4 Fig ) . Overall alterations in proliferation were particularly evident in areas where stem cells and progenitors reside , such as the basal compartment of the seminiferous tubules in the testis and the base of intestinal villi ( Fig 3Af–3Aj , 3Cf–3Cj ) . However , similarly to DDR , testis and kidney marrow showed decreased proliferation with aging regardless of appreciable telomere length variation ( Fig 3Ee and 3Eh ) . In a similar way , decrease of mTL with age did not correlate with apoptotic index in any studied tissue ( p = 0 . 31 for gut , p = 0 . 13 for muscle , p = 0 . 40 for testis , p = 0 . 32 for kidney marrow , Fig 3Ec , 3Ef , 3Ei and 3El ) . Nevertheless , a significant increase of apoptosis with age was observed by the higher TUNEL staining , detected in all 36 month-old WT tissues when compared with 3-month old groups ( p = 0 . 031 for gut , Fig 3Ak–3Ao; p = 0 . 0008 for kidney marrow , Fig 3Bk–3Bo; p = 0 . 0091 for testis , Fig 3Ck–3Co; p = 0 . 0279 for muscle , Fig 3Dk–3Do and S4 Fig ) . Senescent cells gradually accumulate in aging tissues [35] and critically short telomeres determine the onset of replicative senescence in human cells [59] . During aging of primates , the number of cells bearing markers of senescence and telomere dysfunction increases exponentially in the skin [60] . We assessed whether mTL decline would also correlate with the increase in senescence found in aging zebrafish tissues . Our results showed that short telomeres in the gut anticipated the increase in senescence measured by senescence-associated β-galactosidase ( SA-β-gal ) for aging WT and for tert-/- mutants ( p = 0 . 10 , S6 Fig ) . As observed for DDR levels , even though senescence was visible in testis and kidney marrow , there was no correlation of mTL decline with increased senescence with age in these tissues ( S6 Fig ) . Thus , using tert-/- mutant zebrafish mTL as reference , the above analysis establishes the gut and muscle as primary tissues where short telomeres anticipate decreased proliferation and increased DDR with aging . Short telomeres , as defined by tert-/- length , accumulate with zebrafish aging and correlate with activation of persistent DDRs in specific tissues: gut and muscle . We therefore tested whether these cellular phenotypic alterations preceded a decline in tissue integrity . We first looked for changes in tissue inflammation . Aging is characterized by a systemic chronic pro-inflammatory condition with rising levels of TNF , IL1 and IL6 [61–64] . This “proinflammatory state” has been postulated to lead to several chronic degenerative disorders [61 , 62 , 65] . We found that tert-/- zebrafish developed early ( by 6 months ) enteritis and epithelial erosion that was further aggravated in older individuals ( 18% incidence by 12 months , N = 12 in a total of 66 tert-/- mutants analyzed; S7A Fig ) . As short telomeres accumulated in the gut , the incidence of enteritis increased from 0% to 13% in aged WT zebrafish ( 3 month vs . 36 months , N = 30 in a total of 238 zebrafish analyzed , S7A Fig ) . We also looked for general alterations in intestinal structure . The gastrointestinal tract is known to suffer several alterations in microbiota composition , mucosal immune system and regeneration capacities with aging [66–68] . These changes compromise normal organ function and nutrient absorption . We detected a progressive tendency for thickening of the lamina propria and submucosa in the anterior part of the intestine with tert-/- mutants’ aging , from 3 to 12 months , associated with inflammatory cell infiltration ( Fig 4Ab and 4Ad , quantified in 4B ) . WT zebrafish showed significant thickening of the lamina propria and submucosal layers at 36 months ( Fig 4Ae , indicated by asterisk , quantified in Fig 4B ) . This morphological feature correlated well with mTL decline in the gut ( p = 0 . 002 , N = 3 per time point , Fig 4B ) . However , we were unable to find significant changes in general gut villi length in older WT individuals that would correlate with shorter mTL ( S7B Fig ) . Parallel to the gut , decline in telomere length and activation of DDRs in the muscle prompted us to assess whether these could underlie alterations occurring in myocytes with age . Humans are known to lose muscle mass ( also known as sarcopenia ) progressively from adulthood to older ages primarily due to nutrient absorption defects ( 1–2% decrease per year after the age of 50 , [69] ) . Atrophy of type IIa muscle fibers leads to a decay in muscle strength and motor capacity [69] . 12 month-old tert-/- mutants showed significant muscle fiber thinning ( when compared with 12 month WT siblings , Fig 4Ah–4Ai and 4B ) . We observed a 44% reduction in myocyte width in WT zebrafish by the age of 36 months , when compared with 3 months ( p = 0 . 0001 , Fig 4Af–4Aj ) , compatible with progressive sarcopenia . Severe myocyte degeneration and endomysisal edema ( Fig 4Ai–4Aj ) directly correlated with the presence of short telomeres ( <8 . 5Kb , p = 0 . 028 , Fig 4B ) . These results show that short telomeres directly correlate with the degenerative changes associated with aging , particularly intestinal inflammation and sarcopenia . Even though mTL does not appreciably decline to levels that compromise tissue integrity in testis and kidney marrow ( when taking tert-/- mTL as reference ) , we investigated how aging affected tissue homeostasis and function in these organs . Adult tert-/- mutants showed reduced germ cell compartment by 12 months of age ( Fig 4Ak–4An ) . Here , we show that WT male zebrafish display a progressive inability to produce mature spermatozoa with age ( Fig 4Ak–4Ao , L- mature spermatozoa in the lumen of seminiferous tubules ) . In addition , 3% of WT zebrafish after 24 months of age ( N = 7 in a total of 238 zebrafish analyzed , S7C Fig ) displayed hyperplasia of seminiferous epithelium , mostly spermatogonia . Hyperplasia of spermatogonia in tert-/- populations was residual ( N = 1 in a total of 66 zebrafish analyzed ) . These results had not been described before and are consistent with telomerase being essential for expansion of the germ cell compartment in late generation telomerase knockout mice [32] . Mice harboring artificially shortened telomeres display defective spermatogenesis/cell proliferation in testis , failing to produce viable offspring [32 , 70 , 71] . Interestingly , even though we observed a marked decline in percentage of mature spermatozoa with age , this did not correlate with the presence of short telomeres in older WT zebrafish ( Fig 4B ) . Decreased spermatozoa production levels resulted in decline in male fertility ( Fig 4C ) . tert-/- mutants showed a compromised ability ( ~50% ) to fertilize female eggs at 6 months and became completely infertile by 12 months of age ( Fig 4C ) . WT male zebrafish fertility peaked from 6 to 18 months of age and progressively decreased afterwards ( ~50% and ~30% of fertilization capacity by 24 and 30 months , respectively; Fig 4C , p< 0 . 0001 ) . Interestingly , WT zebrafish decreased their ability to stimulate female spawning with age , from 24 months onwards , whereas these defects were already visible in 6-month-old tert-/- males ( S8A Fig ) . Fertility and egg spawning behavior were mildly but significantly affected with age in WT or tert-/- mutant females ( p = 0 . 0231 for WT females and p = 0 . 0357 for tert-/- females , S8B and S8C Fig ) . Thus , although tert-/- mutants anticipate the decline in spermatozoa production and fertility observed in older WT , the development of these phenotypes in a natural aging setting does not seem to depend on the presence of short telomeres in the gonads . We observed that telomeres shortened later in life in the kidney marrow , but this shortening never matched the length of tert-/- mutants ( Fig 1B ) . We tested if , as in testis , tert-/- mutants would recapitulate kidney marrow defects associated with aging . WT zebrafish showed lower levels of red blood cells ( RBC ) with aging ( erythrocytes declined from 17% at 3 months to 6% at 24 months , p = 0 . 0002 , Fig 4D and S9A Fig ) and also a tendency for lower lymphocyte numbers ( 30% at 3 months vs . 10% at 33 months , p = 0 . 0011 , S9A–S9C Fig ) . Conversely , the proportion of myeloid to lymphoid cells gradually increased with age ( 2% at 3 months versus 9% at 33 months , p<0 . 0001 , S9A and S9C Fig ) . In humans the hematopoietic compartment suffers deregulation of stem cell differentiation programs with aging , resulting in higher myeloid to lymphoid progenitor ratios , an event related to “immunosenescence” [72] . An increase in myeloid/lymphoid ratios is usually indicative of innate immune system activation , suggestive of chronic inflammation [73] . Low levels of RBC and alteration in myeloid/lymphoid ratios surged independently of the presence of short telomeres during WT aging ( Fig 4D , S9A and S9C Fig ) . Of these phenotypes , tert-/- mutants only anticipated the low levels of RBC ( p = 0 . 0067 , Fig 4D and S9A Fig ) . tert-/- myeloid/lymphoid and precursors ratios accompanied the tendencies of WT up to 12 months of age ( S9A and S9C Fig ) . We could not detect significant differences in total cell number per blood volume with aging in WT or tert-/- mutants ( S9B Fig ) . Thus , deregulation of cell differentiation programs in the hematopoietic system that accompanies aging is likely to be independent of kidney marrow telomere-length . Nevertheless , telomere shortening accompanies the decline in RBC levels with age . We established that telomere shortening is associated with tissue specific aging phenotypes in zebrafish . Non-tissue specific disease phenotypes also emerged with aging , namely cachexia , infection and cancer . The onset of age-associated pathology correlated with the age at which most tissues started to display short telomeres ( fin , gut and muscle , Figs 1B , 2A and 2B ) . In agreement , we also observed that tert-/- mutants recapitulated these diseases prematurely .
In humans , telomere erosion has been implicated in age-associated diseases [26 , 88 , 89] . However , it is not clear whether telomere shortening is an actual cause of aging and how it contributes towards the development of degenerative phenotypes . The identification of tissues where intrinsic telomere shortening becomes truly limiting for optimal function and homeostasis is crucial for understanding the impact of short telomeres in aging . Moreover , understanding whether tissue specific telomere shortening triggers systemic aging signals will help develop an integrative model of how telomere length influences the aging process as a whole . In our current work , we show that absence of telomerase accelerates diseases of old age , including cancer . We find that zebrafish telomerase mutants show an earlier cancer onset and maintain a close to 40% tumor invasion rate . This is surprising since shutting off telomerase is thought to have evolved as a tumor suppressor mechanism by limiting cell proliferation . How tumors in tert-/- zebrafish succeed in stabilizing chromosome-ends in the absence of telomerase is currently unknown . The simplest explanation would entail the engagement of ALT mechanism , usually activated in the absence of telomerase [90 , 91] . It is possible that the effectiveness of ALT may vary depending on different genetic backgrounds . Absence of telomerase in mouse models may either decrease or increase tumorigenesis depending on the genetic context [33 , 92–94] . Our data shows that , instead of grossly altering tumor incidence , short telomeres accelerate the appearance of spontaneous tumors to juvenile stages in zebrafish , in agreement with previous studies [95] . Accumulation of short telomere induced damage anticipates dysfunction in specific tissues with natural aging . Consistently with telomere shortening affecting mainly proliferative tissues [29 , 30 , 32] , the gut appears as a leading candidate-organ where telomeres shorten over time reaching telomerase mutant length , our reference for critically short telomeres . However , similarly to the gut , the muscle also accumulates short telomeres and DDRs with age , which anticipate severe sarcopenia phenotypes . The rate of telomere shortening in human somatic tissues is not exclusively dictated by the respective rates of proliferation [51] . Reactive oxygen species ( ROS ) cause increased DNA lesions at telomeres that result in telomere shortening [55 , 58 , 96 , 97] . Our data show that ROS levels increase by ca . 12 times with WT zebrafish aging specifically in the muscle , and not in the gut . Thus , whereas cell proliferation probably acts as a primary trigger for telomere shortening in the gut , increased levels of oxidative damage may underlie the decline in muscle telomere length over time . Accordingly , less proliferative tissues of TERT knockout mice also have increased production of ROS due to mitochondrial dysfunction [34] . Alternatively , telomere shortening without significant cell division over time in the muscle could rely on Telomere Rapid Deletion ( TRD ) mechanisms , as previously observed in mammalian cells . Interestingly , both gut and muscle follow a similar behavior of linear telomere decline until 24 months of age with stabilization of length at later ages . Telomere stabilization in older ages has been reported by others [9 , 11] and goes in hand with the existence of selection processes that may favor the elimination of cells with extremely short telomeres . Alternatively , maintenance of shorter telomere lengths at older zebrafish ages may rely on recombination-based ALT mechanisms . Telomere length maintenance by recombination , first described for yeast cells [98] , has been recently found active in mammalian cells , even in the presence of telomerase [99 , 100] . As shown by increased levels of telomeric sister-chromatid exchange ( T-SCEs ) and variation of pq ratios , subtelomeric or telomeric recombination is increased in short telomere cells from telomerase positive mice , Dyskeratosis Congenita patients and in human cancer lines [99 , 100] . Telomeres in the testis and kidney marrow do not reach the critical length of telomerase mutant zebrafish . It , thus , appear that short telomeres do not anticipate these tissues’ dysfunction with aging . We cannot exclude , however , that a minority of cells in these tissues accumulates short telomeres with age and these cannot be detected by methods of whole population analysis . The lack of significant detectable telomere shortening in the testis is consistent with previous work showing that zebrafish gonads retain high telomerase activity levels [47] . Interestingly , testis is the tissue with the highest incidence of cancer in zebrafish , and telomerase is known to be re-activated in 90% of all human cancers [81] . The significant expansion of immature germ cells ( spermatogonia , spermatocytes ) in detriment of mature spermatozoa in WT testis with age could also explain why we do not detect telomere shortening in this tissue . Furthermore , this dramatic alteration in cellular composition may lead to changes in free testosterone levels , which associate with loss of muscle mass during human aging [101] . In contrast , the kidney marrow is the only tissue where significant telomere elongation occurs during development and sexual maturation of zebrafish , from larvae to 3 months . From all WT tissues tested in adult stages , the kidney marrow shows the longest telomeres and the biggest difference between WT and tert-/- telomere length . However , kidney marrow tert-/- telomeres have lengths within the range of other tert-/- tissues . Thus , at least during development , the zebrafish kidney marrow probably has a higher necessity for telomerase activity than other tissues and perhaps retains it in some cells , as in the human hematopoietic compartment [102] . Telomeres still shorten in the kidney marrow with age after 18 months , even though they do not reach telomerase mutant lengths . These data suggest that , similarly to humans , telomere shortening in certain tissues and cell types probably relates to differential restriction of telomerase expression [8] . Tissues where telomeres do not reach telomerase mutant lengths still develop aging phenotypes , namely decline in fertility and anemia . It does not seem likely , however , that these phenotypes arise in a completely telomere-independent manner since they are recapitulated prematurely by telomerase mutants . Thus , aging phenotypes in testis and kidney marrow are due either to: 1 ) non cell-autonomous effects caused by telomere shortening in the gut , muscle and/or unidentified tissues or 2 ) absence of non-canonical functions of telomerase required for proper tissue homeostasis , or a combination of both effects . Regarding the first point , as telomeres shorten the number of senescent cells increase in tissues . These cells may be secreting SASP molecules , such as IL-6 and IL-8 which have been described as capable of influencing growth in neighboring cells and epithelial–mesenchymal transition non cell autonomously [36] . However , non-catalytic telomerase functions have also been described as important determinants of tissue physiology in mammals and zebrafish [103–105] . Emerging evidence implicates a role for telomerase in stimulation of cell proliferation and regeneration , independently of telomere length maintenance , via activation of Wnt signaling [106] . Particularly in zebrafish embryos , knockdown of tert , terc or dkc1 was shown to trigger larval phenotypes that are reminiscent of human DC , such as pancytopenia , impaired hematopoietic differentiation or p53-dependent apoptosis [104 , 105 , 107] . Importantly , these larval premature degenerative phenotypes also develop independently of changes in telomere length [104 , 105 , 107] . Parallel to this work , the decline in cell proliferation , increased apoptosis and anemia we observe in WT aging and in tert-/- tissues could be partly attributed to non-canonical functions of telomerase , as demonstrated to occur in zebrafish larvae . Finally , the observed defects in testis and kidney marrow could still rely on loss of telomere integrity with age that is not detectable using the assays we employed . Alternatively , we cannot exclude that aging associated dysfunction in these tissues ( testis and kidney marrow ) occurs independently of changes in telomere length . In the future it will be important to dissect the mechanisms by which telomere shortening induced stress in one tissue is perceived by other tissues in the rest of the organism . Our work shows that telomere shortening and accumulation of DDR in particular tissues anticipates aging associated dysfunction . As previously proposed [108] , accumulation of short telomeres and high levels of persistent DDR in one specific tissue ( gut ) may be enough to establish the rate of aging in other tissues by non cell autonomous mechanisms ( Fig 6 ) . Strikingly , absence of telomerase triggers premature recapitulation of aging phenotypes in most tissues , even in those where telomeres do not reach tert-/- shortened telomere lengths during aging . Importantly , by using the “percentage of life” instead of absolute age to measure the progression of aging phenotypes , we find surprisingly similar kinetics between the dynamics of WT and tert-/- phenotypes over time , local and systemic ( Fig 6 ) . Thus , rescuing telomere length of aged zebrafish in key tissues ( such as the gut ) may prove sufficient to restore local and systemic homeostasis in old animals . Whether systemic aging signals will be dominant over locally rejuvenated tissues remains to be tested . However , such therapies will likely only be productive before telomeres reach critical length . Previous studies have shown that reactivation of telomerase in tissues that have undergone extensive telomere erosion is a strong potentiator of tumorigenesis and malignancy [109] . Our study suggests a potential therapeutic window of opportunity , in a period that correlates with mid-life in zebrafish , before age-associated defects become apparent . Time- and tissue-dependent telomerase reactivation will enable us to define and test such a useful period for telomerase therapies .
All Zebrafish work was conducted according to National Guidelines and approved by the Ethical Committee of the Instituto Gulbenkian de Ciência and the DGAV ( Direcção Geral de Alimentação e Veterinária , Portuguese Veterinary Authority ) . Zebrafish were maintained in accordance with Institutional and National animal care protocols . The telomerase mutant line tertAB/hu3430 , generated by N-Ethyl-N-nitrosourea ( ENU ) mutagenesis ( Utrecht University , Netherlands; Wienholds , 2004 ) , has a T-A point-mutation in the tert gene . tertAB/hu3430 line ( available at the ZFIN repository , ZFIN ID: ZDB-GENO-100412-50 , from the Zebrafish International Re-source Center—ZIRC ) . The protocols used for outcrossing mutagenized male zebrafish were previously described [30] . The terthu3430/hu3430 homozygous mutant ( tert-/- ) was obtained by incrossing our tertAB/hu3430 strain . WT siblings were used as controls . Genotyping was performed by PCR of the tert gene followed by sequencing [30] . Overall characterization of tert-/- and WT zebrafish was performed in F1 animals produced by tert+/- incross . Due to a male sex bias in our crosses that affected mostly tert-/- progeny , we were unable to obtain significant numbers of females for analysis and so the majority of our data is restricted to males unless otherwise stated . All animals showing signs of morbidity that persisted for up to 5 days , such as inability to eat or swim , or macroscopic lesions/tumors were sacrificed in 200 mg/L of MS-222 ( Sigma , MO , USA ) . TRF analysis was performed as previously described [54] . Briefly , genomic DNA was extracted from freshly isolated tissue using lysis buffer ( Fermentas K0512 ) supplemented with 1 mg/ml Proteinase K ( Sigma , MO , USA ) and RNase A ( 1:100 dilution , Sigma , MO , USA ) . Samples were incubated at 50°C for 18 h in a thermomixer and genomic DNA was extracted by equilibrated phenol-chloroform ( Sigma , MO , USA ) and chloroform-isoamyl alcohol extraction ( Sigma , MO , USA ) . Genomic DNA was quantified and normalized so the same amount of DNA was digested with RSAI and HINFI enzymes ( NEB , MA , USA ) for 12 h at 37°C . Samples were electrophoresed on a 20 cm 0 . 6% agarose gel , in 0 . 5% TBE buffer , at 4°C for 17 h at 110 constant voltage . A 1 . 6 kb telomere probe , ( TTAGGG ) n , labelled with [α-32P]-dCTP using the Prime-it II random primer labelling kit ( Stratagene ) was used for Southern blotting . 3–6 different WT and tert-/- individuals belonging to different age groups were used for quantification of each Southern Blot experiment . Reactive oxygen species ( ROS ) accumulation was assessed by measuring the levels of the oxidized form of the cell-permeant 5-chloromethyl-2’ , 7’- dichlorodihydrofluorescein diacetate ( DCFDA ) ( Sigma ) . Briefly , zebrafish were euthanized with MS-222 and gut and muscle were dissected . Each tissue was homogenized in 100 μl of ROS buffer ( 0 . 32 mM sucrose , 20mM hepes , 1mM MgCl2 and 0 . 5mM Phenylmethanesulphonyl fluoride ) . Homogenates were centrifuged and 20 μl of the supernatant was transferred to a 96-well plate and incubated in 1 μg/ml of DCFDA for 30 minutes . Fluorescence values were measured with a Victor 3 plate reader and normalized to total protein content . N = 3 per time point . Zebrafish were sacrificed by anesthetic overdose , in 200 mg/L of MS-222 ( Sigma , MO , USA ) , fixed for 72 hours in 10% neutral buffered formalin and decalcified in 0 . 5 M EDTA for 48 h . Whole fish were then paraffin-embedded and 3 micrometer midline sagittal sections were stained with hematoxylin and eosin for histopathological analysis . Sections were examined by a pathologist ( TC ) , blinded to experimental groups and microphotographs were acquired in a Leica DM2500 microscope coupled to a Leica MC170 HD microscope camera . A total of 238 WT and 66 tert-/- zebrafish were submitted for histopathological analysis . At least 4 animals from each age group/genotype were analyzed . Sections were deparaffinized , rehydrated and heat-induced antigen retrieval was performed in citrate buffer ( 10 mM Sodium Citrate , pH 6 ) for 20 minutes at 103°C in an oven . Slides were left to cool down for 30 minutes at room temperature ( RT ) , washed three times in dH20 for 5 minutes each and blocked for 1 hour at RT in 0 . 25% BSA in PBST ( Triton 0 . 3% ) . The following primary antibodies were used: rabbit polyclonal antibodies against Proliferation Cell Nuclear Antigen ( PCNA , Santa Cruz , CA , USA , 1:50 dilution ) and Histone H2A . XS139ph ( GTX127342 , USA , 1:500 ) . Incubation with primary antibodies was performed overnight at 4°C , followed by three 10 minute PBS washes . Incubation with the secondary antibody Alexa Fluor 568 goat anti-rabbit ( Invitrogen , UK , 1:500 dilution ) overnight at 4°C was followed by three 10 minute PBS washes , DAPI staining ( Sigma , MO , USA ) and mounting with DAKO Fluorescence Mounting Medium ( Sigma , MO , USA ) . Apoptosis was detected using the In Situ Cell Death Detection Kit ( Roche , SW ) according to manufacturer's instructions . Briefly , deparaffinized slides were incubated with 40 μg/ml Proteinase K in 10 mM Tris-HCl pH 7 . 4 , 45 minutes at 37°C . Slides were washed in 2×5 minutes in PBS and then incubated with TUNEL labelling mix ( protocol indicated by the supplier ) . Washing and mounting were performed as previously described . Images were acquired on Leica SP5 Live Upright confocal microscope ( Leica Microsystems , GER ) equipped with Leica Las AF Lite software and with appropriate configurations for multiple color acquisition . For quantitative and comparative imaging , equivalent image acquisition parameters were used . The percentage of positive nuclei was determined by counting a total of 500–1000 cells per slide , 63x amplification ( N = 3–4 zebrafish per time point/genotype ) . Zebrafish were sacrificed by anesthetic overdose , in 200 mg/L of MS-222 ( Sigma , MO , USA ) . Gut and testis were dissected and placed in 100 μL of DMEM TrypLE supplemented with 10%FBS . Tissues were digested at 37°C for 1 hour and fixed in an equal volume of 10% buffered formalin with 0 . 2% Triton for 2 hours at room temperature , agitating . Tissue cells were cytospinned for 5 minutes at 1000 rpm and slides were left drying overnight at room temperature . Slides were washed in 1xPBS/0 . 1%Triton for 4x5min and incubated in 3%H2O2 for 20min at 37°C . After washing 3 times for 5 minutes in H2O , slides were incubated in acetone at -20°C for 7 minutes . Slides were washed 2 times for 5 minutes and blocked 1 hour at room temperature with PBDX buffer with goat serum ( 50 mL 1xPBS , 0 . 5g BSA , 0 . 5 mL DMSO , 250 μl Triton 10% , 750 μl goat serum ) . Primary antibody ( Histone H2A . XS139ph , GTX127342 , USA , 1:100 ) was added for 1 hour at room temperature and left incubating overnight at 4°C . Primary antibody was washed with 1x PBS/0 . 1% Triton two times , 10 minutes each , followed by four washed ( of 30 minutes each ) with PBDX . Secondary antibody Alexa Fluor 568 goat anti-rabbit ( Invitrogen , UK , 1:500 dilution ) together with DAPI ( Sigma , MO , USA , 1:1000 ) were added to slides for 1 hour at room temperature followed by incubation overnight at 4°C . Secondary antibody was washed with 1xPBS/0 . 05% Tween four times , 15 minutes each wash , followed by fixation in buffered formalin 10% for 20 minutes . Slides were washed in 1xPBS/0 . 05% Tween 2x5 minutes and air-dried . Denaturing was performed for 15 min at 80°C in hybridization buffer ( 70% formamide ( Sigma ) , 25 mM MgCl2 , 1 M Tris pH 7 . 2 , 5% blocking reagent ( Roche ) ) , containing 3 μg/ml Cy-3-labelled telomere specific ( CCCTAA ) peptide nuclei acid probe ( Panagene ) , followed by hybridization for 2 h at room temperature in the dark . The slides were washed with 70% formamide in 2×SSC for 10 minutes , followed by two 10 minutes wash with 2×SSC and PBS . Slides were mounted as previously described and imaged in a Leica HCScreening ( 100x objective ) . For quantitative and comparative imaging , equivalent image acquisition parameters were used . The percentage of positive nuclei was determined by counting a total of 50–100 cells per slide ( N = 3 zebrafish per time point/genotype ) . β-galactosidase assay was performed as previously described [110] . Briefly , sacrificed zebrafish adults were fixed for 72h in 4% paraformaldehyde in PBS at 4°C and then washed three times for 1 h in PBS-pH 7 . 4 and for a further 1 h in PBS-pH 6 . 0 at 4°C . β-galactosidase staining was performed for 24 h at 37°C in 5 mM potassium ferrocyanide , 5 mM potassium ferricyanide , 2 mM MgCl2 and 1 mg/ml X-gal , in PBS adjusted to pH 6 . 0 . After staining , fish were washed three times for 5 minutes in PBS pH 7 and processed for de-calcification and paraffin embedding as before . Sections were stained with nuclear fast red for nuclear detection and images were acquired in a bright field microscope ( Olympus BH2 , LB ) . To quantify SA-β-GAL staining we used an arbitrary scale of signal intensity ( where weaker signal is represented by 1 and stronger signal by 4; N≥3 per time point per genotype ) . Fish were kept in their living tanks until they were randomly selected for breeding . Briefly , breeding pairs were left overnight in external breeding tanks and collection of eggs was performed the following morning . 3–10 independent breeding trials were conducted for each age group and genotype ( using either 6-month old female or male controls , tested previously for successful reproductive ability ) , to evaluate how aging affects male or female reproduction . Only successful breeding trials , defined as events where a female lays a clutch of eggs when paired with a male , were scored . In every trial group , a breeding box including a 6-month old couple was included to ensure the protocol in place guarantees maximum reproductive output . Embryos were collected approximately 2 hours post fertilization ( hpf ) and allowed to develop at 28°C . Assessment of egg fertilization and embryo viability was conducted between 2 and 4 hpf . Single-cell suspensions from head kidney marrow ( HKM ) were prepared from adult zebrafish . Freshly isolated head kidney tissue was placed in a 1 . 5ml eppendorff containing 200μl of complete high-glucose DMEM ( 10% FBS; 1% pen/strep ) on ice . Tissue was maintained in the tube , on ice , while gently disrupted manually with the aid of a plastic pestle . Single-cell suspensions were obtained by gentle pipetting and filtering through a 40μM nylon membrane cell strainer ( Falcon ) . Cells in complete DMEM were incubated for 15’ on ice with 1mg/ml propidium iodide for dead cell exclusion analysis . Single cell suspensions from HKM were examined using a CyanADP flow cytometer ( Beckman Coulter ) , using PI for dead cell exclusion and SSC VS FSC to identify the different cell populations , as previously described [85] . Data were analyzed using FlowJo software Version 9 . 4 . 11 ( TreeStar ) . N = 3–5 zebrafish per time point per genotype . Image edition was performed using FIJI [111] . Statistical analysis was performed in GraphPad Prism5 , using two-way ANOVA test with Bonferroni post-correction for all experiments comparing WT and tert-/- over time . Correlations between phenotypes and telomere size were tested using one-tailed Pearson’s test . Comparison of WT and tert-/- survival curves was performed using a log-rank ( Mantel-Cox ) test . Comparison of WT and tert-/- cancer incidence curves was performed using a Kolmogorov–Smirnov test . Significance of fertility decline in tert-/- mutants was assessed by a Mann–Whitney’s test . A critical value for significance of p<0 . 05 was used throughout the study . | Why , and how , organisms age and ultimately die is a key question of modern biology . Telomeres are considered molecular timekeepers determining cellular lifespans . Within an organism , tissue proliferation is expected to dictate the rate at which telomeres shorten . Using zebrafish , an organism with human-like telomeres , we set out to test whether , in natural aging , proliferative tissues age faster than non-proliferative in a telomere-dependent manner . We found that telomeres shorten with age to a level where they trigger telomere associated DNA damage and culminate in tissue dysfunction , independently of high or low tissue proliferation rates . Specifically , short telomeres accumulate in the gut , a highly proliferative tissue , and in the muscle , a low proliferative tissue , working as direct predictors of cellular damage prior to onset of intestinal inflammation and myocyte degeneration . Based on our data , we propose a model where telomere shortening in these key tissues is sufficient to trigger damage in others and precedes the onset of organism age-associated diseases , namely cancer . Thus , tissue-specific telomere length is limiting for local and systemic physiological integrity , leading to tissue degeneration and disease in aging . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [] | 2016 | Short Telomeres in Key Tissues Initiate Local and Systemic Aging in Zebrafish |
We investigate how the neural processing in auditory cortex is shaped by the statistics of natural sounds . Hypothesising that auditory cortex ( A1 ) represents the structural primitives out of which sounds are composed , we employ a statistical model to extract such components . The input to the model are cochleagrams which approximate the non-linear transformations a sound undergoes from the outer ear , through the cochlea to the auditory nerve . Cochleagram components do not superimpose linearly , but rather according to a rule which can be approximated using the max function . This is a consequence of the compression inherent in the cochleagram and the sparsity of natural sounds . Furthermore , cochleagrams do not have negative values . Cochleagrams are therefore not matched well by the assumptions of standard linear approaches such as sparse coding or ICA . We therefore consider a new encoding approach for natural sounds , which combines a model of early auditory processing with maximal causes analysis ( MCA ) , a sparse coding model which captures both the non-linear combination rule and non-negativity of the data . An efficient truncated EM algorithm is used to fit the MCA model to cochleagram data . We characterize the generative fields ( GFs ) inferred by MCA with respect to in vivo neural responses in A1 by applying reverse correlation to estimate spectro-temporal receptive fields ( STRFs ) implied by the learned GFs . Despite the GFs being non-negative , the STRF estimates are found to contain both positive and negative subfields , where the negative subfields can be attributed to explaining away effects as captured by the applied inference method . A direct comparison with ferret A1 shows many similar forms , and the spectral and temporal modulation tuning of both ferret and model STRFs show similar ranges over the population . In summary , our model represents an alternative to linear approaches for biological auditory encoding while it captures salient data properties and links inhibitory subfields to explaining away effects .
The goal of this paper is to understand the computational principles which underpin neural processing in auditory cortex . In particular , we investigate the hypothesis that neural processing is shaped by the statistics of natural sounds , the physical rules governing how those sounds combine , and the form of the initial processing performed by the ear . It is well known that the outer , middle and inner ear transform an incoming sound pressure waveform into a representation at the auditory nerve which can be approximately described by a filtering stage ( in which the sound is broken into subbands ) , followed by an envelope extraction and compression stage . This approximation to the auditory nerve’s representation of a sound is called a cochleagram and intuitively it can be thought of as revealing the spectro-temporal variations in the energy of the input waveform . It is believed that subsequent stages of auditory processing might decompose this representation into basic “structural primitives” , i . e . , components or building blocks from which natural sounds are composed . Such a representation would provide a basis to support more complex computation at higher levels in the system ( compare , e . g . , [1] ) . The idea of representations in terms of primitives is supported to some extent by in vivo recordings in the primary auditory cortex of mammals which suggests that neurons are most sensitive to structures that are localized in time and frequency [2–6] , but the hypothesis still lacks convincing evidence . One way of investigating the hypothesis that auditory cortex is representing the components of natural sounds is to learn their form from a corpus of natural sounds . A particularly popular approach , which has been used for great success for visual data [7] and subsequently for audio data [8 , 9] , is based on the idea that the stimulus is formed by a linear combination of components which are sparsely activated . However , for auditory stimuli , this “sparse coding” approach is arguably not the most natural one to take for three main reasons . First , a linear mixture of sound pressure waveforms ( formed either from multiple sources in the environment or from a single source comprising a linear mixture of primitive components ) results in a non-linear mixture at the level of the auditory nerve and it seems likely that downstream processing would respect this fact . Second , the cochleagram is non-negative which is not reflected by the standard form of the sparse coding model . Third , sparse coding ( or ICA ) operates most effectively on whitened data ( although this might be due to current algorithmic limitations , rather than a general feature of the approach ) . In the visual system it has been argued that the lateral geniculate nucleus ( LGN ) performs such a whitening step [10] but the initial transformations employed in the auditory system are quite different , making this sort of preprocessing harder to justify . Whitening for cochleagrams would essentially mean that neural activities do not encode energies in frequency bands but deviations from a mean energy relative to energy variances . Adaptation effects to mean and variances over time are well known for regions upstream of the cortex such as the auditory nerve and inferior colliculus [11–14] . However , this adaptation should not be equated with whitening . If it was this would imply that the absence of any signal energy should lead to ( on average ) equally strong responses as energies above the mean . If we do not assume a whitening stage for cochleagrams or a similar preprocessing to obtain mean-free stimuli , then we are confronted with the question: How do measured STRFs with their positive and negative subfields emerge ? In vision , after an assumed whitening stage , stimuli contain positive and negative parts which directly result in components extracted by sparse coding to have negative and positive subfields . For the non-negative energy representation of cochleagrams it is so far unclear how negative subfields can emerge without a whitening stage . Statistical data models not requiring whitening suggest alternative mechanisms commonly referred to as “explaining away effects” which have so far not been linked to negative subfields of neural response properties . As an example for “explaining away” consider the situation of sitting in a park . It is a nice warm day , you have your eyes closed , and are just listening to the sounds around you . There is a small orchestra somewhere with musicians practicing for a concert , and there are birds in the trees . If you now perceive a very short melodic sequence , it may have been generated by a bird or by a musician’s flute . As you are too far away from any of the sources , and as the perceived sequence is too short and unspecific , it is not possible for you to say for sure which of the potential sources may have generated the sound . But you do know that a high probability for one source , e . g . the flute , would mean a low probability for the other . This dependency between the probabilities for the two potential sources given a sound is called “explaining away” . If you were more certain that it was the flute playing ( e . g . , by getting additional visual input ) , the flute would “explain away” the alternative explanation of the sound having been generated by a bird . The statistical models investigated here will have similar explaining away effects but on a lower level of sound processing ( Fig 6 will give a low level example later on ) . The primary statistical model investigated here assumes the data to be non-negative ( and not whitened ) , and it assumes the structural primitives to combine non-linearly . More concretely , we assume structural primitives to combine such that the maximal energy in each time-frequency interval determines the superimposed signal ( Fig 1 shows an illustration ) . To summarize our goal , instead of using the dominating approach of standard sparse coding as statistical model to study neural representation in auditory cortex [8 , 9] , we investigate for the first time a non-linear and non-negative alternative . Our approach is motivated by the observation that alternatives to the assumptions of linear superposition and whitening may be more natural for acoustic data , and it offers an alternative explanation for the inhibitory subfields of STRFs which were previously closely linked to signal whitening .
Animal experiments were done at the Department of Physiology , Anatomy , and Genetics , University of Oxford , performed under license from the United Kingdom Home Office and were approved by the ethical review committee of the University of Oxford . The electrophysiological recordings were made from an adult pigmented ferret under ketamine ( 5 mg/kg/h ) and medetomidine ( 0 . 022 mg/kg/h ) anesthesia . After recording , the animal was killed with 1ml/kg i . v . Pentoject . In the inner ear , sound pressure waves are considered to be broken-down into their frequency components by the cochlea , which then also compresses the frequency response amplitudes to form log-spectrograms resembling cochleagram representations of the input signal . The cochleagrams are then further communicated via the auditory nerve for neural processing and as they arrive in higher brain areas such as the primary auditory cortex , the cochleagrams are believed to get decomposed into elementary components for higher-level processing . We assume that a cochleagram representation y → ∈ R D can be composed as a combination of a ( small ) number of primitive auditory components W → h ∈ R D , which form elements of a large dictionary W = ( W → 1 , … , W → H ) of H components . For such a multi-component encoding scheme , classical modeling approaches such as standard sparse coding [7] or ICA [27 , 28] assume a linear interaction of the components to define a data generation process: y → = ∑ h s h W → h + η → , where s h ∈ R determines the mixing factors for components W → h and η → denotes added noise in the generative process ( which usually is assumed to be zero for ICA ) . However , cochleagrams are a representation of a non-linear interaction between the auditory components , for which a more accurate generative process can be derived from the log-max approximation [15–17] . The log-max approximation implies that the cochleagram of a linear mixture of sound waves can be well approximated by taking the pointwise maximum of cochleagrams computed from the individual waveforms . Fig 1 illustrates the approximation based on the cochleagram model used in this study . The example shows a better match by the point-wise maximum than by a linear combination . Hence , based on the approximation , we can define the following probabilistic generative model for cochleagrams: p ( s →|Θ ) = ∏ h π s h ( 1 - π ) 1 - s h m ( Bernoulli ) ( 2 ) p ( y →|s → , Θ ) = N ( y → ; max h { s h W → h } , σ 2 I ) , ( 3 ) where the max operation is applied element-wise , i . e . , ( maxh{ x→h } ) d=maxh{ xdh } , and where I denotes the identity matrix . Here we assume the factors sh ∈ {0 , 1} to be Bernoulli distributed , whereas the observed noise is assumed to be Gaussian . Eqs 2 and 3 are a version of the MCA generative model [19 , 20] . Parameters of the model are: the frequency π with which a component is activated , the variance of the observation noise σ2 , and the generative components or fields W → h , which we will later relate to STRFs . For notational convenience Θ = ( π , σ , W ) denotes the set of all these parameters . As a control for later numerical experiments with the MCA model , we will also consider a model assuming a standard linear combination of structural primitives . More concretely , we use a model that shares preprocessing , prior , and noise assumption with the MCA model but uses a linear superposition model instead of the point-wise max: p ( y →|s → , Θ ) = N ( y → ; ∑ h s h W → h , σ 2 I ) . ( 4 ) Eq 4 has the standard form of linear sparse coding approaches [7] , and is because of the prior ( 2 ) a form of Binary Sparse Coding ( BSC; [21 , 29 , 30] ) . Given a set of N cochleagrams { y → ( n ) } n = 1 , … , N computed as in Section Cochlear model and spectrogram generation . , we now seek parameters Θ* that optimally fit the MCA model to the data . We use likelihood maximization to find the optimal parameters and apply an approximate version of expectation maximization ( EM; [31] ) for their efficient estimation . The application of standard maximum a-posteriori ( MAP ) based approximations is prohibitively suboptimal for the MCA model because the non-linear interaction of components typically results in multi-modal posteriors . An efficient approximate EM approach which can capture multi-modal posterior structure is , however , provided by Expectation Truncation ( ET; [20] ) . ET can be regarded as a variational EM approach , and it has successfully been applied to MCA [21 , 22 , 32] and many other generative models [33 , 34] . ET approximates the computationally intractable full posterior p ( s →|y → , Θ ) by a truncated one [20]: q ( n ) ( s → ; Θ ) ∼ p ( s →|y → ( n ) , Θ ) δ ( s → ∈ K n ) , ( 5 ) where δ is an indicator function ( i . e . , δ ( s → ∈ K n ) = 1 if s → ∈ K n and zero otherwise ) . If K n is chosen to be small but such that it contains the states with most posterior probability mass , the computation of the expectations in Eq 5 becomes tractable while a high accuracy of the approximations can be maintained [20] . The set K n is , therefore , chosen to consider the subset of the H′ most relevant hidden units for a patch y → ( n ) . Furthermore , at most γ of these H′ units are assumed to be active simultaneously | s → | ≤ γ . Please see Efficient Likelihood Optimization in Supporting Information for a formal definition of K n . Parameter update equations for the MCA model have been derived earlier [19 , 21 , 32] . They are given by: W d h new = ∑n A d h ρ ( s → , W ) q ( n ) y d ( n ) ∑n A d h ρ ( s → , W ) q ( n ) , A d h ρ ( s → , W ) = ( ∂ ∂ W d h W ¯ d ρ ( s → , W ) ) , ( 6 ) W ¯ d ρ ( s → , W ) = ( ∑ h ( s h W d h ) ρ ) 1 ρ , ( 7 ) σ new = 1 N D ∑ n ⟨∥ y → ( n ) - max h { s h W → h } ∥ 2 ⟩ q ( n ) , i i i i π new = 1 H N ∑ n ⟨ | s → | ⟩ q ( n ) , ( 8 ) where the parameter ρ in Eq 7 is set to a large value ( we used ρ = 20 ) and ‖⋅‖ in Eq 8 denotes the L2-norm . The learning algorithm for the MCA generative model is thus given by the equations above with expectation values computed w . r . t . the approximate posterior in Eq 5 . The linear BSC model , Eqs 2 and 4 is trained analogously to the MCA model with parameter update equations as derived earlier ( e . g . , [30] ) . Please see “Efficient Likelihood Optimization” in Supporting Information for more details .
We applied our method to male and female anechoic speeches in English , Japanese , Italian , and German . The data also included recordings of natural sounds such as rustling leaves , clattering stones and breaking twigs . More details about the data acquisition procedure are given in Natural Sound Recordings in Supporting Information . We cut the waveforms of the recordings sampled at 44 . 1 kHz into snippets of 160 ms with a 32 ms overlap . The snippets were then transformed to cochleagram representations following section “Cochlear Model and Spectrogram Generation” . For the gammatone preprocessing we used a 32−channel filterbank with center frequencies ranging between 1000 and 22050 Hz . In this work we used Slaney’s implementation [35] to apply a 4th order gammatone filter . The outputs of the filter were averaged over a 20 ms sliding window with a 10 ms step size . The averaged energies were then compressed through the logarithm ( as described earlier ) to generate 32 × 15 cochleagrams , that is the energy at 32 center frequencies over 15 consecutive time windows . We applied the MCA learning algorithm using H = 1000 generative fields to a set of N = 72800 cochleagrams . Individual cochleagrams were normalized by the L2-norm of their energies . To find the maximum likelihood parameters Θ approximately , we performed 70 EM iterations of the ET based learning algorithm described in “Efficient Likelihood Optimization” . The truncation parameters H′ and γ were set to 10 and 6 , respectively . We initialized each of the components in the W matrix with the mean of the data perturbed by standard Gaussian noise with zero mean and variance set to 1/4th of the variance of the data . Parameter σ was initialized to the square root of the variance of the data and π was set to 30/H where H = 1000 . To minimize the possibility of running into local optima , we applied deterministic simulated annealing [36 , 37] for the first half of the EM iterations with a linearly decreasing temperature from 10 to 1 ( compare [20] ) . As a control , we also trained the linear BSC model analogously to MCA , i . e . , using the same data preprocessing and initialization details as for MCA . Fig 2C shows 100 of the 1000 learned generative fields after the 70 EM iterations . As can be observed , most of the fields are very localized in time and frequency . The generative fields resulting from applying the BSC model are provided in Supplementary S2 Fig . In order to relate the MCA encoding of cochleagrams to neurons in the auditory cortex , we estimate spectro-temporal receptive fields ( STRFs ) from the inference results of the trained MCA model on the natural sound data . In physiological studies , an STRF is the numerically computed estimation of the linear mapping from sound cochleagrams that best predicts a neuron’s response . Similarly we compute STRFs that we consider to be tuned to individual latent components that we learn . To estimate STRFs W ^ * for the MCA model , we seek parameters that minimize the following function: f ( W ^ ) = 1 N ∑ n = 1 N ∑ s → ( n ) ∈ K n p ( s → ( n ) | y → ( n ) , Θ ) ∥ W ^ y → ( n ) - s → ( n ) ∥ 2 + λ ∥ W ^ ∥ 2 , ( 9 ) where y → ( n ) is the nth stimuli , W ^ is the row-dominated matrix of predicted STRFs , and λ is the coefficient for L2 regularization . Here we assume that the neural response to a stimulus will be a sample from p ( s → ( n ) | y → ( n ) , Θ ) , in which case the experimentally measured STRFs will minimize the squared error between W ^ y → ( n ) and s → ( n ) . Our assumption is consistent with interpreting neural responses as posterior samples [38] , and the regularization term corresponds to assuming a zero-mean Gaussian hyperprior for the weights ( compare ridge regression , e . g . , as discussed in [39] ) . The intractable posterior over the latent factors p ( s → ( n ) | y → ( n ) , Θ ) in Eq 9 is truncated to only cover the subspace K n , as defined by the variational approximation technique in Efficient likelihood optimization . By setting the derivative of the cost function ( 9 ) to zero , W ^ can be estimated as: W ^ = ( ∑ n = 1 N ⟨ s → ( n ) ⟩ q n ( y → ( n ) ) T ) ( λ N I + ∑ n = 1 N y → ( n ) ( y → ( n ) ) T ) - 1 ( 10 ) where I is the D × D identity matrix and where 〈 · 〉 q n denotes the expectation value w . r . t . the approximation q n ( s → ; Θ ) of the posterior p ( s → ( n ) | y → ( n ) , Θ ) of the MCA model . The additional term λNI results from a L2-regularization for W in the cost function . Without regularization , the eigenvalues of the data covariance matrix ∑ n y → ( n ) ( y → ( n ) ) T were frequently very close to zero causing numerical instabilities . For the regularization parameter λ , we empirically found that a value in the mid-range of the minimum and the maximum eigenvalues of the data covariance matrix was sufficient to resolve the numerical instability . Corresponding to the generative fields shown in Fig 2C , Fig 2D illustrates the STRF estimates computed from ( 10 ) . We will refer to these estimates as model STRFs from now on . Observe first that many of the model STRFs are localized in time and frequency , a very common feature of receptive fields in the A1 [3 , 40 , 41] . Receptive fields produced by earlier sparse coding models do not as extensively have this punctate character [9 , 42 , 43] . Observe also that many of the model STRFs show flanking inhibition both spectrally and temporally , which is likewise a common feature of A1 receptive fields . However , a difference is that receptive fields of auditory cortical neurons tend to show asymmetry in their temporally flanking inhibition , most inhibition being found in the past relative to the excitatory region . In Fig 3 ( left ) let us first consider 9 exemplary model STRFs , that illustrate various features which are also seen in experimentally recorded A1 STRFs as illustrated on the right-hand-side of Fig 3 ( for how the STRFs were recorded from ferret cortex and estimated see the Supplement ) . Reading the Fig 3 ( left ) from left to right , the first unit shows punctate high frequency excitation , the second two units show punctate mid frequency excitation , and the next two units show punctate low frequency excitation . This illustrates that the units’ spectral tuning are spread over the frequency range , as found in physiology , as shown in Fig 3 ( right ) . The sixth unit illustrates an upward sweep in frequency , and the seventh a downward sweep . The eighth and ninth units illustrate receptive fields that are spread out over frequency and time respectively . Again these four types of STRF are found in A1 , as show in Fig 3 ( right ) . To quantitatively compare the model STRFs and auditory cortical STRFs across the population we took 244 experimentally recorded STRFs from Ferret A1 and AAF ( taken from [45] , see Supporting Information: “Neural Recordings and Real STRFs” ) and compared them to the most frequently used model STRFs ( i . e . , to those fields which were the most probable to be activated across all stimuli ) . For the comparison , a 2D-Fourier transform was applied to each model receptive field and STRF , this provided the modulation transfer function of each receptive field and STRF ( 3 STRFs were excluded as all their values were zero , see Methods ) . Then , for each of the 241 remaining real STRFs and model STRFs the frequency modulation and temporal modulation at which the highest value occurred was taken ( the best scale and best rate , respectively ) . A histogram of distribution of best scale and rate is plotted for the real A1 STRFs in Fig 4A and 4B ( left ) , and for the MCA model STRFs in Fig 4A and 4B ( middle ) . The histogram for the BSC model STRFs is shown in Fig 4A and 4B ( right ) . For Fig 4 we used ( to match the number of neurons we recorded from ) the 241 most frequently used model fields , which represent ≈80% of the overall posterior mass for the MCA model . For comparison , the same histogram but using the 600 most frequently used model fields is shown in the Supplementary S3 Fig ( capturing 97% of the posterior mass for the MCA model ) . S3A Fig ( middle ) is similar to Fig 4A ( middle ) but with more model fields at rate zero . The additional fields of Fig S3 which make up the difference to Fig 4 are , however , four times less likely to be active , which makes Fig 4A ( middle ) more representative for a comparison , see Supplement “Generative Fields and Estimated Model STRFs” for details . In contrast , the histograms for the 241 and the 600 most frequently used BSC fields show comparable percentages of STRFs close to rate zero . Considering Fig 4 , observe that the real STRFs and the receptive fields of the MCA model span a similar range of temporal modulations ( rates ) and a similar range of spectral modulations ( scales ) . Fields tuned to higher scales and fields with higher and lower magnitudes of rate are a bit more frequent for the MCA model than for the experimental data . For the BSC model , the difference of the histogram to the measured data is larger . Significantly more fields have the best rates around zero or at higher magnitudes than the experimental data . The better match of histogram for the MCA model compared to the linear BSC model can be quantified using a χ2 test ( Fig 4C ) . In conclusion , the receptive fields of the MCA model and real STRFs span a similar range of temporal modulations ( rates ) and a similar range of spectral modulations ( scales ) . The model STRFs of the BSC model also span similar ranges of temporal and spectral modulation but this similarity is less pronounced than for the masking-based MCA model . We also examined the tuning width , over frequency and over time , of the excitatory and inhibitory fields of the real and model STRFs . We used the same most frequently active model fields as for Fig 4 , and a tuning width measurement method modified from [46] . For the measurement of frequency tuning width of the excitatory fields , the negative values of the STRFs were set to zero , then the STRF was squared in an element-wise manner and then the STRF was summed over the time bins to give a weighting vector over frequency bands . The excitatory frequency tuning width was then measured as span of frequencies ( in octaves ) whose weighting was ≥ 50% of the highest weighted frequency channel . For the measurement of temporal tuning width of the excitatory fields , the negative values of the STRFs were set to zero , then the STRF was squared in an element-wise manner , and then the STRF was summed over frequencies , to give a weighting vector over time bins . The excitatory temporal tuning width was measured as the number of time bins that were ≥ 50% of the maximum value of the resulting vector , multiplied by the time bin size of 10 ms . The inhibitory frequency and temporal tuning widths were measured similarly but instead the positive values of the STRF were set to zero , rather than the negative values . For a visualization of how they are measured see Supplementary S4 Fig . Observe that for frequency , for the inhibition and to a lesser extent the excitation , the tuning widths of the MCA model STRFs match relatively well the tuning widths of the STRFs of real neurons . For the temporal dimension we see more strongly diverging properties which may have been expected by considering the statistical modeling approach: Like sparse coding or ICA we do focus on the composition of the data points in terms of structural primitives . Our model itself does not contain statistical dependencies in time unlike hidden Markov models or linear dynamical systems would do . As acoustic data does contain such dependencies on multiple time scales , it is likely that neural processing reflects also these dependencies . The discrepancy of temporal modulation in contrast to frequency modulation may therefore be taken as evidence for the auditory cortex capturing the intricate statistical dependencies over time which neither sparse coding , ICA nor the here studied MCA model addresses . The control experiments using BSC support this interpretation . Also for BSC no asymmetry similar to the one of the measured ferret STRFs is observed . Histograms for BSC computed analogously to Fig 5 are given in the Supplementary S5 Fig . In contrast to the histograms of best modulation frequencies , no notable differences between MCA and BSC histograms were observed .
We have shown that the MCA model exhibits a close correspondence to some of the STRF properties of neurons in ferret primary auditory cortex . Like STRFs of the real neurons , the MCA model STRFs show one or a few excitatory regions that are often punctate , being restricted over frequency and often over time . The excitatory regions of the MCA model STRFs are also often flanked by inhibition in frequency and/or time , consistent with real STRFs . The real neurons of our dataset and another ferret cortical dataset [50] show diverse STRFs , likewise the MCA model captures a similar diversity of STRFs with some model STRF broadly tuned over frequency or time , some narrowly tuned , some complex with multiple excitatory regions and some directional with diagonally oriented fields . However , the model STRFs do not capture the fact that inhibitory regions that flank in time tend to occur predominately after excitatory regions , rather than on both sides . This is unsurprising as the MCA model does not have the capacity to reflect causal statistical dependencies in time . MCA shares this property with other ICA-like and sparse coding models ( including BSC ) . It may be noteworthy at this point that already in short-time STRFs , such as we use or are often measured in physiology , the limits of approaches that do not explicitly model dependencies in time are apparent . Measurements and analysis of neural responses in the auditory forebrain of birds [51] suggest that short-time STRFs do represent regularities important for capturing sound regularities over time . There , different types of STRFs have been linked to the processing of different sound properties such as spectral-pitch , rhythm , timbre or periodicity-pitch . Notably , specific functional roles of broad-band STRFs , and of STRFs with inhibition after excitation as well as STRFs with excitation after inhibition have been discussed in this context [51] . Also , the ‘noisy’ type STRFs of Carlin et al [50] with very disordered field structure are not notable in the models here considered . The control model ( BSC ) produces STRFs with many properties similar to the MCA model , and most quantitative differences are relatively small . A main difference is that whereas the MCA model reproduces fairly well the distribution of best spectral and temporal modulation frequencies of real neurons , albeit somewhat overestimating the span of rates and scales , the BSC model shows significantly greater overestimation . On other measures they are similar . The MCA model captures fairly well the frequency tuning widths of real neurons , if underestimating to a degree , however in this capacity it did not perform noticeably better than the BSC model . Curiously , although in ferret data and our models the distribution of frequency tuning widths appears unimodal , in bird auditory forebrain [51] the distribution of frequency tuning widths is bimodal , we speculate as a consequence of the statistics of birdsong . Regarding temporal tuning , birds [51] , our ferret data , and our models all show apparent unimodal distributions of temporal tuning widths . Both the MCA model and the BSC model substantially overestimate the temporal tuning widths of the STRFs of real neurons , which is again unsurprising as neither model has the capacity to reflect causal statistical dependencies in time . Furthermore it should be noted that STRFs are far from a complete description of the tuning properties of auditory cortical neurons . Firstly , auditory cortical neurons show many non-linear properties [52] such as conjunctive AND-gate-like behavior [46] , or amplitude modulation phase invariance [53] . Secondly , neural tuning properties , including STRFs , can also depend to an extent on stimuli used to gather them [45 , 54–59] . Finally , STRFs can also show rapid plasticity depending on the task performed by an awake animal [5] . More generally , it is important to acknowledge that comparing normative models such as MCA to real data is difficult and depends on a number of factors including: details of the training corpus , details of different models of preprocessing and details of the STRF estimation . Any of these factors has an influence on quantitative comparisons as those made in this study . For instance , the data used to optimize a statistical model is unlikely to perfectly match the acoustic statistics experienced by the animals used to obtain the experimental data . Or different STRF estimation techniques applied to meet the requirements of experimental recordings or of the used models will effect the quantitative properties of estimated STRFs . Likewise , different preprocessing models ( which we have not explored ) influence STRF properties ( see [60] for a discussion ) , and have also affected previous work on this topic [8 , 9] . Any preprocessing scheme will , however , agree on cochleagrams being representations of acoustic waveform energies in time-frequency intervals . While such representations may be computed by very complex functions , any energy representations will assume non-negative values . Also strong masking non-linearities of the combination of structural primitives within cochleagram representations are widely agreed on in the literature . Notably , although the generative model here considered incorporates the positivity constraint ( which we believe is biologically important ) , the recognition model nevertheless exhibits inhibitory subfields that arise due to explaining away effects among the components . This result indicates , perhaps counter intuitively , that models with positive generative components can still show inhibitory subfields if STRFs for these components’ generative fields are estimated—a finding which has implications beyond the specific model studied here and beyond the auditory system . More precisely , our study shows that inhibitory subfields can be a direct consequence of the statistical model assumed for explaining the data . Even if the data is non-negative and if the used model assumes non-negative generative fields and non-negative latent activities , inhibitory subfields can emerge directly from explaining away effects , without any additional assumptions . Similar to the introductory example , “explaining away” refers to a dependency between alternative explanations for a given stimulus . For our statistical models , possible explanations of a given stimulus take the form of combinations of generative fields ( which are typically localized in time and/or frequency ) . The co-activation of two similar fields is unlikely ( because of sparsity ) which means that a high probability for one field results in a low probability for the other ( and visa versa ) . Fig 6 aims at providing an intuition why inhibitory subfields emerge because of “explaining away” . Note that the fact that inhibitory subfields do emerge is independent , e . g . , of the combination rule assumed by the statistical model , i . e . , inhibitory subfields can be obtained for non-linear models of generative field combinations ( MCA but also , e . g . , noisy-OR models [61] ) as well as for linear models . For the linear BSC model , we verified such an emergence of negative subfields also for non-negative weights by running additional experiments . While the BSC model we used for controls showed essentially positive weights , negative entries close to zero of the W matrix could be obtained and were obtained in our experiments . To ensure that negative subfields of STRFs also emerge for non-negative weights , we artificially enforced all W entries for BSC to be non-negative in our additional numerical experiments . Also in that case STRF estimation by Eq 10 resulted in negative subfields ( see Supplement “Efficient Likelihood Optimization” for details ) . If measured inhibitory subfields are a consequence of explaining away , then their shapes and the predicted dependencies among hidden neurons change depending on the assumed statistical model . By providing strong evidence for inhibitory subfields to be solely obtainable as a consequence of explaining away , our study offers novel ways of neuro-physiologically evaluating statistical models of neural processing . Here we have compared spectral and temporal modulation as well as temporal and frequency tuning in order to compare different statistical models with data . Comparison of models is made difficult due to the above discussed factors . Significant differences of predicted STRFs can , nevertheless , be obtained if directly comparing statistical models with and without masking non-linearity ( e . g . , Fig 4 ) while all other model properties , training , and preprocessing remained fixed . A step further in the direction of neural evaluation would be represented by a direct in vivo comparison of neural responses to specifically designed stimuli . Given a set of neurons with previously measured STRFs , their responses could be predicted based on different statistical models . These different models will predict different response distributions , and artificial stimuli could be designed to be maximally discriminative between any two statistical models . Based on the results of this study , we predict responses for neurons in A1 which compete to explain an acoustic stimulus to not show a linear anti-correlation ( as predicted by linear models ) . Explaining away resulting from a masking-based model ( such as MCA ) , in contrast , would predict that neurons explaining the same stimulus compete rather in a k-winner-take-all manner , i . e . , small sets of neurons suppress activity in the other neurons with only the maximally active neuron being relevant . For a comparison of explaining away effects between linear models and MCA see e . g . [62] , for k-winner-take-all neural circuits see e . g . [63 , 64] . In this context , let us , furthermore , remark that any neural activity distribution predicated by a model will not only depend on the model for generative field combinations but also on assumed priors , noise model and on the applied approximate inference approach . Furthermore , it will be important which variables of the model are assumed to match any measured neural activity best . Progress in neural recordings , simultaneous recording and stimulus generation , and refined neural modeling may make a direct comparison of statistical models feasible in the intermediate future . A number of normative approaches have been taken to understand auditory spectro-temporal receptive fields as a consequence of stimulus statistics ( e . g . [1 , 9 , 42 , 43 , 47 , 49 , 50 , 65 , 66] ) . Before discussing similarities and differences in relation to the models used here , let us stress that the capturing of stimulus statistics is not the only constraint of importance governing the structure of the nervous system . Biophysical constraints such as energy costs or wiring length are also important , as well as other functional constraints such as the role of particular sounds in an animal’s behavior . Among the stimulus-statistics-based models , the most common approach has been the encoding of spectrogram-like representations of natural sounds subject to a sparsity constraint on the activity of the encoding units . Some sparse normative models balance a constraint for sparsity ( or temporal slowness , [50] ) while forcing dispersal [43] or decorrelation [42 , 50] between the unit responses , and then learn the encoding receptive fields . More relevant for our study are those models which demand sparsity of unit responses while also generatively estimating the spectrograms from the unit activity via learned generative fields [1 , 9 , 47 , 49 , 65] . All the above sparsity and slowness models show some capacity to capture certain characteristics of STRFs . We have made explicit comparison of our model to a linear sparse model in the results ( Fig 4 ) , as it is the standard leading normative model of sensory coding , and we indicate the particular strengths of our model . The model of Carlin et al . [50] is less directly comparable to our model as it does not involve an explicit generative model . While it does in some ways better explain auditory cortical STRFs than a sparse coding model , it is clear that the MCA model captures certain aspects of the neural data that the slowness model of Carlin et al . does not address . Notably , the Carlin et al . model shows a near uniform distribution of best scales up to 2 . 5 cycles/octave , this is in contrast to our neural data ( and that of Carlin et al . ) and the MCA model where the density decays as scale increases ( Fig 4 ) . In general , masking-based non-linearities , i . e . , the dominance of one source in any time-frequency bin , is a property of acoustic data that has frequently been used for acoustic data processing ( e . g . [16 , 67] ) . In contrast , however , for the task of generatively explaining acoustic data by statistically learned structural primitives , almost all contributions in the literature rely on standard linear models . This applies for studies with functional focus ( e . g . , NMF-like [68 , 69] ) as well as for studies explaining neural response properties [1 , 9 , 49 , 65] . The main reason for this strong focus on linear models is presumably related to the challenge of scaling strongly non-linear models to the large sizes required for sensory data . While linear models , e . g . for visual data , are routinely used with hundreds of generative fields / basis functions since about two decades [27 , 70–72] , non-linear models have been trained at large scales only relatively recently [21 , 22 , 62] . Earlier non-linear models , e . g . , based on a noisy-OR non-linearity [61] or the maximum [19] , have not been sufficiently efficient for learning with large numbers of generative fields . While the approach used here does model masking , we do ( as discussed above ) not employ a statistical model that captures regularities in time . Other approaches do consider this important aspect of neural processing [66 , 73 , 74] e . g . , to model longer term amplitude modulation structure of acoustic signals [73 , 74] . Moreover , incorporating additional temporal statistical regularities is clearly important for acoustic synthesis [75] and might therefore be expected to have a strong effect on the neural representation of sound . Among the approaches using assumptions formulated in terms of a statistical model , recent work by Yildiz et al . [47] is closely related to the linear models used in our study . That study , like our approach , seeks to explain acoustic stimuli by combinations of structural primitives . The focus by Yildiz et al . is a specific neural circuit implementation for probabilistic inference and learning . The derivation of the neural circuit relies on a mean field approximation for efficient inference , an adaptive Markovian dynamics , and a divisive inhibitory interaction among neurons representing structural primitives . The interaction of these mechanisms are shown to result in a stimulus representation with the underlying goal of providing a Bayes optimal explanation using combinations of learned generative fields . While this goal is shared with our approach , the assumed linear combination of primitives is the crucial difference of Yildiz et al . 2016 to our non-linear approach , i . e . , they do not model masking . The generative data model underlying Yildiz et al . consequently more closely corresponds to the Binary Sparse Coding ( BSC ) model which we used as a control ( Eqs 2 and 4 ) . However , while Yildiz et al . infer STRFs from the circuit approximation of probabilistic inference , the results of S2 Fig of our study are based on directly inferring model STRFs from the linear BSC model itself . This makes the emergence of inhibitory subfields a direct consequence of the used generative data model , while Yildiz et al . first motivate a divisive form of inhibition to implement approximate probabilistic inference by their suggested circuit . On the other hand , both the here presented study and the study by Yildiz et al . , 2016 , provide evidence for auditory STRFs emerging from probabilistic inference and learning . Also both studies may be regarded as providing evidence for inhibitory subfields being a consequence of explaining away effects , as first hypothesizes by preliminary results obtained for our study [76] . In terms of concrete neural circuits that may realize such inference and learning , the study by Yildiz et al . 2016 goes very significantly beyond the research questions addressed here . On the other hand , in terms of showing that inhibitory subfields are a direct consequence of probabilistic inference , and in terms of using such fields to discriminate between different statistical models , our study significantly goes beyond the work by Yildiz et al . 2016 . Finally , note further technical but potentially import differences of approximate probabilistic inference applied to our and related approaches . The dominating approach for learning representations in terms of structural primitives are maximum a posteriori ( MAP ) approximations [7] , i . e . , the stimulus is represented by the latent state ( i . e . , by the neuron activities ) with the highest posterior probability ( highest p ( s →|y → , Θ ) in our case ) . MAP approximations are both scalable and relatively straight-forward to apply , which makes them being very frequently used also for statistical models of acoustic data ( e . g . , [1 , 9] ) . However , with only maintaining the most probable hidden state for inference , no rich posterior structure is represented: neither correlations , multiple-modes nor any other type of the here very important explaining away effects is captured . In contrast , for our study and for other recent approaches ( e . g . , [47] ) richer posterior representations play an important role . The observation that no previous study using MAP approximations has related inhibitory subfields of STRFs to explaining away effects , indicates that richer posterior representations seem to be required . However , while Yildiz et al . [47] as well as the BSC model used here maintain non-trivial posterior structures , the types of approximations used are different . Yildiz et al . 2016 employ a fully factored variational approximation ( i . e . , mean field ) . Such an approximation essentially assumes a posteriori independence of neural units , which has ( given a stimulus ) a direct impact on the activity dependencies among the stimulus encoding neurons . In contrast , the BSC model ( as well as the MCA model ) uses a truncated EM approximation which does not assume a posteriori independence [20] . The a posteriori independence of mean field has been criticized for introducing biases during learning [77 , 78] while approaches that use truncated EM instead have been favorably compared with mean field [34] . To summarize , we have here shown that statistical models reflecting challenging data properties such as masking-based combinations of structural primitives and non-negativity are applicable to complex sensory data such as cochleagrams . Furthermore , we have found that inhibitory subfields of estimated model STRFs can directly emerge from explaining away effects of the assumed statistical model . This observation may lead to novel tools for the investigation of assumptions underlying probabilistic inference in the auditory cortex , in other sensory areas , and beyond . | The information carried by natural sounds enters the cortex of mammals in a specific format: the cochleagram . Instead of representing the original pressure waveforms , the inner ear represents how the energy in a sound is distributed across frequency bands and how the energy distribution evolves over time . The generation of cochleagrams is highly non-linear resulting in the dominance of one sound source per time-frequency bin under natural conditions ( masking ) . Auditory cortex is believed to decompose cochleagrams into structural primitives , i . e . , reappearing regular spectro-temporal subpatterns that make up cochleagram patterns ( similar to edges in images ) . However , such a decomposition has so far only been modeled without considering masking and non-negativity . Here we apply a novel non-linear sparse coding model that can capture masking non-linearities and non-negativities . When trained on cochleagrams of natural sounds , the model gives rise to an encoding primarily based-on spectro-temporally localized components . If stimulated by a sound , the encoding units compete to explain its contents . The competition is a direct consequence of the statistical sound model , and it results in neural responses being best described by spectro-temporal receptive fields ( STRFs ) with positive and negative subfields . The emerging STRFs show a higher similarity to experimentally measured STRFs than a model without masking , which provides evidence for cortical encoding being consistent with the masking based sound statistics of cochleagrams . Furthermore , and more generally , our study suggests for the first time that negative subfields of STRFs may be direct evidence for explaining away effects resulting from performing inference in an underlying statistical model . | [
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"mec... | 2019 | STRFs in primary auditory cortex emerge from masking-based statistics of natural sounds |
Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection . They achieve this by recruiting factors for actin nucleation , the Arp2/3 complex , and polymerization regulators from the host cytoplasm . Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion , the underlying mechanism of pathogen movement is currently speculative and controversial . Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus , the smallest pathogen yet known to hijack the actin motile machinery . Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators . We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions , with an average of four filaments pushing the virus at any one time . Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo . The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization . Since Listeria monocytogenes , Shigella flexneri , and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move , we suggest they share the same principles of actin organization and mode of propulsion .
The seminal finding of Tilney and Portnoy [1] that Listeria monocytogenes exploits the actin cytoskeleton of infected cells to invade neighboring cells opened a new chapter in motile processes based on actin . Major progress in understanding how L . monocytogenes uses actin to move came from the identification of the Arp2/3 complex [2] as the downstream target promoting actin polymerization [3] and from the subsequent elucidation of the minimal protein cocktail required for propulsion in vitro [4] . Essential in the in vitro motility mix was actin , the Arp2/3 complex , ADF/cofilin , an actin capping protein and an activator of the Arp2/3 complex , ActA on L . monocytogenes , or N-WASP on plastic beads [4] , [5] . Subsequent studies have revealed a growing list of bacterial and viral pathogens that exploit the actin-based motile machinery of infected cells by mimicking or recruiting N-WASP to activate the Arp2/3 complex [6] , [7] or other actin nucleators [8] . To gain more insight into the mechanism of propulsion , several efforts have been directed at establishing the structure of the actin comet tail induced by pathogens . Electron microscopy of plastic-embedded , negatively stained L . monocytogenes comet tails , or critical point dried tails on ActA-coated beads [1] , [9]–[11] showed actin filaments more or less randomly oriented , but the high density of filaments precluded definition of their spatial organization by conventional 2D imaging . Using electron tomography we recently showed that lamellipodia networks comprise subsets of actin filaments linked by branch junctions structurally homologous to those formed from the Arp2/3 complex and actin in vitro [12] . With electron tomography practical limits are set by the thickness of the sample and L . monocytogenes are more than twice as thick as lamellipodia , making them less suitable for structural analysis . The timely finding that a baculovirus species , Autographa californica multiple nucleopolyhedrovirus , just 50 nm in diameter , moves on an actin comet tail in infected cells and bears a minor capsid protein p78/83 that directly activates the Arp2/3 complex [13]–[15] offered an ideal object for resolving comet architecture . In the present report we provide the first structure , to our knowledge , of an actin comet tail driving a pathogen and use this new information to re-evaluate alternative models ( reviewed in [16] , [17] ) of pathogen propulsion by actin .
Lepidopteran cells , the natural host of baculovirus , proved too thick for analysis by electron tomography . However , we found that baculovirus was readily taken up by thinner vertebrate cells of mouse ( B16 melanoma , NIH 3T3 fibroblasts ) , fish ( CAR fibroblasts ) , and human ( HeLa ) origin . Figure 1A and Movie S1 show a B16 melanoma cell expressing GFP-actin that was infected with baculovirus tagged with mCherry [14] . The actin comet tails formed were indistinguishable from those seen in the native host cells , moved with a velocity of up to 50 µm/min , and contained typical tail components ( Figures 1B and S1 ) [11] . From intensity measurements of tails in cells transfected with different combinations of GFP/mCherry-tagged proteins , VASP and ArpC5 co-localized with actin and capping protein β and cofilin trailed behind the actin label ( Figure 1B and Figure S1 ) . By applying cryo-electron tomography to intact cells and cytoskeletons , we previously showed that actin networks in lamellipodia are preserved using a cytoskeleton preparation procedure involving simultaneous extraction with Triton X-100 and fixation in glutaraldehyde [18] . This procedure leads to a marked improvement in filament contrast and resolution as compared to un-extracted cells . Using the same routine with baculovirus infected cells , we could show that the fluorescence intensity profile and length of actin comet tails were retained by this extraction/fixation method ( Figure S2 ) . The ultrastructural organization of baculovirus actin comet tails was resolved in cytoskeletons of infected cells using two complementary approaches involving embedment either in negative stain ( Figure 2 ) or in vitreous ice ( Figure 3 ) . A section of an electron tomogram of a negatively stained baculovirus comet tail in the cytoplasm of a fish fibroblast cytoskeleton and the model derived by filament tracking are shown in Figure 2 ( see also Movie S2 ) . As shown , the comet tail filaments form a fishbone-like array generated through the frequent formation of branch junctions ( highlighted in the insets in Figure 2A ) with an average angle of 75 . 2±8 . 0° ( s . d . , n = 652 ) . From tracking of filaments in the comet tail network , they appeared to be organized into subsets ( averaging 14 . 2±8 . 3 filaments per subset , s . d . , n = 34 ) highlighted in Figure 2B in different colors , each linked by a separate group of branch junctions ( red spots in Figure 2B , C ) . Cross-correlation analysis [19] , [20] of the negatively stained comet tail filaments with reference models of the actin helix ( Figure S3 ) showed that the fast polymerizing , plus ends were directed forwards . The plus ends of the filaments analyzed are highlighted by black spheres in Figure 2C . The filament array belonging to the actin comets was mainly separated in Z from the actin filaments of the cytoplasm ( see additionally Figures S4 and S5 ) , which were typically longer and randomly oriented . Intermingling of the two filament populations also occurred , but it was relatively straightforward to assign filaments to the comets according to additional criteria: 1 , termination within the comet core; and 2 , extension of filaments from the core towards the virus . To circumvent the partial collapse of comet tails in negatively stained preparations , we analyzed them also by cryo-electron tomography . Optimal contrast for filament tracking in actin comets was obtained by imaging them in cytoskeletons of infected cells within cytoplasmic regions that lay over holes in the supporting film ( Figure 3 and Movie S3 ) . Branch junctions with the typical angle were readily identified ( insets , Figure 3A and Movie S4 ) with the comet tail filaments oriented at an average angle of 50 . 9±23 . 7° ( s . d . , n = 485 ) to the core axis . The number of filaments transecting a core region of the comet tail corresponding to the cross-section of the virus ( highlighted in yellow in Figure 3B ) at different axial positions is shown in Figure 3C . These data indicated an average number of filaments involved in pushing of 3 . 9±1 . 4 ( s . d . , n = 252 , range 1–8 ) . Taken together , the data from three negative stain and three cryo-electron tomograms gave an average filament length of 121 . 1±99 . 9 nm ( s . d . , n = 961 ) ( cryo 107 . 8±89 . 8 nm , s . d . , n = 477 , negative stain 134 . 2±107 . 3 nm , s . d . , n = 482 ) , average branch angles of 75 . 2±8 . 0° ( s . d . , n = 652 ) ( cryo 74 . 7±8 . 7° , s . d . , n = 331 , negative stain 75 . 8±7 . 3° , s . d . , n = 321 ) , and an average interbranch spacing of 37 . 7 nm±30 . 1 nm ( s . d . , n = 290 ) ( cryo 37 . 1 nm±32 . 8 nm , s . d . , n = 154 , negative stain 38 . 4 nm±26 . 8 nm , s . d . , n = 136 ) . Figure 3D shows a projection of the 3D model of the comet tail together with ( in translucent grey ) the endogenous actin filaments of the cytoskeleton that were mainly located above and below the comet ( see also Movie S3 and Figure S6 ) . By exposing the p78/83 capsid protein of budded baculoviruses from cell supernatants using detergent ( Figure 4A , B ) we could reconstitute comet tail formation in vitro ( [4] , Figures 4 and 5 ) . In the motility cocktail ( actin , Arp2/3 complex , gelsolin , ADF/cofilin , and profilin ) filament length was influenced most strongly by variations in gelsolin concentration ( Figure 4F , G ) . Addition of VASP to the protein cocktail substantially increased the frequency of particles bearing actin comets . In all cases actin filaments were nucleated at one end of the virus particle and with appropriate concentrations of cofactors , the in vitro comet tails resembled closely those observed in vivo . Cryo-electron tomography was used to determine the three-dimensional organization of the in vitro comet tails ( Figure 5 and Movie S5 ) . As for the in vivo comet tails , we observed a fishbone-like array of filaments linked by branch junctions ( inset and red spheres , Figure 5 ) with four to five filaments in close contact with the rear of the virus ( Figure 5C ) . Cross-correlation analysis [19] of individual filaments in the cryo-tomograms ( Figure S7 ) showed that the plus ends were oriented towards the virus , consistent with the analysis of the in situ comet tails embedded in a negative stain . Using a quasi-static approach in a two-dimensional model ( see Text S1 ) we sought to mimic the intracellular movement of baculovirus and the structural features of the comet tails . We assumed several forces acting on the virus: Cytoplasmic friction , Brownian forces , and the forces exerted by the actin filaments . In the model , actin filaments are simulated as stiff , immobile rods , which can push the virus due to polymerization . We initially considered three variations of the model ( Figure 6A–C ) . In the “tethered” case ( Figure 6A ) actin filaments proximal to the virus surface are continuously tethered , both during pushing and when they lag behind . When lagging behind they exert a pulling force described by a spring connection ( Figure 6A , 2 ) . In the “tethered during branching” case ( Figure 6B ) tethering to the virus occurs only during branching events ( Figure 6B , 2 ) and is described by a spring connection to the Arp2/3 complex . In the “untethered” case ( Figure 6C ) no tethering takes place between the filament plus ends or the branch points with the virus surface . In the latter two cases , the untethered , lagging filaments ( Figures 6B , C and 7 ) may convert into pushing or capped filaments . The primary difference between the three cases is then the degree of pulling forces exerted on the virus by the filament network . In each case , Arp2/3-dependent branches ( marked in red in Figure 6A–C ) are induced by the p78/83 nucleation promoting factor at the rear of the pathogen . Filaments that move laterally off the rear of the virus continue to polymerize ( Figure 6A–C , 4 ) until they are capped ( Figure 6A–C , 5 ) , and those at the rear of the tail de-polymerize from their minus ends ( Figure 6A–C , 6 ) . To compare the electron microscope data with the simulations , we quantified ( 1 ) the angles that filaments subtended with the axis of the comet tails ( Figure 6D , E ) , ( 2 ) the length distribution of the filaments ( Figure 6K ) , and ( 3 ) the number of filaments joined together in subsets ( Figure 2B ) . By an appropriate choice of input parameters ( Table S1 ) we could mimic the measured parameters only for the “tethered” case ( Figure 6F , J , K; summarized in Tables S2 and S3 ) . In the “tethered” situation , the distribution of angles subtended to the trajectory ( 52 . 6±26 . 8° , s . d . , n = 49 , 321 ) compared closely to the value obtained from three cryo-tomograms ( 50 . 9±23 . 7° , s . d . , n = 485 ) , whereas the “tethered during branching” and “untethered” models produced tails with filaments emanating at significantly higher angles ( 64 . 8±42 . 6° , s . d . , n = 58 , 895 , and 59 . 7±41 . 1° , s . d . , n = 51 , 168 , respectively; Figure 6F , G , H , J ) . Pushing the virus along a straight path tends to be dynamically unstable due to its elongated shape [21] . To obtain in the model a persistent and relatively straight movement , an average of 3–4 filaments evenly distributed over the rear of the virus were required at any one time . Due to statistical variation , this condition was not satisfied by a single subset of filaments . Instead , regions on the virus rear , where filaments became sparse , required the engagement of new mother filaments , through de novo nucleation or recruitment from the cytoplasm that then initiated new filament subsets . In the “tethered” case , the simulations generated an average of 11 filaments per subset , comparable to the value determined from the tomograms , whereas for the “untethered” case a new mother filament was required after every 2–3 branching events , leading to an average of 3 filaments per subset . The straightness of tracks adopted by the virus in cells was compared to the simulations by measuring the angular divergence of 200 nm segments along the comet tail axis ( Figure 7 ) . Experimental data were derived from 19 comet tails imaged by conventional 2D electron microscopy ( Figure 7C , D ) . Again , the range of observed turning angles fitted best the values obtained with the “tethered” case simulations ( Figure 7F ) . With simulations run for extended periods of time ( at least 30 min of real time each ) , tracking in the “tethered” model generally mimicked the undulating course of baculovirus observed by live cell imaging and electron microscopy ( Figure 7A , F and Movies S1 and S6 ) , whereas tracks were highly tortuous in the “untethered” simulations ( Figure 7B , F and Movie S6 ) as also previously reported for simulated trajectories of L . monocytogenes [22] . Values of the angular divergence of trajectories from tails acquired by transmission electron microscopy ( 22 . 0±24 . 4° , s . d . , n = 255 angles in 19 individual tails ) and “tethered” simulations ( 17 . 4±14 . 8° , s . d . , n = 1 , 449 ) were significantly smaller than those from “untethered” simulations ( 51 . 2±38 . 0° , s . d . , n = 2 , 681 ) and those from simulations for the case of “tethered during branching” ( 44 . 4±33 . 2° , s . d . , n = 2 , 337 ) ( Figure 7F , Table S3 ) . Although the electron tomography data indicated branching only towards the virus surface , we also considered a “tethered” model in which branching could also occur away from the virus , leading to a subpopulation of filaments unable to engage in pushing . Surprisingly , simulations of this “tethered random branching” case produced relatively straight tracks ( Figure 6I ) with normal deviations ( 24 . 5±20 . 9 , s . d . , n = 1 , 430 , Figure 7F ) but failed to reproduce the typical fishbone-like array of actin filaments . This was reflected in significantly higher filament to trajectory angles as well as in a 40% decrease in the number of filaments per subset . In addition the number of productive , pushing filaments was significantly less ( 3 . 47±1 . 27 , s . d . , n = 3 , 185 , 000 compared to 4 . 10±1 . 25 , s . d . , n = 1 , 965 , 000 ) despite an increase of 20% in the number of filaments per µm tail length .
The resolution achievable by electron tomography is dependent on a number of factors , not least the thickness of the specimen . Baculovirus is at least 20-fold smaller than L . monocytogenes , with a diameter only six times the thickness of an actin filament , making it an ideal object for electron tomography . For cryo-electron tomography , we found that filament tracking and polarity analysis was only possible in energy-filtered tomograms obtained by imaging comet tails in cytoskeletons over holes in perforated films , to reduce background noise . As we show , useful complementary structural information was also provided from electron tomography of negatively stained samples due to higher image contrast and filament detail . Taking into account these different factors we have been able to provide the first 3D structure , to our knowledge , of an actin comet tail propelling a pathogen . Our demonstration of a minimal actin filament cassette for propulsion provides a new framework for analyzing and modeling the underlying mechanisms of actin force production by Arp2/3-complex-dependent actin assemblies . The actin-based propulsion of loads ( from L . monocytogenes to beads ) has already attracted the interest of several groups [16] , . In the case of the baculovirus tail with its limited number of filaments , we can exclude the squeezing mechanism proposed in the active gel model [23] . Mogilner and Oster [25] and Dickinson and Purich [26] provide useful alternative models of how actin filaments may push at the pathogen surface disregarding actin tail architecture . Alberts and Odell's [27] comprehensive in silico model simulates details of the actin filament organization but without knowledge of the actual structure . We chose a tractable modeling approach ( see Text S1 ) using a minimal number of parameters ( Table S1 ) to define the essential set of mechanisms required to simulate the observed filament organization and the motion of the virus in vivo . By comparing alternative mathematical simulations with our experimental data , we may propose key mechanistic features of comet tail propulsion . In particular , comet tail organization and the trajectories of the virus were best explained by assuming continuous attachment of actin filament to the virus surface , supporting the hypothesis that tethering , possibly by VASP ( Figure 1B ) [28] , which is present in the baculovirus tail , and pulling forces between the tail and the pathogen [17] , [29] are needed to stabilize viral movement . A critical parameter influencing the modeling outcome was Brownian motion ( see Text S1 ) . Without it , there was little difference between the simulations for tethered and un-tethered filaments . Since the virus must experience external jostling forces in the cytoplasm , we consider the model featuring Brownian motion and tethering to mimic more closely the situation in vivo . An interesting feature of the model was the prediction that nucleation of filaments on the virus is required not only to initiate movement , but also intermittently to correct sharp turns and a stochastic , regional paucity of pushing filaments . As a consequence , sequential subsets of branched filaments are created , consistent with the tomography data and reminiscent of the organization of actin filaments in lamellipodia [12] . The fishbone-like array of filaments in the comet tail could only be simulated if branching was biased toward the virus surface with the result that all new filaments are involved in pushing . The viral nucleation promoting factor , p78/83 , therefore seems to restrict branching from tethered mother filaments in a biased way , through conformational constraints , or possibly as a result of local bending of actin filaments at the filament–virus interface [30] . The delay of capping protein incorporation in the comet tail corresponding to around 1 s ( Figure 1B ) is consistent with the observed filament length distribution ranging from 10 nm to 800 nm ( Figures 2 , 3 , and 6K ) . The later incorporation of cofilin ( Figure 1B ) is likewise consistent with its preferential binding to ADP-F-actin [31] . Fishbone-like patterns of actin have been described in comet tails induced in cells by vaccinia virus [32] as well as by L . monocytogenes in in vitro motility assays [5] at low gelsolin concentrations . The principles of organization of actin filaments in the baculovirus actin comet tail shown here thus likely apply generally to comets formed on pathogens that hijack the Arp2/3 complex machinery for propulsion and invasion [6] .
The supernatant from Sf9 cells infected with either wild-type or mCherry-tagged baculovirus [14] was harvested after 3–5 d , precleared at 2 , 500 g for 5 min , filtered through a membrane with 0 . 45 µm pore size , then centrifuged at 18 , 000 g for 1 h and re-suspended in 150 µl of baculovirus buffer ( 10 mM HEPES , 0 . 5 M KCl , pH 7 . 4 ) to avoid clustering of the viruses . For in vitro assays , de-enveloped viruses were prepared by adding 2% Triton X-100 ( Fluka ) to the virus suspension and incubating the mixture on a shaker for 15 min at 25°C . The suspension was then centrifuged at 500 rpm to remove large debris and the supernatant centrifuged at 20 , 000 g for 1 h . The resulting pellet was resuspended in 50 µl baculovirus buffer supplemented with 1% BSA and used as stock in the motility assay . The in vitro assay was basically performed as described [4] . Briefly , ADF ( 3 . 7 µM ) , Profilin ( 2 . 5 µM ) , Gelsolin ( 25–200 nM ) , Arp2/3 ( 75 nM ) , G-Actin ( 7 . 6 µM ) , VASP ( 100 nM ) , and the purified , de-enveloped baculovirus were mixed in X-Buffer ( 10 mM HEPES , 100 mM KCl , 1 mM MgCl2 , 0 . 1 mM CaCl2 , pH 7 . 8 ) supplemented with 1% BSA , 2 mM ATP , 4 mM MgCl2 , and 6 . 7 mM DTT . The motility assay was incubated for different times as a drop on a grid in a wet chamber at room temperature . B16 mouse melanoma cells were cultured in Dulbecco's Modified Eagle's Medium ( DMEM ) ( Sigma-Aldrich ) with 10% fetal calf serum ( Thermo Fisher Scientific ) , 1% penicillin/streptomycin , and 1% L-glutamine . B16 cells were transfected as subconfluent monolayer cultures in 30 mm Petri dishes using 2 µg of DNA , 100 µl Optimem ( Invitrogen ) , and 6 µl Fugene ( Roche ) . Before addition to the cells overnight , the transfection mix was incubated for 20 min . The next day , the medium was changed and the transfected cells plated on coverslips coated with 25 µg/ml laminin ( Sigma ) for fluorescence microscopy . Sf9 cells were kept in Grace's insect medium with 10% fetal bovine serum , 1% penicillin/streptomycin , and 1% L-glutamine . Goldfish fin fibroblasts ( line CAR , No . CCL71 , ATCC ) were maintained in Basal Eagle's Medium ( Sigma-Aldrich ) supplemented with 1% nonessential amino acids , 2 . 5% HEPES ( Invitrogen ) , 1% penicillin/streptomycin , 1% L-glutamine , and 12% fetal bovine serum ( Thermo Fisher Scientific ) at 27°C . For transient transfection , subconfluent monolayer cultures on 30 mm Petri dishes were used . The transfection mixture was prepared as follows: 2 µg of DNA and 12 µl of Superfect lipofection agent ( Qiagen ) were mixed in 200 µl Optimem ( Invitrogen ) . After 15 min incubation at room temperature , a further 5% serum-containing medium with transfection mixture was added to the cells for 5 h . The cells were then washed and returned to normal medium . Transfected cells were replated after 24 h on 15 mm coverslips coated with human fibronectin ( Roche ) at a concentration of 50 µg/ml . For virus uptake 150 µl baculovirus stock was added to 2 ml of a subconfluent cell culture growing on coated coverslips or electron microscopy grids and incubated at 27°C or 37°C depending on the cell type , and imaging was performed after 1–3 h . Plasmids used for transfection were mCherry-actin [33] , pEGFP-actin ( Clontech ) , pEGFP-ArpC5 [34] , pEGFP-VASP [35] , pEGFP-Capping Protein β2 [36] , and mCherry-Cofilin [37] . Baculovirus particles tagged with mCherry were prepared as described [14] . Coverslips carrying baculovirus-infected cells were mounted in an open chamber on a heating platform ( Harvard Instruments ) in prewarmed medium . Intracellular virus-induced tails were usually observed after about 20 min postinfection . The samples were observed on an inverted Zeiss Observer epifluorescence microscope equipped with a Perkin Elmer UltraView spinning disc system ( ProSync2 ) . For kymograph analysis , Fiji together with a MatLab-based script were used . Statistical analysis was performed using Prism . Intensity values of tail components were normalized to tail length based on the linear relationship between tail length and speed ( Figure S1E ) . For negative staining electron microscopy , cells were grown on Formvar-coated 200 mesh hexagonal nickel grids ( Agar Scientific ) or 135 mesh NHF15-A gold Finder grids ( Maxtaform ) in standard medium and allowed to spread overnight . After spreading , the samples were washed with cytoskeleton buffer ( 10 mM MES buffer , 150 mM NaCl , 5 mM EGTA , 5 mM glucose , 5 mM MgCl2 , pH 6 . 8 ) , fixed and extracted with 0 . 5% Triton X-100 and 0 . 25% glutaraldehyde in cytoskeleton buffer for 1 min , and kept in 2% glutaraldehyde with 1 µg/ml phalloidin in cytoskeleton buffer at 4°C . For routine inspection in an 80 kV transmission electron microscope ( Morgagni , FEI ) , grids were stained with 70 µl 2% SST including 1 µg/ml phalloidin . For electron tomography , grids were stained with 70 µl 6%–8% sodium silicotungstate ( SST ) including 1 µg/ml phalloidin . For cryo-electron tomography , virus-infected cells were plated in growth medium onto Quantifoil R1/4 , R1 . 2/1 . 3 , and R2/2 perforated carbon films on 200 mesh Au grids , and allowed to spread for 6 h . The cells were fixed as for negative staining and the grids subsequently transferred to forceps in a grid-plunging device ( Leica EM GP ) , supplemented with 4 µl of medium and 10 nm BSA-saturated colloidal gold [12] . During mounting and blotting , the grid was held in a chamber with controlled humidity and temperature ( 95% and 22°C ) and was blotted automatically for 1 . 5–2 s with humidified Whatman No . 1 filter paper applied to the backside of the grid to avoid any contact with the cells . Samples were frozen by plunging into liquid ethane cooled to 80 K by liquid nitrogen . In vitro polymerized actin tails were prepared for electron microcopy as follows . For negative staining the incubation mix was fixed after different times on the 200 mesh Au grids by injecting 2% glutaraldehyde ( final concentration 0 . 5% ) for 4 min and stained with 70 µl 2% sodium silicotungstate ( SST ) . For cryo-electron microscopy , samples were incubated on Quantifoil R3 . 5/1 perforated carbon films on 200 mesh Au grids for 45 min , rinsed with 70 µl X-Buffer , followed by 70 µl X-Buffer with 1∶10 BSA-colloid gold , and without fixation or fixed as above , frozen as described above with blotting times between 1 . 2 and 1 . 6 s . Tilt series were acquired on an FEI Tecnai F30 Helium ( Polara ) microscope , operated at 300 kV , and cooled to approximately 80 K . Automated acquisition of tilt series was driven by SerialEM 3 . x . Normally , the tilt range was −60° to +60° using the Saxton tilt scheme based on 1° increments for negative stain and 2° increments for cryo-samples from 0° tilt , at a nominal defocus value of −5 µm to −7 µm for negative stain and −8 µm to −12 µm for cryo-samples . For negative stain samples , two tilt series around orthogonal axes were recorded on a Gatan UltraScan 4000 CCD camera at on-camera magnifications typically from 23 , 000× to 59 , 000× . For frozen hydrated samples , zero-loss images were recorded on a Gatan MSC 2 k camera on a GIF 2002 using a slit width of 15 eV . The total electron dose for the cryo-tilt series was less than 130 electrons per Å2 , and the primary on-screen magnification was from 20 , 000× to 29 , 000× . Re-projections from the tilt series with gold particles as fiducials for alignment and contrast transfer function corrections were performed using IMOD software [38] . A typical tomogram comprised a z-stack of 60–120 sections of 0 . 4–1 . 5 nm each . The polarity of actin filaments in tomograms of negatively stained and frozen samples was determined using a filament straightening protocol and cross-correlation analysis as described elsewhere [20] . Filaments were manually tracked using IMOD , as previously described [12] . The identity of branch junctions was based on filament tracking analysis , performed by three investigators independently . Filaments were tracked individually and branch junctions identified as blunt intersections of filament pairs subtending an acute angle in the range of 60–90° , as deduced for branch junctions in lamellipodia [12] . An example of the tracking analysis in the cryo-tomograms is shown in Movie S4 . Mathematical modeling was performed using MatLab software . Details of the model are provided in Text S1 . | Several bacteria and viruses hijack the motile machinery of cells they invade to generate networks of actin filaments ( comet tails ) to propel themselves from one cell to another . A proper understanding of the mechanism of propulsion has so far been hampered by a lack of information about the structure of the machinery . Using electron tomography we present here the three-dimensional structure of actin comet tails propelling a baculovirus , the smallest pathogen known to recruit the actin nano-machinery . We show that baculovirus is propelled by a fishbone-like array of actin filaments constructed from subsets linked by branch junctions , with an average of four filaments pushing the virus by their fast polymerizing ends at any one time . Using a stochastic mathematical model we have simulated comet tail organization as well as the tracks adopted by baculovirus inside cells . The simulations support a model of baculovirus propulsion in which the actin filaments are continuously tethered to the virus surface as they grow , branch , and push . Since larger pathogens like Listeria , Shigella , and Vaccinia virus generate comet tails exhibiting the same general morphology and components as those of baculovirus , the basic mechanism of their propulsion is likely a scaled up version of the one described here . | [
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] | 2014 | Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion |
The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O . viverrini infection . During its feeding activity within the bile duct , the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities . Here , we describe the cDNA , gene organization , phylogenetic relationships , immunolocalization , and functional characterization of the cathepsin F cysteine protease gene , here termed Ov-cf-1 , from O . viverrini . The full length mRNA of 1020 nucleotides ( nt ) encoded a 326 amino acid zymogen consisting of a predicted signal peptide ( 18 amino acids , aa ) , prosegment ( 95 aa ) , and mature protease ( 213 aa ) . BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes , including Clonorchis sinensis ( 81% ) , Paragonimus westermani ( 58% ) , Schistosoma mansoni and S . japonicum ( 52% ) , and with vertebrate cathepsin F ( 51% ) . Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host . The Ov-cf-1 gene , of ∼3 kb in length , included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length , the introns from 43 to 1 , 060 bp . The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene , although the gene structure of human cathepsin F is more complex . Unlike Ov-CF-1 , human cathepsin F zymogen includes a cystatin domain in the prosegment region . Phylogenetic analysis revealed that the fluke , human , and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases . A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris . Although the recombinant protease did not autocatalytically process and activate to a mature enzyme , trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1 , releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen . Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures , including vitelline glands , egg , and testis . Ov-CF-1 was detected in bile duct epithelial cells surrounding the flukes several weeks after infection of hamsters with O . viverrini and , in addition , had accumulated in the secondary ( small ) bile ducts where flukes cannot reach due to their large size . A cathepsin F cysteine protease of the human liver fluke O . viverrini has been characterized at the gene and protein level . Secretion of this protease may contribute to the hepatobiliary abnormalities , including cholangiocarcinogenesis , observed in individuals infected with this parasite .
Opisthorchis viverrini is an important human food-borne pathogen endemic in mainland Southeast Asia , predominantly Northeast Thailand [1] , [2] . Infection with this liver fluke parasite causes opisthorchiasis , which is associated with a number of hepatobiliary abnormalities , including cholangitis , obstructive jaundice , hepatomegaly , cholecystitis , cholelithiasis and cholangiocarcinoma . O . viverrini infection induces pathological changes including epithelial desquamation , epithelial and adenomatous hyperplasia , goblet cell metaplasia , inflammation , periductal fibrosis and granuloma formation [3] . Experimental and epidemiological findings implicate O . viverrini infection in the etiology of cholangiocarcinoma ( CCA ) , cancer of the bile ducts ( reviewed in [1] ) . O . viverrini is one of only two metazoan pathogens of humans that is considered a Group 1 carcinogen [4] , [5] . A number of studies suggest that inflammation of the bile ducts caused by O . viverrini infection and induction of endogenous nitric oxide are important factors for cholangiocarcinogenesis [6] , [7] . Other studies have related cell proliferation induced by O . viverrini , its antigens and metabolites , and exposure to exogenous carcinogens such as nitrates , nitrites and even N-nitroso compounds found in fermented or preserved foods such as pla-ra ( traditional Thai fermented fish ) , as factors involved with parasite-associated cholangiocarcinogenesis [8] . The pathogenesis of O . viverrini-mediated hepatobiliary changes , which in turn can lead to CCA , can be attributed to greater or lesser degree to mechanical irritation caused by the liver fluke suckers and to the action of molecules excreted or secreted by the parasite [1] . There may be informative analogies to be drawn between O . viverrini-induced CCA and the development of stomach cancer caused by infection with CagA-positive strains of Helicobacter pylori . In the latter situation , the CagA antigen is delivered by the H . pylori bacterium into gastric epithelial cells , where it undergoes tyrosine phosphorylation . Phosphorylated CagA activates SHP-2 tyrosine phosphatase , causing morphological transformation of the infected cell to the hummingbird phenotype . CagA also destabilizes the E-cadherin/beta-catenin complex to elicit aberrant activation of the beta-catenin signal . These events in signal dysregulation underlie stomach cell metaplasia [9] , [10] . Whereas our understanding of cholangiocarcinogenesis is less advanced than with H . pylori-associated gastric adenocarcinoma , CCA—in like fashion to gastric adenocarcinoma—is an epithelial cell adenocarcinoma associated with a gastrointestinal tract pathogen . In like fashion to CagA-associated stomach cancer , it is conceivable that products secreted or released by O . viverrini into the neighboring bile duct epithelia might promote cholangiocarcinogenesis . The parasite-released mediators might down-regulate apoptosis and/or they may stimulate epithelial cell growth . Accordingly , we have begun to examine the secretome of O . viverrini proteome with a particular interest in proteolytic enzymes since they are prominent components of ES of helminth parasites at large [11]–[13] . Recently we reported the biochemical characterization of cysteine protease activities in extracts of several developmental stages of O . viverrini [14] . We demonstrated that O . viverrini expresses clan CA-like cysteine protease activity with elevated expression in the metacercariae , suggesting that this enzyme activity might participate in larval excystation during mammalian infection . The proteolytic activity was also detected in excretory/secretory ( ES ) products of sexually mature parasites [14] . In the present report , we have identified a transcript and its genome locus encoding a cathepsin F-like cysteine protease from O . viverrini , which we termed Ov-CF-1 . Analysis of the genomic structure of the Ov-cf-1 gene revealed conserved exon/intron boundaries with the gene encoding human cathepsin F , although the human gene exhibits a more complex structure including a zymogen with a cystatin domain found within the pro-segment that is absent from the O . viverrini gene . Phylogenetic analysis revealed that the deduced Ov-CF-1 protease was an orthologue of cathepsin F cysteine proteases , a clade distinct from the cathepsin L proteases described in several related liver and blood flukes . Nevertheless , characterization of a functionally active recombinant form of Ov-CF-1 shows that this enzyme cleaves diagnostic peptides ( Z-Leu-Arg-NHMec , Z-Phe-Arg-NMHec ) that are also cleaved by cathepsin L . Ov-CF-1 is secreted by adult O . viverrini and immunocytochemical studies localized the protease in the cecum of the adult stage of the parasite . Liberation of Ov-CF-1 protease by adult parasites residing in the bile duct may contribute to the pathologies associated with O . viverrini-induced hepatobiliary abnormalities .
Metacercariae of O . viverrini were obtained by digestion with pepsin of flesh of naturally infected cyprinoid fish collected from an endemic area of Khon Kaen province , Thailand . About 100 metacercariae of O . viverrini were used to infect hamsters , Mesocricetus auratus , by stomach intubation , as described [15] . Infection of hamsters with liver flukes and maintenance of hamsters was carried out at the animal facility of the Faculty of Medicine , Khon Kaen University , using procedures approved by the Animal Ethics Committee of Khon Kaen University . The hamsters were euthanized six weeks after infection , after which the adult O . viverrini flukes were perfused from the bile ducts with phosphate-buffered saline ( PBS ) , pH 7 . 2 . The worms were washed with sterile saline , after which genomic DNA ( gDNA ) was extracted using kits from Gentra Systems ( Minneapolis , MN ) for long range PCR or Bio-Rad ( Hercules , CA ) for inverted PCR templates . Eggs of O . viverrini were recovered from tissue culture medium where they had been discharged from adult worms [16] . Total RNA was isolated from adults , eggs and metacercariae of O . viverrini using Trizol [16] , [17] . Contaminating gDNA was removed by treatment of RNA with DNase I ( Promega , Madison , WI ) . For reverse transcription-PCR ( RT-PCR ) , first-strand cDNA was produced with an oligo ( dT ) primer from 1 . 0 µg of total RNA using SUPERSCRIPT III reverse transcriptase ( Invitrogen , Carlsbad , CA ) at 42°C for 60 min . One µl of the resultant cDNA was employed as template in the presence of primers specific for Ov-cf-1 ( 5′-TCGGACCAGTATTGGACCAAG -3′ and 5′-TACGCTGGAAAGCACACAACG ) , with thermal cycling conditions of 30 sec denaturation at 94°C , 30 sec annealing at 55°C and 30 sec extension at 72°C for 30 cycles . Control RT-PCR reactions were performed without reverse transcriptase to ensure that amplified products were derived from cDNA and not contaminating genomic DNA . PCR products were subjected to electrophoresis through 1% agarose and visualized under UV light after staining with ethidium bromide . To search for transcripts encoding Clan CA , Family C1 cysteine proteases of O . viverrini , degenerate primers were designed to target conserved domains of papain-like cysteine proteases . The degenerate forward primer , 5′-TGYGGNTCNTGYTGGGCDTTYTCN targeted the conserved CGSCWAFV residues and the reverse primer , 5′-CCARCTRTTYTTBACDATCCARTA targeted the conserved YWIVKNSW residues ( Figure 1 ) , where B = C/G/T; D = A/G/T; N = A/C/G/T; R = A/G; Y = C/T . These primers were employed to amplify target sequences from cDNA of adult O . viverrini . In addition , specific primers Ov-cf-spf1 ( 5′-TGTGGTTCGTGTTGGGCATTTTCT ) and Ov-cf-spr1 ( 5′-CCAACTGTTTTTGACCGTCCAGTA ) targeting the same regions were designed based on the nucleotide sequence of Clonorchis sinensis cathepsin F ( DQ909018 ) . PCR was performed as follows; 95°C for 5 min followed by 35 cycles of 94°C for 30 sec , 55°C for 30 sec , 72°C for 30 sec and finally , 72°C for 7 min . Fragments of cDNA sequence encoding an O . viverrini cysteine protease were obtained by using rapid PCR amplification of cDNA ends ( RACE ) ; 5′- and 3′-RACE were performed using specific primers Ov-cf-spf1 and Ov-cf-spr1 ( above ) paired with linker specific primers ( Invitrogen ) . Subsequently , a full length transcript termed Ov-cf-1 was obtained by PCR using primers designed from nucleotide sequences obtained from 5′- and 3′-RACE products . Flanking and 5′-UTR sequences upstream of the Ov-cf-1 gene were amplified using inverse PCR ( iPCR ) [18] . O . viverrini genomic DNA ( gDNA ) was digested with Hind III , after which the fragments ( 50 ng/µl ) were circularized using T4 DNA ligase ( New England Biolabs , Ipswich , MA ) . One hundred ng of circularized gDNA fragments was employed as template for amplification of the putative 5′-UTR of the gene . Primers IPCRCPFw ( 5′-CAGGCTCGAATGGGGTAGTTCTGGC ) and IPCRCPRe ( 5′-GCCCGGGCACTATACGAGGAGTTCA ) were derived orientated in reverse direction to the mRNA of Ov-cf-1 ( AY821800 ) . iPCR reactions were carried out in 100 µl in 50 mM KCl , 10 mM Tris-HCl ( pH 9 . 0 at 25° C ) , 0 . 1% Triton X-100 , 1 . 5 mM MgCl2 , 0 . 5 mM dNTP , 0 . 5 U PfuTurboCx Hotstart DNA polymerase ( Promega ) , with thermal cycling conditions of 94° C for 2 min , followed by 30 cycles of 94° C for 60 sec , 58° C for 60 sec and 72°C for 2 min , and a final extension at 72°C for 10 min . Products were cloned into plasmid TOPO XL PCR ( Invitrogen ) and nucleotide sequence of inserts determined . In addition , toobtain a genomic fragment spanning the entire gene locus , the Ov-cf-1 gene locus was amplified from gDNA of O . viverrini with the primers specific for the 5′- ( CPFw , 5′-GACCTTTCGTGTGTTGCGTGTT ) and 3′-termini ( CPRe , 5′-GGGCAAAATATCAACATGGAG ) of the transcript ( GenBank AY821800 ) using LongRange PCR DNA polymerase ( Qiagen , Tubingen , Germany ) . These amplifications were performed in 50 µl reaction volumes using 100 ng gDNA , 1× LongRange PCR buffer , 1× Q-solution , 0 . 4 µM of each primer , 2 . 5 mM MgCl2 , 500 µM of each dNTP , 2 units of LongRange PCR DNA polymerase . After an initial denaturation step at 93°C for 3 min , 40 thermal cycles of 93°C for 15 sec , 50°C for 30 sec , 68°C for 15 min were carried out , followed b a final step of 68°C for 7 min . PCR products were purified ( Gel DNA extraction kit , Real Genomics , Real Biotech Corporation , Taipei , Taiwan ) and ligated to the vector pCR4-TOPO ( Invitrogen ) . The ligation products were used to transform E . coli competent cells ( One shot Mach1-T1; Invitrogen ) which were cultured in the presence of ampicillin . Plasmid DNA was isolated from clones , and the sequence of long range PCR products determined by gene walking with gene specific primers . Bioinformatics analyses to predict potential open reading frames ( ORFs ) , signal peptides , and/or transmembrane domains , conserved domains , and molecular mass were undertaken using ExPASy ( http://expasy . org ) , including the Compute pI/MW tool , Signal P ( www . cbs . dtu . dk/services/SignalP ) , and TMPred 3 . 0 ( www . ch . embrut . org/software/TMPRED_form . html ) . Assembly of contiguous sequences and multiple alignments were performed with BioEdit software version 7 . 0 . 0 , http://www . mbio . ncsu . edu/BioEdit/bioedit . html [19] . Analysis of gene loci using BLAST searches at NCBI was undertaken to determine exon and intron structures and boundaries of the O . viverrini cysteine protease gene , upstream regulatory sequences , and identities of unrelated sequences populating the introns . Positions of splice sites were predicted using NetGene2 program , http://www . cbs . dtu . dk/services/NetGene2/ [20] . For phylogenetic analysis , the amino acid sequence of Ov-cf-1 ( GenBank AY821800 ) was aligned with cathepsin F-like and cathepsin L-like , and related cysteine proteases of a variety of eukaryotes using ClustalW algorithms in the BioEdit software suite [21] . Species names and accession numbers of contributing sequences are provided in the phylogram ( below ) . Cathepsin B from Schistosoma japonicum ( AY222871 ) was used to root the tree . Multiple sequence alignments were calculated with the distance matrix method using Prodist and Neighbor in Phylip software packages to construct neighbor joining trees . Trees were analyzed under 1 , 000 bootstrap resampling values , and drawn with TreeView [22] . The sequence encoding the proform of Ov-CF-1 was amplified from cDNA of adult O . viverrini using the primers ( Ov-cp-f1 ) 5′-CGCGCGGAATTCAGAACTACCCATTCGAG and the ( Ov-cp-r1 ) 5′-CGCGCGTCTAGACGTTTGACAAGGCTGTAGT , which include restriction sites for EcoR I and Xba I ( underlined ) . PCR products were cloned into the yeast expression vector pPICZα ( Invitrogen ) using EcoR I and Xba I restriction sites , and the reading frame confirmed by sequencing . The recombinant plasmid was linearized with Pme I and employed to transform the ×33 strain of Pichia pastoris ( Invitrogen ) by electroporation . Transformed yeasts were selected on 1 mg/ml zeocin ( Invitrogen ) Yeast Peptone Dextrose ( YPD ) plates , screened by PCR using Ov-cf-1 gene specific primers , and positive colonies investigated for protein expression by immunoblotting using a monoclonal antibody specific for the hexa-His tag ( Invitrogen ) . A 1 . 5 litre P . pastoris culture was fermented using a clone that exhibited strong expression . The culture supernatant at 96 hours after induction was concentrated to 200 ml by ultrafiltration through a 10 kDa cut off membrane ( Pall Scientific ) , and buffer exchanged into 50 mM NaH2PO4 , 300 mM NaCl , 10 mM imidazole ( binding buffer ) . Recombinant enzyme was affinity purified on Ni-NTA resin ( Qiagen ) , buffer exchanged into PBS , and protein concentration determined using the bicinchoninic acid assay ( Pierce , Rockford , IL ) . Antiserum against affinity purified Ov-CF-1 was produced by immunization of a male New Zealand White rabbit . Pre-immune blood was collected from the marginal ear vein two days prior to the first injection . For the first immunization , the rabbit was injected subcutaneously with 1 . 0 mg of recombinant Ov-CF-1 emulsified with an equal volume of Freund's complete adjuvant . The second and third immunizations were carried out with 1 . 0 mg recombinant protein formulated with Freund's incomplete adjuvant . Immunizations were conducted on days 1 , 15 and 29 , blood was collected two weeks after the third immunization , and its serum was separated . The specific anti-Ov-CF-1 IgG titer of the rabbit serum was determined by enzyme linked immunosorbent assay , and antiserum was stored at −20°C until needed . Cysteine protease activities were enriched using thiol-sepharose chromatography from ES products and preparations of soluble adult O . viverrini worms ( somatic preparation ) , as detailed [14] . ES and somatic fractions eluted from the thiol-sepharose were sized by SDS-PAGE , electro-transferred to nitrocellulose , and probed with the rabbit antiserum in order to determine the presence of the Ov-CF-1 . O . viverrini adult worms or liver tissue from hamsters infected with O . viverrini ( weeks 1–24 ) were fixed and cut with a microtome into sections of 4 µm [23] . The sections were deparaffinized in xylene , hydrated in a series of ethanol and water , respectively . Endogenous peroxidase was eliminated by incubation of the sectioned tissues in 5% H2O2 in methanol for 30 min . Subsequently , sections were washed in water and PBS , and non-specific staining was blocked by incubation in 5% pre-immunization rabbit serum in PBS for 30 min . Sections were probed with rabbit anti-Ov-CF-1 antiserum or pre-immunization serum from the same rabbit diluted at 1∶100 ( v/v ) in PBS at 4°C overnight . After rinsing 3×5 min with PBS , sections were incubated with HRP-conjugated goat anti-rabbit IgG for 1 h . Sections were rinsed with PBS , 2×10 min , after which bound antibody was detected using the substrate diaminobenzidine ( DAB ) . Sections were counterstained with Mayer's hematoxylin , dehydrated , cleared in xylene and mounted in Permount . Images of sections were recorded using a digital camera ( Nikon DXA 1200 C ) fitted to an Olympus model BX40 compound microscope . To determine whether the recombinant Ov-CF-1 was capable of auto-catalytic activation , 20 µl enzyme ( 100 µg ) was added to 100 µl activation buffer ( 0 . 1 M sodium acetate , pH 4 . 5 , 1 mM DTT , 1 . 25 mM EDTA ) and incubated at 37°C . Aliquots of 10 µl were removed at various time points , transferred into tubes containing 1 µl of 1 mM E-64 to halt the enzymatic reaction . The reaction products were analyzed by 4–12% Bis-Tris NuPage gel ( Invitrogen ) electrophoresis . Trans-processing of recombinant Ov-CF-1 was undertaken by mixing 50 µg purified recombinant enzyme with 5 . 0 µg of recombinant O . viverrini asparaginyl endopeptidase [17] or an activated recombinant Fasciola hepatica cathepsin L ( FhCL1 ) [24] in 0 . 1 M sodium acetate , pH 4 . 5 , 1 mM DTT , 1 . 25 mM EDTA in a total reaction volume of 150 µl . The mixture was incubated for 180 min at 37°C , and samples ( 10 µl ) were removed at intervals for analysis ( as above ) . N-terminal sequencing was performed on recombinant Ov-CF-1 and samples taken at time 180 min following trans-processing with FhCL1 . Following 4–12% Bis-Tris NuPage , proteins were transferred to a polyvinylidene fluoride immobilon-P membrane ( Millipore ) at 120 mA for 45 min . The membrane was washed with distilled water and stained with 0 . 025% Coomassie Brilliant Blue R-250 in 40% methanol , 10% acetic acid . Selected protein bands were subjected to 5 cycles of N-terminal ( Edman ) sequencing at the Biomolecular Research Facility ( BRF ) at the University of Newcastle ( NSW , Australia ) . To analyze the enzyme specificity of Ov-CF-1 and to monitor the course of activation during the trans-processing experiments , we employed three fluorogenic peptide substrates with different residues at the P2 position , Z-Phe-Arg-NHMec , Z-Leu-Arg-NHMec and Z-Pro-Arg-NHMec ( Bachem ) . Assays were performed in 0 . 1 M sodium acetate , pH 4 . 5 , 1 mM DTT , 1 . 25 mM EDTA ( 200 µl ) in 96-well plates . Reaction kinetics were monitored over 150 min at 37°C by measuring the release of the fluorogenic leaving group ( NHMec ) at 370 nm excitation , 460 nm emission wavelength , using a Bio-Tek KC4 microfluorometer . Reactions with each fluorogenic substrate included ( a ) no enzyme control , ( b ) recombinant Ov-CF-1 alone , ( c ) recombinant FhCL1 alone , and ( d ) Ov-CF-1 +FhCL1 .
We undertook PCR investigations targeting conserved active site encoding regions of clan CA peptidases employing ( a ) degenerate primers or ( b ) primers specific for the C . sinensis cathepsin F gene ( DQ909018 ) . The former approach did not amplify products of the expected size whereas DQ909018-specific primers amplified products of ∼500 nt in length . Cloning and sequencing the latter products yielded a single sequence ( not shown ) ; using this information for 5′- and 3′-RACE approaches , we cloned and identified a coding sequence ( CDS ) of 981 bp ( AY821800 ) . Sequence analysis showed identity to cysteine proteases of other trematodes , including C . sinensis ( 85% ) and Paragonimus westermani ( 55% ) ( Figure 1 ) . The ORF encoded a zymogen with a deduced size of 326 amino acid residues ( molecular mass 37 , 325 Daltons , theoretical pI , 5 . 45 ) including a predicted signal peptide of 18 residues in length and a prosegment of 95 residues that encoded a cathepsin F protease . We termed the putative enzyme encoded by this ORF , Ov-CF-1 , and the new gene Ov-cf-1 . These dimensions were similar to the predicted size of the recently reported cathepsin F , CsCF-6 , of the closely related fluke , C . sinensis [13] . The ERFNAQ motif characteristic of cathepsins F and W ( similar to the ERFNIN motif of cathepsins L , S and O , etc . ) was conserved in the prosegment of Ov-CF-1 ( Figure 1 ) . The catalytic triad residues were present as Cys26 , His160 and Asn180 , numbering from the predicted amino-terminal methionine , Met1 , residue of the mature enzyme . Other motifs including six conserved Cys residues likely involved in disulfide bridges were present . The molecular mass of the predicted mature enzyme of 213 amino acids was 23 , 811 . 92 Daltons , with a theoretical pI of 6 . 42 . The mature enzyme included a single , predicted glycosylation site , NGS , at residue 109 . BlastP analysis undertaken with the 213 residues of the mature Ov-CF-1 protease as the query matched to peptidase family C1A , superfamily C1 . Best matches were cathepsin Fs from C . sinensis and Paragonimus . The next best match was to cathepsin F of zebrafish , Danio rerio , NP_001071036 . A TBLASTN search at using the 95 amino acid residues of the prosegment yielded strong matches to the prosegment of cathepsin F-like enzymes from C . sinensis AF093243 [13] and to Paragonimus westermani , DQ016551 [25] and also to schistosome and mammalian cathepsin Fs . ( The transcriptome of O . viverrini indicates the presence of other family C1 cysteine proteases including cathepsins B and L [26] . ) A contiguous sequence of 7 , 368 bp composed of 5′-UTR and the genomic sequence of O . viverrini cysteine protease was assembled from iPCR and long range PCR fragments ( GenBank FJ346536 ) ( Figure S1 ) . The contig extended 4 , 211 bp upstream of the predicted translation start site ( ATG ) of the gene . The entire genomic sequence of O . viverrini cysteine protease characterized from start codon referred to mRNA sequence to the end was 3 , 157 bp . By comparing the genomic and cDNA sequences of Ov-cf-1 and by prediction of the splice sites , a gene structure of seven exons interrupted by six introns was determined for the genomic organization of the Ov-cf-1 gene ( Figure 2A ) . The sizes of exons 1 to 7 were >69 , 69 , 117 , 240 , 267 , 145 and 113 bp , respectively ( Table S1 and Figure 2A–2C ) . The length of introns 1 to 6 were 132 , 43 , 46 , 192 , 1 , 060 and 664 bp , respectively . The structure of cathepsin F-like genes from several species , cathepsin F from S . mansoni , human cathepsin F , and cathepsin L2 from S . mansoni , was compared with that of the Ov-cf-1 gene ( Figure 2B ) since these three other genes were likely to be informative in terms of determination of orthology . Ov-cf-1 has 7 exons while S . mansoni CF1 and human cathepsin F have nine and 13 exons , respectively . As illustrated in Figure 2C , the six exon boundaries are conserved exactly or closely when compared with the exon/intron boundaries in human cathepsin F . Human cathepsin F is substantially longer than O . viverrini cathepsin F , 457 aa compared with 326 aa . The catalytic triad Cys , His , Asn residues were located in exons 4 , 6 and 6 , respectively; in comparison , the catalytic triad C , Cys , His , Asn residues of human cathepsin F are located on exons 6 , 11 and 12 . Exons 1 , 2 and 3 of Ov-cf-1 appear to be orthologous to exons 3 , 4 and 5 of human cathepsin F . Further downstream , the human gene is interrupted by more introns than the O . viverrini gene yet the orthology remains apparent . Thus , exons 6–7 , 8–10 , 11–12 , and 13 of human cathepsin F likely are the orthologues of exons 4 , 5 and 6 , respectively , of Ov-cf-1 . As mentioned , the catalytic Cys , His and Asn residues are encoded by exons 7 , 11 and 12 in the human cathepsin F gene , and exons 4 , 6 and 6 in the Opisthorchis gene , further supporting the orthologous nature of the two genes . The splice donor and acceptor sites of all six introns in the Ov-cf-1 gene confirmed to the established consensus GT at the 5′-end of the intron and AG at the 3′ end [27] ( Table S2 ) . Blastn and tblastx searches were undertaken using the ∼4 , 200 bp upstream of the start codon , ATG . Several matches were obtained , specifically for query residues 1–250 which gave tblastx matches to a genomic sequence of the nurse shark Ginglymostoma cirratum ( AC165195 ) ( tblastx score , 106; E value , 4e-21 ) and the reverse transcriptase of a Penelope-like retrotransposon from Schistosoma mansoni ( BK000685 ) ( score , 100; E value , 2e-19 ) ( Table S1 ) . Similarity searches using tblastx of the intron sequences of ov-cf-1 revealed several matches , including a match of intron five to the gene encoding PHGPx of Clonorchis sinensis and a match for intron six to a trinucleotide repeat containing 6a gene of Mus musculus . Phylogenetic analysis using >75 related sequences ( primarily cathepsin F and cathepsin L like enzymes ) confirmed the relationship of Ov-CF-1 with other cathepsin F proteases ( Figure 3 ) . Ov-CF-1 clustered with more than ten C . sinensis cysteine protease mRNA sequences . It also clustered with five or more cathepsin F-like sequences from the lung fluke , P . westermani , although the Paragonimus orthologues branched separately from the Opisthorchis and Clonorchis cathepsins F . Further , it clustered with the cathepsins F of the schistosomes S . japonicum and S . mansoni , although the schistosome orthologues branched separately from both the Opisthorchis/Clonorchis and the Paragonimus cathepsins F . Together the branches that included these fluke cathepsins F branched separately from mammalian cathepsins F , including human cathepsin F . The branch that included the mammalian cathepsins F clustered with another branch of cathepsin F-like enzymes from a diverse group of eukaryotes including potato , mosquito , C . elegans and trypanosomes . An unusual cathepsin F-like enzyme from the nematode Brugia malayi ( AAT07059 ) [28] formed a basal branch of the cathepsin F-like sequences . Cathepsin L like sequences from Fasciola species , schistosomes , tapeworms , Haemonchus contortus , Aedes aegypti , human , and other species formed a completely distinct clade to the cathepsins F . The entire tree was rooted with the cathepsin B of S . japonicum . RT-PCR revealed that the Ov-cf-1 mRNA was transcribed in eggs , metacercariae , immature and sexually mature forms of O . viverrini , including one- , two- and three-week-old juveniles and adult worms ( Figure 4 ) . Anti-Ov-CF-1 serum was employed to investigate the presence of the cathepsin F in ES products and the organ- and tissue-specific expression of the cysteine protease in sexually mature forms of the O . viverrini liver fluke . Immunoblot analysis of thiol-sepharose enriched cysteine protease activities indicated that Ov-CF-1 was secreted or excreted by adult O . viverrini ( Figure S2 ) . A reactive band at ∼30 kDa present in fractions of both ES and somatic fluke extracts . In addition , there were minor bands of reactivity at ∼37 kDa and 23 kDa in the somatic antigen preparation , and 23 kDa in the ES . Given that there is an N-linked glycosylation site in the mature enzyme , the immunoblot findings indicate that the mature enzyme is glycosylated and migrated at ∼30 kDa . We interpret the additionally bands as the unprocessed zymogen ( ∼37 kDa ) and a non-glycosylated form of the mature enzyme ( ∼23 kDa ) . Together , the immunoblot findings indicated that the antiserum specifically recognized Ov-CF1 . Immunohistochemical localization of Ov-CF-1 revealed strong expression in the gut , vitellaria , egg and testis ( Figure 5B–5D ) . By contrast , control sections of flukes probed with pre-immunization serum showed no or little reactivity ( Figure 5A ) . The intense immunolocalization to the luminal margin of the gut ( Figure 5C ) indicated strongly that the cathepsin F participates in proteolysis of ingested host tissues . In addition , sections through hamster bile ducts containing adult O . viverrini flukes confirmed the localization of Ov-CF-1 in organs and tissues of the fluke , including the gut and , notably , revealed the presence of Ov-CF-1 in epithelial cells lining the infected bile duct ( Figure 6D ) . Ov-CF-1 also had accumulated in secondary bile ducts too small in internal diameter to include an adult fluke ( Figure 6B ) and in Kupffer cells and mononuclear cells lining sinuses of the liver ( Figure 6C ) . Control sections from the same infected hamster probed with pre-immunization serum showed no or little reactivity ( Figure 6A ) . The recombinant Ov-CF-1 purified from yeast culture medium resolved as two major protein bands migrating at 41 kDa and 47 kDa on 4–12% Bis-Tris NuPage gels ( Figure 7A ) . Given the theoretical molecular mass of the recombinant Ov-CF-1 zymogen ( including the c-myc and His6 tags ) of ∼37 . 5 kDa , and given that both the 41 kDa and 47 kDa peptides share identical N-termini ( EFRTT; Figure 7C ) , it is likely that the yeast-expressed enzyme exhibits differential addition of N-linked glycans . This is consistent with the smearing observed around both protein bands ( Ov-CF-1 contains a single predicted N-linked glycosylation site , NGS , at residue 109; Figure 1 ) . The yeast-expressed Ov-CF-1 zymogen displayed modest activity against the protein substrates hemoglobin and gelatin at acidic pH ( 4 . 5–6 . 0 ) ( not shown ) . Assays undertaken with the fluorogenic substrate Z-Phe-Arg-NHMec revealed that the enzyme was active over a broad pH range , 4 . 5–8 . 0 , with optimal activity at pH 5 . 5 ( not shown ) . However , the apparent activity of the zymogen was very low , which is not surprising since the gel analysis did not reveal evidence of a fully processed and activated enzyme ( Figure 7A ) . Both cathepsin L and B cysteine proteases of trematodes such as Schistosoma mansoni and Fasciola hepatica have been shown to auto-catalytically remove the prosegment at low pH to release the fully active mature enzyme ( see [29] ) . In order to investigate whether Ov-CF-1 was likewise capable of auto-catalytic processing , recombinant Ov-CF-1 was incubated for 180 min at pH 4 . 5 , after which the reaction products were analyzed in Coomassie blue-stained gels . Evidence of auto-catalysis of the zymogen was not apparent ( Figure 7A , upper panel ) . When similar reactions were performed in the presence of fluorogenic peptide substrates , no increase in enzyme activity of Ov-CF-1 was detected ( see below , also Figure 8A ) . These data suggested that the recombinant Ov-CF-1 did not auto-catalytically process and activate . Subsequently , we investigated whether Ov-CF-1 could be trans-processed and activated by a functionally active O . viverrini asparaginyl endopeptidase [17] or a fully-activated recombinant cathepsin L cysteine protease from F . hepatica ( termed FhCL1 ) . Ov-CF-1 was incubated with asparaginyl endopeptidase or FhCL1 at a ratio of 10∶1 , at pH 4 . 5 , 37° C , and the reaction products monitored over 180 min by SDS-PAGE . No trans-processing was obvious with the asparaginyl endopeptidase ( not shown ) ; however , in the presence of FhCL1 , the 41 kDa and 47 kDa species of Ov-CF-1 were clipped to progressively faster-migrating bands ( Figure 7A , lower panel ) . By 180 min , a prominent band had accumulated at 30 kDa , together with a minor band of 43 kDa and a small peptide migrating at 5 kDa ( Figure 7B ) . N-terminal sequencing confirmed that the 30 kDa band represented a fully mature enzyme generated by cleavage at Val-Thr ↓ Met ( Figure 7C and 7D ) . Peptide sequencing established that the 43 kDa protein was produced by removal of just three residues ( EFR ) from the N-terminus of the 47 kDa Ov-CF-1 zymogen ( Figure 7C and 7D ) . N-terminal sequencing of the 5 kDa band was inconclusive , but this peptide likely represented a remnant product of the liberated prosegment of Ov-CF-1 ( Figure 7B ) . Figure 8A presents the trans-processing of Ov-CF-1 by FhCL1 in real-time . In these studies , the Ov-CF-1 zymogen displayed very low activity ( 5–10 relative fluorescent units against both peptide substrates Z-Leu-Arg-NHMec and Z-Phe-Arg-NHMec ) . This activity did not significantly increase when the zymogen was incubated alone for 150 min at pH 4 . 5 . However , following addition of FhCL1 to Ov-CF-1 the activity of the cathepsin F increased over the course of the experiment ( Figure 8 ) , demonstrating that exogenous trans-processing of the Ov-CF-1 prosegment generated a highly active , mature enzyme . By subtracting the activity of the FhCL1 when assayed alone against the substrates from that observed for the mixture of Ov-CF-1 and FhCL1 , we estimated that mature Ov-CF-1 was >50 times more active ( after 150 min ) than its zymogen . From the exogenous activation in the presence of three fluorogenic peptides , Z-Leu-Arg-NHMec , Z-Phe-Arg-NHMec and Z-Pro-Arg-NHMec , we obtained information on the substrate specificity of Ov-CF-1 . Ov-CF-1 had a similar specificity for residues in the P2 substrate as FhCL1; both enzymes accommodated the hydrophobic amino acids Phe and Leu but were incapable of cleaving peptides with the bulky residue Pro in the P2 position ( Figure 8A and 8B ) . For FhCL1 , this is consistent with the findings of Stack and co-workers [30] . Initial rates of hydrolysis of the substrates during the linear phase ( 0–30 min ) revealed an affinity by Ov-CF-1 for the substrates in the order Z-Leu-Arg-NHMec >Z-Phe-Arg-NHMec ( Figure 8B ) .
Cysteine proteases have been characterized in numerous infectious pathogens ( e . g . , [31]–[33] ) . In helminth parasites , their functions include excystation , tissue invasion , catabolism of host proteins for nutrition , and immunoevasion [34] , [35] . Given the importance of O . viverrini in the etiology of CCA [1] , [2] , [4]–[6] , there is impetus to characterize parasite antigens , including proteases , involved in the host-parasite relationship and pathophysiology of this food-borne fluke . Previously , we have determined the presence of abundant cysteine protease activity using the diagnostic peptide Z-Phe-Arg-NHMec in the ES products of adult O . viverrini and in other developmental stages [14] and shown that the transcriptome encodes numerous proteases [26] . More specifically , these findings demonstrated that O . viverrini expresses clan CA peptidases and suggested that abundant cysteine protease activity was present in metacercariae where it might be involved in cyst excystation during mammalian infection [14] . Cysteine proteases have been characterized in numerous infectious pathogens ( e . g . , [31]–[33] ) . In helminth parasites , their functions include excystation , tissue invasion , catabolism of host proteins for nutrition , and immunoevasion [34] , [35] . Given the importance of O . viverrini in the etiology of CCA [1] , [2] , [4]–[6] , there is impetus to characterize parasite antigens , including proteases , involved in the host-parasite relationship and pathophysiology of this food-borne fluke . Previously , we have determined the presence of abundant cysteine protease activity using the diagnostic peptide Z-Phe-Arg-NHMec in the ES products of adult O . viverrini and in other developmental stages [14] and shown that the transcriptome encodes numerous proteases [26] . More specifically , these findings demonstrated that O . viverrini expresses clan CA peptidases and suggested that abundant cysteine protease activity was present in metacercariae where it might be involved in cyst excystation during mammalian infection [14] . Here , we report that O . viverrini expresses a cathepsin F cysteine protease throughout its development and that the enzyme is released from the adult parasites which reside in the bile ducts . The cathepsin F may be responsible for the catalytic activity that we described previously and for which an EST was detected [14] , [26] . Earlier studies have shown that both cathepsin L and cathepsin F cysteine proteases are abundant in several flukes , and that these two classes of proteases form distinct but closely related phylogenetic clades [36] , [37] . Phylogenetic studies presented here show that the cathepsin F family of O . viverrini is most closely related the numerous cathepsin F-like transcripts expressed by another liver fluke , C . sinensis , which is informative since this parasite is also associated with cancer of the bile duct [38] . Whereas it is not yet clear whether O . viverrini also expresses and secretes cathepsin L , in addition to cathepsin F , transcripts that could encode cathepsin L are not abundant among the presently available O . viverrini ESTs , despite the fact that transcripts corresponding to several other Family C1 proteases , such as cathepsin B , are available [26] . C . sinensis , like S . mansoni [39] , [40] , S . japonicum [41] and Paragonimus westermani [25] , [42] , [43] , appears to have both cathepsin F and cathepsin L -like enzymes [13] . In conspicuous contrast , Fasciola hepatica and F . gigantica , which also parasitize mammalian bile ducts , have a battery of cathepsin L enzymes yet no recorded cathepsin F [37] , [44] . To date , four orthologous cathepsin F-like proteases of fluke parasites of humans have been functionally characterized . These are SmCF from S . mansoni [39] , [40] , Pw28CCP and related enzymes from P . westermani [25] , [42] , [43] , CsCF-6 from C . sinensis [13] and Ov-CF-1 ( this study ) . Cytochemical studies have shown that the cathepsin L and cathepsin F proteases of schistosomes localize to the gastrodermis of the gut and participate in extracellular digestion of ingested host tissues [40] . Caffrey and colleagues showed that promoter regions of the SmCF gene could drive reporter transgene expression in the gut of adult S . mansoni [45] , thus providing strong support for a specific role for this protease in digestion of host blood [40] . CsCF-6 is also predominantly expressed in the gut of the adult C . sinensis where it is likely has a nutritional role [13] . In F . hepatica , cathepsin L1 and L2 are synthesized and secreted from gastrodermal epithelium and are essential for migration of the juvenile fluke through the intestinal wall and liver capsule , and for digestion of ingested blood and other host tissues [37] , [44] . RNA interference of cathepsin L transcription blocks penetration of host tissues by the juvenile fluke [46] . Thus the localization of Ov-CF-1 in the gut of adult O . viverrini is consistent with findings in these related trematodes and consistent with a generalized function for both cathepsin F and L proteases in the degradation of host blood and other tissues . The immunolocalization micrographs revealed that Ov-CF-1 was also expressed in the vitellaria , testis and eggs ( Figure 5B and 5D ) . Transcripts of Ov-CF-1 were also detected in eggs ( Figure 4 ) . The role of the hydrolase in the reproductive organs of Opisthorchis has yet to be determined , particularly as reports on the immunolocalization of orthologues of cathepsin F in C . sinensis and P . westermani did not describe localization in reproductive structures [13] , [43] . However , it has been reported that SmCL2 , a cathepsin L of S . mansoni , is associated with the reproductive organs where it might activate phenol oxidase , an enzyme involved with eggshell formation , or with the seminal fluid [47] . F . hepatica cathepsin L has also been localized to the vitellaria [44] . It is possible that the cathepsin cysteine proteases in the reproductive structures of trematodes are encoded by different transcripts to those present in the gut but the protein products exhibit immunological cross-reactivity . The immunolocalization investigations also revealed the presence of Ov-CF-1 in the biliary epithelium , and mononuclear cells and Kupffer cells in sinuses of the infected liver , indicating that release of Ov-CF-1 from the flukes into the bile and circulation ( blood ) . It is possible that Kupffer cells and other macrophages phagocytose Ov-CF-1 and present the antigens to T cells , leading to inflammation . In this regard , Ov-CF-1 and other ES components ( which are mitogenic in vitro; Sripa , unpublished ) may stimulate inflammation and proliferation of biliary cells in the vicinity of the adult O . viverrini parasite . Such phenomena may promote cholangiocarcinogenesis . Human cathepsin F and L are a papain-like cysteine proteases , with the MEROPS classification Clan CA , Family C1 , Subfamily A ( C01 . 018 and C01 . 032 , respectively ) [48] . Although the function ( s ) of cathepsin F has not been fully elucidated , it may play a regulatory role in processing the invariant chain that is associated with MHC class II [49] , [50] . Cathepsin L has a wide tissue distribution and is found in lysosomes where its endopeptidase activity is a key catalyst of lysosomal proteolysis , although it also plays specific roles in generation of peptide antigens for the MHC II system and in spermatogenesis . It also has roles in pathological processes including tumor invasion , metastasis and arthritis [49] . Most residues of the prosegment of cathepsin L are conserved in cathepsin F , including the ERFNIN motif ( ERFNAQ in cathepsins F and W ) [51] , [52] . A key structural difference between mammalian cathepsin F and other Family C1 proteases , including cathepsin L , is a cystatin domain within the long prosegment , the physiological role of which has not been established . It has been suggested that the mammalian cathepsin F gene evolved through a gene fusion between an ancestral cystatin and Clan CA cathepsin gene [53] ( Figures 1 and 2 ) . Whereas Ov-CF-1 and related fluke enzymes clearly display sequence identity to mature human cathepsin F , none so far reported include the cystatin domain within the prosegment . Therefore , if their genes represent the progeny of the ancestral cathepsin F gene , the hypothesized gene fusion of cathepsin F and cystatin genes must have occurred after the branching event in the tree of life that lead separately to trematodes ( a Lophotrochozoan clade ) and vertebrates ( Deuterostomia ) . In human cathepsin F , the two splice sites that interrupt exons 1–3 are conserved between cathepsin F and several cystatin genes [53] , providing cogent evidence of a gene fusion during the evolution of human cathepsin F . The genome organization of human cathepsin F includes 13 exons separated by 12 introns; that of Ov-cf-1 is simpler , with seven exons separated by six introns . Because of the absence of a cystatin domain from the fluke cathepsin , exons one and two of the human gene do not have orthologues in the O . viverrini gene . However , downstream of the cystatin sequence , orthology between the human and O . viverrini cathepsin F genes becomes unambiguous; the exon/intron boundaries are strongly conserved between the two genes ( Figure 2C ) . Interestingly , the cathepsin F-like gene Tci-cf-1 from the strongylid nematode Teladorsagia circumcincta , a parasite of the abomasum of sheep , does not encode a cystatin domain in its prosegment , although the prosegment of this nematode cathepsin F is ∼30 amino acids longer than the 95 residue prosegment of Ov-CF-1 and orthologues from other flukes [54] . Cathepsin F is secreted by the parasitic stages of T . circumcincta , including the L4 but not by the free-living larval stages , and is immunogenic . Given that some other nematodes including Caenorhabditis elegans and Brugia malayi do encode a cystatin domain in the prosegment of their cathepsin F orthologues [54] , the evolutionary provenance of the cathepsin F/cystatin gene fusion remains obscure . All cysteine proteases are synthesized as inactive zymogens which include a prosegment [55] . The prosegment lies across the active site , in reverse orientation to normal protein substrates , and prevents premature activation of the zymogen during trafficking and storage . Removal of the prosegment , mediated by protease clipping at the juncture between prosegment and mature enzyme , is essential to allow entry of macromolecular protein substrates into the active site cleft of the hydrolase . Dalton and colleagues proposed that prosegment removal occurs in helminth cathepsins by a two-step route of trans-processing to generate a small pool of activated enzymes which primes a rapid catalytic activation cascade [29] . The initial event might be performed by the same protease or a different protease , such as asparaginyl endopeptidase or cathepsin B or L . Our data showing that recombinant Ov-CF-1 did not undergo autocatalytic activation , despite displaying low-level activity , suggested that native Ov-CF-1 requires trans-processing . Stack and co-workers described how the junction between the prosegment and mature enzyme of human and helminth cysteine proteases consists of a non-conserved , randomly-structured motif that is susceptible to cleavage at several sites , depending on the processing enzyme [24] . Based on the three-dimensional structure of human cathepsin F [50] and multiple sequence alignments with helminth orthologues ( Figure 1 ) , we predict that the protease-susceptible region of Ov-CF-1 prosegment is the peptide PTPQEDVTMD . Since this does not include asparagine residues , it is not surprising that Ov-CF-1 was not trans-processed by the O . viverrini asparaginyl endopeptidase , even though this enzyme occurs in the gut of the adult [17] . Several gut-associated cathepsins of schistosomes and Fasciola do possess asparagine residues at the prosegment/mature enzyme juncture and , consequently , are trans-processed by asparaginyl endopeptidase ( see [29] ) . On the other hand , here we have demonstrated trans-processing of Ov-CF-1 in vitro by the cathepsin L protease FhCL1 of F . hepatica . Cleavage of the prosegment occurred at PTPQEDVT↓MD , positioning a Val at P2 , which is favored by FhCL1 [16] , [56] . This cleavage resulted in a >50-fold increase in Ov-CF-1 activity . The ability of FhCL1 to trans-process Ov-CF-1 suggests that another endogenous Family C1 protease of O . viverrini , e . g . cathepsin B , processes and activates the cathepsin F within the adult gut . Notwithstanding that cathepsins F and L belong to distinct phylogenetic clades [57] ( Figure 3 ) , our studies demonstrated that they share overlapping substrate preferences . Both Ov-CF-1 and FhCL1 accommodated substrates with hydrophobic residues in the P2 position; the P2 residue occupies the S2 subsite of the active site which primarily dictates the specificity of members of Clan CA . Additionally , both enzymes preferred Leu over Phe , whereas neither accommodated the bulky residue , proline . The selectively of FhCL1 for hydrophobic residues is likely the result of specific adaptation to the cleavage of mammalian hemoglobin , a substrate in which 42% of the component amino acids are Leu , Phe , Ala and Val [56] . Our limited substrate specificity analysis , however , has not yet revealed significant differences between the cathepsins F and L , which we suspect do exist . Indeed , we hypothesize that divergence of substrate preferences between cathepsins F and L is of central importance to the virulence and host species range of trematode parasites such as O . viverrini , F . hepatica and S . mansoni . Since phylogenetic divergence of the cathepsin F and L clades must have been driven by positive selection , by extension , elucidation of how this favors degradation of their respective target host macromolecules or tissues will inform our understanding of host-parasite adaptation . Finally , given the extraordinary linkage between a metazoan parasite and a tumor [1] , [4] , [5] , [38] , characterization of the nature and action of secreted proteins of O . viverrini such as cathepsin F may provide insights into liver fluke induced cholangiocarcinogenesis , and indeed fundamental insights into carcinogenesis at large . In addition , cathepsin F may have potential as an intervention target including a vaccine candidate , given recent successes with chemotherapy targeting related enzymes in schistosomes [58] and with vaccines targeting cathepsin L of Fasciola hepatica [59] . Indeed , in view of the recent implementation of an acclaimed vaccination of adolescents against papilloma-virus infection to provide protection from cervical cancer [60] , there is the tantalizing prospect that vaccination to prevent O . viverrini infection could provide protection against another infection-related cancer , liver fluke-induced cholangiocarcinoma . | Opisthorchiasis , oriental liver fluke infection , is a food-borne parasitic disease that afflicts millions of residents in northern Thailand and Laos . Related infections occur in North Asia , including China and Korea . This kind of liver fluke infection is the consequence of eating certain uncooked or undercooked freshwater fish contaminated with the larvae of the parasite Opisthorchis viverrini . Whereas the infection can cause disease in the bile ducts and liver , infection with the oriental fluke can lead to the development of a liver cancer , cholangiocarcinoma ( bile duct cancer ) . Our recent studies have begun to focus on products and metabolites from the parasite that are carcinogenic . Many proteolytic enzymes are known to be secreted by parasites . This report centers on a specific category of protease , termed cathepsin F . We determined here that O . viverrini expresses a cathepsin F in its gut and in other organs . In the liver fluke , cathepsin F likely plays a role in digesting ingested human cells . The gene encoding the parasite enzyme shows evolutionary relatedness to a similar gene in humans . The fluke cathepsin F also is released from the parasite into livers of infected mammals , where it appears to contribute to inflammation surrounding the parasite . In this regard , it may be involved in early events that lead to bile duct cancer . | [
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] | [
"biochemistry/bioinformatics"
] | 2009 | Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini |
The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor ( PRR ) pathways that converge on the induction of type I interferons ( IFNs ) . Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation . Here we show that the 3Cpro cysteine protease of coxsackievirus B3 ( CVB3 ) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein ( MAVS ) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta ( TRIF ) as a mechanism to escape host immunity . We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture . CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3Cpro , occurred at specific sites and within specialized domains of each molecule , and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors . 3Cpro-mediated MAVS cleavage occurred within its proline-rich region , led to its relocalization from the mitochondrial membrane , and ablated its downstream signaling . We further show that 3Cpro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm . Taken together , these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition .
The innate immune system is the first line of defense against pathogen infiltration and is activated by the binding of conserved microbial ligands to pattern recognition receptors ( PRRs ) . Activation of these receptors culminates in nuclear factor ( NF ) -κB and/or IFN regulatory factor ( IRF ) -mediated induction of type 1 interferons ( IFN-α and -β ) , key components of antimicrobial host defenses . PRRs , including Toll-like receptors ( TLRs ) and DExD/H box RNA helicases , signal through an assortment of downstream adaptor molecules to propagate innate immune signaling . TLRs signal through adaptor molecules such as myeloid differentiation factor 88 ( MyD88 ) , Toll/IL-1 receptor domain containing adaptor protein ( TIRAP ) , Toll/IL-1 receptor domain containing adaptor inducing interferon-beta ( TRIF ) , and TRIF-related adaptor molecule ( TRAM ) to activate cellular defenses [1] . These adaptors often display specificity with regard to the TLR family members with whom they interact with and from which they are activated . The specificity of TLR ectodomain-ligand recognition and concomitant specificity in the signaling networks that are engaged by this interaction provides an efficient strategy for microbial recognition . In contrast , activated DExD/H box RNA helicases , which include melanoma differentiation associated gene ( MDA5 ) and retinoic acid induced gene-I ( RIG-I ) , signal to a common downstream adaptor molecule , mitochondrial antiviral signaling [ ( MAVS ) , also known as VISA/IPS-1/Cardif] to activate NFκB and IRF3 [2] , [3] , [4] . MAVS is localized to the mitochondrial membrane and to peroxisomes via a C-terminal transmembrane domain , which is essential for innate immune signaling [5] , [6] . PRR-associated adaptor molecules thus serve critical roles in the activation of cellular defenses associated with microbial recognition . As host cells have developed highly specialized strategies for microbial detection and clearance , it is not surprising that many viruses have evolved strategies to counter these defenses in order to promote their replication and spread . In some cases , virally-encoded proteases directly target components of the innate immune system to abolish antiviral signaling via TLRs and/or DExD/H box helicases . Targeted proteolysis of adaptor molecules serves as a powerful means to eliminate antiviral signaling by suppressing common downstream targets of key innate immune signaling pathways . For example , MAVS is cleaved by the NS3/4A serine protease of hepatitis C virus ( HCV ) [7] , the 3Cpro cysteine protease of hepatitis A virus ( HAV ) [8] , the HCV-related GB virus B NS3/4A protease [9] , and the 2Apro and 3Cpro proteases of rhinovirus [10] . HCV also utilizes the same NS3/4A serine protease to cleave TRIF in order to silence TLR3-mediated signaling [11] . Thus , the targeting of MAVS and/or TRIF by virally-encoded proteases in order to suppress antiviral signaling is emerging as a common theme in the evasion of host defenses . Enteroviruses , which belong to the Picornaviridae family , are small single-stranded RNA viruses that account for several million symptomatic infections in the United States each year . Coxsackievirus B3 ( CVB3 ) , a member of the Enterovirus genus , is associated with a number of diverse syndromes , including meningitis , febrile illness , and diabetes [12] and is an important causative agent of virus-induced heart disease in adults and children [13] , [14] , [15] , [16] . The induction of type I IFN signaling is essential for the control of CVB3 infection , as evidenced by enhanced virus-induced lethality in type I IFN receptor ( IFN-α∼β R ) null mice [17] and increased susceptibility to CVB3 infection in IFNβ-deficient mice [18] . Both TLR3- and MDA5-mediated type I IFN signaling have been implicated in the response to CVB3 infections and mice deficient in either TRIF or MAVS show an enhanced susceptibility to viral infection [19] , [20] , [21] . In this study , we determined the potential mechanisms employed by CVB3 to antagonize type I IFN signaling . We found that infection of cells with CVB3 led to the cleavage of the adaptor molecules MAVS and TRIF . Both MAVS and TRIF were cleaved by the CVB3-encoded cysteine protease 3Cpro , indicating that a single protease suppresses innate immune signaling through two powerful pathways . We found that 3Cpro cleaves specific residues within MAVS and TRIF that render these molecules deficient in type I IFN signaling and apoptotic signaling . Taken together , these data suggest that CVB3 has evolved a mechanism to cleave adaptor components of the innate immune system to escape host immunity .
The induction of type I IFNs is the earliest cellular immune response initiated to combat viral infections and is coordinated by the activation of transcription factors such as interferon regulatory factor ( IRF ) -3 , IRF7 , and NFκB downstream of PRR activation . We found that CVB3 infection of HEK293 cells led to only a modest induction of IRF3 activation as assessed by immunofluorescence microscopy for nuclear translocation ( Figure 1A ) , western blot analysis of nuclear extracts ( Figure 1B ) , and luciferase activity assays for IFNβ ( Figure 1C ) . In contrast , transfection of cells with poly I:C induced pronounced IRF3 activation ( Figure 1A–C ) . We also observed little activation of NFκB signaling in response to CVB3 infection as determined by luciferase activation assay ( Figure 1C ) . Because CVB3 did not elicit a pronounced translocation of IRF3 into the nucleus during infection of HEK293 cells , we investigated the role of several PRRs in mediating CVB3 recognition–TLR3 , RIG-I , and MDA5 . Both MDA5 [22] and TLR3 [19] have been proposed to act as sensors for CVB3 infection . Although infection of cells with CVB3 is sensitive to IFNβ ( Supplemental Figure S1A ) , we observed less enhancement of IFNβ promoter activity as assessed by luciferase activation in CVB3-infected HEK293 cells overexpressing MAVS , MDA5 , RIG-I , and TLR3/TRIF than in uninfected controls ( Figure 1D ) . Instead , we observed the partial ablation of IFNβ promoter activity in response to ectopic expression of MAVS , RIG-I , MDA5 , and TLR3/TRIF in CVB3 infected cells ( Figure 1D ) . We also found that CVB did not induce potent IFNβ production in HEK293 , HeLa , or Caco-2 cells in comparison to VSV controls ( Figure 1E ) . Because CVB3 infection was inefficient at inducing IRF3 , we assessed the pattern of expression of MAVS in CVB3-infected HEK293 cells . By immunoblot analysis , we found that CVB3 infection induced the cleavage of MAVS ( Figure 2A ) . Similar results were obtained in HeLa cells ( Supplemental Figure S2A ) . This effect was specific for CVB3 as infection with VSV did not alter MAVS migration ( Supplemental Figure S2B ) . In uninfected cells , full-length MAVS migrated as a single band of ∼75 kD . However , in cells infected with CVB3 , there was a decrease in the expression level of full-length MAVS and the appearance of a distinct MAVS cleavage fragment migrating at ∼40–50 kD ( Figure 2A ) . Because MAVS cleavage is induced in cells undergoing apoptosis [23] , [24] and CVB3 is known to induce apoptosis in many cell types [25] , [26] , we investigated the role of apoptosis in CVB3-induced MAVS cleavage . We found that incubation of CVB3-infected HEK293 cells with the broad caspase inhibitor z-VAD-FMK and the proteosome inhibitor MG132 had little effect on CVB3-induced MAVS cleavage ( Figure 2A ) . ( The slight reduction in MAVS cleavage observed in the presence of MG132 is likely attributable to a reduction in replication in MG132-exposed cells , consistent with previously published results [27] , [28] ) . The kinetics of MAVS cleavage was also not consistent with apoptosis: MAVS cleavage was evident by 3 hrs post-infection ( p . i . ) whereas apoptosis ( as measured by caspase-3 cleavage ) did not occur until 5–6 hrs p . i . ( Figure 2B ) . MAVS is localized to the mitochondrial membrane via a C-terminal transmembrane domain [5] . We found that CVB3 infection induced a pronounced decrease in MAVS mitochondrial localization as assessed by immunofluorescence microscopy with a mitochondrial marker ( Figure 2C ) . We also found that the expression and mitochondrial localization of ectopically expressed MAVS was significantly reduced in CVB3-infected cells ( Figure 2D , 2E ) . The appearance of cleavage fragments was evident in CVB3-infected cells overexpressing MAVS ( Figure 2D ) . Moreover , we found that mutation of the caspase cleavage site of MAVS ( D429E , [24] ) had no effect on CVB3-induced MAVS cleavage ( Supplemental Figure S2C ) , indicating a caspase-independent mechanism of action . Another common pathway upstream of IRF3 activation is the engagement of TLR3 by viral dsRNA , which is produced as a replication intermediate during viral infection . As we observed cleavage of MAVS in CVB3-infected cells , we sought to determine if CVB3 might also target TRIF , the specific adaptor molecule downstream of TLR3 , to repress IRF3 activation . Similar to our findings with MAVS , we found that TRIF expression was significantly reduced in HeLa cells infected with CVB3 ( Figure 2F ) . Although TRIF can be cleaved during apoptosis [23] , [24] , we found that z-VAD-FMK and MG132 had little effect at antagonizing the CVB3-mediated reduction in TRIF expression ( Figure 2F ) , consistent with our findings with MAVS ( Figure 2A ) . The kinetics of TRIF cleavage also paralleled that of MAVS as we observed a marked reduction in TRIF levels by 3 hrs p . i . ( Figure 2G ) , a time prior to the induction of caspase-3 cleavage ( Figure 2B ) . Ectopically expressed CFP-fused TRIF was also significantly decreased in cells infected with CVB3 and coincided with the appearance of several cleavage fragments ( Figure 2H ) . We next investigated whether cleavage of MAVS and TRIF occurred in cells infected with other enteroviruses including echovirus 7 ( E7 ) and enterovirus 71 ( EV71 ) . Infection of HeLa cells with both E7 and EV71 led to the significant reduction of MAVS and TRIF expression , which corresponded with the appearance of the newly replicated viral protein VP1 ( Supplemental Figure S3A ) . However , in contrast to our findings with CVB3 ( Figure 2A ) , we did not observe the appearance of any significant cleavage fragments in either E7 or EV71-infected cells . This may indicate that the cleavage fragments are short-lived in E7 or EV71-infected cells or that cleavage occurs at different residues within the molecule that alter antibody binding . These results may indicate that members of the enterovirus family target MAVS and TRIF to evade host immunity , but further studies are required to definitively show which members of the enterovirus family utilize this mechanism . CVB3 infections are commonly associated with virus-induced heart disease in adults and children and have been detected in approximately 20-25% of patients with dilated cardiomyopathy and myocarditis [13] , [14] , [15] , [16] . To determine whether MAVS and TRIF are degraded in vivo , mice were infected with CVB3 and the hearts of infected animals were probed for MAVS and TRIF . In contrast to uninfected controls , there was an almost complete absence of both MAVS and TRIF in murine hearts infected with CVB3 ( Supplemental Figure S3B ) . These data indicate that the cleavage of MAVS and TRIF may also occur during CVB3 infection in vivo . Enteroviruses encode specific proteases that are required for the processing of viral proteins and the establishment of replication , but which also cleave a variety of host cell molecules [29] . Because we observed the cleavage of MAVS in CVB3-infected cells , we investigated whether virally-encoded proteases might mediate this effect . We cotransfected HEK293 cells with N-terminal Flag-MAVS and various CVB3 viral proteins fused to EGFP . Of these proteins , we found that expression of the protease 3Cpro was sufficient to induce a significant reduction in MAVS expression ( Figure 3A ) . In fact , in order to observe significant levels of full-length Flag-MAVS ( or cleavage fragments ) in EGFP-3Cpro co-transfected cells , cells had to be transfected with twice as much Flag-MAVS as vector control or other CVB3 viral proteins . The apparent lack of cleavage products in cells overexpressing proteases is a phenomenon that has also been observed for HCV-mediated cleavage of TRIF [11] and likely reflects the high efficiency of cleavage ( which may result from protease overexpression ) and that cleavage fragments are unstable and/or short-lived . For our subsequent studies , we transfected cells with equivalent amounts of MAVS cDNA to compare the level of full-length MAVS in control versus 3Cpro-transfected cells . The cleavage of MAVS required the cysteine protease activity of 3Cpro , as cotransfection of a catalytically inactive N-terminal EGFP-tagged 3Cpro mutant ( C147A ) [30] had no effect on MAVS expression ( Figure 3B ) . In some cases , significant levels of GFP signal alone can be detected in EGFP-3Cpro WT transfected cells which is likely indicative of 3Cpro cleaving itself from the N-terminal EGFP tag . To confirm that 3Cpro was directly cleaving MAVS , we incubated recombinant wild-type or C147A mutant 3Cpro with Flag-MAVS purified by Flag column affinity purification from overexpressing HEK293 cells . Whereas incubation with wild-type 3Cpro induced the appearance of a MAVS cleavage fragment as determined by Flag immunoblotting , the C147A mutant did not induce the appearance of a MAVS cleavage product ( Figure 3C ) . Moreover , whereas expression of wild-type EGFP-3Cpro induced the relocalization of MAVS as assessed by immunofluorescence microscopy , expression of EGFP-3Cpro C147A had no effect ( Figure 3D ) . Because we also observed cleavage of TRIF in CVB3-infected cells , we determined whether 3Cpro was responsible for its cleavage as well . We cotransfected HEK293 cells with TRIF and either EGFP-2Apro , 3A , or -3Cpro . Expression of 3Cpro , but not 2Apro or 3A , led to the cleavage of TRIF , demonstrated by a reduction in the expression of full-length TRIF and the appearance of several TRIF cleavage fragments ( Figure 3E ) . 3Cpro-mediated cleavage of TRIF required the cysteine protease activity of 3Cpro as expression of 3Cpro C147A did not lead to TRIF cleavage ( Figure 3F ) . We also confirmed that 3Cpro was directly cleaving TRIF by incubation of Flag-TRIF purified by Flag column affinity purification from overexpressing HEK293 cells with recombinant wild-type or C147A mutant 3Cpro . Similar to our findings with MAVS ( Figure 3C ) , we found that only recombinant wild-type 3Cpro induced the appearance of TRIF cleavage fragments ( Figure 3G ) . Note that the pattern of TRIF cleavage by in vitro proteolysis assay ( Figure 3G ) differs from our experiments with overexpressed 3Cpro in HEK293 cells ( Figure 3E , 3F ) due to the use of C-terminal CFP- versus N-terminal Flag-tagged TRIF between experiments . Taken together , our data show that 3Cpro directly cleaves both MAVS and TRIF . To assess whether expression of 3Cpro abrogated MAVS-dependent signaling , we transfected HEK293 cells with wild-type or C147A EGFP-3Cpro or vector control , with a luciferase reporter fused to the IFNβ promoter region ( p-125-Luc ) , and with either Flag-MAVS or the caspase activation and recruitment domains ( CARDs ) of MDA5 or RIG-I . Expression of the CARDs of MDA5 and RIG-I alone results in the constitutive activation of type I IFN signaling even in the absence of stimulus [31] . We found that whereas there was pronounced induction of IFNβ activity in cells expressing vector alone or EGFP-3Cpro C147A , expression of wild-type EGFP-3Cpro led to a significant reduction in promoter activity ( Figure 4A ) . We next determined whether 3Cpro attenuated TRIF-mediated signaling . TRIF is involved in the activation of IRF3 and IFNβ induction downstream of dsRNA-TLR3 engagement . While the expression of TRIF and vector control enhanced IFNβ promoter activity , expression of TRIF in combination with 3Cpro significantly impaired IFNβ promoter activity ( Figure 4B ) . We found that 3Cpro-mediated inhibition of IFNβ signaling was indeed occurring upstream of IRF3 activation as coexpression of wild-type 3Cpro and IRF3 had no effect on IRF3-mediated activation of IFNβ promoter activity ( Supplemental Figure S4A ) . Furthermore , we found that expression of wild-type , but not C147A 3Cpro reduced IFNβ activation in response to infection with VSV ( Supplemental Figure S4B ) . In addition to their roles in type I IFN signaling , ectopic expression of MAVS [32] and TRIF [33] potently stimulate intrinsic apoptotic machinery to induce cell death . We found that expression of MAVS or TRIF induced pronounced apoptosis as demonstrated by enhanced Annexin V binding [which identifies the externalization of phosphatidylserine in cells undergoing apoptosis] ( Figure 4C , 4D ) . In contrast , expression of MAVS or TRIF in the presence of 3Cpro potently reduced apoptosis ( Figure 4C , 4D ) . Taken together , these data show that 3Cpro represses both the apoptotic and type I IFN signaling mediated by MAVS and TRIF . 3Cpro preferentially cleaves glutamine-glycine ( Q-G ) bonds in both the viral polyprotein and cellular targets , but may also exhibit proteolytic activity against glutamine-alanine ( Q-A ) bonds , amongst others [29] . In order to identify the residue ( s ) within MAVS cleaved by 3Cpro , we constructed a panel of site directed mutants within MAVS at residues that may serve as 3Cpro cleavage sites ( Q148 , Q211 , and E480 ) ( Figure 5A ) . Of these mutants , only one ( Q148A ) was resistant to 3Cpro-mediated cleavage in HEK293 cells and by in vitro protease assay ( Figure 5B ) . Moreover , whereas wild-type Flag-MAVS was relocalized from the mitochondrial membrane upon expression of EGFP-3Cpro , the Q148A Flag-MAVS mutant retained its mitochondrial localization ( Figure 5C ) . The 3Cpro cleavage site within MAVS ( Q148 ) is located in the proline rich region , which mediates its interaction with a number of signaling molecules including TRAF2 [4] , TRAF3 [34] , TRAF6 [4] , RIP1 [2] , and FADD [2] . We next determined whether the Q148A mutant of MAVS was resistant to 3Cpro-mediated abatement of MAVS signaling . We found that whereas there was a pronounced reduction in IFNβ activity in cells expressing wild-type Flag-MAVS and EGFP-3Cpro , there was no effect of EGFP-3Cpro expression on IFNβ signaling in cells transfected with Q148A Flag-MAVS ( Figure 5D , Supplemental Figure S2D ) . These data show that CVB3 3Cpro cleaves MAVS at Q148 to suppress MAVS signaling . MAVS requires an intact CARD and localization to the mitochondrial membrane ( via a C-terminal transmembrane domain ) to remain functionally active [5] . Because we found that 3Cpro cleaved MAVS at a specific residue ( Q148 ) within the proline rich region , we next determined whether either of the possible 3Cpro-induced cleavage fragments of MAVS would remain active . To that end , we constructed EGFP-fused constructs expressing wild-type MAVS , the N-terminal ( residues 1-148 ) , or C-terminal ( residues 149-540 ) fragments of MAVS that would result from 3Cpro cleavage ( Figure 5F ) . We found that the N-terminal fragment of MAVS ( 1-148 ) no longer localized to the mitochondrial membrane ( Figure 5G ) and induced NFκB or IFNβ signaling significantly less that full-length MAVS ( Figure 5H ) . Whereas the C-terminal fragment of MAVS ( 149-540 ) retained its mitochondrial localization ( Figure 5G ) , it also exhibited significantly less NFκB and IFNβ activation in comparison to full-length MAVS ( Figure 5H ) . These data indicate that 3Cpro-mediated cleavage of MAVS likely inactivates MAVS-mediated downstream signaling by directly cleaving a residue that separates the CARD and transmembrane regions . Overexpressed TRIF forms multimers and localizes to punctate cytoplasmic structures referred to as the TRIF ‘signalosome’ [35] . Downstream components of TRIF signaling localize to signalosomes as a mechanism to stimulate TRIF signaling [35] , [36] . We found that EGFP-3Cpro and EGFP-3Cpro C147A were recruited to TRIF signalosomes when co-expressed with TRIF ( Figure 6A ) . This recruitment was specific for 3Cpro as we did not observe the recruitment of either EGFP-2Apro ( not shown ) or EGFP-3A ( Supplemental Figure S5 ) to TRIF signalosomes . Although TLR3 ( and presumably TRIF ) can localize to endosomal membranes [37] , we did not observe any colocalization of overexpressed TRIF with markers of both early and late endosomes ( Supplemental Figure S6 ) . Because we observed the relocalization of 3Cpro to the signalosome complex , we next determined whether 3Cpro and TRIF interact within this specialized complex . HEK293 cells were transfected with TRIF and either vector ( EGFP alone ) , EGFP-3Cpro wild-type , or EGFP-3Cpro C147A and co-immunoprecipitation studies were performed . We found that whereas EGFP-3Cpro C147A and TRIF co-immunoprecipitated , wild-type EGFP-3Cpro and TRIF did not ( Figure 6B ) . These findings indicate that 3Cpro forms an interaction with TRIF that is likely abolished upon 3Cpro-mediated cleavage . TRIF contains a proline-rich N-terminal region , a Toll/Interleukin-1 receptor ( TIR ) domain , and a C-terminal region . To determine which TRIF domain is responsible for interacting with 3Cpro and recruiting it to the signalosome , we constructed N-terminal ( NT , 1–359aa ) , C-terminal ( CT , 360–712aa ) , and TIR ( 390–460aa ) domain expression constructs of TRIF containing a HA-tag at the N-terminus and a Flag-tag at the C-terminus ( Figure 6C ) . We then coexpressed these constructs with wild-type and C147A versions of 3Cpro and performed fluorescence microscopy and immunoprecipitation analysis . We found that 3Cpro C147A specifically interacted with the C-terminal domain of TRIF , but not the N-terminus ( Figure 6D ) . However , the TIR domain did not mediate the interaction between TRIF and 3Cpro as we observed no co-immunoprecipitation between HA-TIR-Flag and 3Cpro ( not shown ) . Previous studies have shown that expression of the C-terminus of TRIF is required for the formation of the TRIF signalosome [36] . We found that expression of HA-CT-Flag was sufficient to induce the relocalization of 3Cpro C147A to signalosomes ( Figure 6E ) . In contrast , 3Cpro C147A did not localize with either HA-NT-Flag or HA-TIR-Flag ( Figure 6E ) . The formation of tubule-like structures induced by the expression of the TRIF TIR is consistent with previous work by others [36] . Taken together , these data indicate that 3Cpro interacts with the C-terminus of TRIF that is sufficient for its recruitment into the TRIF signalosome . We did not observe any interaction between wild-type 3Cpro and either full-length or C-terminal TRIF ( Figure 6D and 6E ) suggesting that the interaction between TRIF and 3Cpro is diminished following cleavage . Interestingly , we observed the appearance of cleavage fragments of both HA-NT-Flag and HA-CT-Flag when coexpressed with wild-type 3Cpro ( Figure 6D ) . To further define the extent of 3Cpro-mediated proteolysis of the N- and C-terminal regions of TRIF , we coexpressed dually HA- and Flag-tagged constructs of TRIF ( described in Figure 6C ) and wild-type or C147A EGFP-3Cpro and subjected lysates to dual-color ( 700 nm and 800 nm ) immunoblot analysis using a LI-COR Odyssey infrared imaging system and antibodies specific for HA and Flag . This technique could therefore allow for the detection of a variety of TRIF cleavage fragments simultaneously . We found that expression of wild-type 3Cpro ( but not the C147A mutant ) induced the cleavage of both the N- and C-termini of TRIF ( Figure 7A ) . In contrast , we observed no cleavage of the TIR domain ( Figure 7A ) . Additionally , our data indicate that the C-terminus of TRIF is cleaved more abundantly than the N-terminus as we observed a marked decrease in the expression of full-length HA-CT-Flag and the appearance of several HA- or Flag-tag-positive cleavage products induced by 3Cpro overexpression ( Figure 7A ) . The N- and C-terminal regions of TRIF differ in their capacities to induce type I IFN and NFκB signaling—whereas overexpression of the N-terminal region of TRIF activates both IFNβ and NFκB signaling , the C-terminal domain fails to activate IFNβ but potently induces NFκB activation [38] , [39] . Moreover , the C-terminus of TRIF is sufficient to induce apoptosis [33] . The RIP homotypic interaction motif ( RHIM ) at the C-terminus of TRIF is essential for both NFκB and apoptotic signaling [33] , [40] . Because we observed pronounced 3Cpro-mediated cleavage of the C-terminus of TRIF ( Figure 7A ) , we investigated whether NFκB and apoptotic signaling mediated by the C-terminus of TRIF was abolished . We found that expression of wild-type 3Cpro potently abrogated NFκB and apoptotic signaling induced by expression of the C-terminus of TRIF ( Figure 7B , 7C ) . These findings are consistent with those indicating that 3Cpro also inhibits full-length TRIF-mediated apoptotic signaling ( Figure 4C , 4D ) . In order to identify the residue ( s ) within TRIF cleaved by 3Cpro , we constructed a panel of site directed mutants within the TRIF N- and C-terminal domains at residues that may serve as 3Cpro cleavage sites ( Figure 7D ) . [We omitted any potential sites within the TRIF TIR domain as we did not observe 3Cpro-induced cleavage of this domain ( Figure 7A ) ] . We found that a specific residue ( Q190 ) within the N-terminal region of TRIF was targeted by 3Cpro as mutagenesis of this site abolished 3Cpro-induced cleavage ( Figure 7E ) . Because several sites in the C-terminal domain of TRIF can serve as possible 3Cpro cleavage sites , and because these sites lie within close proximity to one another , we mutated these sites simultaneously . We found that simultaneous mutagenesis of four potential 3Cpro cleavage sites ( Q653 , Q659 , Q671 , and Q702 ) was sufficient to prevent 3Cpro cleavage ( Figure 7F ) . These findings are consistent with our observation that the C-terminus of TRIF likely undergoes 3Cpro cleavage at several sites ( Figure 7A ) . Because the N- and C-terminal domains of TRIF function in unique capacities to induce IRF3 , NFκB , and apoptotic signaling , we next explored whether possible 3Cpro cleavage fragments of TRIF could remain functional in these pathways . We constructed EGFP-fused full-length TRIF and various possible cleavage fragments of TRIF ( encoding residues 190-653 , 190-671 , or 190-702 ) . We found that all three possible 3Cpro TRIF cleavage fragments maintained their capacity to activate type I IFN signaling ( as assessed by luciferase assays for IFN- stimulated response element ( ISRE ) , an IRF3-dependent promoter ) ( Figure 7G , 7H ) . In contrast , two of these fragments , 190–653 and 190-671 , lost their ability to activate NFκB or induce apoptotic signaling ( Figure 7G , 7H ) . These data indicate that 3Cpro-mediated cleavage of TRIF may primarily function to suppress TRIF-mediated NFκB and apoptotic signal propagation .
The host innate immune response to viral infections often involves the activation of parallel PRR pathways that converge on the induction of type I IFNs and NFκB activation . Several viruses have evolved sophisticated mechanisms to evade the host innate immune response by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation . Here we show that the 3Cpro cysteine protease of CVB3 targets MAVS and TRIF , two key adaptor molecules in the innate immune response as a mechanism to suppress type I IFN and apoptotic signaling . By targeting these adaptors , CVB3 has evolved a strategy to suppress antiviral signal propagation through two powerful pathways—TLR3 and RIG-I/MDA5 . 3Cpro cleaves MAVS at a specific site within its proline-rich region ( at Q148 ) and suppresses MAVS-mediated induction of type I IFNs and apoptosis . Moreover , 3Cpro targets both the N- and C-terminal domains of TRIF to abrogate its type I IFN , NFκB , and apoptotic signaling capacities . Interestingly , we found that 3Cpro localized to TRIF signalosomes and interacted with the C-terminal domain of TRIF . Taken together , these data highlight the strategies used by CVB3 to evade the host innate immune response . Many viruses target molecules upstream of IFN induction as a means to escape host immunity . Similar to our findings with CVB3 3Cpro , the 3Cpro of HAV directly cleaves MAVS to escape host immunity [8] , but it is not known if HAV 3Cpro also cleaves TRIF . However , although HAV 3Cpro is responsible for mediating MAVS cleavage , the protease must be localized to the mitochondrial membrane via a transmembrane domain within the 3A viral protein in order to facilitate this event [8] . In contrast , CVB3 3A localizes to the ER membrane where it disrupts ER-Golgi vesicular trafficking [41] , [42] and is thus not targeted to the mitochondrial membrane . Our studies indicate that in contrast to HAV , CVB3 3Cpro alone is sufficient to induce MAVS cleavage despite it not being localized to the mitochondrial membrane . Although MAVS and TRIF are potent inducers of type I IFN signaling downstream of PRR activation , they have also been shown to induce apoptotic signaling–another powerful pathway used by host cells to suppress viral replication and progeny release . Enteroviruses are lytic viruses , and as such , possess no known mechanism for progeny release other than the destruction of the host cell membrane . Lytic viruses often develop efficient strategies to tightly regulate host cell death pathways in order to avoid killing the host cell prematurely ( and terminating viral replication ) . CVB3 possesses anti-apoptotic strategies , which are mediated by the 2B and 2BC viral proteins [43] , [44] . In addition , it has been shown that 3Cpro targets the inhibitor of κBα as a means to stimulate apoptosis and suppress viral replication [45] . Our results show that 3Cpro may also serve in an anti-apoptotic capacity to suppress MAVS- and TRIF-mediated apoptotic signaling as a means to tightly regulate host cell apoptotic pathways . The pro-apoptotic signaling mediated by MAVS requires its localization to the mitochondrial membrane and the presence of intact CARDs , but not the presence of an intact proline-rich region [32] . Although 3Cpro cleaves MAVS within the proline-rich region ( Q148 , Figure 5B , 5C ) , this cleavage both induces the relocalization of MAVS from the mitochondrial membrane ( Figure 5C ) and inhibits MAVS signals ( Figure 5D ) . Furthermore , 3Cpro cleavage fragments of MAVS are non-functional ( Figure 5H ) . Thus , the loss of MAVS-induced apoptosis in CVB3 3Cpro-expressing cells is likely the result of both the relocalization of MAVS from the mitochondrial membrane and the inhibition of signaling via the CARD regions . Moreover , CVB3 3Cpro targets the C-terminal region of TRIF , which has been shown to induce apoptosis via direct binding to receptor interacting protein 1 ( RIP1 ) via its RIP homotypic interaction motif ( RHIM ) [33] . Specifically , we found that 3Cpro targeted several sites within the C-terminal domain of TRIF that could effectively remove the RHIM domain , a domain of TRIF known to be critically involved in NFκB and apoptotic signaling ( Figure 7D , F ) . In support of this , we found that 3Cpro cleavage fragments were deficient in NFκB activation and apoptosis ( Figure 7G ) . Taken together , these data indicate that 3Cpro suppresses MAVS and TRIF-induced apoptotic signals both by their direct cleavage and by their relocalization from either the mitochondria or signalosome , respectively . The N- and C-terminal domains of TRIF serve disparate functions in the initiation of innate immune signaling . Whereas the N-terminus of TRIF activates type I IFN induction via the phosphorylation of IRF3 , the C-terminal domain activates NFκB [38] , [39] . Interestingly , we found that 3Cpro cleaves both of these domains—likely as a mechanism to suppress global TRIF-generated signaling capacities . Upon ligand stimulation of TLR3 ( or upon overexpression ) , activated TRIF forms signalosomes enriched in TRIF-associated signaling components including RIP1 and NFκB -activating kinase-associated protein 1 ( NAP1 ) [35] , [36] . We found that 3Cpro localizes to the TRIF signalosome and that expression of the C-terminal domain of TRIF is sufficient to induce this localization ( Figure 6A , 6E ) . Moreover , we found that 3Cpro interacts with the C-terminal domain of TRIF ( Figure 6D ) . However , it remains unclear whether this interaction is direct or mediated via an adaptor molecule that also localizes to the signalosome . Additionally , we found that 3Cpro cleavage of the TRIF C-terminal domain leads to the disruption of TRIF signalosome formation ( Supplemental Figure S7 ) , which is required for the initiation of TRIF-mediated IRF3 and NFκB activation [36] . It is thus conceivable that 3Cpro attenuates TRIF-dependent signaling via direct cleavage , the degradation of the signalosome complex , and inhibition of the interactions between TRIF and downstream molecules that are required to propagate TRIF-dependent signals . Although we found that 3Cpro cleavage fragments of TRIF were deficient in NFκB and apoptotic signaling , they retained their capacity to induce type I IFN signaling ( Figure 7G ) . These data may indicate that the cleavage fragments of TRIF generated by 3Cpro cleavage are short-lived and do not accumulate within the cell . In support of this , we failed to identify TRIF cleavage products induced by CVB3 infection endogenously ( Figure 2F , 2G ) . Alternatively , it remains possible that 3Cpro-mediated disruption of TRIF signaling is not involved in the suppression of type I IFN signaling , but may instead target type II IFN signaling . Previous studies in TLR3 and TRIF deficient mouse models indicate that TLR3- and TRIF-mediated IFNγ production plays an important role in CVB3 infections in vivo [19] . Thus , TLR3 signaling via TRIF to induce type II IFNs may function as a parallel pathway to MDA5 and/or RIG-I-mediated induction of type I IFNs . In this scenario , 3Cpro would suppress the downstream propagation of both type I and II IFN signaling in order to evade host immunity . Viruses often utilize elegant strategies to attenuate innate immune signaling in order to promote their propagation . Here we show that the 3Cpro cysteine protease of CVB3 ( and likely other enteroviruses ) attenuates innate immune signaling mediated by two potent antiviral adapter molecules , MAVS and TRIF . By utilizing a variety of methods to abate MAVS and TRIF signaling , including both cleavage and retargeting from sites of signal propagation , 3Cpro can efficiently suppress both type I IFN and apoptotic signals aimed at clearing CVB3 infections . A better understanding of the mechanisms employed by enteroviruses to suppress host antiviral signaling could lead to the development of therapeutic interventions aimed at modulating viral pathogenesis .
Human embryonic kidney ( HEK ) 293 , HeLa , and U2OS cells were cultured in DMEM-H supplemented with 10% FBS and penicillin/streptomycin . Human intestinal Caco-2 cells were cultured in MEM supplemented with 10% FBS and penicillin/streptomycin . Cells were screened for mycoplasma using a PCR-based mycoplasma test ( Takara Bio USA ) to prevent abnormalities in cellular morphology , transfection , and growth . All experiments were performed with CVB3-RD , expanded as described [46] . Vesicular stomatitis virus ( VSV ) was kindly provided by Sara Cherry ( University of Pennsylvania , Philadelphia , PA ) . Experiments measuring productive virus infection were performed with 0 . 1-1 plaque forming units ( PFU ) /cell for the indicated times . HeLa cells were infected with echovirus 7 and enterovirus 71 at a MOI = 0 . 1 for the indicated times . Mouse infections were performed as described previously [47] and lysates kindly provided to us by Jeffrey M . Bergelson , Children's Hospital of Philadelphia . Plasmid transfections were performed using FuGENE 6 according to the manufacturer's protocol ( Roche Applied Science ) . Following transfection , cells were plated as described above and used 48–72 hrs later . Cells cultured in collagen-coated chamber slides ( LabTek , Nunc ) were washed and fixed with either 4% paraformaldehyde or with ice-cold methanol . Cells were then permeabilized with 0 . 1% Triton X-100 in phosphate buffered saline ( PBS ) and incubated with the indicated primary antibodies for 1 hr at room temperature ( RT ) . Following washing , cells were incubated with secondary antibodies for 30 min at room temperature , washed , and mounted with Vectashield ( Vector Laboratories ) containing 4′ , 6-diamidino-2-phenylindole ( DAPI ) . For detection of apoptosis , cells were washed in cold PBS and incubated with Alexa-Fluor-488 conjugated-annexin V and propidium iodide for 15 min at room temperature . Cells were then washed , fixed in 4% paraformaldehyde , and images captured as described below . Images were captured using an Olympus IX81 inverted microscope equipped with a motorized Z-axis drive . Images were generated by multiple-section stacking ( 0 . 2 mm stacks ) and deconvolved using a calculated point-spread function ( Slidebook 5 . 0 ) . Confocal microscopy was performed with a FV1000 confocal laser scanning microscope ( Olympus ) . Rabbit polyclonal and mouse monoclonal antibodies directed against GFP ( FL , B-2 ) , GAPDH , HA ( Y-11 , F-7 ) and IRF3 ( FL-425 ) were purchased from Santa Cruz Biotechnology . Mouse monoclonal anti-Flag ( M2 ) was purchased from Sigma . Rabbit polyclonal antibodies to TRIF and MAVS ( human and rodent specific ) were purchased from Cell Signaling Technologies or Bethyl Laboratories , respectively . Mouse anti-enterovirus VP1 ( Ncl-Entero ) was obtained from Novocastra Laboratories ( Newcastle upon Tyne , United Kingdom ) . Mitochondria antibody [MTC02] was purchased from Abcam . Mouse anti-enterovirus 71 antibody was purchased from Millipore . Alexa Fluor-conjugated secondary antibodies were purchased from Invitrogen . Flag-MDA5 , Flag-MAVS , and Flag-TRIF plasmids were kindly provided by Tianyi Wang ( University of Pittsburgh ) . pUNO2-hTRIF was purchased from Invivogen . EGFP-2Apro 2B , 2C , 3A and -3Cpro were constructed by amplification from CVB33 cDNA ( kindly provided by Jeffrey Bergelson , Children's Hospital of Philadelphia ) and cloned into the NT-GFP TOPO fusion vector ( Invitrogen ) following PCR amplification . EGFP-fusion constructs expressing cleavage fragments of MAVS and TRIF were generated by PCR amplification from Flag-MAVS or pUNO2-TRIF and cloned into the NT-GFP TOPO fusion vector ( Invitrogen ) . CFP-TRIF was purchased from Addgene ( plasmid 13644 ) . Dual HA- and Flag-tagged TRIF constructs were generated by amplification of TRIF cDNA with primers encoding a N-terminal HA or C-terminal Flag tags and cloned into the XhoI and EcoRI sites of pcDNA3 . 1 ( + ) . Mutagenesis of 3Cpro , MAVS and TRIF constructs were performed using Quickchange mutagenesis kit following the manufacturer's protocol ( Stratagene ) . Primer sequences are available upon request . Cell lysates were prepared with RIPA buffer ( 50 mM Tris-HCl [pH 7 . 4]; 1% NP-40; 0 . 25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM phenylmethanesulfonyl fluoride; 1 mg/ml aprotinin , leupeptin , and pepstatin; 1 mM sodium orthovanadate ) , and insoluble material was cleared by centrifugation at 700×g for 5 min at 4°C . Lysates ( 30-50 µg ) were loaded onto 4–20% Tris-HCl gels ( Bio-Rad , Hercules , CA ) and transferred to polyvinylidene difluoride membranes . Membranes were blocked in 5% nonfat dry milk or 3% bovine serum albumin , probed with the indicated antibodies , and developed with horseradish peroxidase-conjugated secondary antibodies ( Santa Cruz Biotechology ) , and SuperSignal West Pico or West Dura chemiluminescent substrates ( Pierce Biotechnology ) . Immunoblots in isolated mouse hearts and dually HA- and Flag-tagged TRIF constructs were conducted using an Odyssey Infrared Imaging System ( LI-COR Biosciences ) . Tissue homogenized in lysis buffer ( 100 µg ) or whole-cell lysates from transfected HEK293 cells ( 30 µg ) were loaded onto 4–20% Tris-HCl gels , separated electrophoretically , and transferred to nitrocellulose membranes . Membranes were blocked in Odyssey Blocking buffer and then incubated with the appropriate antibodies overnight at 4°C in Odyssey Blocking buffer . Following washing , membranes were incubated with anti-rabbit or anti-mouse antibodies conjugated to IRDye 680 or 800CW and visualized with the Odyssey Infrared Imaging System according to the manufacturer's instructions . For immunoprecipitations , HEK293 cells transiently transfected with the indicated plasmids were lysed with EBC buffer ( 50 mM Tris [pH 8 . 0] , 120 mM NaCl , 0 . 5% Nonidet P-40 , 1 mm phenylmethylsulfonyl fluoride , 0 . 5 µg/ml leupeptin , and 0 . 5 µg/ml pepstatin ) . Insoluble material was cleared by centrifugation . Lysates were incubated with the indicated antibodies in EBC buffer for 1 hr at 4°C followed by the addition of Sepharose G beads for an additional 1 hr at 4°C . After centrifugation , the beads were washed in NETN buffer ( 150 mm NaCl , 1 mm EDTA , 50 mm Tris-HCl ( pH 7 . 8 ) , 1% Nonidet P-40 , 1 mm phenylmethylsulfonyl fluoride , 0 . 5 µg/ml leupeptin , and 0 . 5 µg/ml pepstatin ) , then heated at 95°C for 10 min in Laemmli sample buffer . Following a brief centrifugation , the supernatant was immunblotted with the indicated antibodies as described above . Bacterial expression vectors encoding wild-type or C147A 3Cpro were constructed in pET-SUMO ( which encodes a linear fusion consisting of an N-terminal 6xHis tag for affinity purification followed by SUMO ) following PCR amplification according to the manufacturers protocol ( Invitrogen ) . pET-SUMO-3Cpro constructs were expressed in bacteria and purified by metal chelation resin columns ( Qiagen ) . The resulting 6xHis-SUMO 3Cpro fusion proteins were treated with SUMO protease to create untagged proteins as per the manufacturer's instructions ( Invitrogen ) and then dialyzed . For purification of MAVS and TRIF , 10 cm dishes of HEK2393 cells were transfected with Flag-MAVS or Flag-TRIF and lysed 48 hrs post-transfection . Lysates were purified over anti-Flag affinity gel columns , washed several times , and protein eluted by competition with five washes of 3x Flag peptide using the Flag M purification kit for mammalian expression systems ( Sigma-Aldrich ) . Eluted protein was quantified by BCA protein assay and verified for purity by SDS-PAGE and immunoblot analysis . Activation of the NFκB and IFNβ promoter was measured by reporter assay . Cells were transfected in 24-well plates with p-125 luc ( IFNβ ) or NFκB reporter plasmid together with the indicated plasmids . Luciferase activity was measured by the Dual-Luciferase assay kit ( Promega ) . All experiments were performed in triplicate and conducted a minimum of three times . To measure IFNβ production , the indicated cells were infected with either CVB or VSV and samples of culture supernatant removed at the indicated times . IFNβ levels in culture supernatant were determined by IFNβ ELISA according to the manufacturer's instructions ( PBL Biomedical Laboratories ) . Nuclear extracts were prepared from HEK293 cells after infection with CVB3 for 12 hr . Cells were washed in ice-cold PBS and isolated by incubation in 10 mM EDTA for 10 min . Cells were pelleted at 1000×g for 5 min , washed in ice-cold PBS , and incubated with buffer A ( 10 mM HEPES [pH 7 . 9] , 1 . 5 mM MgCl2 , 10 mM KCl , 0 . 5 mM DTT , 0 . 5 mM PMSF , and 0 . 1% NP-40 ) . The pellets were then resuspended in buffer B ( 20 mM HEPES [pH 7 . 9] , 25% glycerol , 0 . 42 M NaCl , 1 . 5 mM MgCl2 , 0 . 2 mM EDTA , 0 . 5 mM DTT , 0 . 5 mM PMSF , 5-µg/ml leupeptin , 5-µg/ml pepstatin , 5-µg/ml aprotinin ) . Samples were incubated on ice for 15 min before being centrifuged at 10 , 000×g . Nuclear extract supernatants were diluted with buffer C ( 20 mM HEPES [pH 7 . 9] , 20% glycerol , 0 . 2 mM EDTA , 50 mM KCl , 0 . 5 mM DTT , 0 . 5 mM PMSF ) . Data are presented as mean ± standard deviation . One-way analysis of variance ( ANOVA ) and Bonferroni's correction for multiple comparisons were used to determine statistical significance ( p<0 . 05 ) . ( numbers were taken from GenBank at Pubmed ) : mitochondrial antiviral signaling protein ( MAVS ) 57506; Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta ( TRIF ) 148022 , toll-like receptor 3 ( TLR3 ) 7098; Retinoic acid-inducible gene-I ( RIG-I ) 23586; melanoma-differentiation-associated gene 5 ( MDA5 ) 64135; interferon regulatory factor 3 ( IRF3 ) 3661 . | Mammalian cells utilize a variety of defenses to protect themselves from microbial pathogens . These defenses are initiated by families of receptors termed pattern recognition receptors ( PRRs ) and converge on the induction of molecules that function to suppress microbial infections . PRRs respond to essential components of microorganisms that are broadly expressed within classes of pathogens . The relative non-specificity of this detection thus allows for a rapid antimicrobial response to a variety of microorganisms . Coxsackievirus B3 ( CVB3 ) , a member of the enterovirus genus , is associated with a number of diverse syndromes including meningitis , febrile illness , diabetes , and is commonly associated with virus-induced heart disease in adults and children . Despite its significant impact on human health , there are no therapeutic interventions to treat CVB3 infections . Here we show that CVB3 has evolved an effective mechanism to suppress PRR signal propagation by utilizing a virally-encoded protein , termed 3Cpro , to directly degrade molecules that function downstream of PRR signaling . By targeting these molecules , CVB3 can evade host detection and escape antiviral defenses normally induced by mammalian cells . These findings will lead to a better understanding of the mechanisms employed by CVB3 to suppress host antiviral signaling and could lead to the development of therapeutic interventions aimed at modulating CVB3 pathogenesis . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [
"immunology/cellular",
"microbiology",
"and",
"pathogenesis",
"virology/host",
"antiviral",
"responses",
"virology/immune",
"evasion",
"immunology/innate",
"immunity"
] | 2011 | The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling |
Peptidylarginine deiminase IV ( PADI4 ) catalyzes the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline , and this activity has been linked to the repression of a limited number of target genes . To broaden our knowledge of the regulatory potential of PADI4 , we utilized chromatin immunoprecipitation coupled with promoter tiling array ( ChIP-chip ) analysis to more comprehensively investigate the range of PADI4 target genes across the genome in MCF-7 breast cancer cells . Results showed that PADI4 is enriched in gene promoter regions near transcription start sites ( TSSs ) ; and , surprisingly , this pattern of binding is primarily associated with actively transcribed genes . Computational analysis found potential binding sites for Elk-1 , a member of the ETS oncogene family , to be highly enriched around PADI4 binding sites; and coimmunoprecipitation analysis then confirmed that Elk-1 physically associates with PADI4 . To better understand how PADI4 may facilitate gene transactivation , we then show that PADI4 interacts with Elk-1 at the c-Fos promoter and that , following Epidermal Growth Factor ( EGF ) stimulation , PADI4 catalytic activity facilitates Elk-1 phosphorylation , histone H4 acetylation , and c-Fos transcriptional activation . These results define a novel role for PADI4 as a transcription factor co-activator .
The mitogen activated protein kinase/extracellular signal-related kinase ( MAPK/ERK ) pathway couples extracellular signals with a range of intracellular responses including cell growth , proliferation , and differentiation . An important target of MAPK/ERK activity is Elk-1 ( Ets-like protein-1 ) , a member of the ternary complex factors family of ETS domain transcription factors [1] . The ternary complex factors form a complex with serum response factor at the serum response element ( SRE ) and this cis-element is required for regulation of many immediate-early genes by stimulation with growth factors , such as EGF [2]–[6] . Elk-1 is activated by MAPK/ERK via phosphorylation of serine residues 383 and 389 within the transactivation domain [7] , [8] , leading to an enhanced association between Elk-1 and the histone actyltransferase ( HAT ) , p300 [9] . The activated Elk-1-p300 complex then induces a strong p300-mediated acetyltransferase activity on target genes leading to enhanced histone acetylation , chromatin remodelling , and gene activation [8]–[14] . The discovery that PADI4 is a nuclear enzyme which converts histone arginine residues to citrulline [15] provided the first evidence that PADI4 may play a role in gene regulation . This prediction was validated by the findings that PADI4 mediated citrullination of histone arginine residues ( both unmodified and monomethylated ) at the hormone dependent TFF1 promoter [16]–[18] , and at the apoptosis related gene promoters , p21 and OKL38 [19] , [20] , represses gene transcription . However , due to the limited number of target genes identified to date , our understanding of the gene regulatory potential of PADI4 remains far from complete . Therefore , to more fully elucidate the repertoire of PADI4 target genes , in this study we first carried out ChIP-chip analysis using a PADI4-specific antibody in MCF-7 cells . Surprisingly , in addition to the previously described repressive role for PADI4 in transcription , we found that PADI4 is also associated with a large set of actively transcribed genes and appears to function as a transcriptional coactivator of at least a subset of these genes . Computational analysis then found that our PADI4 ChIP-chip data set is strongly correlated with published data sets from activating , but not repressive , markers and that several common transcription factor binding sites are enriched around PADI4 peaks , with the V$ELK1_02 motif being the most significant element . We then demonstrate that PADI4 interacts with Elk-1 and that citrullination of Elk-1 by enzymatically active PADI4 facilitates ERK-mediated phosphorylation of Elk-1 in vitro . Further , we show that either suppression of PADI4 or inhibition of PADI4 activity leads to reduced Elk-1 phosphorylation and histone H4 acetylation on the c-Fos promoter . These mechanistic studies support the hypothesis that PADI4 enzymatic activity potentiates two key features of c-Fos transcriptional activation; Elk-1 phosphorylation and histone acetylation .
The PADI4 antibody utilized for this study was selected because the immunogenic peptide sequence ( N-terminal amino acids 1–15 ) is unique to PADI4 among the PADI family members ( Figure S1A ) . In our study , an overexpressed Flag-tagged version of PADI4 was readily detected in MCF-7 cell extracts by western blot using both the anti-PADI4 antibody and an anti-Flag antibody , indicating accuracy of PADI4 antibody targeting . Furthermore , this antibody recognized PADI4 in the lysates of MCF-7 cells that had been transfected with PADI4 expression vector , but not in cells transfected with any other PADI family members , confirming antibody specificity ( Figure S1B and S1C ) . Additionally as shown in Figure S2 , ChIP analysis found that the PADI4 antibody also bound to the previously reported PADI4 targets , OKL38 and p21 . To identify PADI4 targets in an unbiased genome-wide manner , we performed ChIP in MCF-7 cells using the PADI4-specific antibody and then hybridized the enriched genomic DNA to promoter tiling arrays which spanned 2 . 7 kb of the gene promoter regions ( 2 . 2 kb upstream and 500 bp downstream relative to the TSS ) . The array represented 51 Mbp of genomic DNA , including nearly all known well-characterized RefSeq genes ( 19206 promoters total ) . The raw ChIP-chip signal to input ratios were processed as described in Materials and Methods . Significant PADI4 peaks were defined as the center of three consecutive windows with positive means ( using at least six probes ) and the center window having a mean greater than either adjacent window . The P-values for all the peaks were calculated using the nonparametric Wilcoxon signed-rank test . A total of 1124 peaks of PADI4 genomic binding were identified using a threshold P-value of less than 0 . 016 ( Table S1 ) . The use of this selection criterion was validated by conventional ChIP-qPCR analyses on the randomly selected peaks from the array ( Figure S3 ) which showed a false positive rate ( FPR ) of less than 15% . As demonstrated by the strong correlation between the window averages from the ChIP-chip replicates ( Figure 1A ) , the ChIP-chip results were found to be highly reproducible . To match the identified peaks with all well-characterized RefSeq genes , we examined the distribution of PADI4 peaks throughout the whole promoter region and aligned the peaks to the TSSs for all the genes within the array . Surprisingly , our heat map analysis showed an apparent enrichment of PADI4 in the region surrounding the TSS ( Figure 1B ) . We next determined distribution of the distances of each significant PADI4 peak to the closest TSS and observed that the PADI4 peaks are statistically enriched around the TSSs ( Figure 1C ) , suggesting that PADI4 may be involved in transcription factor mediated gene expression . To explore the relationship between PADI4 and gene expression in more detail , we compared our PADI4 ChIP-chip data sets with gene expression microarray analyses from MCF-7 cells that had been grown under similar conditions ( Affymetrix expression arrays , U133A , GEO accession number GSE9253 ) . We first plotted the average signal intensities for PADI4 around the TSSs for all of the genes in the expression microarray following sorting of these genes into ten distinct expression level subsets by decile ( 1st being lowest 10% and 10th being highest 10% ) . As shown in Figure 2A , most genes with either low or no expression ( 1st decile ) are not bound by PADI4 . The average PADI4 ChIP-chip signal is then seen to increase around the TSS concordant with increasing levels of gene expression , with the 10th decile showing the highest level of PADI4 binding . We then grouped all of the expressed genes ( 5696 ) in the microarray and identified 516 genes ( 9 . 1% ) as having significant PADI4 peaks ( p<0 . 016 , Wilcoxon signed-rank test ) while only 77 ( 1 . 8% ) of the 4333 unexpressed genes showed significant PADI4 peaks , thus indicating a 5 . 1-fold enrichment of PADI4 at expressed genes ( Fisher's Exact Test , p<1×10−50 ) compared to unexpressed genes ( Figure 2B ) . This result supports a positive role of PADI4 in gene transcription . To gain further insight into the biological importance of the PADI4-bound genes , we determined the ontological gene categories by gene function analysis using DAVID ( See Functional classification and annotation of the candidate genes , Genomic data analyses ) . As shown in Table 1 and Table S2 , these genes encode proteins that are involved in a range of processes , with some of the most significant enrichment scores being for genes associated with membrane and organelle lumens , the nuclear lumen , protein catabolism , RNA processing , nucleotide binding , chromatin function , and ribosomal biogenesis . Next , we tested for a functional link between PADI4 and gene expression by evaluating the expression of a subset of PADI4-bound genes in both control MCF-7 cells and in stable short hairpin RNA ( shRNA ) -mediated PADI4 knockdown MCF-7 cells ( Figure 2C and 2D ) . For each gene , we tested both gene expression by reverse transcription ( RT ) -qPCR and PADI4 promoter binding by ChIP-qPCR . Genes with reduced expression in the PADI4 knockdown also showed decreased PADI4 promoter binding while the expression of the GAPDH housekeeping gene ( which is not bound by PADI4 at its promoter ) remained unchanged in the PADI4 knockdown . This analysis further confirms the hypothesis that PADI4 binding is well correlated with transcriptional activation . To further test the hypothesis that PADI4 promoter binding correlates with transcriptional activation , we next compared our PADI4 ChIP-chip dataset with multiple published ChIP-chip datasets for factors that are well correlated with gene regulation ( http://www . ncbi . nlm . nih . gov/geo ) [21] . The spearman correlation was determined between windows of the PADI4 ChIP-chip and identical windows within each of ChIP-chip data sets incorporated in the database . Volcano plots showed both statistically significant positive ( Figure 2E , on the right ) and negative ( Figure 2E , on the left ) correlations between the published ChIP-chip datasets and our PADI4 dataset . For example , as shown in Figure 2F , the PADI4 ChIP-chip output is significantly correlated with output from factors associated with gene activation such as the transcription factors E2F1 and RNAPII [22]–[25] , the chromatin modulating protein PARP-1 [26] and the histone varaiant H2A . Z [27] . On the other hand , the PADI4 ChIP-chip dataset is negatively correlated with datasets from factors associated with gene repression such as the H3me3K9 and H3me3K27 histone modifications and methylated DNA ( 5-meC ) [13] , [28] , [29] . Taken together , this computational analysis again suggests that PADI4 binding plays a role in the activation of a subset of actively transcribed genes . To identify specific transcription factors that might utilize PADI4 to mediate transcriptional output , we computationally explored the promoter regions of genes with significant PADI4 binding ( p<0 . 016 ) to identify putative DNA binding motifs within the bound regions ( See Enrichment of Transcription Factor Binding Sites [TFBS] Analysis , Genomic data analyses ) . The analysis identified a number of highly enriched DNA binding elements for transcription factors such as STAT1/3 , NFY , and E2F , suggesting the possible involvement of these cis-elements in the underlying regulatory control of PADI4 at target genes ( Figure 3A and Figure S4 ) . The transcription factor binding site showing the highest level of significance ( Fisher exact test , p = 5 . 56E-235 ) was V$ELK1_02 , which contains the consensus motif CCGGAA . This motif was originally identified as a binding site for the oncogene , Elk-1 , and has since been found to be bound by other ETS family members such as Ets1 and GABPα [30]–[32] . Interestingly , of the 516 gene promoters showing significant PADI4 binding , 70 genes ( 13 . 56% ) were found to also contain the V$ELK1_02 site adjacent to , or overlapping PADI4 peaks . Further , a ChIP-chip data set is publically available for Elk-1 [33] and a comparison of our PADI4 dataset with this data showed that 15 . 7% of PADI4 peaks are found within 1 . 5 kb of Elk-1 peaks ( Table 2 ) , suggesting that Elk-1 is significantly enriched near PADI4 peaks . This observation suggested that Elk-1 may utilize PADI4 as a cofactor to help mediate transcriptional activation . To begin testing this hypothesis experimentally , we performed independent PADI4 and Elk-1 ChIP experiments using identical promoter primers to test whether Elk-1 and PADI4 bound to the promoters of a subset of genes identified as having PADI4 peaks and the V$ELK1_02 element . Results show that , as predicted , both PADI4 and Elk-1 bound to the same promoter regions of this subset and not to the promoters of a subset of control genes identified as both PADI4 and V$ELK1_02 negative ( Figure 3B ) . To test if PADI4 and Elk-1 might coregulate the expression of this bound subset , we generated a stable Elk-1 knockdown MCF-7 cell line ( Figure 3B , bottom panel , inset ) and then evaluated the expression of the bound and unbound genes in the Elk-1 and PADI4 knockdown lines . We found that PADI4 or Elk-1 depletion in MCF-7 cells reduced the expression of the bound genes while expression of unbound genes ( shaded in gray ) remained unchanged ( Figure 3B ) . These results support the hypothesis that Elk-1 may utilize PADI4 as a co-factor to regulate gene expression . To further test this hypothesis , we next carried out co-immunoprecipitation analysis to establish whether Elk-1 and PADI4 interact . We first confirmed the specificity of the anti-Elk-1 antibody in HEK293 cells by depleting Elk-1 and showing that levels of the anti-Elk-1-reactive band were greatly reduced ( Figure S5 ) . Next , we transiently transfected Flag-tagged PADI4 into 293 cells and immunoprecipitated with anti-Flag antibody followed by western blot analysis using antibodies against both Elk-1 and phospho-Elk-1 ( p-Elk-1 ) . As shown in Figure 3C , a substantial amount of Elk-1 and p-Elk-1 was co-precipitated from cells expressing Flag-PADI4 , but not from cells lacking Flag-PADI4 . Additionally , we also found that levels of p-Elk-1 binding to PADI4 increased following stimulation with EGF ( Figure 3C ) . Reciprocally , we also found that the endogenous Elk-1 proteins were immunoprecipitated with anti-Elk-1 antibody , and the co-precipitated PADI4 proteins were subsequently detected using the anti-PADI4 antibody , while PADI4 was not coimmunoprecipitated with the normal rabbit IgG control ( Figure 3C ) . These results indicated that both Elk-1 and the active form of Elk-1 can physically associate with PADI4 . The interaction of PADI4 and phosphorylated Elk-1 supports the hypothesis that PADI4 may play a direct role in facilitating Elk-1 target gene activation . Perhaps the best-characterized Elk-1 target is the immediate early oncogene c-Fos . The SRE cis element that mediates c-Fos transcriptional activation contains the Elk-1 binding element and is recognized by the Elk-1-containing ternary complex factor [34] . This observation prompted us to examine whether the location of PADI4 on the c-Fos promoter ( as determined by ChIP-chip ) is near the SRE region . Interestingly , PADI4 binding was found to primarily occur in the proximal promoter region of c-Fos , covering the SRE motif ( Figure 3D and 3E ) . This result implied that PADI4 and Elk-1 may also target the same promoter region of c-Fos . Therefore , we further characterized this interaction on the tiled regions of the c-Fos promoter by performing PADI4 , Elk-1 and p-Elk-1 ChIPs in MCF-7 cells ( Figure 3E ) . In line with our hypothesis , we found endogenous PADI4 to be enriched specifically around the SRE motif on the c-Fos promoter but not on promoter regions more removed from the TSS ( ∼500 bp upstream of the SRE or downstream of the TSS ) . We also observed a near perfect overlap between PADI4 , p-Elk-1 , and Elk-1 at the c-Fos promoter by ChIP q-PCR . Given these observations , and that PADI4 interacts with Elk-1 in 293 cells , we also carried out ChIP-re-ChIP analysis using anti-PADI4 antibody for the first round of IP and then anti-Elk-1 for the second round of IP . Results showed that endogenous PADI4 appears to physically associate with Elk-1 at the c-Fos SRE region ( Figure 3E , bottom ) . Given these observations , we next decided to more closely investigate the role of PADI4 in c-Fos regulation to gain mechanistic insight into PADI4's role in transcriptional activation . EGF can activate c-Fos expression in MCF-7 cells [35] and this response is accompanied by activation of Elk-1 via MAPK/ERK phosphorylation , by which the SRE cis element that mediates c-Fos transcriptional activation is recognized [8] , [10] , [11] . Given our finding that PADI4 and active Elk-1 strongly co-occupy the c-Fos promoter , we hypothesized that c-Fos expression may be regulated , in part , by the interplay between PADI4 and Elk-1 . To address this question , we first tested the effects of inhibition of either Elk-1 or PADI4 activity on c-Fos expression following EGF stimulation . We inhibited PADI4 enzymatic activity using a newly developed arginine-based PADI inhibitor , Cl-Amidine [36] , which has been shown to inhibit PADI4 activity at gene promoters in several cancer cell lines [19] , [20] . As shown in Figure 4A , stimulation of MCF-7 cells with EGF ( 50 ng/ml ) for 30 min elicited a dramatic increase of c-Fos mRNA expression . As expected , pretreatment of MCF-7 cells with the ERK inhibitor U0126 abrogated c-Fos activation following EGF stimulation . Interestingly , Cl-Amidine also strongly inhibited c-Fos activation by EGF ( Figure 4A ) and this inhibition was found to be dose-dependent ( Figure S6 ) . We also observed by western blotting that Cl-Amidine globally suppresses Elk-1 phosphorylation in a dose-dependent manner , while having little effect on ERK phosphorylation ( Figure 4B ) . These observations suggests that the effect of Cl-Amidine on c-Fos transcription occurs downstream of ERK kinase , likely at the level of Elk-1 phosphorylation . Note: Given the strong suppressive effect of U0126 in c-Fos expression , we were not able to quantitatively test for possible synergy between this drug and Cl-Amidine . To control for non-specific effects of the drugs on transcription , levels of GAPDH housekeeping gene were also monitored and neither inhibitor altered the expression of this gene . As another test of the collaborative role of PADI4 and Elk-1 in c-Fos activation , we investigated c-Fos expression following EGF stimulation in the Elk-1 and PADI4 knockdown lines . As predicted , depletion of either PADI4 or Elk-1 reduced c-Fos expression upon EGF stimulation ( Figure 4C and 4D ) . We then generated a stable PADI4-overexpressing line and found c-Fos expression to be elevated in this line in both un-stimulated and EGF stimulated conditions ( Figure 4E ) . We also tested whether the observed inhibitory effect of Cl-Amidine on c-Fos transcription is preserved in other breast cancer cell lines by treating BT474 ( ER+ , HER2+ ) and MCF10A-DCIS ( ER− , HER2+ ) cells with the inhibitor . Results showed that Cl-Amidine treatment inhibits EGF-induced c-Fos expression in both of these lines , with the strongest inhibitory effect being observed in the BT474 line ( Figure S7 ) . We next investigated whether PADI4 and Elk-1 might coregulate gene activation on a more generalized template in vitro . For this experiment , we fused full length Elk-1 to the GAL4 DNA-binding domain , and transfected this construct alone or with either WT PADI4 or the PADI4 catalytic mutant ( CS ) into 293 cells . We then tested the effects of these constructs on GAL4-driven luciferase reporter activity . Results showed that while GAL4-Elk-1 did not activate transcription over GAL4 alone , cotransfection of WT PADI4 with Elk-1 resulted in enhanced luciferase activity ( Figure 4F ) . PADI4 enzymatic activity appears to be required for reporter activity because cotransfection of GAL4-Elk-1 with inactive PADI4 did not increase reporter activity above that of Elk-1 alone . We also carried out luciferase reporter experiments using a c-Fos-luciferase reporter system and obtained similar results ( Figure S8 ) . Collectively , the above data support the hypothesis that a functional relationship between Elk-1 and PADI4 on gene promoters potentiates Elk-1 mediated gene transcription . To further test this hypothesis and to investigate whether Elk-1 recruits PADI4 to its target promoters ( or possibly vice versa ) , we then carried out ChIP-qPCR analysis with PADI4 and Elk-1 antibodies in serum-starved MCF-7 cells , in cells treated with EGF , and in EGF-stimulated cells that had been pretreated with inhibitors . Results showed that binding of PADI4 and Elk-1 at the c-Fos SRE promoter region was independent of EGF , U0126 or Cl-Amidine treatment ( Figure 5A ) . Additionally , we found that PADI4 occupancy was decreased at the c-Fos SRE region upon PADI4 knockdown , while depletion of PADI4 did not change Elk-1 occupancy at c-Fos ( Figure 5B ) . On the other hand , depletion of Elk-1 not only caused a decrease in Elk-1 at the c-Fos promoter but also reduced PADI4 enrichment at this site ( Figure 5C ) . The binding of Elk-1 at c-Fos promoter before and after EGF stimulation is consistent with previous reports showing that Elk-1 constitutively binds to c-Fos promoter [9] , [37] . Given that PADI4 has no distinguishable DNA binding motif and that PADI4 has been found to be targeted to specific gene promoters by transcription factors such as p53 [19] , our results suggest that PADI4 recruitment to the c-Fos promoter is Elk-1-dependent . In order to further test how PADI4 regulates c-Fos activity , we next tested the effects of PADI4 inhibition or depletion on Elk-1 phosphorylation and on histone H4K5 acetylation ( Ac-H4K5 ) at the c-Fos SRE following EGF stimulation in MCF-7 cells ( Figure 5 ) . Results showed that EGF treatment resulted in a strong enrichment of p-Elk-1 and Ac-H4K5 at the c-Fos promoter ( Figure 5A ) and that this enrichment was abrogated by pretreatment with the PADI4 inhibitor ( Figure 5A ) or by PADI4 depletion ( Figure 5B ) . Additionally , as expected , both ERK inhibition ( U0126 , Figure 5A ) and Elk-1 depletion ( Figure 5C ) also reduced p-Elk-1 and Ac-H4K5 enrichment at c-Fos promoter following EGF stimulation . The findings that PADI4 interacts with Elk-1 on c-Fos and that PADI4 inhibition suppressed phosphorylation of Elk-1 ( but not ERK ) suggested that PADI4 may regulate c-Fos activation by directly targeting Elk-1 for citrullination . To test the hypothesis , we first investigated whether PADI4 can citrullinate Elk-1 in vitro . Endogenous Elk-1 was immunoprecipitated from MCF-7 cells , treated with recombinant PADI4 ( WT or CS ) , and the resolved proteins were then probed with an antibody that is reactive with citrullinated proteins ( anti-pan-cit , [38] ) . Results ( Figure 6A ) show that the anti-pan-cit antibody was reactive with an appropriately sized band from the anti-Elk-1 immunoprecipitate ( lane 4 ) and was not reactive with proteins from the anti-Elk-1 immunoprecipitate ( lane 3 ) that had been treated with the PADI4 C645S mutant or from proteins immunoprecipitated with IgG ( lanes 1 and 2 ) . Anti-Elk-1 and anti-PADI4 western blots confirmed equal protein loading . These results support the hypothesis that Elk-1 is a target for PADI4 mediated citrullination . Transcriptional activation of c-Fos is initiated following phosphorylation of Elk-1 by ERK kinase . The above findings raised the possibility that citrullination of Elk-1 by PADI4 may facilitate ERK-mediated phosphorylation of Elk-1 . To test this hypothesis , we then performed ERK kinase assays on in vitro translated WT or inactive mutant ( DN ) Elk-1 following pretreatment with either WT or catalytically inactive ( CS ) PADI4 . As shown in Figure 6B , in the presence of activated ERK2 , higher levels of Elk-1 Ser383 phosphorylation were observed following pretreatment of Elk-1 with WT PADI4 ( lane 5 ) when compared to pretreatment with inactive PADI4 ( lane 6 ) . A similar decrease in Elk-1 Ser383 phosphorylation levels was observed with the ERK2 isolated from the non-EGF stimulated JEG3 cells following treatment of Elk-1 with mutant PADI4 , though the overall signal intensities were much lower ( lanes , 1 and 2 ) . As expected , phosphorylation of DN Elk-1 was below the limit of detection ( lanes 3 , 4 , 7 and 8 ) , thus confirming anti-p-Elk-1 antibody specificity . Total Elk-1 levels were also evaluated with anti-Elk-1 antibody to normalize for protein loading . Additionally , the blots were also probed with anti-phosphorylated ERK antibody to confirm that EGF treatment of JEG3 cells activated ERK2 and with anti-ERK2 antibody to further confirm equal protein loading . Collectively , these results support the hypothesis that PADI4 mediated citrullination facilitates phosphorylation of Elk-1 by ERK .
Given previous findings indicating that PADI4 plays a repressive role in gene transcription , we were initially surprised by our global analysis showing that PADI4 binding is primarily correlated with active gene transcription . However , the prediction that PADI4 also appears to play an important role in the activation of a broad range of target genes is supported by the following new lines of evidence . Firstly , we found that the PADI4 binding pattern near the TSSs is highly correlated with actively transcribed genes in MCF-7 cells and that most genes not expressed in the array are not bound by PADI4 . Secondly , our computational ChIP-chip dataset analysis found that PADI4 is highly positively correlated with datasets from factors associated with gene activation and negatively correlated with datasets from factors associated with gene repression . Lastly , our computational analysis of transcription factor binding sites found that PADI4 is significantly enriched on promoters which contain DNA binding elements for a broad range of activating transcription factors . Interestingly , several of the elements identified in our computational analysis are bound by transcription factors such as STAT1 , STAT3 , STAT5 , ETS1 , PAX and E2F , which play important roles in mammary function and in breast cancer [39]–[44] . Thus , the finding that PADI4 binding is enriched near these elements in MCF-7 cells supports the hypothesis that PADI4 plays a role in regulating target gene expression in a range of mammary gland signaling pathways . Given that PADI4 binding was frequently coincident with the Elk-1 DNA binding element , and that PADI4 interacts with Elk-1 , we next focused on investigating the potential relationship between PADI4 and Elk-1 on the c-Fos promoter , in order to better understand how PADI4 may activate gene transcription . Our findings that endogenous c-Fos levels were suppressed in PADI4 depleted and inhibitor-treated lines and elevated in PADI4 overexpressing cells , supported our genome-wide finding that PADI4 plays a role in transcriptional activation . Next , the observations that PADI4 interacts with Elk-1 at c-Fos and that PADI4 facilitates Elk-1-mediated activation of both a ubiquitous , and c-Fos-specific , reporter systems suggested that a functional relationship between Elk-1 and PADI4 confers activation of c-Fos . As to potential mechanisms behind this functional relationship , we found that PADI4 can citrullinate Elk-1 in vitro and that PADI4 enzymatic activity facilitates Elk-1 phosphorylation on c-Fos . Our ERK kinase assay then demonstrated that citrullination of Elk-1 by PADI4 enhances ERK-mediated Elk-1 phosphorylation . Finally , the observation that histone H4K5 acetylation was suppressed at c-Fos following PADI4 inhibition or depletion suggested that the observed PADI4 enzymatic activity on Elk-1 phosphorylation at c-Fos then facilitated histone acetylation and subsequent c-Fos activation . The potential role for PADI4 in c-Fos activation is partially clarified when the above findings are put in the context of what is currently known about the role of Elk-1 in this process . Recent reports have demonstrated that unphosphorylated Elk-1 and p300 form complexes and bind to gene promoters in the absence of stimuli . Following stimulation , Elk-1 is then phosphorylated by ERKs and this phosphorylation event strengthens the interaction between Elk-1 and p300 , leading to enhanced histone acetyltransferase activity at target promoters and gene activation [9] , [14] . In addition to observed Elk-1/p300 interactions , p300 has also been found to be targeted by PADI4 for citrullination [45] . Given these observations , and our findings that PADI4 interacts with Elk-1 and that Elk-1 depletion reduces PADI4 levels at c-Fos , it seems likely that Elk-1 , p300 , and PADI4 are constitutively bound to the c-Fos promoter ( Figure 7 ) . Our current working model then predicts that treatment of MCF-7 cells with EGF then activates PADI4 , which , in turn , citrullinates Elk-1 . Upon citrullination , Elk-1 is then phosphorylated by ERK2 leading to stronger associations between Elk-1 and p300 , increased histone acetylation , and subsequent transcriptional activation [4] , [8] , [10] , [46] . Given that citrullination neutralizes positively charged arginine residues , it seems possible that PADI4-mediated citrullination facilitates ERK-mediated phosphorylation of Elk-1 by allowing this kinase closer access to its target serine residues . As an alternate hypothesis to our working model , Lee et al . [45] has found that PADI4 can also directly citrullinate p300 to enhance p300 activity . Thus , it is possible that a direct functional relationship exist between PADI4 and p300 on c-Fos . However , we found that treatment of p300 with PADI4 did not enhance p300's histone acetyltransferase activity in vitro ( data not shown ) . Additionally , we do not believe that PADI4 is mediating c-Fos activity via histone citrullination as we were not able to detect significant increases of citrullinated histones ( H3 or H4 ) on the c-Fos promoter after EGF stimulation ( data not shown ) . Given these observations , and our findings presented here , the most likely mechanism by which PADI4 is regulating c-Fos is by targeting Elk-1 for citrullination . In conclusion , our genomic analysis has identified a new role for PADI4 in mediating gene transactivation . We present evidence that a broad range of transcription factors may utilize PADI4 as a cofactor to mediate their transactivation functions , thus suggesting that the gene regulatory role for PADI4 is much broader than previously thought . We also provide new mechanistic insights into how PADI4 mediates gene transactivation by showing that PADI4 interacts with and citrullinates Elk-1 and that PADI4 enzymatic activity potentiates two key features of c-Fos activation , Elk-1 phosphorylation and histone H4 acetylation . Studies are currently underway to further dissect the mechanism by which PADI4 regulates gene expression and possibly mammary tumorigenesis .
MCF-7 cells were maintained in DMEM supplemented with 5% calf serum and both HEK293 and JEG3 cells were maintained in DMEM supplemented with 10% fetal bovine serum . Elk-1-depleted MCF-7 and 293 cell lines were generated by transduction with Mission Lentiviral Transduction Particles containing a short hairpin RNA ( shRNA ) construct targeting the human Elk-1 coding sequence ( Sigma SHCLNV-NM_005229 ) . In the control group , cells were transduced with a non-targeting shRNA lentiviral construct ( Sigma SHC002V ) . Cells were selected by medium containing 1 µg/ml puromycin ( Sigma ) . PADI4-depleted MCF-7 cells were generated by transfection with Mission shRNA Plasmid DNA targeting the human PADI4 coding sequence ( Sigma SHCLND-NM_012387 ) . Control cells harboring a non-targeting shRNA lentiviral construct ( Sigma SHC002V ) were generated in parallel . PADI4 overexpression MCF-7 cells were generated by transfection with PADI4-pcDNA3 . 1 ( + ) and selected by medium containing 500 µg/ml G418 ( Gibco ) . Where indicated , Cl-Amidine was diluted in cell culture medium at the final concentration of 200 µM and added to cells for 40 hr prior to harvest . Where indicated , cells were serum starved for 12 hr ( expression analysis ) or 2 hr ( ChIP assay ) and subsequently stimulated with 50 ng/ml EGF for 30 min . When required , cells were pre-treated with MAP kinase inhibitor U0126 ( 10 µM ) for 30 min before EGF stimulation . ChIP was performed essentially as described previously [26] . MCF-7 cells were grown to ∼80 to 90% confluence , cross-linked with 1% paraformaldehyde for 10 min at 37°C , and quenched in 125 mM glycine . Cell lysates were sonicated under conditions yielding fragments ranging from 200 bp to 800 bp . The material was clarified by centrifugation , diluted 10-fold in dilution buffer , and pre-cleared with protein A-agarose beads . The pre-cleared , chromatin-containing supernatant was used in immunoprecipitation reactions with antibodies against PADI4 ( Sigma P4749 ) , Elk-1 ( Santa Cruz sc-22804 ) , p-Elk-1 ( Santa Cruz sc-135646 ) , Ac-H4K5 ( Upstate 07-327 ) , normal rabbit IgG ( Upstate 12-370 ) or without antibodies as a control . Ten percent of the supernatant was saved as reference control . Both the immunoprecipitated genomic DNA and the control DNA were cleared of protein and residual RNA by digestion with proteinase K and RNase ( Roche ) , respectively . The DNA was then extracted with phenol∶chloroform∶isoamyl alcohol and precipitated with ethanol . For gene-specific ChIP analyses , quantitative real-time PCR ( qPCR ) was used to determine the enrichment of immunoprecipitated DNA relative to the input DNA using gene-specific primer sets to the specified regions . Each ChIP experiment was conducted a minimum of three times with independent chromatin isolates to ensure reproducibility . For the ChIP-chip analyses , ChIP was performed in MCF-7 cells stripped in MEM plus 5% charcoal-dextran-treated calf serum for 72 hrs . LM-PCR-amplified immunoprecipitated DNA and the reference DNA were hybridized to a human HG18 RefSeq promoter microarray from Nimblegen . The array contained all known well-characterized RefSeq gene promoters . The promoter region on this array was covered by 50–75 mer probes with a mean probe spacing of 100 bp . The detailed information is available on Nimblegen's website . The genomic data analyses were performed using the statistical programming language R [47] essentially as described previously [21] . All data processing scripts are available upon request . The log2 ratio data from each array was subjected to lowess normalization [48] . The normalized data were scaled to equivalent sum of squares and then the between array mean log2 ratio was determined for each probe . An error model was generated using a 1 kb moving window with 250 bp steps in which both the mean probe log2 ratio and p-values were calculated for each window . The p-values were calculated using the nonparametric Wilcoxon signed-rank test . The TSS-anchored ChIP-chip heat maps were generated using 600 pb windows with 150 bp steps and were visualized ( Figure 1B ) with Java Treeview [49] . For genes with multiple TSSs , the most 5′ site was used . The raw and processed data from the ChIP-chip data described in this manuscript can be found on the GEO website ( http://www . ncbi . nlm . nih . gov/geo/ ) using accession number GSE18755 . Total RNA was isolated from MCF-7 cells using Qiagen RNeasy Mini Kit in combination with on-column DNase treatment ( ABI ) . High Capacity RNA-to-cDNA kit ( ABI ) was used to synthesize the first strand of cDNA . Quantitative real-time PCR ( qPCR ) was performed using Power SYBR Green PCR Master Mix ( ABI ) with gene-specific primers . All target gene transcripts were normalized to β-actin . PADI4 transcription in PADI4-overexpressed MCF-7 cells was assessed using TaqMan Gene Expression Master Mix ( ABI ) with Taqman probes and primers ( PADI4: Hs00202612_m1 and GAPDH: 4352934E , ABI ) . Each experiment was conducted three times with independent isolates of total RNA . The P-values were determined using a two-tailed paired Student's t-test . All of the primers used for the ChIP-qPCR and RT-qPCR assays were listed in Table S3 . PADI4-depleted and parallel control MCF-7 cells ( 90% confluence in 10 cm culture plates ) were collected , washed with phosphate-buffered saline ( PBS ) , and resuspended in 500 µl of buffer A [10 mM HEPES ( pH 7 . 9 ) , 10 mM KCl , 1 . 5 mM MgCl2 , 0 . 34 M sucrose , 10% glycerol , 1 mM dithiothreitol , and protease inhibitor cocktail ( Roche ) ] . Triton X-100 was added ( 0 . 1% final concentration ) , the cells were incubated on ice for 8 min , and nuclei were collected by centrifugation ( 5 min , 1 , 300× g , 4°C ) . The nuclei were washed once in buffer A and lysed for 30 min in buffer B ( 3 mM EDTA , 0 . 2 mM EGTA , 1 mM dithiothreitol , and protease inhibitor cocktail ) , and insoluble chromatin and soluble fractions were separated by centrifugation ( 5 min , 1 , 700× g , 4°C ) . The chromatin fraction was washed once with buffer B and resuspended in sodium dodecyl sulfate ( SDS ) -Laemmli buffer ( and boiled for 10 min ) for Western Blot analysis . Flag-tagged PADI4 in pcDNA3 . 1 ( + ) or control vector were transfected into 293 cells using FuGENE 6 . The whole cell lysates were immunoprecipitated with anti-Flag M2 affinity gel ( Sigma A2220 ) or immunoprecipitated with anti-Elk-1 antibody . Immunoprecipitates were washed and analysed by western blot using anti-Elk-1 , anti-phospho-Elk-1 , and anti-PADI4 antibodies as indicated . HEK293 cells were cotransfected using FuGENE 6 ( Roche ) with 0 . 05 µg of c-Fos-luciferase construct containing the SRE sequence as described previously [55] , 0 . 25 µg of PADI4 ( wild-type [WT] or enzymatically inactive mutant , mutation of Cys645 into Ser645 , [CS] ) in pcDNA3 . 1 ( + ) , and 0 . 25 µg of Elk-1 ( wild-type [WT] or dominant-negative mutant , mutations of Ser383/Ser389 into Ala383/Ala389 , [DN] ) in pSG5 expression vectors [46] . For GAL4-driven luciferase assay , expression vector for the DNA-binding domain of GAL4 ( CMV-GAL4 ) or GAL4-Elk-1 ( full length ) was transfected into HEK293 cells with 5xGAL4-driven luciferase reporter plasmid containing the minimal E1b promoter and plasmids for pcDNA3 . 1 , PADI4 WT and C645S mutant . Lysates were assayed for luciferase activities 24 hours after transfection according to the supplier's protocol ( Dual Luciferase , Promega ) and normalized to protein concentration in the extract . PADI4 proteins were expressed and purified from pET28b-PADI4 or pET28b-PADI4 ( C645S ) mutant using Ni-NTA Protein Purification System ( Qiagen ) according to manufacturer's instructions . The PADI assay was performed essentially as described previously [17] . The expressed Elk-1 was treated with PADI4 in PADI buffer containing 50 mM Tris . HCl , pH 7 . 6 , 4 mM DTT , 4 mM CaCl2 at 37°C for 1 hr . The mutant PADI4 ( CS ) was used as the negative control . Coupled in vitro transcription/translations were performed from Elk-1 constructs using the TNT-coupled reticulocyte lysate system ( Promega ) with T7 RNA polymerase according to the instructions of the manufacturer . Both wild-type ( WT ) and dominant-negative ( DN ) Elk-1 cDNAs are in pSG5 expression vectors [46] . The In vitro ERK kinase assay was performed as described previously [55] . Briefly , after EGF treatment , JEG3 cells ( The cell line is highly sensitive to EGF and has been shown to be good source for activated ERKs [56] ) were washed in 1 ml of ice-cold PBS and lysed in radioimmune precipitation buffer containing 20 mM Tris ( pH 8 . 0 ) , 137 mM NaCl , 10% glycerol , 1% Nonidet P-40 , 0 . 1% SDS , 0 . 5% deoxycholate , 2 mM EDTA , 5 mM sodium vanadate , 5 mM benzamidine , and 1 mM PMSF on ice for 10 min . The cell lysates were collected and cleared by centrifugation . ERK2 was immunoprecipitated by adding anti-ERK2 antibody ( Santa Cruz sc-154 ) and 25 µl of protein A/G-agarose beads to clarified cell lysates . The beads were washed once in 1 ml of radioimmune precipitation buffer; twice in 1 ml of ice-cold Nonidet P-40 wash buffer; and once in 1 ml of kinase buffer containing 20 mM HEPES ( pH 7 . 5 ) , 20 mM MgCl2 , 25 mM β-glycerol phosphate , 100 µM sodium vanadate , 20 µMATP , and 2 mM dithiothreitol . The reaction mixture ( 50 µl ) contained the agarose beads suspended in kinase buffer , and substrate Elk-1 pretreated with PADI4 . Samples were incubated for 30 min at 30°C with frequent mixing and the reaction was terminated with the addition of SDS loading buffer and boiled for 2 min , and analysed by western blot using anti-phospho-Elk-1 ( p-Elk-1 ) , anti-Elk-1 , anti-phospho-ERK ( p-ERK ) ( Cell Signalling E10 ) , and anti-ERK2 antibodies as indicated . | Peptidylarginine deiminase 4 ( PADI4 ) converts positively charged arginine and methylarginine residues on histones to the neutrally charged non-standard amino acid citrulline . We and others have previously shown that citrullination of a small subset of gene promoters , such as the estrogen receptor target , TFF1 , appears to downregulate gene expression . In this study , we looked across the human genome using ChIP-chip to better define the full repertoire of genes that are regulated by PADI4 in breast cancer cells . Surprisingly , we found that PADI4 appears to primarily be involved in gene activation , as opposed to gene repression . Further , we found that PADI4 is likely recruited as a co-activator to these target genes by a range of well-defined transcription factors , such as Elk-1 . With respect to how PADI4 activates gene transcription , we show that PADI4 directly interacts with Elk-1 at its well-defined target , the c-Fos oncogene . Additionally , we found that , following stimulation with epidermal growth factor , PADI4 appears to directly target Elk-1 for citrullination , which in turn leads to increased histone acetylation and gene transcription . These novel genome-wide and gene-specific findings suggest that PADI4 plays a much broader role in gene activation than previously thought . | [
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] | 2011 | Genome-Wide Analysis Reveals PADI4 Cooperates with Elk-1 to Activate c-Fos Expression in Breast Cancer Cells |
Plasmodium falciparum malaria parasites , living in red blood cells , express proteins of the erythrocyte membrane protein-1 ( PfEMP1 ) family on the red blood cell surface . The binding of PfEMP1 molecules to human cell surface receptors mediates the adherence of infected red blood cells to human tissues . The sequences of the 60 PfEMP1 genes in each parasite genome vary greatly from parasite to parasite , yet the variant PfEMP1 proteins maintain receptor binding . Almost all parasites isolated directly from patients bind the human CD36 receptor . Of the several kinds of highly polymorphic cysteine-rich interdomain region ( CIDR ) domains classified by sequence , only the CIDR1α domains bind CD36 . Here we describe the CD36-binding portion of a CIDR1α domain , MC179 , as a bundle of three α-helices that are connected by a loop and three additional helices . The MC179 structure , containing seven conserved cysteines and 10 conserved hydrophobic residues , predicts similar structures for the hundreds of CIDR sequences from the many genome sequences now known . Comparison of MC179 with the CIDR domains in the genome of the P . falciparum 3D7 strain provides insights into CIDR domain structure . The CIDR1α three-helix bundle exhibits less than 20% sequence identity with the three-helix bundles of Duffy-binding like ( DBL ) domains , but the two kinds of bundles are almost identical . Despite the enormous diversity of PfEMP1 sequences , the CIDR1α and DBL protein structures , taken together , predict that a PfEMP1 molecule is a polymer of three-helix bundles elaborated by a variety of connecting helices and loops . From the structures also comes the insight that DBL1α domains are approximately 100 residues larger and that CIDR1α domains are approximately 100 residues smaller than sequence alignments predict . This new understanding of PfEMP1 structure will allow the use of better-defined PfEMP1 domains for functional studies , for the design of candidate vaccines , and for understanding the molecular basis of cytoadherence .
Cycles of exponential parasite growth inside infected red blood cells ( iRBCs ) , followed by lysis , and immediate re-invasion of uninfected RBCs are the hallmarks of malaria caused by P . falciparum . Though seemingly hidden from the human immune system inside the iRBC , P . falciparum expresses a number of proteins on the iRBC surface , extensively remodeling the iRBC , and exposing parasite antigens to the immune system ( for reviews [1]–[6] ) . Major antigens on the iRBC surface include members of the P . falciparum erythrocyte membrane protein-1 ( PfEMP1 ) family , highly polymorphic and modular proteins composed of DBL ( Duffy binding-like ) and CIDR ( cysteine-rich interdomain region ) domains [7] . On each iRBC , a single PfEMP1 is expressed from one of ∼sixty var genes [8] , [9] in the parasite genome . A different var gene can be activated during each cycle of infection , resulting in a new surface PfEMP1 molecule , and the parasite's evasion of antibodies against the previously expressed PfEMP1 [10] . Var genes undergo frequent recombination and gene conversion giving rise in the parasite population to a vast number of variant PfEMP1 proteins [11] that are under immune system selective pressure . PfEMP1 molecules are thought to be constrained in sequence only to maintain binding to host receptors and thus to mediate cytoadherence [12] . Cytoadherence prevents iRBCs from passing through the spleen and thus being cleared from the circulation [13] . Adherence of iRBCs to uninfected RBC ( known as rosetting ) and adherence to the microvasculature ( known as sequestration ) are thought to be primary causes of morbidity , especially in cerebral and placental malaria . iRBCs adhere due to the binding of PfEMP1 to a variety of endothelial receptors among which are CD36 , intercellular adhesion molecule-1 ( ICAM-1 ) , and chondroitin sulfate A ( CSA ) . Almost all parasites isolated directly from patients have the capacity to bind to CD36 [14] . iRBC appear to modulate immune system responses by their binding to CD36 and to other receptors on immune cells in the blood [15]–[17] . With the availability of genomic sequences from several P . falciparum strains , the numbers of DBL and CIDR domains and the extents of their diversity are becoming better known [18] , [19] . Within the ∼sixty PfEMP1 sequences of the 3D7 strain of P . falciparum , there are 130 DBL and 110 CIDR domains [8] , [20] . Though sequence identities among DBL and among CIDR domains are low , short blocks of conserved residues enable the classification of domains into families [21] . PfEMP1 domain families have been named DBLα , DBLβ , DBLγ , DBLδ , DBLε and CIDRα , CIDRβ , CIDRγ [18] and are also consistent with the groupings that arise from sequence similarities as performed by ClustalW [19] . Family groupings are used together with the position of the domain within its particular PfEMP1 to yield domain designations such as DBL1α , CIDR1α , CIDR2β , and so on . As an example , Figure 1A depicts the PfEMP1 molecule from the Malayan Camp ( MC ) strain of P . falciparum that consists of four DBL and two CIDR domains [22] . Correlations of domain families with specific ligand binding have been determined [23] , [24] . In the case of the CIDR families , many domains have been tested for their binding to CD36 . From these experiments , only CIDR1α domains bind CD36; other CIDR domains , even the closely related CIDR1α1 domains , do not [21] , [25] , [26] . Binding to CD36 by RBCs infected with the MC strain of P . falciparum has been studied [27] , [28] . Experiments using labeled PfEMP1 proteins showed that a proteolytically-cleaved portion of PfEMP1 is capable of binding to CD36 . Antibodies against a recombinant protein corresponding to residues 576 to 808 of the CIDR1α domain of MC PfEMP1 block adherence of iRBCs to CD36 [29] . Further analysis of recombinant portions of the MC PfEMP1 molecule identified a 179-residue polypeptide , MC179 ( residues 576 to 754 ) , as a minimal portion of the MC CIDR1α domain that retained binding to CD36 [30] . Recombinant MC179 competes with iRBCs for binding to CD36 under blood flow conditions , resulting in the release of bound RBCs [31] , [32] . CIDR1α domains have been divided into three subdomains: M1 , M2 , and M3 [33] . MC179 corresponds to the M2 subdomain and M1 and M3 are the respective N-terminal and C-terminal portions of the CIDR1α domain . Vaccination with MC179 protects Aotus monkeys from severe infection upon challenge with the MC strain [34] and prevents severe anemia after infection with a heterologous strain [35] . Three-dimensional structures of DBL , but not CIDR domains , have been available , with sequence comparisons revealing a number of conserved hydrophobic residues and cysteines , yet with low overall sequence identities among PfEMP1 domains [7] . We present the structure and analysis of MC179 in the context of the broad sequence diversity of the CIDR domains of the P . falciparum genomes and indicate the insights into overall DBL and PfEMP1 structure that MC179 provides .
MC179 is composed of a bundle of three helices connected by loops and three additional helices ( Figure 1 ) . The first helix ( H1 ) of the bundle is joined to the second helix ( H2 ) through a disordered fourteen-residue loop . At the end of the H2 helix there are seventy residues in three smaller helices , a , b , and c , that form the connection to the third helix ( H3 ) . The “V-shape” of MC179 is made up of the three-helix bundle and the three helices between the H2 and H3 helices , in the order H1-H2-a-b-c-H3 . The H1 and H2 helices make many contacts with each other and are linked by a conserved disulfide bond , Cys 32-Cys 45 , that was not visible in the electron density , but inferred to be present . The H2 and H3 helices also make many contacts with each other and are linked by a conserved disulfide bond , Cys 49-Cys 159 , that is observed in the electron density . The H1 and H3 helices , however , make only a few contacts within 4 Å distance and are not linked by a disulfide . The cysteine-rich area from which the domain gets its name is located near the end of the bundle where three disulfide bonds and one free cysteine are located ( Figure 1D ) . Using mammalian cells transfected to express human CD36 , we confirmed by flow cytometry that the MC179 portion of the CIDR1α domain that had been crystallized was capable of binding CD36 . Adding MC179 to cells expressing CD36 revealed robust binding of MC179 ( Figure 2A and 2B , blue hatched peaks ) . Preincubation of MC179 with soluble human CD36 produced in insect cells diminished the binding of MC179 to the CD36-expressing cells ( Figure 2A , orange peak ) . This indicated that CD36 in solution could compete with cell surface CD36 for the binding of MC179 . Preincubation of the CD36-expressing cells with anti-CD36 antibodies that are known to block the cytoadherence of parasitized RBC [36] prevented the binding of MC179 to CD36 ( Figure 2B , green , red , violet peaks ) . This result signified that the binding of cytoadherence-blocking antibodies to CD36 could interfere with the binding of MC179 to CD36 . The cysteine-richness for which the domain is named stems from the conserved cysteines located on the H1-H2 loop , at the N-terminus of H2 , and at the C-terminus of H3 ( Figure 1D ) . We directly observed four of the seven cysteines and have evidence that six of the seven cysteines in MC179 are in disulfide bonds . The disulfide bond Cys 49-Cys 159 between the H2 and H3 helices was observed in electron density . Two other cysteines ( Cys 45 and Cys 51 ) at the N-terminal end of the H2 helix were also observed in electron density . Difference electron density based on the sulfur anomalous signal in the native X-ray dataset confirmed the observation of each of the four cysteines . The sulfur atoms of cysteines 32 and 41 on the loop between the H1 and H2 helices and of cysteine 168 at the C-terminus of MC179 were not visible . During the preparation of MC179 for crystallization , mass spectrometry detected an adduct of the size of a cysteamine molecule on MC179 . We reasoned that one cysteine had reacted with cystamine ( the oxidized form of cysteamine ) during protein refolding , which implied that there was one free cysteine in MC179 . From comparisons with DBL domains , we concluded that Cys 51 is the single unpaired cysteine in MC179 ( see below ) . In experiments to determine which cysteine possessed the adduct , we deleted Cys 168 by truncating twelve residues from the MC179 C-terminus , resulting in the shorter MC167 construct . After refolding , mass spectrometry of MC167 showed that two cysteines had reacted with cystamine , indicating that the deletion of Cys 168 had generated a second free cysteine . This implied that Cys 168 in MC179 participates in a disulfide bond . Reports that MC179 with Cys 168 substituted by serine and a MC167-like truncated molecule still bind CD36 [30] , [37] encouraged us to produce MC167 crystals and to use them interchangeably with MC179 during the structure determination . As residues 168–179 and the loop between H1 and H2 were not visible in the MC179 structure ( see Methods ) , the observed X-ray structures of MC167 and MC179 are identical . How do MC179 and thus the CIDR1α domains of PfEMP1 molecules bind CD36 ? The effects of mutations chosen based on sequence similarities in several regions of MC179 have been reported and these mutations can now be located and defined structurally . The substitution of Ser for Cys 45 or a double substitution of Ser for both Cys 159 and Cys 168 abolished CD36 binding [30] . Except for those cysteines , point mutations have little effect on CD36 binding , but changing many residues at the same time has a large effect on CD36-binding . CD36-binding was not affected when point mutations within helices H1 and H2 of the three-helix bundle were made or when the residues between Cys 32 and Cys 41 on the loop connecting the H1 and H2 helices were replaced with residues from a non-binding CIDR domain [33] . When several point mutations were placed into the same molecule , CD36 binding diminished from 100% to 63% [21] . CD36-binding was lost when chimeric proteins were made between the MC CIDR1α domain and CIDR domains that do not bind CD36 . When the H1-H2 , a-b-c , and a-b-c-H3 regions of MC179 were substituted in turn with the corresponding sequences from the non-binding CIDR domains , no binding to CD36 was detected [21] , [33] . The MC179 domain appears to function as a whole in binding CD36 and only large changes in the domain have succeeded in affecting binding to CD36 . Three point mutations in the same molecule affected the binding of several CIDR1α domains to CD36 . The wild type CIDR1α domain from the ItG2-CS2 parasite strain does not bind to CD36 , but spontaneous mutations that arose during in vitro culture changed each of the residues in the sequence Gly-His-Arg , resulting in CD36-binding [33] . Sequence comparison aligns Gly-His-Arg with the MC179 sequence , Glu 108-Ile 109-Lys 110 , which is located at the beginning of the b helix and makes contact with the end of the a helix ( Figure 2D ) . Glu 108 lies just above a hydrophobic patch in the right-hand panel of Figure 2D . Replacing Glu-Ile-Lys with the wild type ItG2-CS2 Gly-His-Arg sequence decreased MC179 binding to CD36 by 50% [33] . This suggests that part of the CD36-binding region of MC179 lies within or near the a and b connecting helices . Recently , a chimeric protein ( “1640-f” ) between a non-CD36-binding CIDR1α1 and a CD36-binding CIDR1α molecule exhibited CD36 binding [25] . Based on sequence alignments ( Figure S2 ) and the MC179 structure , the 1640-f chimera contained the H1 , H2 , a , and b helices from the CIDR1α1 ( PFE1640w ) domain and the b , c , and H3 helices from the CIDR1α ( PF10_0406 ) domain . It appears that the 1640-f chimera contained two copies of the b helix . Antibodies raised against the CIDR1α portion of the chimera inhibited CD36 binding by erythrocytes infected with the 3D7 , HB3 , or FCR3 parasite strains , which indicated that there are similar epitopes on CIDR domains that otherwise differ from each other [25] . Where might these cross-reactive epitopes be located ? The c helix has few conserved residues and the conserved residues of the b helix point inward toward the a helix , though they still could be contacted by antibodies . We suggest that the H3 helix may be the location of cross-reactive epitopes since the side chains of some conserved residues of the H3 helix , such as Asp 146 and His 151 , extend out into solvent . Sequences and lengths of the a and b helices in CD36-binding CIDR1α domains vary substantially ( Figure S1 ) , which is difficult to rationalize with CIDR1α binding to the non-polymorphic CD36 protein . In addition , mutations and chimeric molecules have implicated several areas of the MC179 molecule in binding . An understanding of the mechanism of binding CD36 has not been obtained through mutational analysis of conserved residues . That hundreds of CIDR1α sequences still bind CD36 while containing multiple “natural” mutations implies that CD36-binding may derive from the overall structure of the CIDR1α domain . We aligned the MC179-like sequences of the 110 CIDR domains of the P . falciparum 3D7 genome ( Figure S2 ) . For each position in the MC179 sequence , the percent of aligned sequences having the identical residue at that position was calculated [38] . We plotted the percent sequence identity as a gradient of color ( blue ) on a surface depiction of MC179 ( Figure 3 ) . This revealed a conserved region found in all CIDR domains ( Figure 3B ) . This region is located between the H1 and H2 helices near to the N- and C-termini of MC179 and contains the unpaired cysteine , Cys 51 ( Figure 3B ) . Cys 51 is located on the H2 helix and is conserved in all CIDR domains whether CIDR1 or CIDR2 ( Figure S2 ) . The conserved surface shown in Figure 3B likely becomes part of the interior of the CIDR domain in the presence of the M1 subdomain of CIDR1α that is located N-terminal to the MC179 or M2 subdomain [37] . In other words , the blue surface in Figure 3B is predicted to be the interface between the M1 and M2 subdomains because it is conserved in all CIDR domains , is proximal to the MC179 N-terminus , and contains the unpaired , conserved Cys 51 . In the intact CIDR domain , Cys 51 is likely disulfide-bonded to one of the cysteines of M1 . The N-terminal part of MC179 was shown to be important in binding CD36 [30] and MC179-analogous protein constructs from some P . falciparum strains bound CD36 only after the polypeptides were lengthened to include M1 [37] . Examination of the MC179 molecule reveals that the surface on the opposite face of MC179 is little conserved ( Figure 3A ) . The surfaces of the a , b , and c helices are not conserved ( Figure 3 ) and the connecting helices in CIDR1α sequences vary in length . Therefore , a large part of the MC179 surface is not conserved , as most of the residues making up the surface of MC179 are not conserved in other CIDR1α domains . We propose that much of MC179 is exposed at the surface of the PfEMP1 molecule . Of course , MC179 must be exposed at the surface to bind CD36 . How much of it ? Perhaps 1000 Å2 would be a minimum estimate in light of other known protein-protein interactions . MC179 has a total solvent accessible area of 10 , 000 Å2 . Therefore , at least 10% of MC179 is exposed . We detected conserved patches that we attribute to the interface with the M1 subdomain of CIDR1α and to potential CD36 binding ( see below ) . We did not detect a conserved surface that might be an interface with DBL domains , for instance , DBL1α or DBL2 , although the M1 portion may provide a conserved contact surface with DBL1α . It seems likely to us that the CIDR1α domain has only a small surface area in contact with the DBL domains and that more than 10% of the area of MC179 is exposed to the bloodstream . MC179 likely extends out from the PfEMP1 molecule , consistent with the proximity of the MC179 N- and C-termini to the conserved patch predicted to interface with the M1 portion . Persons living in malaria endemic regions only slowly acquire effective antibodies against iRBCs [39] . One or more variable MC179-like surfaces on PfEMP1 molecules would provide a high diversity of antibody epitopes , leading to the slow development of immunity and to the parasite's ability to evade the host immune response . The majority of PfEMP1 molecules from the 3D7 parasite strain contain one CIDR1α domain that binds CD36 and one CIDR2β domain that does not bind CD36 [21] . We mapped the percent identity of the MC179 sequence with the 3D7 CIDR1α domains ( Figure S1 ) and with the 3D7 CIDR2β domains ( Figure S3 ) on separate surface models of MC179 ( Figure 4 ) . This revealed a conserved region located between the H1 and H3 helices that is present in both kinds of domains , but is larger in CIDR1α domains . Every CIDR domain has some identity with MC179 here , accounted for by conserved residues that make up the interior of the three-helix bundle , but are partially exposed at the surface of the molecule . Examples of these are three cysteines ( 45 , 49 , and 159 ) and two leucines ( 19 and 148 ) ( Figure 4 ) . Contributing to this surface in CIDR1α domains are a conserved serine and a conserved glutamic acid . In MC179 , Ser 22 and the charged Glu 152 are both nearly buried and form a hydrogen bond . Except in one PfEMP1 ( PFL0020w ) where Ser 22 is replaced by a cysteine residue , Ser 22 and Glu 152 are completely conserved in CIDR1α domains , but one or both of the residues are absent in other CIDR domains . Ser 22 becomes a tyrosine in most other CIDR domains and Glu 152 becomes a leucine in all other 3D7 CIDR domains ( Figure 4 , middle ) . Although this conserved surface in CIDR1α domains with a nearly buried glutamic acid residue might be a potential interaction surface with CD36 , mutations of Ser 22 to threonine [33] and Glu 152 to leucine [21] have not affected CD36 binding . Additionally , Lys 147 is present in most CIDR1α sequences , within the conserved region shown in Figure 4 . In almost all CIDR2β domains , the residue at the Lys 147 position is a cysteine ( Figure 4 ) . During the first cloning and sequencing of PfEMP1 genes , conserved hydrophobic residues and cysteines observed in the midst of the enormous sequence diversity of these large proteins were used to define the DBL and CIDR domains and to suggest their relatedness [7] . Recent crystal structures of non-PfEMP1 DBL domains [40] , [41] from the P . falciparum erythrocyte-binding antigen ( EBA ) -175 , containing tandem DBL domains F1 and F2 , and the P . knowlesi erythrocyte binding protein ( Pkα-DBL ) , containing a single DBL domain , showed that conserved residues are within DBL helices . We superimposed the MC179 structure on the F1 DBL domain from EBA-175 ( Figure 5A ) , the F2 DBL domain from EBA-175 , and the DBL domain from Pkα ( Figure 6 ) . The three-helix bundle of MC179 structurally matched each DBL C-terminal three-helix bundle , a region that has also been named “subdomain 3” [41] . MC179 and all three DBL structures closely overlaid with root mean squared ( r . m . s . ) deviations between 1 . 6–2 . 0 Å over the 74–77 α-carbon atoms of the three helices . The overlay is best among the H1 and H2 helices of all four proteins and between the regular H3 helices of MC179 and the EBA-175 F1 DBL domain . The H3 helices of EBA-175 F2 and of Pkα-DBL are mixed with irregular secondary structure and take a meandering route along their respective H1 and H2 helices . This structurally conserved three-helix “bundle” is a feature of the DBL and CIDR protein folds and is evidence beyond their sequence similarities for the common ancestry between the two domain families [7] . More than main chain atoms match among the structures , however , as even the conserved side chains in the H1 , H2 , H3 helices of MC179 and in the corresponding helices of the DBL domains are in identical positions . In Figure 5B , six conserved Trp , Phe , and Tyr side chains in the H1 and H2 helices closely overlay between MC179 and the F1 DBL domain . In addition , the Cys 49-Cys 159 disulfide and Cys 45 side chain of MC179 overlay to within 3 . 0 Å of the analogous cysteines of the EBA-175 and Pkα-DBL domains . It is clear that the MC179 and thus the CIDR1α disulfide bonding is the same as in the DBL domains ( Figure 1D and 5B ) . In the MC179 numbering used in the structure , the cysteines involved in disulfide bonds are 32–45 , 41–168 , and 49–159 ( Figure 5B ) . Cys 51 extends into solvent from the H2 helix and is conserved in all CIDR , but not DBL domains . Cysteines 49 and 51 form the Cys-X-Cys sequence motif that is found in most CIDR sequences [18] . Sequence alignments and more recently , detailed modeling studies based on the EBA-175 and Pkα crystal structures , have predicted that the interiors of DBL and CIDR domains would be held together by conserved hydrophobic and cysteine residues and that loops would contain the most polymorphic sequences [42] , [43] . The structural overlay of MC179 on the available DBL domains also reveals the differences in the connections between the helices of the bundle ( Figure 6 ) . A loop of approximately similar length connects the H1 and H2 helices in both MC179 and in the structurally known DBL domains , but the connection between the H2 and H3 helices differs considerably between the two kinds of domains . In the known DBL domains , the H2-H3 connections are about 30 residues in length consisting of a small loop and helix that vary among the three DBL domains . In MC179 , the H2-H3 connection is about 70 residues in length and ranges from 30 to 100 residues in the many known CIDR sequences . While examining the similarities between MC179 and the known DBL domains , it became evident that DBL domains are composed of two three-helix bundles ( Figure 6 ) . As described above , MC179 structurally matches the DBL C-terminal three-helix subdomain , but the DBL subdomain nearer to the N-terminus is also made up of three helices that are connected like the MC179 and C-terminal DBL bundles . The sequence extents of the N-terminal and C-terminal three-helix bundles in the structurally determined DBL domains are: residues 58–159 and 174–282 in EBA-175 F1 , residues 365–463 and 481–596 in EBA-175 F2 , residues 64–165 and 190–304 in Pkα-DBL ( PDB codes 1ZRL and 2C6J ) . In Figure 6 , MC179 and the three DBL domains are colored to highlight bundle helices and connections between helices . MC179 structurally overlays on the DBL C-terminal three-helix bundle , but does not overlay on the N-terminal bundle . However , the helices and the connections between the helices of the N-terminal bundle ( h1 , h2 , h3 ) are similar to those of the C-terminal bundle: the h1 helix ( dark blue ) , a short connecting loop ( gray ) , the h2 helix ( light blue ) , a longer connecting helix ( yellow ) , and the h3 helix ( red ) ( Figure 6 ) . Like the C-terminal bundle , the N-terminal bundle contains a disulfide that connects the h2 helix at its N-terminus with the h3 helix at its C-terminus . The cysteines that compose this disulfide in each DBL domain are: Cys 88-Cys 164 in EBA-175 F1; Cys 396-Cys 471 in EBA-175 F2; Cys 99-Cys 176 in Pkα-DBL . These disulfides pin the h2 and h3 helices to each other and correspond to the Cys 49-Cys 159 disulfide of MC179 and the analogous disulfides of the C-terminal three-helix bundles of DBL domains . Based on structural similarities and conserved residues among the CIDR and DBL domains , we developed sequence expressions to detect the H1 and H2 helices of the three-helix bundles that can be used to locate CIDR and DBL domains in PfEMP1 sequences . These sequence patterns describe the conserved residues that are unique to the H1 and H2 helices . The H1 helix sequence of CIDR and DBL domains satisfies the expression: ( Leu , Phe , Trp , or Tyr ) -X3-Trp-X17 , 18 , or 19-Cys , where “Xsubscript” indicates the number of residues of unspecified type occurring between conserved residues . Several possible hydrophobic residues are indicated in parentheses at some positions . As examples , the H1 helix of MC179 is located with the pattern Phe-X3-Trp-X17-Cys and the predicted H1 helix of the Malayan Camp DBL1α is found with Trp-X3-Trp-X18-Cys ( Figure 7 ) . Different patterns of conserved residues distinguish the CIDR H2 helices and the DBL H2 helices . The H2 helix is found in CIDR domains by the expression: Cys-X3-Trp-X7 or 8-Trp-X6- ( Phe or Tyr ) . This motif is preceeded by an additional Cys-X1- in many CIDR domains . In DBL domains , H2 helices are predicted with this algorithm: Cys-X3-Cys-X2- ( Phe or Tyr ) -X2- ( Leu , Lys , Trp , Tyr ) -X7- ( Phe , Trp , or Tyr ) -X6- ( Phe or Tyr ) . We used the CIDR algorithms for H1 and H2 to detect a CIDR-like domain in the var2csa protein , the product of a relatively conserved gene found in all P . falciparum strains and expressed by most parasites that bind CSA [44] , [45] . For example , the var2csa family member in the 3D7 genome , PFL0030c , contains a CIDR-like domain between the DBL2x and DBL3x domains , two of the six DBL domains of PFL0030c . The H1 pattern of this CIDR-like domain is Leu-X3-Trp-X18-Cys and the H2 pattern is Cys-X3-Trp-X7-Trp-X6-Tyr . We predict a three-helix bundle in this CIDR-like domain that extends from residues 1025 to 1212 of the PFL0030c protein . This CIDR-like domain , termed the ID ( interdomain ) -2 domain , has recently also been predicted through sequence analysis and modeling [43] . In the MC179 crystal , pairs of molecules interact and bury 4300 Å2 of solvent accessible surface area between them ( Figure 8 ) . A crystallographic two-fold symmetry axis relates the two MC179 molecules . Much of the contact between the molecules is made by the helices that connect the H2 and H3 helices including the hydrophobic patch seen in Figure 2D . Helix a residues ( Leu 94 , Leu 95 , Leu 96 , Ile 98 , Ile 99 ) and helix b residues ( Ile 112 , Leu 115 , Leu 116 ) constitute this hydrophobic area on one molecule , interacting with H1 helix residues ( Trp 12 , Val 15 , Leu 19 , Ile 20 , Ile 23 ) and H3 helix residues ( Thr 144 , Lys 147 , His 151 ) of the symmetry-related MC179 molecule . Hydrophobic areas in the third connecting helix , helix c , also contribute to this interaction between molecules in the crystal . Opposite to the hydrophobic patch on the a , b , and c helices is a region made up of negative charges from helix a residues ( Glu 97 , Asp 101 ) , helix b residues ( Glu 113 , Glu 117 ) , and a helix c residue ( Asp 125 ) ( Figure 2E , left side in red ) . These charges are on the surface of the crystallographic dimer . The molecular surface of the crystalline dimer shows the intertwining of the two molecules in a “handshake”-like interaction ( Figure 8B ) . The surface complementarity ( SC ) index of the monomer-monomer interface is high at 0 . 70 [46] and 55 residues ( 220 atoms ) from each molecule take part in the interaction . Residues that when mutated have affected CD36 binding ( His 69 , Glu 108 , Ile 109 , Lys 110 ) contribute to the surface of the MC179 dimer and , except for His 69 , are located near , but not in , the dimer interface ( Figure 8B ) . His 69 is distant from the dimer interface . The interaction between MC179 molecules in the crystal resembles a biological interaction with its large amount of buried surface area and high surface complementarity . In solution , when refolded MC179 was prepared by size exclusion chromatography , we separated molecular species with the mobility of dimers from the desired species with the mobility of monomers and used the monomers for crystallization . In addition , chemical cross-linking experiments indicated the presence of MC179 dimers in solution ( data not shown ) . We conclude that MC179 has the tendency to dimerize in solution . How these dimers of a recombinant fragment of PfEMP1 , if biologically relevant , might function in a PfEMP1 molecule is unclear . Based on the enormous contact area observed in the MC179 “dimer” in the crystal , our hypothesis is that the connecting helices of the MC179 portion of CIDR1α domains bind another molecule , either a PfEMP1 domain or a ligand . This hypothesis that the CIDR connecting helices are involved in binding another molecule extends to our prediction of binding by the proposed connecting helices of the DBL1α domain as described below . Using BLAST with the MC179 amino acid sequence as probe , we aligned the MC179 portions of the approximately 300 CIDR1α sequences from the 3D7 strain , the Ghanaian clinical isolate , the IT strain , and P . reichenowi genomes from the Sanger sequencing center ( http://www . sanger . ac . uk/Projects/Protozoa/ ) . In the alignment , semi-conserved hydrophobic residues implied the presence of the MC179 connecting helices a , b , and c . By their sequences , all CIDR1α domains appear to retain approximately the first two connecting helices , the a and b helices , as predicted by alignment with the MC179 sequence , but the third connecting helix c varies greatly in length ( see 3D7 CIDR1α alignment in Figure S1 ) . These differing CIDR1α sequences and lengths must result in changes in the size of the V-like opening of the CIDR1α domain as seen in the MC179 structure . The sequence composition and length of the three connecting helices ( a , b , and c ) vary even among CIDR1α molecules that bind CD36 ( Figure S1 ) . In some CIDR1α sequences there are two additional cysteine residues , one located about ten residues after the end of the H2 helix and the other located about ten residues before the start of the H3 helix . In the MC179 structure , these two Cα atom positions are separated by about 8 Å , which could be spanned by a disulfide bond . For example , PF10_0001 ( Figure 7 ) , PFD0630c , PFD0005w from the 3D7 strain have such cysteines . We predict that these cysteines form a disulfide bond that could act to stabilize the MC179 structure , by linking the a and b helices . Such a disulfide would prevent movement of helices a and b relative to each other , which would need to be considered in models of how CIDR1α binds CD36 , as PF10_0001 , PFD0630c , and PFD0005w are CD36 binders [21] . Comparisons of the MC179 and DBL X-ray structures with DBL1α domain sequences indicate that DBL1α domains have a longer H1-H2 connecting loop than do the MC179 and DBL domains . Figure 7 presents the DBL1α domain of the MC PfEMP1 as an example . DBL1α domains also have an additional cysteine in the H1 helix and two additional cysteines within the connecting loop to H2 ( Figure 7 ) . The presence of extra cysteines in PfEMP1 DBL domains has complicated the identification of conserved cysteines . The MC179 structure will aid in the comparison of PfEMP1 DBL , non-PfEMP1 DBL , and CIDR domains . From the current knowledge of the MC179 and DBL X-ray structures , it is clear that the C-terminal boundary of DBL1α domains has sometimes been predicted to be at the end of the H2 helix of the three-helix bundle , as determined from conserved residues and the number of residues between them ( Figure 7 ) . This is understandable since after the end of the H2 helix there is little sequence similarity among DBL domains to guide prediction . The MC179 and DBL structures show that a third helix , degenerate helix , or , conceivably , an irregular H3 structure must be present in all DBL and CIDR domains to complete the “bundle” . The structures also predict that there are at least two cysteines at the C-terminus of the third helix that make conserved bonds with Cys 41 and Cys 49 ( MC179 ) or their homologs in the DBL domains ( Figures 5B and 7A ) . This need for two cysteines at the end of the DBL domain can be used to predict the C-terminus of the DBL1α domain and leads to the prediction of MC179-like connecting helices in DBL1α . For example , in the Malayan Camp sequence shown in Figure 7A ( line 8 , DBL1α MC ) , if one proceeds along the DBL1α MC sequence from the end of the H2 helix at conserved residue Tyr 394 ( Figure 7A , red dot ) , a single cysteine is encountered at residue 445 , but a cluster of three cysteines is found at residue 475 ( Figure 7A , DBL1α MC ) . Since two cysteines are required to form disulfide bonds with two conserved cysteines near the start of the H2 helix , the third helix H3 of the DBL1α must include the multiple cysteines around residue 475 in the sequence -Cys-Glu-Ala-Cys-Pro-Trp-Cys- ( -CEACPWC- ) . If the DBL1α domain has a three-helix bundle that ends around position 475 , then the length of the three-helix region is about 70–80 residues longer than in the EBA-175 and Pkα-DBL domains . This leads to the prediction that the DBL1α domain likely has connecting helices between H2 and H3 of about the same length ( 70–80 residues ) as does MC179 ( Figure 7 ) . The definition of a longer DBL1α domain affects the predicted start of the following CIDR1α domain . Due to the longer DBL1α domain , the immediately following CIDR1α domain and its M1 region are predicted to be 70–80 residues shorter . This reasoning implies that the DBL1α C-terminal region has been included in some predictions of the CIDR1α domain . Locating the DBL domain C-terminal boundary in this way by finding the cysteines that end the third helix is applicable to the DBL sequence classes , α , β , γ , δ , ε , and to the analysis of the semi-conserved domain pairings of DBL1α-CIDR1α , DBL2β-C2 , and DBL2δ-CIDR2β that frequently appear in PfEMP1 sequences [18] . The DBL1α-CIDR1α domain pairing or “conserved head structure” is present at the N-terminal end of a majority of PfEMP1 molecules [7] , [8] . As the sequences that connect H2 to H3 in the DBL1α and CIDR1α domains of the MC strain appeared to be similar in length , we examined the lengths of connecting regions in all of the DBL1α and CIDR1α domains that are pairs in the 3D7 genome . We counted the number of residues that connect H2 and H3 for each DBL1α and its associated CIDR1α domain and produced a list of forty-seven pairs of numbers , one pair of numbers from each PfEMP1 with a conserved head structure . Each number is the length of the connecting sequence in a DBL1α or a CIDR1α domain . The lengths in the list are correlated with a 0 . 58 correlation coefficient ( Spearman rank-order ) between them ( p<0 . 0001 ) ( Figure S4 ) . This positive length correlation between DBL1α and CIDR1α implies that the connecting sequences of the DBL1α domains are similar in length to the CIDR1α connecting regions . This may simply reflect the evolutionary relatedness of the two domains , but may indicate similar binding or other function . The H2-H3 connecting helices of DBL1α may extend away from the PfEMP1 molecule , as is the prediction for MC179 based on the present structural analysis . The connecting helices in MC179 extend out from the H1-H3 side of the bundle . In DBL1α , we predict that the connecting helices would also extend out from the H1-H3 side of the bundle . This is distinct from the H1-H2 side that interacts with the N-terminal bundle ( “subdomain 2” [41] ) ( Figures 5A , 6 ) . The overlay of the DBL domain and MC179 in Figure 5A also serves as a model for DBL1α , if one looks at the connecting helices of MC179 as modeling the predicted connecting helices of DBL1α . In addition , it is possible that the DBL1α and CIDR1α domains interact with each other in a manner resembling the MC179 dimer in the crystal . Two additional points support the DBL1α-CIDR1α domain boundary as described here . First , Plasmodium proteins frequently have regions of low sequence complexity inserted in loops and between domains [47] , [48] . In the PFL1960w 3D7 PfEMP1 sequence , there is an insertion of 110 glycine and serine residues after the multiple cysteines that mark the end of the DBL1α H3 helix and before the start of the CIDR1α domain . Binding studies with the CIDR1α domain from PFL1960w showed that it does bind CD36 [21] , indicating that the insertion did not interfere with CD36 binding . This is consistent with the locations of the end of the DBL1α and the beginning of the CIDR1α domain as proposed here . Second , although MC179 is the minimal portion of the MC CIDR1α that binds CD36 , two other recombinant CIDR1α domains bind CD36 only after the domains are lengthened in the N-terminal direction to be longer than MC179 [37] . The minimal lengthening needed to enable these two other CIDR1α domains to bind CD36 extended to just before the end of the DBL1α domains as predicted in this work . This is consistent with the boundary between the DBL1α and CIDR1α domains that does not include the DBL1α H3 helix in the CD36-binding CIDR1α domain . In addition to DBL and CIDR domains , a third type of domain , C2 , is found in PfEMP1 molecules . There are nineteen C2 domains in the 3D7 genome and each is located C-terminal to a DBLβ domain . The A4var PfEMP1 , containing a typical C2 domain , is known to bind ICAM-1 , an endothelial protein that is a ligand of PfEMP1 [49] . When each of the A4var DBL and CIDR domains were expressed using domain boundaries based on sequence conservation , none of the expressed domains bound ICAM-1 [26] . The PfEMP1 domain that bound ICAM-1 was identified by combining DBL2β with the following C2 domain [50] . In subsequent studies to identify the minimal DBL2β-C2 domain that allowed ICAM-1 binding , the C-terminus of the C2 domain was progressively truncated [51] . ICAM-1 binding was lost when a truncation removed , as would be predicted from the current work , the third helix of the three-helix bundle at the C-terminus of the DBL2β-C2 domain . Work with a P . falciparum parasite that binds ICAM-1 also demonstrated that both DBL2β and C2 domains are required for ICAM-1 binding , as neither DBL2β or C2 expressed alone was able to bind ICAM-1 [52] . From these results , we suggest that the C2 domain completes the subdomain 3 of the DBLβ domain , by providing the connecting residues and the third helix of the three-helix bundle that has the cysteines that make disulfides to conserved cysteines in the H2 helix . That the C2 domain contains the third helix of the DBLβ subdomain 3 has been predicted recently from homology modeling [53] . Sequences of PfEMP1 proteins vary enormously to evade the host immune response , while maintaining their binding to endothelial receptors . Conservation of binding to CD36 in the midst of tremendous variation in CIDR1α sequences implies an essential role for binding in parasite survival and in disease outcome for the host . Based on the MC179 structure and the DBL structures known at present , the highly conserved helices and disulfide bonds of the three-helix motif comprise the individual domains of the molecules in the PfEMP1 and EBA families and serve as the scaffolding on which sequence variation takes place . The two helical bundles in DBL domains and the single bundle in CIDR domains imply that a PfEMP1 molecule is not only modular in domain organization , but is a polymer of helical bundles , elaborated by connecting loops and helices . These loops and helices exhibit extraordinary sequence polymorphism and may be regions exposed to the bloodstream and its antibodies . Focusing on the binding of host ligands by PfEMP1 molecules will give insights into vaccine or drug strategies that will affect cytoadherence , help elucidate the ways that the parasite adjusts the functioning of the immune system , and should provide tools to lessen the impact of malaria in endemic areas of the world .
MC179 ( residues 576–754 of GenBank U27338 ) and MC167 ( residues 576–742 ) expression plasmids were transformed into BL21 DE3- ( RIL ) cells , grown to an OD600 of ∼1 , and induced with IPTG to produce insoluble protein ( inclusion bodies ) . To initiate refolding of either MC167 or MC179 , guanidine-solubilized inclusion bodies ( 30 mg ) were then diluted to 45 ml with 3 M guanidine-HCl and pumped ( 0 . 05 ml/min ) into 1 L of refolding buffer containing 400 mM arginine-HCl , 100 mM Tris-Cl , 5 mM cystamine-HCl , 2 mM DTT , and 2 mM NaEDTA . After 24 h , cystamine-HCl ( 50 mM ) was added and allowed to react for 1 h to block free cysteines . The solution was then dialyzed against 10 L of water for 24 h and then against 10 L of 10 mM Tris-HCl pH 8 . 0 for 24 h . Protein was concentrated on CM52 cation exchange resin using 10 mM MES-NaOH at pH 6 . 0 and purified on a Superdex 75 size exclusion column . Protein fractions were concentrated to 6 mg/ml with CentriPrep concentrators . Using the hanging drop method , bipyramidal hexagonal MC167 or MC179 crystals grew in 27% polyethylene glycol ( PEG ) 400 , 100 mM NaCl , 50 mM sodium citrate , pH 4 . 2 , and diffracted to 2 . 7 Å using X-rays from a rotating anode generator . PEG400 ( 30% v/v ) cryoprotected the frozen crystals . Seleno-methionine-containing inclusion bodies were produced in minimal medium ( Athena Enzyme Systems , FL ) using the BL21- ( DE3 ) -X strain that is auxotrophic for methionine . Selenomet-MC167 or MC179 was refolded and purified similarly to native and the presence of selenium was verified by mass spectrometry . Selenomethionine crystals grew in 15% PEG400 , 100 mM NaCl , and 50 mM sodium citrate , pH 4 . 2 . Native , selenomethionine , and heavy atom derivative X-ray data were integrated and scaled with the XDS package [54] ( Table 1 ) . Seleno-methionine positions were found with SHELXD [55] . The native , selenium , ytterbium , and osmium datasets were used in SHARP [56] to find and refine additional heavy atom sites and to estimate protein phases for electron density maps . After density modification with RESOLVE [57] , clear helical segments were observed in the electron density . Using the selenium positions as landmarks , the model was built , and then refined in the CNS package [58] to an Rwork value of 0 . 25 and an Rfree value of 0 . 29 . No dependable model could be built for one N-terminal residue , loop residues 30–43 , and C-terminal residues 168–179 . Using model phases and highly redundant data collected with 1 . 54 Å X-rays , anomalous difference electron density confirmed the positions of each sulfur atom in the model . Models were superimposed with LSQMAN [59] and the SSM server [60] . Figures were produced with PyMOL software [61] and Jalview [62] . Surface area calculations were performed with the PISA server [63] . Coordinates and structure factors have been deposited with accession code 3C64 in the RCSB Protein Data Bank . The binding of recombinant MC179 with a hexa-His tag at its C-terminus to human CD36 , expressed on stably transfected Chinese hamster ovary cells ( CHO-CD36 ) , was assayed with a flow cytometry protocol similar to one described [64] . Briefly , cells were suspended at 106 cells/ml in phosphate-buffered saline ( PBS ) containing 0 . 5% bovine serum albumin and 0 . 1% ( w/v ) sodium azide ( FCA buffer ) . Cells ( 100 µl ) in V-bottomed , 96-well plates were pelleted , then resuspended in 100 µl containing 100 ng/ml MC179 and incubated for 1 h . Following washing with PBS , an Alexa 488-labeled anti-pentahis monoclonal antibody ( 100 µl ) ( Novagen , San Diego , CA ) or an anti-MC179 serum from an immunized Aotus monkey were added at a final concentration of 0 . 8 µg/ml and 1∶100 , respectively . IgG1 isotype mAb ( Becton Dickinson , San Jose , CA ) and Aotus pre-immune serum were included as controls . After incubation for 30 min , cells were washed twice in PBS and Aotus antibodies were detected with Alexa 488-labeled goat anti-human IgG ( Invitrogen , Carlsbad , CA ) ( 100 µl ) diluted 1∶250 in FCA buffer . The cells were then incubated for 30 min , washed , resuspended in 150 µl and analyzed by FACSort ( Becton Dickinson ) . After staining with 0 . 5 µg/ml propidium iodide ( PI ) , PI+ cells were excluded from the analysis . Anti-CD36 antibody clones used were 185-1G2 ( Lab Vision , Fremont , CA ) , CLB-IVC7 ( Sanquin , Amsterdam , Netherlands ) , and 8A6 [36] ( gift from Dr . J . W . Barnwell , Center for Disease Control , Atlanta , GA ) . All the washing steps and incubations were performed at 4°C . Events ( 10 , 000 ) were acquired using CELLQuest software ( version 3 . 3; Becton Dickinson ) and the data from the live cell gate were analyzed by FlowJo software ( version 6 . 4 . 1; Tree Star , San Carlos , CA ) . | Malaria parasites express proteins of the erythrocyte membrane protein-1 family ( PfEMP1 ) on the surfaces of the human red blood cells that they infect . These large proteins vary in sequence extensively , yet bind to host receptors to allow infected cells to adhere to host tissues . PfEMP1 proteins help parasites evade the immune system , as the 60 PfEMP1 genes are expressed one at a time . Sequence comparisons predict that PfEMP1 molecules are modular , made up of Duffy binding-like ( DBL ) and cysteine-rich interdomain region ( CIDR ) domains . Many CIDR domains bind to the human receptor CD36 . We have analyzed the structure of the CD36-binding portion , known as MC179 , of a CIDR domain . The MC179 protein is composed of a bundle of three helices connected by a loop and three additional helices . Based on the structure and sequence similarities , MC179 is a good model for the hundreds of known CIDR sequences . In addition , the MC179 three-helix bundle is remarkably similar to subdomain 3 of the known DBL structures . MC179 provides insight into the relatedness of both kinds of PfEMP1 domains and predicts that the large PfEMP1 molecules are polymers of three-helix bundles and their connecting polypeptides . | [
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] | 2008 | The Cysteine-Rich Interdomain Region from the Highly Variable Plasmodium falciparum Erythrocyte Membrane Protein-1 Exhibits a Conserved Structure |
Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons . Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport , Golgi outposts and endocytic recycling . How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth , however , remains unknown . Using a candidate-based genetic screen in C . elegans , we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis . Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron . RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes . Loss of function mutations of the exocyst complex components exoc-8 and sec-8 , which regulate tethering , docking and fusion of transport vesicles at the plasma membrane , also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles . In rab-10 and exoc-8 mutants , the trans-membrane proteins DMA-1 and HPO-30 , which promote PVD dendrite stabilization and branching , no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles . Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth .
Dendrites and axons are two distinct functional and morphological domains of neurons . Due to the complexity and heterogeneity in the morphology of dendrites , it has been challenging to study the development of dendrites in comparison with the more uniform , simply structured thread-like axons . Previous studies have shown that dendritic growth relies heavily on the secretory pathway and endosomal function[1 , 2] . In Drosophila , loss-of-function mutations in genes encoding the small GTPases Rab1 and Sar1 , which are key regulators of ER-to-Golgi vesicular transport [3] , severely reduce the growth of dendrites . Notably , loss of Rab1 and Sar1 do not diminish axon outgrowth , suggesting that the mechanisms underlying the extension of dendrites and axon are distinct [1] . Furthermore , Golgi outposts , which are primarily found in dendrites but not axons , play an important role in supplying membranes for dendritic branching and growth [1] . These experiments suggest that membrane components generated in the ER and trafficked to the Golgi are essential for dendritic growth . In addition , Rab5 and Rab11-dependent endocytic membrane trafficking has also been implicated in dendrite morphogenesis [2 , 4] . The molecular mechanisms that deliver membranes from the Golgi , Golgi-outposts and endosomes to the dendritic plasma membrane , however , are unclear . To identify the membrane trafficking mechanisms that support dendrite branching and growth , we use the C . elegans multi-dendritic PVD neurons as a model . The PVD neurons exist as a pair , PVDL and PVDR , and they function to detect harsh mechanical forces and cold temperatures [5–7] . Each PVD neuron sits on one side of the animal and has a single axon that extends to the ventral nerve cord , as well as a highly branched dendritic arbor that covers most of the body , except for the neck and head [8] . Recently , the transmembrane leucine-rich repeat protein DMA-1 was identified as a PVD dendritic receptor . DMA-1 recognizes skin-derived pre-patterned cues that promote dendrite stabilization and branching [9–11] . In addition , the claudin-like transmembrane protein HPO-30 also promotes dendrite stabilization [12] . Both DMA-1 and HPO-30 are dendrite specific proteins that are rarely observed in axons . The mechanisms that regulate their sorting and trafficking to the dendritic membranes are still unknown [9 , 12] . The small GTPase Rab10 , which is an ortholog to the yeast Sec4p protein that controls post-Golgi vesicle trafficking , has been shown to mediate polarized membrane addition during axonal growth in mammals . In axons Rab10 is activated by the mammalian ortholog of the Drosophila gene Lethal giant larvae , Lgl1 . The Lgl1 protein dissociates the Rab10-GDI complex [13] . Activated Rab10 then interacts with multiple effector proteins to direct distinct steps of axonal membrane addition . These include an initial interaction with myosin Vb ( MYO5B ) , which controls the biogenesis of post-Golgi Rab10 carriers [14] . Rab10 then binds with c-Jun N-terminal kinase-interacting protein 1 ( JIP1 ) to facilitate anterograde transport of Rab10 cargos [15] . Finally , Rab10 binds myristoylated alanine-rich C-kinase substrate ( MARCKS ) . The Rab10-MARCKS interaction allows the docking and fusion of Rab10 vesicles with the axonal plasma membrane [16] . Unlike extensive studies on the role of Rab10 in axonal growth , it is unclear whether Rab10 is required for dendrite arborization during development . Several studies have shown association between the conserved exocyst complex and Rab10 GTPases [17] . The exocyst complex is composed of eight subunits . In C . elegans these are encoded by the genes sec-3 , sec-5 , sec-6 , sec-8 , sec-10 , sec-15 , exoc-7 and exoc-8 [18] . The exocyst complex functions as the effector of the yeast Rab10 ortholog Sec4p , and facilitates tethering , docking and fusion of secretory vesicles during bud formation [17] . The exocyst complex also associates with Rab10 in renal epithelial cells and may mediate membrane transport to the primary cilium [19] . Both Rab10 and the exocyst complex are further required for the exocytic transport of the glucose transporter Glut4 [20 , 21] . However , whether Rab10 and the exocyst complex function together during other processes , such as dendritic growth and branching , is not clear . Here , we report that loss of the C . elegans rab-10 gene reduces dendritic arborization of the PVD neuron . We show that RAB-10 functions cell-autonomously , and localizes to the Golgi and the early endosomes in the PVD neurons . Further , we find that deficiencies in rab-10 and the exocyst subunits cause accumulation of the dendritic membrane proteins DMA-1 and HPO-30 within intracellular vesicles . We also show that Rab10 and the exocyst complex are required for dendrite arborization in Drosophila , and dendritic spine formation in mammalian neurons . Together , these data suggest that Rab10 and the exocyst complex play a conserved role in controlling Golgi-to-plasma membrane and/or endosome-to plasma membrane trafficking required for dendrite morphogenesis .
To identify potential regulators of membrane trafficking during dendritic branching and growth of the multi-dendritic PVD neuron , a candidate-based genetic screen was performed . We crossed animals harboring mutations in genes encoding proteins important for various membrane trafficking pathways with a PVD neuron-specific fluorescent marker ( F49H12 . 4>gfp or ser2prom3>gfp ) . The morphology of PVD dendritic arbors was then examined . We found that two putative null alleles of rab-10 presented severely abnormal PVD dendrite morphology ( Table 1 and Fig 1A–1E ) . In the proximal region ( including the middle and tail areas ) , rab-10 ( ok1494 ) and rab-10 ( dx2 ) mutant animals contained far fewer dendritic branches compared to wild-type . For example , a count of secondary dendrites within a 100μm long region along the primary dendrite anterior to the PVD cell body in rab-10 ( ok1494 ) and rab-10 ( dx2 ) animals revealed an average of 1 . 1±0 . 3 and 2 . 8±0 . 7 secondary dendrites . In comparison , wild-type animals within this region contained 11 . 1±0 . 5 secondary dendrites ( Fig 1F and 1G ) . Tertiary and quaternary branches were even more affected and were essentially absent in the proximal region of rab-10 ( ok1494 ) and rab-10 ( dx2 ) animals ( Fig 1F , 1H and 1I ) . Interestingly , in the distal area of the PVD , the dendritic branching and growth were minimally affected by loss of rab-10 , indicating that a rab-10-independent mechanism mediates distal dendritic branching ( Fig 1A–1E , 1G–1I ) . The growth of the primary dendrite and axon of the PVD appeared normal in rab-10 deficient animals ( Fig 1A–1E , and S1 Fig ) . rab-10 was also required for the dendritic arborization of the FLP neuron , which covers the head region and has a similar morphology and function as the PVD neuron ( S2 Fig ) [8] . However , rab-10 was dispensable for the growth of unbranched dendrites of OLL , AWB , and AWC neurons , suggesting a specific role of rab-10 in mediating the growth of dendritic branches in multi-dendritic neurons ( S2 Fig ) . Together , these data indicate that RAB-10 is required for the elaboration of branched dendrites in C . elegans . To determine if RAB-10 activity is required within the PVD , we tagged full length RAB-10 at its N-terminus with GFP and expressed this construct under a PVD-specific promoter ( ser2prom3 ) in rab-10 ( ok194 ) animals . GFP::RAB-10 expressed from multicopy extrachromosomal arrays in the PVD neuron fully rescued the morphology of the PVD dendritic arbor in most animals in two independent lines ( Fig 2A–2C ) , indicating that RAB-10 functions within the PVD to promote proximal dendritic arborization . As a GTPase , RAB-10 cycles between the GDP-bound inactive form and GTP-bound active form . To test whether its function in promoting dendrite branching and growth requires GTPase activity , we expressed both GDP-locked ( T23N ) and GTP-locked ( Q68L ) forms of RAB-10 and examined their rescuing ability . Dominant-negative RAB-10 ( T23N ) not only failed to rescue the proximal PVD defects in rab-10 ( ok1494 ) mutants , but also disrupted the distal dendrite arbor in wild-type animals ( Fig 2C , S3 Fig ) . In contrast , constitutively active RAB-10 ( Q68L ) fully rescued the PVD dendrite morphogenesis defects in rab-10 ( ok1494 ) mutant animals ( Fig 2C ) . Over-expressing constitutively active RAB-10 ( Q68L ) did not cause over-growth of dendrites in PVD neuron ( S4 Fig ) , suggesting that other factors limit dendrite growth and patterning . We conclude that RAB-10 functions cell autonomously in the PVD neuron and requires GTPase activity to promote PVD dendrite morphogenesis . RAB-10 orthologs are known to regulate Golgi to plasma membrane vesicle trafficking as well as endocytic recycling events [22–26] . To determine if RAB-10 localizes with either Golgi or endosomes in the PVD neuron we tagged full length RAB-10 with GFPnovo2 , a mutant form of GFP that is brighter . We generated a single copy insertion line using miniMos method to minimize potential ectopic localization of RAB-10 protein induced by over-expression [27 , 28] . Similar to the multicopy line , we crossed this line into the rab-10 null mutant and found that it fully rescued the PVD dendrite arborization defect ( S5 Fig ) . Many intracellular vesicles were labeled by GFP::RAB-10 in the PVD dendrites . Notably , there was a strong correlation between GFP::RAB-10 localization and mCherry::FAPP1-PH ( Golgi reporter ) and a strong correlation of GFP::RAB-10 and mCherry::RAB-5 ( early endosome reporter ) ( Fig 2D and 2E ) [29] . These observations are consistent with previous studies showing RAB-10 localization to the Golgi and early endosomes in the C . elegans intestinal cells [24] . Supporting a role in mediating vesicular trafficking , time-lapse recordings revealed that GFP::RAB-10 labeled vesicles moved bi-directionally along dendrites , consistent with localization to transport vesicles ( S1 Movie ) . Together , these data suggest that RAB-10 might mediate PVD dendrite outgrowth by regulating Golgi-to-membrane trafficking or endosomal membrane recycling events . To attempt to determine whether the PVD dendrite morphogenesis defect in rab-10 mutants might be due to a role for RAB-10 in Golgi-to-plasma membrane trafficking , endocytic recycling , or both , we perturbed each pathway and tested whether it altered PVD dendritic arborization . Consistent with previous studies in Drosophila and rat hippocampus neurons implicating ER-to-Golgi trafficking in dendritic growth , over-expressing dominant-negative RAB-1 ( a key regulator of ER-to-Golgi trafficking ) caused dramatically reduced branching in the PVD ( Table 1 , and S6 Fig ) [1] . In contrast , genetic loss of rme-1 or chat-1 , and over-expression of dominant-negative RAB-5 or RAB-11 . 1 ( all key regulators of endocytic recycling ) [30–33] , had no effect of PVD dendrite morphology ( Table 1 ) . Together , these data suggest a role for RAB-10 in mediating Golgi-to-plasma membrane trafficking that is important for dendrite morphogenesis . Notably , however , we cannot rule the possibility that RAB-10 regulates endosomal recycling independent of rme-1 , rab-5 or rab-11 . 1 . Thus , RAB-10 might also regulate an endosome-to-plasma membrane transport pathway independent of RME-1 , RAB-5 , RAB-11 . 1 function in the PVD neuron that is required for dendritic arborization . Rab GTPases are molecular switches that exert their functions by recruiting and releasing specific effectors [34] . We next sought to identify possible effectors that function with RAB-10 in mediating the branching and growth of PVD dendrites . We first examined EHBP-1 , an Eps 15 domain binding protein that acts as an effector of RAB-10 in endocytic recycling and secretory pathways [26] . Mutant animals of ehbp-1 , however , had normal PVD dendritic arbors ( Table 1 ) . Importantly , we cannot fully exclude the possibility that the PVD dendrite development was rescued by maternally loaded ehbp-1 mRNA or EHBP-1 protein , since we can only examine ehbp-1 homozygous animals derived from heterozygous mothers . We next tested members of the exocyst complex , an established effector of yeast Sec4p with which RAB-10 shares high homology [35] . Notably , loss of the exocyst subunit exoc-8 in viable null mutant animals or loss of sec-8 in the mutant progeny of heterozygous sec-8 mutant mothers , caused a PVD dendrite morphogenesis defect in the proximal but not in the distal region ( Table 1 , and Fig 3A–3F ) [36] . Further , the growth of the primary dendrite and the axon was normal ( Fig 3A–3C , S1 Fig ) . This phenotype was similar to rab-10 mutants , although the growth of secondary dendrites was only mildly affected in exoc-8 and sec-8 mutant worms ( Fig 3A–3F ) . Expression of exoc-8 cDNA under the PVD-specific promoter ( ser2prom3 ) fully rescued the dendritic arborization defect in exoc-8 ( ok2523 ) mutant worms , indicating that the exocyst complex functions in the PVD to promote dendritic arborization ( Fig 3G ) . Homozygous mutants of two other exocyst components , sec-5 and sec-10 , derived from the heterozygous mothers did not show any obvious PVD dendrite defect ( Table 1 ) . To test whether this was due to maternal rescue , we used a newly developed targeted protein degradation system to specifically remove the SEC-5 protein from the PVD [37] . This system takes advantage of cell type specific expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1 , which recognizes proteins tagged with the 36 amino acid ZF1 zinc-finger domain . To selectively remove SEC-5 in the PVD neuron , we drove ZIF-1 in PVD using the ser2prom3 promoter in a sec-5::zf1::yfp knock-in strain ( sec-5 ( xn51 ) ) —a strain where both copies of the endogenous sec-5 genes are tagged with zf1 ( Fig 4A ) [37] . Confirming a role for the exocyst complex in promoting dendritic arborization , depleting ZF1::YFP tagged SEC-5 in PVD resulted in PVD dendrite arborization defects ( Fig 4C–4E ) . In some of these animals , both distal and proximal regions lacked menorah structures , suggesting that sec-5 might be important for the growth of both proximal and distal dendrites ( Fig 4D ) . Control animals expressing ser2prom3>ZIF-1 transgene alone and control animals expressing ZF1::YFP tagged SEC-5 alone had normal PVD dendrite morphology ( Fig 4B and 4E ) . We conclude that the exocyst complex plays a cell-autonomous role in promoting dendritic arborization and that maternal contributions of some of exocyst components contribute to this function . Further , based on the more severe PVD phenotype after ZF-1 tag-directed loss of sec-5 versus the exoc-8 null mutant , our results also suggest that exocyst components have different requirements ( perhaps reflecting their differential necessity for exocyst activity ) during PVD dendrite morphogenesis . Yeast Sec4p and the exocyst complex function together to promote docking and possible fusion of post-Golgi vesicles [17] . Thus , we hypothesized that the PVD dendritic arborization defects in rab-10 , exoc-8 and sec-8 mutants might in part be due to the failure of docking of post-Golgi vesicles . To test this , we built fluorescent reporters for the dendritic membrane proteins , DMA-1 and HPO-30 , transmembrane proteins expressed in PVD neurons that function to mediate dendritic branching and stabilization [9–12] . Consistent with previous studies , DMA-1::GFP and HPO-30::GFP were localized in the dendritic membranes , and in some intracellular vesicles in wild-type animals ( Fig 5A–5C , and S7 Fig ) [9 , 12] . These two transgene reporters likely represent endogenous protein localization , as the transgenes rescued the PVD dendrite arborization defects of dma-1 ( tm5159 ) and hpo-30 ( ok2147 ) mutants ( S5 Fig ) . Consistent with a role in docking and fusion of vesicles , loss of rab-10 and exoc-8 caused severe accumulation of DMA-1::GFP containing vesicles within the growing PVD dendrites ( Fig 5D–5I ) . We quantified the number of vesicular units ( which might be a single vesicle or a vesicle cluster ) in three areas—the distal , middle and proximal regions . We found that wild-type animals contained on average 29 . 6±1 . 9 vesicular units in a 76 . 8μm x 76 . 8 μm area from the middle region of the PVD . In contrast rab-10 ( ok1494 ) and exoc-8 ( ok2523 ) mutant worms contained a dramatic two-to-three fold increase in vesicular units—65 . 6±2 . 8 and 87 . 8±6 . 8 , respectively ( Fig 5J ) . Furthermore , in wild-type animals the DMA-1::GFP containing vesicles mainly localized to the primary dendrites , and rarely appeared in the higher-order dendrites ( including the secondary , tertiary and quaternary dendrites; Fig 5A–5C ) . In contrast , in rab-10 ( ok1494 ) and exoc-8 ( ok2523 ) mutant worms , numerous vesicles appeared in the higher-order dendrites ( Fig 5D–5I and S8 Fig ) . The presence of numerous DMA-1::GFP intracellular vesicles in rab-10 and exoc-8 mutants suggested that delivery of DMA-1 to the membrane might be reduced . To test this idea , the fluorescence intensity of DMA-1::GFP at the surface ( which we presume is predominantly plasma membrane localization ) of the primary dendrites was determined . In the distal region of the dendritic arbor , the intensity of DMA-1::GFP at the primary dendrite in rab-10 ( ok1494 ) and exoc-8 ( ok2523 ) mutant worms was decreased by 63 . 9% and 47 . 8% compared with wild-type animals ( Fig 5K ) . In the rab-10 mutants , reduction of dma-1 by RNAi-mediated knock-down further suppressed distal dendritic arborization , suggesting that the reduced levels of DMA-1 on the surface of distal dendrites is sufficient to promote dendritic stabilization and branching ( S9 Fig ) . In the proximal region , the decrease in DMA-1 was more dramatic . The intensity of DMA-1::GFP at the surface of the primary dendrite in rab-10 ( ok1494 ) and exoc-8 ( ok2523 ) mutant worms was decreased by 89 . 5% and 90 . 3% in the middle regions , and 94 . 4% and 94 . 8% in the tail regions compared to wild-type animals ( Fig 5K ) . HPO-30::GFP showed similar vesicular accumulation and decreased dendrite surface localization in rab-10 ( ok1494 ) and exoc-8 ( ok2523 ) mutant animals ( S7 Fig ) . These results offer compelling evidence that rab-10 and exocyst activity are required for the vesicular delivery of DMA-1 and HPO-3 to the dendritic membrane . The similar phenotypes after loss of exocyst components and rab-10 , as well as functions of these molecules in yeast and vertebrates in delivering vesicles to the plasma membrane [17 , 19 , 35 , 38 , 39] led us to examine whether RAB-10 and exocyst components coexist in vesicles in the PVD dendrites . EXOC-8::GFP and mCherry::RAB-10 strongly colocalized on intracellular vesicles ( Fig 6A ) , indicating that the exocyst might function together with rab-10 to mediate vesicle delivery . Next , we examined the subcellular localization of RAB-10 in animals harboring mutations in the exocyst components exoc-8 and sec-8 . Loss of these exocyst components caused a dramatic accumulation of RAB-10 labeled vesicles in the PVD dendrites ( Fig 6B ) . Wild-type animals contained 39 . 8±2 . 6 GFP::RAB-10 vesicular units in a 88 . 1μm x 88 . 1 μm area from the middle region of the PVD neuron . Worms with mutations in exoc-8 ( ok2523 ) and sec-8 ( ok2187 ) contained over three fold more GFP::RAB-10 vesicular unites—129 . 7±7 . 5 and 127 . 6±2 . 7 , respectively ( Fig 6C ) . To determine whether the accumulated vesicles containing GFP::RAB-10 were the same population observed in exoc-8 ( ok2523 ) mutants carrying DMA-1::GFP or HPO-30::GFP , we expressed DMA-1::GFP or HPO-30::GFP and mCherry::RAB-10 in exoc-8 ( ok2523 ) mutant animals . Confirming these are the same vesicle population , most of the mCherry::RAB-10 labeled vesicles also contained DMA-1::GFP or HPO-30::GFP ( Fig 6D and 6E ) . To test whether RAB-10 functions to recruit EXOC-8 onto vesicles , the EXOC-8::GFP reporter was crossed into the rab-10 loss-of-function mutant . EXOC-8::GFP , however still localized to vesicles in PVD neurons in rab-10 mutants ( S10 Fig ) . Taken together , these results suggest the exocyst complex promotes fusion of RAB-10 carriers within the PVD neuron to facilitate dendritic growth and stabilization , but that the exocyst complex is recruited to these vesicles in a RAB-10 independent manner . To investigate whether Rab10 and exocyst complex-mediated dendritic membrane transport is required for the dendrite morphogenesis in other organisms , we examined Drosophila class IV dendritic arborization neurons and cultured rat hippocampal neurons after loss of rab10 and exocyst components . In Drosophila , RNAi mediated knock-down of rab10 and exo84 significantly reduced dendrite branching and growth ( S11 Fig ) . Compared to animals treated with control RNAi , rab10 and exo84 RNAi targeted loss resulted in a 20% decrease in both the number of total end points and the total dendritic arbor ( S11 Fig ) . ShRNA mediated knock-down of rab10 , sec8 and exoc84 did not alter the total dendrite length in cultured rat embryonic hippocampal neurons , but did lead to a dramatic 67% reduction in the density of dendritic spines at 21 days in vitro ( DIV ) compared to control shRNA treated neurons ( S12 Fig ) . Collectively , we conclude that rab10 and the exocyst complex have important and likely conserved roles during dendrite morphogenesis . Since some higher-order dendrites in the PVD still formed in rab-10 null alleles , we hypothesized that another Rab protein might regulate dendritic branching . To test this idea , we examined the function of the RAB-10 related GTPase RAB-8 [40] . Notably , homozygous viable rab-8 ( tm2526 ) null mutant animals showed normal PVD morphology ( Fig 7A ) . This suggested that if RAB-8 functions in the PVD , it might act redundantly with RAB-10 . To test this idea , we first attempted to create animals with null mutations in both rab-8 and rab-10 . The rab-8 and rab-10 double mutant animals , however , were sterile , which made it challenging to determine PVD morphology [26] . Interestingly , we found that expression of a dominant-negative RAB-8 ( T22N ) in the PVD neuron caused a severe dendrite morphogenesis defect ( S3 Fig ) . In 15% ( n = 40 ) and 12% ( n = 50 ) of transgenic animals ( two independent lines ) harboring the transgene of dominant negative RAB-8 , the dendritic growth in both distal and proximal regions was dramatically reduced ( S3 Fig ) . These results suggest that the dominant negative RAB-8 might act to block the function of both the RAB-8 and RAB-10 proteins ( perhaps through inhibition of a common guanine nucleotide exchange factor ) and that RAB-8 may function redundantly with RAB-10 to promote PVD morphogenesis . To more directly test the idea that RAB-10 and RAB-8 function together in regulating PVD morphogenesis , we took advantage of a newly developed CRIPSR/Cas9-mediated conditional knock-out method and specifically disrupted the function of the rab-10 gene in the PVD neuron and other descendants of the seam cell lineage by restricting Cas9 endonuclease expression using nhr-81 promoter ( Pnhr-81>Cas9 ) [41 , 42] . In three separate lines , approximately 5–10% of animals ( 11 . 6% ( 35/303 ) , 5 . 1% ( 4/78 ) and 4 . 9% ( 3/61 ) ) targeted with conditional PVD knock-out of rab-10 ( which we refer to as rab-10 ( cKO ) ) generated a PVD phenotype . Animals displaying a PVD dendrite arborization defect , showed a similar phenotype as that of rab-10 null mutants ( Fig 7B ) . These results suggest that the CRIPSR/Cas9-mediated conditional knock-out only disrupts the rab-10 gene in the PVD lineage in a small percentage of transgenic animals , but that when it does target rab-10 , it completely perturbs rab-10 function . We crossed the most penetrant rab-10 ( cKO ) line into the rab-8 deletion mutant . We hypothesized that if rab-8 functions redundantly with rab-10 , it should enhance the rab-10 ( cKO ) PVD phenotype . From 302 animals that carried the Pnhr-81>Cas9 and PU6>rab-10-sgRNAs transgene , 27 animals ( 8 . 9% ) showed severely defective PVD dendritic arbors . We measured the total number of secondary dendrites , and found that the rab-10 ( cKO ) rab-8 ( tm2526 ) animals had fewer secondary dendrites than rab-10 ( cKO ) alone ( Fig 7E ) . Further , we observed that 55 . 6% ( 15/27 ) of rab-10 ( cKO ) rab-8 ( tm2526 ) animals had truncated posterior primary dendrites , which was rarely observed in rab-10 ( cKO ) or rab-8 ( tm2526 ) strains ( 2 . 8% ( 1/35 ) and 0% ( 0/25 ) , respectively ) ( Fig 7F ) . Taken together these results suggest that the related GTPases RAB-8 and RAB-10 function redundantly to promote dendritic morphogenesis in the PVD neuron .
Previous work in Drosophila dendritic arborization neurons identified rab1 , sec23 and sar1 , three genes that mediate ER-to-Golgi traffic , as essential regulators of membrane addition during dendritic growth [1] . This study also demonstrated that laser ablation of Golgi-outposts within the dendrites reduced dendrite arborization . Together this work strongly implicated ER-to-Golgi trafficking as well as Golgi outposts as essential for dendrite morphogenesis . These findings left open the question , however , of how membrane trafficking from the Golgi to the dendritic plasma membrane is regulated . Through a candidate-based genetic screen and analysis of the C . elegans multidendritic PVD neuron we have identified the small GTPase Rab10 as a post-Golgi regulator of vesicle trafficking required for dendrite morphogenesis . Rab10 is known to regulate both Golgi-to-plasma membrane vesicle trafficking and endocytic recycling [13 , 22 , 24–26 , 43–45] . Our data suggest that the C . elegans RAB-10 protein primarily promotes post-Golgi trafficking in the PVD neuron to facilitate dendritic growth and branching . We did not observe any obvious PVD dendrite morphogenesis defects after inhibiting numerous important mediators of endocytic recycling . This includes rme-1 and chat-1 mutant animals and animals carrying transgenes for dominant-negative rab-5 or rab-11 . 1 GTPases [30 , 31] . In contrast , animals expressing a dominant-negative rab-1 , which regulates ER-to-Golgi trafficking , showed dramatically reduced PVD dendrite branching and growth . Furthermore , animals harboring exoc-8 and sec-8 mutations , which encode two subunits of the exocyst complex that function to dock secretory vesicles onto fusion sites on the plasma membrane , led to PVD dendrite morphogenesis defects similar to rab-10 [17] . These data strongly implicate a function for the RAB-10 protein in mediating membrane trafficking from the secretory pathway that is crucial for dendritic branching in the PVD . Since we cannot rule out the possibility that RAB-10 might also regulate recycling of endosomes in a RME-1/RAB-5/ RAB-11 . 1-independent manner , we suggest it is possible that RAB-10 might also contribute to PVD morphogenesis by regulating endocytic trafficking ( see Fig 8 ) . Although loss of rab-10 resulted in a dramatic reduction in dendrite arborization in the PVD and FLP neurons , the absence of rab-10 function did not affect the growth of the PVD primary dendrites or the unbranched dendrites of the OLL , AWB and AWC neuron . This might reflect a heavy reliance on post-Golgi trafficking in the PVD and FLP neurons to supply the dramatic expansion in membrane required to form highly branched dendrites . In support of this idea , a recent study in Drosophila revealed that Rab10 and exocyst-mediated membrane trafficking is crucial for the elaborate branching of tracheal terminal cells [44] . It is also possible that rab-10 may transport cargoes that are necessary for the branching and growth of higher-order dendrites . Consistent with this notion , loss of rab-10 severely affected the dendritic transport of DMA-1 and HPO-30 , two dendritic specific transmembrane proteins that are required for the branching and stabilization of higher-order dendrites . The exocyst complex is a well-characterized effector of yeast Sec4p , which shares high sequence homology with RAB-10 [26] . In yeast the exocyst complex functions to target Sec4p secretory vesicles to sites of exocytosis [35] . Suggesting a similar mechanism in the PVD dendrite , we found that loss of the exocyst components exoc-8 and sec-8 caused a dendrite morphogenesis defect that was similar to rab-10 mutants . In animals carrying mutations in these genes , GFP tagged DMA-1 and HPO-30 were sequestered in intracellular vesicles and the dendrite surface expression was greatly reduced . The similarity of the phenotypes suggests that the exocyst complex functions as an effector of RAB-10 to mediate trafficking of secretory vesicles to the dendritic plasma membrane . This idea is further supported by the colocalization of RAB-10 and EXOC-8 on intracellular vesicles in the PVD dendrites . Notably , compared to the PVD dendrite morphogenesis defects observed in the rab-10 mutants , the dendrite phenotypes in exoc-8 and sec-8 mutants were not as severe . This could indicate that another mechanism mediates tethering , docking or fusion of RAB-10 cargo vesicles to the dendritic plasma membrane . However , it is likely that the exoc-8 and sec-8 mutant animals examined were not nulls for exocyst function . While the deletion allele of exoc-8 ( ok2523 ) is thought to completely remove exoc-8 function [36] , it does not lead to lethality , which is associated with loss of all other exocyst subunits . These observations suggest that loss of exoc-8 does not completely eliminate exocyst function [46] . Further , the sec-8 ( ok2187 ) homozygotes were obtained from heterozygous mothers that likely provided maternally loaded sec-8 mRNA or SEC-8 protein . Consistent with this idea , the removal of the exocyst component SEC-5 through ZF-1 tag-mediated degradation often resulted in a complete loss of dendritic branching . We thus suggest that the exocyst complex is the primary effector of RAB-10 . Further , given that loss of SEC-5 lead to a more severe dendrite arborization defect than loss of RAB-10 , it is likely the exocyst complex has functions outside of regulating RAB-10 vesicle trafficking ( possibly RAB-8 vesicle trafficking , see below ) . In rab-10 mutants , the growth and branching of more distal dendrites was minimally affected , suggesting that a rab-10 independent mechanism exists . Notably , expressing dominant negative RAB-10 in PVD caused both distal and proximal dendrite morphogenesis defects . A possible explanation is that dominant negative RAB-10 competes for RAB activators , such as a guanine nucleotide exchange factor ( GEF ) , that interferes with another RAB protein that is essential for distal dendrites . A strong candidate for this other RAB protein is RAB-8 . Although loss of rab-8 had no obvious effect on PVD dendrite morphogenesis , expression of a dominant negative RAB-8 in the PVD caused similar dendritic phenotype to the dominant negative RAB-10 . rab-8 is related to rab-10 , and rab-8 and rab-10 function redundantly in mediating secretion in C . elegans germ cells [26] . Using a newly developed CRIPSR/Cas9-mediated conditional knock-out method , we found that conditional knockout of rab-10 in a rab-8 null mutant showed stronger dendrite arborization defect than loss of rab-10 alone . These observations offer compelling evidence that RAB-10 and RAB-8 function redundantly to regulate PVD dendritic arborization . RAB-10 and the exocyst complex are evolutionarily conserved from C . elegans to humans . We observed that knock-down of rab10 or exo84 caused a significant reduction of the dendrite arbor in the Drosophila class IV dendritic arborization neuron . Knock-down of rab10 , exoc84 or sec8 had no effect on the total length of the dendrites in rat cultured hippocampal neurons , but the number of dendritic spines was greatly reduced . Compared to the PVD dendrite phenotypes of C . elegans rab-10 mutants , knock-down of rab10 in Drosophila and rat neurons showed weaker dendrite morphogenesis defects . This might be due to the use of null alleles of rab-10 in our studies of C . elegans compared to the RNAi strategy that led to reductions but not complete loss of rab10 function in Drosophila and rat . Consistent with this , knock-out of sec5 in Drosophila neurons causes more severe neurite outgrowth defect [47] . Nevertheless , our results imply that rab-10 and exocyst-dependent membrane transport is a conserved mechanism used to build dendritic arbors and dendritic spines during neural development . Dendrite morphogenesis defects are associated with many neurological and neurodevelopment disorders , including Autism spectrum disorders , Alzheimer’s disease and Parkinson’s disease [48 , 49] . Recovery from these diseases will rely on efficient dendrite regrowth and branching to form functional neuronal circuits . Here we identified RAB-10 and the exocyst complex as critical and conserved players during dendrite morphogenesis . Our findings reveal an evolutionarily conserved membrane transport mechanism that can efficiently supply membranes and newly synthesized transmembrane proteins to support rapid dendrite growth and morphogenesis . These findings may help in the development of new therapeutic strategies to help repair damaged neurons in human diseases and during aging .
All animals were euthanized in accordance with the recommendations of the American Veterinary Medical Association and the UCSF Institutional Animal Care and Use Committee . Rats were euthanized with CO2 prior to dissection . After treatment with CO2 , bilateral thoracotomy was performed to ensure the death of the animal . All experimental procedures used comply with regulations adopted by UCSF authorities with support from the Guidelines on Euthanasia of the American Veterinary Medical Association . C . elegans was grown on OP50 Escherichia coli-seeded nematode growth medium agar plates at 20℃ unless otherwise noted . Alleles used are as follows: Linkage group I ( LGI ) : rab-10 ( ok1494 ) , rab-10 ( dx2 ) , exoc-8 ( ok2523 ) , sec-8 ( ok2187 ) , exoc-7 ( ok2006 ) , rab-8 ( tm2526 ) and dma-1 ( tm5159 ) . LGII: sec-5 ( tm1413 ) , sec-5 ( xn51 ) , and rrf-3 ( pk1426 ) . LGIV: sec-10 ( tm3437 ) , and chat-1 ( ok1681 ) . LGV: ehbp-1 ( ok2140 ) , rme-1 ( b1045 ) and hpo-30 ( ok2047 ) . Primer sequences for genotyping are available on request . RNAi experiments were performed as previously described [50] , except that the rrf-3 ( pk1246 ) mutation was used to enhance the RNAi efficacy in the PVD neuron . The dma-1 RNAi clone was obtained from Vidal RNAi feeding library and verified by DNA sequencing [51] . Standard germ line transformation by gonadal micro-injection was used to generate transgenic lines . Plasmid DNAs and fusion PCR products were used at 1–20 ng/ μl , and co-injection marker unc-119 ( + ) or Pmyo-2>mcherry or Pmyo-3>mcherry or Punc-122>rfp or Podr-1>gfp was used at 1–30 ng/ μl . Chromosome integrated stable lines were generated by following gamma ray irradiation protocol [52] . Single copy transgene was generated by following a miniMos-based protocol [28] . Transgenes used are listed in S1 Table . All the plasmids were constructed using standard molecular cloning methods . pPD49 . 26 and pPD95 . 75 were used as vectors . Coding regions of rab-1 , rab-8 and rab-11 . 1 with dominant-negative mutations were amplified from a RAB toolkit [53] . Fusion PCR was performed as previously described [54] . Plasmids and fusion PCR product used are listed in S2 Table . Primers used are listed in S3 Table . pWZ243 Pnhr-81>Cas9 ( 20ng/ul ) , pWZ170 PU6>rab-10-sgRNA #1 ( target DNA sequence was 5’GAAGAGCATGTCATACGGT3’ ) ( 20ng/ul ) , pWZ171 PU6>rab-10-sgRNA #2 ( target DNA sequence was 5’GCAATTTGAAGAGCATGTCATA3’ ) ( 20ng/ul ) , Pmyo-2>mcherry ( 1ng/ul ) and Pmyo-3>mcherry ( 5ng/ul ) were injected into wyIs592 ( ser2prom3>myr-gfp ) worms . Transgenic lines were identified and maintained based on fluorescence of the mCherry co-injection markers expressed in the pharyngeal muscles and body wall muscles . L4 and young adult stage transgenic animals were quantified for PVD dendrite arborization defects . To quantify the percentage of animals with abnormal PVD dendritic morphology ( Table 1 ) , mid-L4 to young adult stage hermaphrodite animals were anesthetized using 1mg/ml levamisole in M9 buffer , mounted on 2% agar pads and examined for PVD morphology using a compound fluorescence microscope ( Carl Zeiss ) with a 63X/1 . 4NA objective lens . Wild-type animals develop many menorah-like structures with higher-order dendrites . An animal was considered abnormal if it lacked more than four menorahs ( either by lacking quaternary dendrites , tertiary dendrites or secondary dendrites ) or if it had truncated primary dendrites . To quantify PVD dendrite and axon morphology , z-stack images of PVD neurons were taken of mid- or late L4 stage animals using either a spinning disk confocal microscope ( Axio Imager; Carl Zeiss ) with a 40x objective lens ( 1 . 4 NA ) equipped with an EM charge-coupled device ( CCD ) camera ( Hamamatsu Photonics ) and a spinning disc confocal scan head ( CSU-10; Yokogawa Electric Corporation ) controlled by Micro-Manager software with 488 and 561 laser lines ( for images showed in Figs 1A–1C , 2A–2B , 3A–3B , and S2A–S2D and S3A–S3B ) , or using a Zeiss LSM710 confocal microscope ( Carl Zeiss ) with a Plan-Apochromat 40X/1 . 3NA objective ( for images showed in Figs 4B–4D , 7A–7D , S2E–S2F , S3C–S3D , S4A–S4B , S5A–S5F , S6A–S6C and S9A–S9B ) . The numbers of secondary , tertiary and quaternary dendrites were quantified using maximum intensity projections generated from z-stack images using ImageJ . Mid-L4 stage hermaphrodite animals were anesthetized using 1mg/ml levamisole in M9 buffer , mounted on 2% agar pads , and then single focus plane images for GFP::RAB-10 labeled vesicles in the primary dendrites were captured using a spinning disk confocal microscope with a 100x objective lens ( 1 . 4 NA ) . Images were taken 1 frame per 0 . 8 second for 1 . 6 minutes ( 120 frames ) . Movies were made using ImageJ . Mid-L4 stage transgenic animals were anesthetized using 1mg/ml levamisole in M9 buffer , mounted on 2% agar pads and dual color images were collected using a spinning disk confocal microscope ( Axio Imager; Carl Zeiss ) with either a 63x ( for images showed in Figs 2D–2E and 6D–6E ) or 100x ( for images showed in Fig 6A ) objective lens ( 1 . 4 NA ) . Colocalization between RAB-10 and RAB-5 , FAPP1-PH and EXOC-8 was quantified using Coloc 2 , a Fiji’s plugin for colocalization analysis ( http://fiji . sc/Coloc_2 ) . The Pearson correlation coefficient index is shown for each group . Z-stacks of ser2prom3>DMA-1::GFP and ser2prom3>HPO-30::GFP fluorescence in distal , middle and tail region of mid-L4 stage worms were taken using a spinning disk confocal microscope ( Axio Imager; Carl Zeiss ) with a 100x Plan-Aprochromat objective lens ( 1 . 4 NA ) . Fluorescence across the primary dendrites was quantified using the “measure” function of ImageJ software from single focal plane images . Background subtraction levels were determined from regions outside of the worms , lacking any GFP signal . Z-stacks of ser2prom3>DMA-1::GFP and ser2prom3>HPO-30::GFP fluorescence in distal , middle and tail region of mid-L4 stage worms were taken using a spinning disk confocal microscope ( Axio Imager; Carl Zeiss ) with a 100x Plan-Aprochromat objective lens ( 1 . 4 NA ) . Number of vesicular units ( either single vesicles or vesicle clusters ) was quantified from images of maximum intensity projections generated from z-stack images using ImageJ . The size of each image was 76 . 8μm x 76 . 8 μm . Z-stacks of ser2prom3>GFP::RAB-10 fluorescence in middle region of mid-L4 stage worms were taken using a spinning disk confocal microscope ( Axio Imager; Carl Zeiss ) with a 63x Plan-Aprochromat objective lens ( 1 . 4 NA ) . Number of vesicular units ( either single vesicles or vesicle clusters ) was quantified from maximum intensity projections generated from z-stacks using ImageJ . The size of each image was 88 . 1μm x 88 . 1 μm . The UAS-Gal4 system was used to express RNAi in the class IV neurons of Drosophila larvae . UAS-RNAi lines are available from Bloomington Stock Center: y[1] v[1]; P{y[+t7 . 7] v[+t1 . 8] = TRiP . JF02058}attP2 ( BL 26289 ) , y[1] v[1]; P{y[+t7 . 7] v[+t1 . 8] = TRiP . JF03139}attP2 ( BL 28712 ) and from the Vienna Drosophila Resource Center: w[1118]; P{GD16778}v46791/CyO; ( v46791 ) , w[1118]; P{GD11816}v30112/TM3; ( v30112 ) . UAS- RNAi lines were crossed to ppktdGFP; ppkgal4 , UAS-dcr2 virgins . Larvae resulting from this cross were imaged at third larval instar . The dendritic arbor was quantified as described [55] . Hippocampal neurons cultured from E19 Long-Evans rats ( Charles River Lab ) were plated at a density of 200 , 000 neurons per 18 mm acid treated glass coverslips ( Fisher ) coated with 0 . 06 mg/ml poly-D-lysine ( Sigma ) and 2 . 5 ug/ml Laminin ( Sigma ) . Neurons were plated using plating media ( Modified Eagle Medium + 10% Fetal Bovine Serum ( Hyclone ) , 0 . 45% dextrose , 0 . 11 mg/ml sodium pyruvate , 2mM glutamine , Reagents from UCSF cell culture facility ) . Cultures were transferred to maintenance media ( Neurobasal Media , Invitrogen+ 0 . 5mM Glutamine+1X B27 , Invitrogen and Penicillin/Streptomycin ) 4 hours post plating . Half of the media was replaced with fresh media every 4 days . All shRNA constructs were made in the pLentilox3 . 7 vector backbone using TTCAAGAGA as the loop sequence . Sequences used for constructing the shRNAs are: 1 ) Rab10 ( NM_017359 . 2 Start position: 2373 ) “TTGACTCTATCATTGTTTA” 2 ) Exoc84 ( NM_139043 . 1 Start position: 310 ) “CGCAGAACCTGAAGCGCAA” 3 ) Sec8 ( NM_053875 . 1 Start position: 887 ) “CCGTTAAAGCCATTAAAGA” . ShRNA transfections were performed using Lipofectamine-2000 ( Invitrogen ) following manufacturer’s guidelines . Transfections were done at DIV7 and DIV14 and fixed 48 hours post transfection at DIV9 and DIV16 , respectively using 4% PFA+4% sucrose at room temperature for 15 min . We did not observe toxicity or neuronal death in any of the shRNA treatment conditions . Neurons were blocked in blocking buffer ( 10% normal donkey serum+ 0 . 2M glycine+ 0 . 1% triton-x100 in Phosphate buffer saline ) for an hour followed by overnight incubation with primary antibodies ( mouse anti GFP , from Roche and rabbit anti-MAP2 from Chemicon ) . Coverslips were washed thrice in PBS and incubated for 2 hours with secondary antibodies ( mouse Alexa 488 and Rabbit Alexa 568 , from Jackson ImmunoResearch ) followed by three washes in PBS before mounting onto slides for analysis using Fluromont mounting media from EMS ) . Imaging of rat hippocampal neurons was performed on Leica Sp5 scanning confocal microscope with Laser lines 488 and 561 using a 40X NA 1 . 25 objective with zoom 0 for dendrite length and zoom 3 . 0 for dendritic spine imaging . Confocal image stacks were acquired at 1024X1024 pixels with 0 . 4micron z spacing between two frames such that the entire depth of the neuron/dendrite was imaged . Analysis was performed on maximum projected image stacks using ImageJ . ShRNA transfected cells were identified by GFP expression . Neurite length was calculated using the ‘Measure’ function of ImageJ and dendrite density was calculated by manually counting the number of spines per 100 microns of dendrite length . One-way ANOVA followed by post-hoc comparisons using the Dunnett’s test was used for all the figures , except S11 Fig ( one-way ANOVA followed by post-hoc comparisons using the Holm-Sidak test was used in this figure ) . Error bar means standard error of mean ( SEM ) for all the figures except S11 Fig ( error bar means standard deviation in this figure ) . | Dendrites are cellular extensions from neurons that gather information from other neurons or cues from the external environment to convey to the nervous system of an organism . Dendrites are often extensively branched , raising the question of how neurons supply plasma membrane and dendrite specific proteins from the source of synthesis inside the cell to developing dendrites . We have examined membrane trafficking in the PVD neuron in the nematode worm C . elegans to investigate how new membrane and dendrite proteins are trafficked . The PVD neuron is easy to visualize and has remarkably long and widely branched dendrites positioned along the skin of the worm , which transmits information about harsh touch and cold temperature to the nervous system . We have discovered that a key organizer of vesicle trafficking , the RAB-10 protein , localizes to membrane vesicles and is required to traffic these vesicles that contain plasma membrane and dendrite proteins to the growing PVD dendrite . Further , our work revealed that a complex of proteins , termed the exocyst , that helps fuse membrane vesicles at the plasma membrane , localizes with RAB-10 and is required for dendrite branching . Together , our work has revealed a novel mechanism for how neurons build dendrites that could be used to help repair damaged neurons in human diseases and during aging . | [
"Abstract",
"Introduction",
"Results",
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"Materials",
"and",
"Methods"
] | [] | 2015 | RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization |
The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis . The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione , T ( SH ) 2] , a low molecular mass cosubstrate absent in the host . In pathogenic trypanosomatids , a single enzyme , trypanothione synthetase ( TryS ) , catalyzes trypanothione biosynthesis , which is indispensable for parasite survival . Thus , TryS qualifies as an attractive drug target candidate . A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species ( Trypanosoma brucei , Trypanosoma cruzi and Leishmania infantum ) . The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species , and/or to avoid assay interference . The screening assay yielded suitable Z’ and signal to noise values ( ≥0 . 85 and ~3 . 5 , respectively ) , and high intra-assay reproducibility . Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors . Compounds displaying multi-TryS inhibition ( N , N'-bis ( 3 , 4-substituted-benzyl ) diamine derivatives ) and an N5-substituted paullone ( MOL2008 ) halted the proliferation of infective Trypanosoma brucei ( EC50 in the nM range ) and Leishmania infantum promastigotes ( EC50 = 12 μM ) , respectively . A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites , which confirmed the on-target activity of these compounds . Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition . Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds , future optimization and screening campaigns should aim at designing and detecting , respectively , more potent and broad-range TryS inhibitors .
Protozoan parasites from the genus Trypanosoma and Leishmania are responsible for diseases affecting humans and their livestock . The zoonotic character of these diseases , which involve different insect species as vectors and wild animals as reservoirs , hamper the implementation of successful control strategies [1] . Immuno-prophylaxis is not yet available and for some species , such as T . brucei spp . and T . cruzi , appears unfeasible due to complex immune-evasion mechanisms [2 , 3] . So far , and probably for several decades ahead , chemotherapy remains as the sole choice of treatment . Only a handful of drugs are available to fight Chagas’s disease ( T . cruzi ) , sleeping sickness ( T . brucei gambiense and T . b . rhodesiense ) and the different forms of leishmaniasis ( Leishmania spp . ) . Unfortunately , they suffer from several drawbacks encompassing low efficacy , resistance and route of administration [4–7] . Moreover , several of these drugs ( e . g . nifurtimox , benznidazole and melarsoprol ) present a non-specific mode of action that accounts for their high toxicity [8 , 9] . Thus , the discovery of new chemical entities targeting specific and indispensable components of parasite metabolism is a priority for trypanosomiasis and leishmaniasis . The thiol-dependent redox metabolism is one of the unique metabolic features that distinguish trypanosomatids from humans and offer reliable molecular targets for selective drug development [10] . An example of genetic hallmarks of trypanosomatids is the lack of genes coding for glutathione reductase and thioredoxin reductase [11–13] , which fuel the major redox systems of most living organisms ( i . e . the glutathione/glutaredoxin system and the thioredoxin system ) with reducing equivalents . Instead , trypanosomatids rely on the low molecular mass thiol N1 , N8-bis ( glutathionyl ) spermidine [trypanothione , T ( SH ) 2] and the flavoenzyme trypanothione reductase for sustaining the intracellular redox homeostasis ( Fig 1 ) . T ( SH ) 2 delivers reducing potential to different redoxin proteins , which , by acting on different targets , control vital functions ( for a review see [10]; Fig 1 ) . Proposed functions of T ( SH ) 2 further comprise neutralization of xeno- and endobiotics ( e . g . methylglyoxal , iron and nitric oxide ) , coordination of iron-sulfur complexes and reduction of ascorbate [14 , 15] . The biosynthesis of T ( SH ) 2 is achieved in two consecutive steps each involving the ligation of a glutathione ( GSH ) molecule by its glycine carboxyl group to the free N1 and N8 amine groups of spermidine ( SP ) . Both reactions are catalysed by the C-terminal ligase domain of trypanothione synthetase ( TryS; EC 6 . 3 . 1 . 9 ) at the expense of ATP ( Fig 1 ) . Some trypanosomatids species harbour ( L . infantum , L . donovani , L . mexicana and T . cruzi ) or express ( Crithidia fasciculata ) a gene coding for glutathionylspermidine synthetase ( GspS; EC 6 . 3 . 1 . 8 ) , which synthesizes the reaction intermediate N8 mono-glutathionylspermidine . The importance of TryS activity for parasite viability has been demonstrated in vitro and in vivo for T . b . brucei [16–19] and L . infantum [20] by means of genetic and pharmacological approaches . In addition , TryS presents several advantages as a drug target candidate: ( i ) it is encoded by a single copy gene [11–13] , ( ii ) the structure of TryS from L . major has been elucidated [21] , ( iii ) TryS has been shown to provide metabolic control to the trypanothione pathway in T . cruzi [22] , and ( iv ) kinetic information is available for several TryS [18 , 22–27] . At an early state of knowledge , the rational inhibitor design was undertaken using GspS of C . fasciculata ( CfGspS ) and Escherichia coli ( EcGspS ) as test enzymes and compounds isosteric with GSH or related transition state analogues as chemical scaffolds [28–34] . Preliminary studies with GSH analogues identified the γ-glutamyl moiety as critical for molecular recognition [28] . Further work revealed that addition of acidic groups to the L-γ-Glu-L-Leu dipeptide resulted in CfGspS inhibitors of reasonable potency such as the phosphonic ( Ki ~ 60 μM ) [29] , the boronic ( Ki ~ 81 μM and Ki* ~ 18 μM ) [34] or the diaminopropionic acid derivative ( Ki ~ 7 . 2 μM and Ki* ~ 21 μM ) [35] . Transition state mimics , previously identified as potent inhibitors of EcGspS [30–32] , proved to be equally active against recombinant CfGspS . Notably , a Gsp-phosphinate derivative was capable to inhibit recombinant TryS from L . major , T . cruzi and T . b . brucei albeit with apparent Ki values 16–40-fold higher than that obtained for CfGspS ( Ki of 18 . 6 nM ) [36] . Unfortunately , these compounds displayed null biological activity against pathogenic trypanosomatids at 100 μM . Nevertheless , this phosphinate remained the only compound able to target TryS from three different pathogenic trypanosomatids . The anti-proliferative activity of GSH derivates ( N , S-blocked GSH diesters , S-2 , 4-dinitrophenyl-GSH ) [37–39] and GSH-related phosphinopeptides [40] ranked from 35 to 0 . 2 μM in different trypanomatids . The poor biological activity exhibited by these inhibitors has been ascribed to their peptidic nature susceptible to hydrolysis by esterases and amidases [37] . Based on the ATP-dependency of TryS , a compound library of protein kinase inhibitors was screened against recombinant CfTryS . This led to the identification of N5-acetamide paullones ( benzo[2 , 3]azepino[4 , 5-b]indol-6-ones ) as potent inhibitors of CfTryS [10 , 41] . Recently , we reported the finding of a related 10-trifluormethylated acetamide derivative of this paullone as nM inhibitor of LiTryS ( IC50 350 nM ) [20] . Information on the activity of these paullones against TryS and parasites from other pathogenic species is lacking . More recently , a HTS campaign against TbTryS with a library of ~ 62000 compounds identified several hits that upon optimization yielded leaders with inhibitory activity against TbTryS in the nM range ( IC50 values of 45 , 95 and 140 nM for DDU86439 , DDD85811 and DDD86243 , respectively ) and EC50 values towards infective T . b . brucei that ranked from 5 to 10 μM . The compounds allowed the chemical validation of TbTryS as drug target [17 , 18] . We here describe the setting-up of a screening technique to detect TryS inhibitors and report the identification and chemical validation of inhibitors from TryS from three major trypanosomatid species ( Trypanosoma brucei brucei: T . b . brucei , Trypanosoma cruzi: T . cruzi and Leishmania infantum: L . infantum ) .
Unless otherwise stated all reagents were of analytical grade and purchased from Sigma-Aldrich , J . T . Baker , Carlo Erba Reagents SA , Gibco , Invitrogen , Life Technologies , Enzo Life Sciencies , Roche . TryS from different trypanosomatids was produced in recombinant form with an N-terminal His-tag . The constructs pET-15b TbTryS [27] , pRSET-B TcTryS and pET-28c ( + ) LiTryS [20] were kind gifts of Alan Fairlamb ( Dundee University , Dundee , Scotland ) , Sergio Guerrero ( Universidad Nacional del Litoral , Santa Fe , Argentina ) and Helena Castro ( Institute for Molecular and Cell Biology , Porto , Portugal ) , respectively . They were used to express TryS of T . b . brucei 427 ( MITat1 . 4 , GenBank accession protein id CAC87573 . 1 ) , T . cruzi strain Tulahuen 0 ( GenBank accession protein id AAO00722 . 1 ) and L . infantum JPCM5 ( GenBank accession protein id CAM69145 . 1 ) . E . coli strain BL21 ( DE3 ) or Tuner ( DE3 ) ( Novagen ) served as expression host . For a detailed description of the expression and purification protocols see S1 Text . Protein concentration was determined using the Bicinconinic Acid assay with bovine serum albumin as standard . The protocols described above yielded 4–8 mg of recombinant TryS per liter of culture medium with ≥ 95% purity and homogeneous specific activity . The kinetic characterization of His-tagged TryS was performed using the LDH/PK assay which couples ATP regeneration to NADH oxidation . The end-point assay based on detection of inorganic phosphate ( Pi ) by the BIOMOL GREEN reagent was used to estimate the apparent Ki for ADP . All reactions were performed at room temperature ( RT , 20–25°C ) and a detailed description of both assays is provided in S1 Text . The apparent kinetic parameters ( KM and Vmax ) were calculated by fitting plots of initial velocity ( v ) vs . substrate concentracion ( [S] ) , determined at saturating concentration of co-substrates , to the Michaelis Menten equation assisted by the software OriginPro 8 . For GSH , the KM and Ki values were determined using the following equation v = Vmax / ( 1 + KM / [GSH] + [GSH] / Ki ) , which considers the nonproductive binding of GSH to the substituted enzyme [42] . The apparent Ki for ADP was estimated from linear fitting of the plots [E]/v vs . [ADP] at different concentrations of ATP , where [E] is enzyme concentration . The compound library involves 144 chemical entities that are clustered by chemical scaffold as follow: ( A ) 6-arylpyrido[2 , 3-d]pyrimidine-2 , 7-diamine derivatives ( APPDA; S1 Table ) developed as ATP-competitive inhibitors of bacterial D-Alanine:D-Alanine ligase [43] and biotin carboxylase [44]; ( B ) 1- ( benzo[d]thiazol-2-yl ) -4-benzoyl-3-hydroxy-5-phenyl-1H-pyrrol-2 ( 5H ) -one derivatives ( BBHPP; S2 Table ) ; ( C ) N , N'-bis ( 3 , 4-substituted-benzyl ) diamine derivatives ( BDA; S3 Table ) that display potent anti-malarial or -trypanosomal/leishmanial activity [45 , 46] and are simplified derivatives of compounds interfering with the parasite’s polyamine metabolism [45 , 47–50]; ( D ) benzofuroxan ( BZ; S4 Table ) [51–55]; ( E ) 4 , 5-dihydroazepino[4 , 5-b]indol-2 ( 1H , 3H , 6H ) -one derivatives ( AI; S5 Table ) , some of which with reported anti-TryS or -T . b . brucei activity [56]; ( F ) 1H-purine-2 , 6 ( 3H , 7H ) -dione ( PD ) , including the 3-butyl-7-methyl-8- ( ( 3- ( trifluoromethyl ) phenylthio ) methyl ) 3 , 4 , 5 , 7-tetrahydro-1H-2 , 6-dione ) ( kindly provided by Dr . Luise Krauth-Siegel , Heidelberg University , Germany; S6 Table ) that was reported as inhibitor of the T ( SH ) 2-dependent oxidoreductase tryparedoxin [57]; ( G ) 2-aminooxazole-5-carboxamide derivatives ( AOCA; S7 Table ) ; ( H ) and several singletons: N , N-dibenzyl-1-ethyl-3-methyl-1H-pyrazole-5-carboxamide; 2-amino-N , N-dibenzyl-4-methyl thiazole-5-carboxamide; prochlorperazine; tert-butyl 8-aminooctylcarbamate; tert-butyl 12-aminododecylcarbamate; N- ( 8-aminooctyl ) acetamide . AAO00722 . 1; CAC87573 . 1; CAM69145 . 1
The kinetic studies of TryS employed in this work were performed with three major aims , first to test the quality of the recombinant enzymes and establish optimal assay conditions , second , to disclose kinetic data previously not addressed for TryS from T . cruzi strain Tulahuen 0 and L . infantum ( strain JPCM5 ) and a reaction product ( ADP ) , and third , to compare the kinetic behavior of different TryS and its implication for enzyme regulation and inhibition . The kinetic data available for TryS from earlier [18; 22–27] and the present work are presented in Table 1 and S1 Fig . LiTryS presented apparent KM values of 166 ± 62 μM for GSH , 1335 ± 167 μM for SP and 42 ± 10 μM for ATP , and a apparent Ki for GSH of 680 ± 160 μM , all of which are in the same order of magnitude as those reported for non-tagged TryS from the related species L . major [26] . The apparent kinetic parameters of TcTryS strain Tulahuen 0 ( KM GSH = 123 ± 23 μM , KM SP = 685 ± 105 μM , KM ATP = 41 ± 6 μM and Ki GSH = 1600 ± 230 μM ) are similar to those published for TcTryS strain Silvio X10 clone 7 [25] and strain Ninoa [22] , except that inhibition by GSH was not observed for TryS from the last strain [22] . The apparent KM values for substrates and Ki for GSH obtained for His-tagged TbTryS ( KM GSH = 135 ± 43 μM , KM SP = 238 ± 51 μM , KM ATP = 18 ± 6 μM and Ki GSH = 242 ± 102 μM ) were almost 3- to 7-fold higher than those reported for the untagged version of this protein ( KM GSH = 23 . 8–56 . 2 μM , KM SP = 37 . 8–92 μM , KM ATP = 6 . 6–8 . 6 μM and Ki GSH = 36 . 5–143 μM ) by different laboratories [24 , 27] , which may be ascribed to different assay conditions ( summarized in S8 Table ) as highlighted in previous publications [23 , 24] . Nonetheless , all values determined here for His-tagged tritryp TryS differed in less than one order of magnitude from those reported for tag-free versions from identical or homologue proteins , hence the recombinant form of the enzymes were rated as suitable for the screening assay . Inhibition by ADP has been reported to occur for CfGspS with a Ki of 80 μM [64] but information is lacking for related enzymes from pathogenic trypanosomatids . As shown here , ADP competed for the ATP-binding site of TbTryS , LiTryS and TcTryS ( S1 Fig ) with apparent Ki values of 40 ± 5 μM , 90 ± 8 μM and 60 ± 6 μM , respectively ( Table 1 ) . It is worth to note that these Ki values represent overall estimates of product inhibititon because the assay conditions used do not allow distinguishing a preferential inhibition of the first or second biosynthetic step catalyzed by TryS . Comparison of the kinetic parameters for TryS from three major trypanosomatid species obtained under similar experimental conditions ( this study ) , shows that the enzyme from African trypanosomes presents KM values for ATP and SP that are remarkable lower ( 2 . 3‒5 . 6-fold ) than those for TryS from L . infantum and T . cruzi ( Table 1 ) . A similar conclusion can be drawn from studies carried out for related TryS in other laboratories [18 , 22 , 24–27] . At variance with Leishmania spp . and T . cruzi , T . brucei spp . is an extracellular pathogen that fully relies on de novo synthesis of polyamines and glycolysis to fulfil its metabolic and energetic needs [58 , 65] . Thus , TryS from African trypanosomes presents kinetic parameters that guarantee the production of the indispensable metabolite trypanothione [16 , 17] under conditions of restricted supply of ATP and SP that the parasite may face during its complex life cycle ( e . g . differentiation , which entails drastic reprogramming of energetic metabolism , and different nutrient availability in vector and host ) . All three tritryp TryS displayed similar KM values for GSH , which contrast with earlier studies reporting a higher affinity of T . b . brucei TryS for this substrate [18 , 22 , 24 , 27] . As previously reported [18 , 23–27] , tritryp TryS were susceptible to substrate inhibition by GSH , although the inhibitory efficiency varied between species as follows: T . b . brucei ( apparent Ki/KM GSH = 1 . 8 ) > L . infantum ( apparent Ki/KM GSH = 4 . 1 ) > T . cruzi ( apparent Ki/KM GSH = 13 . 0 ) . This suggests that the T . b . brucei enzyme is particularly sensititive to inhibition by the substrate GSH . However , the physiological role of this inhibition mechanism , if any , remains questionable because substrate accumulation will further enhance TryS inhibition and GSH cannot surrogate T ( SH ) 2 functions in T . brucei [16] . For the leishmanial and T . cruzi TryS , substrate inhibition will become relevant only at high concentrations of GSH ( e . g . > 1 . 5 mM ) , which , except for a few examples of L . donovani parasites grown to mid-log phase ( e . g . 2 . 27 mM GSH for axenic amastigotes from the strain BOB and 1 . 68 mM for promastigotes from the strain LV9 ) [14] , appears to be a non-physiological condition . Indeed , L . infantum parasites harbouring a single trys allele and with a TryS content about 50% lower than that of wildtype cells show no phenotype in vitro and in infected animals [20] . The Ki ADP / KM ATP ratios for tri-tryp TryS range from1 . 5 to 2 . 2-fold , suggesting that ADP may be a physiological modulator of TryS activity . However , taking into account that the intracellular concentration of ATP for trypanosomatids ( e . g . 2–4 mM and 0 . 58 mM for infective T . b . brucei [65 , 66] and T . cruzi [67] , respectively , and 0 . 87 mM for L . donovani promastigotes [68] ) is > 14-fold in excess with respect to the respective KM values for tritryp TryS ( e . g . 18–60 μM; Table 1 ) and that the ATP/ADP ratio reported or estimated by us for trypanosomatids is between 3 to 10 [65 , 66 , 69–71] , such regulatory role of ADP in T ( SH ) 2 biosynthesis may be questioned . A recent kinetic analysis of TbTryS assisted by computational modeling of the enzymatic mechanism [24] highlighted that the activated enzyme is particularly sensitive to inhibition by GSH and T ( SH ) 2 . Because the experimental setup employed in this study did not consider inhibition by ADP and excluded ADP as an integral component of the enzyme activated complex , the real contribution of this product to TryS inhibition remains to be addressed . The biological activity of the most active inhibitors of TbTryS ( i . e . IC50 ≤ 30 μM; MOL2008 , ZEA10 , EAP1-47 , EAP1-63 , APC1-89 , APC1-99 , APC1-101 , APC1-111 ) and LiTryS ( i . e . IC50 < 1 μM; MOL2008 and FS-554 ) was evaluated against the infective form of T . b . brucei or promastigotes of L . infantum and murine macrophages . Except for the paullone MOL2008 ( EC50 4 . 3 ± 0 . 7 μM ) , all other TbTryS inhibitors presented anti-T . b . brucei activity in the nM range . The most active being the BDA APC1-99 ( EC50 15 ± 1 nM ) , APC1-111 ( EC50 40 ± 1 nM ) , APC1-89 ( EC50 61 ± 1 nM ) and EAP1-63 ( EC50 90 ± 7 nM ) , followed by ZEA10 , EAP1-47 and APC1-101 with EC50 between 200–280 nM ( Table 2 ) . In comparison to paullone FS-554 ( EC50 112 . 3 ± 1 . 1 μM ) [20] , MOL2008 displayed a 10-fold higher anti-leishmanial activity ( EC50 12 . 6 ± 1 . 6 μM ) . The selectivity of the anti-parasitic effect was assessed using murine macrophages . Most compounds , except for MOL2008 , FS-554 and ZEA 10 , presented a selectivity index ( SI ) ≥ 10 with EAP1-63 ( SI = 124 ) and APC1-89 ( SI = 164 ) having the highest selectivity towards bloodstream T . b . brucei ( Table 2 ) . In order to get an insight into the on-target activity of promising compounds , the intracellular thiol content of WT parasites exposed for 24 h to EAP1-47 and MOL2008 was determined . For bloodstream T . b . brucei treated with 50 nM EAP1-47 , T ( SH ) 2 content decreases by 28% while GSH level increases by 39% with respect to untreated cells , which overall resembles the metabolite changes observed for parasites with 48 h RNAi-downregulated expression of TryS ( S11 Table and [16] ) . A compound acting on T ( SH ) 2 metabolism is expected to display an increased cytotoxicity against parasites depleted in TryS . The cytotoxic effect of 100 nM EAP1-47 towards trypanosomes with a TryS content that is 1/3 of that corresponding to non-induced RNAi or wild-type cells was slightly increased ( 1 . 3-fold; Fig 5 ) . For comparison , nifurtimox , a drug inducing thiol depletion [73] , added at 5 μM ( EC50 determined for WT cells ) displayed a 1 . 8-fold increased potency towards TryS-depleted parasites . Strikingly , identical assays performed with the chemically-related and trypanosome-selective compounds EAP1-63 and APC1-99 did not show differences in the potency of these compounds towards WT or TryS-depleted parasites . Taking together , these results shows that EAP1-47 is interfering with T ( SH ) 2 biosynthesis , although the almost two order of magnitude difference between TbTryS inhibition and EC50 indicates that the compound has also other molecular targets in vivo . In contrast , the lack of enhanced cytotoxicity displayed by EAP1-63 and APC1-99 towards the TryS-RNAi induced cell line , is an strong indication that their potent trypanocidal activity is unrelated to interference with T ( SH ) 2 metabolism . On the other hand , L . infantum promastigotes in the log growth phase exposed to MOL2008 at its EC50 ( 12 μM ) for 24 h presented a marked decrease in the intracellular pool of thiols . From two independent experiments , T ( SH ) 2 and GSH content decreases ≥ 90% and > 30% , respectively , upon MOL2008 treatment ( S11 Table ) . Interestingly and at variance with the metabolic changes observed for the genetic ( RNAi ) or chemical ( EAP1-47 ) silencing of TryS in T . b . brucei , inhibition of TryS by MOL2008 did not lead to accumulation of the substrate GSH in L . infantum . Further experiments are required to establish whether the reduction on GSH level is caused by paullone-mediated inhibition of any of the two ATP-dependent enzymes in charge of glutathione synthesis or is a consequence of a species-specific regulatory mechanism triggered by T ( SH ) 2 depletion .
The screening of a compound library consisting of 7 major chemical scaffolds and several singletons against TryS from three major pathogenic species of trypanosomatids at near physiological concentration of substrates led to the identification of 15 inhibitor molecules with μM ( APPDA , BDA , BZ , AI ) and sub-μM ( AI ) potency and a remarkable species-specificity for the molecular target . Despite the high sequence identity between tri-tryp TryS ( e . g . the amino acid sequence identity is 71 . 6% for TbTryS/TcTryS , 64 . 7% for TbTryS/LiTryS and 63 . 4% for TcTryS/LiTryS ) and the almost strict conservation of residues involved in substrate binding [20 , 21] , only 4 compounds were able to target TryS from different species , with only one of them ( a BDA ) inhibiting all three TryS with moderate potency ( 50% at 30 μM ) . This behavior highlights the existence of structural differences between tritryp TryS that determine their specificity for ligands . In support of this observation , single aminoacid substitutions in the homologue enzyme from C . fasciculata were previously reported to produce drastic changes in enzyme activity [23 , 36] and our comparative analysis of the kinetic parameters of tritryp TryS obtained under similar assay conditions reveals considerable differences between species . Early investigations have shown that substitutions at different positions of the paullone scaffold yield nM inhibitors of TryS from C . fasciculata or L . infantum ( N5-substituted AI ) [10 , 20] and potent anti-T . b . brucei agents with moderate inhibition towards TbTryS ( 11-substituted 4-azapaullone ) [56] . By extending the analysis of AI activity towards tritryp TryS , we have confirmed that the inclusion of a N-[2- ( methylamino ) ethyl] acetamide in position N5 confers SP-competitive inhibition of LiTryS activity , since compounds lacking this substitution were almost inactive . From the tritryp TryS evaluated here , LiTryS presented the higher KM for SP , which indicates the enzyme binds less efficiently this substrate probably due to an unfavorable conformation of the polyamine binding site . However , the shape adopted by the SP-binding site of LiTryS appears suitable for accommodating the N5-substituent present in MOL2008 and FS554 . In support of this hypothesis , the trypanosomal TryS displayed a 2- to 6-fold lower KM values for SP and a marked refractoriness to inhibition by N5-substituted paullones ( > 200-fold higher IC50 ) . The opposite behavior was observed for prochlorperazine , a SP-competitive inhibitor of TbTryS [18] . In this case , the degree of enzyme inhibition was inversely proportional to the SP KM value for each TryS , with TbTryS being the most sensitive to inactivation followed by TcTryS , whereas LiTryS was refractory to inactivation ( 300 μM prochlorperazine ) even at a sub-KM concentration of SP ( 240 μM vs . KM of 1335 μM ) . Altogether , these data strongly point to the existence of remarkable differences in the polyamine binding site of tritryp TryS that should be carefully considered for the design of new AI with multi-species activity . In this respect , our study also demonstrated that in spite of the potent anti-T . b . brucei activity [56] , AI substituted with α , β-unsaturated carbonyl chains in position 9 or 11 should be disregarded as ligands for future optimization because of their null to marginal anti-TryS activity . Both N5-substituted paullones identified as potent LiTryS inhibitors displayed an IC50 slope ( Hill coefficient ) below the unity ( ~0 . 35 ) , which suggests binding of the inhibitor to non-equivalent binding pockets or to partitioning of the compounds into an inactive , less potent or inaccessible form at higher concentrations [74] . Since both paullones displayed good solubility in the concentration range tested ( ~1 nM-50 μM ) and produced 100% LiTryS inhibition , we ruled out insolubility or aggregation phenomena as responsible of this behavior . On the other hand , the dose-response plots do not show the presence of a second inflection point that would be indicative of a second binding pocket for paullones . TryS are monomeric but mechanistically complex ( trisubstrate ) enzymes [23 , 24] whose substrate binding sites display large conformational changes during catalysis [21 , 75 , 76] , hence , it is possible that the low Hill slopes reflect an equilibrium between two or more forms of the enzyme that interact differentially with the paullones . Further mechanistic studies are needed to shed light on this issue . From this screening , MOL2008 was the most potent inhibitor identified for LiTryS that , in addition , shows potential for further optimization of its anti-trypanosomal TryS activity ( IC50 ~30 μM ) . The related paullone FS554 was chemically validated in a previous study using transgenic cell lines of L . infantum [20] . Here we show that MOL2008 is almost one order of magnitude more potent towards L . infantum promastigotes than FS554 and , more importantly , that it targets in vivo trypanothione biosynthesis . The future design of MOL2008 analogues should also aim at improving its biological properties , which according to our data are far from optimal ( EC50 in the μM range and SI ≤ 2 ) and suggest off-target effects in host cells . Precedent studies demonstrated that disustituted polyamines are potent antiproliferative agents that interfere with the polyamine metabolism of Plasmodium and African trypanosomes [47–50] . On the basis that the substituted diamine moiety may eventually be recognized as ligand by TryS , a collection of simplified derivatives was screened for their anti-synthetase activity . Two BDA ( EAP1-47 and APC1-111 ) displayed inhibitory activity , albeit moderate ( IC50 ~ 30 μM ) , against multiple TryS . SAR analysis revealed the need to fulfill a specific steric demand on the enzyme interacting pocket . A rational optimization of these compounds seems difficult , since the screening performed here included a wide diversity of derivatives ( mono- and di-substituted , with bulky groups or halogen atoms , and with linker of different length ) that yielded only weak TryS inhibitors . Nevertheless , as in a fragment-based approach , certain moieties of the most active BDA can be selected as substituents of novel or known TryS inhibitors . With few exceptions , most APC derivatives tested here were previously shown to display low to sub-μM potency towards infective T . b . brucei [46] . Our study confirmed and disclosed the potent ( EC50 15–280 nM ) and selective ( SI = 10–164 ) anti-parasitic activity of APC analogues and of two halogenated BDA from the EAP series , respectively , all of which exherted a moderate inhibition of TbTryS ( IC50 ~ 30 μM ) . Interestingly , EAP1-47 induced changes in the intracellular pool of low molecular thiols that overall resembled those triggered by RNAi-mediated silencing of TryS [16] and displayed a moderate increase in its cytotoxicity towards TryS-defficient cells that was not paralleled by the more potent and selective molecules APC1-99 and EAP1-63 . Nonetheless , as pointed out before , the higher in vivo ( anti-trypanosomal activity ) vs . in vitro ( TbTryS inhibition ) activity of EAP1-47 suggests that this compound is targeting other essential molecular target ( s ) in addition to TryS . The positive loop of substrate inhibition of TryS by GSH has a direct impact on the design of inhibitors . On the one hand , it suggests that groups competing with GSH can be excluded from the ligand scaffold to avoid unnecessary substrate competition and to reduce the molecular mass of the inhibitor . On the other hand , it shows that full inhibition of TryS is not required because the phenomenon of substrate inhibition will amplify in vivo the inactivation of the enzyme , which represents a pharmacological advantage of this molecular target . The four APPDA identified as moderate inhibitors of , preferentially , the T . cruzi TryS , are structuraly related to compounds originally designed as inhibitors of bacterial D-Ala:D-Ala ligase . Interestingly , these derivatives presented a 10-fold lower IC50 towards the trypanosomal enzyme than the analogue compounds against the corresponding bacterial target ( best IC50 = 260 μM ) [43] . The APPDA may serve as primary scaffolds to develope analogues with improved anti-TryS activity and , consequently , superior selectivity index . TcTryS , and to minor extent LiTryS , was target of inhibition by a BZ substituted with an imidazolone . A BZ with an additional imidazolone group retained activity against TcTryS but enhanced ATP hydrolysis by LiTryS and TbTryS , indicating that these ligands present a molecular pattern recognized by the active site of the enzymes that should be further investigated . Our work also disclosed several compounds enhancing ATP consumption by trypanosomal TryS . Adding value to the existence of distinctive structural features between tritryp TryS , TbTryS was targeted specifically by large heterocyclic compounds from the BBHPP whereas TcTryS was the most promiscuous enzyme , being activated by a PD , an APPDA and the BZ mentioned above . A single compound , a bis-imidazolone BZ , increased LiTryS activity . According to our results , the activation of ATPase activity in trypanosomal TryS by these compounds takes place only when both co-subtrates , namely GSH and SP , are present in the reaction . More detailed studies are required to establish whether such allosteric activation of TryS is paralleled by an increased production of T ( SH ) 2 . Compounds promoting the non-productive consumption of ATP are attractive candidates for biological evaluation , because the metabolic outcome of their action should be similar to that of true enzyme inhibitors . In summary , this study: ( i ) led to the identification and on-target validation of a potent inhibitor of leishmanial TryS and of chemical scaffolds that can be further developed into inhibitors of wide spectrum against TryS from different trypanosomatid species , ( ii ) highlights the existence of remarkable kinetic and structural differences between tritryp TryS that prompt to obtain 3D structures of TryS from different trypanosomatids in order to guide a structure-based rationale design of multi-TryS inhibitors; iii ) demonstrates the relevance of running a HTS under near physiological concentration of subtrates to minimize the possibility to detect weak competitive inhibitors with low potential to act in vivo | Parasites from the genus Trypanosoma and Leishmania are etiologic agents for a group of neglected diseases with high morbidity and mortality rates in the developing world . Inasmuch as vaccine development is hampered by the successful mechanisms employed by the pathogens to evade the host immune response , chemotherapy remains as a safe option to fight these diseases . However , new drugs with better pharmacological performance ( i . e . safety , efficacy and ease of administration ) than those in current use are urgently needed . The thiol-redox metabolism of trypanosomatids offers an excellent opportunity for the development of more selective and efficacious medicines because it depends on a molecule , trypanothione ( a bis-glutathionyl derivative of spermidine ) , unique and indispensable to the pathogens . Here we report the identification of novel inhibitors of trypanothione synthetase from three major trypanosomatid species of medical and veterinary relevance . Although highly conserved in sequence , trypanothione synthetases display significant species-specifity towards compounds , pointing to structural differences as determinants of ligand selectivity . Most of the active compounds presented two-digit μM inhibitory activity and serve as primary scaffolds to develop more potent inhibitors . Among them , N , N'-bis ( benzyl ) -substituted diamine and paullone derivatives are interesting candidates because of their potent and/or selective anti-trypanosomal and anti-trypanothione synthetase activity . | [
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"pharmacol... | 2016 | Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids |
Gene repression by transcription factors , and glucocorticoid receptors ( GR ) in particular , is a critical , but poorly understood , physiological response . Among the many unresolved questions is the difference between GR regulated induction and repression , and whether transcription cofactor action is the same in both . Because activity classifications based on changes in gene product level are mechanistically uninformative , we present a theory for gene repression in which the mechanisms of factor action are defined kinetically and are consistent for both gene repression and induction . The theory is generally applicable and amenable to predictions if the dose-response curve for gene repression is non-cooperative with a unit Hill coefficient , which is observed for GR-regulated repression of AP1LUC reporter induction by phorbol myristate acetate . The theory predicts the mechanism of GR and cofactors , and where they act with respect to each other , based on how each cofactor alters the plots of various kinetic parameters vs . cofactor . We show that the kinetically-defined mechanism of action of each of four factors ( reporter gene , p160 coactivator TIF2 , and two pharmaceuticals [NU6027 and phenanthroline] ) is the same in GR-regulated repression and induction . What differs is the position of GR action . This insight should simplify clinical efforts to differentially modulate factor actions in gene induction vs . gene repression .
The initial steps by which steroid receptors induce or repress target gene transcription are the same . After steroid binding to the intracellular receptor , the resulting complex is activated/transformed to a form with increased affinity for DNA [1] and is concentrated in the nucleus , where it is recruited to DNA sequences that are usually near the regulated genes . Cofactors and comodulators assist or impede the transcriptional activity of DNA-associated steroid receptors [2 , 3] . Beyond this , it is currently not possible to predict the transcriptional outcome for any specific combination of gene , receptor , and cofactor/comodulator . In most cells , a given steroid-bound receptor will induce one set of genes while repressing another set under otherwise identical conditions . For certain genes , the same receptor-steroid complex activates transcription in one cell line while repressing it in another cell line [4] . Similarly , selected cofactors increase the activity of one steroid receptor while reducing the activity of another receptor [5] . In some cases , different cofactors cause the same gene to be induced or repressed [6 , 7] . In other cases , the same cofactor may interact with the same steroid receptor to augment the induction of one gene but increase the repression of another gene [6–10] . Thus no relationship between induction vs . repression and presence of particular promoter/enhancer bound factors and cofactors has yet emerged [11] . The DNA sequence to which the receptor is recruited , either by direct DNA binding or by tethering to another DNA-bound molecule , can often indicate the resultant activity of induction or repression respectively [11–13] . Even this categorization , though , is not precise as repression can occur from GR binding directly to DNA [14 , 15] and the outcomes can depend upon whether the cofactor binds to DNA-bound glucocorticoid receptor ( GR ) or GR is tethered to DNA-bound cofactor [16] . An unresolved question is whether the underlying mechanism of each transcriptional component is the same or changes with the direction of gene expression output ( i . e . , increase in induction vs . decrease in repression ) . The answers are vital because efforts to modify the responses with selected factor combinations , be it in isolated cells or human patients , will depend critically on whether the mechanism of each component is constant or varies with the specific mixture of factors . Current attempts to address these issues have been inhibited by insufficiently precise methods of analysis . Thus , many cofactors are classified as either coactivators or corepressors based solely upon their ability to increase or decrease respectively , the level of steroid-mediated gene expression [2 , 17] . Unfortunately , while such descriptions are operationally useful , they are mechanistically uninformative . It is well known from enzyme kinetics that an enzymatic inhibitor can increase the total response while an enzymatic activator can lead to decreased output [18–20] . A more precise and quantitative understanding of the mechanisms of gene transcription is required to resolve these issues . Mathematical modeling provides one solution; and , a theory has been developed recently to understand the underlying mechanisms of factor action during steroid-regulated gene induction . The theory is based on the fact that the dose-response curve for gene induction is non-cooperative with a Hill coefficient of one [20] . This shape of the dose-response curve has also been variously described as a Michaelis-Menten function , hyperbolic dose-response , first-order Hill plot , and first-order Hill dose-response . The essential feature is that it is in the mathematical family of linear-fractional functions . The theory enables one to determine the kinetically-defined mechanism of factor action and the position of factor action in the many steps of the overall signaling cascade . This position is specified relative to both another competing factor and a steady-state analogue of a rate-limiting step called the concentration limited step ( CLS ) [20–27] . The concentrations of bound factors after the CLS are negligible compared to their unbound concentrations ( which could be involved in other reactions as long as they are readily available ) . A factor can act kinetically like an enzymatic activator , which we call accelerator , or an enzymatic inhibitor , which we call decelerator , depending on how the factor participates in the reaction [23] ( see Fig . 1 ) . These classifications describe how the factor alters a specific reaction step independently of the direction of change in the observed product . In all cases investigated so far , the reporter acts as an accelerator at the CLS and thus serves as an invariant positional landmark in the otherwise poorly defined landscape of reactions in steroid-regulated gene induction [23 , 25–27] . Here , we extend the mathematical theory and model to GR-regulated gene repression as influenced by varying concentrations of competing cofactors . The relevant mass-action equations have been used to relate the graphs of several reaction parameters derived from the dose-response to the kinetically-defined mechanism and position of action of each of the two competing cofactors . Using the extensively characterized system of GR repression of AP1 induction in U2OS . rGR cells with a transiently transfected synthetic reporter [8 , 15 , 28 , 29] , we show that four factors ( the reporter gene , TIF2 , and two small molecules [30] ) have the same kinetically-defined mechanism and position of action as in GR-regulated gene induction . The difference between induction and repression is the position of GR action . This suggests that many transcriptional cofactors/comodulators are mono-functional and similarly modulate basic steps in gene expression irrespective of the directional change in gene product levels .
Experimentally , the dose-response of gene activity A in steroid-regulated repression has been found to be non-cooperative with a Hill coefficient of one ( see Fig . 2 ) : i . e . A=Amax+Amin/IC50[S]1+1/IC50[S] ( 1 ) where Amax is the maximal activity with no added steroid , Amin is the minimum value of activity with saturating steroid concentrations , and IC50 is the concentration of steroid for half-maximal suppression . Added cofactors can change the parameters Amax , Amin and IC50 while preserving the shape of the dose-response ( 1 ) . Our goal was to develop a theory for gene repression that explains why the dose-response has the shape given in ( 1 ) and what that implies for the actions of the added cofactors that change the parameters . We use the fact that the linear-fractional shape of the dose-response curve puts severe constraints on the possible biochemical kinetic schemes involved in gene repression , as it did for gene induction [20] . We then derive formulas that can be compared to the data to make predictions for the actions of cofactors . Our data for the dose-response curve is based on the contributions from many copies of the induced gene in multiple cells . Hence , it is possible that the dose-response for a single gene could be different from the averaged population response . However , single-molecule imaging experiments for a gene induced by a nuclear receptor show that transcripts are produced in on-off stochastic bursts , where the bursting is well modeled as a stochastic process ( i . e . random telegraph model [31 , 32] ) and the burst probability is similar between cells . The frequency ( stochastic intensity ) of the stochastic bursts of individual genes follows a non-cooperative dose-response with respect to the inducing agonist [31 , 32] . Thus , the mean activity of a single gene follows the same non-cooperative dose-response and our theory explains the behavior of this mean . We first present a theory for which gene induction has a non-cooperative dose-response since the theory for gene repression hinges directly on it . Gene expression involves the binding of molecules , protein , and DNA into complexes that lead to transcription . We model this as a sequence of complex building reactions , Yi−1+Xi↔qiYi , where we call the Y variables products , the X variables accelerators , and the q’s are equilibrium or affinity constants . Decelerators of various types can also inhibit each of these reactions . Fig . 1 shows the general reaction scheme at each step . The accelerator X in Fig . 1 acts like an enzymatic activator and the decelerator D acts like an enzymatic inhibitor [23] . The kinetic scheme is stochastic and characterized by a probability distribution for the reactants . However , to simplify the calculations , we consider the mean field limit obeying the law of mass action and assume that the gene activity is proportional to the mean concentration of one or a set of products . The dose-response curve is the gene activity as a function of the concentration of the initial product [Y0] ( i . e . , agonist steroid ) . The theory can be illustrated by an example of induction with three reactions ( i = 1 , 2 , 3 ) in the absence of deceleration ( which we introduce later ) . Suppose the dose-response is given by [Y3] as a function of [Y0] . The goal is to calculate this function and determine conditions for when it is non-cooperative with unit Hill coefficient . In steady state , the concentrations obey the equilibrium conditions [Yi]=qi[Xi][Yi−1] , i=1 , 2 , 3 ( 2 ) and the mass conservation conditions [X1]+[Y1]+[Y2]+[Y3]=X1T[X2]+[Y2]+[Y3]=X2T[X3]+[Y3]=X3T where XiT is the total concentration of accelerator i . Together they form a system of 6 equations and 7 unknowns . Therefore , any one concentration can be solved in terms of any other . In general , the dose-response for this system will not have unit Hill coefficient [20] . However , a non-cooperative dose response can arise if [Y2] , [Y3]<<[Y1] and [Y3]<<[X3] so that the mass conservation equations become [X1]+[Y1]=X1T ( 3a ) [X2]+[Y2]+[Y3]=X2T ( 3b ) [X3]=X3T ( 3c ) This form for mass conservation can be achieved if the concentration of X2 is limited with respect to its binding affinity , i . e . , q2[X2]<<1 , while the other factors are not limited . This can be achieved biochemically if the products have short lifetimes , which has been observed experimentally [33–35] . We call Step ( 3b ) the concentration-limited step ( CLS ) [20] . All factors following the CLS are in excess ( i . e . , bound concentrations are negligible ) , implying that reactions after the CLS are pseudo-first order . Hence , the CLS is a step where the accelerator concentration is limited with respect to its binding affinity but the accelerator ( s ) following it are not limited . In the Methods , we give a detailed account of the CLS for an arbitrary number of reactions . Substituting ( 2 ) into ( 3a ) – ( 3b ) gives [X1]+q1[X1][Y0]=X1T[X2]+q[X2][Y1]+q3X3Tq2[X2][Y1]=X2T[X3]=X3T Each equation is bilinear in the accelerator and product concentrations . When the accelerator concentrations are substituted back into the equilibrium Equations ( 2 ) , the results are linear-fractional functions between adjacent products: [Y1]=q1X1T[Y0]1+q1[Y0] , [Y2]=q2X2T[Y1]1+q2 ( 1+q3X3T ) [Y1] , [Y3]=q3X3T[Y2] Linear-fractional functions form a group under function composition ensuring that the function of any product in terms of any other product is always linear-fractional [20] . Successive substitution of these functions yields the dose-response: A≡[Y3]=q1X1Tq2X2Tq3X3T[Y0]1+q1[Y0]+q2 ( 1+q3X3T ) q1X1T[Y0] where Amax=q1X1Tq2X2Tq3X3Tq1+q2 ( 1+q3X3T ) q1X1T , EC50=1q1+q2 ( 1+q3X3T ) q1X1T The dose-response can be derived for an arbitrary number of reactions as long as the mass conservation equations are bilinear as in ( 3 ) [20 , 22] . Although , our reactions are reversible and obey detailed balance , the theory can also incorporate dissipative irreversible steps ( see Methods ) [36] . Since [Y3] is proportional to [Y2] and this is true for all concentrations following the CLS , a more general form for the activity is the sum of all of these concentrations . Biophysically , this implies that the final product can arise from each step after the CLS independently . As shown in Fig . 1 , decelerators can interact with accelerators . Consider the competitive decelerator D interacting with X1 via X1+D↔X′1 . If D is in excess , the addition of D leads to one additional equilibrium condition , [X′1]=q′[D][X1] , and a modification to the mass conservation law for X1 ( 3a ) to [X1]+[Y1]+[X′]=X1T . Solving the new equilibrium and mass conservation conditions then gives [Y1]=q1X1T[Y0]1+q1[Y0]+q′[D] The equations for [Y2] and [Y3] are unchanged . Since [Y1] remains a linear-fractional function of [Y0] in the presence of inhibition by D , the dose-response will also remain a linear-fractional function . Activity can also be repressed by a reaction following the third reaction , Y3+X4↔Y4 , which can suppress [Y3] by diverting the product to a less productive pathway . The new reaction changes mass conservation for X2 ( 3b ) to [X2]+[Y2]+[Y3]+[Y4]=X2T which gives [Y2]=q2X2T[Y1]1+q2 ( 1+q3X3T ( 1+q4X4T ) ) [Y1] This is again linear-fractional , yielding [Y2]=q1X1Tq2X2T[Y0]1+q′[D]+q1[Y0]+q2 ( 1+q3X3T ( 1+q4X4T ) ) q1X1T[Y0] which is inhibited by X4T . Since [Y3]=q3X3T[Y2] and [Y4]=q4X4T[Y3] , the most general expression of the activity that preserves non-cooperativity is the sum: A=∑i=24ai−1[Yi] . The activity as a function of the agonist and any other factor is linear-fractional with the form A=V[Y0]1+W[Y0] ( 4 ) where V=q1X1Tq2X2T ( a1+a2q3X3T+a3q3X3Tq4X4T ) 1+q′[D] , W=q1+q2 ( 1+q3X3T ( 1+q4X4T ) ) q1X1T1+q′[D] ( 5 ) V = Amax/EC50 and W = 1/EC50 are linear-fractional functions of the equilibrium constants and the concentrations of all factors ( total concentration of accelerators and free concentration of decelerators ) in the system . From ( 4 ) and ( 5 ) , we see that activity can be repressed by the action of either a decelerator or an accelerator , provided the latter acts after the CLS and the contribution to the activity from [Y4] is less than that from [Y2] and [Y3] . We can write formulas for V and W for an arbitrary number of cofactors . Their functional forms are distinguished by the types of the cofactors ( i . e . , accelerators or one of 6 types of decelerator ) and where they act in relation to each other and the CLS . The formulas for V and W for all the possible combinations of 2 factors are calculated in Dougherty et al . [22] and shown in S1 Table . They are always linear fractional functions of each accelerator or decelerator . They can be used to make predictions of the mechanisms of added factors on the basis of the graphs of V and W vs . the cofactor just as the Lineweaver-Burk plot is used in enzyme kinetics . Finally , it is commonly accepted that transcription factors sometimes form oligomers before they act [17] . Our theory can be generalized to include factors acting through oligomers , provided that they do so non-cooperatively . For example , suppose that an accelerator X first forms an oligomeric complex with another factor Z in the reaction X+Z↔rXZ prior to interacting with a product Yi-1 in a reaction Yi−1+XZ↔qYi before the CLS . If Z is super-abundant compared to X then the concentration of the product as a function of the previous product has the non-cooperative form [Yi]=rqXT[Z]1+r[Z][Yi−1]1+rq[Z]1+r[Z][Yi−1] Hence , for accelerators acting through a hetero-oligomer , the limited factor , X , acts as an accelerator in our current theory while the action of the superabundant factor , Z , saturates with sufficiently high [Z] . A decelerator could likewise act through a hetero-oligomer . We apply the theory to steroid-regulated gene repression by observing that gene activity in induction can be repressed by other factors and still maintain a linear-fractional form . Hence , a linear-fractional dose-response ( 1 ) can arise in repression if the steroid-receptor complex ( GR ) acts as either a decelerator at any position or an accelerator after the CLS of a gene initiated by some other inducer . We compute the dose-response for steroid-regulated gene repression ( 1 ) by substituting the steroid-receptor complex ( GR ) ( or some activated form of GR ) into the formulas for V and W in the dose-response ( 4 ) for a gene activated by another inducer . Note that the experimentally measured dose-response of gene activity with respect to the inducer need not be linear-fractional for this theory to hold . What is required is that downstream steps where the cofactors and GR act have the linear-fractional property . It is well known that steroid binding to GR follows Michaelis-Menten kinetics in terms of [S] [20] . Suppose that GR acts as D in the above example . We substitute [D]=[GR]=G[S]K+[S] into Equation ( 5 ) to obtain V=q1X1Tq2X2T ( a1+a2q3X3T+a3q3X3Tq4X4T ) ( K+[S] ) [Y0]K+[S]+q′G[S] , W= ( q1+q2 ( 1+q3X3T ( 1+q4X4T ) ) q1X1T ) ( K+[S] ) [Y0]K+[S]+q′G[S] Substituting this into ( 4 ) and clearing the fractions results in an activity that is linear-fractional in [S] , which we can write as A=T ( [S] ) U ( [S] ) =T ( 0 ) +T′∗[S]U ( 0 ) +U′∗[S] ( 4’ ) where T=q1X1Tq2X2T ( a1+a2q3X3T+a3q3X3Tq4X4T ) ( K+[S] ) [Y0]U=K+[S]+q′G[ S ]+ ( q1+q2 ( 1+q3X3T ( 1+q4X4T ) ) q1X1T ) ( K+[S] ) [Y0] and the prime signifies derivative with respect to [S] . From this , we can thus surmise that Amax=T ( 0 ) U ( 0 ) , Amin=T′U′ , IC50=U ( 0 ) U′ These three dose-response parameters are determined by the four quantities T ( 0 ) , U ( 0 ) , T′ , and U′ which implies that the combination parameter: AmaxIC50Amin=T ( 0 ) T′ is also linear-fractional . The theory predicts that these four dose-response parameters are always linear fractional and that there are four compatibility conditions between them: a ) the numerator of Amax is equal to the numerator of Amax×IC50/Amin , b ) the numerator of Amin is equal to the denominator of Amax×IC50/Amin , c ) the denominator of Amax is equal to the numerator of IC50 , and d ) the denominator of Amin is equal to the denominator of IC50 . These properties are not expected for arbitrary linear-fractional functions and provide a validity check for the theory . Suppose we are only interested in the influence of an accelerator after the CLS ( i . e . , X3T or X4T ) . We can then write T and U above in terms of the accelerator XT , [S] , and effective constants that depend on the parameters of the hidden reactions: T= ( B1+B2XT ) ( K+[S] ) U=K+[S]+q'G[S]+ ( B3+B4qXT ) ( K+[S] ) From which we immediately obtain Amax=B1+B2XT1+B3+B4XT ( 6a ) Amin=B1+B2XT1+q′G+B3+B4XT ( 6b ) IC50= ( 1+B+3B4XT ) K1+q'G+B3+B4XT ( 6c ) AmaxIC50Amin=K ( 6d ) A cofactor can act like an accelerator or a decelerator before , at , or after the CLS . For two cofactors , such as GR and one other cofactor , there are 5 possible configurations for their action when not acting together at the same step ( e . g . , both before the CLS , one before and one at the CLS , etc . ) . There are 10 total positional combinations since GR can act before or after the other cofactor . There are 3 more configurations where GR acts at the same position as the other cofactor if one is a decelerator while the other is an accelerator . Thus , GR can act as a decelerator in all of these 13 configurations or as an accelerator after the CLS in 5 configurations . This gives a total of 18 possible configurations of GR and one other cofactor . A calculation for T and U can be made for each of these combinations and the results are in S2 Table . What these calculations show is how the dose-response parameters change as a function of differing amounts of added cofactor . The experimental dose-response can be fit to the predictions for each of the 18 cases to see which fits best . However , many of the cases can be eliminated immediately based on qualitative properties of the curves . The dose-response parameters will always be linear-fractional functions with the form y=a+bxc+dx Depending on the parameters , y can appear like a constant , a linear function , a Michaelis-Menten function , or a general linear-fractional function that increases or decreases with x . We also know that y increases with x if ad<bc , decreases if bc<ad , and is a constant if ad = bc . The x value for half-maximal y ( half-maximal concentration ) is c/d , and a/b for 1/y . Using these properties , we see that Amin and Amax in Equations ( 6a ) and ( 6b ) for the three reaction example can either increase or decrease depending on the parameter values but if Amax increases then so must Amin . IC50 in ( 6c ) is an increasing function because the denominator has an extra positive constant term . The graph of Amax×IC50/Amin in ( 6d ) is a horizontal line . Similar predictions for all the possible combinations of GR and one cofactor are summarized in Table 1 . The graph properties in Table 1 represent some sufficient conditions for the predicted mechanisms and position of action and do not represent a comprehensive list of all possible predictions . Our experimental paradigm for steroid-regulated repression is the well-documented GR inhibition of phorbol myristate acetate ( PMA ) induction of a reporter construct ( AP1LUC ) with the human collagenase-3 promoter [15 , 28 , 29] . We usually measured the gene activity for four concentrations of the GR agonist Dex including EtOH in the presence of different concentrations of AP1LUC reporter plus one of three added cofactors: the plasmid for TIF2 or two small molecules ( NU6027 and phenanthroline ) recently identified in a high throughput screen as accelerators of GR transactivation [30] . We present three lines of evidence to support the application and validity of the theory . 1 ) Fig . 2 shows the dose-response curve for GR repression of PMA induction of AP1LUC without ( Fig . 2A ) or with added TIF2 ( Fig . 2B ) . The curves ( including EtOH ) are well fit by the linear-fractional function in Equation ( 1 ) ( solid lines ) as required by the theory . We found that excellent fits of the dose-response data to Equation ( 1 ) could be obtained with four points ( 3 concentrations of Dex plus EtOH ) ( R2 = 0 . 984 ± 0 . 026 [S . D . , n = 160 randomly selected plots] , median = 0 . 993 ) , from which we estimated the dose-response parameters Amax , Amin , and IC50 in the ensuing experiments . 2 ) Figs . 3–5 show plots of Amax , Amin , IC50 , and Amax×IC50/Amin determined from four steroid concentrations for varying amounts of AP1LUC reporter and each of the added cofactors . The parameters are all well fit by linear-fractional functions ( solid curves ) as predicted by the theory . 3 ) We tested if these parameter graphs satisfied the four predicted compatibility conditions using Bayesian model comparison as detailed in the Methods . We found that the Bayesian Information Criterion ( BIC ) is lowest for the predicted model compared to two null models ( see Tables S3 , S4 , and S5 ) , which further validates the theory . Given the confidence that the theory is applicable , we used it to make predictions for the mechanism and position of action for the added cofactors as well as for the reporter and GR . Since the dose-response for repression is derived from the dose-response for induction , how a factor affects the Amax produced by the inducer in GR-regulated repression should be the same as how it alters Amax in GR-regulated gene induction even if the inducer is different in the two cases . That this is so can be seen from the formulas for Amax in induction ( S1 Table ) and repression ( S2 Table ) . In our system , the Amax in gene repression is the response to PMA alone and occurs in the absence of added steroid . All the cofactors we considered ( other than the reporter ) were found to be accelerators after the CLS in steroid-mediated gene induction [22 , 30] . The above example and S2 Table show that a graph of Amax vs . cofactor can inform us of where an accelerator acts in gene repression and these predictions are summarized in Table 1 . We used four concentrations of both AP1LUC and TIF2 in our competition assay to analyze TIF2 action in GR-regulated gene repression in U2OS . rGR cells . The graphs of Amax , Amin , IC50 , and Amax×IC50/Amin vs . TIF2 ( Figs . 3A-D ) all have linear-fractional shapes ( solid lines ) as predicted . The data for Amax vs . TIF2 ( Fig . 3A ) are well fit by Michaelis-Menten functions that have an x-axis intersection coordinate of -46 . 2 ± 26 . 8 ng ( S . E . M . , n = 4 ) of TIF2 plasmid . As is true in the competition assays for gene induction [22] , the interpretation of the graphs for gene repression requires that the x-axis values reflect the total amount of factor , i . e . , the sum of endogenous plus exogenous factor ( also see Methods ) . From quantitative Western blots ( not shown ) , it was determined ( assuming 50% transfection efficiency of cells [22] ) that the endogenous TIF2 is equivalent to 2 . 7 ± 1 . 5 ng ( S . E . M . , n = 3 ) of plasmid . Thus the point of zero endogenous TIF2 is at -2 . 7 ng TIF2 plasmid , which is much more positive than the intersection point of the curves at -46 , despite the large error range . As seen in Equation ( 6a ) , S2 Table ( since T ( 0 ) >0 for XT=0 for all entries where k > CLS ) , and summarized in Table 1 ( entry 5 ) , this is consistent with TIF2 acting as an accelerator after the CLS . Figs . 4 and 5 show that the dose-response parameters are also well fit by linear-fractional functions for the small molecules NU6027 and phenanthroline [30] . Amax and Amin versus both cofactors are lines that intersect at values more negative than the concentration of endogenous chemicals , which is zero . This behavior is consistent with both compounds being accelerators after the CLS ( Figs . 4A&B and 5A&B and entries 5 and 10 of Table 1 ) . The conclusions for TIF2 , NU6027 and phenanthroline being accelerators acting after the CLS in gene repression are consistent with what was observed in gene induction [30] . The traces in the graphs of Amax vs . AP1LUC ( Fig . 3E ) for varying concentrations of TIF2 are all linear , intersecting at the origin . The linear plot of Fig . 3E is preferred over a nonlinear plot ( BIC = 46 . 39 vs . 55 . 56 respectively ) . As can be seen from the example above , in the formulas of S2 Table where k = CLS and summarized in Table 1 ( entry 2 ) , this is consistent with AP1LUC acting as an accelerator at the CLS . Graphs of IC50 vs . AP1LUC ( Fig . 3F ) consist of near horizontal lines ( e . g . , a constant slope ) that decrease in position with added TIF2 ( average slope = -0 . 0016 ± 0 . 0038 , S . D . , n = 4 traces of graph ) . These plots , summarized in Table 1 ( entry 17 ) , are also diagnostic of the reporter AP1LUC being an accelerator ( A ) at the CLS . S1 A&B and S2 A&B Figs . show that Amax and Amin versus AP1LUC for varying concentrations of NU6027 and phenanthroline are again linear through the origin . The graphs of IC50 vs . AP1LUC have a constant zero slope with NU6027 ( = -0 . 0009 ± 0 . 0064 , S . D . , n = 4 traces ) and with phenanthroline ( = 0 . 0067 ± 0 . 0070 , S . D . , n = 4 traces ) ( S1 C and S2 C Figs ) . These imply that AP1LUC is an accelerator at the CLS in the presence of both NU6027 and phenanthroline . Hence , we find that the reporter is always an accelerator at the CLS in both gene induction and repression . Generally , in order to determine the action of a factor , one measures the response to changes of that factor . However , this was not possible with GR because we could only observe the robust repression needed for accurate graphical analyses with the high amounts of stably transfected GR in our experimental system . However , we can still deduce the mechanism and location of GR by comparing the responses to changes in the cofactors to the formulas in S2 Table to see which behaviors for GR are compatible with the observed results . Fig . 3 indicates that Amax increases while Amin , IC50 and Amax×IC50/Amin all decrease vs . TIF2 . As we show in the Methods , this is mathematically possible only if GR acts as an accelerator after TIF2 and TIF2 is an accelerator after the CLS . The results are also summarized in Table 1 ( entries 7 , 19 and 22 ) . Furthermore , from our posterior parameter estimates of our Bayesian model comparison test ( see S3 Table ) , we find that the concentration of TIF2 at half-maximal Amax ( parameter 1 ) is greater than the same for Amin ( parameter 3 ) . This condition is also true for 1/Amax ( parameter 2 ) and 1/Amin ( parameter 4 ) . These conditions further support the above deductions that TIF2 is an accelerator after the CLS and GR acts as an accelerator after both the CLS and TIF2 ( Table 1 , entries 12 and 14 ) . Figs . 4 and 5 show that Amax and Amin are each augmented by increasing concentrations of both NU6027 and phenanthroline . Hence , these cofactors cannot uniquely predict the action of GR . However , they can still be used to test for consistency . Figs . 4C&D show that Amax×IC50/Amin and IC50 are both decreasing versus NU6027 . According to entries 22 and 19 respectively of Table 1 , these graphs support the inference that GR acts as an accelerator after NU6027 , which acts as an accelerator after the CLS . The concentrations of NU6027 at half-maximal Amax and 1/Amax are larger than those of Amin and 1/Amin respectively ( S4 Table ) , which is also consistent with the conclusion that NU6027 acts as an accelerator after the CLS and before GR . Unlike TIF2 and NU6027 , Amax×IC50/Amin and IC50 versus phenanthroline do not exhibit any obvious trends ( Figs . 5C&D ) . However , an examination of the formulas for Amax×IC50/Amin and IC50 in S2 Table shows that there are parameter regimes where Amax×IC50/Amin and IC50 vary so slowly that they would appear constant when the factor acts after the CLS . There was also no significant difference between the half-maximal concentrations of Amax and Amin and their reciprocals for phenanthroline ( S5 Table , mean posteriors ) . Hence , these data neither confirm nor contradict the above conclusion that GR acts as an accelerator after phenanthroline . Therefore , in this system , the reporter ( AP1LUC ) and the added cofactors display the same kinetically-defined mechanisms of action , and at the same positions relative to the CLS , in GR-regulated gene repression and gene induction ( Fig . 6 ) . The only difference is that the position , but not mechanism , of GR action changes in gene repression from that in gene induction .
We introduce a theory for GR-regulated gene repression that is based on first principles . The theory is general but is mathematically solvable only when the dose-response curves for gene repression are linear-fractional ( Fig . 2 ) . The theory accommodates any number of pathway steps , transcription factors , and cofactors that alter the Amax , Amin , and/or IC50 of GR-controlled gene repression . The formation of multicomponent complexes is permitted as long as their concentrations are low or their lifetimes are short , which is biologically reasonable and has been observed for numerous factors [33–35] . The theory could also be generalized to include the action of factors through preformed hetero-oligomeric complexes with other factors . The competition assay for gene repression , like that for GR transactivation [20 , 22 , 23 , 27] , informs the kinetically-defined mechanism of action of each factor ( i . e . , accelerator vs . one of six decelerators ) and the position of factor action relative both to the other competing factor and to the CLS , which again appears to be an invariant marker in the overall reaction sequence ( see below ) . The theory makes specific predictions regarding the graphs of Amax , Amin , IC50 , and Amax×IC50/ Amin vs . one factor with increasing concentrations of a second factor ( Table 1 ) . The utility of the theory has been tested by examining the effects in the competition assay of four factors: AP1LUC reporter , p160 cofactor TIF2 , and two recently identified pharmaceutical modulators of GR transactivation [30] . The theory assumes mass action kinetics in a well-mixed medium . It cannot account for stochastic effects of a single transcription event . However , the averaged amount of mRNA in imaging experiments of a single agonist-induced gene does follow Michaelis-Menten kinetics [31] and may be analyzable by stochastic models [32] . Understanding the connection between our theory and stochastic models is an important future step . In every comparison , the AP1LUC reporter is found to act as an accelerator at the CLS . The CLS corresponds to that step where the concentration of the accelerator is limited compared to its binding affinity but the free concentration of accelerators in reactions after the CLS are in excess compared to their bound concentrations . The CLS can also be thought of as the step in the reaction sequence where an initially limited accelerator is replaced by another limited accelerator , such as GR being replaced by reporter gene . Thus , the species transmitting the input signal undergoes a “baton pass” , as in a relay race , to a new species at the CLS . In induction , the reporter gene also always , and uniquely , acts as an accelerator at the CLS [23 , 25–27] . Thus , reporter action at the CLS is an invariant signpost in both gene induction ( with GREtkLUC ) and gene repression ( with AP1LUC ) about which all other modulating factors and cofactors can be arranged . This leads us to predict that this will also be the case in future examples of gene repression . Experiments with varying concentrations of GR cannot be performed in the present system because only the U2OS . rGR cells with high amounts of endogenous GR gave robust repression . Cells with very low levels of GR , which would permit adding increasing concentrations of GR , did not yield the large fold-repression needed for the high precision measurements of the competition assay ( data not shown ) . Nevertheless , careful examination of the equations for the theory of repression revealed that the position and mechanism of GR action can be deduced from various graphical characteristics with respect to another cofactor . In this manner it can be determined that GR acts as an accelerator after both the CLS and each of the three factors ( TIF2 , NU6027 , and phenanthroline ) . Therefore , the kinetically-defined mechanism of action of GR , like that of the other factors including the reporter , is again the same as that seen during gene induction . What has changed for GR is its position of action . In gene induction , GR acts before the CLS and every other factor so far examined [23 , 25–27] . In contrast , GR functions after the CLS and each of the factors studied here in gene repression . This is not unexpected . Something about GR action must change if the increased response by GR in gene induction is to become a decreased output in gene repression . Furthermore , as the induction of the AP1LUC reporter occurs with added PMA in the absence of GR , it is plausible that GR inhibition might occur at a step downstream of the induction reaction . In this respect , we note that GR represses TNFα induction of IL-8 gene expression by acting after transcription initiation [37] . An accelerator acting after the CLS can increase or decrease the total gene activity depending on the parameters . We propose that repression of AP1LUC results from GR acting as an accelerator to favor a less productive step after PMA-mediated induction , thereby decreasing the level of LUC expression ( Fig . 6 ) . It is important to realize that GR can be found to display identical mechanisms of action in induction and repression while interacting with different factors . The mathematical models for both gene repression and gene induction by GR require the sequential binding of GR monomers as opposed to the binding of preformed dimers . This is supported by the observation of non-cooperative dose-response curves with unit Hill coefficient in both GR-mediated repression ( Fig . 2 ) and induction [20] . The binding of preformed GR dimers would yield a dose-response curve with greater than unity Hill coefficient . The sequential binding of monomers in gene induction to form DNA-bound dimers is further supported by the activity of several dimerization-defective GR mutants [20] and by biophysical studies of other dimerizing proteins that are found to associate with DNA by the sequential binding of monomers to yield DNA-bound dimers [38–43] . Similar experiments with dimerization-defective GR mutants cannot be performed in the current system due to the weak response in cells with very low levels of endogenous GR . However , it currently appears that most , if not all , instances of GR-mediated repression may also involve dimers of GR that are bound or tethered to DNA [44 , 45] . We strongly suspect that , as in gene induction , the association of GR with the DNA of repressed genes also proceeds via the step-wise binding of GR monomers , as required by our mathematical model . How to define the actions of cofactors for gene repression has been complicated by the fact that GR causes changes in total activity that are opposite those seen in gene induction [4–11 , 15] . Therefore , any classification of cofactor action relying on final activity is doomed to ambiguities . The present competition assay defines cofactor activity in an unbiased manner because it considers only what is happening at the position of cofactor action , independent of the direction of changes in final response . Such stepwise considerations can now be achieved independent of the biochemical processes involved and are essential if one desires to selectively modify GR-regulated repression ( and induction ) of specific genes . Using a common set of definitions of cofactor action [23] , we find that TIF2 , NU6027 , and phenanthroline are all accelerators after the CLS in both gene induction [21–25 , 30] and gene repression . Despite apparently identical mechanisms of TIF2 , NU6027 , and phenanthroline in GR-regulated gene repression , there are some graphical differences . This is due to a combination of where they act relative to each other and the specific reaction parameters , which cannot be defined exactly with our current data . Thus identical mechanisms of action do not require precisely identical graphs . Nonetheless , this permits great mechanistic simplifications . It suggests that at least those cofactors that are accelerators act in a constant and modular fashion , independent of the changes in the final product and the inducing agonist . Such common kinetically-defined mechanisms of cofactor action mean that new cofactor actions need not be invoked to account for the different responses in gene induction and repression because the underlying mechanisms are , in fact , the same . It also suggests that manipulation of cofactor action in induction vs . repression can simultaneously affect both pathways . There have been many descriptions of binding partners for TIF2 under conditions where TIF2 reverses a transcriptional response [46–50] . However , it should be realized that those species to which a factor initially binds , or chemically modifies , are unlikely to constitute the position at which factor action is exerted , which is what is revealed by our competition assay . For example , paused RNA polymerase II action occurs at steps downstream from its initial binding [51 , 52] while protein modifications , such as histone acetylation , elicit their effects after the modifying protein has bound . Similarly , the involvement of different cofactor domains in , for example , repression vs . induction , does not require different mechanisms of action . Different domains may alter the strength of a specific cofactor-target interaction or cause similar modifications of different downstream targets [37 , 53 , 54] . Alternatively , the relative importance of two positions of cofactor action may be influenced by domain composition [25 , 26] . In summary , we have described a theoretical model for GR-mediated gene repression that defines factors by their action at a particular step of the overall reaction scheme , as opposed to the final outcome . The theory has been validated by experimental results with five factors . The mechanism and position of action of four factors is qualitatively identical to that previously found in gene induction . The difference is that the fifth factor , GR , is predicted to act before the CLS and various cofactors in gene induction and after the CLS and cofactors in gene repression , presumably by diverting the reaction scheme to , and accelerating , a less productive pathway . The apparent constancy of factor mechanism of action in gene induction and repression means that altering cofactor activities is predicted to simultaneously affect both GR-regulated pathways . Thus , new and uncharacterized pathways will not have to be considered , which will simplify approaches for maximizing desired outcomes . Finally , as for the theory for gene induction [20 , 22] , the current theory for gene repression is general for any gene induction and gene repression process displaying a linear-fractional dose-response and thus could be of use to analyze the mechanisms of other inducible transcription factors .
Unless otherwise indicated , cell growth was at 37°C and all other operations were performed at 0°C . Dexamethasone ( Dex ) , PMA ( phorbol myristate acetate ) , NU6027 , and phenanthroline were purchased from Sigma ( St . Louis , MO ) . Restriction enzymes and T4 DNA ligase were from New England Biolabs ( Beverly , MA ) and the dual-luciferase reporter assay was from Promega ( Madison , WI ) . TIF2 ( Hinrich Gronemeyer , IGBMC , Strasbourg , France ) , and AP1Luc ( Inez Rogatsky , Weill Medical College , Cornell University ) were generously donated . U2OS . rGR cells were grown in DMEM media supplemented with 10% FBS and 0 . 1 mg/ml G418 and seeded at 30 , 000 cells per well in a volume of 300 μl per well in 24-well plates as previously described [29] with the following modifications . One day after seeding in 24-well plates , cells in FBS-free DMEM were transfected with reporter ( AP1Luc ) , 10 ng of phRG-TK Renilla ( Promega ) as an internal control , and the indicated amounts of plasmids for various factors in OPTIMEM plus XTREME Gene HP ( Roche; 0 . 8 μl/well ) . Four hours after transfection , cells were refed with DMEM/10% FBS . The next day , cells were treated with PMA ( 10–25 ng/ml ) and various dilutions of dexamethasone ( Dex ) and chemical . Sixteen hours later , the cells were lysed in lysis buffer and assayed for reporter gene activity using dual luciferase assay reagents according to the manufacturer’s instructions ( Promega , Madison , WI ) . Luciferase activity was measured by a GloMax® 96 Microplate Luminometer ( Promega , Madison , WI ) . The data were normalized to Renilla TK luciferase activity and expressed as a percentage of the maximal response with Dex before being plotted ± S . D . unless otherwise noted . The basic protocol for gene induction [22 , 23] was followed except as noted in the text for 4x4 ( all 16 combinations of 4 concentrations of both factor 1 and factor 2 ) assays with four concentrations of Dex , all in triplicate , for a total of 196 wells . All plots of the data assume a linear increase in factor plotted on the x-axis . When Western blots reveal a nonlinear relationship between the optical density of scanned protein band and the amount of transfected plasmid at constant levels either of total cellular protein , or of β-actin , the linear equivalent of expressed plasmid must be determined as previously described [22] ( see also below ) . The nonlinear plot of OD vs . ng of transfected plasmid is first fit to the Michaelis-Menten formula Amax=m1*plasmid/ ( m2+plasmid ) to obtain constants m1 and m2 . The functional equivalent of the transfected plasmid that gives a linear OD vs . plasmid plot is then obtained from the formula of Plasmid ( linear ) =m2*plasmid/ ( m2+plasmid ) The x-axis value of amount of plasmid in the various graphs is then this “corrected plasmid” value . A linear-fractional dose-response arises when the mass conservation equations are bilinear . The general bilinear form for an arbitrary number of reactions is: [X1]+[Y1]=X1T[X2]+[Y2]=X2T⋮ } Pre-CLS [Xj]+[Yj]+[Yj+1]+⋯+[Yk]=XjT CLS [Xj+1]=Xj+1T⋮[Xk]=XkT } Post-CLS [Xk+1]+[Yk+1]=Xk+1T⋮ } Pre-CLS [Xl]+[Yl]+[Yl+1]+⋯=XlT CLS [Xl+1]=Xl+1T⋮ } Post-CLS where there can be multiple CLS steps . The bilinearity of these equations is easily confirmed by recursively substituting the equilibrium conditions for the products . The reactions can be divided into pre-CLS , CLS , and post-CLS steps . In pre-CLS steps , only the accelerator and its immediate product appear in the mass conservation equation . This can be achieved if all downstream products have much lower concentration than the accelerator and its product , i . e . , [Yj]<<[Yi] , [Xi] , j>i , which is satisfied if [Yi+1]<<[Yi] , [Xi] , i . e . , products become successively smaller . Substituting in the equilibrium conditions gives qi+1Xi+1T[Yi]<<[Yi] or qi+1Xi+1T<<1 . Hence , accelerators are limited with respect to their affinities at pre-CLS steps and the CLS is the last step of this sequence for which the accelerator is limited . At post-CLS steps , the free concentration of accelerator is equal to the total concentration , i . e . , the bound concentration is negligible . This is satisfied if [Yj]<<[Xi] , j≥i , which implies that the concentration of accelerator is in excess compared to the products and the reactions are pseudo-first order . The crucial point for preserving linear-fractional dose-response is that the equilibrium conditions have the form of ( 2 ) and the mass conservation conditions have bilinear form . Any kinetic scheme , reversible or irreversible , that obeys a similar set of equations at steady state will have a linear-fractional dose-response . For example , consider the irreversible reaction P+X →P′ + X and P′→ P . In this “hit-and-run” scheme , the accelerator X interacts transiently with P and modifies it . The modified P then relaxes back to the original state as a first order kinetic process . This reaction could occur for example if P is in an “excited” state , X nudges it to a lower energy state P′ , and energy is expended to pump P′ back to the excited state . The concentrations have kinetics d[P′]dt=kf[P][X]−kr[P′] , d[P]dt=−kf[P][X]+kr[P'] , d[X]dt=0 which has a steady state solution [P′] = q[X][P] , where q = kf/kr , and conservation equations [P]+[P′] = PT , [X] = XT . The equations combine to yield [P′] = q XT[P] , thereby mimicking a post-CLS reaction . Conversely , we could have the irreversible reaction P+X →X′ + P and X′→ X , where the product from the previous reaction now transiently interacts with an accelerator and modifies it to create a new product X′ . In steady state , the new product obeys [X′] = q[X][P] with mass conservation condition [X]+[X′] = XT , or [X]+q[X][P] = XT . The equations combine to yield [X′]=qXT[P]1+q[P] If this reaction were followed by a reaction of the form X′+U→X″+U , X″→ X′ then the mass conservation law for X changes to [X]+[X′]+[X″] = XT . Combining with the additional steady state equation [X″] = q′UT[X′] , we obtain [X′]=qXT[P]1+q[P]+qq′UT[P] which mimics a CLS step . Hence , in this irreversible hit-and-run scheme , accelerators that successively modify a product are akin to post-CLS steps and a reaction where the product switches roles and modifies a factor to make a new product is akin to a CLS . Reversible and irreversible reactions could be combined as long as the steady state conditions resemble the equilibrium conditions ( 2 ) and the mass conservation conditions have bilinear form . Experiments find that Amax increases while Amin decreases with an increase in TIF2 , which acts as an accelerator after the CLS . Experiments also find that IC50 and Amax×IC50/ Amin decrease with increases in TIF2 . We show below that this behavior puts severe constraints on the possible mechanisms and positions where GR can act . Specifically , we need to determine for which cases it is possible for Amax > Amin , and Amax increases while Amin , IC50 and Amax×IC50/ Amin all decrease for increases of a post-CLS accelerator . We examine each possible case for TIF2 acting after the CLS individually . Unless otherwise noted , all experiments were performed in triplicate multiple times . KaleidaGraph 4 . 1 ( Synergy Software , Reading , PA ) was used to determine a least-squares best fit ( R2 was almost always 0 . 95 ) of the experimental data to the theoretical dose-response curve , which is given by the Equation ( 1 ) . This was done by first estimating Amax directly from the data for zero Dex . The data points were subtracted from Amax and the resulting curve was then fit to a Michaelis-Menten function from which Amin and IC50 were determined . The values of n independent experiments were normalized , averaged , and then plotted and analyzed as described in the Supporting Information . The Bayesian Information Criterion was used to determine whether the best fit of Amax vs . AP1LUC data is obtained with linear or Michaelis-Menten plots . The theory predicts that the four dose-response parameters are linear-fractional functions of the cofactor and satisfy four compatibility conditions . To test this , we fitted the theoretically predicted model , with two other “null” models to the data and used the Bayesian Information Criterion to see which model was best . The models tested were 1 ) Predicted Model: Amax=a5[R]a1+[X]a2+[X] , Amin=a6[R]a3+[X]a4+[X] , IC50=a7a2+[X]a4+[X] , Amax=a8a1+[X]a3+[X] 2 ) Permuted Model: Amax=b5[R]b1+[X]b4+[X] , Amin=b6[R]b2+[X]b3+[X] , IC50=b7b3+[X]b2+[X] , Amax=b8b4+[X]b1+[X] 3 ) Unconstrained linear-fractional model: Amax=c9[R]c1+[X]c5+[X] , Amin=c10[R]c2+[X]c6+[X] , IC50=c11c3+[X]c7+[X] , Amax=c12c4+[X]c8+[X] where [X] is the concentration of the added cofactor , the a’s , b’s , c’s are parameters to be fitted to the data , and [R] is the concentration of the reporter , which we know is an accelerator at the CLS . The predicted and permuted models have the same model complexity with 8 free parameters , while the unconstrained model has 12 free parameters . The predicted model is a subset of the unrestricted model . The half maximum of the dose-response parameters are given by the parameter in the denominator . For example , the cofactor concentration for half maximum for the predicted model of Amax is given by a2 , and that of Amin is given by a4 . Conversely , the concentrations of half maximum of 1/Amax and 1/Amin are given by a1 and a3 , respectively . We used a Metropolis-Hastings Markov Chain Monte Carlo ( MCMC ) method [55] to compute the Bayesian posterior probabilities of the various parameters . The likelihood function we used was proportional to exp ( −χ2/2 ) where χ2=−∑p=1k∑i=1n ( yi , pdata−yi , pmodel ) 2σi2 The sum is over all n data points i for each dose-response parameter p out of k total , and the error variance σi , p2 is the experimentally determined standard error variance of replicate experiments . The results for the maximum-likelihood values , and the mean and standard deviation for posteriors for TIF2 , NU6027 , and phenanthroline are in Tables S3 to S5 . The reported results are for 2×107iterations for the models with 8 parameters and 3×107 for the unrestricted model after an even longer transient period to ensure convergence . The longer time for the unrestricted model was to compensate for the extra parameters . We used the Bayesian Information Criterion , BIC = x2 + k ln n , to test which model best fit the data accounting for model complexity . | While the initial steps in steroid-regulated gene induction and repression are known to be identical , the same cannot be said of cofactors that modulate steroid-regulated gene activity . We describe the conditions under which a theoretical model for gene repression reveals the kinetically-defined mechanism and relative position of cofactor action . This theory has been validated by experimental results with glucocorticoid receptors . The mode and position of action of four factors is qualitatively identical in gene repression to that previously found in gene induction . What changes is the position of GR action . Therefore , we predict that the same kinetically-defined mechanism usually will be utilized by cofactors in both induction and repression pathways . This insight and simplification should facilitate clinical efforts to maximize desired outcomes in gene induction or repression . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] | [] | 2015 | Kinetically-Defined Component Actions in Gene Repression |
Tight regulation of the visual response is essential for photoreceptor function and survival . Visual response dysregulation often leads to photoreceptor cell degeneration , but the causes of such cell death are not well understood . In this study , we investigated a fatty acid transport protein ( fatp ) null mutation that caused adult-onset and progressive photoreceptor cell death . Consistent with fatp having a role in the retina , we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants . The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 ( Rh1 ) . Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies . Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance .
Retinal degeneration is a major health concern that affects one in 2000 people worldwide ( http://www . sph . uth . tmc . edu/Retnet/ ) [1] . Although human retinopathies are heterogeneous in physiopathology and severity , they all involve loss of photoreceptor ( PR ) neurons , which leads to blindness . The most frequent retinal disease is retinitis pigmentosa , which is caused by mutations in one or more of at least 54 distinct genes ( Retnet ) . Among them , the most frequently mutated gene is rhodopsin ( rho in mammals ) which is mutated in 30–40% of all cases of autosomal dominant retinitis pigmentosa ( ADRP ) ( Retnet ) [2] . Rhodopsin is the light-sensitive protein of PRs that activates phototransduction . Approximately 100 rho mutations have been identified , and they affect folding , trafficking and activity of the rhodopsin protein . Despite extensive study , the mechanisms of retinal degeneration remain unclear . Retinal degeneration has been studied extensively in Drosophila [3] , [4] , [5] . Many mutations in Drosophila phototransduction pathway genes induce PR degeneration . The mechanism of toxicity of these mutations is related either to a defect in folding , trafficking or activity of the Drosophila Rhodopsin-1 protein ( Rh1 ) , or to an accumulation of toxic Rh1-Arrestin2 ( Arr2 ) complexes , or to a deregulation of the Ca2+ homeostasis [4] , [5] . In addition , mutations can be introduced in Drosophila genes to model human diseases . For example , the rh1P37H allele , which corresponds to rhoP23H , the most frequent mutation of rhodopsin in ADRP , has been successfully introduced into Drosophila and induces PR degeneration [6] . We recently used Drosophila to identify genes involved in PR survival [7] . Using a new method of PR visualization , called Tomato/GFP-FLP/FRT , we screened recessive lethal mutations and found fatpk10307 to be associated with PR cell death . The fatpk10307 mutation consists of the insertion of a P{lacW} element into the open reading frame of the fatty acid transport protein ( fatp ) gene , which has never been characterized in Drosophila . Its closest mammalian orthologs are fatp1 and fatp4 [8] . The mammalian members of the Fatp family of proteins have acyl-CoA synthetase enzymatic activity and facilitate cellular fatty acid uptake [9] , [10] . Each member of the family has a specific expression pattern and function . fatp1 is expressed in muscle , heart , brain , adipose tissue and retina [11] , [12] , and is involved in thermogenesis and obesity . fatp4 is expressed most abundantly in the small intestine , brain , skeletal muscle , heart , skin , liver and kidney [10] , [13] , [14] . Loss of fatp4 in mice or in humans is associated with restrictive dermopathy related to the ichthyosis prematurity syndrome , a skin defect caused by altered lipid and fatty acid compositions [15] , [16] , [17] . In this work , we show that Drosophila fatp is required for PR survival . PR degeneration in a fatp null mutant is adult-onset and progressive . The onset of degeneration in the fatp mutant correlates with the time of expression of fatp in the normal adult retina . We then investigated the mechanisms of PR degeneration in the fatp mutant . We show that PRs in the fatp mutant exhibit an elevated photoresponse that is associated with high levels of Rh1 . We also found that fatp mutant flies have a defect in Rh1 degradation and that reducing the level of Rh1 restored PR survival .
To study the role of fatp in PR viability , we first used RNA interference to reduce fatp expression . fatp-interfering RNA was specifically produced in the retina under the control of eye-specific drivers ( ey-Gal4 and GMR-Gal4 ) ( Figure 1A–1G ) . fatp knockdown led to a progressive loss of PRs , indicating that fatp expression is required for PR viability . To validate these findings , we used the fatpk10307 mutation , generated by the insertion of a 10 . 7 kb P{lacW} element into the first exon of the gene . This mutation is recessive and can be considered a null allele as it completely prevents fatp expression , abolishing the production of fatp mRNA and protein in homozygous fatpk10307 L1 larvae ( Figure S1 ) . As a consequence , fatpk10307 mutant larvae died during the second instar . To study the role of fatp in adult PR and circumvent fatpk10307 larval lethality , we used the Tomato/GFP-FLP/FRT mosaic method [7] . This new method combines mitotic recombination and cornea neutralization techniques [18] , [19] , [20] to allow time-course analysis of homozygous mutant clones in living flies over several days . We evaluated PR presence , identified on the basis of GFP expression , for 14 days from hatching in fatpk10307 mutant retinas ( Figure 1H–1J ) . All fatp mutant PRs were present upon hatching but progressively disappeared starting on day four ( Figure 1H , 1H′ ) . Losses of fatp mutant PRs were statistically significant in the retinas of 8- and 14-day-old adults ( Figure 1H″ , 1H′″ , 1I ) . In contrast , little or no PR loss occurred in neighboring wild-type or heterozygous tissue , indicating that the fatpk10307 mutation was cell-autonomous ( Figure 1H , 1I , 1J ) . The cell-autonomous nature of the PR loss in the fatp mutant ( Figure 1H–1I ) suggested that fatp expression is required for PR viability . To confirm this requirement , we attempted to rescue PR viability in the fatpk10307 mutant with tissue-specific expression of wild-type fatp using the UAS/GAL4 system [21] . fatp re-expression from the rh1 promoter in the R1–R6 PRs fully rescued the fatpk10307 mutant PR phenotype ( Figure 1K–1N ) . These results demonstrate that fatp expression in PRs is required for their survival . Next , we performed structural and ultra-structural analyses of fatp mutant PR in resin-embedded retina sections . We used classical brightfield microscopy to examine PR integrity in whole-eye fatp mutant clones generated by the EGUF/Hid method [22] ( Figure 2A–2E ) . PR loss started after approximately 15 days and increased progressively with age ( Figure 2A–2E ) . Using this approach , PR loss was detected later than with the Tomato/GFP-FLP/FRT method . This may be because the Tomato/GFP-FLP/FRT method is based on the expression and targeting of GFP in the rhabdomere and thus detects early deficits whereas classical histological methods assess only the physical presence or absence of PRs . We also detected progressive and adult-onset PR loss by recording the number of nuclei between the apical and basal layers of fatp mutant retinas in horizontal cryosections ( Figure S2 ) . We then studied fatp mosaic retinas by electron microscopy ( EM ) to characterize further PR degeneration in fatp mutant cells ( Figure 2F–2M ) . On EM images , we could distinguish homozygous fatp mutant PRs from the absence of pigment vesicles at the base of the rhabdomeres and in inter-ommatidial cells ( IOCs ) ( Figure 2F ) . We observed several levels of PR degeneration among fatp mutant cells ranging from normal PRs to PRs that were fully degenerated and engulfed by neighboring IOCs . Most normal PRs displayed no obvious sign that would predict future degeneration ( Figure 2G ) . Some PRs were electron-dense and their cytoplasm was contracted suggesting that they had initiated the degeneration process ( Figure 2H ) . In these PRs , some swelling mitochondria could be observed but the rhabdomeres were still intact . In more advanced stages of degeneration , the rhabdomeres were clearly affected with disorganized microvilli structures ( Figure 2I , 2J ) . In addition , the neighboring IOCs seemed to be activated with a typical spotted pattern in the cytoplasm . At the end of the degenerative process , PRs were phagocytosed by the neighboring IOCs and apparently digested ( Figure 2K–2M ) . Thus , the absence of fatp causes progressive adult-onset PR degeneration . We determined the specificity of fatp expression in developing and adult Drosophila eyes . To do this , we generated an anti-Fatp antibody ( C11-7 ) that was sensitive enough to detect ectopic Fatp and endogenous Fatp on western blots and in immunofluorescence experiments ( Figure 3A–3E ) . No fatp expression was observed in third instar eye imaginal discs ( Figure 3B ) , but we detected Fatp protein in PR cytoplasm juxtaposed to PR rhabdomeres and in IOCs of the adult retina ( Figure 3D , 3E ) . To capture the fatp expression profile , we exploited the fatpk10307 enhancer trap line , which carries the β-galactosidase reporter ( Figure S3 ) . Using a β-galactosidase antibody , we detected β-galactosidase in the nuclei cell layer that includes the outer PRs and accessory cells in adult head cryosections ( Figure S3A–S3D ) . Immunolocalization experiments revealed that small amounts of Fatp consistently co-localized with the neuronal marker ELAV in the outer PRs and that Fatp was also present in apical IOCs ( Figure S3A–S3D ) . Thus , the fatpk10307 enhancer trap line faithfully reproduces the distribution of Fatp and can be used to follow fatp expression . Testing for β-galactosidase activity in this line revealed that fatp was also expressed in the adult optic lobe around the medulla and lamina , in the midgut and in the salivary glands but not in the eye imaginal disc of third instar larvae ( Figure S3E–S3L ) . In conclusion , the absence of fatp expression in the developing eye disc and its presence in differentiated PRs are consistent with fatp being required for the viability of PRs in adult flies . We investigated whether the requirement for fatp expression for PR viability is related to the visual response . First , we tested whether PR degeneration in the fatp mutant is light-dependent . fatp mutant flies reared in normal light conditions exhibited significantly greater PR losses than fatp mutant flies reared in complete darkness ( Figure 4A–4G ) . This indicates that light contributes to PRs loss in the fatp mutant . Then , to examine directly the visual response in the fatp mutant , we performed electoretinogram ( ERG ) recordings on white-eyed flies ( Figure 4H , 4I ) : we used 8-day-old flies , an age at which the integrity of fatp mutant PRs is still intact as observed in resin-embedded retinal sections ( data not shown ) . We obtained ERG recordings that measure the summed responses of all retinal cells . Wild-type flies exhibited a corneal negative receptor potential in response to orange light , which returned to baseline when the light was switched off ( Figure 4H ) . The amplitude of the ERG was higher in fatpk10307 flies than controls , with no apparent difference in the kinetics of the visual response ( Figure 4H , 4I ) . These results suggest that Fatp is a negative regulator of the visual response . We wondered whether PR degeneration in the fatp mutant could be rescued by rh1 mutant alleles as previously described for light-dependent retinal degeneration mutants such as rdgC and norpA [23] . We therefore tested whether PR viability in the fatp mutant could be rescued by rh1G69D and rh1I17 ( ninaEG69D and ninaEI17 ) alleles ( Figure 5 and Figure S5 ) . rh1G69D is a dominant negative allele whereas rh1I17 is a null allele . Although the rh1G69D allele causes retinal degeneration in old flies under constant light exposure , it could be used in our study because these mutants raised under a 12 h day/light cycle did not exhibit retinal degeneration ( Figure 5C , 5H , 5M; Figure S4; and [23] , [24] ) . Using the Tomato/GFP-FLP/FRT method , we compared PR loss in retinas in the single fatpk10307/k10307 mutant to that in fatpk10307/k10307rh1G69D/+ double mutant . PR loss was very much lower in fatpk10307/k10307rh1G69D/+ mutant retinas than in fatpk10307/k10307 mutant retinas ( Figure 5A–5E ) . In resin-embedded eye sections , PR viability was found to be fully rescued in the fatpk10307/k10307rh1G69D/+ double mutant ( Figure 5F–5J ) . EM showed that despite the smaller size of the rhabdomeres , PR ultra-structure was fully rescued in fatpk10307/k10307rh1G69D/+ double mutants ( Figure 5K–5N ) . Similarly , a rescue of PR degeneration was observed in fatpk10307/k10307rh1I17/+ double mutant ( Figure S5 ) . Thus , rh1 mutant alleles rescued PR degeneration in the fatp mutant . We tested whether rh1 is dysregulated in fatp mutant PRs . We assayed Rh1 protein in fatp mutant and control retinas by western blotting ( Figure 6A , 6B ) . The Rh1 content of fatpk10307/k10307 mutant retinas was double that of control retinas . We confirmed that Rh1 was less abundant in fatpk10307/k10307rh1G69D/+ double mutant than fatpk10307/k10307 single mutant retinas ( Figure 6A , 6B ) . These results indicate that Rh1 metabolism is altered in the fatp mutant and that reducing Rh1 levels in fatp mutants protects the PRs . To confirm this conclusion , we examined whether rearing flies on a vitamin A-deficient medium could reduce PR loss in fatp mutant retinas ( Figure 6C–6F ) . Vitamin A is the precursor of the Rh1 chromophore and is required for Rh1 synthesis . A vitamin A-deficient diet rescues retinal degeneration in several mutants , including rdgB , crumbs , and arrestin mutants [25] , [26] , [27] . In retinas of flies reared on a vitamin A-deficient diet , occasional PR degeneration was visible but most rhabdomeres were present ( Figure S6 ) . fatp mutant flies deprived of vitamin A produced no detectable Rh1 protein and the PR loss in these flies was very much lower than that in flies receiving a standard diet ( Figure 6C–6F ) . Thus , reducing the Rh1 level rescued PR degeneration in the fatp mutant . Therefore , PR degeneration in the fatp mutant appears to be due to an over abundance of Rh1 . To test whether high levels of Rh1 are toxic to the PR , we overexpressed rh1 in developing and adult PRs ( Figure S7 ) . Ectopic expression of rh1 with the GMR driver led to the rough eye phenotype , indicating that rh1 overexpression is toxic in the developping eye ( Figure S7A , S7B ) . We also overexpressed rh1 specifically in differentiated R1-6 PRs using the rh1 promoter . rh1 overexpression led to rhabdomere degeneration ( Figure S7C , S7D ) . Thus , we conclude that elevated Rh1 levels in fatp mutant PRs can lead to PR pathogenesis . Next we examined the possible involvement of Arrestin2 ( Arr2 ) in Rh1 toxicity in fatp mutants . Stable Rh1-Arr2 complexes are toxic in adult PRs and are responsible for retinal degeneration in several mutants including norpA , rdgC , rdgB [26] , [28] , [29] , [30] . To examine this possibility , we tested for genetic interaction between fatpk10307 and arr23 mutations ( Figure 6G–6J ) . We found that PRs were rescued in fatp and arr2 double mutant retina , indicating that arr2 is required for the toxicity in fatp mutant PRs . Together with the excess of Rh1 detected in fatp mutant , these observations suggest that toxic Rh1-Arr2 complexes induce PR degeneration in the fatp mutant . We tested whether the elevated Rh1 levels in fatp mutant PRs were due to deregulation of rh1 expression and/or degradation of Rh1 . We first analyzed rh1 expression in fatp mutant retinas using a rh1-lacZ reporter to monitor rh1 transcription . We did not detect higher levels of lacZ in fatp mutant retinas than in control retinas ( ) . This shows that rh1 transcription is not upregulated by the fatp mutation . Presumably , therefore , fatp regulates Rh1 levels post-transcriptionally . To test for the involvement of fatp in Rh1 degradation , we forced Rh1 degradation by illuminating the retina with blue light as previously described [31] . Blue illumination maintains Rh1 in an active conformation and induces its degradation . In white-eyed control flies blue light illumination for 6 h induced a significant loss of Rh1 for the retinas whereas the same treatment of white-eyed fatp mutants did not result in decreased Rh1 abundance ( Figure S8C , S8D ) . This suggests that light induced-Rh1 degradation is impaired in fatp mutant PRs and this may explain why there is more Rh1 in these retinas than controls .
Mutations resulting in inactive Rh1 , impaired visual responses and PR degeneration have been studied extensively [4] , [5] . In this study , we describe the phenotype of fatpk10307 , the first mutation known to exhibit elevated Rh1 levels leading to loss of PRs . We show that fatp expression is required for PR viability in adult Drosophila . In the absence of fatp , PRs degenerate progressively during adulthood . Moreover , we demonstrate that the requirement for fatp in adult PRs is cell-autonomous , which is in agreement with the presence of Fatp in adult PRs and with its absence from the developing eye imaginal disc . The age-dependent PR degeneration in the fatp mutant is reminiscent of Drosophila models of ADRP [3] , [6] . We thus propose that fatp-associated degeneration is a new model of late-onset PR degeneration . Our results indicate that PR death in fatp mutants is a consequence of elevated levels of Rh1 . We demonstrate that reducing Rh1 levels , as a consequence of rh1 mutation or a vitamin A-deficient diet , efficiently restored PR viability in fatp mutants ( Figure 5 , Figure 6 , and Figure S5 ) . Thus , the accumulation of Rh1 is toxic for the PRs in fatp mutants . One possibility is that Rh1 associates with Arr2 , forming toxic Rh1-Arr2 complexes as in norpA , rdgC , rdgB mutants [26] , [28] , [29] , [30] . Indeed , we found that disrupting either rh1 or arr2 rescued PR loss in fatp mutants ( Figure 5 , Figure 6 , and Figure S5 ) . This genetic evidence is consistent with Rh1-Arr2 complexes causing PR degeneration in fatp mutant . Definitive proof of this mechanism requires the direct assessment of Rh1/Arr2 complexes in fatp mutants . Also , we cannot exclude the existence of additional toxic mechanisms . For example , the elevated visual response in fatp mutants ( Figure 4 ) may contribute to PR death because of a defect in Ca2+ homeostasis . We found that Rh1 protein levels were elevated in fatp mutant retinas and that this was probably due to decreased Rh1 degradation ( Figure S8C , S8D ) . fatp may regulate sphingolipid metabolism , which controls Rh1 trafficking and degradation [32] , [33] . In support of this hypothesis , the total ceramide content is higher in the skin of fatp4−/− than control mice [15] . Ceramidase facilitates the endocytic turnover of Rh1 and rescues retinal degeneration in arr2 and phospholipase C mutants [32] , [34] . Similarly , Drosophila Fatp may limit ceramide levels and inhibit endocytic turnover of Rh1 . It is also possible that fatp regulates Rh1 synthesis , but we show that loss of fatp does not affect rh1 gene transcription ( Figure S8A , S8B ) . Alternatively , Fatp may regulate the synthesis or recycling of the retinal chromophore required for Rh1 synthesis . In mammals , Fatp1 inhibits two enzymes of the visual cycle in vitro , LRAT and RPE65 , which respectively produce and consume retinyl-ester , a fatty acid-linked form of the chromophore [12] . A similar visual cycle was recently described in Drosophila [35] , [36] , [37] . Therefore , chromophore synthesis or recycling may be increased in the fatp mutants , resulting in upregulated Rh1 synthesis . In support of this hypothesis , we showed that inhibiting chromophore synthesis in vitamin A-deficient medium fully rescued PR viability in fatp mutant retinas ( Figure 6D–6F ) . Nevertheless , the mechanisms by which fatp may regulate the visual cycle remain to be elucidated . The visual response was higher in the fatp mutant retina than control retina but the mechanisms involved are unclear . Previous work has shown that the ERG amplitude depends on the ratio between the level of Rh1 and the Rh1 kinase activity of gprk1 [38]: decreasing gprk1 activity resulted in higher ratios and elevated ERG amplitudes whereas increasing gprk1 activity resulted in lower ratios and lower ERG amplitudes [38] . Whether there is a disequilibrium between phosphorylated/unphosphorylated forms of Rh1 in fatp mutant retinas remains to be explored . Alternatively , fatp may be required for the production of lipid metabolites that regulate the phototransduction cascade . Indeed , it has been suggested that polyunsaturated fatty acids , which are potential diacyl-glycerol metabolites , act on TRP/TRPL channels [39] , [40] , [41] . Thus , the elevated visual response may be the consequence of a lipid metabolite dysregulation in fatp mutant retinas . In conclusion , fatp mutation is a new model of retinal pathology in flies in which the up-regulation of Rh1 contributes to progressive PR degeneration . Whether a similar pathological mechanism exists in human retinal diseases remains to be determined .
For RNA interference , the UAS-dicer; ey-Gal4 , GMR-Gal4/Cyo; rh1-GFP line ( kind gift of C Desplan ) was crossed with the UAS-RNAi line against fatp ( VDRC #48719 ) . The FRT40A fatpk10307/Cyo and rh1-Gal4 , ey-FLP; FRT40A rh1-tomatoninaC/Cyo; UAS-GFPninaC lines were described previously [7] . The ninaEG69D line ( named rh1G69D in the text ) was previously used in [42] . The rh1I17 ( ninaEI17 ) allele was obtained from bloomington ( BL#5701 ) . Whole-eye mutant clones were generated using the; FRT40A GMR-hid CL EGUF/Cyo; line [22] . The UAS-rh1 and arr23 lines were a kind gift of HD Ryoo and N Colley respectively . To obtain white-eyed flies carrying Pw+ transgenes , we used the pWIZ construct that expresses an iRNA against the white gene [43] . Flies were reared on standard corn medium at 25°C in a 12-h light/12-h dark environment unless noted otherwise . Vitamin A-deficient medium contained yeast ( 12 g ) , agar ( 1 , 5 g ) , sucrose ( 7 , 5 g ) , cholesterol ( 0 , 03 g ) , sodium methyl-4-hydroxybenzoate ( 1 . 15M , 3 . 75 mL ) and propionic acid ( 0 . 72 mL ) in distilled water ( 150 mL ) . fatp cDNA ( SD05207 , Gold cDNAs Collection ) was recovered from BDGP DGRC in pOT2 vector . fatp cDNA was cloned ( XhoI/EcoRI ) into a pUAST-w+-attB transgenic fly vector . Best Gene , Inc ( CA , USA ) generated transgenic lines using PhiC31 integrase-mediated transgenesis [44] . The vector DNA was injected in embryos carrying attP docking sites ( strain 9750 at 65B2 ) . w+ embryos were selected and stable transgenic fly stocks established . CO2-anesthetized flies were placed in a 35 mm cell culture dish half-filled with 1% agarose , covered with water at 4°C and observed using an upright 510 Zeiss confocal fluorescent microscope as described [20] . For the time-course study of age-dependent PR death in single flies , after visualization , each living fly was detached from the agarose , dried and transferred to a vial containing fly food medium . Tangential sections of adult eyes were performed as described [45] . PR viability was determined by counting the number of intact rhabdomeres on retina tangential plastic sections . At least 200 ommatidia from three different animals were scored per experimental condition . Drosophila eyes were dissected and fixed overnight at 4°C in 1 . 5% glutaraldehyde , 1% paraformaldehyde and 0 . 1M PIPES buffer ( pH 7 . 4 ) . After washing , eyes were post-fixed at room temperature in 1% OsO4 , 0 . 1M PIPES ( pH 7 . 4 ) . They were then deshydrated with successive ethanol solutions followed by anhydrous propylen oxyde . Eyes were infiltrated with increasing concentrations of epoxy resin ( EMbed 812 from EMS ) in propylen oxyde for 1 day at room temperature and samples were mounted in pure resin into silicone embedding molds . Polymerization was performed at 60°C for 2 days . Ultrathin sections of 60 nm were stained with lead citrate and examined with a transmission electron microscope ( Philips CM120 ) operating at 80 kV . Two rabbits were immunized with two peptides of Fatp , Peptide 1: YQTSKGRYELLTPQ at the C-terminus of the protein and Peptide 2: NNNSETEKNIPQAK in the middle of the protein . The serum from the two rabbits were pooled and affinity purified . Horizontal eye cryosections were performed using a cryostat microtome ( Microm HM505E ) and deposited on superfrost Plus slides ( Thermo ) . Third instar larval imaginal discs were dissected in 1X PBS . For whole-mount retina , we followed the protocol described in [46] . Briefly , Drosophila heads were bisected in the middle with a scalpel . Brain tissue was removed to expose retina underneath . Cryosections , imaginal discs and whole-mount retinae were fixed in 4% PFA for 15 min . After washing in PBS+Triton X-100 ( 0 . 3% ) , the following antibodies were used in PBS+Triton X-100 ( 0 . 1% ) +Normal Goat Serum ( 5% , Sigma ) overnight at 4°C: anti-fatp C11-7 ( 1/200 ) , anti-ELAV ( 1/500 , DSHB ) , anti-β Galactosidase ( 1/500 , MP Cappell ) . After washing , samples were stained with the following appropriate secondary antibodies: anti-rabbit ( Alexa 488 1/500 , Invitrogen ) , anti-rat ( alexa 633 1/500 , Invitrogen ) . For whole-mount retina , phalloidin-rhodamine ( 1/200 , Sigma ) was also used to stain rhabdomeres . Samples were mounted in DAPI mounting media ( Vectashield , AbCys ) . Fluorescent images were obtained using Zeiss 510 and 710 confocal microscopes . For ERG recordings , white-eyed flies were analyzed . Cold-anesthetized flies were immobilized in clay . A tungsten electrode ( 0 . 5–1 MΩ , Intracell ) was inserted in the back of the head and a glass electrode filled with 3 M KCl ( 2–6 MΩ ) was poked through the cornea . Flies were dark-adapted for 2 min before recording . An orange LED ( 591 nm , 2800 mcd , 40° beam , LY 5436-VBW-1 , Osram , France ) was placed at 1 cm from the head . The flash intensity reaching the eye was 650 µW/cm2 , as measured with a PM100D power meter and S121C photodiode ( Thorlabs , Maisons-Laffitte , France ) . Signals were filtered at 2 kHz and digitized at 10 kHz , using a MultiClamp 700A amplifier , a Digidata 1322A interface and pClamp-8 software ( Molecular Devices , Sunnyvale , USA ) . Flash intensity and duration were controlled through pClamp and the Digidata analog output . Horizontal adult eye sections were performed using a cryostat microtome ( Microm ) and deposited on superfrost Plus slides ( Thermo ) . Third instar laval imaginal discs , midgut and salivary gland were dissected in 1X PBS . Samples were fixed 5 min in PBS 0 . 25% gluteraldehyde . They were stained in a solution of 7 . 2 mM Na2HPO4 , 2 . 8 mM NaH2PO4 , 150 mM NaCl , 1 mM MgCl2 , 3 mM K3[Fe ( CN ) 6] , 3 mM K4[Fe ( CN ) 6] , containing a 1/30 dilution of X-Gal ( 30 mg/ml in dimethyl formamide ) . After washing in PBS , samples were mounted in DAPI mounting media ( Vectashield , AbCys ) . mRNA was extracted from 20 retinas and 40 embryos using QIAshredder and RNeasy Mini kits ( Qiagen ) . 100 ng of mRNA was used to synthesize cDNA using the Enhanced Avian RT First Strand Synthesis Kit ( Sigma Aldrich ) following manufacturer's instruction . Briefly , mRNA is incubated at 70°C for 10 min with dNTP ( 0 . 5 mM ) and oligodT ( 3 . 5 µM ) and then incubated at 50°C for 1 h with 1X buffer , reverse transcriptase enzyme ( 20U ) and RNase inhibitor ( 20U ) . PCR was performed using GoTaq ( Promega , 2U ) in GreenGoTaq Buffer ( Promega , 1X ) with 200 µM dNTP and two pairs of primers ( 200 nM each , fatp forward: GGATTTTTGCTGTGCTCGTC , fatp reverse: ACCACATCGCCCTTTTTGTA , rp49 forward: CGGATCGATATGCTAAGCTGT , rp49 reverse: GCGCTTGTTCGATCCGTA ) . rp49 amplification was used as an internal control . cDNA was first denatured for 5 min at 95°C and amplified during 35 cycles: 95°C for 30 s , 59°C for 30 s , 72°C for 42 s; followed by an incubation at 72°C for 7 min . Amplified cDNA was segregated in 1 . 5% agarose gel . 10 Drosophila adult heads were homogenized in 30 µL Laemmli buffer ( 10% glycerol , pH 6 . 8 0 . 5M Tris , 10% SDS , 1% bromophenol blue , 1% β-mercaptoethanol , 100 mM DTT ) and centrifuged for 30 min at 12 , 000 g . Supernatant was boiled for 5 min and 10 µL was loaded onto a 12% acrylamide gel ( Biorad ) and transferred onto nitrocellulose membranes ( Whatman ) . Anti-Fatp C11-7 ( 1/200 ) , anti-Rh1 ( 1/1000 , 4C5 , DSHB ) and anti-Tubulin ( 1/1000 , Sigma ) antibodies were incubated overnight at 4°C and appropriate HRP-coupled secondary anti-mouse and anti-rabbit antibodies ( 1∶10 000 , Biorad ) were then incubated for 2 h at RT . Chemiluminescent detection was carried out using a ECL kit ( GE Healthcare Life Sciences ) . Protein band quantification was carried out using ImageJ software . For blue light-induced degradation of Rh1 , white-eyed heads were exposed to 10 mW blue light for 6 h and homogenized in Tris-buffered saline ( 20 mM Tris ( pH 7 . 5 ) , 150 mM NaCl ) containing 0 . 5% Triton X-100 and protease inhibitors . After centrifugation , the supernatant was mixed with an equal volume of 2X Laemmli buffer and loaded onto a 12% acrylamide gel as described above . | Normal vision requires precise regulation of the visual response . The deregulation of the visual response can lead to retinal diseases and blindness . The most frequent retinal disease is retinitis pigmentosa , and in 30%–40% of such cases rhodopsin , the light-sensitive protein , is mutated . Approximately 100 rhodopsin mutations have been identified; they affect folding , trafficking , and activity of the rhodopsin protein and induce consequent photoreceptor neuron death . Despite extensive studies , many aspects of retinal degeneration remain unclear . Drosophila is a suitable model organism to study retinal diseases . To understand the mechanisms of retinal degeneration , we studied the previously uncharacterized fatty acid transport protein ( fatp ) gene in Drosophila . We found that flies that lack the fatp gene exhibit a deregulation of the visual response and an adult-onset and progressive retinal degeneration . In addition , we show that such retinal degeneration is due to the death of photoreceptor neurons in which rhodopsin proteins accumulate . In summary , we report a novel fly model of adult-onset retinal degeneration . We uncovered a novel and interesting mechanism by which Fatp , a potential regulator of lipid transport and metabolism , is responsible for the regulation of rhodopsin levels in the photoreceptor neurons . | [
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"gen... | 2012 | Drosophila Fatty Acid Transport Protein Regulates Rhodopsin-1 Metabolism and Is Required for Photoreceptor Neuron Survival |
Progress towards controlling and eliminating parasitic worms , including schistosomiasis , onchocerciasis , and lymphatic filariasis , is advancing rapidly as national governments , multinational NGOs , and pharmaceutical companies launch collaborative chemotherapeutic control campaigns . Critical questions remain regarding the potential for achieving elimination of these infections , and analytical methods can help to quickly estimate progress towards—and the probability of achieving—elimination over specific timeframes . Here , we propose the effective reproduction number , Reff , as a proxy of elimination potential for sexually reproducing worms that are subject to poor mating success at very low abundance ( positive density dependence , or Allee effects ) . Reff is the number of parasites produced by a single reproductive parasite at a given stage in the transmission cycle , over the parasite’s lifetime—it is the generalized form of the more familiar basic reproduction number , R0 , which only applies at the beginning of an epidemic—and it can be estimated in a ‘model-free’ manner by an estimator ( ‘ε’ ) . We introduce ε , demonstrate its estimation using simulated data , and discuss how it may be used in planning and evaluation of ongoing elimination efforts for a range of parasitic diseases .
The World Health Organization ( WHO ) Neglected Tropical Disease ( NTD ) Roadmap [1] advocates targeting several parasitic diseases , including schistosomiasis , onchocerciasis , and lymphatic filariasis ( LF ) , for elimination or control . Control measures generally rely on mass drug administration ( MDA ) and environmental interventions that interrupt the parasites’ life cycles [2] . The WHO has set goals to locally eliminate ( e . g . malaria from the Greater Mekong Region by 2030 [3] ) or globally eradicate ( e . g . yaws by 2020 [4] ) certain NTDs in the coming years , and elimination may also be within reach for onchocerciasis in Guatemala [5]; Chagas disease in parts of Central and South America [5]; lymphatic filariasis in Brazil , Haiti and The Dominican Republic [5]; trachoma in Mexico [5]; and schistosomiasis in China [6] . However , assessing the control effort required to reach elimination in these settings remains a major challenge . The standard mathematical epidemiological approach relies on fitting differential equation models to epidemiological data , and examining model parameters and associated dynamical features . Such models are simple enough to be rapidly solved and yet rich enough to encode many of the mechanisms that give rise to epidemics , e . g . , person-to-person contact processes , incubation times , and infectious periods [7] . However , these models require substantial knowledge of the epidemiological system so that the essential biological processes can be properly mathematically formulated . For this reason , non-parametric approaches ( i . e . , approaches that do not require model parameters to be fitted to data ) that analyze empirical data , such as time series of infection intensity or disease prevalence , have also been proposed to answer policy-relevant questions , such as determining when a disease is close to elimination [8] . A powerful way to use mathematical epidemiology in systems in which the full model cannot be easily specified is to use quantities from well-formulated models to generate model-free analyses of commonly collected data . We refer to analyses that are ‘model-free’ as those that do not require the construction and parameterization of a mechanistic ( or simulation ) model . Here , the effective reproduction number , Reff , is presented as one such candidate quantity for assessing the elimination feasibility of parasitic diseases . Because morbidity associated with parasitic infections is often related to infection intensity ( i . e . , the number or burden of adult parasites or worms harbored by human hosts ) rather than the presence or absence of infection in a particular individual [9] , the effective reproduction number is defined as the number of parasites produced by a single reproductive parasite at a given stage in the transmission cycle , over the parasite’s lifetime . The effective reproduction number is the generalized form of the more familiar basic reproduction number , R0 , which only applies at the beginning of an epidemic . Reff is a function of parasite population density ( it should correctly be referred to as Reff ( W ) but we use the more compact form Reff throughout this article ) and thus changes as the parasite density changes over time [10] . When Reff = 1 , the parasite population is at an equilibrium , where it is expected to be perfectly replaced over the course of its lifetime . Furthermore , Reff > 1 indicates a parasite population expected to increase in size while a population with Reff < 1 would be expected to decrease . The Reff profile ( Fig 1 ) captures the relationship between Reff and parasite population density , providing a summary of system stability across a range of parasite burdens within the human host population . Under the influence of both positive density dependence ( PDD; e . g . the mating probability of dioecious—two gendered—parasites ) and negative density dependence ( NDD ) ( see [10] ) , the Reff profile intersects Reff = 1 twice and has a humped shape . Further examples of PDDs include parasite suppression of the host’s immune response [11] , and of NDDs include host immunity and parasite crowding effects acting upon parasite establishment or fecundity [12] . An endemic equilibrium ( Fig 1 , Weq ) occurs at high parasite burdens where parasite population growth is restricted by NDD ( e . g . , because of crowding , competition , or host immunity ) . The breakpoint ( Fig 1 , Wbp ) occurs at a low parasite burden where strong PDD , also known as an Allee effect [10 , 13] , facilitates parasite elimination due to reductions in parasite reproduction ( e . g . , due to mate limitation ) [10] . The Reff profile can be used to estimate key features of a parasite population such as the rate of rebound following treatment , the expected parasite burden at the endemic equilibrium , or the expected breakpoint parasite population size [10] . Even though the mating probability PDD is often ignored in traditional parasitic models , there are a number of studies that have included this PDD , and breakpoints , in their theoretical analyses [10 , 14–18] . Below the breakpoint ( Fig 1 , Wbp ) , Reff < 1 and the effects of PDD are expected to drive the parasite population to elimination . The breakpoint therefore provides a logical target for intervention campaigns that decrease parasite burden , e . g . , through the use of MDA , as reducing the parasite population below its breakpoint should result in elimination even without further resources devoted to intervention . Detecting the influence of PDD on parasitic disease systems and summarizing its potential influence on the elimination feasibility of sexually reproducing parasites is therefore of great value to public health practitioners developing campaigns to eliminate parasitic diseases from human populations . We hypothesize that parasite populations influenced by PDD exhibit distinct dynamics that are detectable in epidemiological datasets measuring parasite burden or prevalence through the course of an intervention campaign , and that straightforward analysis of these datasets can shed light on the elimination feasibility of parasite populations subjected to regular interventions . To demonstrate this , the Reff profile of the dioecious macroparasitic disease schistosomiasis is estimated , and shown to approximate the rate at which infection intensity rebounds following MDA , a quantity referred to as the Bounce Back Rate , BBR . Furthermore , it is shown that data collected from longitudinal intervention campaigns ( e . g . , annual MDA campaigns conducted in endemic schistosomiasis communities ) can generate time-series of the BBR that can be used to develop an estimator that quantifies the elimination feasibility of the parasite population . The estimator is evaluated by comparing it to the results of commonly used population stability analyses . This estimator is presented as a simple , model-free quantity which can be used to determine the elimination feasibility of a parasitic disease subjected to regular control efforts , and more stringent and systematic collection of data that could be used to derive it is suggested .
The basic schistosomiasis model is given by the following system of differential equations: dSdt=fN ( 1−NC ) ( S+E ) −μNS−12βWHϕγS ( 1 ) dEdt=12βWHϕγS− ( μN+σ ) E ( 2 ) dIdt=σE− ( μN+μI ) I ( 3 ) dWdt=λIρ− ( μW+μH ) W ( 4 ) where the density dependent parameters , ϕ , γ , and ρ , are estimated as: ϕ=1− ( ( 1−WW+κ ) κ+12π∫02π1−cosθ ( 1−WW+κcosθ ) ( 1+k ) dθ ) ( 5 ) γ= ( 1+ ( 1−e−α ) Wκ ) − ( κ+1 ) ( 6 ) ρ=e ( 1−vW−e−vW ) ( 7 ) and N = S + E + I is the total snail population . Parameter and state variable descriptions and values used in the model are shown in Table 1 . The transmission parameters , β and λ , representing man-to-snail and snail-to-man transmission , respectively , are estimated by fitting the full model to epidemiological data collected in a community in the Senegal river basin as described elsewhere [20 , 21] . The aggregation parameter of the negative binomial distribution , κ , is also estimated directly from the data ( S1 Text ) . The degree of parasite aggregation may differ for different stages of the parasite life cycle , but even complex simulation models generally use only a single aggregation parameter [25]; we therefore use a single parameter for the parasite negative binomial distribution to allow simple interpretation of our modelled results . While the BBR can be estimated from the model ( Eq 11 ) , its value lies in its model-free estimation from empirical data . Consider a schistosomiasis intervention program in which an infected human population is treated with annual rounds of MDA . Previous to MDA in year ( or other time frame ) i , mean worm burden , Wprei , in the human population is assessed , while mean worm burden in the human population following MDA , Wposti , is estimated based on the coverage of treatment and the efficacy of the administered anthelminthic drug . Therefore , in what follows , Wposti is the worm burden in the population following MDA in year i , and Wprei+1 is the worm burden in the human population before MDA in the following year i+1 . Using the negative binomial aggregation parameter , κ , the prevalence before , PREVprei , and following , PREVposti , MDA can also be estimated via simple conversion of W to the probability that an individual is infected . The BBR in year i is then estimated using these longitudinal infection or prevalence data as: BBRi= ( 1Wposti ) ( Wprei+1−Wpostiti+1−ti ) ( 12 ) This is the finite-difference estimator of the continuous time BBR ( Eq 11 ) , which is linearly related to Reff , as previously established . Based on this relationship , it is next demonstrated that the underlying dynamics of disease transmission driven by PDD are detectable in empirical datasets by calculating the expression in Eq 12 . From Eq 11 it can be seen that BBR should decrease and eventually become negative as Reff approaches and passes the breakpoint over the course of long-term MDA . Because this behavior will only occur in the presence of strong PDD , longitudinal analysis of BBR can provide information on the presence and strength of PDD and the elimination feasibility of the disease . Specifically , adopting the simplest possible relationship , if BBR is related to time using a simple linear model: BBR ( time ) =ϵ*time ( 13 ) The parameter ε is expected to be positive , or not significantly different from zero , for the PDD-free model , while it should be negative for the model accounting for mating probability and PDD . The coefficient , ε fit to longitudinal BBR data—termed the elimination feasibility coefficient—is estimated using the PDD and PDD-free models run with each of the 100 parameter sets explained above . The ability of ε to identify disease transmission dynamics that are influenced by strong PDD—which are expected to be more susceptible to elimination from MDA-based intervention—is determined by estimating ε for each model for each year 3 < i < 20 in the 20 years of simulated annual MDA . In addition , for each set of estimates of ε derived from the 100 parameter sets and for each year where 3 < i < 20 , one-sided t-tests are used to identify significantly negative values of ε in data derived from the PDD and PDD-free models . An estimate of ε significantly less than 0 provides evidence of the influence of PDD , as it implies BBR is consistently decreasing with successive treatments . The ability of ε to predict the feasibility of elimination in parasite populations influenced by PDD was evaluated by comparing ε to the probability of extinction , P ( e ) —or strictly-speaking local extinction , for parasite control programs , though we will not use the word local in what follows—a common estimate of population viability in ecology and conservation biology [28] . Derived from stochastic models of population dynamics , P ( e ) explicitly incorporates demographic stochasticity , known to have a strong influence on population dynamics [28] . Simulating transmission and annual MDA with a stochastic model across a range of transmission intensities 1 . 3x10-4 ≤ λ ≤ 3 . 0x10-4 , corresponding to the upper and lower bounds of the 95% CI of λ , and parasite aggregation ( 0 ≤ κ ≤ 2 ) therefore provides an estimate of population viability in a variety of transmission scenarios . Stochastic versions of the PDD and PDD-free models were developed using the Gillespie Stochastic Simulation Algorithm [29 , 30] with the adaptivetau package in R [31] . Additionally , random observation noise was added to simulated measurements of worm burden by resampling the estimated mean worm burden with the R function rnbinom to represent imperfect sampling and diagnosis associated with estimates of parasite burden in human host populations [15] . These same resampled worm burden data were then also used to estimate prevalence . Simulated instances ( n = 1 , 000 ) of longitudinal MDA intervention campaigns , as described in section 2 . 2 . 1 , were generated for each of 2500 parameter sets drawn from the ranges of λ and κ above and holding all other parameters to values shown in Table 1 . For each parameter set , the probability of extinction , P ( e ) , was estimated as the proportion of model instances that result in elimination ( i . e . , induced extinction of the parasite population ) out of all 1 , 000 instances . In each simulation , ε was estimated as in Eq 13 and the mean of all 1 , 000 estimates of ε for each parameter set was examined with respect to its correlation with P ( e ) estimates using a simple linear model with logit-transformed estimates of P ( e ) ( thus transformed due to the natural [0 , 1] range of P ( e ) ) : logit ( P ( e ) ) ∝ϵ ( 14 ) Agreement between ε and P ( e ) implies that the model-free elimination feasibility coefficient is a reliable estimate of how likely it is the that a parasite population will be eliminated over the course of a routine MDA campaign . In summary , the higher the positive value of the elimination potential , the further from elimination the system currently lies . As the estimator becomes negative , the system is moving towards elimination and growing negative values indicate progress in this direction . In all modelled parasite prevalence and intensity trajectories that follow , we display the output values from the model unadjusted for diagnostic sensitivity or specificity .
The Reff profile ( Fig 1 ) indicates that , without PDD , Reff → R0 as W → 0 ( Fig 1 , dashed line ) . The disease-free equilibrium ( W = 0 ) is unstable for the PDD-free model , while it is always stable for the PDD model ( Fig 2B , dashed lines ) . As anticipated , the PDD model produces a BBR profile with a humped shape , intersecting a line at BBR = 0 twice: at the breakpoint , Wbp , and the endemic equilibrium , Weq ( Fig 2A , solid line ) . This closely resembles the Reff profile shown in Fig 1 , which can indeed be recovered from the BBR profile through transformation according to Eq 10 . In such a system , as W → 0 , Reff → 0 ( Fig 1A , solid line ) and dWdt < 0 while W < Wbp and BBR < 0 ( Fig 2A & 2B ) . A consequence of the global stability of the endemic equilibrium of the PDD-free model is that the parasite density will always rebound after MDA is halted [19] . In the PDD-free model , dWdt remains above 0 even with repeated MDA driving W → 0 ( Fig 3A , red line ) , with the potential to rebound to pre-treatment infection levels . The low value of W after 20 rounds of treatment in the PDD-free model could mistakenly be interpreted as successful elimination , yet rebound occurs after release of treatment ( Fig 3A , red line ) . By contrast , the PDD model exhibits no such rebound , since repeated MDA suppresses W below Wbp ( Fig 3A , black line ) , restricting transmission in the absence of exogenous parasites added to the system . With respect to bounce back rate , the PDD model yields a steady decline over the period of MDA and beyond caused by a decreasing mating probability as W → 0 , and resulting in the anticipated decline in BBR . As W falls below Wbp , a negative estimate of BBR is also observed ( Fig 3B ) . The PDD-free model shows no such decline in BBR with successive MDA , as the mating probability remains high irrespective of reductions in W , and in fact BBR rises with each round of MDA as the effects of NDD diminish as W → 0 ( Fig 3B ) . The slope of the BBR profile provides an estimate of ε ( Eq 12 ) , the elimination feasibility coefficient and , as anticipated , the PDD model yields a BBR profile with a value of ε < 0 , while the PDD- free model shows ε > 0 . When ε < 0 , elimination is feasible using successive MDA treatments that drive W below Wbp , while ε > 0 implies greater resistance to achieving elimination . Trajectories of prevalence rather than worm burden through the course of annual MDA intervention reveals similar dynamics that yield sustainable elimination in the PDD model and rebound to endemic prevalence in the PDD-free model ( Fig 4A ) . Prevalence based estimation of BBR and ε reveals two key differences from the worm burden based estimation , however . The first is that ε is initially both positive and relatively large in magnitude ( Fig 4C ) as a result of relatively minor changes in the overall prevalence initially induced by MDA . This leads to ε becoming negative only after 7 years of annual MDA . However , as opposed to BBR estimates derived from worm burden data , every estimate of the prevalence-based BBR is < 0 ( Fig 4B ) implying that , at least in the early stages , negative estimates of the prevalence-based BBR may serve as a better indicator of progress towards elimination than prevalence-based estimates of ε . The probability of extinction , P ( e ) , was analyzed in a stochastic modeling framework across a range of realistic transmission intensities and degrees of aggregation of parasites among the human host population , which moderates the strength of PDD . A broad range of transmission scenarios were encapsulated in the tested parameter sets as P ( e ) ranged from 0 to 1 . Fig 5 shows the mean ε for each parameter set and its corresponding value of P ( e ) , each derived from 1 , 000 simulations of the stochastic model . The elimination feasibility coefficient was strongly correlated with logit-transformed P ( e ) ( Eq 14 , R2 = 0 . 94 ) . Additionally , the proposed threshold of ε < 0 as an indicator of feasible elimination appears to be corroborated by corresponding estimates of the probability of elimination with P ( e ) ~ 0 . 25 when ε = 0 and P ( e ) → 1 as ε decreases . At low transmission intensities , the elimination feasibility coefficient remains negative in the presence of any PDD ( i . e . κ > 0; Fig 6 ) . Examining the surface resulting from estimation of ε across a range of transmission intensities and degrees of PDD illustrates that , as transmission intensity increases , ε increases and remains greater than 0 irrespective of the degree of PDD . At these high transmission intensities , single rounds of annual MDA—as were simulated in this experiment—are insufficient to make sustainable reductions in transmission .
Achieving local elimination of global infectious diseases , particularly parasitic worms that cause a variety of NTDs , has become a major focus of the global health community . The availability of chemotherapeutic drugs is at an historic high due to drug donations from large pharmaceutical companies , and to a commitment from multinational NGOs , such as The Bill and Melinda Gates Foundation , to combat NTDs [32] . Yet measuring and tracking progress towards elimination remains challenging [33 , 34] . Here , a model-free estimator of elimination potential for parasites with sexual reproduction and positive density dependence was presented , based on longitudinal measurements of worm burden or disease prevalence in human populations treated with regular MDA . The estimator , ε , is based on observations of the bounce back rate , BBR , of parasitic infections in the host population following MDA , which are shown to be strongly associated with the effective reproduction number , Reff . Negative values of ε derived from worm burden and/or negative values of prevalence-based BBR indicate consistent reductions in transmission that are expected to result in elimination if conditions such as MDA frequency and coverage hold . Short-term estimates of BBR ( measurable following each round of MDA ) and long-term estimates of ε ( measurable following three or more years of well-documented MDA ) can provide a robust estimate of control status and progress towards elimination . Values of BBR derived from worm burden data that approach or reach 0 may indicate reductions in transmission that result in little to no reinfection in the measured time period . Furthermore , negative BBR following MDA may indicate a parasite population that has been driven below its breakpoint and is heading towards elimination due to reduced mating probabilities and below-replacement reproduction ( i . e . , Reff < 1 ) . Negative values of BBR derived from prevalence data may indicate successful reduction in transmission that can be coupled with longer term prevalence-based estimation of ε to indicate progress towards elimination . Meanwhile , high values of BBR ( > 0 . 3 ) may indicate ineffective interruption of transmission and high rates of transmission ( Reff > 2 ) . Since most parasites that infect humans and have sexual reproduction exhibit PDD [19] , ε estimates should tend to be negative as W → 0 in the presence of effective interventions . The elimination feasibility coefficient can therefore serve as a measure of progress and success in an intervention campaign , with negative estimates of ε implying a campaign is achieving sustained success in transmission interruption . On the other hand , positive values of ε could be indicative of a campaign failing to induce sustainable reductions in transmission due to unmeasured , untreated , or otherwise unaccounted for demographic groups [15] , environmental conditions such as temperature and precipitation [35] , and connectivity between transmission sites [36] . Sensitivity analysis of the model revealed the possibility that high , but reasonable , levels of transmission can overwhelm efforts to induce significant reductions in parasite burden using annual MDA alone . Positive estimates of ε and/or high BBR values could indicate such a situation , signaling especially challenging conditions for achieving elimination by MDA alone and possibly necessitating additional sanitation and/or education and/or snail control based interventions . Simultaneous estimation of BBR and ε can provide public health practitioners with a quantitative framework to assess the status of an NTD intervention as it transitions from morbidity control to transmission interruption to elimination . Coupling estimates of ε and BBR may thus be valuable to infer disease dynamics over the course of an intervention campaign . Importantly , however , estimation of ε is reliant on consistent surveys of parasite burden ( that is , measures of infection intensity rather than simple prevalence ) in the human host population over multiple years . Indeed , the viability of any quantitative measure of the progress of control efforts hinges upon the data that are collected before , during , and after the campaign . Here , documentation of infection intensity in the host population prior to treatment , accurate assessment of the treatment coverage , and common assumptions regarding treatment efficacy are all necessary to derive values of Wpre and Wpos or PREVpre and PREVpos that are used to quantify BBR and ε . Such data are available from long-standing , well-documented MDA campaigns , yet rarely are these data made available in public databases [14] . It goes without saying that the collection and reporting of data on parasite burden ( infection intensity ) during MDA campaigns remains a critical priority , particularly as new methods become available to analyze and interpret such data with respect to estimating intervention efficacy . While infection intensity data may be preferred , we acknowledge the great difficulty in collecting parasite or egg counts from infected hosts , and demonstrate how similar methods can be adapted to more commonly and easily collected prevalence data [37] . In the present work , we examined performance of the elimination feasibility coefficient in predicting MDA success in the presence of observation noise , and under the influence of demographic stochasticity . Estimates of ε were compared to the probability of extinction , P ( e ) , a well-established measure of population viability in conservation biology and population ecology which has recently been investigated in relation to elimination strategies in the parasitic disease Onchocerciasis [38] . A strong correlation between P ( e ) and ε was found , including a meaningful threshold at ε = 0 , where there is approximately a 20% chance of elimination under the simulated MDA intervention scenario , and chances increase as ε decreases . These results suggest that the magnitude of the elimination feasibility coefficient may be a valuable indicator of the probability of extinction ( i . e . the success of elimination efforts ) . Future research should explicitly incorporate known sources of heterogeneity that may influence elimination feasibility , beyond those explored here . These should include seasonality , connected transmission sites , untreated demographic groups , and other sources of heterogeneity that can limit the impact of intervention campaigns and threaten their success . Methods to account for these sources of variability and uncertainty have been suggested , such as recent work to develop an accurate predictor of parasite extinction based upon a measure of the variance of disease prevalence over time [39] . Still other work [40] has sought to derive prevalence-based indicators of the elimination probability for STHs using an individually-based stochastic model , finding that end-point prevalence serves as the most reliable indicator of elimination probability . The work represented here adds to these analyses by proposing a potential mechanism underlying the observed effects , and providing additional approaches for estimating elimination probability . As a whole , this body of work demonstrates the utility of considering phenomena such as critical slowing down around population thresholds that is extensively investigated in other fields , but underappreciated for its applications to disease elimination . Critical slowing down is a phenomenon encountered in dynamical systems that are close to phase transitions , initially investigated in physics but carried over to ecology by Holling [41] . Since phase transitions occur in the vicinity of unstable equilibria , where the rates of change of system variables approach zero , we naturally expect this slowing down and we argue here that this is exploitable information in the context of disease elimination . A key remaining question that deserves further theoretical and applied exploration is how to most effectively increase the breakpoint population size of sexually reproducing parasites subject to PDD , and thus increase the population’s susceptibility to elimination , especially when MDA alone appears insufficient to reach elimination due to very high transmission rates . If the breakpoint is very low , then immigration of a single infected individual from outside the treated area could result in the system suddenly returning to the trajectory towards the locally stable endemic equilibrium . Interventions that alter the degree of PDD over time by targeting high risk and/or heavily infected individuals may successfully increase the breakpoint , and interventions such as snail control , sanitation infrastructure , and education campaigns that reduce exposure may also increase the breakpoint by reducing the population’s overall capacity for transmission . The elimination feasibility coefficient , ε , represents a model-free summary estimate of the probability of elimination that can be used to evaluate ‘end game’ control programs aiming for parasite elimination . | Critical questions remain regarding the potential for achieving elimination of helminth ( worm ) infections , and methods are needed to quickly estimate the probability of achieving elimination over specific timeframes . Here , we make use of methods in mathematical epidemiology to propose an estimator of elimination potential , and then apply this estimator of Reff−the number of parasites produced by a single reproductive parasite at a given stage in the transmission cycle , over the parasite’s lifetime—as a so-called ‘elimination feasibility coefficient , ’ or ‘ε . ’ We compare this metric ε to commonly used metrics of population viability for the parasitic disease schistosomiasis . The value of this estimator is that it allows us to determine whether an elimination program is succeeding and how much effort might be required to bring it to completion . | [
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"tro... | 2018 | Estimating the elimination feasibility in the 'end game' of control efforts for parasites subjected to regular mass drug administration: Methods and their application to schistosomiasis |
The recessive lethal mutation flotte lotte ( flo ) disrupts development of the zebrafish digestive system and other tissues . We show that flo encodes the ortholog of Mel-28/Elys , a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex ( NPC ) assembly in amphibian and mammalian cells . Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis . p53 mutation rescues apoptosis in the flo retina and optic tectum , but not in the intestine , where the checkpoint kinase Chk2 is activated . Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae . By contrast , flo mutants were not sensitized to agents that cause DNA double strand breaks , thus showing that loss of Elys disrupts responses to selected replication inhibitors . Elys binds Mcm2-7 complexes derived from Xenopus egg extracts . Mutation of elys reduced chromatin binding of Mcm2 , but not binding of Mcm3 or Mcm4 in the flo intestine . These in vivo data indicate a role for Elys in Mcm2-chromatin interactions . Furthermore , they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress .
Programmed cell death is believed to function in two contexts during early mammalian development . Prior to implantation and near gastrulation , apoptosis eliminates embryonic cells rendered unfit by growth factor deficiency [1] . At other stages , apoptosis serves a morphogenetic role by eliminating cells required for tissue reorganization [2] , [3] . Although apoptosis is normally activated in only a small number of cells of early mammalian embryos , gene targeting experiments have demonstrated the susceptibility of surviving cells . Genomic instability is speculated to be one possible underlying cause of this predisposition , since mutation of DNA repair [4]–[11] and cell cycle checkpoint genes [12] , [13] can activate apoptosis of inner cell mass cells . This susceptibility of embryonic progenitor cells to apoptosis persists through later developmental stages as evidenced by the effect of conditional inactivation of DNA repair and checkpoint genes in specialized cells such as neurons [14] and mammary epithelia [15] . Elys is a conserved mammalian gene that is required for embryonic survival during early development [16] . Embryos homozygous for a null allele of Elys are resorbed at peri-implantation stages ( e5 . 5–e7 . 5 ) and inner cell mass cells from cultured Elys−/−blastocysts undergo apoptosis soon after hatching from the zona pellucida . Elys is expressed throughout the developing mouse embryo and in a wide range of adult tissues [17] , as is human ELYS ( http://www . ncbi . nlm . nih . gov/UniGene/ESTProfileViewer . cgi ? uglist=Hs . 300887#Legend ) . Recently human and frog Elys proteins were shown to be orthologs of the gene encoding Mel-28 , a protein required for nuclear integrity in C . elegans [18] , [19] . Human and frog Elys proteins physically associate with the Nup107–160 nuclear pore protein complex [20] , [21] and localize to kinetochores during mitosis , as has been described for other nuclear pore proteins ( nucleoporins ) . RNAi mediated knockdown of ELYS protein inhibited nuclear pore complex ( NPC ) assembly , thus identifying ELYS as either a component of the NPC that directs its assembly , or a protein that organizes the NPC in the chromatin periphery . In addition to its association with the Nup107–160 nucleoporins , Elys interacts with the Mcm2-7 DNA helicase complex on chromatin derived from Xenopus egg extract [22] . This interaction was proposed to be a mechanism that allows cells to coordinate nuclear assembly with the requirement to shut down replication origin licensing prior to S-phase entry . Here , we show that flotte lotte ( flo ) , a previously described recessive zebrafish mutant with retinal , neural and digestive organ defects [23]–[26] , arises from mutation of zebrafish elys . Strong maternal elys expression enables cells of early flo embryos to survive to larval stages . However , at later stages proliferative cells in tissues in which NPC assembly is disrupted , such as the retina , optic tectum and intestine , undergo cell cycle arrest and ultimately succumb to apoptotic cell death via p53-dependent and p53-independent mechanisms [27] . p53-independent apoptosis in the flo intestine is associated with activation of the Chk2 protein kinase [28] , and initially , normal levels of the DNA damage marker γH2AX [29] , [30] . This suggested that Elys may be needed to resolve replications errors that normally occur in highly proliferative organ progenitor cells . Consistent with this idea , we found that Chk2 activation was required for the survival of homozygous flo larvae , but not their heterozygous or homozygous wild type siblings . Homozygous flo larvae , but not their siblings , were also sensitized to DNA replication inhibitors , but not agents that induce DNA double strand breaks . Finally , we also found that loss of Elys reduces levels of chromatin bound Mcm2 , but not Mcm3 , Mcm4 or phospho-Mcm4 in the wild type or irradiated flo intestine . Together , these and other data support a role for Elys in DNA replication and the cellular response to replication stress , independent of its role in NPC assembly .
Zebrafish flotte lotte ( floti262c; hereafter flo ) is a recessive lethal mutation that was identified as part of a large scale ENU mutagenesis screen [23] . Small eyes , optic tectum degeneration and digestive organ defects are prominent features of the flo mutant phenotype that are first evident in live fish on the fourth day post-fertilization ( Figure 1A–1D ) . In previous work , the flo mutation was shown to disrupt differentiation and survival of 3 of the 4 principal intestinal epithelial cell lineages [24] , [26] , suggesting an effect on organ progenitor cells . Here , we show a comparable though less pronounced effect on retinal development ( Figure 1E–1H ) . Histological analyses revealed that the cellular layers of the flo retinal epithelium were disorganized and contained numerous cells with condensed nuclei typical of apoptotic cells . Acridine orange and TUNEL staining revealed that apoptotic retinal ( Figure 1I–1J ) and intestinal epithelial cells ( Figure 1K–1L ) were already evident before flo mutants were morphologically distinguishable from wild type larvae . Immunohistochemical studies showed that differentiated retinal ganglion and photoreceptor cells were present in flo mutants , albeit at reduced levels compared with wild types , despite retinal disorganization and apoptosis ( Figure 1M–1P ) . These studies suggested that early stages of retinal development proceed normally in flo mutants , most likely as a result of maternally derived mRNA and protein encoded by the affected gene ( discussed below ) . To further assess the effect of the flo mutation on organ progenitors , we also measured cell proliferation in the intestinal and retinal epithelium . Both sets of data were consistent with G1 arrest of rapidly proliferating cells in these tissues ( Tables S1 and S2 ) . In summary , these descriptive analyses suggested a fundamental role for the flo gene in tissue progenitor cells . For this reason , a positional strategy was used to identify the responsible gene . Bulk segregant analysis identified markers on zebrafish chromosome 17 that were linked to the flo locus ( Figure 2 and Figure S1 ) . Analyses of 2629 mutant larvae ultimately identified a zero recombinant marker within a predicted open reading frame that encoded a gene orthologous to mammalian Elys ( also known as AT hook containing transcription factor 1; ATHCF1 ) , a gene recently shown to be required for NPC assembly and nuclear integrity in worms [18]–[21] . Elys is expressed in a wide range of tissues and is essential for early mammalian development [16] . The longest open reading frame of zebrafish elys encodes a predicted protein that consists of 2527 amino acid residues . Computational analysis of the Elys protein revealed two WD-40 repeats and three nuclear export signals in the N-terminus plus a coiled-coil region ( involved in protein-protein interactions ) and several nuclear localization signals in the C-terminus ( Figure 2C and Figure S2 ) . The consensus sequence of the AT hook domain present in mammalian Elys was only partially conserved in the zebrafish ortholog . Sequence analyses of cDNA derived from flo larvae revealed a single base pair mutation encoding a cytidine to thymidine transition that generated a premature stop codon within the predicted zebrafish elys translation product ( Figure 2B ) . This mutation is predicted to generate a truncated Elys protein that lacks 1209 amino acids containing the coiled-coil region and the nuclear localization signals . Protein truncation , coupled with reduced elys expression in flo larvae ( Figure S3 ) , most likely arising from termination codon associated mRNA decay [31] , supports the idea that transcription of the elysti262c allele generates only a small amount of active Elys protein . Antisense knockdown using morpholinos targeting either the elys 5′ UTR , translation initiation codon or the exon 30 splice acceptor ( Figure S4 ) phenocopied the flo retinal ( Figure 2D–2F and 2U ) and tectal defects ( Figure 2G–2I ) . Phenocopy was present in only a minority of embryos injected with single morpholinos ( 10%–12% ) , most likely because of the high levels of maternal elys mRNA relative to the levels of zygotically derived mRNA ( Figure 3A ) . Combined injection of the 5′-UTR and exon 30 splice donor morpholinos led to phenocopy in 30% to 50% of injected embryos ( n = >300 injected embryos ) . All elys-morpholino injected larvae with retinal and tectal apoptosis had minimal exocrine pancreas tissue ( Figure 2S–2T ) as do flo larvae [25] . Similarly , like flo mutants [4] , all of the affected morpholino injected larvae lacked intestinal goblet cells ( Figure 2J–2L ) and had a dramatic reduction in non-enteroendocrine secretory cells and enterocytes ( Figure 2M–2R ) as revealed by previously described monoclonal antibodies [32] . Apoptosis , which is a prominent feature of the flo intestinal phenotype ( discussed below ) , was not evident in the intestine of the elys-morpholino injected larvae despite clear evidence of NPC disruption . Although partial intestinal phenocopy by the Elys knockdown was not unexpected because morpholino knockdowns are often transient , these data raise the possibility that apoptosis caused by loss of Elys function may occur independently of altered NPC assembly seen in flo mutants . The pattern of elys expression in zebrafish embryos and larvae is consistent with nearly all aspects of the flo phenotype ( Figure 3 ) . As mentioned previously , high levels of maternal RNA encoding elys are present in newly fertilized zebrafish embryos ( Figure 3A ) . This could account for the absence of an early zygotic phenotype that was reported in Elys knockout mice . Early zebrafish embryos ( 24 hours post-fertilization; 24 hpf ) show strong elys expression in the brain ( Figure 3B ) , whereas at 48 hpf expression was largely restricted to the retina ( Figure 3C ) , the growth plate of the optic tectum ( Figure 3E–3F ) [33] , and the digestive organs ( Figure 3D ) , tissues that are all highly proliferative at this stage [24] , [34] , [35] . Elys expression in the zebrafish digestive organs persists through 5 days post-fertilization ( dpf ) , when cell proliferation in these organs is low , but at a greatly reduced level compared to 48 hpf , when the rate of cell proliferation is high ( Figure 3G–3H ) [24] . This dynamic expression pattern fits with the recently described role of elys in NPC assembly , since NPC turnover is low in non-proliferating cells [36] . NPC disassembly caused by ELYS knockdown in HeLa cells leads to redistribution of nucleoporins from the nuclear envelope to the cytoplasm [20]–[21] . Immunohistochemical analyses of zebrafish nucleoporins using a monoclonal antibody that recognizes nucleoporins with a highly conserved FG domain [37] showed typical nuclear staining in all wild type zebrafish tissues . By contrast , a dramatic reduction of nuclear FG-nucleoporins was evident in flo intestinal and retinal epithelial cells ( Figure 4A–4F ) . A normal FG-nucleoporin immunostaining pattern was clearly evident in flo skeletal muscle ( Figure 4G–4H ) , pronephric duct epithelia and other non-proliferative tissues ( data not shown ) . The flo retinal NPC defects were evident as early as 36 hpf , and the intestinal NPC defects could be seen at 48 hpf ( Figure S5 ) . FG-nucleoporin immunostainings from flo and elys morpholino injected larvae were nearly identical and closely resembled those of ELYS-deficient HeLa and U2OS cells [20]–[21] thus suggesting partial cytoplasmic redistribution of these nucleoporins . Western analyses of nuclear and cytoplasmic fractions of flo intestinal proteins confirmed these findings ( Figure 4I ) . Similarly , ultrastructural analyses showed a marked reduction in the number of identifiable NPCs in the flo intestinal epithelial cells ( Figure 5 ) and also showed that the flo mutation had no effect on nuclear envelope formation or stability , as was reported in ELYS-deficient mammalian cells [21] . Together , these data confirm a role for zebrafish Elys in NPC assembly . To gain a better understanding of how NPC disruption caused apoptosis and cell cycle arrest in flo intestine and retina , we assayed expression of tp53 and p21 in 50 hpf and 75 hpf flo mutants via real-time quantitative PCR . These experiments showed increased expression of tp53 , p21 and also mdm2 , a negative regulator of p53 whose expression is induced in response to p53 activation ( Figure S6 ) . To further assess the role of tp53 in flo mutants , we injected antisense morpholinos known to target zebrafish tp53 mRNA translation [27] , [38] , [39] into newly fertilized embryos derived from matings of heterozygous flo/+ fish . The tp53 knockdowns restored normal p21 and tp53 expression in flo larvae . We also generated larvae that were homozygous for both the flo mutation and a previously described tp53 mutation that inhibits p53-dependent radiation induced apoptosis [40] . The homozygous tp53 mutation as well as the tp53 knockdowns rescued retinal apoptosis in 50 hpf flo mutants ( n = 25 flo/tp53 double mutants analyzed and >200 tp53 morpholino injected larvae analyzed; Figure 6A–6C ) . Retinal size and architecture was restored or greatly improved by the tp53 knockdown and mutation in most flo larvae ( n>50 , 5 dpf larvae examined; Figure 6D ) , but neither rescued intestinal morphology , differentiation or apoptosis ( Figure 6E; n = 25 flo/tp53 double mutants analyzed at 4 dpf ) . These data show that disruption of NPCs activates the DNA damage response in a tissue-specific manner . Levels of activated Chk1 and Chk2 protein kinases were assayed in the flo intestine to determine whether they could account for tp53-independent apoptosis . Mammalian Chk2 is activated by the ATM kinase in response to double strand DNA breaks and other types of DNA damage [28] . Phospho-Chk2 has been reported to induce cell cycle arrest and apoptosis via both p53-dependent and p53-independent signaling pathways [28] , [41] . Antibodies to phosphorylated and non-phosphorylated mammalian Chk2 recognized 55 kD zebrafish proteins in nuclear extracts from wild type and flo intestine , and morpholino knockdown confirmed the specificity of the anti phospho-Chk2 antibody ( Figure S6 ) . Levels of phospho-Chk2 ( Figure 6F ) , not phospho-Chk1 ( Figure 6G; lanes 4 , 5 ) were elevated in the flo intestine , whereas phospho-Chk2 levels were normal or only slightly elevated in the flo retina ( Figure 6 ) . To get a better understanding of the cause of Chk2 activation in flo intestine , we assayed phospho-Chk1 and phospho-Chk2 levels in response to two types of stimuli , γ-irradiation induced double strand DNA breaks and replication arrest induced by the nucleotide synthesis inhibitor hydroxyurea . Unlike mammalian cells , we found that the levels of phospho-Chk1 in wild type intestine were not increased upon treatment with hydroxyurea or γ-radiation , whereas phospho-Chk2 levels were increased in response to both . ( Figure 6G–6H and data not shown ) . This suggests that intestinal progenitor cells in zebrafish larvae rely on Chk2 rather than Chk1 to activate checkpoints in response to both DNA damage and replication inhibitors . We speculate this may be because endogenous replication stress already maximally activates Chk1 in these rapidly proliferating cells ( a 1 hr incubation with BrdU labels >25% of intestinal epithelial cells; [24] and Table S1 ) . Checkpoint proteins are typically activated in the setting of DNA damage . For this reason , we measured levels of γH2AX , a phosphorylated form of the Histone 2A variant H2AX that accumulates in cells in response to double strand breaks and DNA damage induced by ionizing radiation and other agents [29] , [30] . Western and immunohistochemical analyses showed that γH2AX was either not detectable or present at very low levels in the 75 hpf wild type and flo intestine ( Figure 6I , lanes 1 , 3; and data not shown ) . γH2AX and increased levels of phospho-Chk2 were both detectable , however , in the intestine of 75 hpf wild type and flo larvae 4 hours after γ-irradiation and treatment with hydroxyurea ( Figure 6H–6I ) , thus confirming an intact damage response in the absence of normal Elys function . Together , these data argue that Chk2 activation in the intestine of early flo larvae occurs in the setting of low level DNA damage such as may be expected to occur in the setting of replication stress . To better understand why Chk2 is activated in the flo intestine , we inhibited its function using a commercially available pharmacological inhibitor . Pretreatment of wild type zebrafish larvae with this inhibitor prevented phosphorylation of Chk2 at a conserved serine residue normally phosphorylated by ATM ( Figure S6 ) . An identical phosphorylation response has also been reported to occur with mammalian Chk2 inhibition [42] , thus supporting the activity of this inhibitor against zebrafish Chk2 . Twelve hour treatment with this inhibitor was well tolerated by 4 dpf wild type ( +/+ ) and heterozygous flo larvae , although in two independent experiments the treatment induced apoptosis in the intestinal epithelium of ∼50% of larvae analyzed ( n = 285 larvae ) . By contrast , concomitant treatment was lethal by 24 hours post-treatment for all sibling homozygous flo larvae analyzed ( n = 86 larvae ) , most likely as a result of widespread intestinal injury . Unlike flo , Chk2 inhibition was well tolerated by another zebrafish mutant , slim jim ( 0% lethality of 80 mutant larvae ) in which intestinal epithelial cell proliferation is reduced to a level comparable to flo as a result of altered RNA Polymerase III activity [43] . This lack of effect of Chk2 inhibition in slim jim mutants is expected because in contrast to flo , phospho-Chk2 levels are not elevated by this mutation ( Figure 6F ) . These data argue that checkpoint activation in flo larvae occurs in response to physiological stimuli . To further interrogate the ATM-Chk2 pathway , flo and sibling wild type larvae were treated with a commercially available ATM inhibitor . Overnight treatment of 82 hpf larvae had a profound effect on the development and survival of flo mutants and their siblings; nearly all larvae died or had pronounced cardiac and neural defects ( data not shown ) . Although a modestly more pronounced effect was evident in the flo larvae , the difference in sensitivity to ATM inhibition was minor in comparison to the differential effects of Chk2 inhibition . Treatments with caffeine , an inhibitor of the mammalian ATR-Chk1 signaling pathway was tolerated by flo and sibling larvae , but caused significant developmental delays in both groups ( data not shown ) . Chk2 dependence of flo larvae that have only low γH2AX levels suggested that Elys could be required to maintain replication forks in highly proliferative tissue progenitor cells . To test this hypothesis , we treated wild type and flo larvae with hydroxyurea , a DNA replication inhibitor that can activate Chk2 ( as well as Chk1 ) and H2AX in mammalian cells [44] and zebrafish larvae ( data not shown ) . Homozygous flo larvae were sensitized to hydroxyurea treatment . Overnight or 6 hour exposure to hydroxyurea doses ( 250 mM ) was lethal for all homozygous flo larvae analyzed ( n = 86 larvae ) but was well tolerated by all sibling wild type and heterozygous flo larvae analyzed ( n = 302 larvae ) , as well as slim jim larvae ( n = 60 mutant larvae ) . Survival of flo mutants , but not their wild type siblings or slim jim mutants , was also reduced following treatment with the DNA cross-linker cisplatin ( 10 uM; 12 hour treatment ) as well as UV-irradiation , another cause of replication stress , but not the DNA topoisomerase 1 inhibitor camptothecin , even at doses that caused widespread intestinal apoptosis and wild type larval death ( 5–20 uM; 12 hrs ) , or γ-irradiation ( 30 Gy ) . Together , these data suggest that the Elys protein may be required to prevent replication fork collapse in response to specific types of replication inhibitors . The effects of Chk2 inhibition and replication inhibitors on flo mutants are listed in Table 1 . Recently , the Mcm2-7 complex , a central component of the DNA replication helicase , was shown to bind Elys and promote its loading onto chromatin at the M-G1 transition of the cell cycle [22] . Reconstitution of the NPC complex was postulated to promote the nuclear import of Geminin , a Cdt1 inhibitor , at the onset of S-phase , thus preventing reloading of Mcm2-7 onto chromatin and DNA re-replication . These data were the first to link replication licensing with NPC assembly . Despite the absence of nuclear pores in flo retinal and intestinal epithelial cells , we found no evidence of promiscuous firing of licensed replication origins in these highly replicative cells , as arrest in G1 rather than S phase was evident in both tissues ( Table S2 ) . However , biochemical analyses did reveal an effect of Elys deficiency on Mcm-chromatin interactions . These showed significantly reduced levels of chromatin bound Mcm2 were present in the flo intestine at 75 hpf and 96 hpf ( Figure 7A and Figure S6 ) . By contrast , levels of Mcm3 and Mcm4 in flo mutants were either normal or minimally reduced at both time points ( Figure 7B–7C ) , and Mcm2 levels were only minimally reduced in intestinal cells of slim jim mutants , which have reduced proliferation and undergo cell death ( Figure S6 ) . Levels of phospho-Mcm4 , which are elevated in response to replication stress [45] , [46] were also normal in cells derived from irradiated flo intestines ( Figure S6 ) . Together , these data link Elys deficiency with reduced chromatin loading or maintenance of chromatin binding of selected Mcm2-7 complex proteins .
Nucleocytoplasmic transport enables macromolecules such as messenger and non-coding RNAs , transcription factors , and other proteins to traverse the nuclear envelope . The NPC , which consists of over thirty proteins ( nucleoporins ) , plays an essential role in mediating and regulating this process [36] , [47] . Genetic analyses in fungi , worms , flies and mammals have shown that many nuclear pore proteins are required during early development . This developmental role is thought to largely be a function of the pore's effect on transport . Recent studies however , have identified nucleoporin functions not associated with transport that may influence other cellular processes , such as the function or assembly of spindles and kinetochores , post-translational protein modification , and chromatin organization , which can affect gene transcription and DNA repair [36] , [47] , [48] . The disruption of these processes may play a contributory role to the NPC deficient phenotype . Here , we report the phenotype arising from mutation of zebrafish elys , a gene recently shown to be required for NPC assembly in human cells that was previously known to be essential to early mouse development [16] . We speculate that flo embryos survive early development because of the high level of maternal elys expression . As a result , the effects of the loss of Elys protein function during the later developmental stages , such as organogenesis , can be assayed . The principal findings of our study regarding the role of Elys protein in NPC assembly are in agreement with the reported role of Elys in human cells and in vitro assays of NPC assembly using siRNA knockdown and competitive binding assays [20]–[22] . Using ultrastructural and immunohistochemical analyses , we show that mutation or knockdown of Elys disrupts nuclear pore assembly in rapidly proliferating cells . By contrast , NPC assembly and maintenance appear normal in quiescent cells that stop cycling before stores of maternally derived Elys protein are depleted . These findings conform with studies performed in cultured cells which showed that NPCs are stable until their disassembly during mitosis [49] . The elysflo point mutation introduces a premature termination codon into the Elys cDNA , upstream of the coiled-coil domain , the AT hook region and 3′ nuclear localization signals . Absent an antibody that recognizes the zebrafish Elys protein , we cannot determine if such a protein is actually translated from reduced levels of mRNA we can detect in flo mutants . Furthermore , whether such a truncated protein might retain activity was not addressed in this study . Based on comparison with the NPC phenotype arising form Elys knockdown in cultured cell lines , the elysflo allele appears to be either a severely hypomorphic or null allele . In the future , further analysis of this or other elys alleles may be used to dissect functional domains in the Elys protein that might mediate pore and non-pore related functions ( discussed below ) . Apoptosis is a prominent feature of the flo mutant phenotype . We focused on understanding the cause of cell death in the flo intestinal epithelium because of our prior work using the zebrafish to study digestive organ development . Our experiments showed that apoptosis in the flo intestine was not dependent on p53 , whereas it was in the retina and tectum . Instead Elys deficiency activated Chk2 , a checkpoint protein that can induce cell cycle arrest and apoptosis independently of p53 . Checkpoint activation is an important component of the cellular response to replication stress , because it allows stabilization of stalled replication forks , thereby averting replication fork collapse and activation of apoptotic programs [50]–[51] . In mammalian cells replication stress activates the Chk1 checkpoint protein whereas double strand breaks are considered to be the primary stimulus for Chk2 activation [28] . For this reason , Chk2 activation in flo mutants was unexpected . γ-irradiation of zebrafish larvae induced Chk2 phosphorylation , thus showing an evolutionarily conserved role for this arm of the DNA damage response pathway in zebrafish larvae . Chk2 was also activated by replication stress induced by hydroxyurea , whereas Chk1 phosphorylation was unchanged from its baseline level of activation with either treatment . From this , we conclude that hydroxyurea induced replication stress activates Chk2 in the zebrafish intestine because Chk1 is already maximally activated by endogenous replication errors that occur in these highly proliferative progenitor cells [24] . Although we initially postulated a number of mechanisms to explain how mutation of Elys activated the DNA damage response , several lines of evidence support a model in which Elys protein is required for the repair of replication errors that occur in rapidly dividing cells . First , Chk2 inhibition was lethal to flo larvae even though we found evidence of only low levels of DNA damage in the mutant larvae at this stage ( normal γH2AX levels ) . This argued that checkpoints were activated in response to physiological stimuli; i . e . - replication stress . Second , survival of flo mutants was reduced following treatment with doses of replication inhibitors , such as hydroxyurea , UV-irradiation and cisplatin , that had only minor effects on wild type siblings , or another mutant , slim jim , in which intestinal progenitor cells proliferation is also reduced . These data show that Elys deficient cells cannot tolerate additional genotoxic insults induced by stalled DNA replication forks , as reported for cells carrying mutations of DNA repair genes [44] . In contrast to their sensitivity to replication inhibitors , treatment of flo mutants with γ-irradiation or the topoisomerase inhibitor camptothecin did not compromise survival compared with wild type siblings . Non-sensitivity to these agents , which induce DNA double strand breaks [52] , argues that flo larvae retain the ability to activate repair pathways involving homologous recombination or non-homologous endjoining , whereas mechanisms to maintain replication forks in the setting of nucleotide deprivation ( hydroxyurea ) or intrastrand DNA crosslinks ( UV , cisplatin ) are compromised by Elys deficiency . Collectively , these data argue that sensitivity of Elys deficient cells to hydroxyurea and other agents is not a non-specific effect incurred by cells that lack nuclear pores . Levels of Mcm2-7 complex proteins in eukaryotic cells far exceed the number of active replication origins [53] . In both worms and human cells , excess Mcm2-7 complexes license dormant origins for firing when nearby replication forks are irreversibly stalled , thus preserving DNA replication and cell viability in the setting of replication stress [53] , [54] . Mcm proteins also promote replication fork stability in fission yeast [46] . We found that levels of Mcm2 , but not Mcm3 or Mcm4 , were significantly lower in chromatin preps derived from flo intestinal cells compared with both wild type siblings and slim jim mutants that had a comparable reduction of intestinal cell proliferation . These data argue that reduced Mcm2 levels are caused by Elys deficiency , rather than reduced proliferation , as previously reported [55] . Reduced Mcm2 levels associated with Elys deficiency could arise from an effect on Mcm2-chromatin loading , or maintenance of chromatin bound Mcm2 . We favor an effect on the maintenance of Mcm2-chromatin interaction since a peptide inhibitor of Elys chromatin binding did not affect binding of Mcm2 in Xenopus egg extracts [22] . Mcm protein levels vary with the cell cycle , and we initially considered this as a possible explanation for reduced Mcm2 levels in flo . However , G1 arrest induced by Elys deficiency would be expected to increase rather than decrease levels of chromatin bound Mcm2-7 complex [22] . Mcm levels have been reported to fall when cells enter replicative senescence [56] , [57] . However , senescence is associated with a reduction in the levels of all Mcm proteins [57] , whereas we found that only levels of Mcm2 were significantly reduced in flo mutants . Also arguing against this mechanism , we found that Mcm levels were not reduced in slim jim mutants that had a comparable reduction in the percentage of cycling cells that had arrested in G1 [43] . Finally , reduced Mcm chromatin loading is predicted to occur upon DNA-damage induced degradation of the licensing factor Cdt1 . However , this too is expected to reduce binding of all Mcm proteins . We speculate that reduced levels of chromatin bound Mcm2 prevents firing of dormant replication origins that sustain DNA replication stalled by endogenous replication errors , or stress arising from exogenous agents , such as hydroxyurea . When this compensatory mechanism is impaired , stalled replication forks are more likely to collapse , leading to cell death . Consistent with this idea , mice homozygous for a non-lethal hypomorphic Mcm2 allele have reduced numbers of proliferating progenitor cells in the subventricular zone of the brain [58] . As the surviving progenitor cells cycle normally , their reduced census argues that surviving progenitor cells were able to overcome endogenous replication stress using an alternative salvage mechanism . Interestingly , a comparable effect on progenitor cell survival was noted in this study in the intestine of the Mcm2 mutant mice . Activation of dormant origins of human cells exposed to hydroxyurea is also disrupted by partial Mcm5 knockdown [54] . Unlike zebrafish Elys deficiency , partial Mcm5 knockdown ( 75% reduction ) reduced levels of all other Mcm proteins analyzed , including Mcm2 ( 50% reduction ) , which may explain why survival of these cells was reduced by treatment with hydroxyurea and camptothecin whereas flo mutants were only affected by hydroxyurea . Mcm2-7 proteins form a hexameric complex in vivo , but only the Mcm4 , 6 , 7 subcomplex , and not the Mcm2 , 3 , 5 subcomplex has helicase activity in vitro . Recent work suggests that the Mcm2 , 3 , 5 subcomplex may regulate the association of hexameric Mcm2-7 complex with chromatin , and its helicase activity in vitro , through the phosphorylation of Mcm2 and or Mcm5 by the CDC7/DBF4 kinase [59] . Thus , it is conceivable that reduced Mcm2 in Elys-deficient cells impairs helicase activation at dormant origins when nearby replications forks are stalled by endogenous replication errors . As noted previously , since a peptide derived from the Elys-chromatin binding domain did not impair chromatin loading of Mcm2-7 [22] , we speculate that maintenance of chromatin bound Mcm2 involves interaction with another region of the Elys protein , other members of the NPC or an unrelated mechanism . We recognize that the lack of nuclear pores could play a significant role in the response of flo tissue progenitor cells to both endogenous and exogenous replication stress . Reduced nuclear export stabilizes p53 in response to genotoxic stress [60] , [61] and this could be enhanced in Elys-deficient cells , thus contributing to p53-dependent apoptosis in the retinal and optic tectum . Similarly , mutation of the ALADIN nucleoporin gene has been reported to sensitize cells to pharmacologically induced single strand breaks through a transport mechanism [62] . It is also conceivable that inhibition of NPC assembly affects DNA repair by altering chromatin configuration and secondarily , gene expression , as reported in yeast carrying non-lethal nucleoporin mutation [63] . Indeed , chromatin condensation was evident in Xenopus nuclear extracts depleted of Elys [21] . Electron micrographs of flo intestinal and retinal cells showed no evidence of gross chromatin defects , however , this does not exclude the possibility that zebrafish NPC disruption alters chromatin organization , such as the location of heterochromatin – euchromatin boundaries , which can epigenetically alter gene transcription . Despite these considerations , two experiments from this study argue that a lack of nuclear pores may not be sufficient to affect a cell's ability to respond to replication stress . First , p53-mutation rescued apoptosis in the flo retinal epithelium even though the NPC defect persisted in all of the double mutant cells we analyzed . Second , morpholino knockdown of elys fully phenocopied the NPC and differentiation defects present in flo mutants , but did not cause apoptosis . Thus , in both the eye and intestine , the nuclear pore defect associated with Elys deficiency could be dissociated from apoptosis . These findings are interesting in light of genetic studies in yeast and other fungi that first indicated a role for nucleoporins in DNA repair: non-lethal nucleoporin mutations can cause either sensitization to DNA damaging agents [64]–[66] or synthetic lethality when combined with mutations in DNA repair genes [67] . Given binding of Elys to Mcm proteins [22] , collectively these data suggest a role for Elys , and conceivably other nucleoporins , in chromatin interactions that are important for DNA replication in highly proliferative cells . Although it may be difficult to fully exclude a role for the NPC in the flo phenotype , one way to further address the role of Elys in DNA replication would be to generate elys alleles that preferentially sensitize progenitor cells to replication stress , rather than NPC assembly . This may identify regions of the Elys protein that are required for Mcm-chromatin interactions , but not NPC assembly . We plan to pursue this type of genetic screen and other experiments that can help characterize other interesting features of the flo mutant phenotype , such as the tissue specificity of p53-dependent apoptosis and Chk2 activation , in future studies .
Wild type , heterozygous flo ( floti262c , hereafter flo ) and p53 ( tp53zdf1 ) adult fish were maintained as described [24] . Wild type and flo larvae were treated with hydroxyurea ( Sigma Aldrich ) and the Chk2 inhibitor ( Sigma Aldrich ) at the indicated concentrations for 4 hours and overnight respectively . To identify apoptotic cells , larvae were soaked in acridine orange ( 5 ug/ml ) for 30 minutes , rinsed in embryo media and examined under a fluorescent dissecting microscope ( Olympus MVX 10 ) . Chromosomal localization of the flo locus was performed using bulk segregant analysis as previously described [68] . Details of high resolution genetic mapping of the flo locus are presented as Text S1 . Homozygous flo larvae were identified via PCR amplification of genomic DNA using primers designed to create Afl III and Hph I recognition sites at the site of the flo mutation using dCAPS Finder 2 . 0 ( http://helix . wustl . edu/dcaps/dcaps . html ) . Primer sequences are supplied in Text S1 . RNA recovery and RT-PCR was performed as described [69] . RNA from fifty embryos or dissected tissue samples was used in each experiment . Prior to 4 dpf , flo larvae were identified by molecular genotyping or retinal acridine orange staining . Primers used for RT-PCR reactions are listed in Text S1 . Morpholino antisense oligonucleotides ( Genetools; Open Biosystems ) were injected into one-cell stage wild type embryos as described [68] . In some cases , the morpholinos were also injected into the yolk of 24 or 48 hours post-fertilization ( hpf ) embryos . The sequences of elys and tp53 morpholinos are supplied in Text S1 . Histological and immunohistochemical analyses were performed as described [24] , [25] . Enterocytes and secretory cells were identified using 4E8 and 2F11 monoclonal antibodies [32] . Zn-5 antibody was obtained from the Zebrafish International Resource Center . The 1D1 anti-rhodopsin antibody ( photoreceptor cells ) was a gift of James Fadool . Monoclonal antibody mAb414 ( Covance , Berkeley , CA ) was used to recognize FG-nucleoporins . Nuclear protein was recovered from the intestine or eye of fifty embryos or larvae and pooled . Standard methods for protein recovery and Western analyses were utilized . Detailed descriptions of these methods and the specificity of all antibodies used for Western analyses are presented in Text S1 . Primary antibodies used were rabbit polyclonal anti-Phospho-Chk1 ( Ser345 ) , Phospho-Chk2 ( Ser33 , Thr68 ) , Chk2 and rabbit polyclonal anti-γH2AX ( Ser 139 ) ( Cell Signaling Technology , Danvers , MA ) and mouse mAb414 ( Covance , Berkeley , CA ) , rabbit polyclonal Mcm4 ( Bethyl Laboratories , Montgomery TX ) , Mcm2 and Mcm3 ( BD Pharmingen , San Jose CA ) . All blots are representative of at least triplicate experiments . Wild type and sibling flo larvae ( ∼80 hpf ) were treated with Chk2 inhibitor , ATM inhibitor , caffeine , hydroxyurea , camptothecin and cisplatin at the doses and times indicated in Table 1 . All reagents were purchased from Sigma Aldrich except as listed . For UV irradiation , wild type and flo larvae in embryo media were placed 12 cm from a Sankyo Denki UV germicidal bulb ( 15W ) for 10 minutes . γ-irradiation of wild type and flo larvae were irradiated as previously described [70] . Additional information regarding treatment with replication inhibitors is presented in Text S1 . | DNA replication is a complex process that requires activation of cell cycle checkpoints and DNA repair pathways . Genetic analyses in fungi have suggested that nucleoporins , the proteins that make up the nuclear pore complex ( NPC ) , play a role in the cellular response to agents that disrupt cell proliferation or damage DNA . Here we show that mutation of the Elys nucleoporin causes widespread apoptosis in the intestine and other tissues of zebrafish flotte lotte ( flo ) mutants . Intestinal apoptosis occurs in the absence of the DNA damage marker γH2X , and levels of chromatin bound Mcm2 , a component of the DNA replication helicase , were also reduced in flo mutants . These findings suggested that flo intestinal cells cannot repair endogenous replication errors . Consistent with this idea , flo mutants were highly sensitized to treatment with DNA replication inhibitors such as hydroxyurea , UV irradiation , or cisplatin , but not agents that cause DNA double strand breaks , such as γ-irradiation or camptothecin . These data point to a conserved role for nucleoporins in the cellular response to replication stress in eukaryote cells . | [
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] | 2008 | Mutation of the Zebrafish Nucleoporin elys Sensitizes Tissue Progenitors to Replication Stress |
Sequencing of invasive strains of group A streptococci ( GAS ) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator ( CovR ) protein . However , there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis . The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1 . 50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif . Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure . Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other . Similarly , distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes . Finally , mice that were challenged with GAS CovR isoallelic strains had significantly different survival times , which correlated with the transcriptome and protein-DNA binding studies . Taken together , these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis .
Precise regulation of virulence factor-encoding gene expression is critical to the pathogenesis of a diverse array of bacteria that infect humans [1] , [2] , [3] . Thus , it is not surprising that bacteria possess numerous systems for carefully controlling the expression of virulence factor-encoding genes ranging from alternative sigma factors to small RNAs to two-component gene regulatory systems ( TCS ) [4] , [5] , [6] . TCS consist of a membrane-embedded histidine kinase that responds to environmental stimuli by altering the phosphorylation status of its cognate response regulator protein thereby affecting the regulator's ability to bind DNA and alter gene expression [7] . Thus , TCS act as an efficient mechanism to directly link alterations in the external environment to gene expression , and therefore are critical to the infectivity of numerous major bacterial pathogens [8] , [9] , [10] . Group A Streptococcus ( GAS ) causes a diverse array of infections in humans ranging from colonization and uncomplicated pharyngeal and skin infections to necrotizing fasciitis and toxic shock-like syndromes [11] . GAS has long served as a model for understanding the molecular basis of microbial pathogenesis from the standpoints of both virulence factor content and virulence factor regulation [12] , [13] . One of the key GAS transcription factors is the control of virulence regulator ( CovR ) , a member of the OmpR/PhoB regulator family [14] , [15] . CovR is the response regulator protein of the CovRS TCS and appears to function mainly as a negative regulator by binding to AT-rich DNA regions [14] . The mechanism by which CovR binds DNA appears to differ for various promoters of GAS virulence factor-encoding genes ranging from high-affinity for a single DNA binding site to cooperative binding along long stretches of promoter region DNA [14] , [16] , [17] . GAS strains in which CovR has been inactivated are hypervirulent in mice [18] . Moreover , variation in CovR amino acid content has been identified in GAS strains recovered from humans with invasive infections indicating that alteration in the CovR protein impacts streptococcal virulence [19] , [20] . Also , whole-genome analysis of invasive GAS strains has found significant elevation in the number of single-nucleotide polymorphisms in the covR gene relative to the remainder of the GAS genome [21] . However , although CovR variation has been well-recognized , insight into the functional and clinical impact of CovR amino acid residue replacements has been restricted by the absence of CovR structural data and the limited study of GAS strains that differ from each other by only a single amino acid in CovR [22] . Herein we used a combination of structural , biochemical , and genetic analyses to determine the consequences of clinically occurring single amino replacements in the CovR DNA binding domain . Our data provide functional insight into how distinct CovR single amino replacements result in differential effects on streptococcal virulence thereby extending understanding of the genetic underpinnings of microbial pathogenesis .
Whole genome sequencing of 301 serotype M3 GAS strains recently showed a predominant CovR amino acid sequence which is identical to the most common CovR sequence identified in 223 strains of various GAS M protein serotypes analyzed in a separate investigation [23] , [24] . Thus we will designate this CovR sequence , which is present in the fully sequenced serotype M3 strain MGAS10870 , as wild-type CovR for the remainder of this study [21] . Analysis of published manuscripts and CovR sequences deposited at the NCBI revealed that 36 additional distinct amino acid variants of CovR have been described among clinical GAS isolates ( Table 1 ) . Based on homology with proteins of known structure , CovR can be divided into an N-terminal receiver domain ( amino acids residues 1-121 which include the regulatory phosphorylation site D53 ) and a C-terminal DNA binding domain ( amino acids 134-228 ) connected by a flexible linker [14] , [25] . Single amino acid replacements in CovR occurring in clinical GAS isolates have been observed in the putative receiver and DNA binding domains ( Table 1 ) . Each particular CovR amino acid replacement has occurred in a single clinical isolate except for the R94C , R118C , S154P , and Q216P replacements , each of which have been identified in two distinct strains ( Table 1 ) . To gain insight into the structural and functional consequences of CovR single amino acid replacements , we carried out de novo crystallization experiments on CovR . Despite extensive effort , we were unable to obtain diffraction quality crystals of the full-length protein . It is known that obtaining full-length crystal structures of OmpR/PhoB family members is difficult due to interdomain flexibility [26] . Therefore we determined the crystal structure of the CovR DNA-binding domain ( CovRCD amino acids 134 -228 ) . The structure was solved by the multiple anomalous dispersion ( MAD ) method [27] and refined to 1 . 50 Å resolution ( Figure 1A ) . Selected data collection , processing , phasing , and refinement statistics are given in Table 2 . The final model is comprised of 95 residues and has the topology: β1 ( 138-140 ) -β2 ( 144-148 ) -β3 ( 151-153 ) -α1 ( 157-168 ) -α2 ( 176-183 ) -α3 ( 192-206 ) -β4 ( 216-218 ) -β5 ( 221-224 ) ( residues in parentheses ) . The structure of CovRCD is characterized by a classical helix-turn-helix ( HTH ) motif ( α2-α3 ) that is flanked on either side by antiparallel β-sheets and buttressed by helix α1 ( Figure 1A ) . The HTH-motif together with the C-terminal β-hairpin forms the canonical winged HTH ( wHTH ) DNA-binding motif , which is characteristic of members of OmpR/PhoB family of transcription regulators [15] . Consistent with its homology to OmpR/PhoB family members , structural analysis of CovRCD using DALI [28] identified the Staphylococcus aureus protein WalR ( 41% sequence identity to CovR , Z-scores 15 . 5 , root mean square deviation ( rmsd ) = 1 . 8 Å for 94 amino acids ) as well as the Escherichia coli proteins PhoP and PhoB ( 32 and 39% sequence identity to CovR , Z = 14 . 8 and 13 . 7 , rmsd = 1 . 6 and 1 . 5 Å , for 94 and 91 amino acids , respectively ) as the closest structural homologues . Structural differences between CovRCD and its structural homologues are mainly present in the N-terminal β-sheet and flexible loops whereas the isolated winged helix-turn-helix motif overlays with ∼0 . 9 Å rmsd . Whole-genome sequencing data has indicated that GAS is under evolutionary pressure to vary CovR amino acid composition [21] . Thus , we next sought to analyze the potential functional consequences of single amino replacements in the CovRCD . To this end , we modeled the CovRCD-DNA complex based on the crystal structure of PhoBCD bound to pho-box DNA ( PDB code: 1GXP ) [29] ( Figure 1B ) . The crystal structure of PhoBCD bound to its cognate DNA is the only protein-DNA complex structure available for the OmpR/PhoB family . Consistent with the modeling data having in vivo relevance , the recognition helix α3 and wing of CovR can be docked onto the major and minor grooves of the cognate DNA sequence , respectively , without steric clash ( Figure 1B ) . In this conformation several surface exposed , mainly positively charged , amino acid residues can be positioned favorably to engage in hydrogen bonds and nonspecific polar interactions with the phosphate backbone of the DNA major groove . Such examples include R158 in the first turn of α1 , W184 located on the activation loop that connects α2 and α3 , as well as recognition helix residues N193 , Y198 , Y201 , R200 , and R203 . Moreover , the hydrophobic residues V194 and V197 in the recognition helix are positioned to make van der Waals contacts with the methyl group of the thymine bases present in AT-rich CovR DNA binding sites ( Figure 1B ) . In this conformation , wing residue R219 can insert into the minor groove and is likely to interact with the bases and the negatively charged sugar-phosphate backbone . By combining the DNA-bound CovRCD modeling and sequencing data ( Table 1 ) we can postulate potential functional effects of amino acid residue replacements in the CovR DNA binding domain . For example , SNPs resulting in the amino acid exchanges R158C , W184C , N193H , N193I , and R203S likely interrupt direct contacts with the DNA ( Figure 2A , 2B ) . In contrast , R144 is located on β2 , which is distal to the DNA , and therefore unlikely to be involved in direct DNA contacts . Two proline substitutions ( S154P and Q216P ) have been observed in clinical GAS isolates ( Table 1 ) . These mutations occur at solvent exposed sites with S154P in a loop structure and Q216P at the beginning of wing β4 ( Figure 2A ) . The S154P replacement may result in a local perturbation of the structure of the C-terminus of α1 , which impinges upon the activation loop , thereby affecting the position of the HTH motif . The variant CovR protein Q216P has been shown previously to have a DNA binding defect for a single CovR-regulated promoter [20] . Glycine 222 is located on β5 of the wing ( Figure 2A ) . A mutation of this residue to any other would affect CovR structural integrity by producing a steric clash with the peptide backbone of residues M174 and T175 . Thus , analysis of CovRCD indicates that observed amino acid replacements are likely to detrimentally impact the binding of CovR to DNA via distinct mechanisms in keeping with the known role of CovR as a negative regulator of virulence factor encoding genes . To gain further insight into the functional consequences of single amino acid replacements in the CovR DNA binding domain , we used isoallelic exchange to create three GAS strains that differed from the parental serotype M3 strain MGAS10870 by only a single CovR amino acid replacement ( Table 3 , Figure 3A ) [30] . We chose to study the R144C , R158C , and N193I substitutions because the R144 residue is not predicted to contact DNA whereas the R158 and N193 residues are predicted to contact distinct parts of the CovR DNA binding site ( Figure 2A ) . We also replaced the entire DNA binding domain of CovR with a kanamycin resistance cassette to create the CovR-inactivated strain 10870ΔcovR . There was no observable difference in growth rate in a nutrient-rich medium between the wild-type , CovR-inactivated , and CovR-isoallelic strains ( Figure 3B ) . The growth phenotype of GAS on blood agar plates is influenced by hyaluronic acid ( HA ) capsule production . The level of HA is in part determined by expression of the hasABC operon which is negatively regulated through direct binding of CovR to the hasA promoter [31] . Compared to wild-type , the CovR-inactivated and CovR-isoallelic strains had a hypermucoid phenotype ( Figure 3A ) . Similarly , cell-associated HA levels were higher in the CovR-inactivated and CovR-isoallelic strains compared to wild-type although HA levels in strain CovR-R144C were lower compared to strains CovR-R158C , CovR-N193I , and 10870ΔcovR ( Figure 3C ) . These data are consistent with the isogenic strains having increased hyaluronic acid capsule production due to decreased CovR function as would be predicted given the role of CovR as a negative regulator . As CovR is known to be a global regulatory protein , we next compared the transcriptomes of the wild-type , the CovR-inactivated , and the three CovR-isoallelic strains . Each strain was grown in duplicate to mid-exponential phase ( see Figure 3B for time point of RNA analysis ) which was chosen to facilitate comparison to other published CovR transcriptome data derived from a serotype M3 strain [32] . Compared to the wild-type strain MGAS10870 , we observed distinct transcriptome patterns . Consistent with complete inactivation of CovR , strain 10870ΔcovR had the highest number of genes with differential transcript levels compared to wild-type ( 204 , or approximately 12% of all genes expressed under the tested conditions ) ( Table S1 ) . The CovR-isoallelic strains CovR-R144C , CovR-R158C and CovR-N193I had 69 , 126 , and 156 genes , respectively , with differential transcript levels compared to wild-type ( 4 . 2% , 7 . 8% , and 9 . 6% of all expressed genes , respectively ) . In addition to a difference in the absolute numbers of genes affected , we also observed a difference in the magnitude by which transcript levels varied for affected genes among the CovR-inactivated and CovR-isoallelic strains . Compared to wild-type , the mean fold difference in transcript level for differentially expressed genes was 3 . 01 , 2 . 03 , 2 . 54 , and 2 . 65 for strains 10870ΔcovR , CovR-144C , CovR-158C , and CovR-N193I , respectively ( P<0 . 01 by analysis of variance ) . Similarly , when differences in the datasets were compared using principal components analysis , the wild-type replicates clustered together and the 10870ΔcovR replicates were furthest from the wild-type values ( Figure 4A ) . Strains CovR-R158C and CovR-N193I were clustered near strain 10870ΔcovR whereas the transcriptomes of the CovR-R144C replicates were located between those of the wild-type and the other three strains ( Figure 4A ) . These data are consistent with the R144C replacement having a less severe effect on CovR function compared to the R158C and N193I replacements which is as expected given the key role in DNA binding for R158 and N193 predicted by the structural data ( Figure 1B ) . Virulence factor encoding genes whose transcript levels were significantly different compared to wild-type in the CovR-inactivated and each of the CovR-isoallelic strains included the operon encoding the GAS pilus that is important for adhesion to eukaryotic cells ( spyM3_0098 to spyM3_0102 ) [33] , the gene encoding the plasminogen activating protein streptokinase ( Ska ) [34] , and the operon encoding the cytotoxin streptolysin S ( SLS ) [35] . The pilus genes have not previously been reported to be directly regulated by CovR whereas ska and the SLS-encoding operon are known to be CovR regulated [16] , [17] . Compared to wild-type , the pilus operon and SLS-encoding operon were influenced to a similar degree in each of the other four strains whereas the increase in ska transcript level was greater in the CovR-inactivated , CovR-R158C , and CovR-N193I strains compared to strain CovR-R144C ( Figure 4B ) . Similarly , the transcript level of the gene encoding the superantigen SpeC was increased compared to wild-type in strains 10870ΔcovR , CovR-R158C , and CovR-N193I but was not significantly elevated versus wild-type in strain CovR-R144C ( Figure 4B ) . Taken together , we conclude that single amino acid residue replacements in CovR result in transcriptomes that vary in the total number of genes affected as well as in the degree to which transcript levels are affected depending on the particular amino acid replacement . The microarray data provide insight into global gene expression at one time point in the growth cycle . Given that CovR influence can vary depending on the growth phase [32] , [36] , we next used TaqMan QRT-PCR to assay the transcript level of select genes at multiple time points in the growth cycle . In accord with the observed differences in colony morphology and HA production ( Figure 3 ) , the CovR-inactivated and CovR-isoallelic strains had significantly increased hasA transcript level relative to wild-type , particularly during stationary phase ( Figure 5A ) . However , the increase in hasA transcript level was significantly higher in strains 10870ΔcovR , CovR-R158C , and CovR-N193I compared to strain CovR-R144C consistent with the R144C replacement having a less severe effect on CovR function compared to the R158C and N193I replacements . Similar to the hasA data , strain CovR-R144C had a ska transcript level that was intermediate between wild-type and the other isoallelic strains ( Figure 5B ) . Thus , for hasA and ska , the CovR-R158C and N193I replacements had a more significant effect on transcript level compared to the R144C replacement . For other virulence factor encoding genes , however , the R144C replacement had similar effects to the R158C and N193I replacements . For example , compared to wild-type there were similar increases in the transcript level of sagA and the gene encoding for the IL-8 degrading enzyme SpyCEP [37] for the CovR-inactivated and each of the three CovR-isoallelic strains at all time points tested , although the magnitude of increase varied depending on the growth phase ( Figure 5C , Figure S1A ) . We also observed similar increases in the transcript level of the pilus encoding operon genes spyM3_0099 and spyM3_0100 in each of the CovR-inactivated/CovR-isoallelic strains compared to wild-type ( Figure S1B , Figure 5D ) . Finally , the transcript level of the nra gene , which encodes a protein known to regulate the pilus operon [38] , was similarly increased in each of the CovR-inactivated/CovR-isolallelic strains ( Figure S1 C ) . Taken together , we conclude that distinct single amino acid residue replacements in CovR differentially affect GAS gene transcript levels in a growth phase-dependent and gene-specific fashion . Given that CovR has not previously been shown to directly regulate the expression of the pilus operon , we next examined the pilus promoter region from strain MGAS10870 and the fully annotated serotype M3 strains MGAS315 and SSI-1 [39] , [40] . In these strains , the initial gene of the pilus operon ( cbp ) is divergently transcribed from the gene encoding the regulator protein Nra ( Figure S2 ) . There are five putative CovR consensus binding sites ( i . e . ATTARA sequences ) in the 430 bp cbp/nra intergenic region ( Figure S2 ) . Notably , none of these putative CovR binding sites is present in the analogous region in the fully annotated serotype M1 strain MGAS5005 in which CovR inactivation did not affect transcript level of the pilus operon genes [36] , [41] . Consistent with this observation , electromobility shift assays ( EMSA ) verified direct binding of full-length , recombinant CovR to the cpb/nra intergenic region from the serotype M3 strains MGAS10870 and MGAS315 ( Figure 6A , Figure S3A ) . Recombinant CovR also bound the cpb/nra intergenic region in which the putative CovR binding sites had been mutated as well as the respective region from the serotype M1 strain MGAS5005 , albeit with significantly reduced affinity compared to DNA from the serotype M3 strains ( Figure 6A , Figure S3A ) . Together with previous observations , these data indicate that tight binding of CovR to the cpb/nra inter-genic region is required to influence pilus operon gene transcript levels . Our transcript level data showed that CovR repression of virulence factor expression is relieved to different extents in GAS strains containing distinct SNPs in the covR gene ( Figure 5 ) . Therefore , we next tested the hypothesis that CovR variants resulting from distinct single amino acid replacements have different DNA binding activities . We were able to obtain high yields of soluble , full-length , recombinant CovR-R144C and CovR-R158C ( Figure S4A ) , but because of solubility issues , CovR-N193I was not further investigated . We compared the binding of the phosphorylated wild-type , R144C , and R158C CovR variants to DNA from the promoters of three known CovR-regulated virulence factor-encoding genes: hasA , sagA , and ska . In agreement with previous studies , wild-type CovR∼P bound the promoters of hasA ( Figure 6B ) , sagA ( Figure 6C ) , and ska ( Figure S3B ) at a protein concentration of 0 . 25-0 . 5 µM and showed a complete shift at ∼1 µM [16] , [17] , [31] . CovR-R158C∼P had no detectable DNA binding to all three promoters up to a protein concentration of 5 µM ( Figure 6 , Figure S3B ) . In contrast , CovR-R144C ∼P evidenced minimal binding to the sagA promoter but did display binding to the promoters of hasA and ska , albeit with ∼ 4-10 fold lower affinity compared to the wild type ( Figure 6 , Figure S3B ) . However , the DNA retardation is much less compared to the wt , indicating that a higher molecular weight complex , as seen for wt CovR∼P , cannot be formed with CovR-R144C∼P ( at this concentration ) . Thus , in vitro the DNA binding defect engendered by the CovR-R144C replacement is less severe than the R158C replacement , and the effect of the R144C replacement on DNA binding is different depending on the promoter being studied . These data provide a functional explanation for our finding that the transcriptome of strain CovR-R144C was intermediate between the wild-type and the CovR-R158C strains and that the transcript levels of hasA and ska were less in strain CovR-R144C compared to strain CovR-158C ( Figure 4 , Figure 5 ) . A possible explanation for the impaired in vitro DNA binding activity of the CovR variants is that the recombinant variants are improperly folded . Thus , we analyzed the secondary structure of recombinant , full-length wild-type and CovR variant proteins using circular dichroism ( CD ) . The CD spectra for each of the CovR proteins studied showed characteristics of a mixed α/β protein ( Figure S4B ) . The spectra for wild-type CovR and CovR-R144C were identical indicating that the R144C replacement did not result in a structural change in the absence of DNA . However , compared to wild-type , we observed more pronounced minima at 220 nm and 208 nm for CovR-R158C indicating a higher degree of α helical structure ( Figure S4B ) . One possible explanation for this observation is that in the absence of DNA the N-terminus of α1 is less helical when R158 is present due to electrostatic repulsion between the positive dipole of the helix and positive charge of the guanidinium side chain . Alternatively , when C158 is present the sulphydryl side chain of cysteine might show a significant thiolate character and thus interact favorably with the helical dipole . The CD difference between wild-type and CovR-R158C was also observed in the presence of 1 mM TCEP as a reducing agent suggesting that the introduction of cysteine does not induce intermolecular disulphide bond formation thereby altering the secondary structure ( unpublished data ) . Taken together , we conclude that the recombinant wild-type CovR and R144C and R158C variants are folded similarly but that the R158C replacement induces a slightly more helical structure compared to the wild-type protein in the absence of DNA . In light of our observations regarding the significant effect of CovR variation on global gene expression and protein function , we next tested the hypothesis that GAS strains harboring distinct CovR variants would have differential virulence . To this end , we challenged mice intraperitoneally with 1×107 colony forming units ( CFUs ) of strain MGAS10870 , its CovR-inactivated derivative , and the three CovR isoallelic strains . Each of the tested strains caused some extent of near-mortality in mice ( Figure 7 ) . However , the survival times of the mice infected with the five strains were significantly different ( P<0 . 0001 by log-rank test ) . Specifically , the survival time of mice infected with strain MGAS10870 was significantly longer than mice infected with each of the other four strains ( Figure 7 ) ( P<0 . 05 for MGAS10870 compared to strain CovR-R144C and P<0 . 01 for strain MGAS10870 compared to either strain 10870ΔcovR , CovR-R158C , or CovR-N193I ) . Similarly , the survival time of mice infected with strain CovR-R144C was significantly longer than mice infected with strains 10870ΔcovR , CovR-R158C , or CovR-N193I ( P<0 . 05 for each comparison ) . There was no significant difference in the survival times among mice infected with strains 10870ΔcovR , CovR-R158C , or CovR-N193I ( P = 0 . 35 ) . Thus , we conclude that CovR single amino replacements occurring in GAS clinical isolates significantly impact strain virulence and that the degree of impact differs depending on the particular amino acid substitution . These findings are in accordance with our transcriptome , QRT-PCR , and DNA binding activities showing a CovR-R144C phenotype intermediate between wild-type and strains 10870ΔcovR , CovR-R158C , and CovR-N193I .
It is well recognized that patients with severe , invasive GAS disease can be infected with strains that have variation in the CovR protein ( Table 1 ) [42] . Thus , CovR stands as a paradigm for studying how small-scale genetic changes in infecting microbes can influence disease manifestations . Despite extensive previous investigation of CovR function , however , prior to the work described herein knowledge regarding mechanisms by which single amino acid variation in CovR influences GAS virulence was limited [20] , [22] . A key finding of this work was that GAS strains that contain different single amino acid residue replacements in CovR have distinct transcriptomes and virulence profiles from strains with a wild-type CovR and from each other ( Figure 4A , Figure 7 ) . Specifically , we found that the transcriptome of strain CovR-R144C was different from wild-type and from strains that contained the R158C and N193I amino acid replacements ( Figure 4A ) . Previous studies have examined the effects of a single CovR amino acid replacement and thus could not compare the effects of replacements at distinct sites [20] , [22] . Studies of other microbial proteins that examined effects of single amino replacements found a binary effect on virulence in which particular amino acid replacements either did or did not affect virulence [30] , [43] . Our data show that the effect of CovR single amino acid replacements on virulence occurs on a graded scale with particular replacements ( e . g . R144C ) causing a significant difference compared to wild-type but having a less pronounced effect compared to other replacements ( e . g . R158C and N193I ) . Given the large number of CovR amino acid variations yet to be investigated it is likely that similar differential effects will be observed depending on the particular amino acid involved . The differential effects of the R144C , R158C , and N193I replacements can be explained by a combination of our structural and functional data . The R158C and N193I replacements resulted in a CovR-inactivated phenotype in terms of gene expression ( Figs . 4 and 5 ) and virulence ( Figure 7 ) consistent with the complete lack of DNA binding observed for recombinant CovR-R158C ( Figure 6 ) . Previous mutagenesis studies of OmpR/PhoB family proteins have identified amino acids in analogous positions to CovR R158 and N193 as being important for protein function [44] , [45] , and our structural data suggest a direct role for these two residues in CovR DNA binding ( Figure 2 ) . Conversely , the R144C replacement resulted in a gene expression , DNA-binding , and virulence profile that was intermediate between wild-type and the CovR-inactivated strain . Given the distance from DNA of the R144 residue ( Figure 2A ) , how the R144C replacement affects CovR function is not clear , and the equivalent residue has not been typically identified as critical to the function of OmpR/PhoB family members . Interestingly , introducing a cysteine at the equivalent residue in E . coli OmpR , R150 , also resulted in a differential effect on gene expression as there was decreased OmpR-mediated regulation of the ompC but not the ompF gene [46] . In addition to the previously established head-to-tail conformation , crosslinking experiments indicated a head-to-head arrangement of OmpR dimers in which R150 is part of the intermolecular dimerization interface formed by opposing β-sheets [46] . Thus , the R150C substitution is predicted to affect the function of OmpR dimers arranged in a head-to-head , but not head-to-tail , conformation . The orientation of CovR dimers has been postulated to vary from head-to-head to head-to-tail depending on the promoter [47] . Thus , our discovery that the R144C replacement severely affected CovR function at only particular promoters provides the groundwork for investigating the overall role of the R144 in CovR function as well as further studying different mechanisms by which CovR interacts with diverse promoters . The finding that strain 10870ΔcovR and the CovR polymorphic variants had significantly increased transcript levels of genes in the pilus operon compared to wild-type was a surprise given that multiple CovR transcriptome studies have been performed without identifying the pilus genes as being influenced by CovR [22] , [32] , [36] , [48] , [49] , [50] . These CovR transcriptome studies have mostly been performed in serotype M1 strains except for one study in the serotype M3 strain DSL003 [32] . The nucleotide composition of the pilus promoter region is highly heterogeneous among various GAS strains which may explain the lack of observed CovR influence in previous studies [51] . Given that the pilus regulatory protein Nra is also transcribed from this intergenic region , we cannot say whether CovR directly influences pilus transcription in strain MGAS10870 or is acting indirectly by affecting Nra levels ( Figure S1C ) . A recent study in a serotype M1 strain found that pili diminish GAS virulence in multiple mouse infection models possibly by inducing IL-8 production , neutrophil recruitment , and extracellular DNA-based entrapment [52] . We found that , compared to wild-type , there were increased transcript levels of the gene encoding the IL-8 degrading enzyme SpyCEP and of genes encoding multiple DNase enzymes in the CovR-isoallelic variants ( Table S1 ) . Thus , CovR single amino acid replacements in the serotype M3 strain under study here appear to balance augmented immune activation associated with pilus expression with increased immune evasion via IL-8 degradation and degradation of neutrophil extracelluar traps . It was recently reported that covRS inactivation in the GAS serotype M49 strain NZ131 resulted in increased pilus production [53] . Bioinformatic analysis of the published genome [54] reveals that strain NZ131 also contains five putative CovR binding sites in the nra/cpb intergenic region in this strain , similar to what is observed in strain MGAS10870 . Interestingly , elimination of the ATTARA motifs in the nra/cpb intergenic region only modestly reduced recombinant CovR binding in vitro ( Figure 6A ) suggesting that CovR binding of DNA is necessary but not always sufficient for gene regulation . In summary , we have determined the structure of the DNA binding domain of the key streptococcal global regulator CovR and have discovered that single amino acid residue replacements in CovR can affect CovR DNA binding affinity , GAS global gene expression , and strain virulence differentially . These data provide fundamental insights into the mechanisms by which variation in a global regulatory protein influences infection with a major human bacterial pathogen thereby extending understanding into how virulence factor regulation impacts microbial pathogenesis .
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . The protocol was approved by Methodist Hospital Research Institute Institutional Animal Care and Use Committee ( Protocol Number: AUP-1010-0022 ) . All efforts were made to minimize suffering . The strains and plasmids used in this work are presented in Table 3 , and primers used for isogenic mutant strain creation are listed in Table S2 . Strains were grown in a nutrient-rich medium ( Todd-Hewitt broth with 0 . 2% yeast extract ( THY ) ) at 37 °C with 5% CO2 . When appropriate , kanamycin and chloramphenicol were added at 200 µg/mL and 10 µg/mL respectively . Strain MGAS10870 is a fully-sequenced , invasive , serotype M3 strain that contains a wild-type covRS operon [21] . Strain 10870ΔcovR was created by replacing the entire CovR DNA binding domain in strain MGAS10870 with a kanamycin resistance cassette via insertional inactivation [22] , [55] . Derivatives of strain MGAS10870 that differed only by the presence of a single amino acid replacement were created using the chloramphenicol-resistant , temperature-sensitive plasmid pJL1055 ( gift of D . Kasper ) as described [30] . For each of the CovR strains derived from strain MGAS10870 , sequencing of the entire covRS operon , as well as the mga and emm genes , was performed to check for the presence of spurious mutations that could have developed during the strain construction process ( none were found ) . The nra/cpb operon of the fully annotated serotype M3 strain MGAS315 [40] is presented ( Figure S2A ) to facilitate bioinformatic analysis , but strain MGAS315 was not used as the wild-type strain for this study because it contains a single amino replacement in the CovS protein . CovR and the CovR C-terminal DNA binding domain ( CovRCD ) were amplified from strain MGAS10870 and cloned into pTXB1 as described using primers listed in Table S2 [50] . Recombinant CovR variants were created using site-directed mutagenesis ( Stratagene ) ( primers are listed in Table S2 ) . Overexpression of recombinant forms of the CovR protein was performed in E . coli BL21 grown over night at 18°C . Cells derived from three liters of culture were resuspended in 20 mM Tris-HCl pH 8 . 5 , 500 mM NaCl and purified to >95% homogeneity ( Figure S4 A ) using the IMPACT Protein Purification System ( New England Biolabs ) which allows for recovery of recombinant CovR isoforms lacking any non-native residues and avoids having to recover proteins from inclusion bodies as part of the purification process [50] . After cleavage and elution with 50 mM DTT , proteins were extensively buffer exchanged into 50 mM CAPS pH 10 . 0 , 100 mM NaCl . CovRCD was further purified by size exclusion chromatography on Superdex 75 in 50 mM CAPS pH 10 . 0 , 50 mM NaCl , 1 mM TCEP ( Tris-[2-carboxyethyl] phosphine ) . Proteins were concentrated to ∼25 mg/ml with Amicon centrifugal filter units ( MW cut-off 3kDa ) and protein concentrations were assessed by Bradford assay . Selenomethionine-derivatized CovRCD ( SeMet-CovRCD ) was overexpressed using the methionine inhibition pathway and purified as described for native CovRCD . Crystallization of native and SeMet-CovRCD was carried out at room temperature using the hanging drop vapor-diffusion method with reservoir solutions containing 10–14% PEG 3350 and 50 mM Zinc ( OAc ) 2 . Native crystals were smaller and did not diffract beyond 2 Å resolution while SeMet crystals diffracted to beyond 1 . 50 Å . Crystals were cryo-protected using 30% PEG400 and flash frozen in liquid nitrogen . Multiple wavelength anomalous dispersion ( MAD ) diffraction data using SeMet-CovRCD crystals were collected under cryogenic conditions at the Advanced Light Source ( ALS ) , Berkeley , Beamline 8 . 3 . 1 . Data were processed with MOSFLM [56] and Scala in the CCP4 suite ( Table 2 ) . Crystals take the space group P21 with cell dimensions of a = 30 . 5 Å , b = 36 . 7 Å , c = 38 . 9 Å and α = 90 . 0° , β = 94 . 6° , γ = 90 . 0° . The CovRCD structure was determined to 1 . 5 Å resolution by MAD phasing [27] . Four selenium sites were located using the program SOLVE [57] and solvent flattening and initial model building were performed using RESOLVE [58] . Given the isomorphism of the SeMet-CovRCD and native CovRCD crystals and the significantly higher resolution of the former protein , we refined only the SeMet-CovRCD structure . The asymmetric unit contains one CovRCD molecule . The structure was refined to final Rwork and Rfree values of 21 . 60% and 23 . 64% , respectively , by multiple rounds of model building in COOT [59] and positional and B-factor refinement with CNS 1 . 2 [60] . The resulting model showed excellent geometry ( Table 2 ) and contains residues 134-228 including the initial methionine as well as 107 water molecules . Selected data collection , phasing and refinement statistics are given in Table 2 . Crystallization-related figures were created using Pymol [61] . RNA was purified using an RNeasy mini kit ( Qiagen ) . A custom-made Affymetrix GeneChip that contains 100% of the ORFs of strain MGAS10870 was used for expression microarray ( transcriptome ) studies as described [62] . To compare gene transcript levels between the wild-type and the various derivative strains , a two-sample t-test ( unequal variance ) was applied with a P value of <0 . 05 and a mean transcript level of at least 1 . 5-fold being considered statistically significant . TaqMan real-time QRT-PCR ( primers and probes listed Table S2 ) was performed on an Applied Biosystems 7500 system as previously described [63] . All QRT-PCR samples were performed in duplicate on two separate occasions and analyzed in duplicate . Wt and mutant CovR proteins were phosphorylated with acetyl phosphate as described [17] . The phosphorylation state was verified using Phos-tag Acrylamide gels ( WAKO chemicals , USA , Inc . ) . ∼300 bps of the promoter regions of selected CovR regulated genes were amplified by PCR from indicated GAS strains ( primers are listed Table S2 ) . A variant of the nra/cpb intergenic region in which all five putative CovR binding sites are mutated ( ATTARA -> AGGARC ) was synthesized and cloned into pUC57 by Genscript USA Inc . and PCR amplified as described . The resultant PCR products ( 0 . 25 µg/ml ) were incubated with increasing amounts of phosphorylated wild-type CovR ( 0 . 25–1 µM ) and CovR variant proteins ( 0 . 25–5 µM ) at 37°C for 15 min in TBE buffer containing 89 mM Tris , 89 mM borate , 1 mM EDTA , and 5% glycerol and 10 µg/ml polydI:dC to account for non-specific DNA binding . Samples were then separated by native gel electrophoresis on a 6% TBE gel for 70 min at 110 V and stained with ethidium bromide . Wild type and mutant CovR proteins were diluted to a concentration of 0 . 4 mg/ml in 10 mM potassium phosphate , pH 7 . 5 , 10 mM NaF prior to measurements . Far-UV ( 200–260 nm ) spectra were recorded on a Jasco J-810 spectropolarimeter at 37°C and a scan speed of 20 nm/min . Three scans were averaged and the averaged blank spectra were substracted . Mouse experiments were performed according to protocols approved by the Methodist Hospital Research Institute Institutional Animal Care and Use Committee . 20 female outbred CD-1 Swiss mice ( Harlan-Sprague-Dawley ) were injected intraperitoneally with 1 . 0×107 GAS CFU and monitored for near-mortality . Expression microarray data have been deposited at Gene Expression Omnibus ( GEO ) database at NCBI http://www . ncbi . nlm . nih . gov/geo/accession number GSE32106 . Coordinates and structure factors are deposited in the Protein Data Bank under PDB ID code 3RJP . covR , 3572611; covS , 3572612; slo , 3572764; spyCEP , 3760194; sagA , 3572347; hasA , 3571023; ska , 3571199; speC , 900896; nra , 1008411; cbp , 1008412 | Group A Streptococcus ( GAS ) causes a variety of human infections including invasive disease that can often be deadly . GAS strains that cause serious infections may have alterations in the amino acid sequence of the control of virulence regulator ( CovR ) protein , but mechanisms by which changes in the CovR protein influence GAS disease are not understood . We determined the crystal structure of the CovR DNA binding region and found that alterations in the CovR protein observed in clinical , invasive GAS isolates are likely to disrupt CovR-DNA interaction and overall CovR structure . In accord with the structural data , CovR proteins with a single amino acid change had distinctly different binding affinities for various GAS virulence-factor encoding genes . Similarly , GAS strains that differed by only the presence of a single CovR amino acid change had distinct gene expression profiles . Finally , mice that were challenged with GAS strains that differed by only a single CovR amino acid replacement had significantly different survival times consistent with the idea that alterations in the CovR protein are a key determinant of clinical outcomes in GAS human infections . These findings provide mechanistic insights into how subtle genetic differences can profoundly impact the severity of bacterial infections . | [
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"diseases",
"proteins",... | 2011 | Distinct Single Amino Acid Replacements in the Control of Virulence Regulator Protein Differentially Impact Streptococcal Pathogenesis |
A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface . Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells . Here , we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites . Thus , human monocytes were exposed to three different cancer cell lines ( breast , ovarian , or glioblastoma ) or to live microfilariae ( mf ) of Brugia malayi–a causative agent of lymphatic filariasis . After 2 days of co-culture , monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated ( TNF-α , IL-1β ) , M2-associated ( CCL13 , CD206 ) , Mreg-associated ( IL-10 , TGF-β ) , and angiogenesis associated ( MMP9 , VEGF ) genes . Similar to cancer cell lines , but less dramatically , mf altered the mRNA expression of IL-1β , CCL13 , TGM2 and MMP9 . When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed , monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression . In contrast to exposure to mf , exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells . Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate , they share the ability to alter the phenotype of human monocytes .
A variety of mechanisms used by tumor cells to escape the host’s immune system are similar to those used by some parasites . Both parasites and tumors have developed strategies to escape the immune system by expanding T regulatory cells[1 , 2] , by inducing the production of certain inhibitory cytokines[3 , 4] , or by altering the function of antigen presenting cells ( APCs ) that , in turn , results in diminished ability of these cells to activate T cells[1 , 5 , 6] . Monocytes and macrophages are heterogeneous populations of cells that display high plasticity and are essential for the host innate immune response . Consequently , a change in their function contributes to alterations of immune function that may lead to dysregulation of responses important in limiting cancer progression[7] and constraining some infectious diseases . Based on responses to different stimuli , macrophages can be categorized into classically activated M1 ( type 1 or pro-inflammatory activated by LPS or IFN-γ ( Interferon-gamma ) ) , or alternatively activated M2 ( type-2 anti-inflammatory activated by IL-4 or IL-13 ) [8] . In fact , both parasites and tumors alter the balance of these monocyte /macrophage sub-populations[6 , 9–12] . When recruited to the tumor tissue , monocytes can differentiate into tumor associated macrophages ( TAM ) , a heterogenous population of myeloid cells with both antitumor ( M1 ) and pro-tumor activities ( M2 ) ( reviewed in[13] ) . In fact , TAMs have a wide range of functions including those with beneficial effects , such as phagocytosis of tumor cells and production of cytotoxic factors , [13 , 14] and the more deleterious effects , such as tumor- associated immunesuppression through the expression of inhibitory immune checkpoints PDL1 ( CD274 ) and PDL2 ( CD273 ) [15] . M2 macrophages with both anti-inflammatory and tissue repair functions[16 , 17] , largely driven by IL-4 and/or IL-13 can also be induced by helminth parasites[6 , 18] . Helminth-induced M2 macrophages have been shown to play a role in control of Th1-type inflammation , worm expulsion , and wound healing in murine models[19] . Interestingly , microfilariae ( mf ) of Brugia malayi , the bloodborne stage of one of the helminth parasites that cause lymphatic filariasis in humans , alter monocyte populations somewhat differently in that both M1 and M2 phenotypes are induced[6 , 18 , 20 , 21] . Furthermore , monocyte dysfunction[20 , 22] of either subset in filarial infection is one of the many mechanisms proposed for parasite antigen-specific T cell hyporesponsiveness seen in humans with lymphatic filariasis . Because the regulation of monocyte function plays a critical role in both helminth infection and tumor progression , in the present study we assessed the similarities and differences between parasite and cancer-induced alterations of both phenotype and function of human monocytes in hopes of identifying potential new targets that can be exploited by host directed therapeutics . Breast and ovarian cancers are considered to be among the leading types of cancer in North America and Europe [23 , 24] . Clinical data demonstrate a strong relationship between increased monocyte/macrophage density and poor prognosis in breast and/or ovarian cancers and glioblastoma [25 , 26] . In addition , tumor associated macrophages play an important role in all three cancer types [27–29] . Therefore , in the present study we chose breast , ovarian , and glioblastoma cancer cell lines to compare their effects on human monocytes to that of helminth parasites .
The elutriated monocytes and lymphocytes from leukopacks of healthy adult donors from North America were collected by counterflow centrifugal elutriation under a protocol approved by the Institutional Review Board ( IRB ) of the Department of Transfusion Medicine , Clinical Center , National Institutes of Health ( NIH; IRB 99-CC-0168 ) . The healthy adult volunteers were given informed written consent . Live Brugia malayi mf ( provided under contract with the University of Georgia , Athens , GA ) were collected by peritoneal lavage of infected jirds and separated from peritoneal cells by Ficoll diatrizoate density centrifugation . The mf were then washed repeatedly in RPMI medium with antibiotics and cultured overnight at 37°C in 5% CO2 before use . The breast cancer; MDA-MB-231 ( MDA ) , ovarian cancer; OVCAR-3 ( OVCAR ) , and glioblastoma; U87-MG ( U87 ) cell lines were obtained from American Type Culture Collection ( ATCC ) ( Manassas , VA ) . MDA cells were cultured in Dulbecco’s Modified Eagle’s Medium ( DMEM , ATCC 30–2002 ) containing 10% heat–inactivated fetal bovine serum ( Gemini Bioproducts , Sacramento , CA ) . OVCAR cells were cultured in Roswell park memorial institute medium ( RPMI-1640 , ATCC 30–2001 ) containing 20% heat–inactivated fetal bovine serum . U87 cells were cultured in Eagle’s Minimum Essential Medium ( EMEM , ATCC 30–2003 ) containing 10% heat–inactivated fetal bovine serum . All culture media contained 100 Units/ml penicillin and 0 . 1-mg/ml streptomycin ( [P/S] Biofluids , Inc , Rockville , MD ) . Cells were cultured at 37° C in humidified air at 5% CO2 and were confirmed to be devoid of mycoplasma . Prior to use , these cell lines were stained with 0 . 5uM , 5-chloromethylfluorescein diacetate ( Cell Tracker green CMFDA; Molecular probes ) in serum free media for 30 min followed by washing with PBS . CMFDA labeled cancer cell lines ( MDA , OVCAR and U87 ) were cultured for 24 hours prior to co-culture with monocytes . Human monocytes were cultured at 50 × 106 per 6-well plate in serum-free media RPMI 1640 medium supplemented with 20 mM glutamine ( Lonza ) P/S for 2 h , after which the medium was removed and the adherent cells were harvested . Monocytes were then either cultured alone or exposed to live mf ( 50 , 000 per million cells to reflect physiologically relevant concentrations ) , or to three different CMFDA labeled cancer cell lines at a 1:2 ( cancer cell lines: monocytes ) ratio for 48hrs . For monocyte co-cultures with each cancer cell lines , we chose the media used for culturing the relevant cancer cell line alone . Therefore , for MDA cell line and the co-culture of monocytes and MDA ( mon/MDA ) DMEM complete media; for OVCAR and co-culture of monocytes and OVCAR ( mon/OVCAR ) RPMI complete media; and for U87 and co-culture of monocytes and U87 ( mon/U87 ) EMEM complete media was used . Furthermore , monocytes alone or monocytes exposed to mf were cultured in DMEM complete media as there was no difference between the three-different media in mRNA expression , cell surface expression or viability of monocytes ( S1 Fig ) . After 48hrs , the cells were harvested by cell scraping ( Corning Costar ) and washed once with PBS ( without Ca++/Mg++ ) , counted monocytes were first incubated with human gammaglobulin ( Sigma ) at 10 mg/ml for 10 min at 4°C to inhibit binding of the monoclonal antibody to Fc receptor ( FcR ) and were subsequently labeled with mouse phycoerythrin labeled anti-CD45 mAb ( eBioscience , San José , CA; Cat No . 12-9459-42 ) , at saturating concentrations for 30 min at 4°C . The cells were then washed twice with FACS medium and sorted on FACSAria III , 6-laser , 15-parameter , cell sorter ( Becton Dickinson , Sparks , MD ) by gating on the expression of CD45 and lack of CMFDA ( CD45+/CMFDA- ) . Sorted monocytes were then used for gene expression or functional analysis . After 48hrs of monocytes co-culture with cancer cell lines or mf , exposed and unexposed sorted monocytes were cultured in DMEM media overnight without any stimulation and the production of TNF-α ( Tumor necrosis factor-alpha ) , IP-10 ( Interferon gamma-induced protein ( CXCL10 ) ) , IL-6 , CCL4 ( Macrophage inflammatory protein 1-β ( MIP-1β ) ) and CCL22 ( macrophage derived chemokine ( MDC ) ) in the culture supernatants were measured using a Multiplex human cytokine/chemokine magnetic bead panel kit ( EMD Millipore , Billerica , MA ) and a Luminex 100/200 system ( Luminex , Austin , TX ) . The lower limit for detection for these assays was 3 . 2 pg/ml . Phagocytic activity of the human monocytes was assessed by a phagocytosis assay kit ( Molecular probes; Invitrogen ) with some modifications . Briefly , sorted monocytes ( 1 . 0 X106 cells/well ) were incubated with 108 inactivated E . coli Alexa 488 Bioparticles for 1 hr at 37° C , 5% CO2 in serum free DMEM media . The cells were washed with PBS , incubated for 1 min with 0 . 4% trypan blue to quench any extracellular fluorescence , and washed twice with PBS . Intracellular fluorescence intensity was quantified by flow cytometry . All experiments were done with six replicates . Phagocytic activities of monocytes were expressed as percent phagocytosis relative to that seen with controls . Human monocytes were cultured alone or with mf or three different CMFDA-labeled cancer cell lines ( green; FITC channel ) as mentioned above . After 48 hrs , cells were harvested , washed with PBS and incubated with 10 μl human IgG ( 10 mg/ml; Sigma-Aldrich , St . Louis , MO ) for 10 min at 4°C to inhibit nonspecific binding through FcγRs and then incubated with marker-specific mAb conjugated with PDL2- APC ( eBioscience , Cat No . 17-5888-42 ) , VCAM1 ( Vascular cell adhesion molecule-1 ) -APC ( Biolegend , Cat No . 305810 ) , CD14-APC Cy7 ( eBioscience , Cat No . 47-0149-42 ) , CD45-Pacific blue ( Biolegend , Cat No . 304022 ) , CD206 ( Mannose receptor ) -Percp efluor 710 ( eBioscience , Cat No . 46-2069-42 ) , PDL1-PE-Cy7 or PDL1-APC ( eBioscience , Cat No . 25-5983-42 and Cat No . 17-5983-41 respectively ) , or CD163-PE ( eBioscience , Cat No . 12-1639-42 ) , CD45-PE ( eBioscience , Cat No . 12-9459-42 ) at saturating concentrations for 30 min at 4°C and washed twice with FACS medium . Monocyte cell populations ( CD45+/CMFDA- ) were then identified and gated to measure the expression of cell surface markers . For flow cytometric analysis , 50 , 000 events were acquired per tube using a BD LSRII flow cytometer ( BD Biosciences , San Jose , CA ) . Compensation was performed in every experiment using BD CompBeads ( BD Biosciences ) for single-color controls and unstained cells as negative controls . Data were analyzed using FlowJo Software ( Tree Star , Ashland , OR ) . Nonviable cells were excluded from our analysis on the basis of forward and side scatter . Fold upregulation in Mean Fluorescence Intensity ( MFI ) was measured for all markers . Exposed and unexposed sorted monocytes were used for isolation of total RNA and RT-PCR to measure gene expression . Total RNA was prepared from 8 to 15 independent donors using an RNAEasy minikit ( Qiagen ) . RNA ( 1 μg ) from the cells was used to generate cDNA and then assessed by standard TaqMan assays ( Applied Biosystems Inc . ) using an ABI 7900HT system ( Applied Biosystems , Inc . ) . Briefly , random hexamers were used to prime RNA samples for reverse transcription using MultiScribe reverse transcriptase ( Applied Biosystems Inc . ) , after which PCR products for all genes , as well as an endogenous 18s rRNA control , were assessed in triplicate or duplicate wells using TaqMan predeveloped assay reagents . The threshold cycle ( CT ) , defined as the PCR cycle at which a statistically significant increase in reaction concentration is first detected , was calculated for the genes of interest and the 18S control and used to determine relative transcript levels . Relative transcript levels were determined by the formula 1/ΔCT , where ΔCT is the difference between the CT of the target gene and that of the corresponding endogenous 18S reference . Fold change in gene expression was measured using 2−−ΔΔCT , where ΔΔCT is the difference between the ΔCT of the gene of interest in exposed monocytes and that of the unexposed control . Exposed or unexposed sorted monocytes were cultured with CellTrace carboxyfluorescein succinimidyl ester ( CFSE ) ( ThermoFisher Scientific , Cat No . C34570 ) - autologous and allogeneic lymphocytes ( 1:1 monocyte/T cell ratio ) either in media alone or with 10ug/ml of anti-CD3 ( hOKT3 ) in 24 well tissue culture plates ( Costar , Cambridge , MA ) . Blocking experiments were done using anti-PDL1 ( eBiosciences , Cat No . 16-5983-82 ) and isotype control IgG1 ( eBiosciences , Cat No . 16–4714 ) at a final concentration of 10ug/ml . After 4 days of culture , CFSE-labeled lymphocytes were harvested , and the proliferation of CD4+ and CD8+ T cells was measured by flow cytometry using a combination of APC conjugated anti-CD4 ( BD Biosciences , Cat No . 340672 ) and Alexa Fluor 700-conjugated anti-CD8 ( eBiosciences , Cat No . 56-0086-42 ) . The cells were then analyzed by acquisition of 50 , 000 events/tube using a BD LSRII ( BD Biosciences ) . Compensation was performed in every experiment using BD CompBeads ( BD Bioscience ) for single-color controls and unstained cells . Nonviable cells were excluded from the analysis based on forward and side scatter . CellTrace Violet labeled lymphocytes were further gated on expression of CD4+ or CD8+ , and proliferation was measured by flow cytometry for the dilution of fluorescent dye . Proliferation indices were calculated by using the FlowJo proliferation analysis program ( Tree Star , Ashland , OR ) . Granzyme A expression was measured in CD8+ T cells by staining with pacific blue anti-granzyme A antibody ( Biolegend , Cat No . 515407 ) . Unless noted otherwise , geometric means were used as a measure for central tendency . Omnibus2 normality test was performed to confirm the data is not normally distributed and then the nonparametric Wilcoxon signed-rank test was used for paired group comparisons . All analyses were performed using GraphPad Prism 6 . 0 ( GraphPad Software , Inc . , San Diego , CA ) .
To assess whether cancer cell lines and helminth parasites share similar features in shaping human monocyte gene expression , human monocytes were either exposed to CMFDA- labeled cancer cell lines ( breast cancer; MDA-MB-231 ( MDA ) , ovarian cancer; OVCAR-3 ( OVCAR ) , and glioblastoma; U87-MG ( U87 ) ) or to live mf of Brugia malayi for 48 hours , sorted for CD45+/CMFDA- monocytes and assessed for mRNA expression by RT-PCR . To further assess the phenotype of exposed monocytes , we selected genes associated with inflammatory response ( M1 ) , type-2 response ( type 2/M2 ) , regulatory response ( Reg ) or responses associated with angiogenesis ( Ang ) ( Fig 1A and 1B and S2 Fig ) . Our results indicate that cancer cell lines significantly upregulated genes associated with M1 monocytes including PDL1 , TNF-α , IL-1β , IL-6 , IL-8 , CCL3 , and prostaglandin-endoperoxidase synthase 2 ( PTGS2 ) ( Fig 1A and S2 Fig ) . The expression of genes associated with M2 monocytes ( TGM2 ( Transglutaminase 2 ) , PDL2 , CD206 , CCL13 ) , regulatory monocytes ( IL-10 , TGF-β ) and angiogenesis ( MMP9 ( Matrix metallopeptidase-9 ) , and VEGF ( Vascular endothelial growth factor ) ) were significantly induced in all three different cancer cell line- exposed monocytes when compared to unexposed monocytes ( Fig 1A and S2 Fig ) . Although there were major differences between cancer cell line exposed- and mf-exposed monocytes , mRNA expression of IL-1β , MMP9 , and TGM2 was shown to be significantly upregulated in monocytes following exposure to either stimuli compared to unexposed monocytes ( Fig 1B ) . We next measured monocyte cytokine production ( Fig 2 ) after exposure to cancer cell lines or mf . To further assess the phenotype of exposed monocytes , we selected cytokines associated with inflammatory response ( M1 ) , type-2 response ( type 2/M2 ) , regulatory response or responses associated with angiogenesis ( Fig 2 and S1 Table ) . To this end , human monocytes were either exposed to CMFDA- labeled cancer cell lines ( MDA , OVCAR , and U87 ) or to live mf of Brugia malayi for 48 hours , sorted and rested in media for an additional 24 hours following which cytokine production was assessed in the culture supernatant . As seen in Fig 2 , both cancer cell lines and live mf significantly ( p = 0 . 001 ) upregulated the production of IP-10 and CCL22 , while IL-6 production was significantly upregulated only by cancer cell lines and not by mf ( Fig 2 ) . Among cytokines/chemokines tested the production of TNF-α , CCL4 ( Fig 2A ) , IL-10 ( Fig 2C ) , and VEGF ( Fig 2D ) was not affected by exposure to any of the stimuli . We then determined the phenotype of the monocytes following exposure to either live mf or to the cancer cell lines . The basal expression of selected cell surface markers ( M1 , M2 , inhibitory ) on either human monocytes or cancer cell lines suggest that human monocytes do not express PDL2 ( M2/inhibitory ) , VCAM-1 ( M1 ) , CD206 ( M2 ) , and have a low expression of PDL1 ( M1/inhibitory ) and CD163 ( M2 ) ( Fig 3A , black lines ) . To our interest , after 48 hours exposure to either of the three cancer cell lines , the expressions of PDL1 ( p = 0 . 001 ) , PDL2 ( p = 0 . 003 ) , VCAM-1 ( p = 0 . 003 ) , and CD206 ( p = 0 . 003 ) on monocytes were significantly upregulated ( Fig 3A and 3B ) . Similar to cancer cell lines , but to a lesser extent , live mf significantly upregulated the cell surface expression of each of these markers ( p = 0 . 001 ) with the exception of CD163 ( Fig 3A and 3B ) . Longer exposure ( 5 days ) of monocytes to mf further induced the level of cell surface PDL1 ( S3A and S3B Fig ) . To compare the ability of cancer cell lines and mf to shape the phagocytic function of human monocytes , we measured the ability of monocytes to take up fluorescently labeled E . coli following exposure to mf , or MDA ( Fig 4 ) , or OVCAR and U87 ( S4 Fig ) . Our data clearly show that similar to MCSF-treated monocytes , exposure to MDA ( Fig 4 , P = 0 . 01 ) or to the other two cancer cell lines ( S4 Fig ) significantly induce the phagocytic ability of these cells . However , live mf did not alter the ability of monocytes to phagocytose E . coli bioparticles ( Fig 4 ) . The suppressive effects of tumor-associated macrophages on T cell proliferation have been shown previously[30] . To assess whether these particular cancer cell lines or live mf alter the ability of monocytes to drive T cell proliferation and mediator release , human monocytes were cultured in media alone , or with CMFDA-labeled breast cancer cell lines or live mf of Brugia malayi for 48hr . CD45+/CMFDA- sorted exposed and unexposed monocytes were then co-cultured with CFSE-labeled allogeneic or autologous lymphocytes in the presence of anti-CD3 for an additional 4 days . While exposure to mf did not alter the ability of monocytes to induce T cell proliferation , exposure to MDA and the other two cancer cell lines ( U87 and OVCAR; S5 Fig ) significantly diminished their ability to promote allogeneic and autologous CD4+ ( Fig 5A and 5B ) , and allogeneic CD8+ ( Fig 5A ) T cell proliferation . We next aimed to investigate the role of PDL1 in this diminished T cell proliferative activity . As shown in Fig 5C and 5E , blocking the PDL1 pathway significantly increased the proliferation of allogeneic CD4+ T cells , suggesting that the PDL1 upregulation in cancer cell line exposed- ( but not mf-exposed ) monocytes plays an important role in T cell suppression . Because regulation of cytolytic CD8+ T cells is crucial in controlling tumor progression and growth particularly through the release of granzymes ( reviewed in[31] ) , we studied the effect of cancer cell line exposed monocytes on granzyme A release in CD8+ T cells . As shown in Fig 5D , MDA- and U87- exposed monocytes but not OVCAR- or mf-exposed monocytes significantly decreased the percentage of Granzyme A+ allogeneic CD8+ T cells when compared to the unexposed monocytes . Furthermore , longer exposure of monocytes to mf ( 5 days ) did not result in a decrease in allogeneic or autologous ( α-CD3 dependent ) CD4+ or CD8+ T cell proliferation ( S6A and S6B Fig ) . As well , the further increase in cell surface expression of PDL1 on monocytes after 5 days exposure to mf ( S3 Fig ) did not result in inhibition of T cell proliferation ( S6A and S6B Fig ) .
Within most solid tumors , monocytes and macrophages are the major inflammatory infiltrates that can be recruited to the tumor microenvironment by tumor-derived chemokines , cytokines and other signals[32] . The majority of these infiltrating cells differentiate into TAMs promoting cancer cell proliferation , immunosuppression , and angiogenesis[33–35] . While the immunosuppressive TAMs are activated by IL-4 and IL-13 ( Th2-associated cytokines ) , IL-10 , glucocorticoids and vitamin D3 , and can exert functions similar to M2 macrophages[36] , recent findings suggest that some TAMs are Th1-skewed and resemble M1 macrophages[37] . In murine models , glioblastoma-associated monocytes/macrophages have shown to produce a broad range of cytokines/chemokines with both anti- and pro-inflammatory properties[38] . However , in humans , the same monocytes have phenotypes that are largely anti-inflammatory[39] . In addition , monocytes co-cultured with breast cancer cell lines have shown to have pro-tumor patterns of activities[40] . Therefore , TAMs are not simply restricted to M1 and M2 phenotypes and can represent a spectrum depending on tumor type , location , and microenvironment ( reviewed in[41] ) . In general , helminth parasites are shown to induce an M2 response in monocyte/macrophage populations ( reviewed in[42] ) . Although , it has already been reported that both helminths and tumors alter the function of monocytes/macrophages , there are no studies assessing the similarities and differences between parasites and cancer cells in their effect on the phenotype and function of these cells . Given that both parasites and tumors share features designed to manipulate the host immune response , we aimed to study the similarities and differences between them with respect to monocytes ( S1 Table ) . To do so , we established a comparison between three cancer cell lines ( MDA , OVCAR , and U87 ) and the circulating stage of the helminth parasite , Brugia malayi and then assessed the phenotype and function of human monocytes after exposure to either cancer cell lines or parasites . While we have looked at gene expression and cytokine production of mf- and cancer cell lines-exposed monocytes at various time points ( 5 or 7 days; S7 Fig ) , we chose 48 hours as mf exert its profound effect on DC and monocytes at this time point[6 , 9 , 43 , 44] . In our hands , and in agreement with previous studies[45 , 46] upon exposure of monocytes to cancer cell lines , both pro- and anti-inflammatory genes are induced ( Fig 1 , S2 Fig and S1 Table ) . In fact , all three-cancer cell lines significantly enhanced the monocyte mRNA expression of genes associated with inflammatory responses , Type-2 responses , regulatory responses , and angiogenesis ( Fig 1 and S2 Fig ) . The M2 differentiation of monocytes co-cultured with breast cancer cell lines in transwell has been shown in the past[46] . In our studies , co-culture of monocytes with the supernatant of cancer cell lines resulted in enhanced expression and frequency of CD206 and PDL1 positive cells ( S8 Fig ) . Furthermore , cancer cell lines- exposed human monocytes demonstrated a significant induction of TGF-β , PGE2 and IL-10 as compared to unexposed cells ( Fig 1 ) . These immunosuppressive cytokines are known to be produced by macrophages in the tumor microenvironment to promote tumor growth by maintaining T regulatory cell differentiation[11 , 47] . Moreover , angiogenesis is a key event in tumor growth and progression , and TAMs are the major cell types promoting this event by producing factors such as VEGF in the tumor microenvironment[48 , 49] . For example , the interaction between monocytes/macrophages and ovarian cancer cells results in an increased ability of endothelial cells to promote tumor progression through angiogenesis[33] . Here our data indicate that exposure of human monocytes to all three-cancer cell lines results in significant induction of VEGF and MMP9 ( Fig 1 ) suggesting a phenotype similar to TAMs . One of the major similarities between mf- and cancer cell line- exposed monocytes is the significant upregulation in the mRNA levels of IL-1β ( associated with inflammation or M1 phenotype ) , MMP9 ( associated with angiogenesis ) , and TGM2 ( associated with M2 phenotype ) ( Fig 1B ) . However , the magnitude of this upregulation is less profound in mf-exposed monocytes ( Fig 1A and 1B ) . Another similarity between cancer cell lines and mf is in their regulation of cytokine production . For example , while neither ( mf or cancer cell lines ) induces the production of CCL4 , TNF-α , IL-10 , or VEGF , they both significantly enhance the production of IP-10 and CCL22 in human monocytes ( Fig 2 ) . Both IP-10 and CCL22 are involved in lymphocyte chemotaxis and recruiting regulatory T cells to the tumor microenvironment[50 , 51] . IP-10 also binds endothelial cells and exerts a potent angiogenic activity in tumor settings[52] . While the role of IP-10 in T cell recruitment has been shown with intracellular parasites[53] , the importance of this chemokine in helminth infection is still not fully understood . In humans , CCL22 and CCL18 are the chemokines expressed by M2 macrophage[54 , 55] and mf of Brugia malayi upregulate the mRNA expression of both chemokines[6] and also induce the production of CCL22 ( Fig 2 ) . An important similarity between helminth parasites and cancer cell lines demonstrated here is their ability to upregulate monocyte cell surface expression of inhibitory molecules such as PDL1 and PDL2 ( Fig 3 ) . While unexposed monocytes have low expression of PDL1 , CD163 , and CD206 ( Fig 3A; black solid lines ) , exposure to mf , similar to those of cancer cell lines , significantly upregulate the cell surface expression of PDL1 ( inhibitory ) , PDL2 ( M2/inhibitory ) , CD206 ( M2 ) , and VCAM-1 ( M1 ) ( Fig 3A and 3B ) . Interestingly , while the upregulation in cell surface expression of PDL1 on monocytes was less profound with mf than with cancer cell lines , longer exposure to this parasite further increased the level of PDL1 ( S3 Fig ) . PDL1 and PDL2 are the two ligands for a major immune-checkpoint receptor PD1 ( CD279 ) [56 , 57] . The engagement of PD1 on T cells with its ligands ( PDL1/PDL2 ) on APCs inhibits kinases that are involved in T cell activation[56 , 58] . The majority of lymphocytes that infiltrate tumor microenvironment express PD1 and acquire a phenotype of hyporesponsiveness[59 , 60] . On the other hand , PDL1 is shown to be on most melanoma , ovarian and many other cancer types[61] . In addition to tumors , myeloid cells in tumor microenvironment such as TAMs also express high levels of PDL1 and PDL2[62–67] . Therefore , blockade of this inhibitory pathway is essential in cancer immunotherapy ( reviewed in[62] ) . Our data suggest that monocytes that are exposed to MDA ( Fig 5; and other cancer cell lines , S5 Fig ) have significantly decreased their ability to promote allogeneic CD4+ and CD8+ and autologous CD4+ T cell proliferation as compared to unexposed monocytes ( Fig 5A and 5B ) , suggesting a suppressive phenotype . Interestingly , blocking PD1/PDL1 pathway with anti-PDL1 mAb reversed the suppressed proliferation in allogenic CD4+ but not CD8+ T cells that were co-cultured with MDA exposed monocytes ( Fig 5C ) . Similar to cancer settings , chronic filarial infection with continuous release of parasite antigens is associated with a lack of CD4+ T cell proliferation and production of IFN-γ and IL-2[68] . The role of PD1/PDL1 ( PDL2 ) in regulating T cell response has also been extended to several infections[69] . Recent studies have suggested that macrophage expression of PDL1 is important in regulating T cell responses to influenza infection[70] . In acute malaria the induction of PD1+CTLA4+ effector T cells results in suppressive function and inhibition of other CD4+ T cells[69] . In our study , one major difference between helminth parasites and cancer cells is how they shape monocytes to promote T cell activation ( Fig 5 ) . In contrast to cancer cells , exposure to mf did not diminish the ability of monocytes to promote T cell proliferation ( Fig 5A , 5B and 5C ) . Therefore , blocking PDL1 pathway in mf-exposed monocytes did not have any effect on T cell proliferation . Furthermore , longer exposure of monocytes to mf ( 5 days , S6 Fig ) does not inhibit T cell proliferation . While , it has been suggested that other inhibitory molecules such as CTLA4 can play a role in T cell hyporesponsiveness seen in filarial-infected individuals[71] , how PD1/PDL1 may play a role in this suppression is not known . The ability of cytotoxic lymphocytes to recognize and kill infected or transformed cells is an important part of both innate and adaptive arms of the immune system . In fact cytolytic T lymphocytes are key players in current immunotherapies and promote apoptosis of cancer cells through granule-mediated as well as receptor-mediated mechanisms ( reviewed in[31] ) . Stimulation of these cytolytic T cells through their receptors induces the activation of effector mechanisms including the granule exocytosis pathway releasing granule-associated enzymes ( granzymes ) as well as other factors resulting in target cell death[72] . Granzyme A and B are two important serine proteases that are involved in lymphocyte mediated cytotoxicity[31] . In fact , it has been shown that inhibition in the function of these cytolytic T lymphocytes such as CD8+ T cells by TAM establishes a suppressive microenvironment for the infiltrating immune cells[73 , 74] . Here , we demonstrate that exposure of human monocytes to MDA and U87 cancer cells , but not to OVCAR or mf ( Fig 5D ) significantly downregulated the percentage of Granzyme A+ CD8+ T cells suggesting further suppressive function of these cancer cell associated monocytes . In general , CD8+ T cells can play both effector and regulatory role in parasitic immunity ( reviewed in[75] ) . CD8+ T cells mediated killing activities have been mostly directed and demonstrated against number of intracellular parasites that infect host cells[75] . How CD8+ T cells are regulating immunity against extracellular parasites is not fully understood . In filarial infections , CD8+ T cells exhibited a unique transcriptome in chronically-infected patients when compared to those with relatively acute infections , suggesting an importance of CD8+ cells in this infection[76] . Immune suppression in variety of helminth infections involves regulatory T cells[42] . For example , in onchocerciasis , Granzyme A/B expression was associated with Treg induction and subsequent immune suppression[77] . Induction of Treg were shown both in vitro[78 , 79] and in filarial-infected patients[80 , 81] . One important function of macrophages is their ability to phagocytose[82 , 83] . Macrophage phagocytosis plays a major role in tumor immune surveillance[84] in that antibody- dependent cellular phagocytosis mediated by macrophages contributes significantly to anti-tumor activity[85] . Macrophages that are polarized within the tumor microenvironment have increased phagocytic ability[86] . In the present study , exposure to MDA ( Fig 4 ) and other cancer cell lines ( S4 Fig ) significantly induced the phagocytic ability of human monocytes to phagacytose bioparticles , suggesting that human monocytes exposed to cancer cell lines in vitro behave similarly to TAMs . Our data suggest that despite the fact that helminth parasites and tumor cell lines are extraordinarily disparate , they share the ability to alter the phenotype of human monocytes although the nature of this alteration differed ( see S1 Table ) . Nevertheless , similarities between the two types of stimuli in eliciting macrophage phenotypes similar to that of TAMs were observed , most notably in their ability to drive the surface expression of immune inhibitory molecules such as PDL1 . Finally , utilizing a multidisciplinary approach to understand the mechanisms underlying immune evasion by both tumors and parasites could be beneficial to our understanding in both fields . | Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells including monocytes . In recent years , there has been a large body of evidence in the field of cancer immunotherapy demonstrating that the immune system can be retrained to not only recognize tumor cells but also to eliminate them . Tumor immunology has advanced significantly with the advent of newer tools of molecular immunology and identification of tumor associated biomarkers . In fact , the concepts for immunotherapy as an effective way to reawaken the immune response against tumors that evade the immune system may apply to other diseases . Therefore , using similar approaches in infectious diseases , might lead to activation of the immune system and reversal of immunological ignorance against parasites . We strongly believe that a multidisciplinary approach would encourage crosstalk between different areas of science and will foster and advance research . Here , we investigate how cancer cell lines and helminth parasites shape the phenotype and function of human monocytes . Both cancer cell lines and helminth parasite altered the expression of selected genes associated with inflammatory , regulatory , or type 2 response and upregulated immune checkpoints PDL1 and PDL2 in human monocytes . Our data suggest that despite the fact that helminth parasites and cancer cell lines are different , they share the ability to alter the phenotype of monocytes . | [
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... | 2018 | Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion |
An important determinant of a pathogen's success is the rate at which it is transmitted from infected to susceptible hosts . Although there are anecdotal reports that methicillin-resistant Staphylococcus aureus ( MRSA ) clones vary in their transmissibility in hospital settings , attempts to quantify such variation are lacking for common subtypes , as are methods for addressing this question using routinely-collected MRSA screening data in endemic settings . Here we present a method to quantify the time-varying transmissibility of different subtypes of common bacterial nosocomial pathogens using routine surveillance data . The method adapts approaches for estimating reproduction numbers based on the probabilistic reconstruction of epidemic trees , but uses relative hazards rather than serial intervals to assign probabilities to different sources for observed transmission events . The method is applied to data collected as part of a retrospective observational study of a concurrent MRSA outbreak in the United Kingdom with dominant endemic MRSA clones ( ST22 and ST36 ) and an Asian ST239 MRSA strain ( ST239-TW ) in two linked adult intensive care units , and compared with an approach based on a fully parametric transmission model . The results provide support for the hypothesis that the clones responded differently to an infection control measure based on the use of topical antiseptics , which was more effective at reducing transmission of endemic clones . They also suggest that in one of the two ICUs patients colonized or infected with the ST239-TW MRSA clone had consistently higher risks of transmitting MRSA to patients free of MRSA . These findings represent some of the first quantitative evidence of enhanced transmissibility of a pandemic MRSA lineage , and highlight the potential value of tailoring hospital infection control measures to specific pathogen subtypes .
Methicillin-resistant Staphylococcus aureus ( MRSA ) is responsible for a high burden of morbidity and mortality worldwide [1]–[4] . While community-associated MRSA is becoming increasingly important globally [5] , [6] , in many countries , including the United Kingdom , MRSA remains predominantly a nosocomial pathogen [7] , [8] . The dominant sequence type ( ST ) in Asia is ST239 , and recent analysis of whole-genome sequence data has shown that this ST has distinct lineages in Asia , Europe and South America which probably share a European ancestor [9]–[11] . Little is known about what has enabled this ST to be so successful , or whether its propensity to transmit between hosts differs from other MRSA types in certain settings . A recent concurrent outbreak due to an ST239 MRSA strain ( ST239-TW , subsequently referred to as TW ) and the two dominant endemic UK MRSA types ( ST22 and ST36 , which we refer to as non-TW ) in two linked adult intensive care units in a London teaching hospital provided a rare opportunity to compare the transmissibility of different MRSA types in the same clinical setting [12] . The transmissibility of a potentially emerging pathogen ( the rate at which it spreads from an infected host to exposed susceptible hosts ) is an important factor in determining its success and , in the case of an established pathogen , for estimating how effective interventions must be to bring an epidemic under control [13] , [14] . Quantifying the degree to which strains of a nosocomial pathogen differ in their transmissibility in a particular setting could lead to a better understanding of why major clonal replacements occur . Measuring how such transmissibility changes in response to interventions would allow us to quantify the value of specific control measures , which may vary according to the strain [15] . This could lead to better resource use by allowing us to choose control measures appropriate for the specific strain . Such an analysis has greatest relevance for predominantly clonal organisms , such as S . aureus , where distinct lineages cocirculate over extended periods of time [10] . A fundamental measure of the overall transmission potential of a pathogen in a given setting is the basic reproduction number , . This is defined as the mean number of secondary cases generated by a typical case in a fully susceptible population [16] , [17] . If the transmissibility of each infected host remains constant throughout its infectious period , and if each infected host has an equal chance of infecting each susceptible host , then is simply the product of the mean rate at which an infected host generates secondary infections and the mean infectious period ( provided the two are not correlated ) . The self-sustaining chain reaction that constitutes a major epidemic is possible only if is greater than one . If it is less than one , although there may be some self-limiting chains of secondary transmission following the introduction of an index case ( and quite large clusters become possible as approaches one ) , this will not lead to a sustained increase in cases and , in a large population , only a small proportion of susceptible hosts will be infected [18] . An important related number is the net ( or effective ) reproduction number , . This is defined as the average number of secondary cases generated by a case infected at time , accounting for incomplete host susceptibility to infection and control measures in place . If is greater than one at time , the epidemic will ( on average ) be growing . If is less than one , it will be declining [16] , [19] . These reproduction numbers are central to a mechanistic understanding of infectious disease epidemiology , and a number of methods for estimating them from different types of surveillance data have been devised [16] , [19]–[23] . However , epidemics that predominantly affect hospitalized patients require some special considerations . First , unlike the community setting , the population of those exposed to infection changes rapidly over time as patients are admitted and discharged . Second , most common nosocomial pathogens are bacteria which can be carried asymptomatically over long periods , during which time colonized hosts may have several hospital admissions . This can give rise to distinctive dynamics: in addition to the usual explosive outbreaks , we also see epidemic patterns characterized by a sequence of self-limiting clusters of transmission which , over time , become more frequent and eventually coalesce into an exponentially growing epidemic [24] , [25] . The concept of the single admission reproduction number , , can help in the understanding of these features of hospital epidemics [25] , [26] . is defined as the mean number of secondary cases caused by a typical infectious patient during a single admission to a particular hospital or ward otherwise free of the pathogen . Necessarily , is less than or equal to . However , if and then every outbreak will be locally controlled in the short term , but , with repeated challenges to the hospital , long-term control failure will be inevitable . This results from the persistence of carriage following discharge which , over time , leads to a gradual increase in numbers colonized on admission . To account for changing numbers of susceptibles , we can also define a net single admission reproduction number , . This is analogous to and represents the average number of secondary cases generated during a single hospital/ward admission where not everyone is necessarily susceptible . Direct ascertainment of and would be possible if we could reliably assess who infected whom during a hospital outbreak . In practice , even with detailed surveillance and molecular typing data , there is almost always considerable uncertainty about the true transmission tree . Instead , computationally-intensive approaches based on fitting mechanistic mathematical models to data which account for uncertainty in transmission routes and screening data represent the state-of-the art for analysing nosocomial transmission dynamics [27]–[32] . However , such approaches require detailed data on both susceptible and colonized or infected patients , and an assumption that temporal changes in the transmissibility can be described parametrically by some standard functional form ( most commonly , piecewise constant ) . As currently implemented they do not allow direct estimates of the number of transmission events associated with each patient . The aims of this paper are twofold: to describe a new approach ( method 1 ) for estimating using hospital surveillance data; and to use it to analyse MRSA data from concurrent outbreaks with different MRSA types ( TW and non-TW ) in two linked adult intensive care units ( ICUs ) . The method is simple to use and enables us to track how changes over time without the assumption that changes in transmissibility follow a fixed functional form , and without requiring data on susceptible patients . The method extends techniques for the probabilistic reconstruction of epidemic trees developed for analyzing foot and mouth disease and SARS data [21] , [33]–[35] . We contrast results using this approach with that from a fully parametric mechanistic model ( method 2 ) , which represents an adaptation of previously described parametric models for nosocomial infection to a multistrain system [31] , [32] . This second approach allows to be estimated . It requires more detailed data and stronger assumptions , but allows us to explicitly test hypotheses about how transmissibility is affected by interventions , and how it varies between different wards and subtypes of MRSA . While between-clone and between-ward differences in single admission effective reproduction numbers , , calculated using method 1 may be caused by differences in transmissibility , number of susceptibles , and lengths of stays , with method 2 we assume all MRSA positive patients have the same length of stay distribution and explicitly adjust for different numbers of susceptible patients when calculating .
Under baseline assumptions , on ICU1 there were 282 MRSA importation events ( episodes where patients were assumed to be MRSA positive when admitted to the ICU ) and 132 acquisition events . These comprised of 12 importations and 23 acquisitions with TW MRSA and 270 importations and 109 acquisitions with non-TW MRSA . On ICU2 there were 285 importations ( 25 with TW ) and 166 acquisitions ( 43 with TW ) ( figure 1 ) . Importations with non-TW to the respective ICUs decreased from 0 . 20 and 0 . 19 per day in phase 1 to 0 . 11 and 0 . 13 per day in phase 4 . In contrast , importations with TW MRSA peaked in phase 2 in both ICUs ( at 0 . 03 and 0 . 12 per day ) and were at or below 0 . 01 per day in phases 1 and 4 . Amongst patients who were MRSA positive on admission the median length of stay was 12 days ( inter quartile range [IQR]4 , 18 ) for TW-positive patients and 6 days ( IQR 3 , 13 ) for non-TW patients ( , Wilcoxon rank sum test with continuity correction ) . For patients who acquired MRSA the corresponding numbers were 26 . 5 ( 13 . 25 , 42 . 5 ) for TW patients and 19 ( 12 , 29 . 25 ) for non-TW patients ( ) . There was no evidence that length of stay differed by ward ( ) , or by study phase ( , Kruskal-Wallis rank sum test ) . Over the study period ( January 2002 to April 2006 ) there were three interventions ( referred to as A , B and C ) and these define four study phases . Estimated net single admission case reproduction numbers ( expected number of secondary cases per case during a single ward admission ) associated with each MRSA-positive patient episode are shown in figure 2 ( bottom panel ) together with histograms of case reproduction numbers for each ward and study phase ( top panel ) . These highlight wide between-patient variability which decreases in the second half of phase 4 when transmission is reduced and the TW clone is eliminated . While most patients have a very low expected number of secondary cases , 22 out of 103 patients ( 21% ) with TW MRSA are expected to transmit to at least one other patient . Corresponding numbers for non-TW MRSA are 40 out of 762 ( 4% ) . This proportion was consistently higher for TW MRSA in all four study phases: 25 , 11 , 18 and 31% versus 7 , 0 , 9 and 2% for non-TW MRSA . Aggregating these reproduction numbers into four-week intervals highlights the temporal trends , differences between wards and impact of interventions ( figure 3 ) . In ICU1 these suggest similar patterns of transmission for the different MRSA types for the period prior to intervention C ( a surface antiseptic protocol ) . In contrast , there were marked differences between MRSA types in ICU2 and throughout the study period the four-week averaged reproduction numbers for the TW clone usually exceeded those for non-TW clones when both types were present . These differences are also seen when reproduction numbers are averaged over study phases ( table 1 ) ; the TW clone had a higher reproduction number than the non-TW MRSA in each phase in ICU2 but not in ICU1 . Reproduction numbers for TW MRSA were also more volatile than those for non-TW MRSA in ICU2 . There was evidence from both units to suggest differences between the MRSA types in their response to infection control interventions: while the net reproduction number of non-TW MRSA fell to a low level following intervention C in both ICUs , this was not the case for the TW clone which continued to transmit for several months at pre-intervention levels . Eventually , the TW outbreak came to an end after all patients with TW MRSA were treated empirically with systemic antibiotics ( linezolid ) from 1st September 2004 [12] , [15] . After this intervention , although patients with TW MRSA continued to be imported into the ICUs , only three isolated apparent transmission events occurred ( figure 1 ) . When reproduction numbers for the two ICUs combined were estimated ( allowing for cross transmission between ICUs ) the results suggested the reproduction number of the TW clone was consistently higher than that for the non-TW MRSA and varied little throughout the study period ( table 1 ) . Reproduction numbers for the non-TW clones , in contrast , fell in phase 2 and 4 . These results were not highly sensitive to the assumed strength of coupling between the two ICUs or to the MRSA acquisition assumptions ( supplementary table S2 ) . Results from method 2 showed broad agreement with these findings , but in contrast to method 1 made a priori assumptions about the timing of the changes in transmissibility ( tables 2–3 , figure S1 ) . On both wards , averaging over all phases , there was about a 1 in 400 chance of a given susceptible patient acquiring MRSA from a particular MRSA-positive patient on a particular day ( table 2 ) . When estimates of daily transmission probabilities from a single MRSA positive patient were constrained to take the same values for the TW and non-TW clones but were allowed to vary by ward and study phase , we found clinically significant variation between the four study phases in both ICUs ( table 2 ) . In particular , while estimates were similar in phases 1 to 3 , there was a marked reduction in phase 4 . There was no strong evidence that these joint estimates ( for all MRSA clones ) varied by ICU in any of the study phases ( table 2 ) . These findings were robust to the assumptions made about acquisition events and times ( supplementary table S3 ) . Extending this analysis to allow transmission probabilities to vary with the MRSA type enabled differences between MRSA clones and wards to be quantified ( table 3 ) and allowed hypothesis tests about whether the daily transmission probabilities differed between strains ( thus allowing us to test whether the observed differences in transmissibility found using method 1 could be entirely explained by the longer length of stay of the TW patients ) . In ICU1 no consistent differences were seen . In ICU2 the TW clone had a higher daily transmission probability to susceptible patients in each of the four study phases under the baseline assumption of complete bacterial interference , though confidence intervals were wide and showed considerable overlap . Differences between TW and non-TW MRSA transmission probabilities reached statistical significance at the 5% level for both wards combined and for ICU1 , but not for ICU2 alone , and in only one of the four phases ( phase 4 ) using combined data from both ICUs . This phase corresponded to the introduction of the surface antiseptic bodywash protocol , which was associated with a more than halving of the transmission probability from a patient with non-TW MRSA compared to earlier phases . The fall in the transmission probability for TW MRSA in phase 4 was smaller , and likely to be confounded by the use of linezolid for TW carriers in this phase . Large differences in transmission probabilities for the two MRSA types were also seen in phase 2 ( corresponding to the introduction of hand hygiene promotion ) , but in this case confidence intervals were wider reflecting the short duration of this phase . In other phases differences between TW and non-TW estimates were much smaller . The magnitude of the differences depended on which patients were assumed to be susceptible . Under the baseline assumption that patients colonized with one strain were not susceptible to acquiring another ( complete bacterial interference ) the differences were larger than in the sensitivity analysis where no bacterial interference was assumed ( table 3 ) . This can be explained by the higher prevalence of non-TW MRSA clones; under the assumption of no bacterial interference all non-TW MRSA positive patients would be considered susceptible to infection or colonisation with TW MRSA and vice versa . Changing from complete interference to no interference therefore results in a greater increase in the number of susceptibles available for the TW clones to infect than it does for the non-TW clones . To accommodate these changes , a larger reduction in the daily transmission probability for TW clones is required . Overall , combining data from both wards , the TW clone was estimated to have a daily transmission probability that was between 63 and 100% higher than the non-TW clones in phase 4 , and between 53 and 94% higher in phase 2 ( the lower numbers corresponding to the no bacterial interference assumption ) though the differences only reached significance at the 5% level in phase 4 and only under baseline interference assumptions . Transmission probabilities were broadly similar in the two other study phases . Estimates of the single-admission reproduction number ( ) from the model without background transmission ( and assuming TW and non-TW patients have the same length of stay distribution ) are reported in the supplementary material ( figure S1 ) . Results were robust to the assumptions made about the number and timing of MRSA acquisition events ( supplementary table S4 ) , but fitting a more complex model allowing patient-to-patient transmission and transmission from background sources suggested that the relative importance of patient-to-patient and background transmission could not be reliably identified in such hyperendemic settings without additional data ( supplementary table S5 ) .
Common bacterial nosocomial pathogens have distinct dynamics from typical community pathogens and call for different analytical approaches . Important features of hospital epidemics with such organisms include: i ) a host population that changes rapidly over time in comparison with the timescale of epidemic dynamics; ii ) a high proportion of infected ( or colonized ) hosts who are already infected when they enter the population ( the hospital or ward ) ; iii ) a dominant role for asymptomatic infection so infected hosts can usually only be identified using screening swabs , leading to large uncertainty in the timing of transmission events; iv ) a lack of a well-defined serial interval or generation time ( since asymptomatic carriage can persist for months or years , but transmission is only intermittently observed during hospital admissions ) . The probabilistic tree reconstruction approach described above ( method 1 ) overcame these limitations by using a hazards-based approach applied to patient screening data to assign probabilities to potential source patients for observed acquisition events . Using hazards in this way to reconstruct epidemic trees and estimate reproduction numbers appears to have first been suggested by Kenah et al [36] . Results using this method were supplemented with a maximum likelihood approach ( method 2 ) where the timing of cross-infection events was assumed to be known but which allowed estimation of the daily transmission probability , enabling us to study effects related to study phase and MRSA type while controlling for differences in length of stay . These methods were applied to data from two adjacent general ICUs in which admission and weekly MRSA screens and culture results from clinical samples identified patients admitted with and acquiring MRSA over a four year period . During that time there was sustained transmission with endemic MRSA and a newly introduced TW variant . Both analytical methods supported the hypothesis that intervention C ( the surface antiseptic protocol ) was associated with a sustained reduction in MRSA transmission , and both indicated a reduced effect for the TW clone . Both methods gave point estimates that indicated elevated transmission of TW MRSA compared with endemic strains in all four study phases in ICU2 but not ICU1 . There were , however , some differences: the ward-level reproduction numbers ( method 1 ) tended to indicate greater increased transmission for the TW compared to non-TW MRSA than was seen using method 2 . This reflects the fact that the two methods are quantifying different things: method 1 estimates secondary cases per case , which depends both on transmissibility and the length of ICU stay while carrying MRSA; method 2 , in contrast , estimates only the daily transmission probability from one MRSA carrier to one susceptible patient . This will not be affected by length of stay . Indeed , there was some evidence that patients colonised with TW MRSA ( particularly those colonised on ICU admission ) , had a longer length of stay than those colonised with non-TW MRSA . This may reflect the link between MRSA infection and excess length of stay in this cohort [37] , and the increased virulence of the TW strain which was over four times more likely to cause blood stream infection in colonised patients compared to non-TW MRSA strains in the same ICUs [12] . Even in the absence of an increased rate of transmission to other patients , increased length of stay would lead to a higher single-admission reproduction number . It is possible that such differences in length of stay reflect underlying differences in the characteristics of patients most vulnerable to acquiring the different MRSA types . For example , because the TW outbreak was centred on the two ICUs , patients carrying TW on ICU admission might be more likely than patients carrying non-TW MRSA to have had recent ICU admissions . The TW clones showed a far broader range of antibiotic-resistance than endemic MRSA clones and have previously been shown to preferentially colonise vascular catheters but not carriage sites compared with endemic strains [12] . Taken together , these observations suggest that the TW MRSA could represent a phenotype particularly adapted to transmission in settings , such as ICUs , with high levels of antibiotic usage and patient catheterisation , perhaps at the expense of persistence outside these areas . There is some evidence that such adaptation results from both increased persistence in the ICU ( perhaps by targeting long-stay patients , and causing infections that increase length of stay ) and from an increased daily transmission probability ( particularly in the presence of widespread antiseptic use ) . Caveats , of course , apply: differences in lengths of stays between TW and non-TW colonized/infected patients could be confounded by exposure history ( the recent arrival of the TW clone rather than its biological properties may account for the different patient characteristics ) . Differences in daily transmission probabilities could also be subject to such confounding and could also have arisen by chance ( in all phases – even phase 4 , where the effect size was largest – confidence intervals were wide ) . The mechanisms underlying variations in transmissibility of different MRSA ( and S . aureus ) strains are poorly understood . Reasons for the differences in the two ICUs are also unclear . Chance variation cannot be ruled out , as the formal investigation of transmission potential of different MRSA types was , in part , motivated by perceived differences in transmissibility ( using the same data ) , and the usual limitations of post hoc analyses therefore apply . Also , although the analyses accounts for demographic stochasticity , there may also be important sources of environmental stochasticity which are not accounted for . It seems unlikely that the difference in TW transmission in the two ICUs can be explained by colonized staff: a universal staff screening programme failed to detect the TW clone during the outbreak [12] . Differences in infection control practice also seem unlikely but cannot be ruled out: the two wards share the same infection control policies and staff pool , with medical and nursing staff rotating between units at 3–6 monthly intervals , though only physiotherapy , radiology and pharmacy staff worked across both units at the same time . It is possible that the built environment influences MRSA transmission . ICU2 was last refurbished in 1969 , retaining a mixture of original materials including wood , and has much less open space , only eight sinks , and one side room , whereas ICU1 was refurbished in 1999 to an open plan configuration with better space utilization , 19 sinks and three side rooms . The reduced availability of sinks , side rooms and space to circulate may have adversely affected the ability to carry out infection control practice or cleaning , although it is unclear why this should only affect TW MRSA , which was not detected on environmental screening during the outbreak [12] . Despite anecdotal reports that some lineages of S . aureus strains have an enhanced epidemic potential in hospital settings [38] , objective assessments of between-strain variation in transmissibility are largely lacking . Such variation is nonetheless to be expected given the large degree of phenotypic variation in different S . aureus and MRSA clones , and the dominance of a small number of MRSA lineages [39] . One of the few instances where the nosocomial transmission potential of different subtypes of the same nosocomial pathogen have been quantified comes from a comparison of the onward transmission from patients admitted to hospitals in the Netherlands carrying MRSA [40] . In this case , because MRSA introductions were infrequent ( as MRSA prevalence in hospitals in the Netherlands is below 1% ) and contact tracing extensive , the secondary cases could be assigned to distinct clusters of transmission following identified introductions . This allowed the authors to use methods based on a branching process model to estimate the single admission reproduction number , [41] . It was found that newly admitted ST398 MRSA strains ( which are commonly associated with livestock production ) had a greatly reduced propensity to spread compared with other MRSA sequence types , with an value ( 95% CI ) of only 0 . 16 ( 0 . 04–0 . 40 ) , about one sixth of the corresponding value for non-ST398 MRSA . The authors concluded that less stringent control measures were likely to be sufficient to control ST398 MRSA clones than those needed for non-ST398 MRSA types . Such methods would not have been applicable for our data , and the first method used here to quantify the transmissibility of different strains ( method 1 ) instead built on recent approaches to estimate reproduction numbers by probabilistically reconstructing epidemic trees . Such tree-reconstructions have used simple rule-based methods , for example assigning sources from a candidate list based on proximity data [33] , more formal semi-parametric methods using partial likelihoods and assuming a known serial interval distribution [21] , [34] , and , most recently , semi-parametric hazard-based approaches [36] . Hazard-based approaches have some advantages over the first two methods: they avoid some of the arbitrary assumptions of the rule-based approaches , do not require knowledge of the serial interval distribution , and can avoid biases that arise from the fact that the serial interval distribution changes over the course of an epidemic . Advantages over approaches based on fitting a full transmission model include fewer assumptions , in particular with regard to the functional form of changes in the transmission potential over time . In this respect , tree reconstruction approaches have some similarities with other semi-parametric approaches that make use of survival analytical methods , such as the approach adopted by Wolkewitz et al . , who derived non-parametric estimates of a time-varying transmission rate changed over time using a Martingale-based method [42] . An important difference in the current approach is that we are specifically interested in estimating how the distribution of the number of secondary cases resulting from each case changes over time . The method 1 approach described here also makes relatively low demands for data ( with no information required for patients who do not become colonized or infected ) , has a low computational burden , and can be easily adapted to cope with co-circulating subtypes as in the application here . This approach is appropriate when the daily probability of a patient acquiring MRSA is small , as in this case reconstructed epidemic trees will be approximately independent of this probability . This approximation is likely to be reasonable for all but the most explosive outbreaks . For example , using the exact formula we found that changing this probability from a baseline of 0 . 005 to 0 . 001 and 0 . 025 changed the estimated mean reproduction numbers for each phase and MRSA type by less than 3% . Two assumptions underlying the analytical approaches used here are i ) that new MRSA acquisitions can be explained by patient-to-patient spread within the units ( which is likely to be mediated by contacts with transiently colonized healthcare workers ) and ii ) that risk of transmission increases in line with colonization pressure ( the number of patients with MRSA on the ward ) . While these assumptions are supported by observational and quasi experimental studies [43] , [44] , it would be desirable to more rigorously challenge them . Unfortunately , unpublished simulation studies and analysis here with a more complex model allowing different transmission routes ( table S5 ) both suggest that the ability to identify the relative importance of background and patient-to-patient transmission may be limited in hyper-endemic settings in the absence of more discriminatory typing data . The inability of our typing methods to reliably distinguish between non-TW MRSA types , or to identify genetic variants of the TW clone therefore represent important limitations of this work . High resolution genotyping data would enable more definitive assessments of who infects whom , and therefore allow us to quantify the risks of transmission of different MRSA subtypes in different wards at different times with greater certainty . Figure 1 confirms that not all acquisition events can be explained by transmission from a known MRSA positive patient from the same ward . The combined-ICU analysis , allowing for between-ward transmission , is able to account for some MRSA acquisitions where no known source was present on the same ward , and this explains why combined ICU estimates of the reproduction number are sometimes outside the range of individual ICU estimates ( table 1 ) , but unknown MRSA sources are also likely to be present in the patient population [32] . A full model-based analysis using data augmentation ( which estimates model parameters and latent parameters that represent “unobserved” - or augmented - data , typically using Markov chain Monte Carlo methods for fitting ) could account for such unknown sources . Such an approach retains some important advantages for analysing typical surveillance data . These include the ability to account for imperfect swab sensitivity and for uncertainty in the number and timing of acquisition events , circumventing the need to make arbitrary assumptions about which patients were colonized on admission to a ward . In the present context such an analysis would allow us to explicitly account for the change in the screening protocol in November 2004 . Since this involved screening more body sites , it is likely to have increased screening sensitivity and led to increased detection of MRSA , potentially biasing the estimated effect of intervention C . To date , however , no published work has adapted such approaches to cope with multiple co-circulating subtypes . The method 2 used here can be thought of as a simplified version of such an approach ( in that it is based on a fully-specified mechanistic transmission model ) but it avoids the complexities of data augmentation by assuming the epidemic process is perfectly observed . An important area for future work will be to extend data augmentation methods to cope with carriage of multiple types . Such approaches have been developed for the sequential carriage of community pathogen subtypes [45] . Addressing issues of co-colonisation with different subtypes may be particularly important for some nosocomial pathogens , and neglecting such effects is a potential source of bias . In our analysis here we considered two possibilities – complete bacterial interference ( where one strain completely inhibits the acquisition of another ) , and no bacterial interference . The reality may lie somewhere between these two extremes . Such an analysis will be complicated by the fact that routinely-used laboratory methods are not well-suited to detecting the simultaneous carriage of multiple types [46] , and sensitivity for detecting a second type will not , in general , be the same as sensitivity for detecting a single type .
Ethical approval for this research was granted by the NHS National Research Ethics Service , South East Research Ethics Committee . All data were analyzed anonymously . Anonymised data from two 15-bed adult general intensive care units ( ICU ) within a 1050-bed teaching hospital in London , United Kingdom , were collected between 1st January 2002 and 30th April 2006 as described elsewhere [12] , [15] . Dates of admission and discharge and MRSA culture results from screen and clinical samples were analysed for all 4 , 570 consecutive patient admissions to both ICUs . Infection control policies were in place including specifying hand hygiene between patient contacts and use of contact precautions for known MRSA colonized patients throughout . On this background three main new MRSA control interventions were introduced: intervention A ( introduced on 15th July 2003 ) was an education campaign to promote hand hygiene and barrier nursing; intervention B ( introduced on 15th October 2003 ) was isolation of known MRSA colonized patients in side rooms or in patient and nursing cohort pairs; intervention C ( introduced on 26th April 2004 ) was a surface antiseptic protocol which included daily chlorhexidine bodywashes for known MRSA positive patients , and daily triclosan bodywashes for other patients . The three interventions defined four study phases for analysis: phase 1 from 1st January 2002 to 14th July 2003; phase 2 from 15th July to 14th October 2003; phase 3 from 15th October 2003 to 25th April 2004; and phase 4 from 26th April 2004 to 30th April 2006 . Patients were swabbed for MRSA carriage on admission and every Monday morning . Swabs were taken from nose , axillae and perineum until 1st November 2004 , when additional rectal and throat samples were included ( a change associated with an approximate 30% increase in the proportion of patients identified as carriers on admission to ICU ) [47] . Clinical samples were collected when infection was suspected . S . aureus colonies were identified using a combination of catalase positivity , Staphaurex ( Remel Europe Ltd . , Dartford , England ) and/or salt mannite positivity with confirmation by a tube coagulase test . Methicillin resistance was determined by disc testing . Screen samples were identified using a selective mannitol broth technique [47] . TW MRSA was defined initially by its distinctive and extensive antimicrobial resistance pattern , sequence typing and microarray analysis [12] . More extensive typing of available admission and acquisition isolates has shown all antimicrobial resistance patterns defined TW isolates to belong to CC8/239 and non-TW isolates to be ST22 and ST36 [15] . When TW and non-TW MRSA isolates were recovered from the same patient , only the first type recovered was considered . This was , however , rare: two patients had both types recovered from pooled screening sites; nine had both types from sputum; and seven had both types from wounds . Thirteen patients had both types recovered from different sites . Further details of patient characteristics , interventions , swabbing sites and microbiological procedures have been described elsewhere [12] , [15] . We analyse the data using two separate approaches which we refer to as method 1 and method 2 . In both analyses we define a new MRSA acquisition to have occurred if a patient has a negative admission screening swab , a subsequent MRSA positive screen or clinical sample while in the ICU and more than 48 hours after being admitted to the ward , and no prior MRSA positive isolate in the 90 days preceding ICU admission . Patients with any MRSA positive samples taken within 48 hours of admission are assumed to be positive on admission ( MRSA importations ) . A patient who is believed to be neither colonized nor infected on a given day is assumed to be susceptible to becoming colonized or infected by either MRSA type ( see supplementary material for further details ) . In the first approach ( method 1 ) , which probabilistically reconstructs the epidemic tree , we assume that the acquisition occurred one or more days before the first positive screening swab . In the second approach ( method 2 ) we assume a new acquisition to have occurred on day if a patient has his or her first MRSA positive swab on day , following a negative MRSA admission screening swab during the same ward admission . We also assume i ) that once MRSA positive , a patient remains so until ward discharge ( hence no information from swab results after the first positive is used ) , and ii ) MRSA-positive patients only become potential sources for transmission to other patients after their first positive swab , unless they are assumed to be positive on admission , in which case they are potential sources from their date of admission . For patients readmitted to one of the wards following ward discharge , we apply the same criteria that we use for first time admission to determine admission colonisation status . We use a time unit of one day , and take dates of admission and discharge to represent the first and last whole days of a patient admission . | Different strains of hospital pathogens may differ in their ability to spread between patients and respond differently to control measures . Attempts to quantify such between-strain variation are lacking in high prevalence settings . We analysed data from concurrent outbreaks with different MRSA strains in two adult intensive care units . MRSA is usually carried by patients asymptomatically , and most of our data came from routine screening swabs used to detect such carriage . We divided strains into two groups: common United Kingdom strains and strains from a type often found in Southeast Asia . We developed a new method to estimate how transmission changes over time and compared results with those from an adaptation of a previously described approach . An advantage of the new method is that it makes weaker assumptions about the process generating the data . The methods gave broadly similar results: the introduction of daily antiseptic bodywashes for all patients was the only intervention associated with a substantial fall in transmission , but this intervention was less effective for the Asian strain . This work should be useful for assessing the between-strain variation in the transmission of other hospital pathogens , and for assessing the impact of interventions on patient-to-patient transmission . | [
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"epidemiol... | 2012 | Quantifying Type-Specific Reproduction Numbers for Nosocomial Pathogens: Evidence for Heightened Transmission of an Asian Sequence Type 239 MRSA Clone |
Several viruses have been found to encode a deubiquitinating protease ( DUB ) . These viral DUBs are proposed to play a role in regulating innate immune or inflammatory signaling . In human cytomegalovirus ( HCMV ) , the largest tegument protein encoded by UL48 contains DUB activity , but its cellular targets are not known . Here , we show that UL48 and UL45 , an HCMV-encoded inactive homolog of cellular ribonucleotide reductase ( RNR ) large subunit ( R1 ) , target receptor-interacting protein kinase 1 ( RIP1 ) to inhibit NF-κB signaling . Transfection assays showed that UL48 and UL45 , which binds to UL48 , interact with RIP1 and that UL48 DUB activity and UL45 cooperatively suppress RIP1-mediated NF-κB activation . The growth of UL45-null mutant virus was slightly impaired with showing reduced accumulation of viral late proteins . Analysis of a recombinant virus expressing HA-UL45 showed that UL45 interacts with both UL48 and RIP1 during virus infection . Infection with the mutant viruses also revealed that UL48 DUB activity and UL45 inhibit TNFα-induced NF-κB activation at late times of infection . UL48 cleaved both K48- and K63-linked polyubiquitin chains of RIP1 . Although UL45 alone did not affect RIP1 ubiquitination , it could enhance the UL48 activity to cleave RIP1 polyubiquitin chains . Consistently , UL45-null virus infection showed higher ubiquitination level of endogenous RIP1 than HA-UL45 virus infection at late times . Moreover , UL45 promoted the UL48-RIP1 interaction and re-localization of RIP1 to the UL48-containing virion assembly complex . The mouse cytomegalovirus ( MCMV ) -encoded DUB , M48 , interacted with mouse RIP1 and M45 , an MCMV homolog of UL45 . Collectively , our data demonstrate that cytomegalovirus-encoded DUB and inactive R1 homolog target RIP1 and cooperatively inhibit RIP1-mediated NF-κB signaling at the late stages of HCMV infection .
Human cytomegalovirus ( HCMV ) , which belongs to the β-herpesvirus subfamily , typically causes asymptomatic infections in immunocompetent individuals . HCMV is ubiquitously but latently distributed throughout the world . However , infection of pregnant women often causes congenital infection , and reactivation from latent infection in immunocompromised individuals can lead to life-threatening complications [1] . HCMV is a large , enveloped , double-stranded DNA virus and its 235 kb genome encodes for at least 165 proteins [2] . A structural feature unique to herpesviruses is the presence of a protein layer , called the tegument , between the capsid and the envelope . Upon initial fusion of the viral envelope with the host cell plasma membrane , many of these tegument proteins are delivered into the cytoplasm and the nucleus , where they perform diverse functions including activation of viral gene transcription and antagonization of host intrinsic and innate immunity . Viral tegument proteins are also thought to be involved in capsid transport and virion egress [1 , 3 , 4] . The herpesvirus deubiquitinase ( DUB ) was first discovered as an N-terminal fragment of the 336 kDa UL36 tegument protein ( also known as VP1/2 ) of herpes simplex virus-1 ( HSV-1 ) [5] . This DUB domain is conserved in the UL36 equivalents of other herpesviruses [6] . Interestingly , the herpesvirus DUBs bear no structural homology to known eukaryotic DUBs , although the key amino acid residues in the active site are highly conserved [7] . The herpesvirus DUBs appear to play a key role in regulating innate immune and inflammatory signaling . HSV-1 UL36 and Kaposi’s sarcoma-associated herpesvirus ( KSHV ) ORF64 interact with and deubiquitinate TRAF3 and RIG-I , respectively , inhibiting IRF3 activation [8 , 9] . Epstein-Barr virus ( EBV ) BPLF1 targets TRAF6 and inhibits its ubiquitination , downregulating NF-κB activation during lytic infection [10] . BPLF1 is also known to deubiquitinate PCNA and inhibit trans-lesion synthesis at DNA damage sites [11] . In HCMV , the UL48-encoded tegument protein is a homolog of HSV-1 UL36 and possesses DUB activity . The UL48 DUB was identified using a suicide substrate probe specific for ubiquitin-binding cysteine proteases in virus-infected cells [12] . This DUB , mapped to the first ~350 amino acids of the N-terminal region of pUL48 , contains both ubiquitin-specific carboxyl-terminal hydrolase activity and isopeptidase activity that cleaves ubiquitin K11 , K48 , and K64 linkages [13 , 14] . Mutations in active site residues ( C24 and H162 ) completely abolish DUB activity , and the virus containing the UL48 ( C24S ) gene shows moderately reduced growth in culture cells , demonstrating that the DUB activity of HCMV can influence viral replication [14] . Like the UL36 DUBs of HSV-1 and Pseudorabies virus ( PRV ) [15 , 16] , the UL48 DUB has autocatalytic activity that regulates its own stability [17] . The N-terminal DUB-containing region from UL48 is required for virion stability and efficient virus entry , although the associated DUB activity appears not to be critical [17] . Despite accumulating evidence that DUB activity and the DUB-containing region of UL48 are required for efficient viral growth , whether the UL48 DUB , like the equivalents of α and γ-herpesviruses , plays a role in regulation of innate immune or inflammatory signaling is not known yet . UL48 contains the nuclear localization signal ( NLS ) that is essential for viral growth [17 , 18] , suggesting a critical role of UL48 in the nucleus or in routing of the capsid to the nuclear pore as seen in infection with HSV-1 UL36 mutant virus [19 , 20] . The nuclear factor-kappa B ( NF-κB ) transcription factors are critical regulators of host cell’s early responses to viral infection [21] . HCMV infection can induce NF-κB’s transcriptional activity , which subsequently drives expression of a number of different proinflammatory cytokines and chemokines . There is increasing evidence that HCMV can inhibit NF-κB signaling during lytic infection . This negative regulation may be necessary to suppress excessive immune responses that are detrimental to viral infection . Although viral downregulation of NF-κB signaling in the early stages of infection has been confirmed , it is believed that late virus functions are critical to suppress NF-κB signaling . UL26 is an early viral protein that initially localizes to the nucleus , but becomes cytoplasmic as the infection progresses and eventually localizes to virion assembly sites [22 , 23] . UL26 is known to antagonize NF-κB activation induced by TNFα by attenuating IKK phosphorylation and subsequent IκBα degradation [24] . The HCMV UL45 gene encodes an inactive homolog of cellular ribonucleotide reductase ( RNR ) large subunit ( R1 ) , which is contained in the tegument . The function of UL45 still remains largely unknown . Analysis of the VR1814-derived bacmid ( FIX-BAC ) reconstituted recombinant virus showed that the UL45-null mutant virus normally grows in endothelial cells [25] but is defective in viral particle accumulation at low multiplicities of infection ( MOI ) and in spreading in fibroblasts [26] . The growth defect of UL45-null virus did not appear to result from a reduced dNTP supply [26] . In mouse CMV ( MCMV ) , M45 , a homolog of HCMV UL45 , interacts with mouse receptor-interacting protein kinase 1 ( mRIP1 ) and inhibits mRIP1-mediated signaling pathways , including activation of NF-κB after stimulation of TNFR1 and TLR3 [27] . M45 also blocks mRIP1-independent NF-κB activation and cytokine production after stimulation of IL-1R and TLRs and binds to and induces lysosomal degradation of NEMO by targeting it to autophagosomes [28] . Furthermore , M45 is also known to inhibit cytosolic DNA sensor DAI-mediated NF-κB activation through RIP homotypic interaction motif ( RHIM ) -dependent interaction [29] . Meanwhile , M45 acts as a suppressor of virus-induced or TNFα-induced cell death [30 , 31] . M45 inhibits a caspase-independent form of programmed necrosis ( necroptosis ) , which depends on the adaptor kinase RIP3 and DAI [32 , 33] . It is not known whether UL45 has activity similar to M45 in regulating the host’s immune responses . In this study , we identified RIP1 as a cellular target of HCMV-encoded deubiquitinase UL48 . We provide evidence that RIP1 is also targeted by UL45 , an HCMV-encoded inactive homolog of cellular RNR R1 , and that UL48 and UL45 cooperatively suppress RIP1-mediated NF-κB signaling at the late stages of viral infection .
Since RIP1 is a critical mediator of NF-κB signaling and K63-linked polyubiquitination of RIP1 is an important process involved in this signaling , we tested whether the HCMV UL48 deubiquitinase targets RIP1 . When CoIP assays were performed in co-transfected 293T cells , UL48 was found to interact with RIP1 ( Fig 1A and 1B ) . RIP1 has three domains: the N-terminal kinase domain ( KD ) , the central intermediate domain ( ID ) , and the C-terminal death domain ( DD ) ( Fig 1C ) . Three RIP1 deletion constructs ( ΔKD , ΔID , and ΔDD ) were employed for similar CoIP assays to determine the UL48-binding region of RIP1 . The results revealed that both the intermediate and death domains of RIP1 are required for efficient RIP1 binding ( Fig 1D and 1E ) . The intermediate domain contains the TNFα-induced Lys63 ( K63 ) -linked polyubiquitination site ( K377 ) [34] and the RHIM . To further investigate the effect of the RHIM and ubiquitination of RIP1 on the pUL48-RIP1 interaction , RHIM-deleted ( ΔRHIM ) and K377R mutants were also used in CoIP assays . The results showed that pUL48 still interacts with these ΔRHIM and K377R mutant RIP1 proteins ( Fig 1F and 1G ) . Taken together , these results demonstrate that UL48 interacts with RIP1 through the intermediate and death domains , but this interaction does not require presence of the RHIM and polyubiquitination at K377 of RIP1 . Luciferase reporter assays were performed to investigate the effect of UL48 on RIP1-mediated NF-κB activation . UL48 expression effectively inhibited RIP1-mediated NF-κB activation in 293T cells ( Fig 2A ) . HCMV permissive HF cells were more sensitive to apoptotic cell death induced by RIP1 overexpression than other cell types . Therefore , similar reporter assays were performed in HF cells with co-transfection of CrmA , an inhibitor of caspases 1 and 8 . The results showed that the NF-κB activity induced by RIP1 expression was effectively inhibited by UL48 in HF cells ( Fig 2B ) . TNFα treatment of HF cells moderately increased NF-κB activity , but this elevation was also inhibited by UL48 ( Fig 2C ) . We next investigated whether the DUB catalytic activity of UL48 is required for this suppression using the C24S mutant in which the Cys24 active site was replaced with Ser [14] . The results of reporter assays showed that the inhibitory effect of UL48 ( C24S ) on RIP1-mediated and TNFα-induced NF-κB activation was reduced to 30% to 60% that of wild-type UL48 ( Fig 2D to 2F ) . The expression levels of wild-type and C24S mutant UL48 proteins were comparable and the RIP1 modification ( probably ubiquitination ) was reduced by wild-type UL48 but not by C24S mutant in transfected cells ( S1A Fig ) . These results suggest that the DUB activity of UL48 is largely required for inhibition of RIP1-mediated NF-κB activation . UL48 was first shown to interact with UL45 , an inactive homolog of cellular RNR R1 , in yeast two-hybrid interaction assays [35] . We also observed this interaction in CoIP assays ( Fig 3A and 3B ) [17] . Given that UL48 interacted with both RIP1 and UL45 and that M45 , an MCMV-encoded homolog of UL45 , interacted with mRIP1 [27 , 31] , the potential for interaction of UL45 with RIP1 was also tested . The results of CoIP assays revealed that UL45 interacts with RIP1 ( Fig 3C and 3D ) . CoIP assays using the mutant RIP1 constructs revealed that UL45 interacts with RIP1 through the intermediate domain ( Fig 3E and 3F ) . The RHIM was reported to mediate the interaction between M45 and mRIP1 [31] . However , the UL45-RIP1 interaction did not seem to require the RHIM because UL45 did not contain an apparent RHIM , and a RHIM-deleted RIP1mutant also effectively bound to UL45 ( Fig 3G ) . Furthermore , the K377R mutant RIP1 still interacted with UL45 , demonstrating that the UL45-RIP1 interaction does not require polyubiquitination at K377 of RIP1 ( Fig 3H ) . When UL45 , UL48 , and RIP1 were co-expressed , both UL48 and RIP1 were co-precipitated when UL45 was immunoprecipitated ( Fig 3I ) . Similarly , UL45 and RIP1 were co-precipitated with UL48 ( Fig 3J ) , and UL48 and UL45 were co-precipitated with RIP1 ( Fig 3K ) . These results suggest the possibility that both UL48 and UL45 simultaneously target RIP1 and that they may form a complex . We investigated whether , like UL48 , UL45 inhibits RIP1-mediated NF-κB activation . In reporter assays performed in 293T and HF cells , NF-κB activity induced by RIP1 expression was effectively inhibited by UL45 ( Fig 4A and 4B ) . UL45 also inhibited NF-κB activation induced by TNFα treatment in HF cells ( Fig 4C ) . We further investigated whether UL48 and UL45 cooperatively affect RIP1-mediated NF-κB activation . In reporter assays , co-expression of UL48 and UL45 more effectively inhibited NF-κB activation by RIP1 than UL45 alone , and this cooperative effect of UL48 and UL45 was largely diminished when the UL48 ( C24S ) mutant was used ( Fig 4D and 4E; S1B Fig ) . This inhibitory effect of UL48 and UL45 was also observed in TNFα-induced NF-κB activation in HF cells ( Fig 4F ) . Collectively , our data demonstrate that UL48 and UL45 may cooperate to inhibit RIP1-mediated NF-κB activation , and that the deubiquitinating activity of UL48 contributes to this repression . To address the significance of the RIP1 targeting by UL48 and UL45 during HCMV infection , recombinant Toledo viruses that do not express UL45 ( UL45-null ) or express HA-tagged UL45 ( HA-UL45 ) were produced using bacmid mutagenesis ( S2 Fig ) . The growth curves of wild-type , UL45-null , and HA-UL45 viruses were compared in HF cells at an MOI of 0 . 1 or 2 . The viruses released in the culture supernatants and associated with the cells were collected and pooled at various time points after infection , and titers of the infectious progeny virions were determined by infectious center assays . At an MOI of 0 . 1 , the peak titers of UL45-null virus on day 7 was seven-fold lower than those of wild-type and HA-UL45 viruses ( Fig 5A , left ) , while , at an MOI of 2 , UL45-null virus produced about three-fold fewer progeny virions on day 5 than wild-type and HA-UL45 viruses ( Fig 5A , right ) . The results of immunoblotting assays showed that the reduced growth of the UL45-null virus at low MOI correlated with the low level accumulation of viral late proteins , such as the late forms encoded by UL44 and the pp28 ( encoded by UL99 ) true late protein ( Fig 5B , left ) , while this difference was reduced at an MOI of 2 ( Fig 5B , right ) . We also observed the reduced accumulation of the SUMO-modified forms of IE2 in UL45-null virus infection , which was often observed in mutant viruses with a moderate growth defect [14 , 36] . Collectively , the analysis of UL45-null and HA-UL45 virus growth patterns suggests that UL45 contributes to viral growth in cultured cells at low MOI by affecting the late stages of the virus life cycle and that HA-UL45 virus has a similar growth pattern as wild-type virus . Since each of UL48 , UL45 , and RIP1 was co-precipitated with the other two proteins in co-transfected cells , the possible formation of a complex containing these three proteins during viral infection was investigated using HA-UL45 virus . When HF cells were infected with HA-UL45 virus , immunoprecipitation of HA-UL45 co-precipitated both UL48 and RIP1 ( Fig 6A ) and immunoprecipitation of UL48 co-precipitated both HA-UL45 and RIP1 ( Fig 6B ) . In a control CoIP experiment , however , immunoprecipitation of abundant pp65 viral protein did not co-precipitated UL48 and RIP1 , although it unexpectedly pull-downed HA-UL45 ( Fig 6C ) . These results suggest that RIP1 may exist in a protein complex containing UL48 and UL45 . Furthermore , the result of gel filtration chromatography demonstrated that in HA-UL45 virus-infected cells , the RIP1 fractions shifted to higher molecular mass fractions compared to those in mock-infected cells , and that these high molecular mass fractions ( >400 kDa ) contained HA-UL45 and UL48 ( Fig 6D ) . These results suggest that both UL48 and UL45 interact with RIP1 , and a complex containing UL48 , UL45 , and RIP1 may be produced during HCMV infection . We also investigated the localization patterns of UL48 , HA-UL45 , and RIP1 in cells infected with HA-UL45 virus . Human γ-globulin was used as a blocking agent for HCMV Fc receptors , which are recognized by rabbit antibodies in virus-infected cells at the late stages of infection ( S3 Fig ) . When triple-label IFA was performed to visualize UL48 , HA-UL45 , and pp28 at 96 h after infection , UL48 and HA-UL45 were largely colocalized with pp28 in the cytoplasmic compartments , while a small fraction was also detected in the nucleus as punctate forms ( Fig 7A ) . Since pp28 is localized in the cytoplasmic virion assembly complex ( cVAC ) [37] , the association of UL48 and HA-UL45 with the cVAC was further investigated by co-staining GM130 , a Golgi-associated cellular marker for the cVAC [38] . The results demonstrated that UL48 and HA-UL45 are largely colocalized with GM130 in the cVAC ( Fig 7B ) . We next performed similar triple-label IFA to visualize UL48 , HA-UL45 , and RIP1 in virus-infected cells . In mock-infected cells , endogenous RIP1 was detected at very low levels throughout the cells as a diffuse form . However , in virus-infected cells , RIP1 was slightly stabilized and accumulated in the UL48 and HA-UL45-containing cVAC ( Fig 7C ) . In two-color merge images , RIP1 appeared to be more effectively colocalized with HA-UL45 than UL48 ( S4 Fig ) . These IFA data indicate that RIP1 is colocalized with UL48 and UL45 in the cVAC , supporting that UL48 and UL45 form a complex with RIP1 during viral infection . RIP1 plays a key role in NF-κB signaling after TNFα treatment and HCMV inhibits NF-κB activation in the late stages of infection . Therefore , we investigated the effects of UL48 and UL45 on TNFα-induced NF-κB activation in the late stages of infection . We previously produced the DUB-defective UL48 ( C24S ) mutant virus using the UL/b’ region-deleted Towne strain [14] , while the UL45-null mutant in this study was produced in the Toledo strain . As a preliminary experiment , we compared the levels of NF-κB activation after TNFα treatment in cells infected with HCMV Toledo and Towne for different time periods . We found that phosphorylation of the NF-κB p65 subunit at serine residue 536 peaked 48 h after infection and began to decrease from 72 h in both Toledo and Towne virus-infected cells , while the phosphorylated p65 levels were higher during Toledo virus infection than during Towne virus infection ( Fig 8A ) . The latter is consistent with the earlier reports demonstrating that the UL/b’ region in the Toledo virus and other clinical isolates sensitizes cells to TNFα signaling by upregulating cell surface expression of TNFR [39 , 40] . Since UL45 and UL48 are expressed with early-late kinetics , their effects on TNFα-induced NF-κB activation were assessed 72 h after infection with wild-type or mutant viruses . In mock-infected cells , TNFα treatment for 5 or 15 min increased phosphorylation of p65 and IKKα/β . In Toledo virus infection , HA-UL45 virus less effectively upregulated TNFα-induced p65 and IKKα/β phosphorylation compared to UL45-null virus , demonstrating that UL45 expression inhibits TNFα-induced NF-κB activation ( Fig 8B ) . In the case of Towne virus infection , wild-type virus resulted in less TNFα-induced p65 and IKKα/β phosphorylation compared to UL48 ( C24S ) virus ( Fig 8C ) . When we introduced the UL48 ( C24S ) mutation in HA-UL45 Toledo virus , similar higher levels of p65 and IKKα/β phosphorylation were observed in UL48 ( C24S ) -containing HA-UL45 virus compared to HA-UL45 virus ( S5A and S5B Fig ) . In the experiments using UL48 ( C24S ) mutant viruses , the levels of UL48 ( C24S ) were lower than those of wild-type UL48 . Therefore , it is not clear whether increased p65 and IKKα/β phosphorylation in mutant virus infection was due to the lack of DUB activity or due to the reduced protein level . However , since UL48 ( C24S ) mutant protein that was expressed at a comparable level to wild-type protein largely lost the NF-κB repressing activity in reporter assays ( see Figs 2D and S1A; Figs 4D and S1B ) , it is likely that the lack of UL48 DUB activity rather than the reduced level of UL48 ( C24S ) largely contributed to the increase of TNFα-induced NF-κB activation . Supporting this idea , both HA-UL45 and RIP1 were still localized in the cVAC in HA-UL45/UL48 ( C24S ) virus-infected cells ( S5C Fig ) . Collectively , our analysis of UL45 and UL48 mutant viruses demonstrate that both UL45 and UL48 are involved in inhibition of TNFα-induced NF-κB activation in the late stages of infection . The effect of UL45 and UL48 on TNFα-induced NF-κB activation was further investigated in HF cells co-transfected with UL45 and UL48 expression plasmids ( Fig 9A ) . TNFα treatment increased phosphorylation of p65 in cells transfected with empty vector . However , TNFα-induced p65 phosphorylation was significantly impaired in cells transfected with UL45 or UL48 . UL26 , which was previously reported to antagonize NF-κB activation [24] , also inhibited TNFα-induced p65 phosphorylation , but UL45 and UL48 did so more effectively , suggesting that UL45 and UL48 are potent inhibitors of NF-κB signaling . To investigate the molecular mechanisms by which UL48 and UL45 inhibit TNFα-induced NF-κB activation , we assessed the effect of UL48 and UL45 expression on the levels of K63-linked polyubiquitination of RIP1 . Co-transfection/ubiquitination assays were performed using plasmids expressing ubiquitin ( K63 only ) or ubiquitin ( K48 only ) in which all lysine ( K ) residues except K63 or K48 within ubiquitin are mutated to arginine to allow for the formation of only K63-linked or K48-linked ubiquitin chains . We found that UL48 inhibited RIP1 polyubiquitination through the K63 or K48 linkages , while UL45 did not affect RIP1 polyubiquitination ( Fig 9B ) . When UL48 and UL45 were co-expressed , UL45 increased the activity of UL48 to cleave K63-linked polyubiquitin chains of RIP1 ( Fig 9C ) . To address the role of UL45 in RIP1 ubiquitination in the context of viral infection , HF cells were infected with UL45-null or HA-UL45 virus and ubiquitination levels of endogenous RIP1 were examined . The results showed that the ubiquitination level of RIP1 was higher in UL45-null virus infection than in HA-UL45 virus infection ( Fig 9D ) . These results demonstrate that UL48 inhibits TNFα-induced NF-κB activation through deubiquitination of RIP1 and that UL45 promotes this UL48 activity . We also found using CoIP assays that the interaction of UL48 with RIP1 in co-transfected cells was increased when UL45 was co-expressed , demonstrating that UL45 promotes the binding of UL48 with RIP1 ( Fig 10A ) . Furthermore , when HF cells were infected with HA-UL45 virus , RIP1 was re-localized to the cVAC in all cells showing HA-UL45 in the cVAC; however , when cells were infected with UL45-null virus , this RIP1 re-localization to the cVAC was not observed ( Fig 10B ) . Taken together , these results demonstrate that UL48 inhibits TNFα-induced NF-κB activation through deubiquitination of RIP1 and that UL45 promotes the binding of UL48 with RIP1 and re-localization of RIP1 to the UL48-containing cVAC . Since the DUB-containing tegument proteins and R1 homologs are conserved in other herpesviruses , we further investigated whether RIP1 is targeted by DUB and R1 encoded by other human α- and γ-herpesviruses . The results of CoIP assays using HSV-1 proteins showed that UL36 ( DUB ) did not interact with RIP1 , while UL39 ( R1 ) bound to RIP1 as previously reported [41] ( S6A and S6B Fig ) . In similar assays using KSHV proteins , neither ORF64 ( DUB ) nor ORF61 ( R1 ) interacted with RIP1 ( S6D and S6E Fig ) . Furthermore , the interaction between DUB and R1was not observed with HSV-1 and KSHV proteins in CoIP assays ( S6C and S6F Fig ) . These results demonstrate that targeting RIP1 by viral DUB occurs in HCMV but not in HSV-1 and KSHV , whereas targeting RIP1 by viral R1 homolog is shared by both HCMV and HSV-1 . We further examined whether the RIP1 targeting by viral DUB and R1 homolog is conserved in β-herpesviruses . In CoIP assays with MCMV proteins , both M48 ( DUB ) and M45 ( R1 homolog ) interacted with mRIP1 ( S7A and S7B Fig ) . Furthermore , M48 interacted with M45 ( S7C Fig ) , suggesting that the activity of viral DUB and R1 homolog to target RIP1 and interact with each other may be preserved in β-herpesviruses ( S7D Fig ) .
In this study , we identified RIP1 as a cellular substrate of the HCMV-encoded UL48 DUB . We showed that UL48 interacts with RIP1 , cleaves its K48- and K63-linked polyubiquitin chains , and inhibits NF-κB activation induced by RIP1 overexpression or TNFα treatment . The inhibitory action of UL48 on NF-κB signaling largely required its DUB activity , although the catalytically inactive mutant protein still resulted in modest inhibition of NF-κB activation . Other herpesvirus-encoded DUBs , such as HSV-1 UL36 , KSHV ORF64 , and EBV BPLF1 , have been shown to target TRAF3 , RIG-I , and TRAF6 , respectively [8–10] . Therefore , our findings support a notion that the herpesvirus-encoded DUBs target key regulators of host innate immune and inflammatory signaling . We found that MCMV M48 interacted with mRIP1 , while HSV-1 UL36 and KSHV ORF64 did not interact with RIP1 , and that the interaction between viral DUB and R1 homolog was observed with MCMV proteins but not with HSV-1 and KSHV proteins . These suggest that RIP1 targeting by viral DUB and the interaction between DUB and R1 homolog may be conserved in β-herpesviruses . We also showed that UL45 , an inactive viral homolog of cellular RNR R1 , interacts with RIP1 and inhibits NF-κB activation mediated by RIP1 overexpression or TNFα treatment . In MCMV infection , M45 , a homolog of UL45 , was shown to interact with mRIP1 to inhibit mRIP1-mediated NF-κB activation or cell death . M45 interacted with mRIP1 in a RHIM domain-independent manner and inhibited TNFα-induced activation of NF-kB , p38 MAPK , and caspase-independent cell death [27] . A role of the RHIM-dependent interaction of M45 with RIP1 in suppressing cell death was also reported [31] . Furthermore , the M45 required RHIM-dependent interaction with DAI and RIP3 in order to block DAI-induced NF-κB activation [29] and RIP3-mediated necroptosis [32 , 33] . Our data demonstrate that HCMV UL45 interacts with RIP1 independent of the RHIM since UL45 , which does not contain an apparent RHIM , bound to a RHIM-deleted RIP1 mutant . Interestingly , unlike the R1 homologs encoded by α- and γ-herpesviruses , HCMV UL45 or other β-herpesvirus R1 homologs are catalytically inactive due to the absence of most residues known to have a catalytic role [42] . Why β-herpesviruses encode inactive R1 homologs is not fully understood . However , our findings , together with the above-mentioned previous works with MCMV M45 , point to the possibility that the inactive R1 homologs of β-herpesviruses have been preserved to regulate RIP1-associated activities . M45 was also reported to target NEMO and induce its lysosomal degradation [28] . Whether UL45 also targets NEMO needs to be further investigated . HCMV has been shown to suppress robust activation of NF-κB signaling during the late stages of infection and viral early and late proteins appear to be involved in this regulation [43–45] . Recently , UL26 , an early protein , was found to inhibit NF-κB activation by attenuating IKK phosphorylation in TNFα-treated cells [24] . The results of the present study reveal that UL48 and UL45 , expressed at delayed-early or late times after infection , are potent suppressors of NF-κB activation in the late stages of infection . In particular , the results of our transfection assays demonstrated that UL48 and UL45 more effectively suppress TNFα-induced NF-κB activation than UL26 . Therefore , at least three viral proteins appear to downregulate NF-κB signaling in the late stages of infection . We observed that infection with the Toledo virus containing the UL/b’ region led to greater NF-κB activation after TNFα treatment than infection with the Towne virus lacking this region . It has been shown that the UL138 gene in the UL/b’ region is responsible for this greater sensitivity of Toledo by upregulating cell surface expression of TNFR and this UL138 activity may contribute to TNFα-induced reactivation of virus from latently infected cells [39 , 40] . Notably , our results demonstrated that the lack of UL45 expression or inactivation of UL48 DUB still increased TNFα-induced NF-κB signaling in Toledo virus infection . HCMV appears to differentially regulate NF-κB activation during the early and late phases of infection and between lytic and latent infection . Therefore , it is not surprising at all that HCMV has been evolved to have several genes that positively or negatively regulate NF-κB activation [46] . We demonstrated that , although UL48 and UL45 are separately capable of targeting RIP1 and inhibiting RIP1-mediated NF-κB signaling , they have a cooperative effect on the regulation of NF-κB signaling . Although UL48 was previously shown to interact with UL45 in yeast two-hybrid interaction assays and co-transfection/CoIP assays [17 , 35] , we for the first time provide evidence that this interaction occurs in virus-infected cells . In co-transfection assays , UL45 increased the activity of UL48 to cleave K63-linked polyubiquitination of RIP1 and interact with RIP1 . More importantly , the ubiquitination level of endogenous RIP1 was higher in the late stages of infection during UL45-null virus infection compared to infection with HA-UL45 virus . One possible mechanism for the cooperative activity of UL45 is that UL45 binding to RIP1 alters RIP1 conformation or localization , converting it to a better substrate for UL48 DUB activity . Indeed , we observed that UL45 promoted the RIP1 binging by UL48 in co-transfected cells and re-localization of RIP1 to the UL48-containing cVAC in virus-infected cells . It would also be intriguing to test whether UL45 targets other UL48 substrates and promotes UL48’s DUB activity toward them . Accumulation of deubiquitinated RIP1 in cells is a key step for the switch from pro-survival to pro-apoptotic function of RIP1 . Our observation that UL48 and UL45 cooperatively increase the level of deubiquitinated RIP1 raises a question whether the expression of UL48 and UL45 sensitizes cells to apoptosis . We investigated this possibility by comparing anti-Fas/cycloheximide-induced cleavage of poly ( ADP-ribose ) polymerase ( PARP ) between wild-type and UL48 ( C24S ) mutant virus-infected HF cells . However , we did not observe higher level of PARP cleavage by wild type virus than mutant virus . We reason this to the expression of strong viral inhibitor of caspase-8-induced apoptosis ( vICA ) [47 , 48] that works at downstream of RIP1 . Both UL48 and UL45 are viral tegument proteins that are present within virions and delivered into host cells upon virus entry . Therefore , these proteins may also regulate NF-κB signaling immediately after virus entry . The interaction of these proteins may also play a role in the nuclear egress of the capsid or virion maturation in the cytoplasm , but this awaits further investigation .
Primary human foreskin fibroblast ( HF ) ( American Type Culture Collection; ATCC PCS-201-010 ) and human embryonic kidney ( HEK ) 293T cells ( ATCC ) were grown in Dulbecco’s modified Eagle’s medium ( DMEM ) supplemented with 10% fetal bovine serum , penicillin ( 100 U/ml ) , and streptomycin ( 100 μg/ml ) in a 5% CO2 humidified incubator at 37°C . Stocks of the Towne wild-type and UL48 ( C24S ) mutant viruses were prepared as described previously [14] . To produce UV-inactivated HCMV ( UV-HCMV ) , the virus stock was irradiated with UV light three times at 0 . 72 J/cm2 using a CL-1000 cross-linker ( UVP ) . For infection experiments , HF cells were infected with virus at specified multiplicities of infection ( MOI ) . At the indicated times , the growth medium was collected and combined with lysates prepared from the cell layer by freezing and thawing three times , clarified by centrifugation , and stored at -70°C until assayed for infectivity . The wild-type UL45 DNA ( Towne strain ) was cloned into pENTR vectors ( Invitrogen ) . Plasmid expressing Myc-tagged UL45 was produced by transferring the DNA to a pCS3-MT ( with a 6Myc tag ) -based destination vector using LR Clonase ( Invitrogen ) . Plasmids expressing Myc-tagged UL48 ( wild-type or C24S mutant ) were described previously [14] . Plasmid expressing hemagglutinin ( HA ) -tagged UL48 was generated on a pSG5 background using Gateway technology as previously described [17] . Plasmid for flag-tagged RIP1 was provided by Jaehwan Song ( Yonsei University ) . The wild-type RIP1 DNA was cloned into pENTR vectors ( Invitrogen ) . Plasmid for HA-tagged RIP1 was produced by transferring the DNA to a pSG5-based destination vector using LR Clonase ( Invitrogen ) . Plasmids encoding HA-tagged ΔKD , ΔID , ΔDD , ΔRHIM , and K377R mutant RIP1were generated by PCR in the same background . Plasmids expressing HSV-1 UL36 ( pUL36-EGFP-N2 ) and KSHV ORF64 ( pcDEF-Flag-ORF64 ) were provided by Prashant Desai ( Johns Hopkins University ) and Kyung-Soo Inn ( Kyung Hee University ) , respectively . The HSV-1 UL39 and KSHV ORF61 genes were PCR cloned from viral DNAs into pENTR vector and plasmids expressing myc-tagged proteins were produced on a pCS3-MT-based destination vector using LR Clonase . pENTR vectors containing mouse RIP1 and MCMV M45 were produced by PCR from pCMV2-Flag-mRIP1 and pcDNA-M45-HA , respectively , that were provided by Wolfram Brune ( Leibniz Institute for Experimental Virology ) . pENTR vector containing MCMV M48 was also produced by PCR from pEGFP-N1-M48 that was gifted by Jason W . Upton ( University of Texas at Austin ) . pSG5 plasmids expressing HA-mRIP1 and HA-M45 and pCS3-MT plasmids expressing Myc-M45 and Myc-M48 were produced by transferring the DNAs into appropriate destination vectors using LR clonase . Plasmids for HA-Ub and CrmA were provided by Hongtae Kim ( Sungkyunkwan University ) and Kyeong Sook Choi ( Ajou University ) , respectively . Mouse monoclonal antibody ( MAb ) 810R , which detects epitopes present in both IE1 and IE2 , was obtained from Millipore . Chicken egg yolk anti-UL45 IgY antibodies were gifted from Andrea Gallina ( University of Milano ) . Rabbit polyclonal antibody ( PAb ) raised against the synthetic peptide corresponding to UL48 residues 278 to 295 ( anti-UL48-N ) was provided by Wade Gibson ( Johns Hopkins University School of Medicine ) [14] . Mouse MAbs against p52 ( encoded by UL44 ) and pp28 ( UL99 ) were purchased from Advanced Biotechnologies . The anti-pp65 ( UL83 ) mouse MAb was purchased from Virusys . The anti-HA rat MAb ( 3F10 ) either conjugated with peroxidase or labeled with fluorescein isothiocyanate ( FITC ) , anti-HA mouse MAb ( 12CA5 ) , anti-HA rat MAb ( 3F10 ) , and anti-Myc mouse MAb ( 9E10 ) conjugated with peroxidase were purchased from Roche . The anti-flag mouse MAb M2 was obtained from Sigma . Mouse MAbs for RIP1 ( C-12 ) and p65 NF-κB subunit were purchased from Santa Cruz Biotechnology and Ab Frontier , respectively . The anti-phospho-p65 ( S536 ) and anti-phospho-IKKα/β rabbit MAbs were obtained from Cell Signaling Technology . Mouse MAbs for α-tubulin and β-actin were purchased from Sigma . Rabbit PAbs against NF-κB p50 and HDAC2 were purchased from Upstate and Zymed , respectively . Secondary antibodies such as FITC-labeled donkey anti-mouse IgG , Rhodamine Red-X-conjugated donkey anti-mouse IgG , Rhodamine Red-X-conjugated donkey anti-rabbit IgG , and Cy5-conjugated donkey anti-rat IgG were obtained from Jackson ImmunoResearch Laboratories , Inc . The HCMV ( Toledo strain ) bacterial artificial chromosome ( BAC ) ( Toledo-BAC ) clone was kindly provided by Hua Zhu ( Rutgers-New Jersey Medical School ) . The Toledo BAC clones encoding the UL45stop ( UL45-null ) and HA-tagged UL45 ( HA-UL45 ) genes were generated using a counter-selection BAC modification kit ( Gene Bridges ) . Briefly , the rpsL-neo cassette DNA was PCR-amplified using LMV1708/1731 primers containing homology arms consisting of 50 nucleotides upstream and downstream of the target region plus 24 nucleotides homologous to the rpsL-neo cassette . The amplified rpsL-neo fragments with homology arms were purified and introduced into E . coli DH10B containing the wild-type Toledo-BAC clone for recombination by electroporation using a Gene Pulser II ( Bio-Rad ) . The intermediate Toledo-BAC constructs containing the rpsL-neo cassette were selected on Luria broth ( LB ) plates containing kanamycin . Next , the UL45stop fragments for replacing the rpsL-neo cassette were amplified by PCR using LMV1732/1733 primers . The amplified fragments were recombined into the Toledo-BAC DNA containing the rpsL-neo cassette , and the UL45-null Toledo-BAC was selected on LB plates containing streptomycin . The HA-UL45 Toledo-BAC clone was also generated from the UL45-null Toledo-BAC by the same method using LMV1494/1495/1733 primers . LMV primers used for bacmid mutagenesis were as follows: LMV1708 , 5’-TCACTTTATTGAAATCTACCTGATTTCTTTGTTATTTTCCTCGTAAACTTGGCCTGGTGATGATGGCGGGATCG-3’; LMV1731 , 5’- GCCGTCGGGAGACGGCGACTCGGGACGCCAACTGACGACGCCGCCACCACTCAGAAGAACTCGTCAAGAAGGCG-3’; LMV1732 , 5’- TCACTTTATTGAAATCTACCTGATTTCTTTGTTATTTTCCTCGTAAACTTATGAATCCGGCTGACGCGGA-3’; LMV1733 , 5’-GCCGTCGGGAGACGGCGACTCGGGACGCCA-3’; LMV1494 , 5’-ATGTACCCATACGATGTTCCAGATTACGCTATGAATCCGGCTGACGCGGACGAGGAACAGCGGGTGTCCT-3’; and LMV1495 5’-TCACTTTATTGAAATCTACCTGATTTCTTTGTTATTTTCCTCGTAAACTTATGTACCCATACGATGTTCC-3’ . Electroporation was used to introduce the Toledo-BAC DNA into HF cells . For each reaction , HF cells ( 2 x 106 ) in 400 μl of resuspension buffer were mixed with 5 μg of Toledo-BAC DNA , 2 μg of plasmid pCMV71 encoding pp71 to enhance activation of the major immediate-early ( MIE ) promoter , and 1 μg of plasmid pEGFP-C1 to monitor electroporation efficiency . Electroporation was done at 1300 V for 30 ms using a Microporator MP-100 ( Digital Bio Technology ) , and the cells were transferred to T-25 plates . When the surviving cells reached confluence , cells were transferred at a dilution of 1:2 into new flasks . 293T cells were transfected via polyethylenimine ( PEI ) ( Sigma ) . A mixture of plasmid DNA and serum-free media was mixed with PEI . The volume of PEI used was based on a 3:1 ratio of PEI ( μg ) :total DNA ( μg ) . The mixture was kept at room temperature for 20 min and then added dropwise to cells . HF cells were transfected via OmicsFect ( Omics Bio ) or via electroporation . Electroporation was performed at 1300 V for 30 ms using a Microporator MP-100 ( Digital Bio ) . The transfection efficiency of HF cells using electroporation under these conditions was about 50 to 60% ( S1C Fig ) . Cell lysates were harvested and prepared by sonication in 1 ml CoIP buffer ( 50 mM Tris-Cl [pH 7 . 4] , 50 mM NaF , 5 mM sodium phosphate , 0 . 1% Triton X-100 , containing protease inhibitors [Sigma] ) using a Vibra cell microtip probe ( Sonics and Materials ) for 10 sec ( pulse on: l s , pulse off: 0 . 5 s ) . The clarified cell lysates were incubated for 16 h with the appropriate antibody at 4°C . A total of 30 μl of 50% slurry of protein A- and G-Sepharose ( Amersham ) was added . After incubation for 2 h at 4°C , the mixture were pelleted and washed several times with CoIP buffer . Each sample was analyzed by SDS-PAGE and immunoblotting with the appropriate antibody . The DNA-transfected cells or virus-infected cells were harvested with phosphate-buffered saline ( PBS ) and total cell extracts were prepared by boiling the cell pellets in sodium dodecyl sulfate ( SDS ) loading buffer . Equal amounts of the clarified cell extracts were separated by SDS-PAGE , and then transferred onto a nitrocellulose membrane ( GE Healthcare Life Sciences ) or PVDF membrane ( Millipore ) . The membrane was blocked for 1 h in PBST ( PBS plus 0 . 1% Tween 20 [Sigma] ) containing 5% skim milk and then washed with PBST . After incubation with the appropriate antibodies , the proteins were visualized by standard procedures using an enhanced chemiluminescence system ( Roche ) and x-ray film ( Kodak ) . Cell extracts were prepared and loaded onto a Superose6 10/300 GL column ( GE Healthcare ) pre-equilibrated with CoIP buffer . The proteins were eluted at 0 . 5 ml/min . Each fraction ( 15 μl ) was analyzed by immunoblotting with antibodies for RIP1 , HA-UL45 , and UL48 . Apparent molecular mass was evaluated after column calibration with standard proteins [thyroglobulin ( 669 kDa ) , ferritin ( 440 kDa ) , aldolase ( 158 kDa ) , conalbumin ( 75 kDa ) , and ovalumin ( 44 kDa ) ] in the Gel Filtration Calibration Kit ( GE Healthcare ) . Cells were fixed in cold methanol for 5 min and rehydrated in cold PBS . Cells were first incubated for 30 min with human γ-globulin ( Sigma ) ( 2 mg per ml ) at 37°C to block non-specific binding of antibodies to HCMV-induced Fc receptors [49] . They were then incubated for 1 h with primary antibodies in PBS at 37°C and then for 1 h with appropriate secondary antibodies labeled with FITC , Rhodamine Red-X , or Cy5 . Antibodies were incubated together for triple labeling . The slides were examined and photographed with a Carl Zeiss LSM710Meta confocal microscope system . Diluted samples were used to inoculate a monolayer of HF cells ( 1 x 105 ) in a 24-well plate . At 24 h post-infection , cells were fixed with 500 μl of cold methanol for 10 min . Cells were then washed three times in phosphate-buffered saline ( PBS ) , incubated with anti-IE1 rabbit PAb in PBS at 37°C for 1 h , followed by incubation with phosphatase-conjugated anti-rabbit IgG antibody in PBS at 37°C for 1 h . Finally , the cells were gently washed in PBS and treated with 200 μl of developing solution ( nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate ) at room temperature for 1 h according to the manufacturer's instructions . The IE1-positive cells were counted in at least three to five separate fields per well under a light microscope ( 200X magnification ) . Cells were collected and lysed by three freeze-thaw steps in 200 μl of 0 . 25 M Tris-HCl ( pH 7 . 9 ) plus 1 mM dithiothreitol . Cell extracts were clarified in a microcentrifuge , and 20 μl of each extract was incubated with 350 μl of reaction buffer A ( 25 mM glycyl-glycine [pH 7 . 8] , 5 mM ATP [pH 7 . 5] , 4 mM EGTA [pH 8 . 0] , 15 mM MgSO4 ) and then mixed with 100 μl of 0 . 25 mM luciferin ( Sigma ) in reaction buffer A . A TD-20/20 luminometer ( Turner Designs ) was used for a 10-s assay of the photons produced ( measured in relative light units ) . Cells were collected and lysed by three freeze-thaw steps in 200 μl of 0 . 25 M Tris-HCl ( pH 7 . 9 ) plus 1 mM dithiothreitol . Cell extracts were clarified in a microcentrifuge . Each reaction mix was prepared in a 96-well plate including 20 μl of extract plus 100 μl of Z-buffer ( 0 . 06 M Na2HPO4 , 0 . 04 M NaH2PO4 , 0 . 01 M KCl , 0 . 001 M MgSO4 , 0 . 05 M β-mercaptoethanol ) plus 20 μl of o-nitrophenyl-β-D-galactopyranoside ( ONPG [Sigma]; 4 mg/ml in sterile water ) . Reactions were incubated at 37°C until the yellow color developed . The reaction was stopped by adding 50 μl of 1 M Na2CO3 . The β-galactosidase absorbance of each reaction was read at 420 nm . 293T cells were co-transfected with plasmids expressing target or effector proteins and plasmids expressing HA-ubiquitin . At 24 h after transfection , cell pellets were re-suspended with 2% SDS lysis buffer containing protease inhibitors ( Sigma ) and boiled for 10 min . Cell lysates were diluted ten-fold with CoIP buffer and sonicated using a Vibra cell microtip probe . The clarified cell lysates were incubated with 30 μl of 50% slurry of anti-Flag M2 affinity gel ( Sigma ) for 16 h at 4°C . The mixture was pelleted and washed several times with CoIP buffer . The bound proteins were boiled and analyzed by SDS-PAGE and immunoblotting with the appropriate antibody . To detect endogenous RIP1 ubiquitination in virus-infected cells , the mock-infected or virus-infected cells were treated with 5 mM N-ethylmaleimide ( NEM ) for 30 min before they were harvested . Cells were lysed in PBS containing 1% SDS and 5 mM NEM and then boiled for 10 min . Cell lysates were sonicated and immunoprecipitated with anti-RIP1 antibody ( BD Biosciences ) . For immunoblotting , proteins were transferred to PVDF membranes and denatured using 6 M guanidine-HCl containing 20 mM Tris-HCl ( pH 7 . 5 ) , 1 mM PMSF , and 0 . 5 mM β-mercaptoethanol for 30 min at 4°C . Ubiquitinated RIP1 was identified by HRP-conjugated anti-ubiquitin antibody ( Biomol ) . Samples were compared using Student’s t-test , and p-values <0 . 05 ( * ) <0 . 01 ( ** ) and <0 . 001 ( *** ) are indicated in the figures . | Activation of NF-κB signaling leads to expression of proinflammatory cytokines and chemokines and plays a key role in regulating innate immune response and inflammation to virus infection . HCMV upregulates and downregulates NF-κB signaling during the course of infection . Upregulation of NF-κB signaling may promote viral gene expression or viral dissemination , but its downregulation may be necessary for suppression of excessive immune responses . Recently , it was demonstrated that viral late functions downregulate TNFα- and IL-1β-induced NF-κB activation . However , the viral proteins involved and the underlying mechanisms are not understood . In the present study , we demonstrate that two HCMV proteins , the largest tegument protein harboring deubiquitinase activity and the inactive homolog of cellular ribonucleotide reductase large subunit , cooperatively inhibit RIP1-mediated NF-κB signaling at the late stages of infection . This study for the first time identified RIP1 as a substrate of viral deubiquitinase and highlights the importance of the negative regulation of NF-κB during virus infection . | [
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"imm... | 2017 | Cooperative inhibition of RIP1-mediated NF-κB signaling by cytomegalovirus-encoded deubiquitinase and inactive homolog of cellular ribonucleotide reductase large subunit |
A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments . Drosophila legs include two types of joints: the proximal or “true” joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature . These joints are not only morphologically , functionally and evolutionarily different , but also the morphogenetic program that forms them is distinct . Development of both proximal and distal joints requires Notch activity; however , it is still unknown how this pathway can control the development of such homologous although distinct structures . Here we show that the bHLH-PAS transcription factor encoded by the gene dysfusion ( dys ) , is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints . In the presumptive tarsal joints , Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective and the expression of the RhoGTPases modulators , RhoGEf2 and RhoGap71E , thus directing key morphogenetic events required for tarsal joint development . When ectopically expressed , dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death . This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes . We also identified a dedicated dys cis-regulatory module that regulates dys expression in the tarsal presumptive leg joints through direct Su ( H ) binding . All these data place dys as a key player downstream of Notch , directing distal versus proximal joint morphogenesis .
Throughout evolution , animal appendages have diversified to display very different morphologies , which are indicative of their diverse functionality such as locomotion , feeding or environment exploration . One of the keys of the evolutionary success of arthropods ( from Greek árthron , “joint” , and pous i . e . “foot” ) , the most diversified group of animals , is the acquisition of joints that allow the articulation of their appendages . Appendages are external projections from the body wall , which require the formation of a proximo-distal axis ( PD ) that is specified de novo during development from previously established antero-posterior and dorso-ventral axes . In Drosophila , thoracic appendage primordia are specified during embryogenesis by the expression of the homeobox gene Distalless ( Dll ) [1] . During larval development , the PD axis is generated by the juxtaposition of cells that express two signaling molecules , wingless ( wg ) and decapentaplegic ( dpp ) . They regulate the expression of the leg “gap genes” Dll , dachshund ( dac ) and homothorax ( hth ) dividing the leg into distal , medial and proximal domains , respectively ( reviewed by [2] ) [3]–[8] . Subsequently , the distal leg is further subdivided into more discrete domains of tarsal-specific gene expression in response to the Epidermal Growth Factor Receptor ( EGFR ) pathway activity [9] , [10] . The Drosophila adult leg is divided in 10 segments ( coxa , throchanter , femur , tibia , five tarsal segments and the claw or pretarsus ) , which are articulated due to the presence of joints in between them . A common event during joint formation is the positioning of Notch ligands Delta ( Dl ) and Serrate ( Ser ) by the combined action of the leg “gap genes” and PD tarsal genes in concentric rings at the proximal end of each segment [11]–[14] . Notch pathway activation adjacent and distal to the Dl and Ser expression domains involves the proteolytic cleavage and release of the intracellular fragment of Notch ( NICD ) that acts as a transcriptional co-activator with proteins of the CBF1-Suppressor of Hairless ( Su ( H ) ) family [15] . In the leg joints , Notch pathway activation , visualized by the expression of its target genes Enhancer-of-split mβ ( E ( spl ) mβ ) and big brain ( bib ) , mediate leg segmentation and growth . Therefore , mutant clones for components of the Notch signaling pathway that span two leg segments are associated with joint and growth defects [12] , [14] , [16] , [17] . While Notch activity is absolutely required for all joints , proximal and distal joints are functionally , morphologically and evolutionarily different [18]–[24] . Proximal joints , also known as “true joints” , such as the tibial/tarsal joint , are asymmetrical and motile due to muscular attachments . In contrast , distal joints , the ones that separate the tarsal segments , are radially symmetrical , not motile and depend on a different molecular mechanism for their development [20] , [22] . Moreover , several genes are specifically expressed in the “true joints” such as the odd-skipped ( odd ) family members odd , drumstick ( drm ) and sister of odd and bowl ( sob ) , and others are restricted to the tarsal ones , like deadpan ( dpn ) or tarsal-less ( tal ) [14] , [25] , [26] . Two morphogenetic processes , apoptosis and changes in cell shape , contribute to joint formation [20] , [27] . Apoptosis in the legs involves the Jun kinase ( JNK ) -mediated activation of the pro-apoptotic gene reaper ( rpr ) in response to sharp boundaries of Dpp activity [20] , [28] . The early expression of dpp in the leg disc in dorsal-anterior cells is later refined into a segmented pattern of incomplete rings with higher levels at the distal edge of each segment [20] . It has been suggested that a confrontation of cells with different levels of Dpp pathway activity at the distal end of tarsal segments triggers cell death via JNK activation at both sides of the activity discontinuity [20] , [28] . Interestingly , the cell death-mediated mechanism is required for joint architecture only in the tarsal segments [20] . Changes in cell shape are in part mediated by the modulation of RhoGTPases activity [27] . RhoGTPases function as molecular switches ( active GTP-bound state and inactive GDP-bound state ) that regulate a variety of developmental processes such as cytoskeletal dynamics , cell migration , cell polarity , cell-cycle progression , vesicle trafficking and cytokinesis [29] . A subset of RhoGEFs and RhoGAPs , proteins that regulate Rho activity , are specifically expressed in all joints or restricted to tarsal joints , and the downregulation of some of them produce defects in leg joint formation [27] . An important and yet unresolved question is to understand how the same signaling pathway , Notch , could direct the formation of homologous but morphogenetically distinct structures , such as the different joints along the PD axis of the leg , which will require the deployment of different genetic programs for their formation [11] , [12] , [14] , [20] , [22] . In this work we characterize the expression , function and regulation of the Npas4/NXF vertebrate ortholog dysfusion ( dys ) . dys encodes a basic-helix-loop-helix PAS containing ( bHLH-PAS ) transcription factor required for embryonic tracheal development [30]–[32] . Here we describe a novel and essential role for dys during leg joint formation . We show that dys is absolutely required for tarsal joint formation while it is dispensable for proximal joints . Dys regulates the expression of the pro-apoptotic genes rpr and head involution defective ( hid ) and the Rho GTPase regulators RhoGAP71E and RhoGEF2 . dys expression at the presumptive tarsal joints is controlled by a dedicated cis-regulatory module ( CRM ) , directly regulated by Notch through binding of Su ( H ) . In summary our results provide a molecular explanation of how Notch can regulate the formation of different types of joints along the leg PD axis .
Proximal and distal leg joints are not only morphologically and functionally different , but also the mechanisms that sculpt them have evolved independently [18]–[22] . Since the Notch pathway is required for both types of joints , other factors should contribute to the differential development of distal versus proximal joints . To understand how the tarsal joints are genetically distinguished from the proximal joints , we have searched in the flylight database for CRMs exclusively active in rings in either tarsal or ‘true’ joints [33] ( see Materials and Methods ) . We identified two overlapping sequences within the dysfusion ( dys ) genomic locus that drove very similar or identical GFP reporter gene expression in concentric rings at the tarsomeres in third instar and prepupal leg imaginal discs ( Figure 1A and Figure S1 ) . We also detected GFP expression in distal rings in the antenna for these two CRMs ( Figure S1 ) . Using an available antibody for Dys [30] , we confirmed that dys is expressed at the distal end of each tarsal segment in prepupal and third instar leg discs , with the exception of the boundary between the most distal tarsus and the pretarsus , which accounts for all the four tarsal joints ( Figure 1B and 1C ) . We also detected an incomplete ring of expression at the distal tibia that was not reproduced by any of the two CRMs identified ( Figure 1A and 1B ) . The specification of joints is controlled by the local activation of the Notch pathway [11] , [12] , [14] . Notch downstream targets and its ligands Dl and Ser are expressed in complementary concentric rings along the PD axis of the leg . The Notch targets bib and E ( spl ) mβ are restricted to the distal end of each segment ( joint domain ) , while the Notch ligands , Dl and Ser , are restricted to proximal adjacent cells ( inter-joint domain ) [11] , [12] , [14] . To confirm that dys expression is restricted to the presumptive joints we compared its expression with that of bib , E ( spl ) mβ and Dl . We found that dys coexpress with both joint markers at the distal-most cells of tarsal segments 1 to 4 , while its expression is distal to Dl localization ( Figure 1B–1E ) . In this manner , dys expression is restricted to Notch-responsive cells of the tarsal region , suggesting a potential role for dys in the development of tarsal joints downstream of Notch . To analyze in detail the role of dys during leg morphogenesis we first used a combination of two null dys alelles , dys2/dys3 , which produces some escapers ( less than 1% of the flies ) ( see Materials and Methods ) . All dys2/dys3 adult flies display a complete absence of tarsal joint formation with a small shortening of the tarsal region ( Figure 2A and 2B ) . Interestingly no defects were observed in most proximal joints ( including the tibial/tarsal joint where a half-ring of dys expression is detected ) or in the tarsus/pretarsus joint ( Figure 2A , 2B and Figure S2A and S2B ) . In addition we detected the loss of the joint between the a5 and the arista in the antenna ( Figure 2D and 2E ) . We also expressed a dys-RNAi construct that efficiently reduced Dys protein levels ( Figure S2C ) . dys knockdown in the Dll domain ( Dll-Gal4/UAS-dys-RNAi; distal tibia to claw ) abolish joint formation in the tarsal segments without affecting the tibial/tarsal or tarsus/pretarsus joints just as described for dys2/dys3 mutant legs ( Figure 2C ) . To test whether dys is sufficient to induce joint-like structures in the leg , we ectopically expressed dys in an anterior row of cells along the PD axis of the leg disc using the patched ( ptc ) -Gal4 line . We restricted dys ectopic expression to mid-third instar stage using the Gal80ts technique ( see Material and Methods ) . dys misexpression induces the formation of cuticle folds along the PD axis of the leg that resembles ectopic joint formation ( Figure 2F ) . These joint-like structures are more evident in the tarsal region , although we also detected ectopic folds in more proximal domains such as the tibia or the femur ( Figure S2D ) . These phenotypes are very similar to the ones described for ectopic Notch pathway activation in the leg [12] , [14] ( see Figure 3M ) . Although we can not conclude that ectopic dys is able to induce the complete joint architecture , which would include the ball-and-socket structure , we were able to observe a phenotype that recapitulates some major aspects of joint formation such as indentation of the cuticle and fold formation . Taken together , our results suggest that dys is necessary for tarsal joint formation and sufficient to induce joint-like structures . dys expression is restricted to Notch responding cells in the presumptive tarsal leg joints , and its function is required for tarsal joint formation . To analyze the relationship between the Notch pathway and dys , we first tested if dys expression requires Notch activity . We found that Notch downregulation abolishes dys expression ( ptc-Gal4/UAS-Notch-RNAi; Figure 3A ) , while Notch activation promotes dys expression ( ptc-Gal4/UAS-NotchICD; Figure 3B ) . Interestingly , although Notch activation was driven within the ptc domain along the entire PD axis of the leg , dys ectopic expression is restricted to the tarsal segments , suggesting that dys is a downstream target of Notch exclusively in the tarsal region . Next , we tested whether dys is required for the expression of two Notch targets , bib and E ( spl ) mβ . When Dys function is reduced or eliminated using dys-RNAi or the dys2/dys3 allelic combination , respectively , we observed a strong downregulation of bib-lacZ expression in the tarsal segments , while E ( spl ) mβ-CD2 is only slightly downregulated or remains unaffected ( Figure 3C–3F . ) . Interestingly , the expression of Dl is not altered in dys2/dys3 mutants ( Figure 3G ) , indicating that neither Dl expression nor Notch activation require Dys function . In contrast , bib-lacZ expression does depend on Dys , although we do not know the basis of this regulation . To study the functional relationships between the Notch pathway and dys , we first analyzed the ability of Dys to promote the formation of joint-like structures in a Notch mutant background . We induced dys ectopic expression in the ptc domain using the tubGal80ts technique in a hemizygous background for a Notch temperature-sensitive allele ( Notchtsa ) ( see Material and Methods ) . Notchtsa mutants reared at 17°C ( permissive temperature ) and shifted to 29°C ( restrictive temperature ) at late third instar show a complete absence of tarsal joints ( Figure 3I compared to 3H ) . As previously described , temporarily restricted dys misexpression in a wild type background induced cuticle invaginations in a joint-like fashion ( Figure 3J ) . In Ntsa; ptc-Gal4/UAS-dys; tubGal80ts flies shifted to 29°C at late third instar we found a uniform and continuous tarsal cuticle with no joints , characteristic of Notch mutants , and a fold running along the PD axis ( compare Figure 3I and 3J with 3K ) . In the corresponding leg discs , ectopic dys expression induced the formation of an epithelial fold along the PD axis , both in wild type and in Ntsa mutant backgrounds ( compare Figure S2F and S2G with S2E ) . We also performed the reverse experiment , activating the Notch pathway in a dys null background . We found that in dys2/dys3 mutant legs , forced expression of NICD in the ptc domain still retains the capacity to make a fold even though endogenous tarsal joints are not formed ( compare Figure 3L and 3M with 3N ) . This phenotype is very similar to that produced by the ectopic expression of NICD in a wild type background ( Figure 3M ) . This result suggests that forced Notch pathway activation in the absence of dys could be inducing other effector genes that play key roles in the formation of joint-like structures different from the tarsal ones . All together , these results demonstrate that dys is downstream of Notch during tarsal joint formation . Epithelial cells at the presumptive joints undergo apical constriction and form characteristic folds that prefigure the future joint [22] , [23] . The formation of epithelial folds involve cells immediately distal to the E ( spl ) mβ-CD2 expression domain ( Figure 4A ) . In contrast , in dys mutant prepupal legs these cells distal to the E ( spl ) mβ-CD2 domain fail to reproduce these shape changes compared to control legs ( compare Figure 4B and 4A ) . Two processes help sculpt the joint structure , namely JNK-mediated apoptosis driven by the pro-apoptotic gene rpr [20] and cell shape changes mediated by the Rho-family of GTPases [27] . The expression of RhoGef2 and RhoGap71E is restricted to the tarsal segments where they are coexpressed with a single row of Dys-positive cells and are extended distally to a row of Dys-negative cells ( Figure 4C and 4D ) . Downregulation of Dys levels with a dys-RNAi in the engrailed ( en ) or the hedgehog ( hh ) domains lead to compartment cell autonomous loss of RhoGap71E and RhoGef2 expression , respectively ( Figure 4E and 4F ) . Next , we compared the expression of dys with the pro-apoptotic genes rpr and hid . We found that the expression of these two genes in the prepupal leg discs is restricted to the distal end of each tarsal segments , where they are coexpressed with dys ( Figure 5A , S3A ) [20] , [34] . Interestingly , as previously described for RhoGap71E and RhoGef2 , we observed that rpr and hid are also expressed in a row of cells distal to dys expression ( Figure 5A and S3A ) . Next we investigated if the expression of rpr and hid depends on dys . The downregulation of Dys levels in anterior cells along the PD axis with a dpp-Gal4 line or in the posterior compartment with en-Gal4 , strongly reduced or eliminated the expression of rpr and hid , respectively ( Figure 5B and S3B ) . Furthermore , forced expression of dys in the posterior compartment for 24 hrs using the tubGal80ts technique is sufficient to promote cell autonomously rpr and hid expression in the inter-joint domain ( Figure 5C and S3C ) . This ectopic activation of the pro-apoptotic genes rpr and hid is accompanied by an increase of cell death in larval and prepupal leg discs , as visualized by the presence of activated Caspase-3 ( DCas-3 ) ( Figure 5D and S3D ) . To test if the downregulation in the expression of the pro-apoptotic genes observed after reducing Dys levels is associated to a decrease in cell death , we compared the number of DCas-3 positive cells in wild type and dys mutant legs ( see Material and Methods ) . To determine the precise location of the apoptotic cells during joint formation , we separately counted DCas-3 positive cells within the E ( spl ) mβ domain and at the gap between two E ( spl ) mβ domains ( termed here as “fold” domain ) . In wild type prepupal legs we found a significant increase in the number of DCas-3 positive cells in the “fold” domain compared to the E ( spl ) mβ one , while in dys mutant legs such significant difference was not observed ( Figure 5E–G ) . Interestingly , while the total number of apoptotic cells per segment was comparable between wild type ( average = 13 , 8 ) and dys mutant legs ( average = 14 , 8 ) , the distribution of DCas-3-positive cells in the joint was altered . dys mutant legs have approximately the same number of apoptotic cells in the E ( spl ) mβ than in the “fold” domains ( Figure 5E–G ) . It has been proposed that sharp discontinuities of Dpp activity trigger JNK-mediated apoptosis through the activity of rpr [20] , [28] . Therefore we first compared P-Mad ( a readout of Dpp signaling ) levels with bib expression , which , as we have shown previously , is coexpressed with Dys in the tarsal joints . P-Mad in prepupal legs forms a dorsal ring-like pattern with higher levels coincident with bib-expressing cells ( Figure S3E ) . Next we tested if the absence of rpr expression observed in dys-RNAi legs could be due to a failure in the generation of Dpp activity borders . ptc-Gal4; UAS-dys-RNAi prepupae legs show downregulation of P-Mad levels , suggesting that dys is required for the correct formation of sharp Dpp activity boundaries at the tarsal joints ( Figure 5H ) . Our results suggest that Dys is a downstream effector of Notch signaling during tarsal joint development , which activates the expression of the pro-apoptotic genes rpr and hid . To confirm these results , we tested if in the absence of Notch activity Dys is still able to induce the transcription of rpr . We found that the depletion of Notch function for 24 to 48 hrs before dissection , using the Notchtsa allele , was sufficient to completely abolish rpr-lacZ expression in the leg disc ( compare Figure 5I with 5J ) . As expected , Notchtsa; ap-Gal4/UAS-dys; tubGal80ts larvae switched to 29°C , 24 to 48 hrs before dissection show ectopic rpr-lacZ expression in the ap domain of the leg imaginal discs ( a ring in tarsal segments 4 and part of 5 ) while no rpr-lacZ expression is detected in more proximal tarsal rings ( Figure 5K ) . These results confirm our previous observations that indicate that Dys is epistatic to Notch during tarsal joint development . In summary , these experiments suggest that dys is necessary for the correct expression of the pro-apoptotic genes rpr and hid and the RhoGTPase modulators RhoGap71E and RhoGef2 , being both key events during tarsal joint morphogenesis . Tango ( Tgo ) is a bHLH-PAS transcription factor that is able to form heterodimers with multiple bHLH-PAS proteins including Spineless ( Ss ) , Trachealess ( Trh ) and Dys [32] , [35] , [36] . In the absence of any of its partners , Tgo localizes in the cytoplasm while in the presence of a partner bHLH-PAS protein , Tgo and its companion form a complex that is translocated into the nucleus where it is functional [36] . During embryonic tracheal development , Tgo dimerizes with Dys to activate the transcription of tracheal fusion target genes [32] . To investigate if the Dys function dependency on Tgo also exists for tarsal joint formation , we analyzed Tgo protein localization and Tgo loss of function phenotypes during joint development . In prepupal leg imaginal discs , Tgo protein shows nuclear localization specifically where dys is expressed , at the presumptive tarsal leg joints ( Figure 6A ) . Moreover , a 24 hr pulse of ectopic expression of dys ( ptc-Gal4/UAS-dys; tubGal80ts ) induced the nuclear localization of Tgo , while in cells where we reduced Dys levels ( en-Gal4; UAS-dys-RNAi ) Tgo fails to accumulate in the nucleus of presumptive tarsal joint cells ( Figure 6B and 6C ) . tgo loss of function phenotypes are characterized by fusions and deletions of tarsal segments without affecting proximal ones , phenotypes very similar to those of ss and trh null mutant flies [35] , [37] . Since Tgo interacts with Ss to activate transcription , much of tgo tarsal leg defects were attributed to Ss phenotypes [35] . We decided to test if some of the tgo leg phenotypes described could be also due to Dys's inability to activate transcription in the absence of its partner . Tgo knockdown specifically at the presumptive tarsal joints using a tgo-RNAi driven by the GMR_13D07-Gal4 line ( see Material and Methods ) disrupted joint formation , a phenotype very similar to dys mutant legs ( compare Figure 6D with 6E and Figure 2B ) . As described for dys loss-of-function conditions , we found strong bib-Z downregulation in the presumptive tarsal joints in dpp-Gal4; UAS-tgo-RNAi flies , while the proximal or the most distal ones remain unaffected ( Figure 6F ) . The same effect was observed in the expression of rpr and RhoGap71E after reducing Tgo activity ( Figure 6G ) . All these phenotypes could be due to Dys's inability to activate transcription in the absence of Tgo . To test this possibility we have generated ectopic expression clones of dys in cells that are also mutants for tgo ( see Material and Methods ) . As previously described above , dys ectopic expression in the leg activates rpr in a cell autonomous manner . As expected for a Tgo-obligated transcriptional co-activator , dys ectopic expression clones mutant for tgo are not able to induce the expression of rpr ( Figure 6H ) . All together these results demonstrate that Dys functions with Tgo and both together regulate the expression of their target genes such as rpr . We have screened 11 DNA fragments derived from the Janelia Gal4 data base that cover the 5′ region and the introns of the dys genomic locus for expression in the leg imaginal disc [33] . Only two overlapping sequences , located between exons 2 and 3 , GMR_13D07 and GMR_13B03 , drive expression of a GFP reporter in a ring-like pattern that resembles dys expression in the leg ( Figure 1A and S1 ) . The overlapping sequence ( 640 bp long ) , cloned in a nuclear lacZ reporter vector ( see Material and Methods ) , contains the information necessary to reproduce dys expression pattern in the tarsus ( Figure 7B ) . We have previously shown that Notch acts upstream of dys and , in agreement with our genetic results , dys640-lacZ expression is disrupted in Notch knockdown prepupal leg discs ( Figure S4A ) . Moreover , ectopic expression of NotchICD activates dys640-lacZ expression , although this activation is restricted to the tarsus , just as described for dys endogenous expression ( Figure S5B ) . To gain further insights in the molecular regulation of dys , we divided the dys640 CRM in two overlapping fragments and studied their expression using the same lacZ reporter construct ( Figure 7A ) . The dys640A-lacZ fragment included two putative Su ( H ) binding sites conserved among different Drosophila species , and it is expressed weakly in patches of cells that do not overlap with dys expression ( Figure 7C and 7E ) . Fragment dys640B-lacZ does not include these Su ( H ) sites and resulted in a weak but consistently extended expression of lacZ in the tarsal inter-joint domain , with low levels in dys expressing cells ( compare Figure 7D with 7B ) . The two candidate binding sites were also tested for their ability to bind Su ( H ) in an electrophoretic mobility shift assay ( EMSA ) ( Figure 7E and 7F ) . Su ( H ) binds to both putative sites and this binding was abolished when the sites were mutated ( Figure 7F ) . To further assess the contribution of the identified binding sites to dys640-lacZ expression , we mutated each individual binding site and the combination of Su ( H ) -1 and 2 in vivo in a transgenesis reporter assay . Mutation of either of these sites in isolation , dys640Su ( H ) -1 and dys640Su ( H ) -2 , significantly reduced but did not eliminate lacZ expression ( Figure 7G–I ) . Interestingly we observed a slight derepression of lacZ signal in the inter-joint domain of all tarsal segments , being stronger in the fourth tarsal segment for the two constructs . The combined mutation of the two sites , dys640Su ( H ) -1+2-lacZ , resulted in an overall weaker expression than mutating each site separately and a derepression of lacZ in the inter-joints throughout the tarsal region and in the distal portion of the tibia ( compare Figure 7J with 7G ) . Taken together , these results indicate that dys is a direct target of Notch through Su ( H ) binding to the dys640 CRM . We propose that in the absence of Notch signaling , as in the tarsal inter-joint region , Su ( H ) binds to dys640 CRM repressing dys expression . Conversely , Notch activation at the presumptive joint cells leads to loss of Su ( H ) repression , probably through a displacement of co-repressors and recruitment of co-activators , therefore converting Su ( H ) DNA-bound in a positive input to activate dys expression .
In a search for genes expressed exclusively either in distal or proximal leg joints we identified the gene dys . dys is coexpressed with bib and E ( spl ) mβ and it is distal to the Notch ligand Dl in four rings at the presumptive tarsal joints and in an incomplete ring at the presumptive tibial/tarsal joint . Legs mutant for dys do not develop tarsal joints and have instead a smooth continuous cuticle , without defects in other proximal joints or in the most distal one , the tarsus/pretarsus joint . In dys mutant prepupal leg discs the characteristic apical constriction of cells and the subsequent fold at the presumptive joint are lost . Conversely , ectopic expression of dys is able to induce ectopic folds along the leg that resemble joint-like structures , a phenotype very similar to those observed after misexpression of the activated form of Notch [12] , [14] . Several experiments suggest that dys is a Notch target that is indispensable for tarsal joint development: ( 1 ) Notch directly regulates dys expression ( see below ) . ( 2 ) The expression of the Notch targets bib and rpr absolutely require dys function , even when the Notch pathway is still active . ( 3 ) Dys is able to induce the expression of rpr and the formation of joint-like cuticle invaginations in the absence of Notch signaling . All these results place dys genetically downstream of Notch in the development of tarsal joints . Our results show that dys expression at the presumptive tarsal segments is controlled by a dedicated CRM 640 bp long that integrates Notch signaling through direct Su ( H ) binding . This is , to our knowledge , the first characterized Notch direct target described for leg joint development . Interestingly , the mutation of the two identified Su ( H ) consensus sites , dys640-lacZSu ( H ) -1+2 , resulted in lacZ derepression in the inter-joint domain of the tarsal segments although at lower levels compared to normal signal observed in the presumptive joints in dys640-lacZ control legs . These results are in favor of the “default repression” model in which Su ( H ) associates with co-repressors in the absence of Notch signaling to repress target gene transcription [39] ( Figure 8A ) . In the event of Notch activation , co-repressors are displaced by NICD , so Su ( H ) binding could lead to target gene transcription through the recruitment of co-activators . Therefore , dys fulfills the two predictions of the model to occur in the absence of Su ( H ) binding: ( 1 ) target genes will be derepressed and ( 2 ) their expression will be reduced in their normal expression domains . These two predictions can be validated in the dys640Su ( H ) 1+2 and dys640B gene reporter constructs , where Su ( H ) binding is compromised or lost . In both cases we observed a consistent derepression of lacZ expression in the inter-joint domain of the tarsal segments , although with weak levels . We also observed reduced lacZ expression at the presumptive joint domain in the dys CRM with the two Su ( H ) sites mutated or the dys640B reporter gene compared to the intact dys640 . This characteristic is specially evident in the dys640B construct that lacks the two described Su ( H ) sites and probably others not identified in our analysis . Enhancers are logic integrators of positive and negative inputs that drive precise temporal and spatial gene expression . In the case of dys , two previously identified CRMs drove reporter gene expression in all tracheal fusion cells in the embryo [40] ( Figure 7A ) . For dys expression in the leg we have identified a different CRM that integrates Notch input and is only active in the tarsal presumptive joints . Although Notch pathway is active in rings along the PD axis of the leg , dys expression is restricted to the distal region , suggesting that other inputs are required to give dys its positional specificity along the PD axis . Thus , it is probable that tarsal transcription factors bind to dys640 CRM to restrict its activity ( Figure 8A ) . Some candidates are bric à brac ( bab ) , rotund ( rn ) or ss , which are expressed in the four tarsal segments where dys is specifically active [41]–[43] . Joint development involves a complex developmental program that leads to the formation of flexible structures connecting leg segments . At least two processes play key roles during tarsal joint development: epithelial morphogenesis controlled by the activity of RhoGTPases , and JNK-dependent programmed cell death [20] , [27] . Several RhoGTPases modulators , RhoGaps and RhoGefs , which have a restricted expression pattern , are regulated by Notch and their downregulation affects joint formation [27] . Another important morphogenetic event is the formation of sharp Dpp signaling boundaries at the presumptive tarsal joints that triggers JNK-dependent localized cell death through rpr activation [20] . Interestingly P-Mad and rpr segmented expression depends on Notch activity , while ectopic formation of new Dpp signaling borders feedback to the system and activate downstream Notch targets such as E ( spl ) mβ [20] . Therefore , Notch controls tarsal joint morphogenesis in part through Dpp pathway and RhoGTPases regulation . Although a relationship between the Dpp pathway and the expression of RhoGTPases regulators has not been described , it is possible that these genes could also be regulated by sharp Dpp discontinuities , as is the case for rpr . Our results place dys directly downstream of Notch in the presumptive tarsal joints , regulating the Dpp pathway and the expression of rpr , hid , RhoGef2 and RhoGap71E ( Figure 8B ) . One interesting and unresolved question is how Dys can control the expression of rpr , hid , RhoGap71E and RhoGef2 non-autonomously . We have described that these genes are expressed in a row of Dys-positive cells and in an additional Dys-negative distal row of cells ( see Figure 4C , 4D , 5A , S3A and 8B ) . Interestingly , Dys downregulation blocks rpr , hid , RhoGap71E and RhoGef2 expression both in dys expressing and non-expressing cells , while dys misexpression activate rpr and hid exclusively in a cell autonomous manner . Our results suggest that Dys could regulate rpr in two ways . The first one would be a direct activation by Dys and the second one an indirect effect through P-Mad and JNK pathway regulation that also regulates rpr [20] . Therefore , sharp Dpp discontinuities lead to JNK-mediated rpr expression while Notch activation in the presumptive tarsal joints activates dys expression that also regulates rpr cell autonomously . The cross talk between the Notch and Dpp pathways might be important to ensure a robust activation of rpr expression at the tarsal joints , and dys could be a key player in the communication between these pathways ( Figure 8B ) . A similar mechanism to that described for rpr could regulate the expression of hid , RhoGap71E and RhoGef2 in the presumptive tarsal joints . It would be of interest to study if Dys controls the same subset of genes used for joint morphogenesis in other developmental contexts where it is expressed ( e . g . embryonic tracheal fusion or leading edge cells ) [30] . The role of Rho GTPases during tracheal development has been previously described [44] , [45] but its implication in tracheal fusion and the possible role of Dys as a regulator of Rho GTPases in this process remains to be elucidated . Another important observation is the discrepancy we observed between the expression of the pro-apoptotic genes and the number of DCas-3 positive cells . While in dys misexpression experiments we observed a positive correlation between the activation of rpr/hid and the increase of apoptotic cells , in dys mutant legs this relationship is more difficult to find . In wild type prepupal legs , the number of DCas-3 cells is significantly increased in cells distal to E ( spl ) mβ domain , while in dys mutants , this difference is not observed: there is a significant increase of apoptotic cells in the E ( spl ) mβ domain and a reduction , although not significant , in the “fold” domain . Therefore , our results suggest that in the absence of dys the apoptosis is not preferentially localized in any part of the joint , while in the presence of dys , this apoptosis is bent towards the cells distal to the E ( spl ) mβ domain . We propose that this imbalance of apoptosis is necessary for the correct formation of the tarsal joints . An important question in developmental biology is how a given signaling pathway is able to direct different morphogenetic programs . For example , the Dpp and the Sonic Hedgehog ( Shh ) pathways pattern the fly wing and the ventral neural tube , respectively , in a morphogene concentration-dependent manner , activating different sets of target genes ( reviewed in [46] ) . In the case of the Notch pathway , target genes are regulated cell autonomously and depending on the cellular context a different set of downstream genes is regulated by Notch [15] . In the leg disc , the Notch pathway is similarly activated in 10 concentric rings along the PD axis that prefigure the future joints [11] , [12] , [14] , [23] . Although all joints are homologous structures , distal joints differ from proximal or “true joints” not only in the absence of muscular attachments but also in their morphology , evolutionary origin and in the morphogenetic program that sculpt them [18] , [20] , [22] . Moreover , while each proximal joint has a unique morphology , all distal joints display the same ball and socket organization . Therefore , if Notch is absolutely required for all joints , the question arises of how Notch can be directing the formation of two very different yet homologous structures like distal and proximal joints . One possible scenario is that Notch controls joint morphogenesis through the activation of different sets of downstream effectors along the PD axis of the leg . Several Notch downstream target genes have been characterized to be required in all joints , such as dAP-2 [16] , while others are restricted to the proximal or “true joints” . Members of the odd-skipped family of zinc finger transcription factors are expressed in all joints of the leg except the tarsal joints and they might act redundantly to regulate the development of these proximal joints [21] , [47] . Other genes are expressed exclusively in the tarsal joints like tal , dpn or dys . dpn , as dys , also encodes for a bHLH transcription factor expressed in the tarsal joints , although we did not find a phenotype in several dpn mutant allelic combinations or knocking down Dpn levels using a dpn-RNAi construct ( data not shown ) [26] . Interestingly , as it occurs in the embryonic trachea where Dys and another bHLH-PAS transcription factor , Trh , function in non-overlapping cells [30] , in the leg these proteins are also present in distinct domains [37] . It would be interesting to study if the cross-regulation described in the trachea for Trh and Dys also exists in the leg . Based on the expression and requirements for dys in the joints , we propose a model in which Notch directs the formation of the different joints by the PD-restricted activation of target genes such as odd in proximal joints and dys in the distal ones .
dys2 and dys3 , as well as the UAS-dys strains are described in [30] , [31] and GMR_13D07- and GMR_13B03-Gal4 and the rest of Janelia enhancer/GAL4 lines are described in the flylight database ( http://flweb . janelia . org/cgi-bin/flew . cgi ) [33] and are all publicly available at Bloomington Stock Center . dpn mutant alelles , dpn7 , dpn6 and dpnDef3D5 were kindly provided by Antonio Baonza . The reporter lines bib-lacZ and E ( spl ) -mβ-CD2 [12] were used for assessing Notch pathway activation . To study the relation between dys and the Notch pathway , we used the UAS-NotchICD [48] , and UAS-Notch-RNAi lines [49] . The Notch thermosensitive mutant allele ( Notchtsa ) allowed us to knockdown Notch activity when the flies are shifted to the restrictive temperature ( 29°C ) [50] . For loss- and gain-of-function experiments , we employed the Gal4 lines ptc-Gal4 , dpp-Gal4 , Dll-Gal4MD212 , ap-Gal4 , hh-Gal4 and en-Gal4 and the tubGal80ts allele , all-available at Bloomington Stock Center . We used the reporter lines rpr-4kb-lacZ ( rpr-lacZ ) [51] , hid-lacZW05014 [52] , RhoGAP71E- and RhoGEF2-lacZ ( Bloomington Stock Center ) . The lines dys- , tgo- and dpn-RNAi are available at the Vienna Drosophila Resource Center ( VDRC ) . RNAi knockdown experiments were performed on a UAS-Dcr-2 background [53] . To generate tgo mutant clones we utilized the null allele tgo5 ( Bloomington ) and the MARCM technique , which allowed us to simultaneously eliminate tgo function and express dys cell autonomously . The detailed genotype is: yw hs-flp , tub-Gal4; UAS-dys; FRT 82B tubGal80/FRT 82B tgo5 . Loss of function clones were created by heat-shocking the larvae for 1 hour at 37°C 48 to 72 hrs after egg laying . dys gain-of-function experiments were performed using the Gal4-tubGal80ts system , which allowed us temporal restriction of UAS-dys expression to mid-third instar stage . 24 hrs collection of hh- , en- , ap- or ptc-Gal4/UAS-dys; tubGal80ts flies were maintained at restrictive temperature ( 17°C ) until mid-third instar stage , when the fly vials were shifted to the permissive temperature ( 29°C ) . Larva and prepupae were dissected between 24 to 48 hrs later . Larval and prepupal leg discs were fixed and stained following standard procedures . As primary antibodies we used rabbit and mouse anti-βGal , rabbit anti-Dys ( a gift from L . Jiang and S . T . Crews ) , rabbit anti-DCas-3 ( cleaved Drosophila Dcp-1 , Cell Signaling Technology ) , rabbit anti-P-Mad ( kindly provided by G . Morata ) . Mouse anti-Dl , anti-Dlg and anti-Tgo are from Developmental Studies Hybridoma Bank , University of Iowa . TRITC-phalloidin and Phalloidin-Atto 647N were used to stain F-actin ( Sigma Aldrich ) , and secondary antibodies were coupled to Red-X , FITC and Cy5 fluorocromes ( Alexa Fluor Dyes , Invitrogen ) . To determine the levels of cell death in E ( spl ) -mβ and “fold” domains , we have performed Z-stack imaging of wild type ( n = 8 prepupae leg discs ) and dys2/dys3 mutants ( n = 10 prepupae leg discs ) and counted the number of D-Cas3 positive cells on each domain with the aid of the Fiji software . We selected for this analysis the joints between tarsal segments 2/3 and 3/4 . The 640 bp overlapping DNA sequence between the GMR_13D07 and GMR_13B03 lines as well as the different mutant conditions were cloned in the HLz attB plasmid vector , which expresses a nuclear lacZ reporter under the control of the cloned sequence [6] . The primers used were the following for each reporter line ( restriction sites are underlined and restriction enzyme used is noted in brackets ) : dys640: Forward: 5′-cagtcctaggCCAAGCCGATGAGCCATTCCATACC-3′ ( AvrII ) Reverse: 5′-cagtagatctCCACTCTGGAGCAAACCACACCGAA-3′ ( BglII ) dys640A: Forward: 5′-cagtcctaggCCAAGCCGATGAGCCATTCCATACC-3′ ( AvrII ) Reverse: 5′-cagtagatctTTCTGCTGATTTTCTTCTTTAGGTT-3′ ( BglII ) dys640B: Forward: 5′-cagtcctaggCTCTCCATGGTTAAGCTCAGACTAA-3′ ( AvrII ) Reverse: 5′-cagtagatctCCACTCTGGAGCAAACCACACCGAA-3′ ( BglII ) Putative Su ( H ) binding sites were identified on the basis of a bioinformatics analysis combining data from the JASPAR CORE Insecta database ( http://jaspar . genereg . net/ ) and the Target Explorer tool [54] . Mutagenesis of the Su ( H ) putative binding sites was performed using the QuikChange Site-Directed Mutagenesis Kit ( Stratagene ) . We used the following primers: dys640Su ( H ) -1: Forward: 5′-TCGATCCAAGAACCAAGTCcgagaccAATTTCCGTACACACACAA-3′ Reverse: 5′-TTGTGTGTGTACGGAAATTggtctcgGACTTGGTTCTTGGATCGA-3′ dys640Su ( H ) -2: Forward: 5′-GGAGGAAGAAAAAACTCAGtggagacagCAAATTAAGATAATCG-3′ Reverse: 5′-CGATTATCTTAATTTGctgtctccaCTGAGTTTTTTCTTCCTCC-3′ dys640-lacZ reporter construct was inserted both in the 2R ( 51D ) and 3R ( 86Fb ) chromosomal locations . To allow proper comparison , all the dys640-lacZ versions ( dys640-lacZ , dys640A-lacZ , dys640B-lacZ , dys640Su ( H ) -1-lacZ , dys640Su ( H ) -2-lacZ and dys640Su ( H ) -1+2-lacZ ) were inserted in the same location . Confocal settings were kept constant when imaging wild type and mutant versions of dys640-lacZ , so lacZ expression levels are comparable between these conditions . An incomplete form of Su ( H ) , that bears the DNA binding domain [55] was translated in vitro using the TNT T7 Quick master MiX kit ( Promega ) and tested for binding with a series of labeled dsDNA probes . 50 ng of each sense oligonucleotide were labeled following standard procedures with γ-32P ATP , and then hybridized with the complementary “cold” oligonucleotide . Wild type and mutant probes , where nucleotides at consensus Su ( H ) binding site were mutated , were generated for the two identified Su ( H ) sites . The designed oligonucleotides were: Su ( H ) -1 WT Forward: 5′-CCAAGTCATGGGAAAATTTCC-3′ Reverse: 5′-GGAAATTTTCCCATGACTTGG-3′ Su ( H ) -1 mut Forward: 5′-CCAAGTCcgagaccAATTTCC-3′ Reverse: 5′-GGAAATTggtctcgGACTTGG-3′ Su ( H ) -2 WT Forward: 5′-GGAGGAAGAAAAAACTCAGTTTCGCACGCAAATTAAGATAATCG-3′ Reverse: 5′-CGATTATCTTAATTTGCGTGCGAAACTGAGTTTTTTCTTCCTCC-3′ Su ( H ) -2 mut Forward: 5′-GGAGGAAGAAAAAACTCAGtggagacagCAAATTAAGATAATCG-3′ Reverse: 5′-CGATTATCTTAATTTGctgtctccaCTGAGTTTTTTCTTCCTCC-3′ Mutated Su ( H ) sites are noted with lower case letters . Wild type and mutant adult flies were collected and their legs and heads dissected without any fixation and avoiding moisture prior to preparation for SEM . The preparation of the samples and Scanning Electron Microscopy was performed at the Microscopy Unit at Universidad Autónoma de Madrid . | One of the keys of the evolutionary success of arthropods , the most diversified group of animals , is the acquisition of joints that allow the articulation of their appendages . Two main kinds of joints with different morphologies and evolutionary origin are found in the fly leg: the proximal or “true” joints that are motile due to muscular attachment and the distal joints that are immotile . A common event during joint formation is the activation of the Notch pathway at the presumptive joints along the leg proximo-distal axis . In this work we investigated how the same pathway , Notch , can control the formation of such homologous although different structures . We described that the transcription factor Dysfusion ( Dys ) is a Notch target required for distal joint development and that is sufficient to induce joint-like structures when ectopically expressed . Dys controls two important morphogenetic events that direct tarsal joint development such as programed cell death and epithelial cell shape . Moreover , we identified a regulatory DNA sequence that controls dys expression in the tarsal segment by direct binding of the transcriptional effector of the Notch pathway Su ( H ) . Thus , Notch controls the development of proximal vs distal joints by the recruitment of specific downstream target genes such as dys . | [
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"devel... | 2014 | The bHLH-PAS Transcription Factor Dysfusion Regulates Tarsal Joint Formation in Response to Notch Activity during Drosophila Leg Development |
As a devastating Ebola outbreak in West Africa continues , non-pharmaceutical control measures including contact tracing , quarantine , and case isolation are being implemented . In addition , public health agencies are scaling up efforts to test and deploy candidate vaccines . Given the experimental nature and limited initial supplies of vaccines , a mass vaccination campaign might not be feasible . However , ring vaccination of likely case contacts could provide an effective alternative in distributing the vaccine . To evaluate ring vaccination as a strategy for eliminating Ebola , we developed a pair approximation model of Ebola transmission , parameterized by confirmed incidence data from June 2014 to January 2015 in Liberia and Sierra Leone . Our results suggest that if a combined intervention of case isolation and ring vaccination had been initiated in the early fall of 2014 , up to an additional 126 cases in Liberia and 560 cases in Sierra Leone could have been averted beyond case isolation alone . The marginal benefit of ring vaccination is predicted to be greatest in settings where there are more contacts per individual , greater clustering among individuals , when contact tracing has low efficacy or vaccination confers post-exposure protection . In such settings , ring vaccination can avert up to an additional 8% of Ebola cases . Accordingly , ring vaccination is predicted to offer a moderately beneficial supplement to ongoing non-pharmaceutical Ebola control efforts .
The Ebola outbreak in West Africa has resulted in unprecedented morbidity and mortality . As of March 4 2015 , the World Health Organization ( WHO ) had reported 23 , 914 cases and 9 , 792 fatalities in countries with widespread transmission [1] , with Liberia and Sierra Leone having been most profoundly impacted . Ebola transmission occurs via direct human-to-human contact with body fluids from symptomatic patients . An elevated viral load in late-stage symptomatic or deceased victims can also put family members and funeral attendees at risk of post-mortem disease transmission [2 , 3] . The health ministries in Liberia and Sierra Leone have been implementing intensive contact-tracing procedures , where patients or their relatives are interviewed to identify people with whom they came into close contact after developing symptoms . Contacts who are healthy but might have been exposed are monitored for 21 days , the maximum duration of the Ebola incubation period [4] . Contacts who present with Ebola symptoms , such as fever , are transported to isolation clinics [5] . Given that Ebola is transmitted directly between close contacts , the social clustering of individuals can be fundamental to the success of intervention strategies [6–8] . Several Ebola vaccine candidates have been developed in the past decade [9 , 10] , some of which have already been found to be safe and immunogenic in Phase 1 clinical trials [11] . One of these , a recombinant vesicular stomatitis viruses ( rVSV ) vaccine , conferred protection to non-human primates when administered immediately following exposure to an otherwise lethal dose of Ebola virus [12] . An alternate vaccine formula based on the chimpanzee adenovirus type 3 ( ChAd3 ) , developed in partnership between the National Institute of Allergy and Infectious Disease and GlaxoSmithKline , together with the rVSV vaccine are currently entering Phase 2/3 clinical trials in West Africa [13] . In addition , the WHO has deemed it ethical to use experimental vaccines in the current Ebola emergency situation [14] . Even with the scale up in production [15] , the supply would be insufficient for mass vaccination of affected countries , given a combined population of over twenty million people . Consequently , the judicious prioritization of vaccine recipients is essential to maximize vaccine impact . Aside from vaccinating healthcare workers who are at high occupational risk of contracting Ebola [16 , 17] , a vaccination strategy to reduce community-wide transmission has yet to be evaluated . Evaluating Ebola vaccination strategies is pertinent not only to the current epidemic , but also in mitigating future outbreaks . The targeting of the exposed contacts of infected individuals , a strategy known as ring vaccination [18 , 19] , is efficient for controlling rare pathogens [20] . For example , ring vaccination proved to be an effective strategy for smallpox eradication [18 , 19] . Furthermore , ring vaccination could be seamlessly incorporated into the contact tracing efforts underway in the affected countries . To evaluate the effectiveness of Ebola ring vaccination in West Africa , we developed a mathematical model that approximates disease progression in a realistic contact network . We predicted that the marginal benefit was greatest in settings where there are more contacts per individual , greater clustering , or insufficient resources for effective contact tracing . However , we found that ring vaccination provides moderate marginal benefit beyond current non-pharmaceutical interventions .
To determine the effectiveness of ring vaccination and case isolation of Ebola in West Africa , we modeled the transmission between close contacts by using the pair-approximation methodology which simulates disease propagation through a network ( Fig 1 , S1 Text ) [6 , 21] . Specifically , we tracked both the number of susceptible individuals ( [S] ) , latently infected individuals ( [E] ) , infectious individuals ( [I] ) , removed individuals ( [R] ) , as well as the number of contacts between epidemiological states . For example , [SI] denotes the number of contacts between susceptible and infectious individuals . We denoted the average number of contacts per individual in the network by k . To account for empirical mixing patterns between individuals during the Ebola outbreak in West Africa [22] , we considered clustering of individuals . Clustering can be defined as the extent to which individuals who are in contact with each other share other contacts in the network and is quantified by the clustering coefficient ( ϕ ) . A susceptible individual becomes latently infected ( E ) at rate β[SI] per day , where β is the transmission rate . An individual remains in the latent period for an average duration of 1/σ days until becoming symptomatic and infectious ( I ) . An infectious individual will transition to the removed state ( R ) ( i . e . recovered or deceased ) at rate δ , where 1/δ days is the average duration of the infectious period . In addition , we incorporated case isolation by removing a percentage of infectious individuals ( ψ ) from the community at rate γ per day ( Table 1 ) . For our base case analysis , we used k = 5 . 74 as the mean number of contacts and ϕ = 0 . 21 for the clustering coefficient , derived from contact tracing data collected by the Liberian Ministry of Health and Social Welfare [22] . We also considered clustering coefficients of 0 . 10 and 0 . 40 ( Table 1 ) , consistent with previous studies on human contact networks [23–26] . In addition , we accounted for possible under-reporting of contacts by considering higher values of k ( Table 1 ) . During contact tracing , an infected contact in the latent period moves to the observed state ( TE ) while a contact in the symptomatic period enters the isolated state ( TI ) at a daily contact tracing rate of τ per isolated case . Once an isolated individual has recovered and is no longer infectious , they transition to the TR state ( S1 Text ) , while the individuals contacts are followed for an additional 1/ω days ( Table 1 ) . We defined the contact tracing efficacy by the probability of identifying an infected individual before transmission occurs ( τ/ ( τ+ν ) ) , where 1/ν is the average serial interval ( Table 1 ) . We assumed that the base case contact tracing efficacy was 40% , consistent with empirical estimates [27 , 28] . We deemed the current Ebola epidemic to be eliminated at the point where incidence became lower than 0 . 025 cases per day , which corresponds to no new cases over a 42 day period [29] . d [ S ] d t = - β [ S I ] d [ E ] d t = β [ S I ] - τ ( [ E T I ] + [ E T R ] ) - σ [ E ] d [ I ] d t = σ [ E ] - ( 1 - ψ ) δ [ I ] - ψ γ [ I ] - τ ( [ I T I ] + [ I T R ] ) d [ R ] d t = ( 1 - ψ ) δ [ I ] + ω [ T R ] d [ T E ] d t = τ ( [ E T I ] + [ E T R ] ) - σ [ T E ] d [ T I ] d t = τ ( [ I T I ] + [ I T R ] ) - δ [ T I ] + σ [ T E ] + ψ γ [ I ] d [ T R ] d t = δ [ T I ] - ω [ T R ] d [ S I ] d t = - β ( [ S I ] + ( 1 - ϕ ) k - 1 k [ S I ] 2 [ S ] + ϕ k - 1 k N k [ S I ] 2 [ I I ] [ I ] 2 [ S ] ) - τ ( ( 1 - ϕ ) k - 1 k [ T I I ] [ S I ] [ I ] + ϕ k - 1 k N k [ T I I ] [ S I ] [ S T I ] [ S ] [ I ] [ T I ] ) -τ ( ( 1 - ϕ ) k - 1 k [ T R I ] [ S I ] [ I ] + ϕ k - 1 k N k [ T R I ] [ S I ] [ S T R ] [ S ] [ I ] [ T R ] ) +σ [ S E ] - ( 1 - ψ ) δ [ S I ] - ψ γ [ S I ] We assumed that a proportion of the contacts of isolated cases identified through contact tracing are vaccinated . Experimental studies indicate that several vaccine candidates facilitate recovery of latent infection in non-human primates if administered within two days post-infection [12 , 30] . We used a vaccine efficacy ( ɛ ) of 100% as our base case scenario and consider a range of vaccine efficacies from 5% to 95% ( S1 Text ) . We investigated two scenarios: 1 ) vaccination must be administered pre-exposure in order to confer protection such that only susceptible individuals ( S ) who are uninfected can be protectively vaccinated , and 2 ) vaccination is efficacious with both pre- and post-exposure administration such that vaccine protection can be conferred to individuals in both the susceptible ( S ) and latently infected ( E ) [30] . The use of a post-exposure vaccine in the equations below is represented by χ = 1 , otherwise χ = 0 . Susceptible and latently infected individuals are vaccinated at the same daily contact tracing rate τ per isolated individual ( S1 Text ) . When a susceptible individual is vaccinated they remain unprotected ( P ) until vaccine-mediated immunity is acquired 1/υ days later . d [ S ] d t = - β [ S I ] - τ ( [ S T I ] + [ S T R ] ) d [ E ] d t = β [ S I ] - τ ( [ E T I ] + [ E T R ] ) - σ [ E ] d [ I ] d t = σ [ E ] + σ [ F ] - ( 1 - ψ ) δ [ I ] - τ ( [ I T I ] + [ I T R ] ) - ψ γ [ I ] d [ R ] d t = ( 1 - ψ ) δ [ I ] + χ τ ( [ E T I ] + [ E T R ] ) + ω [ T R ] + υ [ P ] d [ P ] d t = τ ( [ S T I ] + [ S T R ] ) - β [ P I ] - υ [ P ] d [ F ] d t = β [ P I ] - τ ( [ F T I ] + [ F T R ] ) - σ [ F ] d [ T E ] d t = ( 1 - χ ) τ ( [ E T I ] + [ E T R ] ) + τ ( [ F T I ] + [ F T R ] ) - σ [ T E ] d [ T I ] d t = τ ( [ I T I ] + [ I T R ] ) - δ [ T I ] + σ [ T E ] + ψ γ [ I ] d [ T R ] d t = δ [ T I ] - ω [ T R ] To account for the recent decline in Ebola incidence [31 , 32] , we used a piecewise approach to calibrate our model , fitting both the early growth phase of the epidemic and the later phase of reduced transmission ( S1 Fig , S1 Table , S1 Text ) . We estimated the rate of transmission per infectious contact ( β ) , the date of Ebola emergence into the population ( t0 ) , the initiation of intervention scale up ( tS ) , and reduced transmission mediated by factors other than intervention , such as behavior change ( ξ ) . From the date of intervention scale up , we assumed that case isolation was implemented at our base case contact tracing efficacy . Using a least squares fitting algorithm , we estimated these four parameters from weekly confirmed incidence data in Liberia and Sierra Leone , respectively ( S1 Table and S1 Text ) . We fit the model to confirmed incidence data from June 8 , 2014 to January 4 , 2015 for Liberia [31] and from May 11 , 2014 to January 4 , 2015 for Sierra Leone [32] ( S1 Table and S1 Text ) . However , on December 20 , 2014 , the deployment of international aid began in Sierra Leone and considerably reduced transmission [33–35] . Thus , we considered a second reduction in transmission from this time point ( S1 Text ) . To validate the calibration of Liberia and Sierra Leone , we forecasted the incidence of Ebola to March 8 , 2015 and calculated the correlation fit value to the observed incidence ( S1 Fig , S1 Table ) .
We estimated that intervention efforts were initially scaled up on September 5 , 2014 in Liberia and October 8 , 2014 in Sierra Leone . Our model predicts that under status quo intervention the total number of confirmed Ebola cases in Liberia will be 3 , 899 cases and 10 , 425 cases in Sierra Leone ( Fig 2 ) . If ring vaccination using a prophylactic vaccine had been combined with the initial scaling up of non-pharmaceutical interventions , four additional cases could have been averted in Liberia and 36 cases could have been averted in Sierra Leone , corresponding to relatively low marginal benefits of 0 . 21% and 0 . 48% additional cases averted , respectively ( S2 Fig ) . By contrast , for a vaccine that can confer post-exposure protection , we estimate that 107 cases could have been averted in Liberia and 477 cases could be averted in Sierra Leone ( Fig 2 ) , corresponding to marginal benefits of 4 . 7% and 6 . 5% , respectively ( S3 Fig ) . We found that a prophylactic vaccine had minimal impact on the number of symptomatic individuals identified by contact tracing ( S4 Fig ) . However , if the vaccine confers post-exposure protection , then ring vaccination is predicted to reduce the number of symptomatic individuals identified by contact tracing by 40 in Liberia and 137 in Sierra Leone compared to case isolation alone , thereby reducing requirements of isolation units in hospitals ( S4 Fig ) . We conducted sensitivity analysis with respect to contact tracing efficacy , average number of contacts per individuals ( k ) , and the clustering coefficient ( ϕ ) ( S2 Text ) . Varying contact tracing efficacy from 5% to 50% , the predicted epidemic size without ring vaccination ranged from 3 , 874 to 3 , 988 confirmed cases in Liberia and 10 , 246 to 11 , 048 confirmed cases in Sierra Leone ( Fig 2 ) . Implementing ring vaccination with a prophylactic vaccine is expected to achieve the greatest marginal benefit when contact tracing efficacies are between 20% and 28% in both Liberia and Sierra Leone ( S2 Fig , S2 Text ) . Specifically , the greatest marginal benefit of ring vaccination can be up to 0 . 29% in Liberia and up to 0 . 64% in Sierra Leone , depending on the vaccine efficacy ( S2 Fig ) . However , a vaccine with post-exposure protection is estimated to provide up to 6% marginal benefit in Liberia and 8% in Sierra Leone ( S2 Text , S3 Fig ) . These results suggest that ring vaccination provides moderate benefit to case isolation under a range of contact tracing efficacies and vaccine efficacies . Our sensitivity analysis of the social mixing patterns ( k and ϕ ) show that the marginal benefit of adding ring vaccination rises with more contacts per individual or increasing clustering ( S2 and S5 Figs , S2 Text ) .
We demonstrated that a combination of contact tracing , case isolation and ring vaccination could effectively reduce Ebola transmission . In Liberia , as well as in past Ebola outbreaks , implementing case isolation along with change in human behavior dramatically reduced transmission [36–39] , which commonly occurs when people perceive infection risk associated with specific behaviors . Our results suggest that in this current context of reduced transmission , ring vaccination offers only moderate additional benefit , which is consistent with findings from a previous cholera model [40] . Although the incremental benefit of ring vaccination could be relatively moderate , the marginal benefit of ring vaccination is expected to increase with the average number of contacts per individual , the clustering coefficient or lower contact tracing efficacies . Therefore , ring vaccination is particularly useful in regions where contact tracing is logistically challenging . In addition , a vaccine that can confer post-exposure efficacy is much more effective for use in ring vaccination strategies than a vaccine that can only confer protection prophylactically . An additional benefit of ring vaccination that is more pronounced for a vaccine that confers post-exposure protection than for an exclusively prophylactic vaccine is the reduction in the number of symptomatic individuals who need hospitalization . The 2014 Ebola outbreak in West Africa has affected both rural and urban communities [41] , which differ in accessibility for contact tracing and hospital facilities for case isolation . There is also regional variation in the number and clustering of contacts [42] . Our analysis demonstrates that the benefit of adding ring vaccination to case isolation is influenced by the extent of clustering in a population . Specifically , we found ring vaccination provided the greatest marginal benefit to case isolation in highly clustered populations , such as crowded homes or schools [23–26 , 43] . As social networks can be highly complex systems consisting of densely connected communities [44–46] , future studies should evaluate not only transmission patterns within communities but also transmission patterns between rural and urban areas in order to tailor ring vaccination to specific locations . Clinical trials are underway to evaluate Ebola vaccine candidates , but there is currently considerable uncertainty regarding the efficacy post-exposure and prophylactically . Our analysis of vaccine efficacy , ranging between 5–100% , demonstrates a diminishing marginal contribution with declining vaccine efficacy . For example , at a hypothetical efficacy of 50% ring vaccination could have averted up to 214 cases , whereas a vaccine with an efficacy of 75% up to 360 cases of Ebola could have been averted . Our modeling approach is based on a deterministic approximation to a stochastic network model . Consequently , our model did not capture the stochastic effects that become pronounced in the eradication phase of an outbreak [6] and for ring vaccination strategies that include second-order ring vaccination , where contacts of exposed individuals are vaccinated in addition to the exposed contacts of the isolated case . We would expect an increase in the marginal benefit of ring vaccination if both first-order and second-order vaccination were implemented because vaccination would be administered ahead of the wave of transmission . Thus , our results are conservative with regard to second-order ring vaccination . Ebola has similar family and household transmission as smallpox . During the final eradication phase of smallpox , public health authorities relied on case isolation and ring vaccination [18 , 19 , 47 , 48] . Because smallpox is more infectious than Ebola [2 , 49 , 50] , we conservatively expect that case isolation and ring vaccination would likewise be a practical approach to eliminate Ebola and contain future outbreaks , especially with limited vaccine supplies . The combined implementation of ring vaccination and case isolation can be an effective approach in curtailing an Ebola outbreak . Our model predicts that ring vaccination offers moderate benefit to case isolation , with the greatest benefit occurring where there are more contacts per individual or greater clustering among individuals , and when contact tracing has low efficacy or vaccination can confer post-exposure protection . | Public health efforts for controlling the 2014–2015 Ebola outbreak in West Africa have focused on contact tracing and isolation of symptomatic individuals . In addition , substantial resources have been committed to scaling up the production of experimental vaccines . Ring vaccination—the vaccination of the contacts of an infected individual—was successfully implemented to achieve smallpox eradication . Ring vaccination is particularly feasible and effective in settings where the supply of vaccines is limited and disease incidence is low . Using a disease transmission model , we evaluated the benefit of adding ring vaccination to case isolation in Liberia and Sierra Leone . We found that ring vaccination could have averted up to 126 cases in Liberia and 560 cases in Sierra Leone , thereby saving lives and intervention resources . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [] | 2015 | Harnessing Case Isolation and Ring Vaccination to Control Ebola |
Large fractions of eukaryotic genomes contain repetitive sequences of which the vast majority is derived from transposable elements ( TEs ) . In order to inactivate those potentially harmful elements , host organisms silence TEs via methylation of transposon DNA and packaging into chromatin associated with repressive histone marks . The contribution of individual histone modifications in this process is not completely resolved . Therefore , we aimed to define the role of reversible histone acetylation , a modification commonly associated with transcriptional activity , in transcriptional regulation of murine TEs . We surveyed histone acetylation patterns and expression levels of ten different murine TEs in mouse fibroblasts with altered histone acetylation levels , which was achieved via chemical HDAC inhibition with trichostatin A ( TSA ) , or genetic inactivation of the major deacetylase HDAC1 . We found that one LTR retrotransposon family encompassing virus-like 30S elements ( VL30 ) showed significant histone H3 hyperacetylation and strong transcriptional activation in response to TSA treatment . Analysis of VL30 transcripts revealed that increased VL30 transcription is due to enhanced expression of a limited number of genomic elements , with one locus being particularly responsive to HDAC inhibition . Importantly , transcriptional induction of VL30 was entirely dependent on the activation of MAP kinase pathways , resulting in serine 10 phosphorylation at histone H3 . Stimulation of MAP kinase cascades together with HDAC inhibition led to simultaneous phosphorylation and acetylation ( phosphoacetylation ) of histone H3 at the VL30 regulatory region . The presence of the phosphoacetylation mark at VL30 LTRs was linked with full transcriptional activation of the mobile element . Our data indicate that the activity of different TEs is controlled by distinct chromatin modifications . We show that activation of a specific mobile element is linked to a dual epigenetic mark and propose a model whereby phosphoacetylation of histone H3 is crucial for full transcriptional activation of VL30 elements .
Our present view on transcriptional regulation has substantially advanced in recent decades . The classical model , that the presence of promoter sequences and the availability of transcription factors determine the expression status of corresponding genes , has been extended to a model , in which the accessibility of the DNA is central to transcriptional control . In eukaryotes , DNA is packed and compacted into chromatin with the nucleosome consisting of DNA and histone proteins as the basic unit . The degree of compaction – either into inaccessible heterochromatin or open euchromatin – has major implications for the transcriptional potential of associated DNA . A way to regulate chromatin accessibility is the posttranslational chemical modification of histone proteins . It can alter chromatin structure and switch genes from a transcriptional repressed to an active state and vice versa [1] . The best-studied histone modification is the acetylation of N-terminal lysine residues , which correlates with open chromatin and active gene transcription . In contrast , histone deacetylation is linked to gene silencing and histone deacetylases ( HDACs ) are considered as transcriptional co-repressors [2] . Only recently , a more general role of histone deacetylation during transcriptional regulation is emerging [3] . The use of chemical HDAC inhibitors and knock out/down technologies resulted in the identification of an increasing number of target genes controlled by histone acetylation in different cellular contexts [4]–[6] . Based on sequence homology , mammalian HDACs have been divided into four classes , of which class I enzymes seem to fulfil more basic cellular functions whereas class II enzymes are thought to play specialised cell-type specific roles . HDACs are often found to interact with proteins important for stable gene silencing via DNA methylation and constitution of heterochromatin , but also with transcriptional regulators mediating only transient repression . Crosstalk between histone acetylation and other epigenetic marks is an important feature of HDAC function [7] . Hence , HDACs are central components of multiple silencing complexes containing additional enzymatic activities such as DNA and histone methylation . Recent annotation of multiple complete genomes has revealed that a large fraction of eukaryotic genomes consists of repetitive sequences , mainly derived from transposable elements ( TEs ) [8] , [9] . Most of those sequences are remnants of once active TEs now incapable of transposition because of their host-mediated inactivation followed by subsequent functional erosion and the accumulation of mutations and deletions . However , some elements remain intact and constitute a constant threat to the integrity of the host genome . Potentially functional elements can act as insertion-mutagens via targeting protein coding genes or causing chromosome breakage . Even transpositionally inactive TEs have the potential to trigger illegitimate recombination and genome rearrangement , or to influence neighbouring genes by causing alternative splicing , premature termination , and modulating gene expression patterns [10] , [11] . Recent estimations suggest that TEs provoke around 0 . 1% ( in humans ) or 10–12% ( in mice ) of all spontaneous germ line mutations , and an increasing number of reports document the contribution of somatic transposon activity to pathological situations [11]–[13] . Consequently , organisms were challenged to evolve multiple lines of defence to restrict TE activity , targeting crucial steps in their life cycle [11] , [14] . One of the most efficient host-directed strategies is to block TE transcription . This is achieved applying epigenetic mechanisms such as DNA methylation , modification of chromatin structure , and the action of small RNAs [13] , [15]–[19] . Different subclasses of small RNAs ( siRNAs , rasiRNAs and piRNAs ) seem to guide and target the silencing machinery , while chromatin modifiers and DNA methyltransferases ( DNMTs ) accomplish the transformation into a heterochromatic , transcriptionally silent state [20] . Numerous proteins , involved in the establishment , maintenance and read-out of DNA methylation and chromatin modification patterns , are crucial for TE silencing in germline and somatic cells [13] . However , the exact contribution of DNA methylation , chromatin remodelling , modification of histones and their interplay has not been completely resolved . Analysis in several model organisms revealed that the different silencing-mechanisms are of varying importance depending on the host organism . Some ( e . g . yeast and Drosophila ) lack an efficient DNA methylation machinery and therefore depend on alternative silencing pathways , while others ( e . g . mammals and plants ) widely use DNA methylation in concert with other mechanisms to silence TEs [18] , [21] . Despite the tight collaboration of multiple silencing layers , numerous TEs maintain their capacity to escape host-mediated silencing under certain conditions . Different types of internal and external stimuli are able to trigger TE activity in a wide range of organisms . Many of those stimuli can be subsumed as cellular stress and the correlation between TE activity and stress has been in the focus of interest for several decades [22] , [23] . Recent examples of mammalian TEs , showing increased transcriptional activity upon external stimuli , include members of all major TE orders: SINEs [24] , [25] , LINEs [26] , and LTR elements [27] . To better understand the role of histone acetylation in transcriptional regulation of TEs in mammalian somatic cells , we monitored the expression of selected , potentially active TEs in mouse fibroblasts with impaired histone deacetylase capacity . In our present study we found that chemical inhibition of class I and II HDACs , but not genetic inactivation of HDAC1 alone , resulted in local hyperacetylation and expression of a defined subset of VL30 LTR retrotransposable elements . Furthermore , we show that not only histone acetylation , but histone phosphorylation in concert with acetylation ( phosphoacetylation ) was triggering transcriptional induction of VL30 elements . We suggest a model , in which external stress signals cause the activation of MAP kinase pathways , ultimately leading to the phosphoacetylation of histones and efficient transcription of VL30 elements .
To confirm that our experimental setup indeed perturbed epigenetic states in mouse fibroblasts , we performed the following experiments: firstly , we monitored the expression levels of proteins targeted by chemical or genetic inhibition . Protein expression levels of the class I deacetylases HDAC1 , HDAC2 , HDAC3 and the maintenance DNA methyltransferase DNMT1 were surveyed via Western blot . As shown in Figure 1A , loss of HDAC1 in immortalized fibroblasts led to upregulation of HDAC2 , its closest homologue . This compensatory mechanism may buffer some effects caused by the loss of HDAC1 , but still total cellular HDAC activity was diminished by about 30% in HDAC1−/− compared to HDAC1+/+ cell lines ( data not shown ) , demonstrating an important enzymatic function of HDAC1 in fibroblasts . In our experimental setup TSA treatment for 24h did not influence expression of HDAC1 , HDAC2 or HDAC3 , whereas DNMT1 levels were slightly decreased . A reduction of DNMT1 levels after histone deacetylase inhibitor treatment has been previously reported [28] , [29] . Aza-dC treatment resulted in slightly reduced amounts of DNMT1 , consistent with earlier reports showing that Aza-dC can trap DNMT1 by covalently linking the enzyme to DNA [30] or lead to proteasomal degradation of the DNMT1 protein [31] . Protein expression of HDAC1 , HDAC2 and HDAC3 remained unaffected . Next , we evaluated effects on the biological targets of HDACs and DNMT1 , i . e . the acetylation states of histones and CpG methylation levels of DNA . To examine CpG methylation levels at defined targets we employed the MS-SNuPE technique [32] . We quantified DNA methylation levels of three CpG sites within the regulatory LTR region of an IAP retrotransposons , highly methylated in somatic cells [33] . Whereas neither loss of HDAC1 , nor chemical inhibition of HDACs with TSA for 24h affected levels of DNA methylation , Aza-dC treatment for 24h severely reduced DNA methylation of the IAP LTR region to 50% ( Figure 1B ) . Furthermore , we analysed histone acetylation levels in wildtype fibroblasts , either untreated ( HDAC1+/+ ) or treated with TSA and Aza-dC and HDAC1-deficient cells ( HDAC1−/− ) . To that end we extracted histones and performed Western blot analyses using antibodies recognising acetylated histones . Previous reports had shown that in murine embryonic stem cells genetic inactivation of Hdac1 was accompanied by hyperacetylation of histones H3 and H4 , detectable with antibodies recognising acetylated histone H3 ( H3ac ) and acetylated histone H4 proteins ( H4ac ) [34] . In our fibroblast system , however , we failed to detect global hyperacetylation of histones in HDAC1−/− cells as compared to HDAC1+/+ cells using such H3ac or H4ac antibodies ( Figure 1C ) ; only when we probed the blots with antibodies specifically recognising the acetylation state of individual lysine residues , we detected weak hyperacetylation occurring at defined residues ( data not shown ) . In contrast to this limited site-specific response , inhibition of all class I and II HDACs with TSA led to a robust and global hyperacetylation detectable with H3ac and H4ac antibodies . Finally , interfering with DNA methylation by Aza-dC treatment moderately enhanced global H3 and H4 acetylation levels ( Figure 1C ) . After defining global effects of HDAC and DNMT inhibition in our cell system , we moved on to specifically address the role of histone acetylation in the regulation of TEs . We analysed the acetylation state of chromatin associated with defined TEs in wildtype fibroblasts without treatment ( HDAC1+/+ ) , or treated either with TSA or Aza-dC , and HDAC1-deficient fibroblasts ( HDAC1−/− ) . To monitor their acetylation status , we performed chromatin-immunoprecipitation ( ChIP ) experiments with H3ac and H4ac antibodies . Furthermore , we included antibodies recognising the C-terminal part of histone H3 to survey nucleosome occupancy and unspecific rabbit IgGs to control the specificity of immunoprecipitation reactions . Quantitative Real-Time PCR analyses allowed the quantification of H3 and H4 acetylation levels associated with a representative selection of ten murine TEs , including a DNA transposon ( Tigger ) , non-LTR retrotransposons ( LINEs , SINEs ) , and LTR retrotransposons ( VL30 , IAP , ETn , MERVL , MT , ORR1 ) ( Table 1 ) . We chose LINEs and SINEs , because they constitute the majority of murine TEs and populate the genome in more than 660 , 000 and 1 . 5 million copies , respectively . LTR elements are less abundant , but harbour the most active TEs within the mouse lineage; MERVL , MT and ORR1 elements are expressed during early embryogenesis [35] and VL30 , IAP and ETn elements have been responsible for a number of germ line transposition events [12] . The transposon Tigger was included as representative of the class of DNA elements , which is functionally extinct in vertebrates . As shown in Figure 2A ( upper panel ) , loss of HDAC1 did not influence histone H3 acetylation at any transposon type analysed as compared to wildtype cells . In contrast , TSA treatment was sufficient to specifically enhance H3 acetylation at SINE B1 and B2 elements and VL30 LTR elements , the latter ones near the promoter as well as throughout the body of the element . Histone H4 acetylation of TE-associated chromatin was analysed similarly in a subsequent series of experiments . Again , loss of HDAC1 did not alter histone acetylation at tested transposons , but TSA-induced inhibition of all class I and II HDACs led to increased H4 acetylation at all repeat families investigated ( Figure 2A; lower panel ) . Thus , TSA treatment exhibited differential effects on histone H3 and histone H4 acetylation of TEs . Whereas H3 histones were found hyperacetylated at specific elements only , H4 hyperacetylation occurred as a global response . Interestingly , the elements hyperacetylated at H3 upon TSA treatment , SINEs and VL30 , also showed highest enrichment of H4 acetylation compared to other TEs . Next , we analysed histone acetylation levels upon loss of DNA methylation . Incubation of cells with Aza-dC increased H3 acetylation at SINE B1 and VL30 elements ( Figure 2B , upper panel ) , although to a lesser extent than TSA . Furthermore , IAP element promoter sequences exhibited slightly increased H3 acetylation levels in response to Aza-dC . Finally , Aza-dC treatment resulted in elevated H4 acetylation levels of SINEs , VL30 and IAP elements ( Figure 2B; lower panel ) . Since hyperacetylation of histones in general correlates with transcriptional activation , we tested whether increased histone acetylation levels were mirrored by changes in transcriptional activity of TEs . Therefore we monitored mRNA levels of the ten mouse transposons , listed in Table 1 , in untreated wildtype fibroblasts ( HDAC1+/+ ) , TSA or Aza-dC treated wildtype cells , and HDAC1-deficient fibroblasts ( HDAC1−/− ) by real-time PCR analyses . We also analysed the expression of another LTR element , MuLV , which comprises endogenous as well as exogenous family members [36] ( Figure S1 ) . As shown in Figure 3 , upper panel and Figure S1 , loss of HDAC1 did not influence TE expression . In contrast , general HDAC inhibition via TSA strongly increased VL30 element expression in this fibroblast cell line ( Figure 3 , upper panel ) , as well as in other commonly used fibroblast cell lines such as Swiss 3T3 and NIH/3T3 ( data not shown ) . Enhanced activity of VL30 elements correlated with hyperacetylation of VL30 chromatin upon TSA treatment . This suggests that modulation of histone acetylation regulates VL30 element activity . Expression of SINE B1 , the second element showing H3 and H4 hyperacetylation after TSA treatment , was modestly increased . Besides IAPs showing a moderate two-fold increase , all other TE candidates investigated did not exhibit major changes in transcriptional activity . This expression pattern mirrors the unchanged local acetylation state of histone H3 , but not the one of histone H4 acetylation , which was elevated upon TSA treatment . Therefore , H3 acetylation of the transposons analysed correlates with transcriptional activity , whereas H4 hyperacetylation is not sufficient to trigger transcriptional activation . The role of DNA methylation in transposon silencing is well established and it has been shown earlier that loss of DNA methylation in somatic cells correlates with VL30 , IAP and MuLV expression [37]–[39] . Indeed , Aza-dC treatment in fibroblasts caused highly elevated VL30 , IAP and MuLV mRNA levels , but had no effect on other TEs tested ( Figure 3 , lower panel and Figure S1 ) . However , it was surprising that the majority of murine elements tested could not be reactivated with Aza-dC . This finding suggests that those transposons are not permissive for transcriptional activation in our fibroblast system , either because they lack regulatory features necessary for efficient transcriptional activation or additional mechanisms contribute to their silencing [40] . ChIP analyses of TE-associated chromatin showed that two elements that were highly expressed upon Aza-dC treatment ( VL30 and IAP elements ) also gained hyperacetylation after DNMT inhibition ( Figure 3 , lower panel ) . SINE B1 and SINE B2 elements , both showing increased acetylation upon Aza-dC treatment , were not or only modestly activated . In summary , we could establish a correlation between the transcriptional activity of TEs and the acetylation state of associated chromatin . In particular histone H3 acetylation correlated well with increased expression of certain TEs . Furthermore , we identified VL30 elements as the single candidate transposon efficiently regulated via acetylation of associated chromatin . Retrotransposition is a complex process depending on multiple steps , each of which can be targeted by regulatory mechanisms [11] . Alterations in RNA stability , translation efficiency , nuclear shuttling , editing of template RNA , or integration efficiency can have severe impact on the rate of successful transposition . Treatment of cells with the HDAC inhibitor TSA has strong effects on the chromatin state . Since chromatin constitutes the substrate for the last step of transposition , i . e . integration , we tested whether histone modification patterns might influence transposition efficiency , independent of TE transcription . Genetic screens in yeast have uncovered numerous host factors either restricting or enhancing transposon activity [41]–[45] . Strikingly , a substantial fraction of the proteins identified has a role in chromatin biology; for instance , SIN3 and RPD3 , both major components of yeast HDAC complexes , were found to restrict Ty1 activity and modulate target site selection . Interestingly , SIN3 and RPD3 do not act on the transcriptional level , as they do not alter Ty1 mRNA levels [45] . To test whether changed histone acetylation levels induced by TSA treatment might have similar effects in mammalian cells , we determined transposition levels of four mammalian retroelements in untreated and TSA treated cells . To this end we took advantage of an experimental system described earlier [46]–[48] , in which episomally delivered retrotransposons are marked with a neo-transgene , which gets activated only after its successful retrotransposition ( Figure S2A ) . Following this approach we quantified the transposition efficiency of murine IAP and MusD LTR retrotransposons , and murine and human L1 elements in untreated and TSA treated cells . In order to avoid potential interference with endogenously transcribed IAP , MusD and L1 elements , we performed the experiments in the human U2OS osteosarcoma cell line . In addition , we monitored transcript levels of episomally delivered retroelements via real-time PCR ( Figure S2B ) . As presented in Figure S2C , TSA treatment led to a 2- to 4-fold increase in transposition rates , a moderate increase compared to findings reported elsewhere , where mutation of TE restricting factors in yeast led to an increase between 5 and several hundred times [45] . In our system , enhanced transposition was accompanied by a 2- to 3-fold increase in TE mRNA abundance . These data suggest that TSA treatment led to enhanced expression of marked elements , which in turn resulted in increased transposition rates . Therefore , under our experimental conditions , inhibition of deacetylases by TSA was not sufficient to influence TE transposition rates independent of TE expression , suggesting transcription as the rate-limiting factor for transposition . In contrast to single copy genes , TEs exist in multiple copies dispersed throughout the genome . Individual elements within a TE family are different in respect of genomic environment , influencing chromatin status and transcriptional potential . Furthermore , transposons are under high selective pressure and mutations mediated by host and/or transposon-intrinsic factors lead to the generation of pools of elements with individual sequence specificities within given TE families . Therefore , a family of elements is a collection of similar , but heterogeneous individuals , differing in external and internal features . Upon external signals increased transcriptional activity could originate from the upregulation of all TEs within a family , of a subgroup of elements exhibiting specific features , or even a single copy with unique intrinsic properties . In order to distinguish between these different scenarios , we aimed to determine the chromosomal origin of VL30 and IAP derived transcripts detected in untreated , TSA and Aza-dC treated wildtype fibroblasts . RNA from three independent biological samples each ( untreated , TSA , Aza-dC ) was collected , reversely transcribed , and subjected to PCR amplification . To amplify IAP derived transcripts , primers annealing in the highly conserved gag region were used ( Figure S3A ) . For VL30 transcript amplification primers within the 3′ UTR and 3′LTR R region were chosen ( Figure 4A ) . To exclude a bias due to individual PCR reactions , six independent PCR reactions for each condition were pooled , PCR products were cloned and sequenced . As control , the same procedure was also performed with genomic DNA as template . This approach allowed us to amplify chromosomal copies of both candidate elements and to estimate the diversity of IAP and VL30 elements in our cell system . Each sequenced clone corresponding to an individual transcribed ( or genomic ) TE was then analysed as follows: to identify genomic elements serving as potential sources of transcription , sequences were blasted against the annotated murine genome ( http://www . ncbi . nlm . nih . gov/blast/Blast . cgi ) . Genomic sequences exhibiting at least 98% identity to the cloned sequences were considered to show sufficient homology to serve as potential sources of transcription and were analysed further . If more than one genomic sequence showed identity >98% , the sequence with the highest E-value was chosen . That way ∼90% of the cloned sequences were assigned to distinct genomic elements ( IAP: 115 of 127 clones; VL30: 111 of 122 clones ) . Individual characteristics , such as chromosomal localisation and length were examined for each element as summarised in Table S1 . Next , we defined whether the transcribed IAP and VL30 elements were derived from multiple genomic sources , a small number of elements , or even unique elements . Therefore , we analysed the diversity of the identified genomic TEs , constituting bona fide sources of transposon transcription . Following this approach , each IAP transcript could be assigned to a different genomic element , indicating that IAP transcripts originated from multiple loci throughout the genome ( see chromosomal origins and individual features of elements listed in Table S1 ) . In untreated cells , VL30 transcripts were of heterogeneous origin , with a broad level of sequence diversity similar to the chromosomal VL30 elements that were amplified from genomic DNA . In contrast , upon TSA and Aza-dC treatment , VL30 transcripts could be assigned to only four and two genomic loci , pointing towards a limited number of VL30 elements serving as source of expression . TSA treatment led to the accumulation of transcripts related to the genomic loci NT_039207 4840bp on chromosome 2 ( 36 of 41 sequences ) , NT_039341 4902bp on chromosome 6 ( 3 of 41 ) , NT_039462 5167bp on chromosome 8 ( 1 of 41 ) , and NT_039649 4919bp on chromosome 17 ( 1 of 41 ) . Aza-dC treatment resulted in transcripts similar to NT_039207 4840bp on chromosome 2 ( 13 of 26 sequences ) and NT_039341 4902bp on chromosome 6 ( 13 of 26 ) ( Table S1 ) , Alignment of VL30 sequences directly obtained after cloning and sequencing further showed that transcripts assigned to the genomic locus on chromosome 2 ( NT_039207 4840bp ) were almost identical , indicating a single locus as transcriptional source . In contrast , transcripts assigned to the locus on chromosome 6 ( NT_39341 4902bp ) were slightly divergent , demonstrating that they originate from a group of related VL30 elements , of which only one copy is annotated in the published mouse genome ( data not shown ) . The long terminal repeat regions of LTR elements are enriched with regulatory sequences important for transcriptional regulation . Therefore , we determined the structure of the LTR regions associated with expressed IAP and VL30 elements in more detail . First , we analysed the LTRs associated with transcribed IAP elements , which revealed a marked length polymorphism between different LTRs: the shortest LTR , identified with an IAP element expressed in untreated fibroblasts , comprised 322bp , whereas the longest LTR , found in an IAP element expressed after Aza-dC treatment , was 456bp long . Alignment of IAP LTR sequences disclosed that this difference was due to the presence or absence of nucleotides in a highly variable CT-dinucleotide-rich stretch within the R region of the LTR ( Table S1 and Figure S3B ) . The region encoded a varying number of tandem repeat sequences containing a 13bp core sequence ( TCTCTCTTGCTTC ) , previously described as polymorphic marker for different IAP subtypes [49] . Interestingly , the average length of this stretch was significantly longer in LTRs associated with IAPs expressed upon Aza-dC treatment ( Figure S3C ) , indicating that upon loss of DNA methylation an increased number of tandem repeats might be favourable for expression . As shown in earlier studies the structure of the VL30 LTR region is crucial for stimulus mediated activation of VL30 expression ( reviewed in [27] ) . Based on their highly divergent U3 regions , VL30 LTRs have been classified into four distinct subtypes: I , II , III and IV [50] . To allocate the VL30 elements isolated in our transcript analysis to the four subtypes , we isolated the 5′LTR sequences from the genomic sequences , aligned them together with VL30 LTR reference sequences ( NVL3: subclass I , BVL1: subclass III , NVL1/2: subclass IV ) and generated a phylogenetic tree . As depicted in Figure 4B , all elements including the references were located in three distinct clades corresponding to subclass I , III and IV elements . Elements expressed in untreated cells mainly comprehend subtype III containing LTRs , which also associate with most genomic elements ( Figure 4B and Table S1 ) . As described above , transcripts arising upon TSA and Aza-dC treatment , mainly originate from two types of loci: one VL30 insertion on chromosome 2 ( NT_039207 4840bp ) and several other loci showing high similarity to another element on chromosome 6 ( NT_039341 4902bp ) . Interestingly , whereas the VL30 element on chromosome 6 is flanked by LTRs containing subtype I U3 regions at both ends , the element located on chromosome 2 exhibits an unusual structural feature: it is associated with two different LTRs , a 5′ LTR containing a U3 region belonging to subtype I , but a 3′ LTR containing a U3 region of subtype IV ( Figure 4A , lower part ) . Transposon transcription depends on the 5′ LTR , therefore we conclude that expression of this VL30 element is controlled by LTR sequences comprising a subtype I U3 region . 40 out of 41 transcripts isolated from TSA treated fibroblasts were associated with a LTR containing a subtype I U3 region , suggesting that this LTR design might be favourable for VL30 expression upon TSA treatment . However , 36 of those 40 were derived from a single genomic source . Therefore we wanted to clarify if increased VL30 transcription upon TSA treatment is exclusively caused by activation of this particular locus mirroring a locus specific phenomenon or if it reflects a general feature of subtype I elements . We surveyed the chromatin signature at the 5′LTRs of five genomic VL30 elements via ChIP using flanking primers: the highly responsive hybrid element on chromosome 2 ( NT_039207 4840bp ) , two other subtype I elements ( NT_039649 4919bp , NT_039341 4902bp ) , one subtype III ( NT039589 5225bp ) and one subtype IV element ( MW_001030889 5248bp ) ( Figure 4C ) . Interestingly , we detected increased levels of H3K4me3 ( a mark for active promoters ) upon TSA treatment at all five VL30 elements , with the highest levels at the hybrid element , followed by the two other subclass I elements ( Figure 4C ) . The subclass III and IV element showed the lowest response . A similar pattern could be observed for H3ac and H4ac . In addition we monitored the repressive histone mark H3K9me2 , which was low in all elements and remained unchanged upon TSA treatment . Another repressive mark , H3K27me3 showed variable levels with no clear correlation with VL30 subtypes . Together these data suggest an increased responsiveness to activation of the hybrid element , followed by other subclass I elements and finally subclass III and IV elements . Based on the finding that VL30 elements respond to histone acetylation , we examined the potential activation mechanism for VL30 elements in detail . In the recent past VL30 elements have been studied extensively and others showed that VL30 elements expression is highly sensitive to a plethora of internal and external stimuli [27] , [39] , [51] . Interestingly , many of those stimuli can be described as internal or external stress in the broadest sense , and a considerable number among them is able to trigger signalling cascades such as the ERK or p38 pathways . Signalling via these pathways ultimately leads to phosphorylation and activation of several transcription factors but also to phosphorylation of histone H3 . Phosphorylation of serine 10 at histone H3 ( H3S10p ) during interphase is a rare event that correlates with the activation of specific target genes [52] , [53] . Importantly , the presence of the H3S10ph mark at promoter-associated chromatin coincides with acetylation of neighbouring lysines K9 and K14 leading to a dual histone modification termed phosphoacetylation [54]–[56] ( Figure 5A ) . Histone H3 phosphoacetylation correlates with the activation of the so called “immediate early” genes c-fos and jun-B , as well as late inducible genes such as HDAC1 [57] . The finding that the HDAC inhibitor TSA was sufficient to highly induce VL30 expression in logarithmically growing fibroblasts , where ERK pathways are activated through the action of growth factors , prompted us to ask whether both stimuli , histone acetylation and histone phosphorylation , were required for full VL30 activation . To answer this question , we performed all following experiments with serum-arrested Swiss 3T3 fibroblasts . Serum withdrawal arrests cells in G0 and eliminates mitotic H3 phosphorylation , a modification that targets the majority of histone H3 molecules during mitosis and is associated with chromatin condensation . Furthermore , the absence of the serum-dependent activity of different kinase pathways allows to selectively activate specific signalling cascades , thereby triggering H3 phosphorylation . To induce the ERK pathway in fibroblasts , cells were arrested in G0 for 72h and then treated with foetal calf serum ( FCS ) . Alternatively , anisomycin ( Aniso ) was used to activate the p38 stress pathway . Additionally cells were treated with TSA to induce hyperacetylation . To analyse simultaneous phosphorylation and acetylation of histone H3 we used an antibody that recognises histone H3 only in the presence of the dual H3S10phK14ac mark ( Figure 5A , Figure S4 ) . Figure 5B shows the effect of different compounds on histone H4 acetylation and histone H3 phosphoacetylation in G0 arrested Swiss 3T3 fibroblasts . As expected , TSA led to the accumulation of hyperacetylated histones as detected by the H4ac antibody; H3 phosphoacetylation was low in untreated and TSA treated G0 cells and slightly induced upon Aniso or FCS treatment . Combined treatment with Aniso/TSA or FCS/TSA led to enhanced phosphoacetylation of histone H3 . It was shown previously that , even upon Aniso/TSA stimulation , the fraction of phosphoacetylated histone H3 is only minute [52] , [58] and that the mark is localised to a restricted number of genomic loci in Aniso/TSA treated fibroblasts ( Simboeck E . , unpublished results ) . Hence we tested , if phosphoacetylated histones were associated with VL30 elements . To this end we isolated chromatin from G0 arrested fibroblasts that were untreated or treated with TSA , Aniso , or Aniso/TSA . We performed ChIP experiments using the H3S10ph/H3K14ac antibody or an unspecific control antibody and surveyed phosphoacetylation levels of the VL30 5′LTR regulatory region via semi-quantitative PCR . Strikingly , we could detect phosphoacetylated histones at VL30 elements at low levels when cells were treated with Aniso alone , and at higher levels upon Aniso/TSA treatment , but not in untreated or TSA-treated cells ( Figure 5C ) . Additional ChIP experiments with resting cells activated with growth factors , containing serum in combination with TSA ( FCS/TSA ) , demonstrated the presence of phosphoacetylation also upon activation of the ERK pathway , together with induction of hyperacetylation ( Figure 5D ) . In summary , activation of the p38 or ERK pathway together with induction of hyperacetylation resulted in the establishment of a phosphoacetylation mark at VL30 LTRs . In agreement with these findings we detected an increased presence of the H3S10phK14ac mark at individual VL30 insertions in TSA-treated proliferating HDAC1+/+ fibroblasts suggesting a direct link between this dual histone modification and transcriptional induction of particular VL30 elements ( Figure S5 ) . Next , we examined the effect of the different stimuli on VL30 expression . We isolated RNA from arrested 3T3 Swiss fibroblasts exposed to the aforementioned inducers , reversely transcribed them and quantified VL30 levels with Real Time PCR . Whereas single treatment of cells with TSA , Aniso , or FCS led to a modest increase in expression , combined treatment with Aniso/TSA or FCS/TSA resulted in robust induction , suggesting a synergistic effect between histone phosphorylation via p38 or ERK activation and histone acetylation ( Figure 5E–5G ) . To directly test whether activation of the ERK and p38 pathways is linked to H3 phosphoacetylation and VL30 activation , we took advantage of the protein kinase inhibitor H89 . This compound was used at a concentration ( 10µM ) , shown to efficiently block histone H3 phosphorylation without affecting transcription factor phosphorylation [53] . As presented in Figure 6A , inhibition of kinase activity via H89 abolished the formation of a phosphoacetylation mark upon Aniso/TSA treatment in arrested Swiss 3T3 fibroblasts . Equally important , H89 treatment also prevented transcriptional induction upon Aniso/TSA treatment ( Figure 6B ) , indicating that H3S10p/H3K14ac does not only correlate with transcriptional induction mediated by Aniso/TSA treatment , but is necessary for proper VL30 expression . Finally , we want to propose the following model for VL30 regulation , which integrates many of the previous findings about VL30 induction: external stimuli activate the p38 ( stress ) or the ERK ( growth signal ) pathway leading to activation of effector kinases such as MSK1/2 . By local phosphorylation of H3S10 at responsive VL30 elements together with the induction of hyperacetylation , a phosphoacetylation mark is established leading to full transcriptional activation ( Figure 7 ) .
Having monitored global changes mediated by TSA and Aza-dC treatment , we focused our interest on the interrelation between histone acetylation and TE regulation . We determined histone acetylation levels of a representative selection of TEs in HDAC1−/− background , upon TSA and Aza-dC treatment . Subsequently , we correlated these data with the expression levels of the corresponding TEs . This analysis revealed the following key findings: ( 1 ) only three out of eleven TEs tested were significantly activated by Aza-dC treatment . Inhibition of DNA methyltransferases in fibroblasts strongly enhanced the expression of IAP , VL30 and MuLV LTR elements . These data indicate that a block in DNA methylation was sufficient to activate transcription of some , but not all TEs in fibroblasts . Efficient transcriptional activation of other TEs might depend on activating cofactors absent in fibroblasts . This is consistent with the finding that many TEs exhibit tissue- or cell type-specific expression patterns [67]–[69] . Alternatively , additional layers of silencing mechanisms directed against specific TE families could be present in fibroblasts in the absence of DNA methylation . ( 2 ) Enhanced expression roughly correlated with increased acetylation . This was obvious for VL30 elements , which were highly expressed upon TSA and Aza-dC treatment , and for IAP elements , which were induced after treatment with Aza-dC: both LTR elements exhibited hyperacetylation when activated . Remarkably , loss of DNA methylation at IAP elements was accompanied by increased acetylation , whereas HDAC inhibition by TSA did not result in IAP-associated histone hyperacetylation . This suggests a model , in which histone acetyltransferase ( HAT ) activity is tethered to IAPs only upon loss of DNA methylation , which otherwise might prohibit the binding of transcription factors recruiting HATs . Hence , HDAC inhibition in cells with intact DNA methylation was not sufficient to induce local hyperacetylation . The situation of VL30 element regulation was strikingly different . Inhibition of HDAC activity led to robust hyperacetylation , pointing towards a direct involvement of HDACs and HATs in the transcriptional control of VL30 elements . Finally , we observed that SINE elements gained hyperacetylation upon Aza-dC and TSA treatment , which was accompanied with mild effects on transcription . However , SINEs posses some peculiar features , making them distinct from other TEs: they are short , highly repetitive sequences , which tend to accumulate nearby and within genes and their regulatory regions . Therefore , it is difficult to unambiguously assign the increase in histone acetylation and RNA abundance to direct activation of SINE elements and not to the activation of Aza-dC- or TSA-responsive target genes containing SINE element sequences . ( 3 ) TSA treatment led to hyperacetylation of histone H4 independent of transcriptional activity . Histone H4 specific HATs seemed to readily acetylate lysine residues of H4 when the dominant HDACs were inhibited , suggesting their regular presence at silent TEs . In contrast , acetylation of histone H3 seemed coupled with transcription , pointing towards a model , in which histone H3 acetylation might stimulate the recruitment of the transcription machinery . Alternatively , histone H3 specific HATs were recruited together with the transcription machinery . Interestingly , Koch and colleagues recently showed that although H4ac and H3ac were both enriched around the transcriptional start site , only H3ac could be used as a highly predictive marker for gene activity [60] . One might speculate that differential HAT recruitment reflects a functional difference between histone H3 and histone H4 acetylation . In addition to their role in transcription where both modifications act synergistically , H4 ( de ) acetylation could have other biological functions not described so far . Examining IAP and VL30 transcripts in untreated fibroblasts clearly showed that expressed TEs originate from multiple loci dispersed throughout the genome . However , upon treatment with different stimuli , certain transposon subfamilies were preferentially expressed . We could show that these inducible elements harboured characteristic DNA sequence features within the LTR , the regulatory region of the element . IAPs were more efficiently activated by Aza-dC , when they carried increasing numbers of DNA sequence repeats , containing putative binding sites for the transcription factor CCAAT/enhancer-binding protein alpha ( C/EBPα ) ( identified with the Alibaba2 . 1 transcription factor binding site program using the TRANSFAC 4 . 0 database ) . Strikingly , Ishihara and colleagues , who characterised IAP elements active upon irradiation of mouse acute myeloid leukaemia ( AML ) cells , isolated IAP elements with especially long stretches of C/EBPα binding sites [70] ( Table S1 , AB099818 ) . In contrast , IAP elements responsible for recent integration events in the mouse germline [12] harboured only one TF binding site ( Table S1 , X04120 and AxinFu ) . Therefore , the availability of the transcription factor might influence the choice of which IAP subtypes are expressed in different cellular contexts . Treatment with TSA or Aza-dC led to preferential expression of one particular VL30 element associated with a U3 subtype I LTR . This individual element also showed particularly high levels of active histone marks ( H3K4me3 , H3ac , H4ac , H3S10phK14ac ) upon TSA treatment . Other elements driven by a subtype I LTR giving rise to transcripts showed intermediate enrichment levels , whereas the analysed subclass III and IV elements , which were hardly expressed under these conditions , exhibited lowest levels of active chromatin marks . Nilsson and Bohm earlier proposed a model stating that the enhancer design of VL30 elements determines tissue specific expression and the response to external stimuli [50] . Our data support this model . Subtype I U3 promoters seem to be specifically susceptible to activation in this cell type . This could also be observed in arrested Swiss 3T3 fibroblasts . When treated with Anisomycin and TSA , 5 out of 14 ( 36% ) transcripts were derived from an element with a subtype I U3 region; when left untreated we could only detect transcripts derived from subtype III elements ( 7 out of 7 ) ( Table S1 ) . Nevertheless , it remains unclear , which features distinguish the highly expressed hybrid element from other VL30 elements . Examination of the genomic environment did not reveal any obviously unusual architecture , the closest protein coding genes being located 764kb upstream and 16kb downstream of the element . Our finding that VL30 elements could be reactivated with Aza-dC was in accordance with DNA methylation as the dominant silencing mechanism for TEs and supported by previous reports [39] . However , it could not account for earlier findings illustrating that a multitude of other stimuli without link to DNA methylation was able to trigger VL30 expression [27] , [39] , [51] , [71] . Therefore , DNA methylation might be only one of several independent silencing pathways . Alternatively , Aza-dC treatment could be the cause for indirect effects leading to VL30 upregulation independent of DNA methylation levels at VL30 elements . Interestingly , loss of Dnmt1 in mouse brain tissue is not sufficient to influence VL30 expression ( S . L . and C . S . , unpublished data ) , although other stimuli have been reported to efficiently trigger VL30 in neurons [71] . This observation supports a model that Aza-dC might be an indirect trigger for VL30 activation . Other mammalian TEs also respond to external signals , e . g . SINE elements can be reactivated via stress as well as Adenovirus infection [24] , [25] , LINEs by benzo ( a ) pyrene [26] . In cancer cells , TE silencing is impaired probably due to altered DNA methylation patterns [72] , [73] , and several human diseases are associated with a relaxation of TE repression [74] . Nonetheless , the regulation of VL30 expression is unusual among mammalian retrotransposons; a diverse repertoire of stimuli is able to highly induce transcription in several cell systems such as fibroblasts , keratinocytes , haematopoietic , and neuronal cells [27] , [39] , [51] . In contrast to many other TEs , VL30 elements seem to exist in a default state , which is “poised for transcription” , without stringent silencing mechanisms . External stimuli can easily flip the switch to “active transcription” . There are several feasible explanations for this exceptional situation: ( 1 ) it may mirror the unusual life cycle of VL30 elements . They can be packed into MuLV ( murine leukaemia virus ) viral particles and get horizontally transferred , rather than vertically propagated as endogenous parasites [27] . For such a life cycle it is essential to be efficiently transcribed in somatic cells . Of special benefit could be a system that allows the elements to “sense” the physiological situation of the host . Stressful conditions might trigger high-level transcription and increase the chance to escape the host successfully via virus-mediated horizontal exit strategies . ( 2 ) Song and colleagues recently showed that VL30 RNA can interact with the DNA-binding protein PSF and modulate PSF-mediated repression of target genes [75] , [76] . Therefore , VL30 elements could have acquired physiological functions in murine cells and might be necessary for the regulation of certain host genes ( a process , called molecular domestication [77] ) . Another novel finding of our study is the positive impact of histone H3 phosphoacetylation on VL30 transcription . For several years , mechanistic links between histone acetylation and histone phosphorylation caused by different stimuli , including stress and growth factors , have been known . For example , immediate early genes such as c-myc , c-fos , c-jun , TFF1 [54]–[56] , [78] , but also others [57] , depend on the induction of histone H3 phosphoacetylation . In a series of experiments we could show that VL30 expression was synergistically activated by stimuli leading to histone phosphorylation ( FCS , anisomycin ) and histone acetylation ( TSA ) ; full transcriptional activity was correlated with the formation of phosphoacetylated histone proteins at the VL30 LTR . Inhibition of the nucleosomal response by H89 abolished VL30 phosphoacetylation and expression , indicating that the formation of this dual histone mark was necessary for activation caused by FCS/TSA or anisomycin/TSA and that MSK1/2 might be the responsible kinase responsible for phosphorylation . In summary , we collected data supporting a model that VL30 transposable elements are novel phosphoacetylation target genes . Still many details about the exact mechanism of VL30 regulation via dual histone modification are unclear , and it remains to be determined which are the responsible HAT ( s ) , HDAC ( s ) and phosphatase ( s ) regulating dynamic acetylation and phosphorylation . In this context it will be interesting to investigate putative connections between enhancer design , transcription factor binding , and recruitment of histone modifiers dependent on the VL30 subtype . The link between TE transcription and transposition is fundamental . Production of an adequate amount of mRNA is an essential premise for all subsequent steps in the lifecycle of a TE . Having monitored the influence of HDAC inhibition on transcription of several TEs , we also examined , if there is an influence on subsequent events resulting in altered transposition activity . We used an experimental system , which allowed us to survey transposition rates of episomally delivered LTR and LINE elements upon HDAC inhibition . We detected only minor differences in the frequency of retrotransposition events when cells were treated with TSA , i . e . transposition events were slightly increased upon TSA treatment . We also observed a comparable enrichment of the transcript . Therefore , we could not detect obvious effects on transposon activity exceeding effects coherent with enhanced transcription . Final answers on the issue of histone- and protein-acetylation , influencing the process of transposition in mammals , will certainly demand further and more detailed investigations . HDAC inhibitors are promising anticancer drugs because they can change gene expression patterns in cancer cells , leading to cell cycle arrest or apoptosis [4] . More recently , their use for the treatment of non-cancer diseases has also been suggested [79] , [80] . Our finding that HDAC inhibition can reactivate VL30 elements in mice might indicate that other TEs or non-coding elements could also respond to an epigenetic therapy in humans . Therefore , careful evaluation of potential effects beyond the deregulation of single copy genes will be an important issue for future studies . An interesting aspect – along with other dangers arising from increased TE activity – will be their emerging potential to modulate cellular regulatory networks . An exiting example for such a pathway conserved in mice and humans is the interaction of the PSF protein and VL30 RNA in mice and other non-coding TE-derived RNAs in human cells , respectively , affecting tumourigenesis [81] .
Wildtype ( HDAC1+/+ ) and corresponding HDAC1−/− fibroblasts cell lines were established from E13 . 5 embryos carrying two floxed alleles of the Hdac1 gene [82] . Primary fibroblasts ( mixed 129SvOla/Bl6 , more than 80% being of Bl6 origin ) were isolated and immortalised following the 3T3 immortalisation protocol [83] . Subclones were either transfected with a control vector or a vector encoding the cre recombinase leading to the deletion of exon 6 encoding the catalytic core of HDAC1 . Cells were maintained in DMEM supplemented with 10% foetal calf serum ( FCS ) and gentamycin . For growth arrest of cells , Swiss 3T3 fibroblasts were serum starved for 72h in DMEM containing 0 . 2% FCS . The following inhibitors were used in this study: trichostatin A ( TSA ) ( 50 ng/ml [166 nM]; Wako Pure Chemical Industries ) was applied for 3–24h; 5′Aza-2′-deoxycytidine ( Aza-dC ) ( 1µM ) for 24h followed by additional incubation of cells for 24h without inhibitor; anisomycin ( Aniso ) ( 50 ng/ml [189 nM]; Sigma ) for 1–6h , and H89 ( 10 µM; Alexis Biochemicals ) for 1h 15min . H89 was added 15min prior to anisomycin . Plasmids carrying a tagged IAP , MusD , L1 . Md or L1 . Hs retrotransposon ( described in [48] ) were transfected into logarithmically growing U2OS cells maintained in DMEM with 10% FCS and gentamycin . Cells were split 24h prior to the transfection procedure and 1×105 cells were seeded on a 6-well plate . Transfection was performed with 1µg of plasmid DNA and Lipofectamine ( Invitrogen ) following the manufacturer's instructions . After 6h , the medium was replaced by DMEM supplemented with 10% FCS and gentamycin . If required , 66nM TSA was added for 24h after transfection . To monitor expression levels of the transfected constructs , cells were lysed 24h post transfection with TRIzol reagent ( Invitrogen ) , RNA was isolated , DNase treated , reversely transcribed and subjected to quantitative RT-PCR as described above . The following primers were used for normalisation: GAPDH-for TCT TCT TTT GCG TCG CCA G , GAPDH-rev AGC CCC AGC CTT CTC CA and determination of episomal TE expression: Neo-for ATC AGA CAG CCG ATT GTC TG , Neo-rev CAG TTC CGC CCA TTC TCC G . For the analysis of transposition rates , cells were transferred to a 10cm dish 24h post transfection and cultured for 1 week , split and reseeded on 10cm dishes ( 5×105 per dish ) . Selection for neo-positive cells was started 24h later by adding 500µg/ml G418 to the growth medium . After 2 weeks , cell clones were fixed with 70% EtOH , stained with 0 . 1% methylene blue/0 . 5M Na-acetate solution and counted . Histone and total protein preparation for Western blot analysis was performed as previously described [57] . The following antibodies were used: HDAC1 polyclonal antibody ( Millipore 06-720 ) , HDAC2 3F3 monoclonal antibody ( Millipore 05-814 ) , HDAC3 ( Abcam ab16047 ) , DNMT1 ( Abcam ab13537 ) , β-actin ( Sigma A5316 ) , H3ac ( Millipore 06-599 ) , H4ac ( Millipore 06-598 ) , H3K4me3 ( Millipore 07-473 ) , H3S10pK14ac ( Millipore 07-081 and a rabbit serum raised against the H3 peptide ARTKQTARKS ( ph ) TGGK ( ac ) APRKQL in collaboration with Eurogentec , Belgium ) and c-H3 ( Abcam ab1791 ) . Densitometric quantification of Western blots was performed with the Imagequant software . For Dot blot analysis , 1 microliter of each peptide ( 0 . 2 mg/ml and 0 . 04 mg/ml in water ) was spotted on a PVDF membrane , antibody incubation and detection was performed by standard Western blot methods . To quantify DNA methylation levels at defined DNA sequences , we employed the MS-SNuPE technique as described elsewhere [32] . Primers for PCR: bs-IAP-rc-for GTT TTA GTA TAG GGG AAT GTT AGG GTT; bs-IAP-rc-rev ACC AAA AAA AAC ACC ACA AAC CAA AAT CTT CTA C . Primers for the SnuPE reaction: rcS789 GTA GTT AAT TAA GGA GTG ATA; rcS628 AGA TTT TTG TGT TGG GAG T; rcS894 GAA GAT GTA AGA ATA AAG TTT TGT . Preparation of soluble chromatin and chromatin immunoprecipitation assays were carried out as described previously [57] . Antibodies for immunoprecipitation of modified histones were obtained from Millipore ( H3ac 06-599 , H4ac 06-598 , H3S10p/H3K14ac 07-081 ) or generated in collaboration with Eurogentec , Belgium ( H3S10p/H3K14ac ) , the antibody recognising C-terminal histone H3 was ordered from Abcam ( c-H3 ab1791 ) . H3K9me2 and H3K27me3 antibodies were a kind gift of T . Jenuwein , MPI Freiburg . As negative control unspecific IgGs from preimmunsera were used . Quantification of precipitated material was performed via quantitative Real-Time PCR employing an iCycler iQ system from Bio-Rad , SYBER-green ( Molecular probes ) and Taq DNA Polymerase ( Fermentas ) or via semi-quantitative PCR using a Biometra T3 thermocycler and the GoTaq PCR Master mix from Promega followed by visualisation of DNA fragments on an ethidium bromide stained 2% agarose-TAE gel . Primers have been described elsewhere [15] , [84] or can be found in Table S2 . RNA was extracted with TRIzol Reagent ( Invitrogen ) , possible genomic DNA contamination was removed using Turbo DNase ( Ambion ) and cDNA was synthesised with the iScript cDNA Synthesis Kit ( Bio-Rad ) . For quantification of transcripts we performed RT-PCR on an iCycler iQ system from Bio-Rad using SYBER-green ( Molecular probes ) and Taq DNA Polymerase ( Fermentas ) . Primers sequences have been described previously [15] , [84] and are given in Table S2 . To characterise TE transcripts , RNAs from three independent biological samples from untreated , TSA and Aza-dC treated fibroblasts were harvested , extracted with TRIzol Reagent ( Invitrogen ) , DNase treated ( Turbo DNase , Ambion ) , reversely transcribed ( iScript , Bio-Rad ) and used for PCR amplification in doublets ( Biometra T3 thermocycler , GoTaq PCR Master mix from Promega; primers see Table S2 ) . As control , PCR reactions were also performed with genomic DNA as template , amplifying genomic elements . The resulting six PCR reactions for each condition were pooled ( to exclude a bias due to PCR reactions ) , loaded on an ethidium bromide stained agarose-TAE gel , eluted and purified using the Wizard Plus SV Gel and PCR Clean Up System ( Promega ) . DNA fragments were cloned into the pGEM-T vector system ( Promega ) and transformed into E . coli ( DH5α ) . Positive clones were grown in LB liquid medium , plasmid DNA was extracted using the Wizard Plus SV Miniprep DNA Purification System ( Promega ) and sequenced . | The majority of genomic sequences in higher eukaryotes do not contain protein coding genes . Large fractions are covered by repetitive sequences , many of which are derived from transposable elements ( TEs ) . These selfish genes , only containing sequences necessary for self-propagation , can multiply and change their location within the genome , threatening host genome integrity and provoking mutational bursts . Therefore host organisms have evolved a diverse repertoire of defence mechanisms to counteract and silence these genomic parasites . One way is to package DNA sequences containing TEs into transcriptionally inert heterochromatin , which is partly achieved via chemical modification of the packaging proteins associated with DNA , the histones . To better understand the contribution of histone acetylation in the activation of TEs , we treated mouse fibroblasts with a specific histone deacetylase inhibitor . By monitoring the expression of ten different types of murine mobile elements , we identified a defined subset of VL30 transposons specifically reactivated upon increased histone acetylation . Importantly , phosphorylation of histone H3 , a modification that is triggered by stress , is required for acetylation-dependent activation of VL30 elements . We present a model where concomitant histone phosphorylation and acetylation cooperate in the transcriptional induction of VL30 elements . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"genetics",
"and",
"genomics/epigenetics",
"molecular",
"biology/histone",
"modification"
] | 2010 | Epigenetic Regulation of a Murine Retrotransposon by a Dual Histone Modification Mark |
Endoplasmic reticulum ( ER ) stress is an important modifier of human disease . Genetic variation in response genes is linked to inter-individual differences in the ER stress response . However , the mechanisms and pathways by which genetic modifiers are acting on the ER stress response remain unclear . In this study , we characterize the role of the long chain fatty acid elongase Baldspot ( ELOVL6 ) in modifying the ER stress response and disease . We demonstrate that loss of Baldspot rescues degeneration and reduces IRE1 and PERK signaling and cell death in a Drosophila model of retinitis pigmentosa and ER stress ( Rh1G69D ) . Dietary supplementation of stearate bypasses the need for Baldspot activity . Finally , we demonstrate that Baldspot regulates the ER stress response across different tissues and induction methods . Our findings suggest that ELOVL6 is a promising target in the treatment of not only retinitis pigmentosa , but a number of different ER stress-related disorders .
Phenotypic heterogeneity is common in simple and complex diseases and is the driving force behind the Precision Medicine Initiative [1–4] . Genetic variation among individuals accounts for much of this heterogeneity , but the identity and nature of modifying genes or variants is largely unknown [4 , 5] . These modifying variants are often cryptic and may not influence the physiology or visible phenotypes of healthy individuals , but can alter the expression of disease phenotypes . Understanding the role of modifiers and the pathways in which they function will enable the development of patient-specific therapeutic approaches . One such process is the response to endoplasmic reticulum ( ER ) stress , which occurs when misfolded proteins accumulate in the lumen of the ER . This activates the Unfolded Protein Response ( UPR ) in an attempt to return the cell to homeostasis . Failure to do so will eventually result in apoptosis [6] . The UPR is controlled by the activation of three sensors located in the ER membrane: IRE1 , PERK , and ATF6 [7] . IRE1 is the most highly conserved of these sensors and contains an endonuclease domain responsible for the non-canonical splicing of Xbp1 . The spliced Xbp1 transcript is translated and the protein translocates to the nucleus , where it activates the expression of chaperones and other genes involved in resolving ER stress [8 , 9] . IRE1 also cleaves a number of additional mRNA targets , which reduces the quantity of newly translated proteins into the ER . This process is known as Regulated IRE1 Dependent mRNA Decay ( RIDD ) and is commonly linked with increased cell death [7 , 10 , 11] . When PERK is activated , it phosphorylates the translation initiation factor eIF2α , which decreases the translation of most mRNA transcripts with canonical translation initiation mechanisms [7] . This reduces the protein-folding load of the ER and allows for the upregulation of select transcripts involved specifically in the UPR , such as ATF4 [7] . ATF6 , the third transmembrane activator of the UPR , is transported to the Golgi upon the sensing of misfolded proteins and cleaved , whereupon the cytosolic portion of ATF6 travels to the nucleus to act as a transcription factor , binding ER stress response elements to further upregulate key players in the UPR [7] . Chronic ER stress leads to apoptosis , tissue degeneration , and dysfunction . In rare cases , primary mutations of key ER stress pathway components cause Mendelian syndromes [12–16] . More commonly , ER stress can exacerbate disease , including obesity [17] , neurological disease [18] , retinal degeneration [7] , and some cancers [19] . Experimental genetic or pharmacological manipulation of ER stress levels can alter disease phenotypes , suggesting that inter-individual differences in ER stress responses may influence disease severity . Indeed , human [20] , mouse [21] , and Drosophila [22 , 23] show extensive genetic variation in their response to ER stress . Understanding the role of genetic variation in modulating ER stress pathways may provide more therapeutic targets and improve the accurate identification of high risk patients . In a previous study , we crossed the Rh1G69D model of retinitis pigmentosa ( RP ) into the Drosophila Genetic Reference Panel ( DGRP ) to identify genetic modifiers of this disease [23] . In this model , retinal degeneration is induced by overexpression of misfolded rhodopsin in the developing larval eye disc [24] , resulting in chronic ER stress and apoptosis . The DGRP is used to study the genetic architecture underlying complex traits . There are approximately 200 inbred DGRP strains , each strain representing a single , wild-derived genome . Thus , the DGRP captures genetic variation that is present in a natural , wild population . Importantly , we have the whole genome sequence of each strain , allowing phenotype-genotype studies [25] . The degree of degeneration induced by this model varied substantially across the strains of the DGRP . Using an association analysis , we identified a number of promising candidates that modify this RP phenotype and have putative roles in the ER stress response [23] . In this study , we demonstrate that Drosophila Baldspot , one of the candidate RP modifiers identified in our genetic variation screen , is both a disease modifier of ER stress-induced retinal degeneration and a more general modifier of the ER stress response across different physiological contexts . Baldspot , which is orthologous to mammalian ELOVL6 , is an ER-associated long chain fatty acid elongase which catalyzes the extension of palmitate to stearate [26 , 27] . We show that loss of Baldspot rescues retinal degeneration by reducing ER stress signaling . Loss of Baldspot elongase activity reduces IRE1 and PERK activity and downstream apoptosis , without affecting the misfolded protein load . Strikingly , in the absence of Baldspot activity , dietary supplementation of stearate bypasses the need for Baldspot to increase UPR activation . Finally , we show that Baldspot regulates the ER stress response in a number of different genetic and chemically-induced ER stress models across multiple tissue types , making Baldspot a potentially wide-reaching modifier of ER stress-associated diseases . Understanding how modifier genes alter disease outcomes is the first step to developing personalized therapies .
In previous work , we overexpressed a misfolded rhodopsin protein in the developing eye disc ( GMR-GAL4>UAS-Rh1G69D ) , the larval precursor to the Drosophila retina and eye , in the 200 strains from the DGRP [23] . Using a genome-wide association approach , we identified SNPs that are associated with degree of retinal degeneration . One of these candidate SNPs , Chr3L:16644000 ( BDGP R5/dm3 ) , is located in intron four of Baldspot . Baldspot is the only Drosophila orthologue of mammalian ELOVL6 , a member of the very long chain fatty acid elongase family of enzymes ( Fig 1A ) [28] . Strains carrying the minor allele ( C , 18880 ± 3010 pixels ) exhibit more severe Rh1G69D–induced retinal degeneration as compared to strains carrying the major allele ( T , 21822 ± 2294 pixels; P < 0 . 005 ) ( Fig 1B ) . This is also apparent when examining the qualitative range of degeneration in strains containing each of these alleles ( Fig 1C ) . Based on the position of this SNP in an intron , we predict that it will affect the timing or levels of Baldspot expression . We also predict that it is exerting its effect in the small subset of cells that are GMR-expressing . This is very difficult to assess , as these cells make up a very small proportion of the eye disc . It is also difficult to know when , during development , this SNP is affecting expression . Nevertheless , we examined the effect of this SNP on Baldspot expression in adult flies and in the larval brain/eye-imaginal disc complex . Expression of Baldspot is unaffected by variation at this SNP in whole adult flies ( not containing the Rh1G69D transgene ) ( S1 Fig ) . In larval brain-imaginal disc complexes , there is not a significant increase in Baldspot expression from strains carrying the C allele and expressing the Rh1G69D transgene ( C , 1 . 15 ± 0 . 03 as compared to T , 1 . 01 ± 0 . 09; ) ( S1 Fig ) . While there is not a detectable substantial difference in expression between the alleles , the possibility remains that altering the expression or activity of Baldspot may impact degeneration and the causative pathways . To test whether eliminating Baldspot expression can modify Rh1G69D-induced retinal degeneration , we expressed an RNAi construct targeted against Baldspot in the GMR-GAL4>UAS-Rh1G69D background . Expression of this construct results in a strong , significant reduction in Baldspot mRNA ( ~7% of controls; P = 3 . 5 x 10−3 ) ( S2 Fig ) . We found that loss of Baldspot expression results in partial rescue of Rh1G69D-induced retinal degeneration ( Rh1G69D/Baldspoti ) as compared to controls expressing only the Rh1G69D misfolded protein and no RNAi ( Rh1G69D controls ) ( Fig 2A ) . Quantification of eye size demonstrates a significantly larger , less degenerate eye in Rh1G69D/Baldspoti flies ( 13742 ± 913 pixels , n = 20 ) as compared to Rh1G69D controls ( 11432 ± 688 pixels , n = 20 ) ( P = 5 . 3 x 10−11 ) . We found a similar effect using another , independent RNAi strain ( S2 Fig ) . Loss of Baldspot on a wild-type background ( 26478 ± 1191 pixels , n = 20 ) resulted in no significant change in eye size or phenotype as compared to genetically matched controls ( 26582 ± 1110 pixels n = 20 ) ( Fig 2B ) . We conclude that loss of Baldspot expression provides significant rescue of Rh1G69D-induced retinal degeneration . Rescue of eye size in the Rh1G69D model is often accompanied by reduction in apoptosis [29 , 30] . Based on this and the rescue effect we observed , we hypothesized that apoptosis in the Rh1G69D eye discs would also be reduced upon loss of Baldspot expression . Indeed , we found that apoptosis , as measured by terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) staining , is significantly reduced in Rh1G69D/Baldspoti eye discs as compared to Rh1G69D controls ( 101 ± 95 TUNEL-positive cells in Rh1G69D/Baldspoti and 226 ± 69 TUNEL-positive cells in controls; P = 1 . 8 x 10−3 ) ( Fig 2C and 2D ) . Excessive ER stress in the Rh1G69D eye discs leads to apoptosis and degeneration and abnormal , degenerate adult eyes . To determine if the decrease in apoptosis we observed was due to changes in the ER stress response , we measured the activity of IRE1 and PERK , two of the transmembrane sensors of misfolded proteins in the ER . Because the ATF6 branch of the UPR is mostly unstudied in Drosophila , we are unable to draw any conclusions about the activity of this branch of the UPR . Under conditions that induce ER stress , Xbp1 transcript is spliced by IRE1 . The in-frame Xbp1 transcript is translated and acts as a transcription factor , inducing genes involved in the UPR [8 , 9] . The level of spliced Xbp1 transcript is directly proportional to the degree of activation of the ER stress response . To measure IRE1 signaling and Xbp1 splicing in the absence of Baldspot expression , we used a previously characterized transgenic Xbp1-EGFP marker [24 , 31–33] . In this transgene , the 5’ end of the Xbp1 mRNA is expressed under the control of the UAS promoter . The 3’ end of the gene , downstream of the IRE1 splice site , has been replaced with sequence encoding EGFP . As with the endogenous Xbp1 transcript , the in-frame , spliced Xbp1-EGFP transcript , and subsequently the Xbp1-EGFP fusion protein , increases as ER stress signaling increases . Thus , EGFP signal increases as ER stress signaling increases . As previously reported , expression of UAS-Xbp1-EGFP in Rh1G69D controls results in increased EGFP , indicating high levels of ER stress ( Fig 2E ) [24 , 31–33] . When UAS-Xbp1-EGFP is expressed in Rh1G69D/Baldspoti larvae eye discs , we see a significant reduction in EGFP signal ( 0 . 59 ± 0 . 13 in Rh1G69D/Baldspoti relative to controls , 1 . 00 ± 0 . 18; P = 9 . 6 x 10−4 ) , indicating reduced ER stress signaling ( Fig 2E and 2F ) . However , Rh1G69D/Baldspoti eye discs display no significant change in rhodopsin protein levels ( 0 . 78 ± 0 . 33 in Rh1G69D/Baldspoti relative to controls , 1 . 00 ± 0 . 44 in controls; P = 0 . 286 ) ( Fig 2E and 2F ) , suggesting that loss of Baldspot expression reduces IRE1/Xbp1 signaling without affecting the accumulation or degradation of misfolded Rh1 protein . IRE1-dependent cell death is primarily triggered by the Jun Kinase ( JNK ) signaling cascade when IRE1 has been chronically and strongly activated [7] . To measure activation of JNK signaling , we monitored expression of puckered ( puc ) , a well characterized JNK target , using a LacZ-tagged allele ( puc-LacZ ) . While puc-LacZ is induced in Rh1G69D controls ( 1 . 00 ± 0 . 41 ) , it is significantly reduced in Rh1G69D/Baldspoti ( 0 . 16 ± 0 . 19 relative to controls; P = 9 . 5 x 10−3 ) ( Fig 2G and 2H ) . This reduction in JNK signaling correlates with a reduction in cell death in the absence of Baldspot and could be due to reduced signaling through the IRE1-JNK signaling axis . However , changes in JNK signaling can also be achieved through a number of alternative pathways and influence other downstream processes besides apoptosis [34 , 35] . Our current analyses do not distinguish between these possibilities . We also examined the effect of Baldspot loss on the PERK branch of the UPR . When PERK is activated , it phosphorylates the translation initiation factor eIF2α , which decreases the translation of most mRNA transcripts with canonical translation initiation sequences [7] . We monitored P-eif2α levels by Western blot in brain/eye-imaginal disc complexes isolated from Rh1G69D control and Rh1G69D/Baldspoti larvae . P-eif2α levels were substantially reduced in samples isolated from Rh1G69D/Baldspoti compared with Rh1G69D controls , indicating that PERK activity is reduced in the absence of Baldspot ( Fig 2I ) . Our data demonstrate that loss of Baldspot expression reduces IRE1 and PERK signaling without altering misfolded protein levels . This is consistent with previous reports demonstrating that the cellular concentration of stearate is associated with increased activation of the ER stress response through direct activation of IRE1 and PERK , independently from misfolded proteins [36–38] . IRE1 and PERK both contain a conserved domain that is embedded in the ER membrane and can detect and respond to changes in membrane lipid composition , leading to increased activation of these sensors [39] . Together , this suggests that the modifying effect of Baldspot is linked to its fatty acid elongation activity . Baldspot converts palmitate to stearate [26 , 27] and the absence of Baldspot function should result in lower levels of stearate . We hypothesized that lower levels of stearate underlies the reduced ER stress response and retinal degeneration we observed , and that this signaling could be restored if stearate were supplemented during development . To test this , we raised Rh1G69D/Baldspoti and Rh1G69D control flies on media with or without 10% stearate and measured eye size in adults . This diet has no effect on eye size in wild-type individuals ( Fig 3A ) . Stearate supplementation resulted in increased degeneration and reduced eye size in Rh1G69D/Baldspoti flies ( 14033 ± 1553 pixels ) as compared to those raised on standard media with no stearate ( 16846 ± 1699 pixels; P = 1 x 10−7 ) ( Fig 3B ) . This increase in degeneration in Rh1G69D/Baldspoti flies was accompanied by an increase in IRE1 activity and spliced Xbp1 , as evidenced by an increase in Xbp1-EGFP expression in eye discs ( 1 . 26 ± 0 . 08 on stearate relative to standard media , 1 . 00 ± 0 . 10; P = 3 . 4 x 10−3 ) ( Fig 3C and 3D ) . Stearate supplementation had no effect on eye size , retinal degeneration , or Xbp1-EGFP signal in Rh1G69D control flies ( 13478 ± 1197 pixels on stearate and 12873 ± 1006 pixels on standard media; P = 0 . 502; 0 . 83 ± 0 . 15 on stearate relative to standard media , 1 . 00 ± 0 . 21; P = 0 . 171 ) ( Fig 3B–3D ) , likely because of the high level of ER stress signaling already present . High stearate levels are sufficient to induce ER stress signaling and bypasses the need for Baldspot activity . Because Baldspot is ubiquitously expressed ( www . flyatlas2 . org/ ) , it is possible that Baldspot’s modifying role in the ER stress response is generalizable across different contexts and tissues . We first tested whether Baldspot modifies ER stress in another tissue . Because wing size is a visible and easily scorable trait , we used the MS1096-GAL4 driver to express Rh1G69D in the developing larval wing disc ( wing-Rh1G69D ) . The Rh1G69D protein acts as a misfolded protein , inducing ER stress only in that tissue . The wing disc gives rise to the adult wing , and perturbations in cellular homeostasis in this tissue result in small , misshapen wings [40] . Expression of wing-Rh1G69D produces a phenotype similar to a vestigial wing ( Fig 4A ) . When the RNAi construct targeted against Baldspot was concurrently expressed in the wing disc ( wing-Rh1G69D/Baldspoti ) , this vestigial phenotype was partially rescued . The adult wings showed an increase in size and an increase in unfolding upon eclosion ( Fig 4A ) . While this phenotype is variable in the degree of degeneration and unfolding of the wing , we do see a significant increase in 2D wing area and wing length in the absence of Baldspot ( area: 100769 ± 9903 pixels in wing-Rh1G69D/Baldspoti and 57681 ± 30912 pixels in controls; P = 0 . 043; length: 633 ± 44 pixels in wing-Rh1G69D/Baldspoti and 378 ± 183 pixels in controls; P = 0 . 039 ) ( S3 Fig ) . Similar to what we observed in the eye discs , this partial rescue was associated with a decrease in the level of Xbp1-EGFP in the wing disc ( 0 . 76 ± 0 . 17 in wing-Rh1G69D/Baldspoti relative to controls , 1 . 00 ± 0 . 14; P = 6 . 0 x 10−3 ) ( Fig 4B and 4C ) . We also measured JNK activation in these wing discs by monitoring the puc-LacZ transgene . Again , as we saw in eye discs , LacZ expression is significantly reduced in wing discs lacking Baldspot ( 0 . 64 ± 0 . 12 in wing-Rh1G69D/Baldspoti relative to controls , 1 . 00 ± 0 . 12; P = 4 . 7 x 10−3 ) ( Fig 4D and 4E ) . It appears that loss of Baldspot modifies UPR signaling and degeneration through the same mechanisms across different tissues and is not eye disc-specific . To test whether Baldspot modifies ER stress signaling by pharmacological induction , we used tunicamycin to induce ER stress . Tunicamycin inhibits N-linked glycosylation in the ER and results in massive misfolding , ER stress , and a robust UPR [41] . Treatment with tunicamycin in Drosophila larvae results in a robust activation of the ER stress response [33 , 42] . We acutely exposed 2nd instar control larvae ( Tub-GAL4 , no RNAi ) and larvae with ubiquitous knockdown of Baldspot ( Tub-GAL4>Baldspot RNAi ) to tunicamycin or control DMSO and scored survival to pupation as a proxy for the physiological ability to recover from ER stress . To account for any larval lethality due to loss of Baldspot , we normalized the survival of larvae on tunicamycin to the DMSO treatment . Strikingly , larvae lacking Baldspot were less susceptible to tunicmycin-induced lethality than control larvae ( Fig 4F , S1 Table ) , as evidenced by increased survival to pupation ( P = 9 . 2 x 10−4 ) . This was accompanied by reduced Xbp1 splicing ( S4 Fig ) . We conclude that the modifying effect of Baldspot expression on the ER stress response is generalizable across different tissues and methods of ER stress induction . Because our data indicates that Baldspot regulates Ire1/Xbp1 signaling and IRE1-dependent JNK signaling , we next tested whether Baldspot regulates another key IRE1 function: Regulated IRE1 Dependent Degradation of mRNAs ( RIDD ) [10 , 11 , 43] . In addition to Xbp1 splicing , the RNase activity of activated IRE1 is also important for the degradation of a number of ER-associated transcripts through RIDD . To test if RIDD activity is affected by loss of Baldspot expression , we measured levels of known RIDD target mRNAs , under ER stress , in control cells and tissues with or without Baldspot expression . We employed S2 cell culture and treated S2 cells with dsRNA targeting either Baldspot or EGFP as a control . This treatment resulted in a near complete reduction in Baldspot transcript levels ( ~7 . 8% of control; P = 1 . 3 x 10−9 ) ( Fig 5A ) . We used Dithiothreitol ( DTT ) to induce ER stress in S2 cells . DTT is a reducing agent that breaks disulfide bonds in proteins , resulting in misfolded proteins and a strong ER stress response [44] . As expected , treatment of cells with DTT increased Xbp1 splicing ~3-fold , indicative of ER stress . In agreement with our in vivo studies in the Rh1G69D fly , this splicing was significantly reduced in the absence of Baldspot , with only ~2-fold increase ( P = 0 . 023 ) , indicating that IRE1 signaling is disrupted ( Fig 5B ) . We also monitored PERK activity by measuring levels of P-eif2α , which increase in control cells upon treatment with DTT ( Fig 5C ) , as expected ( P = 0 . 021; N = 3 ) . In cells treated with Baldspot DsRNA , there was no increase in P-eif2α levels upon DTT exposure ( P = 0 . 954; N = 3 ) ( Fig 5C ) . Due to a lack of antibody , we could not monitor ATF6 activity in S2 cell culture . It appears that loss of Baldspot expression in S2 cells has a similar effect on ER stress response as observed in our in vivo model . We next measured IRE1 RIDD activity in the absence of Baldspot in S2 cells . When RIDD is activated , ER-localized mRNAs are cleaved by the RNase domain of IRE1 , leading to a reduction in protein translation into the ER [10 , 43] . A number of these targets have been well-characterized in Drosophila , including the transcripts sparc and hydr2 . Both of these mRNAs are degraded upon the initiation of ER stress [43 , 45] . We tested the effect of Baldspot knockdown on these targets after treatment with DTT . Hydr2 expression is significantly reduced in control cells treated with DTT ( 0 . 71 ± 0 . 07; P = 2 . 0 x 10−3 ) , but not in those treated with DsRNA against Baldspot ( 0 . 91 ± 0 . 16; P = 0 . 41; P = 0 . 021 ) ( Fig 5D ) . However , we found that sparc is equally degraded upon DTT treatment in cells treated with DsRNA against either Baldspot ( 0 . 74 ± 0 . 09; P = 2 . 0 x 10−5 ) or EGFP ( 0 . 71 ± 0 . 06; P = 1 . 9 x 10−5 ) ( Fig 5D ) . Non-RIDD targets , such as Actin5C , were unaffected ( Fig 5D ) . The fact that only some RIDD targets are affected is in agreement with our other results: loss of Baldspot activity partially reduces IRE1 functions , including RIDD .
The ER stress response has the potential to be an important modifier of human disease . ER stress is activated in a wide variety of diseases ranging from Mendelian degenerative diseases to complex metabolic disorders [7] . The role of the ER stress response is complex and in some cases , a stronger response is beneficial and in other cases , a strong response underlies the disease . In some forms of retinitis pigmentosa , like the focus of this study , loss of vision is a direct result of the ER stress-induced cell death [46] . Neurodegenerative diseases such as Parkinson’s , Alzheimer’s , Amyotrophic Lateral Sclerosis ( ALS ) , and prion disease are all associated with the accumulation of cytoplasmic misfolded proteins that can activate a secondary ER stress response and subsequent cell death [19] . In diabetes and obesity , misfolded proteins and the ER stress response are at least partially responsible for the cellular dysfunction that is a hallmark of the disease state [47] . In some cancers , however , cells selectively upregulate ER stress pathways associated with survival , while inhibiting those associated with cell death [48] . Genetic variation that alters the activation of the ER stress response may modify disease in the population . We and others have shown that genetic variation can influence how and to what extent the ER stress response is activated . Genetic variation in Drosophila [22 , 23] , mouse [21] , and human cells [20] can impact the expression of genes involved in ER stress pathways . Furthermore , this variation in expression has been linked to differences in the UPR and the downstream activation of cell death [21–23] . It is apparent that cryptic genetic variation drives these extensive inter-individual differences in the ER stress response [21] . Stress or disease conditions reveal the effect of this otherwise silent genetic variation . The same variation that drives differences in this important stress response might also influence disease heterogeneity . Indeed , modulating the ER stress response is an effective way of modulating disease outcome in models of human disease . [49–54] . In this study we show that loss of Baldspot/ELOVL6 activity can affect the outcome of retinal degeneration and has the potential to be a broader regulator of ER stress-associated diseases . As a 16-to-18 carbon fatty acid elongase in the ER membrane , this enzyme is responsible for increasing the concentration of C18 stearate , a saturated long chain fatty acid [28 , 37] . Increases in stearate concentrations in the ER membrane can induce and enhance ER stress , and is associated with disease outcomes in mammalian systems [36–38] . Here , we show that reducing the activity of Baldspot/ELOVL6 reduces the degree to which the UPR and apoptosis are induced in models of ER stress . Our study shows that Baldspot/ELOVL6 has a modifying effect on the IRE1 and PERK branches of the ER stress response . This is consistent with observations that increased long chain fatty acids in the ER membrane impacts IRE1 and PERK signaling [38 , 39] . Future research will explore the Baldspot/ELOVL6 interaction with ER membrane composition and how this alters IRE1 and PERK oligomerization and function . It is particularly interesting to note that RIDD is only partially affected in the absence of Baldspot . In Drosophila , the RIDD activity of IRE1 targets most ER-localized mRNAs [43] . In contrast , mammalian RIDD is much more selective , and only mRNAs with specific signal sequences and RNA secondary structures are degraded by IRE1 [55] . More studies on how these processes are regulated in Drosophila and mammals is necessary to understand the differences in RIDD between species as well as the conservation of Baldspot/ELOVL6 function . These studies will enhance our understanding of how these pathways are regulated in different disease contexts . Therapeutics that reduce ELOVL6 activity could conceivably be used to treat or delay progression in a variety of ER stress-associated diseases . In RP patients with mutations that cause the accumulation of misfolded rhodopsin , similar to our Drosophila model , reduction of ELOVL6 expression might reduce cell death , delaying vision loss . We present evidence that the modulation of the ER stress response through Baldspot/ELOVL6 activity may impact the severity of diseases beyond RP . Particularly intriguing is that Baldspot/ELOVL6 can alter the ER stress response when stress is induced by different conditions . Regardless of the mechanism by which ER stress is induced , inhibiting ELOVL6 activity reduces IRE1 and PERK signaling . In fact , in a recent study of the db/db mouse model of diabetes , loss of ELOVL6 alters insulin sensitivity and the expression of ER stress genes [26] . It is likely that reducing Baldspot/ELOVL6 expression would also alter complex diseases where ER stress contributes to degeneration and cell death . In some cases , even a small delay in degeneration could dramatically improve the quality of life . We have identified Baldspot/ELOVL6 in Drosophila as a novel modifier of the ER stress response . Its potential as a therapeutic target extends beyond RP to any number of ER stress-associated disorders . The identification of Baldspot in a natural genetic variation screen [23] suggests that variation in expression of this gene is well tolerated under healthy conditions and might be amenable to modulation . Identifying variable elements of the ER stress response pathway may prove to be a successful strategy to nominating new modifiers with broad applications to many ER stress-associated diseases .
Flies were raised at room temperature on standard diet based on the Bloomington Stock Center standard medium with malt . The strain containing GMR-GAL4 and UAS-Rh1G69D on the second chromosome ( GMR>Rh1G69D ) has been previously described [23] . The following strains are from the Bloomington Stock Center: MS1096-GAL4 ( 8696 ) , Baldspot RNAi ( 44101 ) , control attP40 ( 36304 ) . The puc-LacZ enhancer trap is available from Kyoto ( 109029 ) . A second Baldspot RNAi line ( Vienna Drosophila Resource Center: 101557KK ) showed similar rescue of the original Rh1G69D phenotype . Most analysis was performed in the Bloomington Stock Center RNAi line . The strains containing the UAS-Xbp1-EGFP transgenes were a gift from Don Ryoo ( NYU ) [24] . For eye and wing images , adult females were collected under CO2 anesthesia and aged to 2–7 days , then flash frozen on dry ice . Eyes were imaged at 3X magnification using a Leica EC3 camera . Wings were dissected away from the body , then imaged at 4 . 5X magnification using the same camera . Eye area was measured in ImageJ as previously described [23] . Eye discs and wing discs were dissected from wandering L3 larvae in cold 1X PBS , then immediately transferred to cold 4% PFA on ice . Tissues were fixed in 4% PFA for 15–20 min , then washed in 1XPAT ( 0 . 1% TritonX100 ) prior to blocking with 5% normal donkey serum . Tissues were stained with primary antibodies for rhodopsin ( 1:50 , Developmental Studies Hybridoma Bank #4C5 ) , GFP ( 1:2000 , Thermo-Fisher #A6455 ) , and LacZ ( 1:20 , Developmental Studies Hydbridoma Bank #40-1a ) . Apoptosis was monitored using the ApopTag Red In Situ Apoptosis Detection Kit ( Millipore #S7165 ) . Tissues were mounted in Slowfad Diamond Antifade Mountant ( ThermoFisher #S36967 ) and imaged with an Olympus FV1000 confocal microscope . For GFP blots , 5 L2 larvae were homogenized in 1X Laemmli/RIPA buffer containing 1X protease inhibitors . For P-eif2α blots , protein from S2 cells or brain/eye-imaginal discs was isolated in 1X Laemmli/RIPA buffer containing 1X protease inhibitors ( Roche cOmplete Mini EDTA-free protease inhibitor tablets ) , as well as the phosphatase inhibitors Calyculin A and okadaic acid . Equivalent amounts of protein were resolved by SDS-PAGE ( 10% acrylamide ) and transferred to PVDF membrane by semi-dry transfer . Membranes were then treated with either 5% BSA or 5% milk protein block in 1XTBST prior to immunoblotting . Blots were probed with antibodies for GFP ( 1:5000 , Thermo-Fisher #A6455 ) , tubulin ( 1:2000 , Developmental Studies Hybridoma Bank #12G10 ) , P-eif2α ( 1:1000 , abcam #32157 ) , and Pan-eif2α ( 1:500 , abcam #26197 ) . Blots shown are representative of at least three biological replicates , and quantification was performed using Image J software . Crosses were set up on the control , standard media alone , or standard media with 10% stearate supplementation . To make stearate supplemented media , standard media was melted in the microwave and kept at 98°C on a stir plate . Stearate ( 10% final concentration ) was added to this media and mixed until lipids were homogenous with the media . The standard control diet received the same treatment , but no additional stearate was added . To determine retinal degeneration , flies were aged and imaged as described above . Antibody staining was performed on the eye imaginal discs of L3 larvae , also as described above . Crosses to generate the indicated genotypes were set up on egg caps containing yeast paste . L2 larvae were then treated with either 10 μg/mL Tunicamycin ( diluted 1:1000 from a 10 mg/mL stock solution ) or 1:1000 DMSO in Schneider’s media for 30 minutes at room temperature . The larvae were then washed in 1XPBS twice and placed on solid media containing 6% yeast and 1% agar . Viability was determined by survival to pupation . Survival for each genotype was normalized to the DMSO-treated control condition . Each replicate represents ~25–60 larvae per genotype; a total 180 to 250 larvae per genotype were analyzed across all replicates . Protein was isolated from five L2 larvae expressing Xbp1-EGFP , one hour after treatment was concluded . A Western blot was performed to analyze Xbp1-EGFP levels . DsRNA was generated using the MEGAscript T7 Transcription kit ( ThermoFisher #AM1334 ) , with primers for EGFP ( F: TTAATACGACTCACTATAGGGAGACCACAAGTTCAGCGTGTCC and R: TTAATACGACTCACTATAGGGAGAGGGGTGTTCTGCTGGTAGTG ) and Baldspot ( F: TTAATACGACTCACTATAGGGAGAATCCGCCCAGGTTCATCTCG and R: TTAATACGACTCACTATAGGGAGAGTCACATCTCGCAGCGCAAC ) . S2 cells were treated with DsRNA against EGFP ( as a control ) or against Baldspot at a density of approximately 2 x 106 cells/mL in a 24-well plate . Cells were incubated with DsRNA for 4–7 days before being split and treated with either DMSO as a control or DTT . Concentrations of DTT varied with experiments: 0 . 5 mM for 1 hour ( Xbp1 splicing ) , 2 . 0 mM for 3 hours ( RIDD target analysis ) , or 2 . 0 mM for 4 hours ( P-eif2α detection ) . RNA was isolated from cells using either Trizol/chloroform extraction ( Xbp1 splicing ) or the Direct-zol RNA Miniprep Kit ( qPCR ) and used to generate cDNA ( Protoscript II , NEB ) . Protein was isolated from cells as described above and levels of P-eif2α normalized to Pan-eif2α as a loading control compared between matched DMSO or DTT-treated S2 cells . Knockdown of Baldspot was confirmed using qPCR ( primers: F: TGCTGGTCATCTTCGGTGGTC and R: ACGCAGACGGAGTGGAAGAG ) . Xbp1 splicing was evaluated from the cDNA using PCR ( primers for Xbp1: F: TCAGCCAATCCAACGCCAG and R: TGTTGTATACCCTGCGGCAG ) . The spliced and unspliced bands were separated on a 12% acrylamide gel and the proportion of these bands quantified using ImageJ software . RIDD target levels were analyzed by qPCR ( sparc primers: F: AAAATGGGCTGTGTCCTAACC and R: TGCAGCACAATCTACTCAATCC; hydr2 primers: F: CGCATACACGACTATTTAACGC and R: TTTGGTTTCTCTTTGATTTCCG; Actin5C primers: F: ATGTGTGACGAAGAAGTTGCT and R: GAAGCACTTGCGGTGCACAAT; rpl19 primers: F: AGGTCGGACTGCTTAGTGACC and R: CGCAAGCTTATCAAGGATGG ) . Transcript levels were normalized to rpl19 and compared between matched DMSO or DTT-treated S2 cells . Statistics were calculated using R software . P-values were determined using ANOVA for eye size , fluorescence levels , transcript levels in qPCR , and protein levels by Western blot . Tukey multiple testing correction was used for the fatty acid feeding experiment and RIDD target analysis . Bonferroni correction was used for Xbp1 splicing in S2 cells . A pairwise T-test was performed for larval tunicamycin treatment and P-eif2α response to DTT in S2 cells . A cutoff of P = 0 . 05 was used for significance . | Differences in genetic background drives disease variability , even among individuals with identical , causative mutations . Identifying and understanding how genetic variation impacts disease expression could improve diagnosis and treatment of patients . Previous work has linked the endoplasmic reticulum ( ER ) stress response pathway to disease variability . When misfolded proteins accumulate in the ER , the ER stress response returns the cell to its normal state . Chronic ER stress leads to massive amounts of cell death and tissue degeneration . Limiting tissue loss by regulating the ER stress response has been a major focus of therapeutic development . In this study , we characterize a novel regulator of the ER stress response , the long chain fatty acid elongase Baldspot/ELOVL6 . In the absence of this enzyme , cells undergoing ER stress display reduced cell death , and degeneration in a Drosophila disease model . Feeding of excess fatty acids increases degeneration to original disease levels , linking the regulatory activity of Baldspot to its enzymatic activity . Finally , we demonstrate that Baldspot can alter the ER stress response under a variety of other ER stress conditions . Our studies demonstrate that Baldspot/ELOVL6 is a ubiquitous regulator of the ER stress response and is a good candidate therapeutic target . | [
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... | 2018 | Baldspot/ELOVL6 is a conserved modifier of disease and the ER stress response |
The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection . IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors . Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97 , results in loss of IE2 expression , subsequent suppression of early and late gene expression and , ultimately , failure in virus replication . RNAseq analysis showed increased levels of IE1 splicing , with a corresponding decrease in IE2 splicing following VCP knockdown . Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression , including UL112/113 . Furthermore , Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus . Finally , we show that NMS-873 , a small molecule inhibitor of VCP , is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection .
Human cytomegalovirus ( HCMV ) is a highly prevalent herpesvirus , infecting 30 to 100% of the global population depending on the socio-economic status . Although normally asymptomatic in healthy individuals , HCMV infection is a significant cause of morbidity and mortality in immunocompromised populations , individuals with heart disease and recipients of solid organ and bone marrow transplant . HCMV is also the leading cause of infectious congenital birth defects [1–9] . During infection , HCMV initiates a programmed cascade of gene expression , resulting in production of infectious virus . Two of the first genes to be expressed are the major immediate early ( MIE ) genes IE1 ( IE72 ) and IE2 ( IE86 ) . The MIE proteins have multiple roles during infection including transactivation of viral genes , which drives replication and virus production [10–12] . Because of this , they are thought to play a pivotal role in controlling the switch between latent and productive infection in HCMV [13 , 14] . While IE1 is required for efficient virus replication at low multiplicity of infection [13 , 14] , IE2 expression is essential , with deletion resulting in non-viable virus [15] . IE1 and IE2 are generated from the same primary transcript by differential splicing and alternative polyadenylation [10 , 12 , 16] . They share the first three exons , with splicing to the fourth or fifth exon determining expression of IE1 or IE2 transcript , respectively ( Fig 1 ) . Independent polyadenylation signals exist downstream of both exon four and exon five . Such genomic arrangements , that require terminal exon skipping , are considered relatively unusual in the host cell , with specific factors and mechanisms involved in regulating the process not fully understood [17] . Valosin containing protein ( VCP ) belongs to the hexameric AAA ATPase family and plays a pivotal role in ubiquitin mediated signaling through remodeling target proteins , often leading to proteosomal degradation [18] . VCP contains two ATPase domains , which hydrolyze ATP to generate the energy required to remodel or unfold target proteins . Through this action , VCP is able to segregate target proteins from associated cellular membranes or larger protein complexes . Once segregated , the target protein is relocalised or degraded via the proteosomal complex . VCP can also affect which proteins are modified through its interaction with multiple ubiquitin regulatory co-factors , making VCP a central signalling hub for ubiquitin mediated regulation . In addition to ubiquitin , VCP has been linked to other post-translational modifiers such as SUMO and Nedd8 [19 , 20] . As such it is linked to a wide range of biological functions , including protein quality control , autophagy , chromatin remodeling , DNA damage response and more recently RNA processing [21] . Functionally , VCP plays a central role in protein homeostasis by facilitating proteosomal degradation of misfolded or damaged proteins as part of the endoplasmic reticulum associated degradation pathway ( ERAD ) . In addition to its role in protein degradation , VCP has been linked to non-degradative functions involving removal and relocation of proteins from membranes and protein complexes . Examples include removal of Aurora B protein from mitotic chromosomes , removal of transcription factors from chromatin and disassembly of RNP complexes [22 , 23] . It has also been linked to a number of functional roles in virus replication including entry of Sindbis virus and replication of poliovirus [24–26] . While VCP plays a role in US11-specific degradation of MHC class I protein during HCMV infection [27] a direct role for VCP in the replication of HCMV has not previously been reported . Using a focused siRNA screen , we identified VCP as an essential factor for HCMV replication . We show that VCP is essential for the onset of virus replication and knockdown of VCP results in changes in alternative splicing of the MIE transcripts , loss of IE2 expression and ultimately failure in virus replication .
Previously , we identified the host gene ATP6V0C as a critical factor in HCMV virus production [28] . ATP6V0C is a component of the vacuolar ATPase , and among other functions , is involved in membrane organization [29] . To identify additional host membrane organization factors involved in HCMV replication we performed a focused siRNA screen against 160 host genes involved in membrane organization using pools of four siRNAs against each target . Primary human fibroblast cells were double transfected with each siRNA pool in 96 well format . Two days post-transfection , the cells were infected with a low passage HCMV strain , TB40E , expressing GFP from an SV40 promoter . GFP levels were monitored over a period of seven days by fluorometry to determine virus infection and replication levels ( S1 Table ) . Analysis of the screen indicated both increased and decreased virus replication ranging from 88% reduction in GFP levels to a 84% increase ( Fig 2A ) . Z-score analysis based on three biological repeats of the siRNA screen identified five clear outliers that resulted in reproducible reductions in virus replication , as measured by GFP fluorescence intensity ( Fig 2B ) . Of these , knockdown of VCP resulted in the largest negative Z-score . To validate that the observed phenotype was due to knockdown of VCP , rather than potential off-target effects , the original siRNA pool was deconvoluted to test the four individual siRNAs against VCP . All four siRNAs resulted in reduced VCP expression and reduced HCMV replication based on GFP fluorescence ( Fig 2C ) . The third siRNA of the pool generated less efficient VCP knockdown , and corresponded with less inhibition of HCMV in the siRNA assay , providing additional evidence that knockdown of VCP results in a direct reduction in virus replication . These results indicate that the initial observed phenotype is due to specific knockdown of VCP rather than off-target artefactual effects of the siRNAs . Although GFP expression serves as an effective read-out for virus replication , loss of GFP signal may not necessarily reflect reduced virus production . To determine the direct effect of VCP knockdown on HCMV virus production , one-step growth curves were generated . Primary human fibroblast cells were transfected with siRNA pools targeting VCP or a negative control non-targeting siRNA . Two days post transfection cells were infected at a multiplicity of infection ( MOI ) of three with TB40E-GFP . Supernatant was collected at 24-hour time points for seven days total . Knockdown of VCP resulted in significant reduction in HCMV replication , with viral titers over four log10 lower in cells depleted of VCP compared to negative control cells at day seven ( Fig 2D ) . No amplification of infectious virus was observed in cells knocked down for VCP , suggesting a complete block in virus replication . Cell viability assays showed a moderate decrease in cell viability at 5 days post transfection with VCP siRNA compared to negative control siRNA , although visual inspection of cells did not indicate gross cytotoxicity and the reduction is not sufficient to account for the substantial reduction in virus replication ( S1 Fig ) . Further characterization was performed to determine at what stage of the HCMV replication cycle VCP is required . Analysis of GFP fluorescence at 48 hours post infection ( HPI ) indicated that all cells were infected , demonstrating that VCP expression is not required for virus entry , translocation of genome to the nucleus or initial transcription of the viral genome ( Fig 3A ) . Western blot analysis was performed to measure the levels of each of the major kinetic classes of HCMV genes , including immediate early ( IE1 and IE2 ) , early ( pp52 ) and late ( pp28 ) , to define when disruption in virus replication occurs ( Fig 3B ) . In control cells , protein expression of each class of virus gene is clearly observed . However , in cells knocked down for VCP , neither IE2 nor downstream viral gene expression was detected . Strikingly , despite being encoded by the same primary transcript , IE1 expression levels were higher relative to control cells , up until day four post infection . This data indicates that VCP expression is required for the onset of HCMV replication and suggests knockdown of VCP affects expression of the major immediate early genes . Because knockdown of VCP resulted in specific loss of IE2 but not IE1 , we hypothesized that VCP may play a role in IE2 protein stability , transcript stability or regulation of alternative splicing and polyadenylation . To determine whether VCP was required for IE2 protein stability we inhibited the two main pathways of protein degradation using MG132 ( proteasome inhibitor–ubiquitin dependent ) and pepstatin A or E64 ( protease inhibitors–ubiquitin independent ) in the context of VCP knockdown ( Fig 4A ) . If VCP regulation of IE2 was due to protein degradation , inhibition of these pathways should rescue IE2 protein levels . Neither MG132 , pepstatin A , nor E64 rescued the loss of IE2 proteins levels following knockdown of VCP . Instead , higher concentrations of MG132 phenocopied VCP knockdown in control cells , indicating that proteosomal function is required for IE2 but not IE1 gene expression . As VCP targets ubiquitinated proteins for proteosomal degradation , this result suggests that an intermediate protein , targeted by VCP , may inhibit IE2 expression and is consistent with previous publications showing inhibition of the proteasome reducing IE2 expression and HCMV replication [30] . To determine whether regulation of the MIE genes was occurring at the RNA level , Northern blot analysis was performed . Fibroblast cells transfected with siRNA against VCP or control siRNA were infected 48h post transfection at high MOI with TB40E and total RNA harvested at 24 , 48 and 72 HPI . As shown in Fig 4B , knockdown of VCP resulted in a substantial reduction of IE2 RNA levels at 48 and 72 HPI , including the smaller IE60 and IE40 species . This result was confirmed by qRT-PCR , using primers specific for IE1 and IE2 transcript . Supplemental Fig 2A demonstrates the clear difference in the kinetics of IE1 and IE2 transcript accumulation in control cells , with IE1 transcript levels rapidly increasing following infection , while IE2 levels initially increase , then plateau between 12 and 24 HPI , before accumulating to substantially higher levels from 24 HPI onwards . This accumulation fails to occur following knockdown of VCP , where levels of IE2 do not substantially increase after 24 HPI , in contrast to control cells ( S2B and S2D Fig ) . MIE transcription is also affected at earlier time points with levels of both IE1 and IE2 reduced at 6 HPI and increased at 24 HPI ( S2C and S2D Fig ) . An increase in IE1 and IE2 RNA levels at 24 HPI may be a result of increased MIE promoter activity due to the associated loss of IE2 protein expression at early time points , as IE2 is a negative regulator of the MIE promoter . IE1 transcript levels decrease rapidly 24 to 48 HPI , while protein levels remain high , suggesting that IE1 protein is stable and the increase in IE1 transcription at 24 hours may contribute to the prolonged increase in IE1 protein levels . These results suggest that VCP may be affecting other aspects of MIE transcription . However , Northern , Western and qRT-PCR data are all consistent with a substantial effect on IE2 expression from 24 HPI onwards . To determine whether loss of VCP results in specific destabilization of IE2 transcript , RNA levels were measured following treatment of cells with actinomycin D 48 HPI . Actinomycin D inhibits RNA polymerase , blocking new transcription of IE1 and IE2 , allowing for monitoring of transcript stability over time . Total RNA was harvested at the indicated time points and IE1 and IE2 transcript levels determined by Northern blot analysis . Calculation of the half-life of IE1 and IE2 transcripts indicated that stability of both IE1 and IE2 modestly increased by 1 . 8 and 1 . 5 fold respectively , suggesting loss of VCP does not result in IE2 transcript destabilization ( Fig 5 ) . Defining changes in alternative splicing of IE1 and IE2 is challenging as multiple factors , including promoter activity , RNA stability and viral genome amplification can all contribute to an increase or decrease in total IE1 and IE2 mRNA levels . To determine whether knockdown of VCP altered differential splicing of the MIE region , RNAseq analysis was performed . This allows a direct comparison of read counts from the first three shared exons of the MIE transcripts to the IE1 and IE2 specific exons four and five , respectively . This controls for changes in promoter activity and effects of genome amplification . Therefore , based on our current understanding of RNA processing , changes in the absolute ratio of exon four or five to the shared exons must be due to changes in splicing or changes in RNA stability . Cells were transfected with VCP siRNA or a negative control siRNA and infected with HCMV at an MOI of three . Total RNA was harvested at 24 , 48 and 72 HPI and subjected to strand-specific , paired-end , Illumina sequencing . The annotated exons of the major immediate early region were clearly apparent from the mapped reads in all six samples and reads corresponding to the splice junctions were consistent with previous data for the region , indicating splicing between shared exons one to three and splicing between exon three and four , resulting in IE1 transcript , and three and five resulting in IE2 transcript ( S3 Fig ) . Consistent with the Northern blot and qRT-PCR data , MIE transcription increased at 24 HPI but decreased at 48 and 72 HPI following knockdown of VCP ( S4 Fig ) . Furthermore , splicing of the MIE primary transcript is heavily biased towards IE1 at 24 hours , with IE2 splicing increasing as the infection progresses ( S4A Fig ) . To determine whether knockdown of VCP alters the balance of splicing between IE1 and IE2 , the proportion of total reads mapping to the five exons of the IE region were calculated ( S2 Table ) . Fig 6A represents the absolute difference in exon frequencies from VCP knockdown samples compared to negative control samples . At 24 HPI , knockdown of VCP has no effect on the relative proportion of reads mapping to exon four and five . However , by 48 HPI there is a clear increase in the relative proportion of reads mapping to exon four in VCP knockdown samples , with a corresponding decrease in exon five read frequencies . In contrast knockdown of VCP has no effect on the relative proportion of reads mapping to the shared exons , indicating reduced splicing from exon three to five ( IE2 ) and a corresponding increase in splicing from exon three to four ( IE1 ) . Knockdown of VCP also resulted in lower frequencies of read pairs spanning exon three to five splice junction ( IE2 ) and a corresponding increase in read pairs spanning exon three to four splice junction ( IE1 ) ( Fig 6A and 6B , S4B Fig and S3 and S4 Tables ) . These results show that knockdown of VCP alters splicing of MIE transcripts resulting in a failure to switch from IE1 splicing to IE2 splicing . To determine whether knockdown of VCP resulted in more generalized effects on virus transcript splicing , we analysed read counts for other well-characterized HCMV spliced transcripts ( S5 Fig ) . Based on exon counts , only UL37 showed a similar pattern to MIE transcripts in response to VCP knockdown where absolute exon frequencies for UL37 exon 1 increased with a corresponding decrease in exon 3 read frequencies . However , unlike the MIE transcripts , analysis of paired-end reads did not support substantial splicing between UL37 exon 1 to exons 2 and 3 ( S3 Fig ) . Instead , exon 1 and exons 2 and 3 of UL37 are likely to be predominantly independent transcriptional units with differing kinetics , with exon 2 and 3 requiring IE2 expression for transactivation . To determine the effect of VCP knockdown on global viral gene expression , read counts were mapped to the entire viral genome . As shown in Supplemental Fig 6 total read counts mapping to the viral genome were relatively similar at 24 HPI . However , while viral gene expression increased in control cells by approximately 6 . 5 fold between 24 and 72 HPI , expression in VCP knockdown cells only increased by 3-fold , indicating a general reduction in viral gene expression following VCP knockdown . This is unsurprising given the vital role IE2 protein plays in transactivation of viral gene expression . To determine whether knockdown of VCP results in a similar effect on all viral genes or differential effects , total reads were mapped to individual viral open reading frames and viral gene expression compared between control cells and VCP knockdown cells ( Fig 7 and S5 Table ) . At 24 HPI moderate reductions in viral gene expression can be observed , with a subset of transcripts expressed at relatively higher levels following VCP knockdown . In particular , expression levels of UL36 and UL37 , UL112/113 and the MIE transcripts UL122 ( IE2 ) and UL123 ( IE1 ) were relatively higher at 24HPI in VCP knockdown cells compared to control cells . However , at 48 and 72 HPI there is more profound global suppression of virus gene expression in VCP knockdown cells . This is particularly apparent for the non-coding genes RNA 4 . 9 and 5 . 0 and other genes that are highly up regulated at later stages of infection , including UL85 , UL86 , UL100 and UL102 . Despite the loss of IE2 expression , the expression of a subset of viral genes remained equivalent to control levels following knockdown of VCP ( Fig 7 and S7 Fig ) . These include UL21A , UL36 and UL38 , UL111A and UL112/113 , UL123 ( IE1 ) and UL144 . This is particularly surprising for UL112/113 which has previously been identified as a target for IE2 transactivation [31 , 32] . Previous studies have shown that the cell cycle impacts on the regulation of MIE alternative splicing with IE2 splicing and virus replication blocked at the G2/M phase [33] . Furthermore , cyclin dependent kinases are required for efficient IE2 expression and cyclin A2 overexpression inhibits IE2 splicing and virus replication [34] . To determine whether knockdown of VCP results in alterations in cell cycle , which in turn regulates MIE splicing , cells were stained with propidium iodide following VCP knockdown . The results clearly show that knockdown of VCP has no gross effect on cell cycle control ( Fig 8 ) . Consistent with the majority of cells being in G0/G1 phase , cyclin A2 levels remained undetectable by western blot in control and VCP knockdown cells ( S8 Fig ) . These results show that regulation of MIE splicing by VCP is not a consequence of alterations in cell cycle control and instead represents a novel regulatory pathway . To determine whether the effect of VCP on MIE expression could be the result of a simple delay or inhibition of progression in virus replication , we compared MIE protein and transcript levels following treatment of cells with Ganciclovir , a well characterized drug , which inhibits viral DNA synthesis . While both Ganciclovir treatment and VCP knockdown result in substantial reductions in IE2 gene expression , only knockdown of VCP results in a corresponding increase in IE1 gene expression and relative changes in IE1 and IE2 splicing ( S9 Fig ) . Furthermore , in contrast to VCP knockdown , IE2 and pp52 expression could be detected four days post infection following Ganciclovir treatment . These data suggest alterations in MIE levels , caused by VCP knockdown are not an artifact of delayed virus replication . Previous studies have demonstrated that VCP plays an important role in the cytoplasm of HCMV infected cells , mediating US11-dependent degradation of MHC class I , by stripping the protein from the ER membrane in a ubiquitin dependent manner [27] . To determine the cellular localisation of VCP during HCMV infection , primary fibroblast cells were infected at high MOI with HCMV , fixed at 24-hour time points and stained for VCP . As shown in Fig 9 , VCP displayed dynamic temporal cellular localisation during HCMV infection . In uninfected cells , VCP staining resulted in diffuse signal throughout the cell . However following infection with HCMV , distinct puncta can be observed in the nucleus of infected cells . By 48 HPI VCP was consistently found within two large puncta within the nucleus , which increased in size by 72 hours . Such staining is characteristic of the viral replication compartments . Following infection with HCMV the viral genomes are deposited adjacent to ND10 domains before forming two distinct replication compartments within the nucleus , characterized by colocalisation of viral DNA , DNA replication proteins and IE2 protein [35] . Therefore , VCP localisation to the virus replication compartments in the nucleus coincides with increased IE2 expression . Given that VCP is essential for HCMV replication , we investigated whether small molecule inhibitors against VCP are viable antiviral candidates . Because VCP is a potential anti-cancer target , a number of effective small molecule inhibitors of VCP have been developed . NMS-873 is a highly potent and selective inhibitor of VCP [36] . To determine whether treatment of infected cells with NMS-873 results in the same phenotype as VCP siRNA knockdown , cells were pretreated with NMS-873 or DMSO and infected at high MOI with HCMV . Total protein was harvested every 24 hours for a total of five days and subjected to western blot analysis . As shown in Fig 10A , treatment with 1μM of NMS-873 recapitulated the phenotypic effects of VCP siRNA , with loss of IE2 transcript and protein expression . In contrast to siRNA knockdown of VCP , treatment with NMS-873 did not result in increased IE1 expression . This may be due to additional off target effects of the drug compared to siRNA knockdown or a consequence of inhibiting the activity of VCP compared to directly knocking down the protein . Next , we compared the antiviral activity of NMS-873 to Ganciclovir , currently the most commonly used HCMV antiviral . Primary human fibroblast cells were pretreated with different concentrations of Ganciclovir , NMS-873 or DMSO control , with cells and supernatant harvested seven days post infection for plaque assay . As shown in Fig 10B , NMS-873 and Ganciclovir clearly inhibited the production of infectious virus . However , NMS-873 displayed a higher level of potency at ten-fold lower concentrations , with an IC50 of 0 . 13 μM compared to 1 . 3 μM for Ganciclovir . Co-treating cells with NMS-873 at the same time as infection also resulted in reduced IE2 levels and a similar reduction in infectious virus ( Fig 10C and S10A Fig ) . In contrast , treating cells 24 HPI did not reduce IE2 expression or virus production to the same extent ( Fig 10C and S10B Fig ) . While immediate early and early viral gene expression was not substantially affected by treatment of cells with NMS-873 at 24 hour post infection , expression of the late gene pp28 was substantially reduced , with infectious virus reduced 100 fold , suggesting VCP may also be involved in late processes of virus replication . This is consistent with the pleiotropic nature of VCP and its potential to affect HCMV replication in a multitude of ways . Treatment with NMS-873 also showed little toxicity at effective concentrations ( Fig 10D ) , suggesting NMS-873 and small molecule inhibitors of VCP show significant potential as HCMV antiviral therapies .
VCP plays a pivotal role in ubiquitin-dependent signaling through remodeling target proteins , often leading to proteosomal degradation . As such it is linked to a wide range of biological functions , including protein quality control , autophagy , chromatin remodeling and more recently RNA processing [21] . Following systematic screening of host genes involved in membrane organization , we identified VCP as a critical host factor for HCMV replication . Further characterization demonstrated that knockdown of VCP resulted in an initial increase in MIE transcription , followed by a substantial reduction in the expression of the major immediate early transcript IE2 . Strikingly , despite being expressed from the same primary transcript , IE1 levels increased following VCP knockdown . Our data also suggests that VCP plays a role during late stages of virus replication as inhibition of VCP 24 HPI did not substantially affect IE2 expression but still resulted in a two-log reduction in infectious virus . Given the functionally pleotropic nature of VCP , it is unsurprising that knockdown results in multifactorial effects on the virus and potentially reflects the diverse roles of ubiquitin modulation and signaling during virus infection . Viral gene expression during HCMV replication occurs through a tightly regulated cascade mechanism with expression of the MIE genes IE1 and , in particular , IE2 , required for transactivation of early viral genes and subsequent late viral gene expression . Due to the central role of the MIE proteins in driving replication of the virus they have been suggested to play a pivotal role in regulating the balance between productive and latent infection . IE1 and IE2 are generated from the same primary transcript by alternative splicing , sharing the first three exons , while splicing to independent final exons through the relatively unusual process of terminal exon skipping [10 , 12] . Consistent with previous publications [33 , 34] , our data shows that IE1 expression levels increase more rapidly than IE2 , due to preferential splicing to the IE1 terminal exon 4 , resulting in lower levels of IE2 transcription , which plateaus between 12 and 24 HPI ( S2 , S3 and S4 Figs ) . As infection progresses , splicing to IE2 terminal exon 5 increases , with a corresponding decrease in IE1 splicing . Regulation of alternative splicing of the MIE transcripts is therefore crucial for determining whether replication of the virus progresses , as expression of IE2 is dependent on this switch in splicing . Although the timing and precise splicing pattern of the MIE region has been well documented , how the splicing is regulated and the downstream consequences for virus replication are poorly understood [10] . However , it is likely to depend on a complex interplay of cellular and viral factors and the architecture of the MIE transcriptional region [37] . Expression of the MIE transcripts and their splicing are intimately linked to multiple factors including cell cycle , cell type and differentiation status [13 , 38 , 39] . Suppression of MIE transcripts during HCMV infection of myeloid cultures is thought to be key for establishing latent infection . Conversely , activation and differentiation of latently infected cells results in up-regulation of MIE gene expression and subsequent reactivation . During infection of fibroblast cells , expression of MIE transcripts and replication are closely linked to cell cycle status , with MIE expression suppressed during S/G2 phase [40 , 41] . This suppression is due in part to cyclin A2 , which alters splicing of the MIE transcripts in a manner similar to knockdown of VCP , resulting in loss of IE2 expression and failure in virus replication [33] . While expression of cyclin A2 results in a similar phenotype , our data shows that VCP knockdown does not cause gross changes in the cell cycle or increased cyclin A2 expression , indicating an alternative mechanism of action . Although the majority of research on VCP has focused on its cytoplasmic activity , recent studies have identified equally important and diverse roles for VCP in the nucleus . The association of VCP with the viral replication compartments early in infection suggests that the nuclear functions of VCP may be playing a critical role in HCMV replication . Studies in yeast and mammalian cell lines have demonstrated that VCP modulates the association of factors with chromatin , leading to regulation of cell cycle , transcription , DNA replication and DNA damage response , all of which could potentially affect virus replication and MIE expression [21] . Previous reports have shown that HCMV induces the host DNA damage response , with response factors including ATM , γH2AX and E2F1 required for efficient HCMV replication and IE2 expression [42] . γH2AX also colocalises with the viral replication compartments in the nucleus . VCP acts downstream of ATM and is required for the removal of ubiquitinated factors associated with dsDNA break regions , allowing repair to proceed [21] . If knockdown of VCP disrupts the DNA damage response during HCMV infection , this could contribute to the observed loss of IE2 expression and inhibition of virus replication . Reports are also emerging that VCP plays a role in transcript stability and regulation of splicing . HuR is a RNA binding protein that stabilizes transcripts by binding to canonical AU rich regions within 3’UTRs . Following ubiquitination of HuR in response to stress signals , VCP removes HuR from associated transcripts resulting in transcript destabilization [43] . In Drosophila , VCP has been linked to dendrite pruning in neuronal cells [44] . Expression of mutant forms of VCP results in incorrect splicing of a gene important to dendritic pruning , MICAL , possibly through mis-localisation of the RNA binding protein TDP-43 . Given the known mechanism of action of VCP it is likely that any effects on MIE splicing are indirect , occurring through targeting of a ubiquitinated intermediate protein . One possible model is that a negative regulator of IE2 splicing exists at early stages during HCMV infection , which promotes IE1 expression at the expense of IE2 expression . Based on the transcriptional architecture of the major immediate region , such a factor could regulate MIE splicing by promoting polyadenylation of the IE1 transcript , thereby inhibiting read-through to the IE2 final exon . The factor would be ubiquitinated and removed by VCP between 12 and 24 HPI , allowing read-through of the first polyadenylation site and splicing of the IE2 transcript . This would be consistent with the initial plateau in IE2 transcription and localisation of VCP to the replication compartments at 12 to 24 HPI , where spliceosome and proteasome components have also been reported to colocalise [30 , 45] . The NMS-873 data also supports this model , with inhibition of IE2 expression only effective with either pre-treatment or addition of the drug at the same time as infection . Treatment at 24 HPI was ineffective at blocking IE2 expression , suggesting a window in which VCP activity is required for regulation of MIE splicing . Interestingly , blocking de novo protein translation by cycloheximide treatment , effectively rescues IE2 transcription following NMS-873 treatment , suggesting the inhibitory factor is induced by virus infection ( S11 Fig ) . Although not common , there are a number of examples of terminal exon skipping in the human genome , which would be analogous to the proposed model . Maturation of B cells to differentiated plasma cells results in a switch from predominantly membrane bound IgM to secreted IgM . Studies have shown that increased expression of the polyadenylation factor cstF-64 promotes polyadenylation upstream of the exons required for membrane bound IgM resulting in increased expression of the secreted form [46 , 47] . Tissue specific regulation of calcitonin peptide is also regulated through differential polyadenylation of an embedded terminal exon [48] . Here , the splicing factor SRSF3 ( SRp20 ) binds to a polyA enhancer sequence and directs polyadenylation of the upstream polyA site through recruitment of polyA factors . Restricting SRSF3 activity results in removal of the upstream terminal exon by splicing and enhanced expression of the downstream terminal exon . In these examples splicing is directly linked to cellular differentiation , in the case of IgM , and tissue specificity , in the case of calcitonin . These factors are fundamental to the regulation of HCMV latency with cell type specificity determining the site of latency and differentiation of latently infected cells linked to reactivation [13 , 14] . Given the central role of IE2 in determining virus replication , linking regulation of MIE splicing to cellular differentiation and tissue specificity would be a potent mechanism of regulating the establishment , maintenance and reactivation of latency in HCMV . Experiments are currently underway to determine whether regulation of MIE splicing by VCP follows a similar paradigm to cellular examples and whether such regulation is involved in HCMV latency . Finally , as VCP is clearly essential for virus replication , small molecule inhibitors of VCP are potentially attractive antiviral candidates . HCMV-related illness accounts for more than 60% of diseases associated with solid organ transplant patients . Prolonged treatment , especially in patients with severely suppressed immune systems , greatly increases the risk of antiviral resistance [49–51] . Very few antivirals have been developed for use against HCMV since the licensing of Ganciclovir , and of these , the same viral genes are targeted , reducing the likely usefulness of these drugs against resistant strains [52] . An alternative strategy for the development of novel antivirals involves targeting of host genes required by the virus for successful replication . VCP has been identified as a potential anti-cancer target and as such a number of small molecule inhibitors have been developed , the most potent of which is NMS-873 [36] . We show that NMS-873 is 10 times more potent than Ganciclovir at equivalent concentrations , with little sign of toxicity at active levels . Whether the drug is toxic in vivo remains to be determined . Deletion of VCP in transgenic mice is non-viable and naturally occurring mutations in humans are linked to severe developmental defects [21 , 53] . However , transient inhibition of VCP by small molecule inhibitors may be a viable treatment option , especially in patients where resistance to current antivirals poses a significant risk to health .
Normal Human Dermal Fibroblasts ( NHDF; Gibco ) were maintained in Dulbecco’s modified high glucose media ( DMEM; Sigma ) supplemented with 10% fetal bovine serum ( FBS; Gibco ) and 1% penicillin-streptomycin ( Invitrogen ) . A low passage HCMV strain TB40E , which expresses GFP from an SV40 promoter was used for siRNA library screening , western blot analysis , and northern blot analysis . Laboratory adapted HCMV strain AD169 was used for immunofluorescence experiments . The Human siGENOME siRNA Library that targets 140 membrane trafficking genes ( 4 siRNAs per gene; Dharmacon , Inc . ) and 20 other selected genes were included in the primary screen . In brief , NHDFs were seeded in 96-well plate a day before siRNA transfection . Next day , cells reached 90–95% confluency and were transfected with siRNA twice ( 4 hours apart between first and second transfections ) using Lipofectamine RNAiMAX ( Invitrogen ) according to the manufacturer’s protocol . Transfected NHDFs were incubated for 48 hours and then infected with GFP expressing TB40E virus at an MOI of three . GFP intensity was monitored every 24 hours with Synergy HT microplate reader ( Biotek ) . The entire screen was performed in duplicate and repeated three times . VCP gene identified from the primary screen was subsequently silenced with four individual siRNAs to eliminate the possibility that the phenotype was associated with off target effects . Cells were lysed with RIPA buffer ( 0 . 1% SDS , 1% Triton X-100 , 1% deoxycholate , 5 mM EDTA , 150 mM NaCl , and 10 mM Tris at pH 7 . 2 ) containing protease inhibitor cocktail ( Roche ) . Ten micrograms of the total lysate was separated in 10% SDS-polyacrylamide gels and transferred to PVDF membranes ( Millipore ) . Primary antibodies used in this paper are mouse anti-CMV IE1/2 monoclonal antibody ( MAB8131 , Millipore ) , mouse anti-CMV pp52 monoclonal antibody ( CH16 , Santa Cruz Biotechnology ) , mouse anti-CMV pp28 monoclonal antibody ( CH19 , Santa Cruz Biotechnology ) , rabbit anti-VCP polyclonal antibody ( H-120 , Santa Cruz Biotechnology ) , and mouse anti-β-Actin monoclonal antibody ( Abcam ) . Blots were probed with primary antibody ( 1:500–1:2000 ) diluted in 5% dehydrated milk in Tris Buffered Saline ( TBS ) and subsequently the HRP-conjugated secondary antibodies ( Pierce ) at 1:5000 . Blots were washed in TBS three times , incubated with chemiluminescent substrate ( SuperSignal West Pico; Thermo Scientific ) according to the manufacturer’s protocol , and exposed in G:Box ( Syngene ) for visualization of bands . Total RNA was isolated by using Trizol solution according to the manufacturer’s protocol . Northern blot analysis for IE1 and IE2 mRNAs was conducted using total RNA that was separated on a 1 . 2% agarose formaldehyde gel and then transferred using Whatman TurboBlotter Rapid Downward Transfer Systems . IE1 and IE2 probes were generated by PCR using cDNA from TB40E infected NHDFs ( as template ) and labelled with Amersham Rediprime II DNA labelling system ( GE Healthcare ) with the following primers ( IE1: TCAAACAGATTAAGGTTCGAGTGG , and ATCCACATCTCCCGCTTATCCTCG; IE2: TCATGGTGCGCATCTTCTCCACC , and TTACTGAGACTTGTTCCTCAGGTCC ) . After hybridization and wash , the membranes were exposed to autoradiography film with an intensifying screen ( Biomax Transcreen HE , Kodak ) for visualization of bands . Cells were transfected and infected as described above . Total RNA was harvested using Trizol according to manufacturers guidelines . Total RNA was submitted to Edinburgh Genomics for generation of TruSeq stranded libraries and subjected to HiSeq high output v4 125PE sequencing . The strand-specific RNA-seq reads were mapped to a combined version of the human ( hg38 ) and human herpesvirus 5 ( KF297339 ) genomes using the HISAT spliced read mapper ( PMID:25751142 ) . Only valid read pairs mapped together in the correct orientation were retained ( i . e . carrying a SAM flag of one of 83 , 99 , 147 or 163 ) and a custom Perl script was used to count the number of these reads mapping to each exon as well as the number of read pairs spanning different combinations of exons . NMS-873 ( Xcess Biosciences ) and Ganciclovir ( Cambridge Bioscience ) were dissolved in DMSO and added to the cell culture at a working concentration 24 hours before HCMV infection . Viability for NMS-873 treated cells was assessed using the CellTiter-Glo luminescent cell viability assay kit ( Promega ) and CellTiter-Blue ( Promega ) 120 hours after NMS-873 addition . MG132 ( Cambridge Bioscience ) , Pepstatin A ( Sigma ) , and E64 ( Sigma ) were dissolved in DMSO and added to the cell culture 24 hours after HCMV infection . 100 μg/ml cycloheximide ( in DMSO , Sigma ) was added to the cell culture 30 minutes before HCMV infection to block protein biosynthesis . Laboratory adapted HCMV strain AD169 infected cells were fixed and permeabilized in Methanol:Acetone solution ( 1:1 ) for 7 minutes , and then blocked with 5% human serum in PBS for 30 minutes . Primary and secondary antibodies were diluted with 5% human serum in PBS . Cells were washed with PBS after primary and after secondary antibody incubations . Primary antibodies used in this paper are mouse anti-CMV IE2 monoclonal antibody ( 12E2 , Santa Cruz Biotechnology ) , rabbit anti-VCP polyclonal antibody ( H-120 , Santa Cruz Biotechnology ) at 1:500 . Alexa-fluor-647 conjugated goat anti-mouse or Alexa-fluor-488 conjugated goat-anti-rabbit IgG secondary antibodies were diluted 1:1000 . All images were acquired with Zeiss LSM 710 confocal microscope fitted with 63X/1 . 4 oil-immersion objective lens . Total RNA was isolated by using Trizol solution according to the manufacturer’s protocol followed by DNase ( Turbo DNA-free kit , Ambion ) treatment , and then reverse transcribed with poly T primers using High Capacity cDNA Reverse Transcription Kit ( Invitrogen ) . Real-Time PCR was carried out using by Taqman assays with pre-designed gene-specific primer/probe set ( Applied Biosystems ) on Rotor gene 3000 ( Corbet Research ) . Custom primer/probe set are CGTCAAACAGATTAAGGTTCGAGTGG , CCACATCTCCCGCTTATCCTCG , and 56-FAM/CATGCTCTG/ZEN/CATAGTTAGCCCAATACACTTCATCTC- CTCG/3IABkFQ for IE1 , and ATGGTGCGCATCTTCTCCACC , TTACTGAGACTTGTTCCTCAGGTCCTG , and 56-FAM/CAGGCTCAG/ZEN/GGTGTCCAGGTCTTCGGGAGG/3IABkFQ for IE2 . Cells were harvested with trypsin , washed with PBS , and followed by fix with 70% cold ethanol for 30 minutes at 4°C . Then the fixed cells were washed twice with PBS , treated with RNaseA , and labelled with 10 μl of propidium iodide ( 1 mg/ml stock solution , Sigma ) . Cell cycle analysis was first carried out using BD LSRFortessa X20 , and then further analyzed using FlowJo software . | Viruses are obligate intracellular pathogens , meaning that they are completely dependent on the host cellular machinery to replicate . Identifying which host genes are necessary for virus replication extends our understanding of how viruses replicate , how cells function and provides potential targets for novel antivirals . Here , we show that a cellular factor called valosin containing protein ( VCP ) is essential for human cytomegalovirus replication . We demonstrate that VCP is required for the expression of an essential virus gene called IE2 . Finally we show that a chemical inhibitor of VCP is a potent antiviral against human cytomegalovirus , demonstrating the potential for VCP inhibitors as novel therapeutics against this virus . | [
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"genetics",... | 2017 | The host ubiquitin-dependent segregase VCP/p97 is required for the onset of human cytomegalovirus replication |
The most established model of the eukaryotic innate immune system is derived from examples of large effect monogenic quantitative resistance to pathogens . However , many host-pathogen interactions involve many genes of small to medium effect and exhibit quantitative resistance . We used the Arabidopsis-Botrytis pathosystem to explore the quantitative genetic architecture underlying host innate immune system in a population of Arabidopsis thaliana . By infecting a diverse panel of Arabidopsis accessions with four phenotypically and genotypically distinct isolates of the fungal necrotroph B . cinerea , we identified a total of 2 , 982 genes associated with quantitative resistance using lesion area and 3 , 354 genes associated with camalexin production as measures of the interaction . Most genes were associated with resistance to a specific Botrytis isolate , which demonstrates the influence of pathogen genetic variation in analyzing host quantitative resistance . While known resistance genes , such as receptor-like kinases ( RLKs ) and nucleotide-binding site leucine-rich repeat proteins ( NLRs ) , were found to be enriched among associated genes , they only account for a small fraction of the total genes associated with quantitative resistance . Using publically available co-expression data , we condensed the quantitative resistance associated genes into co-expressed gene networks . GO analysis of these networks implicated several biological processes commonly connected to disease resistance , including defense hormone signaling and ROS production , as well as novel processes , such as leaf development . Validation of single gene T-DNA knockouts in a Col-0 background demonstrate a high success rate ( 60% ) when accounting for differences in environmental and Botrytis genetic variation . This study shows that the genetic architecture underlying host innate immune system is extremely complex and is likely able to sense and respond to differential virulence among pathogen genotypes .
Our understanding of host/pathogen interactions is largely driven by examples of large-effect , qualitative resistance involving a small subset of host genes focused on detection and signal responses to the pathogen . However , many host/pathogen interactions are predominantly quantitative in nature involving an unspecified number of genes with moderate to small effects involved in detection and response to the attacking pathogen . Quantitative resistance is characteristic of host resistance to endemic pathogens that are ubiquitous within a host’s environment and create a persistent selective pressure on the innate immunity of the host [1] . This constant selective pressure from endemic pathogens prevents the development of highly susceptible host alleles and may canalize the genetic variability of many genes involved in resistance . This differs distinctly from epidemic pathogens that are extremely scarce between outbreaks allowing for the development of resistance genes with large-effect alleles that can spread throughout the host population [2 , 3] . The continuous nature of quantitative resistance suggests that there may be alleles at a number of genes that can provide long-term , durable resistance to these pathogens . However , this lack of large effect natural alleles and the large number of genes that are thought to be involved in quantitative resistance complicates our understanding of involved biological functions and pathways . The resistance mechanisms utilized by the host may also depend upon the lifestyle of the pathogen . Quantitative resistance is often seen in response to necrotrophic pathogens , such as Aspergillus fumigatus in humans [4 , 5] , southern leaf blight ( Bipolaris maydis ) in maize [6] , and pitch canker ( Fusarium circinatum ) in loblolly pine [7] . However , there have been very few quantitative resistance loci actually cloned and the underlying genetic architecture or molecular mechanisms associated with this natural variation is relatively hidden [6] . Comparatively , qualitative resistance is commonly observed in specialist , biotrophic pathogens where host individuals can easily be categorized into susceptible and resistant genotypes for a single strain of the pathogen . Examples include Vibrio cholerae in mammals [8] , Xanthomonas oryzae in rice [9 , 10] , and Pseudomonas syringae in Arabidopsis and tomato [11 , 12] . The large-effect polymorphisms involved with qualitative resistance lend themselves nicely to investigation through Mendelian genetics allowing for the identification of the individual causative genes . Currently , the vast majority of qualitative resistance genes identified using natural allelic variants in Arabidopsis have been either direct or indirect receptor proteins , including receptor-like kinases ( RLKs ) and nucleotide-binding site leucine-rich repeat proteins ( NLRs ) [13–18] . The QTL studies leading to the cloning of these genes typically found large and small effect loci but for experimental reasons , focused solely on the cloning of large effect loci , leaving the molecular identity of the small effect loci uncovered . In contrast , naturally variable alleles in downstream components of qualitative resistance , such as mitogen-activated protein kinases , transcription factors , and proteases/lipases , have largely not been found underlying the large effect qualitative loci . These downstream components have predominantly been identified through the use of careful molecular assays , including mutant screens , protein co-immunoprecipitations , and yeast one/two hybrid assays . Expression QTL analysis investigating the impact of major effect R genes showed that these large effect loci function via a vast array of downstream loci , but it was not clear if genetic variation in these downstream loci also contributed to disease resistance [19–22] . Thus , while it is clear that variation in qualitative resistance works through the upstream receptors and signaling pathways , it is unclear whether these loci and/or pathways play a key role in the variation of quantitative resistance . To empirically measure quantitative resistance and identify the genes that control variation in this resistance , we are using the endemic plant fungal pathogen Botrytis cinerea to conduct a genome-wide association ( GWA ) study with the model host plant Arabidopsis thaliana . Botrytis cinerea is a necrotrophic fungal pathogen of plants that is found on every continent with a temperate climate and is thought to have a pan-global natural range prior to human agriculture [23] . This endemic pathogen has a large amount of standing genetic variation in all currently studied populations [24–28] and has an extremely large host range that spans non-vascular and seedless plants to most true eudicots [29–31] . There are currently no known large-effect qualitative resistance alleles identified in any plant for resistance to B . cinerea and quantitative studies in structured mapping populations have shown that resistance is highly polygenic with significant epistasis that is highly dependent on genetic variation between Botrytis isolates [32–34] . Thus , B . cinerea is a highly useful model pathogen that allows us to both identify the underlying mechanisms of quantitative resistance in innate immunity and to assess how pathogen genetics influences these loci . For the plant host , we are using the model plant Arabidopsis thaliana to measure quantitative resistance to this endemic fungal necrotrophic pathogen . Arabidopsis thaliana has already provided a wealth of information for understanding host resistance within pathology including some loci identified to control resistance to a variety of pathogens via natural alleles as well as forward and reverse mutant screens [35–37] . Specifically , the use of natural variation in the host has allowed the identification of a small set of novel genes involved with B . cinerea resistance including an RLK specific for mannitol perception [36 , 38 , 39] . To gain a broader view of quantitative resistance and the potential mechanisms involved , we are utilizing the rich genetic resources of natural variation across Arabidopsis accessions [40] to conduct a GWA study and identify individual genes and biological processes associated with resistance to B . cinerea . Natural populations used in GWA studies have several advantages over structured breeding populations , such as increased allelic variants per locus and higher recombination between loci allowing for finer mapping [41] . However , these populations can be limited if natural selection imparts a strong structure on the underlying allelic variation such that some variants or combinations are missing from the population . GWA has already been proven to be effective by recapitulating results from previous qualitative resistance studies [42] and by identifying new genes controlling defense mechanisms within Arabidopsis [43] . GWA has also been applied to a few non-model or genomically complex host species [6 , 7] to identify novel components of quantitative resistance , many with unknown function . However , these studies were unable to validate if these genes affect quantitative resistance . By working with the model plant host A . thaliana , we can combine GWA hits with the vast store of gene expression studies to help identify co-expression modules that are involved with quantitative resistance and provide putative function to genes with unknown function as we have previously done for defense metabolism [43] . In this study , we measured quantitative resistance in a collection of natural A . thaliana accessions individually infected with one of four phenotypically and genetically diverse B . cinerea isolates . We quantified resistance by both lesion size and camalexin production as a quantitative biochemical marker of the plant’s defense response . Our study shows that quantitative resistance can involve thousands of genes linking to a wide variety of cellular processes ranging from hormone signaling and reactive oxygen signaling that have previously been associated with disease resistance to development and RNA processing , which to our knowledge , has not been associated with disease resistance . The data also suggest that there is a hierarchical nature to related disease phenotypes with some genes contributing to both lesion development and defense response while others appear to be specific to one aspect of quantitative resistance . In addition , our study also illustrates that quantitative resistance of the plant host is highly specific to the pathogen’s genotype . Thus , mapping for quantitative resistance can provide very different answers depending on which isolate is used and suggests that even quantitative resistance will have difficulty in providing durable resistance within host populations .
Arabidopsis accessions have a wide range of flowering times that can affect the ontogenic status of the plant and influence plant/pathogen interactions [42 , 44] . Thus , we wanted to choose our GWA population to minimize any ontogenic influence on quantitative resistance while still maintaining genetic diversity . As such , we filtered a population of 241 accessions to a collection of 96 individuals based on similar flowering time ( 63 . 1 ± 0 . 95 ( s . e . ) days to flowering under short-day conditions [42] ) . This filtering should both alleviate issues with differential rate of development across the accessions and minimize any potential influence of flowering time in our mapping . Importantly , this collection includes several natural accessions that are the parents of defined breeding populations that have been screened for resistance to B . cinerea ( i . e . Bay-0 , Sha-0 , and LeR-1 ) [33 , 34] . Another potential concern for GWA mapping within a natural population is the effect of population structure and rare alleles that result in spurious associations between the phenotype and an allele [45–47] . To assess the level of population structure in our population , we performed a hierarchical clustering analysis of the kinship matrix for 215k SNPs for both the 241 accessions and the ontogenically-filtered collection of 96 accessions . The analysis showed that by simply filtering on flowering time in a growth chamber , we were able to remove the majority of large genetic outliers as well as the majority of population structure ( Fig 1 and S1 Fig ) . Further , while the remaining accessions cluster into five major genomic subgroups , these subgroups do not appear to cluster to a specific geographical region ( S2 Fig ) . While growth chamber measured flowering time is correlated with geography , there is still significant variation within each region and our filtering appears to have removed the extreme flowering individuals and chosen a subpopulation that encapsulates the majority of the geographic range of the species . The final population contains a total of 115 , 301 SNPs with a minor allele frequency > 0 . 2 spanning a total of 19 , 352 genes tested ( S1 Table ) . This population selection should reduce the influence of correlated ontological phenotypes and potentially the false positive rate from rare alleles and population structure , which we tested empirically and describe below ( Fig 1 ) . To investigate the basis of quantitative resistance and how genetic variation in the host and pathogen influence the ability to identify the underlying loci , we infected the above 96 accessions with four phenotypically diverse Botrytis cinerea isolates chosen based on their diversity in previous studies [33 , 48–51] using a randomized complete block design with four replicates in each of two independent experiments . We measured lesion size at 72 hours post infection using a digital image analysis pipeline developed in the R statistical environment using the EBImage and CRImage packages [52 , 53] . Additionally , all leaves were collected at 72 hours to be assayed for camalexin , a key phytoalexin providing defense against Botrytis , via HPLC to provide a measure of plants quantitative defense response to the interaction . F-tests derived from separate generalized linear models ( GLMs ) for both camalexin content and lesion size revealed that there was a statistically significant interaction between the genotype of the plant host and genotype of the isolate ( Table 1 ) . This indicates that quantitative disease resistance within the host is highly isolate dependent . In contrast , the effect of the experimental replicate , individual plant , and interactions of treatment:plant were non-significant for camalexin showing that the biological effects drive most of the phenotypic variance for this experiment ( S2 Table ) . While there was a significant effect of experiment and individual plant on lesion size , we were able to account for this using the model ( Table 1 ) . We chose to analyze each quantitative resistance in response to each pathogen genotype separately since both traits show a statistical significant interaction between the host and pathogen genotypes . This should both simplify the analysis and avoid confounding the results across the isolates . Another concern for genetic mapping in natural populations is the ability of population structure to drive false positive associations . To address this concern we conducted additional GLMs for both camalexin and lesion area for each isolate separately to determine how much phenotypic variance is attributable to the residual population structure . The F-tests revealed that the residual population structure accounted for a significant amount of variation for camalexin in response to all four isolates but was not significant for lesion area ( S2 Table ) . Importantly , however , the proportion of variance ( η2 ) attributable to the phylogenetic group was on average only 7% of the level of phenotypic variation attributable to differences among the accessions ( S2 Table , Fig 2 ) . Model corrected means for genetic mapping were derived from this model using this population to remove potential effects derived from residual population structure . Notably , the distribution of these model corrected means were largely unimodal with only slight skewing that did not require transformation given the large sample size ( S3 and S4 Figs ) . This approach should help reduce false positive associations attributable to the general population structure . To test if there is a relationship between lesion size and camalexin induction across the host/pathogen genotype combinations within this dataset , we calculated Spearman’s rank correlations among the phenotypes . This analysis showed that correlation between camalexin production and lesion area is significant but weakly negative ( S5 Fig ) , as previously found [33 , 34] . Within this analysis , camalexin production between the different isolates showed a higher correlation across accessions than was observed for lesion size . This suggests that the genetic variation for camalexin response may be more similar across the accessions than is the genetic variation for lesion size . Interestingly , the basal camalexin concentration prior to infection had no correlation with either induced camalexin content or lesion size , indicating that basal levels of camalexin are poor predictors of induced camalexin or disease resistance within this collection of accessions . To conduct the GWA mapping and identify individual candidate genes associated with quantitative resistance in Arabidopsis , we used the model corrected means from the full experimental model described above in a separate ridge-regression GWA model . Ridge regression has the advantage over single marker procedures , such as EMMA [46] , of testing all SNPs in single model by treating each SNP as a random effect and avoids issues with multiple test corrections [54] . This benefit comes at the expense of obtaining a direct p-value for each SNP but does provide estimates of the heteroscedastic effect of each SNP . We calculated a significance effects threshold by randomly permuting the phenotypes among the genotypes 1 , 000 times and pulling the 99th percentile from the distribution of effects derived from the permutations to identify significant SNPs . Because this permutation maintains relationship of genotypes within the matrix , it maintains any population structure across the permutations and should help to control for spurious effects caused by population structure . The genetic map for the accessions included a total of 115 , 301 SNPs with a minor allele frequency ( MAF ) > 0 . 2 that covered 19 , 352 unique genes ( S1 Table ) . We identified a total 20 , 476 SNPs associated with camalexin accumulation and 23 , 199 SNPs associated with lesion size across the different isolates ( S4–S8 Figs and S3 Table ) . As some SNPs may be false-positive associations due to genetic linkage with the true casual SNP , we defined significantly associated genes as having ≥2 SNPs that crossed with the permuted threshold within the predicted mRNA coding region , similar to the previous approach by our lab [43] . Using this definition , we identified a total of 2 , 982 genes associated camalexin production and 3 , 354 genes associated with lesion area ( S4–S8 Figs and S3 Table ) . Approximately one third of the genes identified were found to associate with both camalexin and lesion size . This supports the idea that , while they are related measures of quantitative resistance , there are still differences in the genetic architecture underlying the phenotypes ( S9 Fig and S3 Table ) . Notably , most of the genes identified as candidates for controlling resistance to the different isolates were unique to the specific isolate with camalexin only having 38 genes in common among the four isolates ( Fig 3A and S3 Table ) and lesion area having only 9 genes in common ( Fig 4A and S3 Table ) . Of the genes that were common among the isolates , none of these genes are shown to be in common for both camalexin and lesion area , and most are currently understudied with unknown functions ( S3 Table ) . To test if our significance threshold or whether our two-SNP approach is biasing the overlap of candidate gene hits , we utilized different significant thresholds ( 0 . 95 , 0 . 975 , 0 . 99 and 0 . 999 ) as well as requiring a single SNP or two SNPs and reassessed the presence or absence of overlap amongst the candidate genes . This showed that the pattern of candidate genes showing little overlap across isolates was consistent across all permutation thresholds and SNP calls tested ( S11 Fig ) . The fact that most candidate genes consistently appear unique to a subset or individual isolates at a variety of thresholds supports the idea that quantitative resistance to B . cinerea is isolate specific and host signaling/response pathways may depend on the different collections of PAMPs and effectors produced by the different pathogen genotypes . To test if the GWA approach is identifying novel genes or is largely uncovering genes previously identified as being involved with B . cinerea resistance , we used a collection of 101 genes previously identified as providing differential resistance to B . cinerea within Arabidopsis when functionally disrupted [55 , 56] . These studies were largely conducted using a single isolate of B . cinerea and not collections of diverse genotypes . We then tested how many of these 101 genes known to affect resistance to B . cinerea within Arabidopsis were also found in our collection of GWA candidates . Of these genes , 41 genes did not have two SNPs with a MAF > 0 . 2 in our population , and therefore could not be identified in our study ( S4 Table ) . Of the remaining 59 known quantitative resistance genes , only 12 genes had two or more significant SNPs associated with either camalexin or lesion area and were usually only identified as a candidate gene in either one or two of the four total isolates ( S4 Table ) . These genes include RLM3 , RST1 , NPR1 , VSP2 , ATG2 , PAD4 , LYK3 , COI1 , LYK4 , Erecta ( ER ) , AXR1 , and BOI ( S4 Table ) . Previous work has shown that key resistance regulatory genes that have been found by mutagenesis are likely under purifying selection [57] . The 37 known causal genes that were not identified in the GWAS are likely to have insufficient genetic or functional variation amongst the natural accessions to be found in a GWA approach . Thus , out of the 3 , 504 genes that we identified as possible candidates for controlling quantitative resistance , only 12 have been previously linked to B . cinerea resistance . A current model of quantitative resistance either directly or indirectly implies that genetic diversity in R-genes , including receptor-like ( RLKs ) and Nod-like receptors ( NLRs ) , are the main drivers of quantitative resistance . To address this hypothesis with our list of candidate genes associated with quantitative resistance , we looked for enrichment in associations with genomic lists of potential RLK and NLR R-genes [58 , 59] . Using a hypergeometric test , we found a clear enrichment in NLRs , and to a lesser extent RLKs , for both camalexin and lesion size amongst our candidate genes ( S5 Table ) . A total of 40 R-genes were identified in both the camalexin and lesion area mapping . All of these genes are unknown , with the exception of one RLK ( NIK3 ) and three NLR proteins ( WRKY19 , RLM3 , and DAR4 ) ( S5 Table ) . While these known genes all have been identified in different aspects of disease resistance , RLM3 is the only gene that has been previously characterized as conveying resistance to a broad range of fungal pathogens including Botrytis [60] . While there is a significant over-representation of these associated R-genes , they only account for a small percentage of the total number of associated genes in the study ( 5 . 5% of camalexin genes and 6 . 2% of lesion area genes ) ( S3 and S5 Tables ) . Thus , while it appears that some R-genes do underpin a component of the measured quantitative resistance phenotype , they do not define the vast majority of genes identified in this study . To identify what other cellular functions may be involved with quantitative variation in the host innate immune system , we conducted an unbiased gene ontology ( GO ) enrichment of biological processes using all associated genes ( S10 Fig and S6 Table ) . This analysis revealed that genes associated with variation in camalexin production are enriched in transmembrane transport and cell cycle . Alternatively , genes associated with variation in lesion area are enriched in more diverse biological processes including defense response , oxidation-reduction processes , and nucleic acid mismatch repair mechanisms ( S6 Table ) . Genes associated with lesion area were also enriched in microsporogenesis-related processes , which is likely related to callose deposition as both pollen coats and lesion perimeters show increased callose deposition [61 , 62] . Both camalexin and lesion area also shared some similar enriched biological processes , which include methylation-dependent chromatin silencing , transmembrane transport , mismatch repair mechanisms and categories related to modification of the cell wall ( S10 Fig and S6 Table ) . Again , these results were unchanged by utilizing different significance thresholds or candidate gene calling methodology . Thus , a GO analysis implicates a variety of cell related processes that are linked to variation in both measures of quantitative resistance , illustrating the complexity of defense response . The above results show that natural variation in quantitative resistance involves novel genes far beyond R-genes and other known defense genes . Given this multi-locus architecture of quantitative variation in the Arabidopsis innate immune system , we worked to identify candidate networks controlling this variation . To do this , we filtered all associated genes through the ATTEDII co-expression database [63 , 64] to identify potential modules of candidate defense genes that are co-regulated . This filtering could also help reduce the number of false associations from the genetic mapping due to linkage and we have previously utilized this approach to identify genes associated with Arabidopsis defense metabolism with a >70% rate of successful validation [43] . Co-expression of two genes was defined as having a mutual rank ( MR ) < 15 and the putative biological function of co-expression networks was determined using GO-enrichment analysis against all testable genes using our pipeline at multiple statistical thresholds from the GWAS data ( 0 . 95 , 0 . 975 , 0 . 99 , and 0 . 999 ) ( S14 Fig ) . The camalexin and lesion area associated genes produced approximately the same number of networks , 25 and 21 networks with >10 genes respectively , but the camalexin networks were on average larger ( 36 nodes ) than lesion area networks ( 24 nodes ) ( Figs 3B and 4B ) . This is in contrast to the fact that there are more GWA candidate genes for lesion area than for camalexin . Notably , this suggests that the difference in network size between the two phenotypes may be due to the overall transcriptional complexity of the candidate genes . Utilizing the higher permutation thresholds ( 0 . 999 or greater ) to call candidate genes led to a greatly diminished ability to identify any co-expression networks since too few candidate genes remained at the higher significance thresholds to create meaningful networks . Additionally , lower thresholds appeared to collapse co-expression networks in to large single networks that are difficult to interpret from a biological perspective ( S14 Fig ) . Thus , we chose to continue with the 0 . 99 permutation threshold analysis . GO analysis of the five largest co-expression networks for each phenotype identifies a variety of enriched biological functions . The largest network ( I ) associated with camalexin production was not enriched for GO categories in disease resistance but is enriched for GO categories connected to cell cycle , vesicle trafficking , and protein/metabolite turnover ( Figs 3B and 5A; S7 Table ) . The two largest hubs within the network are an uncharacterized transducin-like ( At3g50590; degree = 12 ) and an uncharacterized phosphoinositol kinase ( At2g1790; degree = 9 ) . The second largest camalexin network ( II ) is enriched in GO categories typically associated with the plant defense response ( Figs 3B and 5B; S7 Table ) , and includes the salicylate receptor ( NPR1 ) . Interestingly , NPR1 is proximally located to several large hubs within this network , including the NLR protein ZAR1 ( At3g50950; degree = 8 ) , a SAM-dependent methyl transferase ( At1g55450; degree = 8 ) , and an unknown RLK ( At4g08850 , degree = 8 ) . Camalexin-associated network III is an extremely inter-connected network with few well characterized genes , but appears to be involved with production and tolerance of reactive oxygen species ( S7 Table ) . Network IV is a small network ( n = 48 ) that appears to include genes that regulate DNA methylation ( S7 Table ) and contains two major hubs that are well-studied Arabidopsis genes; ERECTA ( At2g26330 , degree = 6 ) and the histone 2A protein HTA11 ( At3g54560 , degree = 6 ) ( S3 Table ) . Lastly , genes contained in camalexin-associated network V ( n = 46 ) include important enzymes for cell wall formation , including cellulose synthase 10 ( At2g25540; degree = 4 ) and uncharacterized pectin-esterase hub ( At5g49180; degree = 7 ) . However , GO enrichment of camalexin network V showed enrichment in RNA processing ( S7 Table ) , which suggests that cell wall modification and RNA processing may be commonly co-regulated . Thus , natural variation in genes involved with a variety of host processes can have quantitative implications on a host’s ability to produce camalexin during pathogen infection . Co-expression networks of genes associated with lesion area were structurally similar to networks associated with camalexin , but were considerably smaller ( S8 Table ) . The largest network associated with lesion area ( I; n = 129 genes ) was enriched in GO categories related to gravitropism , cell cycle , and leaf development ( Fig 4B; S7 Table ) , and includes a highly interconnected group of hubs that appear to be involved with RNA/DNA binding and protein degradation ( At1g33680 , At2g28540 , At3g50590 , At4g38600 , At5g35430 , At5g62000 ) . Interestingly , the uncharacterized transducin-like protein ( At3g50590 ) was a major co-expression hub in both the lesion area network I ( degree = 9 ) and the camalexin network I ( degree = 12 ) . This indicates that this WD40-repeat protein may play a key role in coordinating protein complexes at the plasma membrane during infection . The second largest network ( II; n = 78 ) associated with lesion area is enriched in GO categories traditionally associated with the innate immune response in Arabidopsis ( S7 Table ) . Despite the observation that natural variation in the jasmonate or salicylate hormone receptor was not associated with lesion size , lesion network II contains a large portion of genes with putative annotations related to defense and includes two of the major hubs from camalexin network II ZAR1 ( At3g50950; degree = 4 ) and the uncharacterized SAM-dependent methyltransferase ( At1g55450; degree = 4 ) . Lesion network III was enriched in GO categories related to autophagy and catabolism and included the large hubs that are known for being involved with amino acid and lipid metabolism ( ASP4; At1g62800 and 2OG; At3g50210 ) ( Fig 4B; S7 Table ) . The next largest network ( IV ) is also linked to catabolic and metabolic processes as it is enriched in GO categories linked to catabolism and one of the largest hubs is homogentisate 1 , 2-dioxygenase , a well-studied enzyme in the tyrosine catabolic pathway ( HGO; At5g54080 , degree = 7 ) ( Fig 4B; S7 Table ) . Thus , these two networks suggest that variation in catabolic processes is linked to plant defense . Lastly , lesion network V is largely comprised of unknown and uncharacterized genes , including the two largest hubs within the network ( At1g10020 , At4g03110 ) . Including the unknown genes , this network is enriched for GO categories related to trehalose and polysaccharide metabolism and includes genes like SUGAR_TRANSPORTER_1 ( STP1; At1g11260 ) and CELLULOSE_SYNTHASE_LIKE_C4 ( CSLC4; At3g28180 ) , which suggests that this network is related to polysaccharide metabolism and callose wall deposition . Thus , these co-expression networks illustrate the broad array of mechanisms that may be variable and influence quantitative resistance to Botrytis cinerea in Arabidopsis thaliana and they provide new gene targets and testable hypotheses for future experiments . Given the modular construction of the co-expression networks and the common concern with false positives within GWA data , we selected several genes that were present within co-expression networks and a set of genes that were not within co-expression networks to validate their involvement in quantitative resistance to B . cinerea . All of these genes were required to have at least two SNPs crossing the 0 . 95 threshold as previously published [43] . Specifically , we selected a total of 22 genes associated with either camalexin production or lesion area whose homozygous and viable mutants could readily be obtained ( S9 Table ) . In addition , to test if the lack of GWA hits on known candidate genes was potentially caused by our selection of isolates , we selected three genes previously identified to be associated with quantitative resistance to B . cinerea , but were not found in either association studies , including PAD3 [65] , TGA3 , and ANAC0155 [55] . Individual gene knockouts in a Col-0 background were obtained from both the Arabidopsis Biological Resource Center , as well as several other laboratories that regularly work with these mutants , and each line was tested for homozygosity . Similar to the GWA experiments , each mutant and a wildtype Col-0 control was grown in a randomized complete block design with three experimental replicates and four independent biological replicates . At five weeks of growth , the first five mature leaves of each plant was removed and infected with either one of the previous isolates ( Apple517 , B05 . 10 , Supersteak , UKRazz ) or a mock inoculation . Infected leaves were imaged at 72 hpi and collected for camalexin measurement via HPLC . Pictures were analyzed using the same image analysis pipeline described above for the GWAS . Of the 24 genotypes planted , we were unable to obtain validated and viable homozygous seed for seven mutant lines ( S9 Table ) . Mutant genotypes for seven of the 15 remaining genes tested demonstrated significant differences for camalexin production in comparison to wildtype , and mutant alleles in six of the 15 genes showed altered disease resistance as measured by lesion area ( Tables 2 and S10–S11 ) ( Figs 5 and 6 ) . Of the six lines that altered lesion development , five of them had increased lesion size in the knockout lines while the CESA2 mutant actually showed increased resistance to B . cinerea in comparison to WT ( S10 Table ) . As such , we can identify both susceptibility and resistance genes via this approach . Interestingly , there was less evidence of isolate specificity within the single gene analysis than we found using the accessions , which could indicate that the isolate specificity is more environmentally variable as suggested by the three-way interaction of environment with isolate and plant genotype ( Tables 2 and S11 ) . Alternatively , this could imply that the highly polygenic nature of resistance among the accessions complicates the ability to compare measures isolate specificity between individual genotypes and accessions . However , it should be noted that including multiple isolates to test every gene did increase our level of replication and likely benefited our power to detect a phenotypic difference above what would have happened with a single isolate . To assess the impact of significance thresholds on our validation efforts , we compared our validation candidates with the presence of SNPs at different significance thresholds . There were no SNPs in any of the candidate genes at a 0 . 999 significance threshold indicating that this threshold would have a high false-negative error rate with this set of genes , which would result in missing the nine genes that did validate . Of the 15 genes tested , six had one or more SNPs at the 0 . 99 threshold and these genes validated at a slightly higher rate than the genes with just two or more SNPs at the 0 . 95 threshold ( 67% v 56% when using all traits ) ( S12 Table ) . Interestingly , there was no difference in the validation rate between genes with a 0 . 99 threshold SNP that were within networks or absent from these networks ( 67% in both cases ) ( S12 Table ) . While this suggests that most of the increase in validation rate was achieved by requiring two or more SNPS , there were only three genes in these two groups and more tests would be required to assess if the network approach helped identify true positives . While the genes with a 0 . 99 threshold validated at a slightly higher level than genes with two or more SNPs at the 0 . 95 threshold , the fact that 56% of the genes with the two SNPs at the lower threshold also validated shows that the 0 . 99 threshold still imparts a high false-negative error rate . Thus , this test provides some empirical insight into how to set a significance threshold for calling candidate genes to attempt and validate depending upon the level of false positive and false negatives that a researcher is willing to accommodate in their experimental design .
One current theory of evolution of innate immunity is that it is primarily driven by the presence/absence of R-gene combinations , including RLKs and NLR proteins [2 , 3 , 66 , 67] . This would imply that natural variation in quantitative resistance is merely the differential ability of host genotypes to directly detect the presence of a pathogen via PAMPs , or to indirectly detect the impact of pathogen effectors , and trigger the appropriate downstream response . Next generation genomic re-sequencing efforts have difficulty in resolving genetic variation at resistance gene clusters , which could potentially cause us to underestimate the contribution of R-genes to natural variation in quantitative resistance [68–71] . However , the effect of polymorphisms at known R-gene clusters is not drastically larger than other loci identified ( S13 and S14 Figs; S12 Table ) and thus would not lead to a significant alteration in the resulting conclusion . While we did see enrichment in the putative receptor RLKs and NBS-LRR proteins in genes associated with quantitative resistance , they only account for a small proportion of the genes implicated in quantitative variation in the innate immune response . Interestingly , we observed a much stronger enrichment in cytoplasmic localized NLR proteins relative to the plasma membrane bound RLKs . Taken with the fact that Botrytis cinerea does not have a type III secretion system or form haustoria , this implies that resistance to B . cinerea in Arabidopsis is primarily driven by indirect pathogen detection by monitoring of host cellular components . Therefore , it appears that it may be more efficient for the plant host to monitor the health of its own cellular components rather than attempting to directly detect the myriad potential pathogen PAMPs and effectors to determine its infection state . This may also help explain why genes were implicated in an isolate specific manner as different genotypes of the pathogen may have differential effects on host specific processes . In addition , this may also help explain why endophytic microbes that are phylogenetically related to plant pathogens do not elicit an immune response in the host . In contrast to studies looking at quantitative resistance in defined breeding populations [32–34 , 72 , 73] , we were able to identify a vastly larger pool of candidate genes that could be validated with a 60% success rate ( Table 2 ) . This GWA method was likely more successful than previous efforts to map quantitative resistance loci for multiple reasons related to the specific experimental design and statistical methods used . For instance , by removing a large portion of the large genetic outliers and major population structure from the host mapping population , we were left with a core set of natural Arabidopsis accessions that share a large fraction of common allelic variants and have reduced cryptic population structure . In addition , the use of the ridge regression mapping method allows for testing all polymorphisms within the population in a single model by treating them as random variables . This obviates the need for post-hoc multiple testing corrections that can arbitrarily increase false negatives . However , this makes it difficult to assign significance and p-values for SNP-association since it is difficult to assign accurate degrees of freedom for each variable . To resolve this issue , we used a permuted a significance threshold to identify heteroscedastic SNP effects that are larger than can be expected by a random association . This method identified thousands of moderate effect SNPs associated with a change in quantitative resistance relative to the population mean , which likely represent an upper bounds on the number of causal loci for this population . These are more loci than have been identified in previous studies , and thus necessitates developing methods to filter results and test for false positive associations . To address this , we reduced the number of loci into gene co-expression networks derived from all publically available microarrays in Arabidopsis [64] . These smaller collections of genes are both genetically associated with Botrytis resistance and are typically expressed at the same time , in the same tissues , and under the same treatments . This implies that genes within a network function together during the transition to an immune response during pathogen attack . However , it should be noted that both genetic mapping and co-expression analyses have known issues with false-positive associations due to the underlying assumptions in each method . For instance , genetic linkage can falsely implicate a gene that is directly adjacent to a causal gene in genetic mapping analyses and gene co-expression is often insufficient to imply co-function . However , since these limitations represent different and independent assumptions , we leveraged the two methods against each other to filter out false positives within each method and obtain a more detailed biological interpretation of the results . Thus , we were able to achieve a much more accurate picture of quantitative resistance by selecting a population with reduced structure using appropriate statistical approaches , and leveraging complementing datasets . Importantly , our study shows that the genetic background of the fungal isolate plays a significant role in identification of plant disease resistance genes . While this has long been shown in presence/absence of functional alleles in several biotrophic pathogens , the importance of the pathogen genotype for quantitative , necrotrophic interactions is only recently being rediscovered [74] . Here , we attempted to use the genetic and phenotypic diversity in B . cinerea to challenge the innate immune system of Arabidopsis in as wide a range as possible . This enhanced our ability to detect genes associated with quantitative resistance as the most of gene networks are made up of genes identified as candidates for resistance to individual isolates . In addition , we were able to further enhance our ability to identify genes associated with quantitative resistance by mapping multiple phenotypic descriptions of infection . For example , the uncharacterized putative methyltransferase At4g01880 was only identified by mapping for camalexin associated loci in response to the B05 . 10 isolate . However , a T-DNA knockout of At4g01880 significantly influenced both camalexin and lesion area for multiple isolates upon single gene validation . These findings argue for analysis of multiple phenotypes and isolates to differentially probe the host innate immune system . Further , since there was very little overlap between the genes identified among the isolates , this argues that our estimates of the genes identified in this study are a more conservative estimate and that we have not identified all genes associated with quantitative resistance . Lastly , this isolate specificity has potential long term implications concerning the design and development of durable resistance for quantitative pathogenic interactions . The isolate specificity in genes associated with quantitative resistance seem to imply that there is no single network common to resistance among genetically distinct isolates of the pathogen . However , individual pathogen genotypes appear to affect different loci of genes that are commonly co-expressed . This implies that each isolate is a core set of genes associated with quantitative resistance . However , these components can only be discovered through the use of multiple , phenotypically distinct pathogenic isolates . Overall , quantitative resistance appears to be dependent on the efficient functioning of several cellular processes and must be able to deal with a number of potential inputs . This study has generated several new broad hypotheses to explore , such as what is the impact of developmental genes on quantitative resistance and are the core components of quantitative resistance functionally canalized ? The large list of candidate genes we identified in this study as altering quantitative resistance provides the raw genetic material to begin asking numerous functional questions with regard to quantitative resistance including 1 ) how does a host interpret the biological inputs to understand that it is infected , 2 ) how does a host weight those inputs relative to other environmental and physiological inputs , 3 ) how does a host chose to respond to a particular pathogen and is the response canalized for all similar pathogens , 4 ) is the host or the pathogen the larger determining factor in the interaction . By developing approaches to study these putative quantitative resistance networks in combination and in isolation , we can begin to understand how this intricate system functions across diverse pathogens and environments . In addition , a better understanding of quantitative resistance should help breeding efforts to for improved disease resistance .
A collection of 96 natural accessions of Arabidopsis thaliana were selected from a larger population of 241 accessions based on similar flowering time ( 63 . 1 ± 0 . 95 ( s . e . ) days to flowering ) [42] . Using accessions with similar flowering time limits the potential effects of ontogenic variation of relative plant age across our population while , for an unknown reason , also removing extreme genetic outliers ( Fig 1 and S1 Fig ) . Seeds of each accession were cold stratified in 0 . 1% Phytagar for 4 days at 4°C in the dark . Three seeds per accession were sown on soil ( Sunshine Mix #1 , Sun Gro Horticulture , Agawam , MA ) randomly in 104-cell flats and were covered with a clear humidity dome . Seeds were allowed to germinate and seedlings were thinned to one plant per cell at one week of age . Seedlings were allowed to mature for 5 weeks at 22°C with 10 hours of full spectrum light . A total of four flats were planted and the experiment was repeated two weeks after the initial sowing , which allowed for a total of 8 independent biological measurements per accession in a 4-by-2 randomized complete block design . Plants were watered bi-weekly as needed . At five weeks of growth , the first five mature leaves were collected for infection and placed on growing flats lined with 1% Phytagar . The spores of five-day-old sporulating cultures of four phenotypically and genotypically diverse Botrytis cinerea isolates ( Apple517 , B05 . 10 , Supersteak , UKRazz ) [50 , 51] on peach slices were collected in 1/2x grape juice , counted using a hemacytometer , and diluted to 10 spores/μL . Detached leaves were infected with 4 μL of the spore solution , which allows for approximately 40 spores per leaf , and leaves from each plant were randomly assigned either a 1/2x grape juice control or one of the isolate spore dilutions as previously described [33 , 34 , 75] . This level of inoculation allows for lesion progression that is sufficiently moderate to optimize the ability to quantify the infections progression over time . Infected leaves were incubated at room temperature for 72 hours , at which point pictures were taken of the expanding lesion using an 18 Mp high resolution T3i Canon camera outfitted with an EF-S 10-22mm f/3 . 5–4 . 5 USM ultra-wide angle lens , and infected leaves were collected for chemical analysis . Pictures of developing lesions were taken every 24 hours at a final resolution of approximately 10 pixels per mm . Lesion areas were measured using a semi-automated image analysis script using the open-source R statistical environment [76] enabled with the EBImage and CRImage packages [52 , 77] . Leaf objects were automatically identified as green , highly saturated objects in the image and lesions were identified as brown , low saturation object in the leaf . Image masks were generated for both the leaf and lesion , which were manual refined by a technician to ensure proper object calling . The area of these leaves and lesions were then automatically measured in the number of pixels per lesion and converted to area using a 1 cm reference contained within each image . Camalexin was extracted and measured as previously described [75] . Briefly , the leaves were ground in 400μL of 90% methanol , centrifuged to remove precipitates , and the supernatant was transferred to a fresh plate . A total of 50 μl of the extract was run on an Agilent Lichrocart 250–4 RP18e 5 μm column using an Agilent 1100 series HPLC . Camalexin was detected using a DiodeArray ( DAD ) at 330 nm and with a fluorescence detector at emission 318 nm/excitation 385 nm . Separation was achieved using the following program with aqueous acetonitrile: 5 min gradient from 63% to 69% acetonitrile , 30 sec gradient from 69% to 99% acetonitrile , 2 min at 99% acetonitrile and a post-run equilibration of 3 . 5 min at 63% acetonitrile [75] . Purified camalexin was used to produce a standard curve to identify and quantitate camalexin . To select Arabidopsis accessions for the GWA population , publically available SNP data was collected from the Arabidopsis 1 , 001 genomes project [40 , 70 , 78–80] for all accession available on campus . The EMMA package in R [46] was used to generate the kinship matrix for all related accessions . Accessions were filtered based on common flowering time [42] ( 63 . 1 ± 0 . 95 ( s . e . ) days to flowering ) , which also removed the most extreme genetic outliers . Relative genetic groups were qualitatively determined . Experimental and treatment effects on each phenotype were determined using a multivariate linear model . To explore the experimental effects on our phenotypes , we used one of two linear models: Cametaip = β0 + Ee + Tt + Aa + T ( I ) ti + A ( P ) ap + A:Tat + A:T ( I ) ati + T:Ptp +εetaip Lesioneiap = β0 + Ee + Ii + Aa + A ( P ) ap+ A:Iai+ εeiapeiap The main fixed effects are denoted as E , T , A , I , and P , which represents the experimental block , infection treatment , accession background , isolate background , and plant respectively . In addition , e = 1 , 2; t = 1 , 2; a = 1 , … , 95; i = 1 , … , 5; and p = 1 , … , 400 . The residual error was assumed to be normal ( ɛ ~ N ( 0 , σ2 ) ) . Infection treatment was not included in the lesion model as no lesions were observed on mock treated tissue and control tissue was not included in the dataset . Both models showed a highly significant interaction between the accession genetic background and the isolate genetics background ( Table 1 ) , as such , all downstream analysis was conducted within an isolate background . Broad-sense heritability ( H2 ) and genetic grouping was calculated as a proportion of the explained variance for each isolate using the following equation: yefga = β0 + Ee + F ( E ) ef + Gg + A ( G ) ga + + εefga where the main effects are denoted as G , A , E , and F to represent group , accession , experiment block , and flat block respectively , and g = 1 , … , 5; e = 1 , 2;f = 1 , … , 4; and a = 1 , … , 95 . Again , residual error was assumed to be normal ( εgaef ~ N ( 0 , σε2 ) ) . To look for a relationship between camalexin production and lesion area , we used the non-parametric Spearman’s rank correlation test using model corrected means for each Arabidopsis accession within each isolate treatment . Since very little variance for each phenotype can be attributed to the genotypic structure of the population , we opted for a ridge regression approach [54] . This approach models the effects of all polymorphisms in a single model by treating them as random effects , which avoids the use of post-hoc multiple testing procedures . From this model , the heteroscedastic effects ( HEM ) were extracted for each polymorphism in lieu of SNP-based best linear unbiased predictors in accordance with Shen , 2013 [54] . Since determining the degrees of freedom for random variables is difficult , and thus corresponding p-values , a significant effect threshold was determined by randomly permuting the phenotypic means across the accession backgrounds 1 , 000 times and taking the 99th quartile . Individual genes were called associated with the phenotype if they had at least 2 significant SNPs in their coding region . A . thaliana genes associated with quantitative resistance to any of the four B . cinerea isolates were condensed into co-expression networks determined using the ATTEDII database , as described by Chan , 2011 . Briefly , Pearson’s correlation coefficients and mutual ranks ( MR ) were collected for all pairs of significantly associated genes in the database . Interactions were filtered by MR rank < 15 . Putative function of the top 5 largest networks was qualitatively determined by the function of the well-studied genes in their membership . Several candidate genes from many of the larger networks were selected for downstream validation . Gene ontology ( GO ) enrichment for biological processes within the large 5 networks for each phenotype was determined using the Bioconductor packages org . At . tair . db , topGO , and goProfiles in the R statistical environment . The unbiased approach compared all associated genes against all genes that could potentially be called associated ( at least two SNPs with MAF > 0 . 2 ) within this population . R-gene enrichment was determined using a hypergeometric test comparing associated RLKs [58] and NBS-LRR [59] proteins with all R-genes that could be called in the background of the unbiased approach . Single gene T-DNA knockout lines in a Col-0 background were obtained and genotyped to be homozygous using standard PCR-based indel markers developed from the SALK Institute Genomic Analysis Laboratory ( http://signal . salk . edu/tdnaprimers . 2 . html ) . Plants were grown , infected , and phenotyped for lesion size and camalexin against all four B . cinerea isolates and a mock control as described above . Plant were sown in a complete block design with three experimental replicates and four biological replicates in each block , thus providing 12 measurements per A . thaliana mutant / B . cinerea isolate combination . An F-test was conducted for each T-DNA line relative to Col-0 using one of the following GLMs: Cametgi = β0 + Ee + Tt + Gg + T ( I ) ti + G:Tgt + G:T ( I ) gti +εetgi Lesioneig = β0 + Ee + Ii + Gg + G:Igi+ εeig where the main fixed effects are denoted as E , T , G , and I , which represents the experimental block , infection treatment , genetic background , and isolate background respectively , with e = 1 , 2; t = 1 , 2; g = 1 , 2; and i = 1 , … , 5 . The residual error was assumed to be normal ( ɛ ~ N ( 0 , σ2 ) ) . | The innate immune system is an important driver of quantitative resistance among all eukaryotes and is the primary immune system for many multicellular organisms , including plants , fungi , and insects . While some components of the innate immune system are known , many components are still undiscovered . Here we use genome wide association ( GWA ) mapping in a natural population of the Arabidopsis thaliana host against four phenotypically and genetically distinct isolates of the fungal pathogen Botrytis cinerea to describe and validate components of the plant innate immune system . Using both the induced production of a known defense compound , camalexin , and lesion area as quantified outputs of the plant innate immune system , we found approximately 2 , 982 and 3 , 354 genes associated with quantitative resistance respectively . Genes associated with variation in the plant immune system were largely dependent on the pathogen genotype , indicating the presence of specificity in pathogen detection and response by the host . Associated genes , which were filtered into co-expression networks followed by GO enrichment analysis , showed both known and novel biological processes associated with pathogen response . Validation of associated genes using single gene knockouts in a common genetic background indicates an ability to validate causal genes with a 60% validation rate . | [
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... | 2016 | The Quantitative Basis of the Arabidopsis Innate Immune System to Endemic Pathogens Depends on Pathogen Genetics |
Mass azithromycin distributions have been shown to reduce mortality among pre-school children in sub-Saharan Africa . It is unclear what mediates this mortality reduction , but one possibility is that antibiotics function as growth promoters for young children . 24 rural Ethiopian communities that had received biannual mass azithromycin distributions over the previous four years were enrolled in a parallel-group , cluster-randomized trial . Communities were randomized in a 1:1 ratio to either continuation of biannual oral azithromycin ( 20mg/kg for children , 1 g for adults ) or to no programmatic antibiotics over the 36 months of the study period . All community members 6 months and older were eligible for the intervention . The primary outcome was ocular chlamydia; height and weight were measured as secondary outcomes on children less than 60 months of age at months 12 and 36 . Study participants were not masked; anthropometrists were not informed of the treatment allocation . Anthropometric measurements were collected for 282 children aged 0–36 months at the month 12 assessment and 455 children aged 0–59 months at the month 36 assessment , including 207 children who had measurements at both time points . After adjusting for age and sex , children were slightly but not significantly taller in the biannually treated communities ( 84 . 0 cm , 95%CI 83 . 2–84 . 8 , in the azithromycin-treated communities vs . 83 . 7 cm , 95%CI 82 . 9–84 . 5 , in the untreated communities; mean difference 0 . 31 cm , 95%CI -0 . 85 to 1 . 47 , P = 0 . 60 ) . No adverse events were reported . Periodic mass azithromycin distributions for trachoma did not demonstrate a strong impact on childhood growth . The TANA II trial was registered on clinicaltrials . gov #NCT01202331 .
Undernutrition is thought to contribute more to the global burden of disease than any other risk factor . [1] Poor nutrition potentiates the effects of infections such as diarrhea , respiratory infections , and malaria , leading to worse outcomes and higher mortality . [2 , 3] Infectious diseases in turn lead to poor growth . [4] It is conceivable that antibiotics could have an important role for breaking this cycle of malnutrition and infection . For example , a randomized trial demonstrated less stunting and underweight in HIV-infected children who took daily co-trimoxazole compared to those taking placebo . [5] This idea is not new; antibiotics have long been thought to be effective growth promoters for animal husbandry . [6] Mass azithromycin treatments have recently been shown to reduce childhood mortality in sub-Saharan Africa , although the causal pathway for the mortality reduction is unclear . [7 , 8] Azithromycin is a broad-spectrum antibiotic with efficacy against a wide array of pathogens that cause respiratory disease , diarrhea , and malaria , so it is possible that azithromycin prevents mortality by directly clearing these infections . [9–11] Alternatively , if azithromycin caused height and weight gain in children , it is possible that the improved growth could be partly responsible for the survival benefit . In a recent cluster-randomized trial , we randomized communities that had been treated with four years of mass azithromycin for trachoma to either continued antibiotics or cessation of treatment . Cluster-randomization , which was chosen for the primary trachoma outcomes , allowed assessment of both the direct and spillover effects of antibiotics . [12] We recognized that this trial provided an opportunity to assess whether mass azithromycin distributions had an effect on childhood growth , and consequently added height and weight as secondary outcomes . We compared anthropometric measurements at the individual level to determine whether mass azithromycin increased childhood growth .
The study had approval from the University of California , San Francisco; Emory University; the Ethiopian Ministry of Science and Technology; and the Food , Medicine , and Health Care Administration and Control Authority of Ethiopia . The study was carried out in accordance with the Declaration of Helsinki and overseen by a Data Safety and Monitoring Committee appointed by the National Institutes of Health-National Eye Institute . Verbal informed consent was obtained in Amharic from community leaders before randomization and from the guardians of all children post-randomization; verbal consent was approved by all institutional review boards and was used due to the high levels of illiteracy in the study area . This study describes a secondary outcome from a parallel-group cluster-randomized clinical trial for trachoma ( TANA II; clinicaltrials . gov #NCT01202331 ) performed in the Goncha Siso Enese woreda ( district ) of the Amhara region in Ethiopia . [13] The trial was conducted in communities that had been treated with 8 biannual mass azithromycin distributions for trachoma as part of an earlier trial ( TANA I; clinicaltrials . gov #NCT00322972 ) . [14] In the initial TANA I trial ( June 2006 until November 2009 ) , 72 contiguous subkebeles ( sub-districts ) were randomized to 1 of 6 different trachoma treatment strategies . [14] Rural subkebeles were eligible for enrollment if they were located within a 3-hour walk from the farthest point accessible to a four-wheel drive automobile . As part of the TANA I trial , 12 subkebeles were randomized to receive biannual mass azithromycin distributions ( every 6 months , ±1 month ) . Each subkebele consisted of approximately 4–6 government-defined demographic units known as state teams; all state teams in the subkebele were treated identically . In TANA II ( November 2010 until May 2013 ) , two randomly selected state teams from each biannually treated subkebele were randomized to either continued biannual mass azithromycin ( N = 12 state teams ) or cessation of antibiotics ( N = 12 state teams ) . Anthropometric indices were measured as a secondary outcome in these 24 state teams 12 and 36 months after randomization , providing a randomized controlled trial comparison of childhood growth in communities treated with mass azithromycin distributions versus no treatment . No changes were made to the anthropometry portion of the study after its addition to the main trial . The trial was reported according to CONSORT guidelines ( S1 Checklist ) . Details of the study design were pre-specified in a trial protocol ( S1 Protocol ) . This study employed pair-matched cluster-randomization with matching based on the TANA I subkebele . A biostatistician ( TCP ) used the statistical package R ( R Foundation for Statistical Computing , Vienna , Austria ) to randomize 12 pairs of state teams from each of 12 subkebeles , with one member of the pair allocated to intervention and the other to control . A study coordinator enrolled the communities and participants and assigned the intervention . Allocation was concealed by enrolling all communities before randomization and administering the intervention to the entire community population . Although study participants were not masked to their cluster’s treatment assignment , the study personnel responsible for anthropometric assessments were not informed of the treatment allocation or study hypothesis . Local health extension workers performed a population census each year to enumerate all community members eligible for treatment . Because birthdates are imprecise in this part of Ethiopia , census-takers recorded age in years . During scheduled mass treatment visits , these same health workers offered a single dose of directly observed oral azithromycin ( 20mg/kg height-based approximation for children , 1g for adults ) to all community members except for children under 6 months of age , self-reported pregnant women , and those known to be allergic to macrolide antibiotics , each of whom were instead offered a six-week course of twice daily tetracycline ophthalmic ointment . Each community was treated twice per year ( Fig 1 ) . Antibiotic coverage during mass treatments was assessed from the preceding census . Although study participants from all communities continued to receive routine government health services during this time , no other studies or interventions were performed during the study period . No adverse events were reported through routine passive surveillance activities; active monitoring of adverse events was performed during the trial and is reported elsewhere . [15] We performed anthropometric assessments after the month 12 and month 36 treatments . All children aged 0–59 months identified from the most recent census were offered height and weight measurements , conducted in a central area in each state team . We recruited anthropometrists from the local communities , and conducted a 2-day training session using methods recommended by the World Health Organization ( WHO ) . Anthropometry teams demonstrated high levels of reproducibility for all measurements . [16] We used a portable stadiometer ( Shorr Productions , LLC , Olney , MD , USA ) to measure height or length and a Seca 874 floor scale ( Seca GmbH & Co . KG , Hamburg , Germany ) to measure weight . Each measurement was taken in triplicate , with the median used as the official measurement . The pre-specified primary analysis for the anthropometric outcomes was a post-test only comparison of height adjusted for age and sex in children not old enough to be eligible for treatment during the first trial ( i . e . , aged 0–36 months at the 12-month visit and 0–59 months at the 36-month visit ) . Repeated measures for height were modeled as a function of treatment arm , time , age in years ( as a continuous variable rounded to whole years ) , and sex in a mixed effects linear regression , with state team and individual modeled as random effects . Heteroskedasticity was modeled by allowing independent residual errors over each year of age . The p-value was estimated with a Monte Carlo permutation test stratified by subkebele to account for the randomization strategy ( 10 , 000 permutations of the likelihood ratio comparing models with and without the treatment term ) . Intraclass correlation coefficients ( ICCs ) were calculated from the model to assess cluster-correlation of the primary outcome . Similar models were constructed for the secondary underweight analysis ( weight adjusted for age and sex ) . We purposefully did not use z-scores in the primary analysis given the imprecision of reported ages in the study area . For all outcomes , we performed intention-to-treat analyses . Sample size calculations for the trial were based on the primary trachoma outcomes and set at 12 communities per arm; including 18 children per community would provide 80% power to detect a 1 cm difference between the two arms assuming an average height of 90 cm ( standard deviation 10 cm ) , ICC of 0 . 02 , correlation coefficient of 0 . 9 for the relationship of height with age and sex , equal cluster sizes , and a two-sided alpha of 0 . 05 . [17] All analyses were performed using Stata 14 . 2 ( Statacorp , College Station , TX ) except for permutation tests , which were done in R 3 . 4 . 0 ( R Foundation for Statistical Computing , Vienna , Austria ) .
The study was conducted from November 2010 until May 2013 . Characteristics of the two treatment arms were balanced at baseline ( Table 1 ) . The biannual treatment group received 7 mass azithromycin treatments during the continuation trial; the control group received none . Table 2 shows the median antibiotic coverage at each distribution for the under-5 age group . We obtained anthropometric measurements on 282 of 387 ( 73% ) eligible children aged <3 years at the 12-month visit and 455 of 591 ( 77% ) eligible children aged <5 years at the 36-month visit , with 207 children having measurements at both visits . As shown in Table 3 , children who were eligible but did not participate in the anthropometric assessment tended to be younger than those who participated . The mean height and weight in each treatment arm at the two time points are shown in Table 4 , stratified by age . After adjusting for age , sex , and study visit , the mean height was 84 . 0 cm ( 95%CI 83 . 2–84 . 8 ) in the azithromycin-treated communities and 83 . 7 ( 95%CI 82 . 9–84 . 5 ) in the untreated communities . In the primary pre-specified analysis adjusted for age , sex , and time point , children in the azithromycin-treated communities were on average 0 . 31 cm taller ( 95%CI -0 . 85 to 1 . 47 ) than those in untreated communities ( P = 0 . 60 ) . The ICC derived from the main statistical model suggested mild clustering of height measurements within communities ( ICC 0 . 05 , 95%CI 0 . 02 to 0 . 13 ) . The weight outcome is summarized in Table 4 . After adjusting for age , sex , and study visit , the average weight was 10 . 8 kg in the azithromycin-treated communities and 10 . 7 in the untreated communities—a non-significant difference ( mean weight 0 . 09 kg heavier in the azithromycin arm , 95%CI -0 . 20 to 0 . 39; P = 0 . 54; ICC 0 . 07 , 95%CI 0 . 03 to 0 . 15 ) . Additional sensitivity analyses using all <5 year-old children at both outcome visits ( i . e . , including children from the 12-month visit who had been treated in the previous trial ) had similar results ( S1 File ) .
We failed to find an association between mass azithromycin distributions and childhood growth . Although on average children from communities treated with biannual mass azithromycin distributions over a 3-year period were slightly taller than children from untreated communities , the differences between the two treatment groups were not consistently statistically significant across different regression model parameterizations and of small magnitude . This trial assessed the hypothesis that azithromycin functions as a growth promoting agent for preschool children . The specific mechanisms through which antibiotics may promote growth are unknown , though several theories have been proposed . [18] Antibiotics could treat or prevent infections that would otherwise divert metabolic resources . Antibiotics may also alter intestinal absorption by clearing pathogenic organisms and changing the composition of the gut microbiome , both of which may ultimately mitigate the role of bacterial infection in environmental enteropathy . [19 , 20] Alternatively , antibiotics like azithromycin are known to have direct anti-inflammatory properties , which could potentially affect growth . It is conceivable that improvements in growth could translate into improved childhood survival . It is thought that growth and nutrition are important modulators of infectious diseases . Better nutrition may enhance the ability of the immune system to clear infections , and thus make a child less susceptible to severe complications of infection . Indeed , growth promotion is one theory for the improved survival observed following mass azithromycin distributions . [7 , 8] While this study does not provide evidence that mass azithromycin impacts childhood growth , it is still possible that azithromycin treatments affect growth but at a magnitude smaller than could be detected by the present study . For example , the results of the present study are consistent with a meta-analysis that estimated antibiotics to confer an additional 0 . 04 cm of linear growth per month . [21] Previous randomized trials have studied the impact of antibiotics on childhood growth in the context of a specific disease , such as severe acute malnutrition , HIV , diarrhea , cystic fibrosis , or vesicoureteral reflux . [22–25] Notable among these is a placebo-controlled trial of daily prophylactic co-trimoxazole therapy for HIV-infected children in Zambia that found improved linear growth and decreased mortality in antibiotic-treated children . [5] While many of the other studies failed to find an effect of antibiotic therapy on height measurements , they were of relatively short duration and focused on changes in weight instead of height . [22–25] Other trials have assessed whether community antibiotic distributions affect growth parameters . Two placebo-controlled studies performed in Guatemala , one of which treated school-aged children with aureomycin and another which treated preschool children with metronidazole , found improved height and weight metrics in children treated with antibiotics . However , these studies may be subject to type I error since they allocated a single cluster of children to the antibiotic intervention but performed analyses at the individual level . [26 , 27] Several recent cluster-randomized trials testing more frequent vs . less frequent mass azithromycin distribution strategies for trachoma have failed to detect a difference in height or weight between the different dosing groups . [17 , 28 , 29] The control groups in those studies were not ideal in that they too received antibiotics—albeit at lower frequencies—and thus may have received a benefit of antibiotic therapy . The present study is an improvement over these previous reports in that no programmatic antibiotics were given in the control group , providing a better assessment of the effectiveness of antibiotics alone . Mass antibiotics provide selection pressure for antibiotic-resistant organisms , which increases the community prevalence of resistance . [30] Although the clinical importance of such resistance is unclear given the low usage of macrolide antibiotics in developing countries , the potential for antibiotic resistance should be taken into account wherever implementation of mass azithromycin distributions is considered . [31] Several limitations to this study should be noted . We conducted a post-test only analysis with two follow-up visits . Baseline and more frequent anthropometric measurements would have increased the precision and added to the statistical power of the study . Ages were not known with great accuracy in this study , since families in this region of Ethiopia do not keep health cards and do not record dates of birth . Imprecision in age could have biased the study if age was differentially recorded between treatment arms or if cluster-randomization left an imbalance of ages in the two groups . This study was cluster-randomized since it was performed in the context of community antibiotic treatments for trachoma . An individually randomized trial could have been powered to detect an even smaller difference between children treated with and without antibiotics . On the other hand , the cluster-randomization better approximates the effectiveness of programmatic azithromycin and allows for the possibility of both direct and spillover effects of antibiotics . [12] Finally , this study was conducted in a region of Ethiopia with hyperendemic trachoma that had already received several rounds of mass azithromycin distributions . The generalizability of the findings to an antibiotic-naïve population or a population with milder or no trachoma is unclear . In conclusion , mass azithromycin distributions have proven extremely effective at reducing the burden of ocular chlamydia and may also have important ancillary benefits , like reducing the prevalence of respiratory infections , diarrhea , malaria , and skin infections—as well as childhood deaths . [9–11 , 32] Although we hypothesized that childhood growth promotion might be an additional benefit of mass azithromycin distributions , the present study was unable to provide strong evidence of such an association . | Mass distribution of a single dose of the broad-spectrum antibiotic azithromycin twice per year to pre-school children in Sub-Saharan Africa has been shown to reduce childhood mortality . The mechanism by which azithromycin reduces mortality is currently not clear , especially since the antibiotic is not targeted to sick children but rather given to all children in the community whether or not they have an infectious disease . In this study , we report the height and weight of children enrolled in a trial in Ethiopia in which communities were randomized either to twice annual mass azithromycin distributions for blinding trachoma or to no treatments . After accounting for age and sex , children from azithromycin-treated communities were on average slightly taller at the 12- and 36-month study visits than those from untreated communities , but the difference was not statistically significant . | [
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"... | 2019 | Linear growth in preschool children treated with mass azithromycin distributions for trachoma: A cluster-randomized trial |
Seed size is a key determinant of evolutionary fitness in plants and is a trait that often undergoes tremendous changes during crop domestication . Seed size is most often quantitatively inherited , and it has been shown that Sw4 . 1 is one of the most significant quantitative trait loci ( QTLs ) underlying the evolution of seed size in the genus Solanum—especially in species related to the cultivated tomato . Using a combination of genetic , developmental , molecular , and transgenic techniques , we have pinpointed the cause of the Sw4 . 1 QTL to a gene encoding an ABC transporter gene . This gene exerts its control on seed size , not through the maternal plant , but rather via gene expression in the developing zygote . Phenotypic effects of allelic variation at Sw4 . 1 are manifested early in seed development at stages corresponding to the rapid deposition of starch and lipids into the endospermic cells . Through synteny , we have identified the Arabidopsis Sw4 . 1 ortholog . Mutagenesis has revealed that this ortholog is associated with seed length variation and fatty acid deposition in seeds , raising the possibility that the ABC transporter may modulate seed size variation in other species . Transcription studies show that the ABC transporter gene is expressed not only in seeds , but also in other tissues ( leaves and roots ) and , thus , may perform functions in parts of the plants other than developing seeds . Cloning and characterization of the Sw4 . 1 QTL gives new insight into how plants change seed during evolution and may open future opportunities for modulating seed size in crop plants for human purposes .
Seeds represent the vehicle by which plants vie for evolutionary success . A key feature of seeds is their size , which in turn is one of the most variable traits in the plant kingdom . Seeds range in weight from less than 1 microgram in the Coral-root orchid ( Corallorhiza maculate ) to more than 10 kg in the Coco-de-mer palm ( Lodoicea maldivica ) . This large range can be observed not only among taxa , but also within taxa . For example , contained in the genus Solanum are a set of 9 cross compatible species closely related to the tomato . Despite their close taxonomic affinities , these species show a 10-fold range in seed size suggesting a rapid rate of evolutionary change ( Figure 1 ) . Why plants vary so much in seed size is not known . However , evolutionists and ecologists have long noted this great variation and hypothesized its importance in adaptation . In terms of survival , there are both risks and benefits for a species to increase ( or decrease ) seed size . Because maternal resources are limited , a species has to “decide” whether to invest energy into a few large seeds or many small seeds [1] , [2] . Intra- and interspecific studies of offspring fitness in plant communities have demonstrated that plants producing a small number of large seeds often have higher tolerance to drought [3] , herbivory [4] , shading [5] , and nutrient-deficient soils [6] . However , plants producing a large number of small seeds exhibit superior colonization abilities with the advantage of dispersal due to the abundance of seeds and higher likelihood to escape from predation [7] , [8] . Scientific interest in seed size relates not only to its importance in evolution , but also to crop domestication . Crops domesticated for consumption of their seeds ( e . g . soybean , wheat , sunflower ) often produce seeds significantly larger than their wild ancestors [9]–[12] . It is likely that early humans consciously selected for larger seeds in an effort to increase yield and improve harvest efficiency . However , seed size also increased during domestication in crops not harvested for their edible seed . For example , domesticated tomatoes produce seeds up to several fold larger than their wild ancestors ( Figure 1 ) [13] . Likewise cultivated squash ( Cucurbita pepo ) produce seed more than two fold larger than their wild counter parts [14] . Why seed size increased during domestication in crops not consumed for their seeds is unclear . However , it has been conjectured that seed size increased in these species due to indirect selection for greater seedling vigor and germination uniformity under field production [15] . Despite the importance of seed size in plant evolution and crop domestication , relatively little is known about the genetic and molecular processes underlying natural variation in seed size . Most of our knowledge comes from quantitative trait mapping studies which have revealed a fairly large number of QTL affecting seed size in a variety of plants – e . g . Arabidopsis [16] , rice [17]–[22] , soybean [23] , [24] , sunflower [11] , [25]–[28] . However , most of these studies have not gone beyond the mapping stage and hence provide little insight into the developmental and molecular mechanisms underpinning seed size variation . The exception is rice where three seed size QTLs have been recently cloned . These encode a previously unknown RING-type E3 ubiquitin ligase [29] , a putative transmembrane protein [30] , and deletion of a gene of unknown function [17] . Tomato is one of the few species not domesticated for edible seeds , where extensive QTL mapping for seed size has been conducted . Over the past 25 years , mapping studies involving crosses between the cultivated tomato and related wild species have revealed approximately 20 QTLs which account for most seed size variation [13] , [31]–[34] . Different subsets of these QTLs were identified in different studies . However , a common feature of all studies was that a major QTL on chromosome 4 ( referred to as Seed weight 4 . 1 or Sw4 . 1 ) invariably accounted for a large portion of the genetic variation for seed size . Sw4 . 1 is responsible for up to 25% of the total phenotypic variation in segregating populations and up to 54% of the seed weight variation in crosses between nearly isogenic lines [13] . The conservation of Sw4 . 1 across tomato species , and its potential role in the evolution and domestication of cultivated tomato , makes Sw4 . 1 a prime candidate for characterization and cloning . Thus the objective of this study was to uncover the genetic , developmental and molecular mechanisms underlying modulation of seed size by the Sw4 . 1 QTL .
The size or weight of a seed can potentially be affected by the genotype of three different plant parts/tissues: a ) the female plant bearing the fruit which contains the developing seed and contributes the testa; b ) the triploid endosperm which nourishes the developing embryo and c ) the diploid embryo . A maternal effect would be caused by a substantial contribution of the maternal genotype from the testa ( a ) and/or endosperm ( b ) to the seed development , while a zygotic effect would be attributed to the equal contribution of maternal and paternal genotypes from the zygote ( c ) . To differentiate maternal from zygotic effects , a reciprocal cross experiment was conducted using a pair of nearly isogenic lines ( NILs ) ( see Material and Methods ) . In Cross 1 , an inbred line homozygous for a “large-seed” ( L/L ) allele from S . lycopersicum was used as the female in a cross with a nearly isogenic line ( NIL ) homozygous for the “small-seed” ( S/S ) allele from S . pimpinellifolium . In Cross 2 , the reciprocal cross was performed using the S/S as the female parent . For Cross 1 , the F1 seed would develop on a maternal plant of the L/L genotype , whereas with Cross 2 , the seed would develop on a maternal plant of the S/S genotype . If Sw4 . 1 exerts its effect on seed weight through the maternal parent , F1 seed from Cross 1 should be significantly larger than F1 seed from Cross 2 . The results from these experiments revealed that reciprocal crosses result in seed indistinguishable in weight: 3 . 07 mg ( Cross 1 ) versus 3 . 10 mg ( Cross 2 ) ( P = 0 . 99 ) . Whereas self-pollination of the same parents , L/L and S/S , resulted in seeds weighting 3 . 34 mg and 2 . 63 mg respectively ( P<0 . 005 ) . The equivalency in seed weight for the reciprocal F1s suggests that Sw4 . 1 does not influence seed weight through any significant effect exerted by the genotype of the maternal environment ( i . e . , fruit ) . It should be noted that reciprocal crosses would result in triploid endosperm with different parental allelic dosage ( L/L/S versus L/S/S ) . Such differences in allelic dosage might also cause differences in seed weight between seed produced from reciprocal crosses . The fact that no such differences were observed suggests that the genotype of the developing embryo is most likely the major point of control in the differential seed weight associated with Sw4 . 1 alleles . By comparing the seed weight values of the self-pollinated L/L and S/S NILs , it was estimated that the additive gene effect of Sw4 . 1 is approximately 0 . 36 mg [ ( LL-SS ) /2] . As a result , it is estimated that the genetic effect of a single “large-seed” ( L ) allele is to increase seed weight by 14% – a value very similar to what was reported previously [33] . Using these same parental values , as well as data from the F1 seed lots , it was also possible to calculate the degree of dominance ( d/a or k ) for interaction of the L and S alleles . It is thus estimated that the alleles interact in a largely additive manner ( d/a = −0 . 21 ) . This result suggests that the change in seed size affected by the two alleles is not due to a loss-of-function ( e . g . deletion ) at the locus , in which case a dominant-recessive gene action would be observed . This result is consistent with results from the gene expression experiments to be presented later . In an effort to identify the time during development at which Sw4 . 1 alleles modulate seed size , a comparative developmental study was conducted on the large-seeded ( L/L ) NIL and small-seeded ( S/S ) NIL . Fruit size was also measured to determine whether Sw4 . 1 may also affect this character . Developmental plots for the two NILs are shown in Figure 2 . No differences were observed for the NILs with respect to fruit size at any time during development . It is therefore concluded that Sw4 . 1 alleles specifically modulate changes in seed size , and that these changes are not the indirect effect of modulations in fruit size . These results are consistent with the earlier showing that the Sw4 . 1 effect on seed size is exerted largely through the genotype of the zygote and not the maternal plant . Beginning at 10 days after pollination ( DAP ) , seed size ( as measured by width and length ) was consistently greater for the L/L NIL than for the S/S NIL . For example , seeds from the L/L NIL were on average 23% longer than those of the S/S NIL at 15 DAP ( P = 0 . 008 , Figure 2 ) . At maturity , seeds of the L/L NIL were 17% longer than seeds of the S/S NIL ( P = 0 . 001 ) . Overall , L/L NIL showed the greatest change in seed size , relative to the S/S NIL , in the period from 10 DAP to 15 DAP , suggesting that Sw4 . 1 exerts its largest differentiating effect during this early period of seed development . In an effort to determine the stage of embryo development corresponding to this critical period , paraffin cross sections were prepared from seeds at 10 , 15 and 20 DAP . Microscopic examinations of the sections revealed that the embryos are globular at 10 DAP and torpedo-shape to curving at 15 DAP . These stages are similar to those previously reported by Lersten [35] and correspond to the initiation of rapid deposition of starch and lipids into the endospermic cells . An examination of sections of mature seed from the L/L and S/S NILs revealed that the L/L NIL produces seed that are increased with respect to the size of both the embryo ( P = 0 . 014 ) and endosperm ( P = 0 . 002 ) relative to those of the S/S NIL . However , no difference was observed between the two NILs with respect to the ratio of endosperm to embryo ( P = 0 . 956 ) . In both NILs the embryo accounts for the largest portion of the total seed with an embryo∶endosperm ratio of 4∶1 . These results suggest that Sw4 . 1 modulates changes in the amount of both embryo and endosperm tissue during seed development . Germination tests of the L/L and S/S NILs revealed that both give rise to highly viable seed ( germination rates of 97%±3 . 1 versus 96 . 3%±2 . 1 , respectively ) . Therefore , while Sw4 . 1 affects seed size , it appears to have no detectable affect on seed viability as measured by germination percentage . Another way to characterize seed viability is by germination rate . Germination rate is defined as the speed of germination , which is often associated with seedling vigor [36] . When the L/L and S/S NILs were subjected to germination rate tests , no difference was observed ( P = 0 . 106 ) . However , since these tests were done under laboratory conditions , we cannot rule out the possibility that allelic variation at Sw4 . 1 does affect seed germination under natural or field conditions . A high-resolution genetic mapping study was conducted in an effort to establish the molecular basis for Sw4 . 1 . This study was facilitated by the availability of the L/L and S/S NILs from which large F2 mapping populations could be derived . The first such F2 population was screened with the markers CT50 and T891 which flank the Sw4 . 1 QTL ( Figure 3A ) . DNA from individuals showing recombination between these two markers was further screened by an additional 23 markers within the region , allowing the Sw4 . 1 QTL to be resolved to an 11 cM interval between markers T877 to TG2 ( Figure 3B ) . Within this interval the highest LOD score was observed for marker CT97 , 53% of the variation in seed weight was associated with this marker . To further resolve the position of Sw4 . 1 , 140 additional individuals , derived from a F2-heterozygous individual , were then screened for all markers ( including 9 additional markers ) in the 11 cM interval . From this mapping , Sw4 . 1 was further resolved to a 7 cM interval between marker T877 and T725 ( Figure 3C ) . Within this interval , seed weight showed the strongest association with marker S1 . The position of Sw4 . 1 was further delimited to a 2 . 4 cM interval ( between markers ST4 and T725 ) by the screening of an additional 1 , 000 progeny ( Figure 3D ) . The S1 marker again showed the strongest association with seed weight explaining 41% of the seed weight variation . The S1 marker was then used to isolate and sequence a 130 kb BAC ( LE_HBa0077O05 ) from the Sw4 . 1 region of chromosome 4 . The markers SS4 and ST4 , derived from the end sequence of this BAC , were subsequently used to screen an additional 9 , 000 F2 plants and identify 25 recombinants within the BAC interval . However , sufficient seed for analysis was obtained for only 13 of the recombinant individuals . The R2-plot for the seed weight association among the 13 recombinants indicated that the cause of the Sw4 . 1 QTL resides within the central portion of this BAC ( Figure 3E ) . To gain further precision on the location of Sw4 . 1 within the BAC , selfed progenies from 10 selected recombinants were examined . These individuals contained crossovers between markers SSR1S2 and G18 ( ∼73 kb apart ) - where the maximum marker association with seed weight was localized by fine mapping ( Figure 3E , 4 ) . A comparison of seed weight from homozygous recombinant and non-recombinant progeny allowed positioning of the Sw4 . 1 QTL relative to the crossover point in each stock ( Table 1 , Figure 4 ) . Two independent crossover events ( 05T427-200 and 06T662-255 ) delineated the cause of the Sw4 . 1 QTL to a 38 kb interval extending from markers dABC20665 to S106 indel1 ( Table 1 , Figure 4 ) . The exact position of the crossover in each of these recombinant stocks was established by PCR sequencing through the crossover boundary region for each stock . As a result , the cause of the Sw4 . 1 QTL could be further narrowed to a smaller , 23 kb interval ( Figure 4 ) . Based on annotation , this 23 kb interval contains a single gene encoding a putative ATP binding cassette ( ABC ) transporter protein ( gene 11 in Figure 4 ) . ABC transporters represent a super family of ATP-binding cassette proteins found in a wide range of species [37] . They are used in transmembrane transport of diverse substances – including peptides , sugars , lipids , heavy metal chelates , polysaccharides , alkaloids , steroids , inorganic acids and glutathione conjugates [38]–[42] . The Arabidopsis genome contains at least 129 ABC transporter-like genes , and a number have already been investigated with regards to function [43] . As an aid to annotation of the tomato ABC transporter gene , and in the hope of gaining possible insights into its function , an effort was made to determine which of these Arabidopsis ABC transporter gene ( s ) might be orthologous to the tomato gene associated with Sw4 . 1 . Synteny has proven a very powerful method for establishing orthology [44] . A Reciprocal Best Match ( RBC ) approach [45] was thus used to identify microsyntenic genomic region ( s ) between Arabidopsis and tomato for the Sw4 . 1 region . With this approach , an effort was made to identify the putative orthologs of the 12 genes annotated in the tomato BAC containing the ABC transporter gene ( Table S1 ) . As a result , three microsyntenic blocks were identified in Arabidopsis – two blocks located on chromosome 5 and one on chromosome 4 ( Figure 5 ) . Only the syntenic block on chromosome 4 contains an ABC transporter ( At4g39850 ) ( Figure 5 ) . It is therefore concluded that the Arabidopsis ABC transporter gene At4g39850 is orthologous to the tomato ABC transporter associated with Sw4 . 1 . It is worth noting that At4g39850 is located at the same chromosomal position as a QTL associated with variation in both seed length and width in a cross between Arabidopsis ecotypes , raising the possibility that this gene may also underlie natural variation in seed size in this species [16] , [46] . Sequence alignment of Arabidopsis At4g39850 cDNA with the genomic sequences of both the L allele ( S . lycopersicum ) and S allele ( S . pimpinellifolium ) allowed the prediction of intron and exon boundaries in the tomato gene ( Figure 6 ) . It also allowed prediction of the full-length tomato ABC transporter protein , which is characterized by 4 functional domains , two of which are ATP-binding cassette ( ABC ) or nucleotide binding folds ( NBFs ) and two of which are hydrophobic integral membrane domains ( TMDs ) ( Figure 6 ) [43] . While both the length of coding region and the intron positions are highly conserved between the tomato and Arabidopsis orthologs , the introns are more variable in length and are generally longer in tomato than Arabidopsis ( data not shown ) . Several T-DNA insertion mutants have been isolated for the Arabidopsis ortholog At4g39850 . Some of these mutants affected seed size [47] . However , the Arabidopsis mutants were associated with an increase in seed size , whereas in tomato the RNAi transgenics produced smaller seeds . Another difference between Arabidopsis and tomato , is that some of the Arabidopsis mutants also affected seed germination , whereas no germination effects were observed for Sw4 . 1 in tomato . Further , one of the mutants ( cts-2 ) produced seeds with significant higher levels of fatty acids . These results led to the suggestion that the Arabidopsis ABC transporter protein might be involved , not only in lipid metabolism during germination , but also in lipid accumulation during seed development – possibly explaining why the mutants produced larger seeds [47] . In this regard , it is worth noting that in tomato the Sw4 . 1 QTL produces its largest effects during the stages of seed development associated with lipid deposition ( see previous section , Figure 2 ) . Thus , it is possible that the tomato ABC transporter gene modulates seed size by controlling the accumulation of lipids during seed development . Transformation experiments were used to test whether the ABC transporter gene has the ability to modulate seed size . This gene is quite long ( due to many introns ) – spanning more than 20 kb from the start to stop codon ( Figure 6 ) . The large size of the gene , absence of efficient enzymatic sites for cloning and lack of a full-length cDNA , precluded complementation analysis with the full-length genomic copy of the gene . Therefore , a gene silencing approach , via RNAi hairpin formation , was employed as an alternative strategy for testing the potential role of the ABC transporter gene in determining seed size . Efficient post-transcriptional silencing has been reported when 3′UTR regions are targeted for RNAi machinery [48] , [49] . Hence , a 278 bp fragment from the 3′UTR of the ABC transporter gene was inserted in the pHELLSGATE2 ( Invitrogen ) binary vector , the pSP13-1 construct ( Figure S1 ) . Sequence specificity was assessed by blasting the 278 bp fragment against the tomato unigene database in the SGN website ( www . sgn . cornell . edu ) . The retrieval of a unique unigene that corresponds to this ABC transporter gene suggests this sequence is specific to this gene and therefore there would present a low risk to silence other genes in the same ABC transporter family . This construct was transformed into both the L/L and S/S NILs . Multiple independent T0 and non-transgenic controls were then analyzed for both NIL sets . L/L transformants were highly fertile , yet produced seeds weighing on average 38% less than those from the non-transgenic controls ( P = 0 . 03 , Table 2 ) . S/S transformants also produced smaller seed ( 11% less heavy ) than the non-transgenic controls , however the statistical difference did not quite reach statistical significance ( P = 0 . 09 , Table 2 ) . It is worth noting that transformation/complementation with two other genes from the BAC ( annexin and a gene of unknown function – genes number 6 and 7 , respectively , in Figure 4 ) did not show statistical difference between transgenic and non-transgenic plants ( data not shown ) . The transformation experiments thus appear to corroborate the results from high-resolution mapping – both pointing to the ABC transporter gene as the cause of the Sw4 . 1 QTL . Comparing the sequence of the L and S allele , within the 23 kb Sw4 . 1 interval , revealed 79 SNPs and single indels located in introns , 10 in the promoter region and 6 in exons ( Figure S2 ) . In the exons , 2 non-synonymous changes were observed ( Figure 6 ) . Either of these might be causal to the phenotypic effects rendered by the L and S alleles . However , neither substitution is located in a conserved functional domain ( e . g . nucleotide binding folds or hydrophobic integral membrane domains ) ( Figure 6 ) . Among the polymorphisms in non-coding regions , a few are worth mentioning as possible causal candidates for the Sw4 . 1 QTL . One is a 12 nt indel approximately 1 . 5 kb upstream in the 5′ promoter and the second , a 24 nt indel in the first intron ( Figure 6 ) . Either of these indels might cause a change in expression – as could the many other small nucleotide differences observed in non-coding regions of the two alleles . In an effort to determine the expression pattern of the ABC transporter gene , and especially whether the L and S allele differ in regulation/expression during seed development , a set of semi-quantitative RT-PCR experiments were conducted . The first experiment revealed that this gene is expressed at high levels in seeds , and lower levels in shoots and roots ( Figure 7A ) . High expression of the ABC transporter gene in developing seeds is further evidence that this gene is the cause of the Sw4 . 1 QTL . The subsequent experiment compared expression of the ABC transporter gene in both the L and S NILs at 10 , 15 and 20 DAP – the time when the major change in seed size is observed ( see previous section ) ( Figure 7B ) . Two conclusions can be drawn from this second experiment . First , the ABC gene is expressed in both the L and S NILs – ruling out a loss-of-function as the cause of seed size variation associated with Sw4 . 1 . Second , the failure to detect any major change in expression of the ABC transporter gene between the L and S NILs during seed development would seem to rule out a gross change in the regulation as the cause of the Sw4 . 1 QTL ( Figure 7B ) . However , we cannot exclude the possibility of a difference in translational/post-translational regulation or small changes in spatial or temporal regulation as the cause of QTL effect . For example , in tomato it was previously shown that a modest change in the timing of allele expression can cause major QTL effects on fruit size [50] . The results presented herein point to natural variation in an ABC transporter gene as a major cause of the change in seed size that differentiates the cultivated tomato from related wild species . While prior studies in a number of plants have reported the effects of induced mutations on seed size , this example is among the few in which the cause of changes in seed size in nature populations has been pinpointed . Further , the ortholog to this gene in Arabidopsis has been identified through homology/synteny . Results from QTL mapping and mutagenesis studies are consistent with this gene also playing a role in determining seed size in Arabidopsis . However , further studies are required to clearly establish whether this ABC transporter gene operates in a similar manner in both tomato and Arabidopsis . Also , it remains to be established whether variation in ABC transporter genes is a major cause of seed size variation in natural populations of Arabidopsis or other plant species . Having identified the cause of the Sw4 . 1 QTL in tomato may open he door to addressing these questions in the future .
Two Solanum lycopersicum nearly isogenic lines ( NILs ) , with alternative alleles at the Sw4 . 1 locus , were the origin of all genetic stocks used in this study . TA209 carries the large-seeded ( S . lycopersicum ) allele at Sw4 . 1 locus . TA2080 is isogenic with TA209 , but carries a 55–84 cM segment of chromosome 4 of S . pimpinellifolium LA1589 ( Figure 3A ) containing the small-seeded allele of Sw4 . 1 [13] , [33] . TA2080 was developed via marker assisted selection during 5 sequential backcrosses of LA1589 into TA209 followed by a single selfing generation ( BC5S1 ) . An F2 population , segregating for Sw4 . 1 , was then developed from a cross between TA209 and TA2080 . A derived , shorter introgression NIL , derived from TA2080 , was used for the reciprocal cross experiments . Likewise , the S/S NIL ( TA3820 ) used for transformation experiments was also a shorter derivative of TA2080 . Detailed description of the construction of both derived subNILs can be found in Orsi [46] . The sequence of all markers used in this study can be found in Table S2 . Plants were grown in pots in the greenhouse in 12 randomized blocks . Each block was comprised of the following Sw4 . 1 genotypes and type of pollination: L/L: selfing , S/S: selfing , L/S: selfing; L/L: used as the female in crosses to S/S , S/S: used the female in crosses to L/L . Each plant was either selfed or crossed manually . At maturity , 5 normal fruit ( no blossom end rot , non-parthenocarpic ) were harvested from each plant and the seed extracted . The fruit were weighed and the average weight per fruit was recorded . From a pool of seeds from 5 fruits , 50 healthy seeds were randomly sampled . From these , an average seed weight was calculated for each plant . A comparison of the least squares means was performed using the adjustment for multiple comparisons Tukey-Kramer ( SAS enterprise guide 3 . 0 ) . Effects of Sw4 . 1 on seed development and fruit size: Ten pairs of L/L and S/S NIL plants were grown in the greenhouse . Two to three fruits were harvest from each plant at each of the following stages: anthesis ( 0 days after pollination –DAP ) , 5 DAP , 10 DAP , 15 DAP , 20 DAP , 25 DAP , 30 DAP , 35 DAP and 40 DAP . Because of the microscopic size of ovule and developing seeds , it was not possible to collect mass ( weight ) data . Instead , all traits were recorded as spatial metrics ( e . g . , length , width ) . Both fruit and seed were scanned using a HP ScanJet ( 1200 dpi ) . Length and width measurements were then extracted from the images using the software Tomato Analyzer Version v . 1 . 2 [51] . A second independent experiment , of identical design , was then conducted . However , based on results from the first experiment , fruit were collected only at 10 DAP , 15 DAP and 20 DAP – periods associated with most rapid changes in seed development . For the verification of seed developmental stages , seeds from 10 , 15 and 20 DAP were fixed , dehydrated and embedded in paraplast ( Sigma ) as described by [52] . Role of Sw4 . 1 in determining the proportion of embryo to endosperm in mature seed: Five pairs of L/L and S/S individuals were grown in the greenhouse . Five mature fruits were harvested from each individual and the seeds extracted . A random sample of 10 normal seeds was then drawn from each individual . Each individual seed was then dissected longitudinally into halves and the images digitalized under a dissecting scope ( ZeissStemi 2000-CS attached to 3CCD camera MTI ) using the software Scion Image ( www . scioncorp . com ) . The areas of the entire seed , embryo and endosperm were manually delineated and measured using the software ImageJ 1 . 31 v ( http://rsb . info . nih . gov/ij/ ) . Ten pairs of L/L and S/S individuals were grown in the greenhouse . One hundred normal seeds from a pool of 10 fruits of each genotype were germinated in Petri dishes on filter paper saturated with distilled water . The seeds were scanned prior to the germination process for seed length measurement ( Tomato Analyzer v . 1 . 2 ) . The number of germinated seed was recorded on a daily basis until no additional seed germination was obtained . For these experiments , a seed was considered germinated once the root tip had emerged . Tests of heterogeneity on the number of germinated seeds at days 3 , 4 and 5 ( when the majority of seed germination was observed for all the genotypes ) were performed for the detection of possible differences in germination rate between NILs ( Minitab 15 ) . The genetic stocks used for the progeny analysis were derived from sub-NILs selected from each selected recombinant within the BAC ( Figure 4 ) . Ten homozygous recombinant and ten homozygous non-recombinant individuals were selected , via marker analysis , from selfed seed of selected recombinants ( Table 1 , Figure 4 ) . The selected progeny were grown in a completely randomized design in the greenhouse . From each plant , seeds were extracted from 5 fruits and pooled . From each pool , 50 normal seeds were randomly selected and weighed . For each family , statistical comparisons for seed weight were made between the 10 recombinant and 10 non-recombinant progeny from each family using a one-tailed t-test ( Minitab 15 ) ( Table 1 ) . BAC LE_HBa0077O05 , isolated with marker probe S1 , was annotated using the automated annotation tools developed by SGN and refined manually through BLAST searches against EST libraries ( Solanaceae , coffee , Arabidopsis ) . Arabidopsis genes , putatively orthologous to genes in the tomato BAC , were identified using the Best Reciprocal Matches ( RBM ) in BLAST comparisons [45] . Based on these putative orthologs , it was possible to identify region in the Arabidopsis genome showing conserved microsynteny with the Sw4 . 1 region of tomato chromosome 4 . A 38 kb segment , delimited by markers dABC20665 and S106indel1 and known from progeny analyses to encompass the Sw4 . 1 QTL , was also sequenced via PCR from the genome of S . pimpinellifolium LA1589 – the small-seeded parent of the original mapping population . The objective of the sequencing was to identify polymorphisms that might be causal to the Sw4 . 1 QTL . Sequence analysis was performed with DNASTAR Lasergene software and alignments with BioEdit version 7 . 0 . 9 . 0 using ClustalW . In order to further narrow the Sw4 . 1 interval , PCR-based sequencing was performed on the two recombinant individuals that define the left ( 05T427-200 ) and right ( 06T661-255 ) side of the 38 kb interval . By sequencing across the exact crossover point in each recombinant , it was possible to narrow the location of the Sw4 . 1 QTL to a 23 kb interval ( Figure 4 ) . | Given fixed resources , plants have a choice whether to produce many small seeds or a few large seeds . In terms of reproductive fitness , there are costs and benefits to both strategies . As a result , plant species vary more than 100 , 000-fold in both seed size and seed output . The current study focuses on understanding the molecular and developmental basis of a single genetic locus ( or quantitative trait locus ) that determines seed size between the cultivated tomato and its wild relatives . We show that the cause of size variation can be traced to a gene encoding an ABC transporter protein . The gene apparently exercises its control on seed size through expression in the developing seeds and not the mother plant that nurtures those seeds . A comparison with the model plant Arabidopsis thaliana suggests that the ABC transporter identified in tomato may also control seed size in other plants , opening research opportunities for understanding plant adaptation and for potentially modulating seed size in crop plants for human purposes . | [
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] | 2009 | Natural Variation in an ABC Transporter Gene Associated with Seed Size Evolution in Tomato Species |
Human immunodeficiency virus type 1 ( HIV-1 ) interactions with myeloid dendritic cells ( DCs ) can result in virus dissemination to CD4+ T cells via a trans infection pathway dependent on virion incorporation of the host cell derived glycosphingolipid ( GSL ) , GM3 . The mechanism of DC-mediated trans infection is extremely efficacious and can result in infection of multiple CD4+ T cells as these cells make exploratory contacts on the DC surface . While it has long been appreciated that activation of DCs with ligands that induce type I IFN signaling pathway dramatically enhances DC-mediated T cell trans infection , the mechanism by which this occurs has remained unclear until now . Here , we demonstrate that the type I IFN-inducible Siglec-1 , CD169 , is the DC receptor that captures HIV in a GM3-dependent manner . Selective downregulation of CD169 expression , neutralizing CD169 function , or depletion of GSLs from virions , abrogated DC-mediated HIV-1 capture and trans infection , while exogenous expression of CD169 in receptor-naïve cells rescued GSL-dependent capture and trans infection . HIV-1 particles co-localized with CD169 on DC surface immediately following capture and subsequently within non-lysosomal compartments that redistributed to the DC – T cell infectious synapses upon initiation of T cell contact . Together , these findings describe a novel mechanism of pathogen parasitization of host encoded cellular recognition machinery ( GM3 – CD169 interaction ) for DC-dependent HIV dissemination .
Myeloid dendritic cells ( DCs ) are potent antigen presenting cells that monitor their immediate environment for invading pathogens . The ability of these sentinel DCs to sample and process antigen , and present processed antigen to naïve T cells is critical to the development of effective adaptive immune responses . HIV , in turn , has evolved to evade antigen presentation pathways and exploit DC biology to facilitate its dissemination within the infected host . DC-dependent HIV-1 trans infection of CD4+ T cells is an efficacious HIV dissemination mechanism [1] , [2] that has been hypothesized to provide virus particles evasion from host innate and adaptive immune responses [3] . HIV-1 capture by DCs and access to the DC-dependent trans infection pathway has long been thought to be exclusively dependent on the interactions of the mannosylated virus envelope glycoprotein gp120 with C-type lectin receptors ( CLRs ) [4] , such as dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin ( DC-SIGN ) [5] . In addition to HIV-1 capture , virus targeting to tetraspanin protein positive non-lysosomal compartments that allow for virus persistence and evasion from lysosomal degradation pathways in DCs [6] , [7] , [8] has also been reported to be dependent on DC-SIGN [9] , [10] . In contrast , knock-down of DC-SIGN expression in DCs using shRNAs [11] , or blocking DC-SIGN function using neutralizing antibodies [12] , [13] have failed to attenuate DC-mediated HIV-1 capture or trans infection . Furthermore , while DCs upon maturation downregulate cell surface expression of DC-SIGN and CLRs , HIV-1 capture and trans infection efficiency is dramatically enhanced upon maturation over that observed with immature DCs [7] , [14] , [15] . Interestingly , capture of HIV-1 particles by DCs can occur in a gp120-independent manner , and this virus capture mechanism is also enhanced upon maturation of DCs [7] , [14] . Furthermore , capture of gp120-deficient virus-like particles by mature DCs can result in localization within non-lysosomal compartments and trafficked to DC – T cell infectious synapses upon initiation of T cell contacts [7] . These findings highlight the existence of a virus particle associated host encoded HIV-1 capture mechanism in DCs . In addition to gp120 , HIV-1 particles also incorporate host cell derived constituents in their lipid bilayer . Assembly of HIV-1 particles within plasma membrane lipid rafts [16] , [17] results in production of virions with a unique surface exposed host cell derived proteome and lipidome [18] , [19] , [20] . Interestingly , selective depletion of virion-associated glycosphingolipids ( GSLs ) , either by targeting GSL biosynthesis pathways by small molecule inhibitors [7] , [14] or by siRNAs targeting GSL biosynthetic enzymes in virus producer cells [21] can attenuate gp120-independent capture of HIV-1 particles by mature DCs . In support of this hypothesis , we and others , have recently demonstrated that gp120-independent capture of HIV-1 by DCs is dependent on the expression of the ganglioside , GM3 ( α2 , 3-sialylated GSL ) in the virus particle membrane [21] , [22] . The data presented in this report demonstrate that the IFNα-inducible sialic-acid-binding immunoglobulin-like lectin ( Siglec-1 ) , CD169 , is the receptor that mediates the gp120-independent , GM3-dependent interaction of HIV-1 with DCs and defines a novel molecular mechanism ( virus particle associated GM3 binding by CD169 ) by which HIV-1 transits through the DC to arrive at the infectious synapse and may provide a novel conserved host-derived target ( GM3 – CD169 interaction ) for intervention strategies .
To identify the DC-receptor required for GM3-dependent HIV-1 capture , primary peripheral blood CD14+ monocytes , immature DCs , and DCs activated with TLR2 ( Pam3CysK4 ) , TLR3 ( poly ( I:C ) ) , or TLR4 ( E . coli LPS ) agonists , or stimulated with the cytokines , IFNα or TNFα were tested for their ability to capture Env-deficient HIV-1 Gag-eGFP virus like particles ( VLPs ) . While monocytes failed to capture VLPs , differentiation of monocytes into DCs resulted in a low level of VLP capture ( Figure 1A ) . Furthermore , stimulation of immature DCs with TLR3 or TLR4 agonists dramatically enhanced VLP capture ( Figure 1A ) , similar to the previously observed maturation induced enhancement in infectious virus capture by DCs [7] , [14] , [15] . But surprisingly , activation of DCs with Pam3CysK4 ( TLR2 agonist ) resulted in no appreciable increase in VLP capture over that observed with immature DCs ( Figure 1A ) . While signaling downstream of all TLRs is conceptually similar and can result in maturation of DCs , individual TLRs trigger different signal transduction pathways with the key distinction being that TLR3 and TLR4 signaling can induce both pro-inflammatory and type I IFN responses via a TRIF-dependent pathway while TLR2 receptors induce only a pro-inflammatory response [23] . Interestingly , direct exposure of immature DCs to IFNα alone but not TNFα , resulted in a similar level of VLP capture to that observed with LPS or poly ( I:C ) -stimulated DCs ( Figure 1A ) , suggesting HIV-1 capture by DCs is dependent on the presence of an IFNα-inducible factor . We and others have demonstrated that capture of HIV-1 by LPS-matured DCs is dependent on virion incorporation of terminal α2 , 3-sialylated GSL , GM3 [21] , [22] . Interestingly , this IFN-inducible , GSL-dependent capture mechanism of VLPs was specific to myeloid cells and was exquisitely sensitive to proteolytic cleavage of DC surface expressed proteome ( Supplementary Figure S1 ) . The sialoadhesin , Siglec1 or CD169 , a member of the sialic acid binding immunoglobulin superfamily of lectins [24] , is a type I IFN-inducible myeloid cell specific protein , with binding specificity for gangliosides that contain terminal α2-3 linked terminal sialic acid residues , such as GM3 [25] . While peripheral blood CD14+ monocytes do not express CD169 ( Figure 1B ) , expression on monocytes can be induced upon exposure to IFNα in vitro and in vivo [26] , [27] , [28] . Interestingly , some studies have also reported inducible expression of CD169 on DCs in vitro [29] . We sought to determine if peripheral blood monocyte-derived DCs express CD169 and whether CD169 functions as the DC receptor responsible for GSL-dependent capture of HIV-1 . Differentiation of monocytes to immature DCs resulted in a low level of CD169 expression by day 6 post-initiation of differentiation ( ∼25 , 000 ABS/cell; Figure 1B and C ) , with a dramatic enhancement in cell surface CD169 expression upon subsequent stimulation with LPS ( ∼125 , 000 ABS/cell; Figure 1B and C ) . In contrast cell surface expression of DC-SIGN was inversely correlated with VLP capture phenotype , in that immature DCs expressed high levels of DC-SIGN ( >2 , 000 , 000 ABS/cell; Figure 1B and D ) , while cell surface DC-SIGN expression was downregulated upon maturation with LPS ( ∼600 , 000ABS/cell; Figure 1B and D ) . It should be noted that measurement of cell surface DC-SIGN expression by QFACS is an approximation , since DC-SIGN ABS values fell beyond the linear range of the standards employed in the assay , and , hence were derived via extrapolation of the standard curve beyond the highest standard ( 300 , 000 ABS ) . Interestingly , in strict correlation with the observed type I IFN-dependent enhancement of VLP capture by DCs ( Figure 1A ) , enhancement of CD169 expression was only observed upon stimulation of immature DCs with poly ( I:C ) , LPS or IFNα but not with Pam3CysK4 or TNFα ( Figure 1E ) , To determine if capture of HIV-1 by mature DCs is dependent on CD169 specifically , immature DCs were transduced with lentivectors expressing CD169-specific shRNA , DC-SIGN-specific shRNA or scrambled control shRNA prior to initiation of maturation with LPS . CD169 expression was effectively suppressed upon transduction with CD169-shRNA ( ∼80% decrease in cell surface CD169 expression; ( Figure 2A and Supplementary Figure S2A , C ) , while DC-SIGN cell surface expression was downregulated ( ∼50% ) upon transduction with DC-SIGN shRNA ( Figure 2B , and Supplementary Figure S2B , D ) , though transduction with either shRNA had negligible impact on cell surface expression of CD86 ( Figure 2C ) . Interestingly , knock-down of CD169 expression on DCs robustly inhibited capture of VLPs ( Figure 2D ) , Env-deficient HIV-1 ( Figure 2E ) , or infectious HIV-1 particles ( Figure 2F ) , while decrease in DC-SIGN had negligible impact on virus capture by mature DCs ( Figure 2D , E and F ) . To achieve high efficiency transduction of immature DCs with shRNA expressing lentivectors , pretreatment with SIVmac Vpx containing VLPs was required that resulted in low level of DC maturation ( data not shown ) . Hence , we utilized neutralizing antibodies ( nAb ) to define the role of CD169 in HIV-1 capture by immature and mature DCs . Pre-exposure to α-CD169 nAb alone blocked virus capture ( Figure 2G ) and trans infection to autologous CD4+ T cells ( Figure 2H ) by immature and mature DCs , while α-DC-SIGN nAb alone had modest inhibitory effects in immature DCs and failed to block HIV-1 capture by mature DCs ( Figure 2G and H ) , consistent with previously published results [11] , [12] , [13] , [30] . While pre-incubation with a combination of α-DC-SIGN and α-CD169 nAbs completely abrogated HIV-1 capture and trans infection by immature DCs , there was no additional attenuation observed with the combination of α-DC-SIGN and α-CD169 antibodies to that observed with α-CD169 antibody alone in mature DCs ( Fig . 2G and H ) . Though DCs also express additional Siglecs , such as Siglec-7 and -9 with specificity for α2-8 and α2-3 sialic acid containing glycans , respectively [24] , capture of VLPs by mature DCs was only inhibited upon pre-incubation with CD169 nAb but not α-Siglec-7 or α-Siglec-9 nAbs , even upon desialylation of DC surface ( Supplementary Figure S3 ) , that can result in unmasking of sialic acid binding sites on Siglecs [24] . These results suggest that CD169 is largely responsible for DC-mediated HIV capture and trans infection . Capture of HIV-1 by mature DCs can result in sequestration of virus particles in teraspanin positive non-lysosomal compartments , that might be connected to the DC surface by deep membrane invaginations , and transferred to T cells upon initiation of DC – T cell contacts [2] , [6] , [7] , [8] , [31] . To determine if CD169 mediates HIV-1 trafficking to DC – T cell infectious synapses , cell surface distribution of CD169 on mature DCs was determined by confocal microscopy . While CD169 distribution on mature DCs displayed punctate staining in the absence of virus ( Figure 3A ) , there was clear evidence of redistribution of the receptor within 10 min of exposure to HIV Gag-mCherry VLPs , with 27% of CD169 associated with virus particles ( Figure 3B ) . By 120 min post exposure , CD169 underwent a dramatic polarization ( Figure 3C ) that resulted in 81% of CD169 co-localized with VLPs at the DC – T cell infectious synapse ( Figure 3D ) . HIV Gag-mCherry VLPs showed nearly 45% co-localization with CD169 by 10 min post exposure ( Figure 3B ) , and increasing after 2 h of virus exposure to 70% or 84% in the absence ( Figure 3C ) or presence ( Figure 3D ) of autologous CD4+ T cells , respectively . We verified similar patterns of co-localization between CD169 and infectious virus particles ( Lai-iGFP ) and again observed the spatial concentration coincident between CD169 and viral particles within 2 h post virus exposure ( Supplementary Figure S4A and B ) . While CD169 has been hypothesized to phagocytose sialylated pathogens [32] , mechanism of endocytosis remains unclear . Interestingly , HIV-1 particles were trafficked to non-lysosomal compartments at the cell periphery , since no co-localization was observed between VLPs and endosomal markers , EEA1 and LAMP1 ( Supplementary Figure S4D and E ) , nor between VLPs and cell surface proteins CD9 and CD45 , at 10 min or 120 min post virus exposure ( Supplementary Figure S4C , F and G ) . These results suggest that interaction of HIV-1 particles with CD169 on mature DC surface leads to virus particle localization at the cellular periphery that might correspond to the previously described deep plasma membrane invaginations [8] , [31] and upon initiation of DC – T cell contacts , trafficked to the DC – T cell infectious synapse . Even though IL-4/GM-CSF pathway of DC differentiation from peripheral blood monocytes is an efficient in vitro method to generate immature DCs [33] , IL-4 is an anti-inflammatory cytokine [34] and high levels of IL-4 are unlikely to be present during host response to acute virus infections . In contrast , during acute SIV infection , a rapid influx of type I IFN-producing plasmacytoid DCs into the genital mucosa has been noted [35] . Furthermore , under conditions of chronic immune activation as a consequence of a compromised gut epithelial barrier , a hallmark of HIV-1 infection in vivo , a low-level of circulating type I IFNs has been observed [36] . Such inflammatory conditions provide strong signals for rapid influx and differentiation of monocytes into tissue inflammatory DCs [37] , [38] , [39] , [40] . Interestingly , peripheral blood CD14+ monocytes stimulated with IFNα and GM-CSF are differentiated into inflammatory DCs ( referred to as IFN-DCs ) that can successfully prime naïve T cells and bias the T helper response towards a Th1 type [41] , [42] . We sought to determine the interactions of HIV-1 particles with DCs differentiated from monocytes under pro-inflammatory conditions . Interestingly , IFN-DCs expressed high levels of CD169 ( 150 , 060±49 , 827 ABS/cell ) but not DC-SIGN ( 20 , 298±16 , 147 ABS/cell ) ( Figure 4A and Supplementary Figure S5A ) , in contrast to DCs differentiated in the presence of IL-4 and GM-CSF for 3 days ( IL4-DCs ) that expressed high levels of DC-SIGN ( 1 , 848 , 000±364 , 359 ABS/cell ) with little to no expression of CD169 ( 20 , 040±3 , 604 ABS/cell ) ( Figure 4B and Supplementary Figure S5B ) . While there was some variation in the expression of CD169 and DC-SIGN between donors , these two DC populations provided phenotypically distinct subtypes that expressed either CD169 or DC-SIGN . CD169+ IFN-DCs , but not DC-SIGN+ IL4-DCs captured VLPs in a dose-dependent manner ( Figure 4C ) . Furthermore , depletion of GSLs from virus particles via PDMP ( competitive inhibitor of glucosylceramide synthase ) treatment of virus producer cells ( Supplementary Figure S6 ) [14] , resulted in attenuation of capture of both VLPs and HIV/Lai-Bal virus particles by IFN-DCs ( Figure 4C and D ) . Interestingly , capture of VLPs and HIV/Lai-Bal particles by DC-SIGN+ IL4-DCs was significantly lower in comparison to that exhibited by CD169+ IFN-DCs ( Figure 4C and D ) . Cellular source of virus has been hypothesized to play a significant role in the ability of HIV-1 particles to interact with DC-specific attachment factors [14] , [43] , [44] . The virus producer cell phenotype had no impact , since capture of HIV/Lai-Bal derived from either HEK293T cells or PBMCs by IFN-DCs was dependent on GSLs ( Figure 4D and Supplementary Figure S7 ) , in a CD169-dependent manner ( Figure 4E ) . Even though IFN-DCs expressed CD4 and CCR5 ( Supplementary Figure S5A ) , these cells were refractory to productive infection ( Figure 4F ) . Rather , captured particles were transferred to autologous CD4+ T cells , with a greater efficiency than IL4-DCs ( Figure 4F ) , and with a stringent requirement for GSLs in the virus particle membrane ( Figure 4F ) . In addition to monocyte-derived DCs , we sought to determine if CD169 dependent interactions of DCs with HIV are also conserved in circulating blood DCs . since previous studies have demonstrated DC-SIGN or CLR-independent capture and trans infection of HIV-1 by these cells [45] , [46] . BDCA1+ myeloid DCs directly isolated from PBMCs expressed CD169 ( Figure 5A ) , and expression of CD169 was enhanced upon stimulation with IFNα ( Figure 5B ) . Capture of VLPs ( Figure 5C ) , Env-deficient , or infectious HIV-1 particles ( Figure 5D ) by myeloid DCs was also increased upon IFNα stimulation , while challenge with GSL-depleted particles ( Figure 5E ) or pre-incubation with α-CD169 nAb ( Figure 5F ) attenuated capture . These results suggest that HIV capture and access to the trans infection pathway by both inflammatory and myeloid DCs is dependent on the expression of CD169 on the cell surface and GSLs in the virus particle membrane . To determine if CD169 expression alone is sufficient for mediating HIV-1 capture and trans infection , CD169-deficient Raji B cells were engineered to constitutively express CD169 ( Figure 6A ) . While parental Raji cells did not bind Env-deficient HIV-1 particles ( Figure 4B ) [5] , [47] , CD169 expression rescued the ability of these cells to capture VLPs ( Figure 6B ) , and Env-deficient or infectious HIV-1 particles ( Figure 6C ) . Furthermore , exogenous expression of CD169 on Raji cells resulted in efficient capture of HEK293T ( Figure 6D ) and PBMC-derived virus particles ( Figure 6E ) , and capture efficiency was significantly attenuated upon GSL depletion from virus particle membrane ( Figure 6D and E ) . Interestingly , Raji/CD169 cells efficiently transferred captured HIV/Lai-Bal virus particles to CD4+ T cells in a GSL dependent manner ( Figure 6F ) . These results suggest that expression of CD169 alone defines the ability of DCs to capture and transfer HIV-1 particles . Our previous studies had demonstrated binding of GM3 and GM1 ( terminal α2 , 3 sialic acid residue containing GSLs ) liposomes by mature DCs [21] . In additional support of a role for CD169 as the primary attachment factor on DCs responsible for α2 , 3 sialylated GSL recognition , Raji/CD169 cells preferentially bound GM3 or GM1 containing liposomes , over that observed with parental Raji cells ( Figure 7A ) , while no enhancement in liposome binding was observed with Raji/CD169 cells compared to Raji cells upon challenge with liposomes containing either GalCer or GQ1b ( terminal α2 , 8 sialic acid residues ) ( Figure 7A ) . Furthermore , Env-deficient HIV-1 capture by Raji/CD169 cells was competitively inhibited by GM3-containing liposomes ( Figure 7B ) , similar to previous findings with mature DCs [21] . The extracellular region of CD169 , like other members of the Siglec family , consists of a single V-set immunoglobulin ( Ig ) -like domain that contains the sialic acid binding motif [24] , [48] . We mutated the critical Arg residue in the extracellular N-terminal V-set domain ( R116A ) shown previously to be essential for sialic acid binding by Siglecs [25] , [48] , and a control Arg residue ( R96A ) , not predicted to be required for sialic acid recognition . Induced expression of CD169 in HEK293T cells ( Figure 7C and Supplementary Figure S8A ) rescued VLP ( Supplementary Figure S8B ) and infectious virus capture ( Figure 7D ) . Though total and cell surface expression of both mutants ( R96A and R116A ) was equivalent to that of wild type CD169 in transiently transfected HEK293T cells ( Supplementary Figure S8A and Figure 7C ) , there was a complete loss of VLP ( Supplementary Figure S8B ) and infectious HIV-1 capture ( Figure 7D ) and transfer ( Figure 7E ) by only the sialic acid binding deficient CD169/R116A mutant . Siglec1 or CD169 is extremely well-conserved amongst various mammalian species , with the highest similarity observed in the N-terminal extracellular domain [24] . In particular , amino acids in the V-set Ig-like domain essential for sialic acid recognition are identical between human and mouse CD169 proteins [25] . Interestingly , similar to HIV-1 particles , GM3 is also enriched in the lipid bilayer of the simple retrovirus , murine leukemia virus ( MLV ) [18] . Furthermore , capture of HIV-1 particles by mature DCs can be competitively inhibited by ecotropic-MLV particles [7] . Since animal models can prove invaluable for the study of CD169 function in vivo , we wanted to determine if murine CD169 could also function as a retroviral attachment factor that can mediate GM3-dependent virus capture and transfer . Transient expression of murine CD169 in HEK293T cells resulted in high cell surface expression ( Figure 8A ) and robust HIV Gag-eGFP ( Figure 8B ) and MLV Gag-YFP VLP capture ( Figure 8C ) . To determine if capture of MLV Gag-YFP VLPs was also dependent on GSLs , GSL-depleted MLV Gag-YFP VLPs were produced from PDMP-treated HEK293T cells ( Supplementary Figure S9A and B ) . Interestingly , capture of retroviral VLPs by murine CD169 was also dependent on presence of GSLs in the virus particle membranes ( Figure 8B and C ) . Furthermore , mature DCs ( Figure 8D ) and Raji/CD169 cells ( Supplementary Figure S9D ) could also capture MLV Gag-YFP VLPs in a GSL and human CD169-dependent manner . We next determined if both human and murine CD169 could transfer captured HIV-1 particles to CD4+ T cells and MLV particles to murine cells , a mechanism of retroviral trans infection . HEK293T cells transiently transfected with human or murine CD169 were exposed to single cycle of replication competent luciferase expressing HIV-1/Lai-Bal or ecotropic-MLV ( MLV-E ) particles , washed and then co-cultured with human CD4+ T cells or MLV-E susceptible Rat-2 cells . Interestingly , we observed robust transfer of HIV-1 particles to CD4+ T cells ( Figure 8E ) and transfer of ecotropic MLV particles to Rat-2 cells ( Figure 8F ) by murine CD169 . Finally , mDCs also transferred MLV-E particles to Rat-2 cells and this transfer mechanism was dramatically decreased if capture of MLV-E particles by mDCs was carried out in the presence of anti-CD169 blocking antibody prior to initiation of co-cultures ( Figure 8G ) . Collectively , these results suggest that recognition of α2 , 3 sialylated GSL , GM3 , on retroviral particles by CD169 is essential for access to DC-mediated retrovirus trans infection pathway .
The results presented in this report define a novel mechanism of type I IFN-dependent enhancement of HIV-1 capture by DCs and subsequent access to the DC-mediated trans infection pathway . Maturation of DCs with TLR ligands that induce TRIF-dependent type I IFN-signaling pathway , differentiation of monocytes to DCs in the presence of IFNα , or exposure of blood myeloid DCs to IFNα , resulted in enhanced expression of a HIV-1 attachment factor , CD169 , that facilitated explosive virus replication in DC – CD4+ T cell co-cultures . In agreement with our findings , Izquierdo-Useros , et al , also reported ( while our manuscript was under review ) that Env-independent , ganglioside-dependent capture of HIV-1 particles by LPS-matured DCs required CD169 expression [49] . These findings are in direct contrast to a long-standing hypothesis in the literature that has suggested an absolute requirement for DC-SIGN in mediating DC-mediated HIV-1 trans infection [5] . While exogenous expression of DC-SIGN in B cell lines does result in robust HIV-1 trans infection [5] , [47] , whether DC-SIGN is the HIV trans infection factor in DCs has been a matter of considerable debate [11] , [12] , [13] , [14] , [45] . Immature DCs express high levels of DC-SIGN ( Figure 1C ) , but most of the particles captured by immature DCs have previously been reported to be endocytosed rapidly and degraded [50] , [51] , and targeted towards antigen presentation pathways in a DC-SIGN dependent manner [52] , [53] . The results in this study also failed to confirm a significant role for DC-SIGN in immature DC-mediated HIV-1 trans infection ( Figure 2G ) , in agreement with previously published studies that have observed at best a two-fold attenuation in immature DC mediated virus capture and trans infection upon virus exposure in the presence of anti-DC-SIGN neutralizing antibodies [12] , [13] , [54] , [55] , [56] . In contrast , knock-down of DC-SIGN expression nor blocking DC-SIGN function with neutralizing antibodies in mature DCs had no impact on virus capture ( Figure 2 ) . Furthermore , maturation of DCs with LPS resulted in downregulation of DC-SIGN ( Figure 1 ) , but an upregulation of CD169 and a significant enhancement in maturation-dependent HIV-1 capture and trans infection . It is possible that localization of DC-SIGN itself at the immature DC – T cell immunological synapse [54] for generation of stable synaptic junctions is necessary for optimal immature DC , but not mature DC-mediated HIV-1 trans infection . While CD169 is essential for HIV-1 capture by mature DCs , whether CD169 is also necessary for the formation of stable mature DC – T cell synaptic junctions remains to be determined . In contrast to trans infection , DC-SIGN has also been thought to enhance productive infection of DCs [51] , [57] , [58] , [59] . Indeed , DC-SIGN dependent recognition of HIV gp120 might concentrate HIV-1 particles on the DC surface such that the probability of virus interaction with CD4 and CCR5 and productive infection in cis is enhanced [57] , [60] , though the potential benefit to HIV-1 replication is questionable . In addition to the observation that HIV-1 fusion to DCs dramatically declines upon maturation [61] , intracellular environment of DCs is antagonistic to productive virus replication , presumably because of presence of SAMHD1 and other yet-to-be defined IFN-induced myeloid cell-specific anti-viral factors [62] , [63] , [64] , [65] . In contrast , HIV-1 interactions with CD169 target captured virus particles to the trans infection pathway that is amenable to robust virus replication . Thus , incorporation of sialylated GSLs in the virus particle membrane , we hypothesize , is a unique example of molecular mimicry by HIV-1 of “self” recognition pathways that aid virus dissemination and might be the HIV-1 evasion strategy from DC intrinsic virus restriction factors . Unlike other members of the Siglec family , which mostly interact with sialylated ligands in cis , because of the extensive cell surface sialylation that masks interactions in trans [24] , CD169 is uniquely positioned to participate in both host-pathogen interactions and cell-to-cell interactions in trans , because of the 17 Ig-like domains that extends the receptor away from the cell surface [24] . In addition to mediating binding and endocytic entry of Trypanosome cruzi [66] , Campylobacter jejuni [67] and porcine respiratory and reproductive syndrome virus , PRSSV [32] , [68] , results presented in this report suggest that capture of the simple retrovirus , MLV , is also dependent on CD169 and is facilitated via surface exposed terminal α2-3 sialoglycoconjugates . Interestingly , the capture of HIV-1 by CD169 results in virus localization in non-lysosomal compartments ( Figure 3 and Supplementary Figure S4 ) , nature of which still remain under debate [2] , [6] , [7] , [8] , [31] , [69] , [70] . While no endocytic or signaling motifs have been defined in the cytoplasmic tail of CD169 [25] , distinct pathogen localization upon CD169 ligation might reflect the requirement for co-factor ( s ) driving trafficking of diverse pathogens to unique intracellular compartments . Although CD169 is extremely well conserved in various mammalian species ( from mouse to human ) , the physiologically-relevant sialylated ligand involved in CD169-dependent cell-to-cell adhesion and the biological role of CD169 in mammalian cells are not well defined . The extensive similarity between the murine and human CD169 proteins , especially in the extracellular domain , and our findings on MLV and HIV-1 interactions with human and murine CD169 ( Figure 8 ) , suggest that GSL-dependent capture of retrovirus particles is conserved across species . Furthermore , similar to the mechanism of HIV-1 trans infection , captured MLV particles were transferred to virus-susceptible cells in a CD169-dependent manner . While mouse myeloid DCs have previously been shown to capture and transfer HIV-1 particles to human CD4+ T cells [1] , the virus attachment factor on murine myeloid DCs that mediates retroviral infection in trans has not been defined . Whether murine CD169 fulfills this function on murine myeloid DCs and whether CD169+ DCs can mediate dissemination of MLV in vivo remains to be determined . Interestingly , expression of murine CD169 has been reported predominantly on subsets of tissue resident and inflammatory macrophages lining the subcapsular sinus in peripheral lymph nodes and the spleen [24] , with isolated descriptions of CD169 expression on migratory DCs in B cell follicles [71] . Thus , examination of the role of CD169 in MLV infection in vivo might provide valuable insights into the mechanisms of retroviral parasitization of antigen acquisition , antigen presentation and T cell stimulation functions of macrophages and DCs . Human CD169 has a much broader expression pattern as it has been detected systematically on myeloid-derived cells . In addition to human DCs ( described in this report , and [29] , [49] , [72] ) , monocyte-derived macrophages ( MDMs ) [73] and IFNα-exposed monocytes were also reported to express CD169 [26] . In contrast to our findings , CD169 on MDMs and IFNα-exposed monocytes was reported to bind soluble sialylated HIV-1 gp120 [73] . While theoretically possible , the limited number of Env trimers on virus particle surface [74] might preclude high efficiency interaction with CD169 , considering that the binding affinity of Siglecs , including CD169 , for their ligands is estimated to be in the micromolar ( Kd ) range . Interestingly , avidity of Siglec binding to ligands can be greatly enhanced upon challenge with geometrically arrayed , multivalent ligands [75] , such as those when presented on virus capsid surface . Thus , in contrast to virus particle-associated Env , the high valency [18] , [21] and , presumably , unique spacing of GM3 molecules on the surface of HIV-1 particles , might provide a multivalent scaffold for high efficiency binding to CD169+ DCs . We propose that there is a hierarchical order to ligand choice by DC-associated CD169 with virus capsid-associated GM3 providing the primary interaction interface , and that cell-intrinsic variability in surface localization and expression level of CD169 between DCs and macrophages might account for putative cell-type differences in HIV-1 particle-associated ligand specificity for CD169 . While CD169 expression on macrophages can enhance productive infection by HIV-1 [73] , whether CD169+ macrophages can also capture and trans infect HIV-1 in a GM3-dependent manner remains to be determined . A cardinal feature of HIV-1 infections in vivo is high level of chronic immune activation , even observed in individuals on highly active antiretroviral therapy . Many factors have been attributed to cause HIV-associated immune activation including virus infection as well as translocation of microbial products from the intestinal lumen to the systemic circulation , and the release of high levels of pro-inflammatory cytokines , such as IL-6 , TNFα and type I IFN [36] . Recent studies with the SIV – macaque model systems have demonstrated that intravaginal application of SIVmac results in recruitment of plasmacytoid dendritic cells ( pDCs ) to the vaginal mucosa that upon detection of virus produce chemokines and type I IFN [35] . Furthermore , rapid and transient elevations in systemic IFNα have also been observed in acute HIV-1 infection [76] . While HIV-1 replication can be potently inhibited by type I IFNs at different steps of the viral life cycle in vitro [77] , paradoxically , high tissue and plasma levels of IFNα as a consequence of overactive innate immune responses have been shown to correlate with rapid disease progression [36] . Intriguingly , exposure of HIV-1 infected cells to immune activation stimuli can result in increased incorporation of GM3 within HIV-1 particles derived from these cells [21] . Furthermore , recruitment of monocytes and myeloid DCs to peripheral tissues and subsequent IFNα-dependent differentiation and induction of CD169 on DCs might result in enhanced GM3-dependent virus interaction with CD169+ DCs , and access to DC-mediated trans infection pathway . Thus , utilization of the GM3 – CD169 axis of recognition is a subversion mechanism by which HIV utilizes DC functions to promote its replication .
This research has been determined to be exempt by the Institutional Review Board of the Boston University Medical Center since it does not meet the definition of human subjects research , since all human samples were collected in an anonymous fashion and no identifiable private information was collected . Human CD169 was amplified from total RNA derived from IFN-α stimulated THP1 cells by overlap RT-PCR using the following primer sets: CD169 sense ( TGTTAACATATGGCACAAGAACCTGCTATGG ) , CD169 internal-antisense ( GCCTCAGTCAGGATGCGGCAGGCGTAAAGGGCAGCATCAGTT ) , CD169 internal-sense ( AACTGATGCTGCCCTTTACGCCTGCCGCATCCTGACTGAGGC ) and CD169 antisense ( AGATATCTAGACAACACCACTGGTCAGCC ) using Superscript III RT-PCR first strand synthesis kit ( Invitrogen ) and Phusion High Fidelity PCR ( NEB ) . Mouse CD169 was amplified from total RNA derived from RAW264 . 7 mouse macrophage cell line by overlap RT-PCR using the following primer sets: muCD169 sense ( ATAAGAATGCGGCCGCATGCACCTGGGCAC ) , muCD169 internal-antisense ( AGCTCCGTTCAGGTACCAGGAGAAG ) , muCD169 internal-sense ( AGCTCCTGACACTCGCTTCTCC ) and muCD169 antisense ( CGTCTAGACAGAGAGCAGCAACCACTTCC ) . Overlapping CD169 PCR fragments were first cloned into the pSC-B blunt cloning vector ( Agilent Technologies ) and then subsequently into the lentivector , pHIV-dTomato ( Addgene ) using EcoRI and XbaI restriction enzymes for human CD169 and NotI and XbaI restriction enzymes for mouse CD169 ( pCD169-IRES-dTomato ) . Human CD169 was also cloned into a retroviral expression vector , LNCX ( LNC-CD169 ) . Point mutations in human CD169 orf , R116A ( Arg→Ala ) and R96A ( Arg→Ala ) were introduced by site-directed mutagenesis ( Quikchange; Agilent Technologies ) . All clones were verified by sequencing . Stable expression of CD169 in Raji B cell line was accomplished by transduction either with VSV-G pseudotyped pCD169-IRES-dTomato lentivector and cell sorting for dTomato expression , or LNC-CD169 retroviral vector followed by G418 selection . CD169 was transiently expressed in HEK293T cells by calcium phosphate-mediated transfection , and protein expression confirmed by western blot analysis and/or FACS . Human cell lines , HEK293T , Raji and TZM-Bl , have been described before [13] , [14] , [21] , [47] . Rat-2 , a rat fibroblast cell line , was obtained from ATCC . PBMC , isolated from leukupaks of healthy donors , were activated with 2% phytohemagglutinin ( PHA ) ( Invitrogen ) and 50 U/ml rhIL-2 ( Roche , NIAID ARRRP ) in RPMI/10%FBS . Primary monocyte derived DCs were differentiated from CD14+ peripheral blood monocytes , as described previously [14] . Immature DCs were matured by incubation with either LPS ( 100 ng/ml ) , poly ( I:C ) ( 25 µg/ml ) , or Pam3CysK4 ( 100 ng/ml ) for 2 days . Alternatively , DCs were matured by addition of cytokines , IFNα ( 1000 U/ml ) or TNFα ( 10 ng/ml ) for 48 h . To generate IFN-DCs , CD14+ monocytes were cultured in the presence of GM-CSF ( 1 , 400 U/ml ) and IFNα ( 1000 U/ml ) for 3 days [42] . Autologous activated CD4+ T cells were derived as described previously [21] . Blood myeloid DCs were positively isolated from PBMCs using BDCA-1 conjugated magnetic beads ( Miltenyi ) and cultured in RPMI/10%FBS containing 20 U/ml GM-CSF or stimulated with 1000 U/ml IFNα for 48 h prior to use . The following antibodies ( all from Becton Dickinson ) were used for DC immunophenotyping analysis: PE-conjugated CD14 , CD11c , HLA-DR , CD83 , CD4 , and CCR5; and FITC-conjugated α-CD86 and α-DC-SIGN; Alexa488- or Alex647-conjugated α-CD169 ( AbD Serotec ) , and isotype controls . FACS analysis was performed as described before [21] . CXCR4-tropic HIV/Lai , CCR5-tropic HIV/Lai-Bal and luciferase expressing HIV/Lai-Bal-luc provirus clones have been described previously [78] . To generate HIV/Lai-iGFP and HIV/Lai-iGFPΔenv molecular clones the eGFP fragment flanked by HIV-1 protease cleavage sites from HIV/NL43-iGFP ( gift of Dr . Benjamin Chen , Mt Sinai School of Medicine ) was exchanged into HIV/Lai . HIV Gag-eGFP and HIV Gag-mCherry expression plasmids that express codon-optimized Gag-eGFP and Gag-mCherry fusion proteins have been described previously [7] . MLV Gag-YFP expression plasmid has been described previously [79] . HIV Gag-eGFP and MLV Gag-YFP VLPs and infectious viruses were either derived via calcium phosphate mediated transfection of HEK293T cells , as described previously [21] . Luciferase expressing , single cycle of replication competent MLV particles ( MLV-E/luc ) were derived via co-transfection of HEK293T cells with LNC-luc ( luciferase expressing retroviral expression vector ) and pCL-Eco ( MLV packaging vector that expresses MLV Gag , Pol and ecotropic Env glycoproteins; Imgenex ) plasmids . To derive GSL depleted virus particles , HEK293T cells were pre-treated with 10 µM PDMP ( Calbiochem ) for 24 h prior to transfection , and cells were maintained in the presence of PDMP ( 10 µM ) during the course of transfection [14] . To produce PBMC-derived viruses , cells were infected with VSV-G double Env pseudotyped HIV/Lai-Bal virus particles in the presence or absence of PDMP ( 10 µM; cells pre-treated for 2 days ) , and the drug concentration was maintained for the duration of the infection . Cell-free supernatants were harvested 3 and 6 days post infection and stored at −80°C until further use . To determine the infectious titer of the PBMC and HEK293T cell derived viruses , 104 TZM-Bl cells were infected with limiting dilutions of virus stocks . Cells were lysed 48 h post infection , and cell lysates were analyzed for luciferase activity using a commercially available kit ( Promega ) . Production and quantification of HIV Gag-eGFP or MLV Gag-YFP particles from transiently transfected HEK293T cells in the presence or absence of PDMP was confirmed by quantitative LICOR-western blot analysis using goat α-GFP polyclonal antibody . To quantify virus particle incorporation of GSLs , virus particles ( 100 ng Gag-eGFP or MLV Gag-YFP ) derived from untreated or PDMP-treated HEK293T cells were incubated with 2 µg cholera toxin subunit B ( CtxB ) -biotin ( Sigma ) , coupled to 25 µl streptavidin coated 2 . 8 µM dynabeads ( Invitrogen ) in 1% casein/PBS ( Pierce ) at RT for 1 h with rotation , washed twice with 0 . 1% casein/PBS and beads-associated virions lysed with SDS-sample buffer . The amount of immunoprecipitated Gag-eGFP or MLV Gag-YFP was determined using quantitative western blot analysis with recombinant GFP ( Roche ) as standards . Membranes were probed with goat α-GFP ( Novus Biologicals ) followed by donkey α-goat-IgG-IRDye 800CW ( Li-Cor , Lincoln , Nebraska ) . To measure GM3 incorporation in HIV/Lai-Bal virus particles , virions were stained with α-GM3 mAb [80] and Alexa594-conjugated goat α-mouse IgG ( Invitrogen ) . Quantification of GM3 staining in virus particles derived from untreated or PDMP ( 10 µM ) -treated virus producer cells was determined , as described previously [21] . The p24gag content and gp120 incorporation in HIV-1 particles derived from HEK293T or PBMC was determined using mouse α-gag mAb ( Clone p24-2 , NIH AIDS Research and Reference Reagent Program , contributed by Dr . Michael Malim ) and rabbit α-gp120 polyclonal antibody ( gift of Dr . Nancy Haigwood ) and staining visualized by goat α-mouse-IRDye 680CW or goat α-rabbit-IRDye 800CW ( Licor ) , respectively . Membranes were imaged with an Odyssey scanner ( Licor ) and the amount of gp120 or p24gag or Gag-eGFP in the samples was quantified using recombinant HIV-1 Bal gp120 ( ARRRP ) or HIV-1 IIIB p24 ( ABL , Rockville , MD ) as standards , respectively . Lentivirus particles expressing CD169 , DC-SIGN or scrambled shRNAs were produced via transient transfections of HEK293T cells with lentiviral vectors ( Pierce , Rockford IL ) and packaging plasmids psPAX2 and pMD2G ( Addgene ) . Immature DCs , 2 days post initiation of differentiation from CD14+ monocytes , were infected with lentiviral vectors by spin-inoculation in the presence of SIV Vpx containing VLPs , as described previously [21] . Cells were washed with PBS 24 h post transduction and cultured for 48 h . DCs were stimulated with 100 ng/ml LPS and vector-transduced cells were selected with 2 µg/ml puromycin for 48 h prior to use . To assess expression of CD169 and DC-SIGN on shRN-transduced cells , membranes were probed with a mouse α-CD169 antibody ( 7D2 , Novus Biologicals ) and a mouse α-DC-SIGN antibody ( DC28 , NIH AIDS Research and Reference Reagent Program Catalog #5443; contributed by Dr . R . W . Doms ) , respectively . As loading controls , either actin or HSP70 was detected using rabbit α-actin ( SIGMA , St . Louis , MO ) or mouse α-HSP70 ( Stressgen , Victoria , BC ) antibodies , respectively . Infectious virus and HIV Gag-eGFP and MLV Gag-YFP VLP capture assays and HIV and MLV trans infection assays were performed as described previously [21] . Briefly , cells ( 1×105 ) were incubated with virus ( 20 ng p24gag unless noted otherwise ) or VLPs ( 2 ng p24gag ) for 2 hr at 37°C in raw RPMI media . Cells were washed 3× with 1×PBS and analyzed for capture using either p24gag ELISA or FACS analysis . Virus capture was quantified by ng/ml of p24gag associated with cell lysates using a p24gag ELISA that has been described previously [14] . Capture of fluorescent VLPs was detected by FACSCalibur and quantified for percent positive cells . For transfer assays , cells ( 1×105 ) were incubated with virus particles at 37°C for 2 hr , washed 3× with 1×PBS and co-cultured with CD4+ T cells ( for HIV-1 transfer ) or Rat-2 cells ( for MLV transfer ) at a 1∶1 cell ratio in complete RPMI media . To competitively inhibit virus binding , DCs ( 1 . 0×105 cells ) were pre-incubated with 20 µg/ml final concentration of α-CD169 ( 7D2 , Novus Biologicals , Littleton CO ) , α-DC-SIGN ( 14EG7 , NIH AIDS Research and Reference Reagent Program Catalog #11423 ) , murine IgG2b or IgG1 isotype controls ( eBioscience ) for 30 minutes at room temperature prior to virus exposure . To determine the effect of cell surface-associated sialic acid residues on VLP capture , mature DCs were treated with neuraminidase that efficiently cleaves α2-3 , α2-6 , and α2-8 sialic acids ( NEB ) for 1 h at 37°C prior to challenge with HIV Gag-eGFP VLPs , as described previously [21] . Fluorescent unilamellar liposomes containing GSLs were produced as described previously [21] . For liposome capture and virus inhibition assays , sodium azide-treated cells ( 105 per well ) were incubated with liposomes for 45 min at 37°C , and cells either fixed and processed for FACS analysis , or challenged with HIV/Lai-iGFPΔenv virus particles ( 20 ng p24gag ) for 1 h at 37°C . Virus capture was determined by measuring cell-associated p24gag by ELISA . To determine the number of CD169 and DC-SIGN molecules on DCs , antibody-binding sites ( ABS ) were quantified using a microbeads kit ( Bangs Laboratories ) . Beads ( 105 ) and cells were stained with saturating amounts of FITC-conjugated α-DC-SIGN or IgG2b isotype control ( both from BD ) , or Alexa647-conjugated α-CD169 or IgG1 isotype control ( both from AbD Serotec ) for 30 minutes on ice . Stained populations were detected using a LSRII ( BD ) , and the data analyzed using FlowJo software ( Treestar ) . For analysis , auto-fluorescence signals from unstained samples were subtracted from GMFIs of stained samples , and ABS of CD169 , DC-SIGN or isotype controls on DCs were calculated from standard curves generated from stained beads . To visualize virus localization , mature DCs ( 1 . 5×105 ) were incubated with 10 ng Gag-mCherry VLPs or 500 ng of Lai-iGFP virus particles at 37°C for 10 minutes or 2 h . For conjugate analyses , DCs were incubated with virus particles for 1 h , washed and co-cultured for an additional 1 h with autologous CD4+ T cells ( 1∶1 DC∶T ratio ) pre-labeled with 5 µM CMAC ( CellTracker Blue , Invitrogen ) . Cells were fixed with 1% PFA , blocked with 20% NHS , and probed with Alexa-488 conjugated α-CD169 or stained with α-CD45 , α-CD9 , α-CD81 , α-EEA-1 , α-Lamp1 mAbs ( all antibodies from BD ) or isotype controls and detected with Alexa488-conjugated 2°Abs ( Invitrogen ) . Confocal Z-stacks were captured with an Olympus DSU spinning disk at 60× and processed with ImageJ software . For visualization , images were deconvolved , background subtracted , and flattened for maximum intensity . All virus capture and transfer values and FACS MFIs are expressed as means ± SD . Statistical significance was determined by Student's one-tailed t test , and significant P values ( <0 . 05 ) are indicated on the figures . To quantify co-localization analysis , single z-stack slices from 10 independent cells were assessed using Mander's coefficient and the average ratio of red ( Gag-mCherry ) associated with green ( CD169/isotype control/CD45 ) +/− SD is reported . | Dendritic cells ( DCs ) are one of the initial cellular targets of HIV-1 and can play a crucial role in determining the course of virus infection in vivo . While sentinel functions of DCs are essential for establishment of an antiviral state , HIV-1 can subvert DC function for its dissemination . One of the mechanisms by which DCs can mediate virus spread is via the trans infection pathway whereby DCs capture HIV-1 particles and retain them in an infectious state without getting infected , and pass these infectious particles to CD4+ T cells upon initiation of cellular contacts . In this report , we demonstrate that expression of Siglec-1or CD169 , on DC surface is responsible for capture of HIV-1 particles by binding the ganglioside , GM3 , present in the virion lipid bilayer . This interaction between CD169 and GM3 targets captured virus particles to non-degradative compartments and resulted in retention of virus particle infectivity within DCs . Upon initiation of T cell contacts with virus-laden DCs , HIV-1 particles were trafficked to the DC – T synaptic junctions and transferred to T cells for establishment of productive infection . These studies define a novel host-encoded receptor – ligand interaction that drives HIV-1 dissemination and can be used for development of novel anti-viral therapeutics . | [
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] | 2013 | Interferon-Inducible Mechanism of Dendritic Cell-Mediated HIV-1 Dissemination Is Dependent on Siglec-1/CD169 |
There is an urgent need for the development of new antifungal agents . A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery . We show that Candida albicans , as well as other Candida species , are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C . elegans intestinal track . Importantly , key components of Candida pathogenesis in mammals , such as filament formation , are also involved in nematode killing . We devised a Candida-mediated C . elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities , while synchronously screening against toxic compounds . The assay is performed in liquid media using standard 96-well plate technology and allows the study of C . albicans in non-planktonic form . A screen of 1 , 266 compounds with known pharmaceutical activities identified 15 ( ∼1 . 2% ) that prolonged survival of C . albicans-infected nematodes and inhibited in vivo filamentation of C . albicans . Two compounds identified in the screen , caffeic acid phenethyl ester , a major active component of honeybee propolis , and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis . The whole-animal C . elegans assay may help to study the molecular basis of C . albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth . Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as “probe compounds” and may have antifungal activity against other fungi .
The discovery of substantial commonality between microbial pathogenesis in mammals and nonvertebrate model hosts , such as the nematode Caenorhabditis elegans , has provided the foundation for the development of high-throughput genetic analysis of microbial virulence factors in live animal models ( reviewed in [1 , 2] ) . We sought to extend the use of the C . elegans infection models to identify chemical compounds with antifungal activity against Candida species , the most common human pathogenic fungus . Candida spp . are the fourth most common cause of nosocomial blood-stream infections , and disseminated candidiasis continues to have an attributable mortality rate of over 25% [3] . The mortality rate for device-associated Candida infection can be even higher [4] . In the United States , the overall excess cost attributable to candidemia is estimated at $1 billion per year and the average cost of candidemia for a single episode is about $40 , 000 ( 1997 United States dollar ) [5 , 6] . However , efforts to identify new antifungal compounds have been hindered by the fact that most compounds that have antifungal activity in vitro are also toxic to mammalian cells . A facile bioassay compatible with high-throughput screening technologies , which simultaneously evaluated libraries of chemical compounds for antifungal activity and host toxicity in the context of a whole-animal Candida infection model , could solve some of the main obstacles in current antifungal discovery . Here , we show that Candida albicans , as well as other Candida strains , can kill C . elegans . We used these findings to develop a C . elegans-based infection model that is performed in standard 96-well plates in liquid media , thereby enabling automated addition of compounds . An important feature of the assay is that the first assay endpoint is nematode survival , which automatically eliminates highly toxic compounds that affect nematode viability . Moreover , important components of Candida pathogenesis in mammals are involved in nematode killing , and filamentation becomes apparent as the nematode dies , providing a second clinically relevant endpoint . We demonstrate how this model enables the screening of a chemical library for compounds that prolong nematode survival or inhibit Candida filamentation . Importantly , the screen identifies compounds that prolong survival in a hematogenous murine model of candidiasis .
C . elegans nematodes eat microorganisms , but they die when fed a variety of human bacterial and fungal pathogens [1 , 2] . In the laboratory , C . elegans-killing assays are typically carried out by transferring nematodes from lawns of Escherichia coli strain OP50 ( their normal laboratory food ) to lawns of pathogenic bacteria or yeast grown on solid agar media . However , screening chemical libraries using an agar-based C . elegans-killing assay is not readily compatible with the use of robots for filling assay plates and pin transfer of compounds , homogeneous distribution of chemicals in the medium , or the use of automated plate readers or automated screening microscopes to monitor nematode survival . We therefore sought to develop a C . elegans–Candida spp . -killing assay that could be performed in liquid media . We found that when wild-type L4 stage N2 nematodes were fed Candida spp . on solid brain–heart infusion ( BHI ) medium for 2 h and then were transferred to a liquid medium consisting of 20% BHI and 80% M9 minimal medium , the longevity of the worms was significantly reduced compared to worms not infected with Candida ( Figure 1A ) . We tested different times of exposure to Candida spp . on solid BHI medium ( from 5 min to 24 h ) and in all cases the longevity of the worms was significantly reduced compared to uninfected worms ( unpublished data ) . Although Candida spp . killed wild-type C . elegans in liquid medium , the killing did not occur rapidly enough to prevent the production of progeny produced by 3- and 4-d-old hermaphroditic nematodes . The presence of a brood in the assay mix made it difficult to score the viability of the infected parents . Indeed , in the assay described above and shown in Figure 1A , it was necessary to painstakingly remove progeny nematodes that survived beyond the L1 or early L2 developmental stage . Production of progeny during the assay had another undesirable consequence in the scoring of the assay; namely , Candida-infected wild-type nematodes exhibited significant matricidal death ( over 30% ) involving the premature hatching of eggs in the C . elegans uterus ( which is not necessarily directly associated with an infectious-like process ) around day 3 of the experiment . To avoid these problems associated with progeny production , we substituted C . elegans glp-4 mutants for wild-type in the Candida spp . killing assay . C . elegans glp-4 mutant animals have normal morphology and brood sizes at 15 °C , but do not make gonads and are unable to produce eggs at 25 °C [7–10] . As shown in Figure 1A , Candida spp . also killed glp-4 nematodes , but the rate of killing was significantly slower than for wild-type worms ( Figure 1A ) , similar to results reported previously for the killing of glp-4 and other sterile mutants by bacterial pathogens . Although glp-4 mutant worms could have potentially been used in an antifungal screening assay , we sought to increase the rate of killing of the glp-4 mutant by utilizing glp-4;sek-1 double mutant worms . SEK-1 encodes a mitogen-activated protein kinase kinase that functions directly upstream of the C . elegans homolog of the mammalian p38 mitogen-activated protein kinase that was shown previously to be an important component of the C . elegans defense response to pathogens [7 , 11] . As shown in Figure 1A , the C . elegans glp-4;sek-1 double mutant was significantly more susceptible to Candida than the glp-4 mutant ( p < 0 . 001 ) , suggesting that the C . elegans homolog of the mammalian p38 mitogen-activated protein kinase ( which is involved in mammalian response to candidiasis [12] ) plays a significant role in the C . elegans response to Candida . This finding is consistent with the observation that sek-1 mutants are more susceptible to a variety of pathogens [7 , 11] . When glp-4 and glp-4l;sek-1 mutants were moved from OP50 plates to pathogen-free liquid media , their survival was not significantly different from N2 worms in the presence of E . coli in liquid media ( unpublished data ) . Notably , when we exposed glp-4;sek-1 worms to DAY185 for 2 h and then moved to lawns of an E . coli OP50 strain that expresses green fluorescent protein ( GFP ) , over 70% of the worms had no GFP-expressing bacteria in their intestine , suggesting that the grinder is still operational , at least in the majority of the nematodes . Because the glp-4;sek-1 mutant was similarly susceptible to a variety of C . albicans strains as well as to the non-albicans strains Candida krusei and Candida parapsilosis ( Figure 1B ) , because it did not produce progeny , and because we found it compelling to study Candida pathogenesis in immunocompromised nematodes ( in some ways analogous to candidiasis in immunocompromised humans ) , we utilized the glp-4;sek-1 strain in all of the further studies described below . Candida yeast cells have the ability to develop filaments that are usually differentiated to hyphae ( long continuous germ tubes separated by true septin rings ) or pseudohyphae ( chains of distinct cells that fail to separate ) . Development of human candidiasis involves adhesion to the substratum ( which allows the yeast to colonize surfaces , such as the gastrointestinal tract and intravascular catheters ) , development of biofilm , and formation of hyphae and pseudohyphae [13–15] . Filamentation is instrumental in Candida virulence in mammals; nonfilamentous mutants of C . albicans are highly attenuated [16] . Candida hyphae invade host tissues and exert mechanical force through cells and tissues , enabling escape of C . albicans from the host bloodstream [17 , 18] . Notably , there is significant coregulation between adherence , biofilm , and filament pathways [15 , 19 , 20] . For example , genes involved in biofilm formation are required for hyphal development , while filaments are a prominent feature of C . albicans biofilms that support biofilm integrity [4 , 21–23] . Persistence in the intestine appears to be a critical first step during Candida infection in C . elegans . Indeed , nematodes exposed to Candida lawns are unable to clear the Candida infection when they are transferred to Candida-free liquid media ( Figures 2 and S1 ) . Even when nematodes were exposed to Candida lawns for as little as 5 min and then transferred to pathogen-free liquid media or lawns of E . coli OP50 , 100% of the nematodes remain colonized ( unpublished data ) . As a result , Candida cells fill the nematode pharynx and intestine ( Figure S1B and unpublished data ) . Also , we placed the worms on lawns of C . albicans strain MLR62 , a strain that constitutively expresses GFP , and then we moved them onto the non-GFP parent strain DAY185 . Over a 24-h period , the GFP-expressing Candida cells were displaced by non-GFP-expressing cells , suggesting that Candida cells are not too big to be defecated . To evaluate the hypothesis that biofilm formation and filamentation are involved in C . elegans killing , we infected C . elegans with C . albicans kem1 and suv3 mutants that are defective in hyphal production [24] . As shown in Figure 3 , C . elegans killing by kem1 ( strain MLR74 ) and suv3 ( strain GKO443 ) mutants was significantly attenuated compared to the isogenic parental strain DAY185 ( p = 0 . 0008 and p < 0 . 0001 , respectively for the two mutants , compared to the wild-type strain DAY185 ) . There was no significant difference in killing between the wild-type and strains in which the kem1 and suv3 mutations had been reconstituted with wild-type alleles by homologous recombination ( unpublished data ) . Notably , it appears that the interface between solid and liquid is important for filamentation , as it is not observed during Candida–C . elegans interaction on solid media ( unpublished data and [25] ) . Because the C . albicans biofilm defective mutants that were used in our studies were also defective in filamentation , the study of the role of Candida biofilm in C . elegans pathogenesis requires further experimentation . Interestingly , in the absence of C . elegans , none of the Candida species tested in the liquid assay formed filaments . Nevertheless , after nematodes died , extensive filamentation was observed in the colonized worms with numerous filaments exiting through the nematode cuticle ( Figure 4A and 4B ) . Moreover , after the integrity of the cuticle was breached , the yeast cells did not dissipate within the liquid medium , but rather remained connected , resulting in C . elegans “ghosts” in which the transition from mostly yeast-like Candida cells to the filaments outlined where the cuticles used to be ( Figure 4C–4F ) . We used a C . albicans reporter strain that expresses yeast-enhanced GFP ( yEGFP3 ) from the promoter for the gene Hyphal Wall Protein 1 ( HWP1 ) to evaluate whether true hyphae are formed during the infectious process in C . elegans . This strain confers developmental expression of yEGFP3 in germ tubes and hyphae , but not in yeasts or pseudohyphae [18 , 26 , 27] . As shown in Figure 5 , yEGFP3 expression was observed in filaments as they penetrated the nematode cuticle , confirming that C . albicans forms hyphae in the course of C . elegans infection . No yEGFP3 expression was detected prior to filamentation . To develop a system that allows high-throughput screening for antifungal compounds , we first evaluated whether established antifungals prolong the survival of nematodes infected with Candida spp . For this , we used different C . albicans isolates as well as non-albicans strains , including those used as reference strains by clinical microbiology laboratories [28] . Notably , all strains tested were susceptible to amphotericin B and caspofungin , but had different susceptibilities to fluconazole [24 , 26 , 29 , 30] ( Table 1 ) . We found that antifungals that are active against a particular Candida strain resulted in prolongation of C . elegans survival when the nematodes were infected with the relevant Candida strain ( Figure 6A–6D ) . Caspofungin , a fungicidal agent that is active against Candida biofilm [31 , 32] , and also the fungicidal agent amphotericin B , were the most active agents tested in prolonging nematode survival to C . albicans strain MLR62 ( Figure 6A ) , C . krusei American Type Culture Collection ( ATCC ) #6258 ( Figure 6B ) , C . parapsilosis ATCC#20019 ( Figure 6C and 6D ) , and C . albicans ATCC#90028 ( unpublished data ) . In the case of C . krusei ATCC#6258 , the most resistant to fluconazole among the strains we tested , caspofungin and amphotericin B were more effective than fluconazole ( in both cases p < 0 . 0001; Figure 6B ) . Antifungal agents were also effective at lower concentrations; for example , caspofungin was effective at concentrations as low as 1 μg/ml in the case of C . albicans strain MLR62 ( p < 0 . 0001 ) . Since filament formation was only seen after the Candida cells overwhelmed and killed the nematode , there were significantly fewer nematodes with filaments in the wells that contained antifungal compounds ( unpublished data ) . Interestingly , at high concentrations , the beneficial effect of fluconazole was lost and the nematodes died faster than untreated infected worms . Fluconazole up to 32 μg/ml was effective in prolonging survival of nematodes exposed to the fluconazole-susceptible strain C . parapsilosis ATCC#20019 , but at higher concentrations ( 100 μg/ml ) nematode survival was diminished , even compared to the nematodes in the untreated control group ( p = 0 . 01; Figure 6D ) . Probably this toxicity is present at even lower concentrations , but the beneficial effect from the antifungal activity outweighs the toxic effect . For example , when nematodes were exposed to the fluconazole-resistant strain C . krusei ATCC#6258 , fluconazole at 32 μg/ml not only had no effect on nematode survival , but killing in the fluconazole group was significantly faster than untreated worms ( p = 0 . 02; Figure 6B ) . Taken together , these observations suggest that the C . elegans–Candida model can be used for the concurrent screen for antifungal activity and host toxicity . We also evaluated the effect of antifungal agents in reducing fungal burden in the nematode intestine . For example , in a representative experiment , after 48 h , untreated C . elegans infected with C . albicans MLR62 had an average of 170 . 8 ± 63 . 5 colony-forming units ( cfu ) per worm , whereas caspofungin-treated animals ( at 8 μg/ml ) had almost cleared the infection with an average of 0 . 3 ± 0 . 2 cfu per worm ( p < 0 . 001 ) . Fluconazole and amphotericin B also significantly decreased the concentration of Candida cfu in the nematodes , and microscopic examination of the worms confirmed that treatment with antifungals was effective in clearing the worm intestine of visible Candida cells ( unpublished data ) . To facilitate adaptation of the C . elegans–Candida-killing assay to screening chemical libraries for antifungal compounds , we used C . albicans strain MLR62 that expresses GFP ( linked to the constitutively active TEF1 promoter ) and that exhibits similar killing kinetics to the parent strain DAY185 in the C . elegans assay . Using this strain , nematodes exposed to compounds that have significant antifungal efficacy exhibited sinusoidal movement and no green fluorescence in the intestine at the endpoint of the assay ( Figure 7A ) , whereas nematodes exposed to compounds without antifungal efficacy did not move , were rod-shaped , exhibited high levels of intestinal fluorescence ( Figure 7B ) , and developed filaments ( Figure 7C ) . As an initial test of the C . elegans–Candida compound screening assay , we utilized libraries of compounds made available through the Institute of Chemistry and Cell Biology ( ICCB ) at Harvard Medical School that include known compounds that affect diverse cellular pathways as well as the United States Food and Drug Administration approved drugs that are known to be safe and bioactive in humans ( http://iccb . med . harvard . edu ) . We screened a total of 1 , 266 compounds and identified 15 ( ∼1 . 2% ) that prolonged nematode survival and completely or almost completely inhibited filamentation ( Table S1 ) . An additional 57 ( ∼4 . 5% ) compounds prolonged nematode survival but had no or minimal effect on filamentation ( Table S1 ) . To the best of our knowledge , the C . elegans screen identified all of the compounds among the 1 , 266 tested with established , clinically documented antifungal activity against Candida ( Table S1 ) . Among the known antifungals , ketoconazole and butoconazole were among the most effective compounds identified ( Table S1 ) . We selected three compounds identified in the screen , which are not established antifungals , for further analysis . By design , we selected compounds that have a positive safety profile in mammals and exhibit variable efficacy in the C . elegans assay , including compounds that both affected or did not affect filamentation . The three compounds selected for further evaluation were caffeic acid phenethyl ester ( CAPE ) , enoxacin , and lapachol ( Figure 8 and Table S1 ) . CAPE is a major active component of propolis , a natural antimicrobial resinous product derived from plants , which is collected and used by honeybees to seal cracks or large potentially infectious objects ( such as carcasses ) that honeybees cannot remove from their hives ( [33 , 34] and Table S1 ) . Enoxacin , a fluoroquinolone antibiotic , was shown previously to marginally enhance the in vitro antifungal activity of amphotericin B and mepartricin [35] . Lapachol is a naphthoquinone found in the seeds and heartwood of tropical plants that exhibits anti-leishmanicidal activity [36–38] . Enoxacin did not inhibit C . albicans filament formation in the C . elegans-killing assay . To determine whether CAPE , enoxacin , and lapachol play a role in mammalian virulence , we evaluated their efficacy in an established murine model of systemic Candida infection . After tail vein injection of 1 × 106 blastospores , both CAPE and enoxacin prolonged the survival of mice inoculated with C . albicans strain DAY185 ( Figure 9 ) . Among these three compounds , CAPE , which was the most effective agent in the C . elegans screen , was also the most effective agent in the murine assays ( p = 0 . 008 , compared to the control of mice that received placebo ) . Notably , intravenous enoxacin was effective in once-a-day dosing of 25 mg/kg ( p = 0 . 03; 12 mice per group ) , whereas CAPE was not statistically effective in once-a-day dosing ( unpublished data ) . Lapachol ( 150 mg/kg ) was not active in the mouse model ( unpublished data ) , consistent with previous work that suggested that lapachol may be transformed into inactive metabolite ( s ) or neutralized in mammals [36–38] . All three compounds were also evaluated in vitro for their activity in Candida biofilm formation . CAPE strongly inhibited both filamentation and biofilm formation ( Figures 10 and S2 and Text S1 ) . Interestingly , lapachol also exhibited a modest effect on biofilm formation , suggesting that metabolites of lapachol that are not transformed into inactive metabolite ( s ) or neutralized in mammals [36–38] may be useful antifungal agents . Of note is that the antifungal activity of the three compounds tested is most likely not due to their ability to inhibit the growth of yeast . Among the three compounds , only CAPE inhibited growth of C . albicans in vitro , and that was at a minimum inhibitory concentration ( MIC ) of 64 μg/ml , which was higher than the concentration ( 33 μg/ml ) used in the C . elegans screen . Moreover , CAPE was effective in the C . elegans–Candida assay to a concentration as low as 4 μg/ml ( unpublished data ) . Enoxacin and lapachol did not inhibit the growth of C . albicans strain DAY185 at the highest concentration tested ( 256 μg/ml ) . To evaluate for toxicity , CAPE , enoxacin , and lapachol were evaluated in vitro using sheep erythrocytes [7] and in vivo using glp-4;sek-1 worms in liquid media with E . coli OP50 . Neither CAPE nor lapachol hemolyzed the red blood cells , as compared to DMSO and Triton X-100 controls , though enoxacin exhibited a marginal amount of hemolysis; however , none of the three compounds had any acute toxicity to uninfected C . elegans glp-4;sek-1 worms ( unpublished data ) .
The objective of the studies detailed in this paper was to develop a semi-automated , high-throughput , whole-animal C . elegans assay that can be used to identify chemical compounds with antifungal activity . Key features of the C . elegans assay are that it allows concurrent evaluation of toxicity and antifungal activity and that it identifies compounds that target important pathways associated with human Candida infection , including filament formation . Moreover , the assay allows the study of Candida cells that are in non-planktonic form and identification of antifungal compounds in a system where both the pathogen and the host can be genetically manipulated . The C . elegans whole-animal screen has several advantages compared to in vitro screens that use planktonic cells . Some virulence traits are induced only in the host , and therefore the identification of compounds that are effective against these virulence traits may require detection in vivo . Also , the whole-animal approach provides two relatively unambiguous assay endpoints in the survival/death of the worms and filamentation , and allows the use of liquid-handling robots for filling assay plates and for pin transfer of compounds from library stock plates to assay plates . The current rate-limiting step in the assay is the manual scoring of worm viability and filament formation . Thus far , scoring of live/dead and filamented/nonfilamented worms has been done by direct human observation of movement and morphology of the nematodes on a microscope or by reviewing microscopic images . A quantifiable automated scoring assay that is compatible with the use of high-throughput automated screening microscopes is currently under development and would greatly increase the throughput of the assay . Also , the delivery of a standard number of worms in each well would further enhance the uniformity of the assay . This is possible using a commercially available worm-dispensing robot . Moreover , in the future , we believe that it will be possible to robotically dispense both Candida and C . elegans to the wells , thereby eliminating the labor-intensive infection and wash steps . The C . elegans assay has many advantages compared to screens using mammalian models . The study of pathogenesis in mammals is complicated by difficulty of handling , long reproductive cycles , small brood sizes , complexity of mammalian hosts , high cost , and ethical considerations . In vivo models for Candida biofilm are particularly cumbersome and expensive , as they usually entail the placement of vascular catheters in rabbits [39] or rats [40 , 41] . Overall , the whole-animal C . elegans screen can help identify compounds that most likely would not be identified by in vitro screens or would be too costly and inefficient using an in vivo mammal approach . In addition to the Candida assay , members of our group have screened libraries of chemical compounds to identify those that have antimicrobial activity against C . elegans that are persistently infected with the human opportunistic pathogen Enterococcus faecalis [7] . However , it should be noted that the use of C . elegans for the identification of antimicrobial agents also has obvious limitations . First , it is unlikely that the use of C . elegans ( or other invertebrate model hosts ) will completely eliminate toxic compounds , even though there is a reasonable correlation between the toxicities of a variety of compounds in mammals and C . elegans [7 , 42–44] . In addition , the small size that makes C . elegans less expensive and amenable to high-throughput screens makes it very difficult to use this model to study pharmacokinetic variables such as impact of protein binding , absorption , and distribution that are automatically assayed in other , larger animal models . In addition to providing a facile system for screening chemical libraries for antifungal compounds , the experiments detailed above support the hypothesis that natural selection in nonmammalian hosts may have aided the evolution and maintenance of virulence-related genes in pathogenic human fungi [8 , 45] . In previous work from our laboratories , we showed that Cryptococcus neoformans , another important human opportunistic fungal pathogen , is also a potent C . elegans pathogen , supporting previous conclusions that the pathogenic potential of C . neoformans may have evolved as a by-product of avoiding predation by soil organisms , including nematodes and amoebae that feed on yeasts [46] . Thus , even though C . albicans is not necessarily found in the same environment as C . elegans , other fungal pathogens are found in the soil , and the interactions between C . elegans and fungi in general can be seen as a representation of the evolutionary arms race between fungi and soil-borne predators such as C . elegans [47] . Interestingly , however , C . neoformans and Candida use different approaches to kill C . elegans . Cryptococcal cells , which are larger than Candida cells , do not persist in the nematode intestine , and nematodes exposed to cryptococcal lawns are able to defecate the cryptococcal cells upon transfer to liquid media and thus can clear the cryptococcal infection ( [8] and unpublished data ) . In contrast , Candida , a common colonizer of the intestinal track of humans , establishes a persistent infection in the C . elegans intestine , forms biofilm , dissolves nematode tissues , and breaks through the nematode cuticle by forming an impressive network of filaments ( Figures 2 , 4 , and 5 ) . It is expected that the C . elegans–Candida-killing assay will not only identify compounds that affect the growth of the pathogen in vitro , but also compounds that affect the virulence of the pathogen in vivo , or , even immuno-modulate the host . For example , CAPE is known to inhibit NfkappaB [48] in mammals , and murine mouse studies suggest that it has immunomodulatory effects in vivo [49 , 50] . Since C . elegans appears not to have an NF-kB-like protein , if CAPE also affects C . elegans immunity , the mechanism presumably does not involve NF-kB inhibition . Since the compounds identified through this screen have no obvious similarities with current classes of antifungals and thus may have a different mechanism of action , combination of these compounds with established antifungal agents could have additive or synergistic action that should be studied further .
The Candida strains used in these experiments are summarized in Table 1 or described in the text . Yeast cultures were maintained on yeast peptone dextrose ( YPD ) ( Difco , http://www . vgdusa . com/DIFCO . htm ) , agar , or as frozen stocks . The C . elegans strains were propagated on E . coli strain OP50 or E . coli strain HB101 using established procedures ( detailed below and in [7 , 51] ) . Candida strains were inoculated in 2 ml of YPD and grown at 30 °C for 24 h . Lawns were prepared by spreading 10 μl of each culture on 35-mm tissue culture plates ( BDFalcon , http://www . bdbiosciences . com ) containing solid BHI media ( Difco ) with kanamycin ( 45 μg/ml ) , ampicillin ( 100 μg/ml ) , and streptomycin ( 100 μg/ml ) . The plates were incubated at 30 °C for 24 h followed by 25 °C for 24 h . glp-4;sek-1 and glp-4 worms were grown at 15 °C until they reached the L2 stage , and then they were transferred to 25 °C for 24 h . N2 wild-type worms were grown at 15 °C for 72 h , until they reached the L4 stage . Approximately 100 worms were picked onto each lawn and allowed to feed for 30 min to 2 h . The worms were washed off the plates with M9 buffer and allowed to crawl on unseeded BHI plates to remove yeast cells from their cuticles . Roughly 70–80 worms were then picked to wells in a six-well microtiter dish that contained 1 . 5 ml liquid medium of 79% M9 buffer , 20% BHI , 10 μg/ml cholesterol in ethanol , and 90 μg/ml kanamycin . The plates were incubated at 25 °C overnight and then examined at 24-h intervals for survival . Worms were considered dead and removed when they did not respond to being touched by a platinum wire pick . C . albicans strains DAY185 and HGFP3 lawns were prepared , and C . elegans glp-4;sek-1 worms exposed to C . albicans as described above for the killing assays . The worms were then transferred to the liquid media described above in both the presence and absence of 32 μg/ml fluconazole . At 24 , 48 , 144 , and 192 h , 20–40 worms exposed to DAY185 were stained in 200 μl of 10 μM FUN-1 ( Invitrogen , http://www . invitrogen . com ) and 25 μg/ml Concavalin A-Alexafluor 488 ( Invitrogen ) for 45 min [52] . Pictures were taken with a confocal laser microscope ( TCS NT , Leica Microsystems , http://www . leica-microsystems . com ) . FUN-1 is a fluorescent yellow dye that is absorbed by metabolically active fungal cells and fluoresces red when illuminated with 480 nm ( fluorescence emission ) . Concanavalin A-Alexafluor ( fluorescence emission at 519 nm ) is a fluorescent green dye that binds to polysaccharides and stains filaments . MLR62 and DAY185 lawns were prepared as above . An OP50-GFP strain was grown in LB media in the presence of ampicillin ( 100 μg/ml ) , instead of streptomycin and spotted on nematode growth media ( NGM ) agar plates . For the first experiment , sek-1;glp-4 worms were exposed to MLR62 for 1 h , washed thoroughly , and then pipetted onto DAY185 for 2 h . The worms were then moved to pathogen-free liquid media and photographed on days 0 and 1 with the confocal laser microscope mentioned above . For the second experiment , sek-1;glp-4 worms were placed on DAY185 for 2 h , thoroughly washed , and then pipetted onto OP50-GFP NGM plates . Worms were thoroughly washed and then photographed with a confocal laser microscope at 2 , 24 , and 48 h . Additionally , living and dead worms on day 1 were moved to pathogen-free liquid media in the presence of kanamycin ( 90 μg/ml ) and monitored for hyphae formation . The number of C . albicans cfu in C . elegans was quantified based on the protocol detailed in [45 , 53] with a few modifications . C . albicans strain MLR62 lawns were prepared on BHI as described above . C . elegans glp-4;sek-1 worms were exposed to the lawns for 30 min and then moved to liquid media and incubated at 25 °C in the presence or absence of antifungal agents . At appropriate time points , three groups of ten worms each were washed twice in 8-μl drops of M9 medium on BHI agar plates , in order to remove surface Candida . Each group of ten worms was then disrupted using a Pellet Pestle Motor ( Kontes , http://www . kimble-kontes . com ) and plated on YPD agar containing kanamycin ( 45 μg/ml ) , ampicillin ( 100 μg/ml ) , and streptomycin ( 100 μg/ml ) . The plates were incubated for 48 h at 30 °C and colonies were counted . Statistical significance was determined by the Mann-Whitney test using STATA6 statistical software ( Stata , http://www . stata . com ) . A p-value of less than 0 . 05 was considered to be significant . For testing the efficacy of chemical compounds , C . albicans strain MLR62 was inoculated into 2 ml of YPD and grown at 30 °C for 24 h; 10 μl of the culture was spread on 35-mm tissue culture plates ( BDFalcon ) containing BHI agar ( Difco ) with kanamycin ( 45 μg/ml ) , ampicillin ( 100 μg/ml ) , and streptomycin ( 100 μg/ml ) . The plates were incubated at 30 °C for 24 h . C . elegans animals at the L4 developmental stage ( grown as detailed above under C . elegans liquid medium killing assays ) were transferred from a lawn of E . coli HB101 onto C . albicans lawns on BHI medium , incubated at 25 °C for 30 min , and then pipetted in 50 μl into 96-well plate wells that contained 100 μl liquid media ( 79% M9 buffer , 20% BHI media , 10 μg/ml cholesterol in ethanol , and 90 μg/ml kanamycin ) . C . elegans glp-4;sek-1 nematodes were pipetted into 96-well plates that contained compounds from chemical libraries . Library 1361 was assembled by Biomol . It contains well-characterized compounds that affect many different aspects of cellular pathways . The NINDS custom collection ( plates 501–503 ) was put together by MicroSource Discovery Systems for the National Institute of Neurological Disorders and Stroke ( NINDS ) , the Huntington Disease Society of America ( HDSA ) , the Amyotrophic Lateral Sclerosis ( ALS ) Association , and the Hereditary Disease Foundation ( HDF ) . It mostly contains FDA-approved drugs . The Prestwick library ( plate 1569 ) contains compounds that are known to be safe and bioactive in humans . The majority of the compounds are marketed drugs ( details available at http://iccb . med . harvard . edu ) . On day 6 , nematodes were evaluated for survival , filament formation , and optical density . The plates were incubated at 25 °C and examined on day 6 for survival with a Nikon SMZ645 dissecting microscope , and images were obtained using a CellWorx High Content Cell Analysis System ( Applied Precision , http://www . appliedprecision . com ) at 4× magnification . Dead worms were counted but not removed . Worms were considered dead if they had developed filamentation , were rod-shaped , or did not respond to the well being tapped . Each compound was also scored by the number of dead worms in the well that developed hyphea ( Table S1 ) . Compounds were considered to have completely or almost completely inhibited filament formation if fewer than 25% of the dead worms in the well developed hyphea . Compounds were categorized as having a minimal effect on filament formation if 25%–75% of the dead worms developed hyphea . The compounds that had no effect on filament formation allowed over 75% of the dead worms to form hyphea . Approximately 25 nematodes were used to analyze each chemical compound tested . Two controls were used in all plates . The positive control was the antifungal caspofungin ( which allows >75% survival at day 6 ) . Phosphate-buffered saline ( PBS ) was the negative control ( <25% of nematodes in PBS are alive at day 6 ) . In this pilot screen , we considered a compound as a “hit” when survival in the compound well was 50% and above of the median survival of the control wells . Cells were maintained in YPD ( 1% yeast extract , 2% peptone , and 2% dextrose ) at −80 °C until used . The MIC was evaluated for each of the above compounds according to the Clinical and Laboratory Standards Institute ( formerly the National Committee for Clinical Laboratory Standards , NCCLS ) microdilution protocol ( M27-A ) [28] . CAPE , enoxacin , and lapachol were evaluated for hemolysis of sheep erythrocytes as described by Moy et al . [7] . DMSO and Triton X-100 were used as controls . To test whether CAPE , enoxacin , and lapachol were toxic to the worms at the concentration used in the hemolysis assay , glp-4l;sek-1 L4s were prepared as described above . The nematodes were moved from E . coli OP50 to pathogen-free liquid media containing 2% DMSO or 2% Triton X-100 or 100 μg/ml CAPE , enoxacin , or lapachol . The worms were incubated for 4 h at 25 °C; at hourly intervals dead worms were counted and removed . BALB/c mice ( 22–24 g , female , Charles River Laboratories ) were used in all studies . In brief , C . albicans strain DAY185 was grown at 30 °C with shaking overnight in YPD to late-log phase . The yeast cells were centrifuged , washed in PBS , and re-suspended to a concentration of 5 × 106 blastospores/ml ( measured with a hemocytometer ) . 200 μl of this suspension were inoculated into the lateral tail vein ( 1 × 106 blastospores ) . In all mammalian experiments , the concentration of yeast cells in the inoculum was confirmed by plating serial dilutions and enumerating cfus 24–48 h later . The murine protocols were approved by the Massachusetts General Hospital Committee on Research , Subcommittee on Research Animal Care and special attention was given to minimize the suffering of the mice . The effect of CAPE , lapachol , and enoxacin on biofilm growth on silicone pads was evaluated as described by Richard et al . [24] and Nobile et al . [41] . The C . albicans strain DAY185 was inoculated in 2 mL of YPD and grown at 30 °C for 24 h . The DAY185 culture was diluted into SD media plus 50 mM glucose to an OD600 of 1 . 0 . After the 90-min incubation and PBS wash , caspofungin ( positive control ) , CAPE , enoxacin , and lapachol were added to fresh media at 2 μg/mL , 30 μg/mL , 13 . 3 μg/mL , and 16 . 1 μg/mL , respectively . The silicone pads were visualized as reported in [24 , 29] . DAY185 cells were incubated at 37 °C for 60 h on silicone squares in SD media with 50 mM glucose . Biofilms were stained with 25 μg/ml Concavalin A-Alexafluor 488 ( Invitrogen ) for 1 h in the dark . Pictures were taken with a confocal laser microscope ( TCS NT , Leica Microsystems ) . The biofilm dry mass was evaluated as described by Nobile et al . [29] . Statistical significance was determined by the Mann-Whitney test using STATA 6 . A p-value of less than 0 . 05 was considered to be significant . Murine and C . elegans-killing curves were plotted , and estimation of differences ( log-rank and Wilcoxon tests ) in survival analyzed with the Kaplan-Meier method performed using STATA 6 . A p-value of less than 0 . 05 was considered to be significant .
The protein sequences from the TrEMBL database ( http://www . ebi . ac . uk/trembl ) discussed in this paper are HWP1 ( Q59TL7 ) and SEK-1 ( Q95Y19 ) . | Candida spp . are among the most significant causes of nosocomial infections , and disseminated candidiasis continues to have an attributable mortality rate of over 25% . For this reason , we have developed a liquid media assay using the model nematode Caenorhabditis elegans as a model organism for Candida infection . The worms are infected on solid media lawns and then moved to pathogen-free liquid media . Unless antifungal compounds are added to the wells , the majority of worms die within 3–4 d . This model is similar to the infection process in humans , in that Candida cells are able to produce filaments , which are essential for the infection process in humans . We used this pathogen model to create a semi-automated , high-throughput screen using C . elegans to evaluate the antifungal effectiveness of many types of chemical compounds . Through this process , we have identified three compounds that we show have varying degrees of antifungal activity in C . elegans , in vitro , and in mice . | [
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] | 2007 | Antifungal Chemical Compounds Identified Using a C. elegans Pathogenicity Assay |
Human neurons express the innate immune response receptor , Toll-like receptor 3 ( TLR3 ) . TLR3 levels are increased in pathological conditions such as brain virus infection . Here , we further investigated the production , cellular localisation , and function of neuronal TLR3 during neuronotropic rabies virus ( RABV ) infection in human neuronal cells . Following RABV infection , TLR3 is not only present in endosomes , as observed in the absence of infection , but also in detergent-resistant perinuclear inclusion bodies . As well as TLR3 , these inclusion bodies contain the viral genome and viral proteins ( N and P , but not G ) . The size and composition of inclusion bodies and the absence of a surrounding membrane , as shown by electron microscopy , suggest they correspond to the previously described Negri Bodies ( NBs ) . NBs are not formed in the absence of TLR3 , and TLR3−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice . These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication . This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit .
Toll-like receptors ( TLR ) are innate immune receptors that recognise and respond to the presence of PAMPS ( pathogen associated molecular patterns ) encoded by pathogens [1] . TLR3 is a type I intracellular transmembrane protein that contains a large leucine-rich repeat ( LRR ) in the extracellular region and a Toll/Il-1 receptor homology ( TIR ) signalling domain in its cytoplasmic region . TLR3 can detect the presence of and respond to exogenous and endogenous RNA molecules: dsRNA of viral origin , mimicked by polyriboinosine-polyribocytidylic acid ( polyI∶C ) ; mRNA; and ssRNA ( polyinosinic acid ) [2] , [3] , [4] . Upon ligand binding , TLR3 signals via a MyD88-independent signalling pathway involving the adaptor molecule TRIF/Ticam-1 [5] , [6] . TRIF can induce activation of IRF3 and NF-κB , notably through the interaction with TRAF6 and RIP1 [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] . TLR3-dependent activation leads to the expression of genes encoding proinflammatory cytokines , chemokines and IFN-α/β . TLR3 is present within the central nervous system ( CNS ) [2] . High levels of TLR3 are found in glial cells [15] , [16] and neurons in disorders of the brain , neurodegenerative diseases and viral infections [17] , [18] . The high levels of TLR3 in the CNS suggests an important role in the response to neuronal injury and/or viral infection [19] , which may involve mechanisms other than those limited to the innate immune response . Indeed , TLR3 has been described as a negative regulator of axonal growth [20] . In non-neuronal cells ( dendritic cells ( DCs ) or epithelial cells ) , TLR3 is found in intracellular compartments — sometimes small perinuclear structures ( 300–500 nm ) , such as those observed after overexpression [21] , [22] , or others identified as multivesicular bodies ( MVBs ) [16] , [21] . Intracellular localisation of TLR3 appears to be crucial for its activation [23] , [24] . In neuronal cells , TLR3 is mainly intracellular [16]–[18] . However , the precise intracellular localisation of TLR3 in neuronal cells is largely unknown . Rabies virus ( RABV ) almost exclusively infects neurons , where it triggers interferon , inflammatory and antiviral responses [17] , [25] . The virus particle binds cell-surface receptors and follows the endosomal pathway , allowing the release of viral nucleocapsid ( NC ) into the cytoplasm . The life cycle of the virus then progresses in the cytoplasm , with transcription of the five N , M , P G and L viral genes and the replication of negative- and positive-polarity genomes [19] , [26] . Moreover , RABV infection induces the formation of cytosolic protein aggregates called Negri Bodies ( NBs ) . NBs are characterized by the accumulation of viral NC proteins [27]–[29]; they also contain endothelial nitric oxide synthase ( eNOS ) [30] . NBs resemble inclusion bodies and/or aggresomes seen in several neurodegenerative disorders [31] . Aggresomes are defined as pericentriolar cytoplasmic inclusions resulting from the aggregation of misfolded and/or ubiquitinated proteins [32] , [33] . The aggresomal subcellular compartment sequesters proteins produced in excess following a cellular stress such as a viral infection . These proteins are then targeted to proteasomes and/or autophagy components present in this area . Some viruses may use aggresomes to enhance replication [34] , [35] . In this study , we further analysed the production , function and spatiotemporal location of neuronal TLR3 , and notably its association to the endosomal compartment . We carried out these studies either in cultures of human neuronal cells ( neuronal precursor Ntera-2clD/1 , post mitotic neurons NT2-N or neuroblastoma cell line SK-N-SH ) or in TLR3−/− mice using the pathogenic RABV strain CVS .
Human post-mitotic neurons ( NT2-N ) produce several TLRs , including TLR3 [17] , [19] . TLR3 mRNA levels were analysed by semi-quantitative RT-PCR in the neuronal precursors Ntera-2clD/1 , from which NT-2N cells are derived , and in a human neuroblastoma cell line ( SK-N-SH ) . An amplified product of 527 bp was detected in the three types of neuronal cells ( Figure 1A ) . We then sequenced the whole TLR3-encoding gene open reading frame by direct sequencing . We found that human neuronal TLR3 sequence from NT2-N ( sequence ID: DQ445682 ) was strictly identical to those previously identified in other human cells ( GenBank accession: NM_003265 ) . These findings strongly suggest that TLR3 in neuronal cells could be functional . Yang et al . reported the existence of a TLR3 isoform in primary human astrocytes and glioblastoma [36] . This isoform is produced by deletion of an intron-like sequence within exon IV of the gene and is co-produced with wild-type TLR3 . The presence of this isoform in Ntera-2clD/1 , U373MG ( astrocytoma ) and CHME ( microglia ) was tested by semi-quantitative RT-PCR experiments using pairs of primers spanning the intron-like sequence in exon IV ( Figure 1B , upper gel ) . This isoform was not detected in the three cell lines tested . Moreover , no quasi-species of wild-type transcripts or transcripts with deleted sequences were observed after RABV infection of SK-N-SH neuroblastoma ( Figure 1B , lower gel ) . The effect of infection on TLR3 levels was monitored by immunoblotting using two distinct antibodies ( Ab ) directed against the NH2 terminal of the TLR3 ectodomain ( sc-Q18 and IMG-315A ) . Cellular extracts were prepared from Ntera-2clD/1 , either non-infected ( NI ) or infected with RABV for 12 , 24 or 48 h . We detected a 100 kDa band corresponding to full size TLR3 protein in all cell extracts ( Figure 1C data shown for IMG-315A Ab ) . The amount of neuronal TLR3 was not affected by RABV infection even 48 h after infection ( Figure 1C ) . These results were consistent with those obtained by cytofluorimetry ( two upper right panels of Figure 1D , data shown for Sc-Q18 Ab ) , with no difference in proportion of cells displaying fluorescently labelled total TLR3 observed between NI and infected Ntera-2clD/1 cells ( 40 and 39% respectively ) . In experiments performed without cell permeabilisation ( surface fluorescence ) , TLR3 was not detected at the surface of NI or infected cells ( lower right panels of Figure 1D ) . Thus , TLR3 is not expressed at the surface , at least at detectable levels , and infection does not cause TLR3 to relocate towards the cell surface . Similar findings were obtained with the SK-N-SH neuroblastoma ( data not shown ) . These data indicate that human neuronal cells produce full-length TLR3; this protein was not upregulated by RABV infection and had an intracellular localisation in both NI and infected cells . Intracellular localisation of TLR3 was further investigated in Ntera-2clD/1cells by immunocytochemistry and confocal microscopy . In the absence of infection , TLR3 was found within small vesicles throughout the cytoplasm . Co-immunocytochemistry analysis using Ab directed against the NH2 terminal of TLR3 ( Sc-Q18 ) and against either CD63 , a marker of late endosomes and/or multivesicular bodies ( left panels of Figure 2A ) , or a marker of early endosome ( EEA1 ) ( upper panels of Figure 2B ) revealed that a proportion of TLR3 was associated with endosomes , mainly in late endosomal vesicles ( colocalisation index of 0 . 55 and 0 . 86 for early and late endosomal markers , respectively; Figure 2C ) . A small proportion of TLR3 was found associated with Golgi ( GM130 ) and with the endoplasmic reticulum , ER ( calnexin ) ( data not shown ) . Thus , in neuronal cells , TLR3 protein was located mainly within the early/late endosomal compartment . Two days after RABV infection , the endosomal distribution of TLR3 was conserved ( as shown in Figure 2A , left panel , and Figure 2B , lower panel ) . However , colocalisation coefficients between TLR3 and CD63 were slightly lower in RABV-infected than in NI cells ( 0 . 86 and 0 . 79 for NI and RABV-infected cells , respectively ) . This decrease may result from the appearance of a new site of TLR3 intracellular localisation . TLR3 became concentrated in large perinuclear aggregates in RABV-infected cells ( arrow heads in Figure 2A and 2B ) . This observation was reproduced using three distinct TLR3-specific Ab ( Sc-Q18 , Sc-C20 and FITC-conjugated Ab IMG-315C ) and in cultures of NT2-N ( Figure S1 ) and SK-N-SH ( data not shown ) . Perinuclear aggregates were not stained by anti-CD63 or anti-EEA1 Ab , or by OKT9 , a marker of recycling endosomes . This suggests that the RABV-induced aggregates were distinct from the endosomal compartment . RABV-induced TLR3 aggregates were not associated with Golgi ( GM130 ) or the ER ( calnexin ) ( data not shown ) . This aggregation of TLR3 was not observed when cells were infected with herpes simplex virus type 1 or Sendai virus , or when cells were treated for 24 h with rIFN–β ( 500 IU/ml ) ( data not shown ) . These observations suggest that RABV triggers perinuclear aggregation of TLR3 , distinct from the endosomal compartment . Confocal microscopy analysis of RABV-infected cells co-stained with Ab directed against TLR3 and RABV nucleocapsid NC revealed that RABV NC was located in perinuclear inclusions , with the great majority displaying the same localisation pattern as TLR3-containing aggregates ( Figure 3A , TLR3 in red , NC in green , ) . Viral NC ( comprising N , P , L proteins and the viral RNA genome ) seemed to form a ring around a TLR3 core . TLR3-positive inclusions were further analysed by confocal microscopy ( Figure 3B ) and imaging ( Figure 3C ) . Modelling of deconvoluted confocal images with 3D Imaris® software confirmed the particular spatial organisation of TLR3/NC aggregates ( Figure 3C ) . The reconstruction of images of a typical 48 h TLR3/NC aggregate is shown in Figure 3B . The round-shaped perinuclear structure has a total diameter of 3 . 0 µm with an NC coating 0 . 5 µm thick . TLR3/NC aggregates were also observed using monoclonal antibody ( mAb ) specific for viral P or N protein , confirming that the main protein constituents of NC were present in the aggregates ( Figure 4A ) . In contrast , immunocytochemistry performed with a mAb directed against the RABV G protein — associated with the RE or Golgi vesicles in the cells — showed that TLR3 aggregates were not associated with RABV G protein ( Figure 4B ) . Co-immunoprecipitation performed with Ab directed against NC and TLR3 ( Q18 and C20 Ab ) gave negative results ( data not shown ) , suggesting that interactions between TLR3 and viral NC were either weak or indirect . The kinetics of RABV NC aggregate formation were analysed in Ntera-2clD/1 at 12 , 24 and 48 h post-infection ( pi ) and stained with anti-NC Ab ( Figure 4C ) . NC aggregates were detected as early as 12 h pi , with an average diameter of 1 . 0 µm ( Figure 4E ) . The size of aggregates increased as infection progressed , reaching an average size of 3 . 0 µm by 48 h pi ( Figure 4D , left panel ) . Number of inclusions per cell was analyzed by Acapella software in 24 h RABV-infected SK-N-SH cells ( Figure 4D , right panel ) . Majority of cells harbour one or two perinuclear TLR3 aggregates 24 h after infection . Later in the infection ( 48 h after infection and thereafter ) , the average number of inclusions per cell increases ( as described below ) , suggesting that NB formation is a highly dynamic process . These observations suggest that RABV triggers formation of perinuclear TLR3 aggregates -surrounded by a halo of viral N and P proteins- which number and size are modulated during infection . TLR3 has been described for its ability to bind viral RNA . We carried out hybridisation experiments using a fluorescent probe specific for the RABV genome leader sequence to determine whether the TLR3/NC aggregates contained viral RNA ( Figure 5 ) . We did not detect fluorescence in the absence of probe ( Figure 5B ) or in NI cells ( Figure 5D ) . The fluorescent probe stained perinuclear structures ( Figure 5A–5C ) , corresponding to TLR3/NC aggregates , forming discrete ovoid structures visible in transmission images , as described above ( Figure 3 ) . Intensity of fluorescence was variable , suggesting that accessibility of the viral RNA may differ among aggregates . These observations show that TLR3/NC aggregates also contain RABV genomic material . Immunocytochemistry experiment using J2 mAb specifically recognizing dsRNA [37] , [38] , [39]—including endogenous cellular dsRNA [40] , [41]—revealed accumulation of dsRNAs in NBs ( Figure S2 ) . The presence of viral N and P proteins and genomic material within the aggregates suggests that they could be NBs . NBs are found both in the neurons of rabid brain and in vitro in RABV-infected neuronal cells , in the form of accumulated viral material around the nucleus . Electron microscopy analysis of ultra-thin sections of SK-N-SH 48 h after RABV infection showed that amorphous aggregates ( usually one or two per cell ) , with an average diameter of 3 . 0 µm and without a membrane , were indeed present and displayed perinuclear localisation ( Figure 5E ) . We did not find such aggregates in NI cells ( data not shown ) . We used electron microscopy to analyse cells treated with Triton X-100 ( 0 . 1% ) and stained with Ab directed against NC or TLR3; cellular structures were not preserved . Aggregates were strongly stained ( DAB accumulation ) with both Ab , indicating they contained both TLR3 and RABV NC proteins ( Figure S3 ) . Electron microscopy also showed that the aggregates were located in particular cell areas characterised by the accumulation of surrounding mitochondria ( M ) , rough endoplasmic reticulum ( rER ) and , in some cells , ‘nests’ of viral particles ( Figure 5F ) . These areas , with their particular composition resembled the viral factories previously described for other viruses . Thus , TLR3/NC aggregates may function as a storage or construction area for viral NC . Overall , these observations suggest that TLR3/NC aggregates observed in RABV-infected neuronal cells correspond to previously described NBs . Due to their perinuclear localisation , size and protein composition , it is possible that NBs represented virus-mediated aggresomes . Aggresomes are associated with molecular chaperones such as Hsp70 and surrounded by a cage of cytoskeleton proteins such as vimentin and α-tubulin [32] , [33] , [42] . Thus , we studied the presence of aggresome characteristics in NBs . Examination of the distribution of α-tubulin and vimentin in RABV-infected cells revealed that a ring of α-tubulin indeed surrounded the NC-positive aggregates ( Figure 6A , 1–3 ) . However , vimentin was not closely associated with NBs ( Figure 6A , 4–6 ) . The chaperone Hsp70 was present at the periphery of the NBs ( Figure 6B ) . Aggresomes are often formed at the microtubule organising centre ( MTOC ) , which can be visualised using Ab directed against γ-tubulin [43] . Double staining of MTOC and NC proteins clearly demonstrated that NBs are not localized at the MTOC ( Figure 6C ) . When cells are lysed in the presence of detergent , aggresomes are retained in the insoluble ( IS ) fraction , together with cytoskeletal proteins such as vimentin , whereas proteins in the cytoplasm or associated with the ER — such as calnexin — appear in the soluble ( S ) fraction . S and IS fractions were prepared from Ntera-2clD/1 cells infected with RABV for 48 h , and were separated by SDS-PAGE . The IS fraction prepared from RABV-infected cells did not contain calnexin or α-tubulin . These proteins were present in the S fraction , as expected , with the IS fraction containing vimentin ( Figure 6D , upper ) . Immunostaining revealed discrete NBs ( positively stained for both RABV NC and TLR3 ) in the IS fraction ( Figure 6D , lower ) , indicating that NBs are detergent-resistant structures . Given the major role of microtubules in the formation of aggresomes , we analysed their role in the formation of NBs . We treated RABV-infected SK-N-SH cells ( 24 h pi ) with colcemid ( Figure 7A ) — an inhibitor of microtubule polymerisation — diluted in PBS vehicle , or with vehicle alone or without any reagent . Effect of colcemid on size and number of NBs per cell was analyzed 24 h post treatment -corresponding to 48 h post infection- ( Figure 7B and 7C ) . As shown in Figure 7B , colcemid treatment modified the size of NBs . Overall , in colcemid-treated cells there are more cells with large NB ( range between 200 and 350 pixels ) than cells exhibiting small NB ( range 50 to 150 pixels ) . In the meantime , colcemid treatment drastically modified the distribution of NB per cell ( Figure 7C ) . In absence of treatment , 24 h after infection RABV-infected cells harboured 2 or 3 NBs per cell ( as shown in Figure 4D ) . This number rises up to more than 7–8 NBs per cell 48 h after infection ( Figure 7C ) . Colcemid treatment blocks this increase since colcemid treated cells have less NBs per cell than non-treated cells . These observations indicate that microtubule polymerisation play a role in the dynamic of NBs particularly after their formation , as suggested by the increase in number of NBs per cell between 24 and 48 h pi . These data support a role of microtubules in the outcome of NBs . NBs from cells treated with colcemid were twice as small as those from cells treated with vehicle alone ( Figure 7B ) , supporting a role for microtubules in NB formation . Overall , our findings showed that NBs are resistant to detergent , involve microtubules in their outcome and are surrounded by the cellular α-tubulin and chaperone network . These features are reminiscent of aggresomes . However , NBs were not associated to the MTOC and not surrounded by a vimentin ‘cage’ . Thus , they do not represent canonical aggresome structures . TLR3 was found at the centre of NBs . Thus , we analysed the involvement of TLR3 in the formation of NBs in TLR3 silencing experiments . Plasmids encoding emerald GFP and miRNA targeting the TLR3 gene ( miTLR3 ) were delivered by nucleofection ( Amaxa program Q-001 ) into Hek293A Qbiogene cells . Non-specific miRNA ( miNEG ) was used as a control . Nucleofection efficiency ( ≥90% ) was controlled using a plasmid encoding the GFP protein ( provided by the Cell Nucleofector Kit V , Amaxa ) . Cells were infected with RABV 24 h after nucleofection . 48 h pi , RNA was extracted or cells analysed by immunocytochemistry . TLR3 mRNA levels were compared between miTLR3 and miNEG cultures . As shown in the left panel of Figure 8A , the level of TLR3 mRNAs was decreased by 60% in miTLR3 compared to miNEG cells . Cytofluorimetry analysis of TLR3 ( sc-Q18 Ab ) stained cells showed that these reduced mRNA levels were associated with a reduced intensity of fluorescence ( Figure 8A , right panel ) , indicative of reduced TLR3 protein production in miTLR3 cultures . Moreover , the reduced TLR3 levels were accompanied by a 10-fold reduction of viral transcription ( Figure 8B , left panel ) and RABV N protein expression as shown by cytofluorimetry ( Figure 8A , right panel ) , with N protein present in 77% of miNEG cells and 59% of miTLR3 RABV-infected cells . Transitory nucleofected cells with the miTLR3 and miNEG vectors expressing emerald-GFP were assayed for expression of Em-GFP fluorescence by FACS ( data not shown ) and immunocytochemistry ( Figure 8C ) , showing that the intensity of Em-GFP fluorescence is not homogenous among cells in the same population . Similar data were obtained on stable cell lines selected for resistance to blasticidin which all possess the expression plasmid but nevertheless exhibit a heterogenous Em-GFP fluorescence ( data not shown ) . Cells were immunostained with anti-TLR3 ( red ) and anti-N-RABV mAb ( blue ) showing that the miTLR3 population of cells exhibits some TLR3-negative cells ( Figure 8C , lower panel ) in contrast to the miNEG population ( Figure 8C , upper panel ) . In the TLR3-negative cells , NB formation is abolished . Immunocytochemistry analysis of individual cells ( such as the green GFP cell on the right in Figure 8D ) revealed that NBs — stained red for TLR3 and blue for RABV N protein — were not present in miTLR3 cells . These observations strongly suggest that TLR3 is required for NB formation and RABV N expression . To determine whether the cytoplasmic domain of the TLR3 protein encompassing the signalling TIR domain was involved in NB formation , we stable transfected HEK cells with a plasmid encoding a TLR3 gene with a deleted cytoplasmic domain ( pZero-hTLR3-HA ) . Cells transfected with the pZERO-hTLR3 plasmid are therefore unable to signal via TLR3 because the absence of the TIR domain does not allow binding of TRIF to TLR3 . As shown in Figure S4B , NBs were still formed and viral transcription was not modified in pZERO-hTLR3-expressing cells ( transitory experiments ) . Similar data were obtained with stably transfected cell lines selected for their resistance to puromycin ( data shown in Figure S4 are representative of both conditions ) . Furthermore , the adaptor TRIF could not be detected in NBs ( Figure 4C ) , suggesting that activated TLR3 molecules were not present in NBs . Thus , the ectodomain of TLR3 appears to be crucial for the formation of NBs . TLR3 may therefore favour virus multiplication . Assuming that TLR3-NBs are required for virus multiplication , we would expect RABV infection to be impaired in the brain of TLR3-deficient mice ( TLR3−/− ) and subsequently these mice would be expected to have a higher survival rate than WT mice . We compared the progression and outcome of RABV infection in TLR3−/− with WT mice . TLR3−/− ( n = 9 ) and control WT ( n = 8 ) C57Bl6 mice were injected with a dose of virus expected to kill 3/4 of the mice . Mice were observed for 15 days post infection . At day 13 pi , only 37% of the WT mice were alive , whereas 66% of the TLR3−/− mice survived ( Figure 9A ) . Progression of the disease in the two groups of mice is illustrated by the cumulative clinical curves ( Figure 9B ) . Clinical score was significantly decreased in TLR3−/− mice compared to WT mice . Finally RABV neuroinvasiveness was compared in these mice by measuring the amount of RABV genome in the brain of RABV-infected WT and TLR3−/− mice at day 11 . Levels in NI brain were given a reference value of 1 , for calibration . RABV genome level in TLR3−/− brain was markedly lower than in WT mice ( 20 times lower ) ( Figure 9C ) . All together , these strongly support a role of TLR3 in RABV infection .
Our study demonstrates that TLR3 is localised in the cytoplasm of non-infected neuronal cells , where it is associated mainly with the endosomal compartment . TLR3 subcellular localisation is notably altered by RABV infection , with TLR3 found within viral-induced inclusions identified as RABV-induced Negri Bodies ( NBs ) . The presence of TLR3 in the core of viral NBs , surrounded by a ring of viral N and P proteins , appears to be crucial for NB formation . This finding demonstrates a novel function of TLR3 , a molecule mostly known for its immune functions . In the absence of infection , reactivity with anti-CD63 antibody and weak signal from EEIA-expressing early endosomal structures showed neuronal TLR3 to be confined to the endosomal compartment , associated mainly to the late endosomes and multivesicular bodies . TLR3 is rarely detectable within the Golgi apparatus or at the endoplasmic reticulum . Indeed , we observed the canonical distribution pattern of TLR3 in human non-infected neuronal cells , as previously described for human DCs or fibroblasts [21] , [22] . The subcellular distribution of TLR3 is markedly altered in RABV-infected cells , being localised in perinuclear inclusion bodies , the NBs . These NBs contain viral NC but do not include the envelope viral proteins [27] . Relocation of TLR3 during viral infection has only previously been described for respiratory syncitial virus ( RSV ) . In epithelial cells , RSV infection increases the TLR3 levels and targets TLR3 to the membrane [44] . In neuronal cells , RABV infection did not increase TLR3 protein levels , or target TLR3 to the membrane . Given that RABV infection did not increase the total pool of TLR3 protein , it is possible that the TLR3 is recruited from pre-existing cytoplasmic- , ER- or endosome-associated TLR3 by NBs . However , NBs were not stained by endosomal or ER markers , suggesting that endosome- or ER-associated TLR3 is not involved in NB formation . NBs observed by electron microscopy did not reveal the presence of any membrane surrounding the NBs . Thus , it is unlikely NBs are autophagolysomes or lysosomes . Confocal microscopy and 3-D imaging revealed that NBs have a highly organised structure , with a TLR3-containing core surrounded by a halo of viral N and P proteins . The central position of TLR3 suggests that NB formation may be initiated by the aggregation of TLR3 molecules . A primordial role of TLR3 in NB formation is consistent with the absence of NBs from cells in which TLR3 had been silenced . TLR3 is a horseshoe-shaped solenoid with 23 leucine-rich repeats ( LRRs ) located in the ectodomain of the molecule . Similarly to other solenoid proteins including polyQ , TLR3 may have an intrinsic capacity to form aggregates [45] . NBs formed even in the absence of the cytoplasmic domain of TLR3 , suggesting a major role for the TLR3 ectodomain in NB formation ( Figure S4 ) . PolyQ proteins such as mutant huntingtin protein form inclusions consisting of an inner dense core of aggregated huntingtin surrounded by a ring structure composed of sequestered cellular proteins [46] , [47] . Experiments involving the sequential expression of polyQ followed by expression of these cellular proteins led to the conclusion that the ring structure results from the subsequent recruitment of cellular proteins at the exterior surface of an initial polyQ core aggregate . The organisation of NBs may follow a similar pattern . In this case , NBs would result from the initial association of TLR3 molecules followed , in a second step , by the accumulation of viral proteins , forming a ring into which cellular proteins could also be inserted . This would be consistent with the concentric organisation of NB structure and observation of the cellular protein eNos is recruited in NBs in RABV-infected brain . However , the NB structure is distinct from polyQ-initiated aggregates . In particular , TLR3 in NBs is still accessible to Ab . This has not been observed for huntingtin . These observations suggest that the viral ring of protein in NBs is more porous than the ring surrounding the ordered huntingtin aggregate structure . Consequently , NB and polyQ aggregates may have distinct properties . The recruitment and sequestration of cellular proteins in polyQ aggregates may lead to the functional depletion of cellular functional proteins , possibly underlying toxic properties of these aggregates in the cell . This is not necessarily the case for NBs . The nature of the interaction between TLR3 and viral proteins in NBs remains unclear , since attempts to coimmunoprecipitate TLR3 and NC have failed . NBs mimic aggresomes but do not exhibit the full set of characteristics of these stress-induced cellular processes . NBs are associated to the chaperone Hsp70 and to the microtubule network , reminiscent of the two principle features of aggresomes [48] . However , the marked redistribution of vimentin fibres observed for example in GFP-250-induced aggresomes , and in Theiler's or reovirus-induced aggresome-like inclusions [49] , [50] was not observed in RABV-infected neuronal cells . In contrast to most aggresomes , such as those formed in HSV2 infection [51] , NBs were not associated with the microtubule organising centre ( MTOC ) . The effect of inhibition of microtubule depolymerisation , by addition of colcemid , on the distribution and size range of NB may indicate that microtubules are required for NB outcome . The finding that NBs do not display all the characteristics of aggresomes has also been observed for inclusions formed in African swine fever virus ( ASFV ) , which have been described to function as “viral factories” . Viral factories are areas of cytoplasm where viral components and cellular components supporting viral replication are concentrated [51] , [52] . The possibility that NBs function as dynamic structures involved in viral multiplication , as proposed by Lahaye et al . ( submitted ) , is supported by electron microscopy pictures showing that NBs are found closely associated with newly synthesised viral particles . TLR3 would thus be an essential component for virus multiplication . Consistent with this notion , we showed that NBs were absent from cells with TLR3 silencing and that TLR3−/− mice were less severely infected by RABV than WT mice . Further studies are now required to show whether RABV production is promoted by TLR3 . A major function of TLR3 is to sense and respond to viral infection . The presence of TLR3 in the core of NBs can be seen as an attempt to inactivate TLR3 function . Sequestration of TLR3 into NBs could reduce the cellular innate immune response . The absence of TRIF –adaptor of TLR3- in NB could support the hypothesis that TLR3 molecules in NB are inactive- . The presence of viral RNA in NBs may reflect an interaction of TLR3 with viral components at an early stage of infection , when the virus enters the cell through the TLR3 decorated endosomal compartment . TLR3 could thus be involved in recruitment of viral NC from the endosomal compartment , before aggregation in NBs . However , the absence of endosomal markers within the NBs remains unexplained . In addition , the presence of dsRNAs in NBs may suggest that NBs can also sequester dsRNAs and thus may play a role in the innate immune response to RABV infection . Alternatively , given that TLR3 can trigger neuronal apoptosis [53] , the sequestration of TLR3 could be seen as an attempt of RABV-infected neuronal cells to escape TLR3-induced apoptosis . Tanaka et al , 2004 showed an uncoupling of α-synuclein-/synphilin-1-positive aggregate formation and apoptosis [54] . They showed that aggregation of α-synuclein-/synphilin-1 seemed to promote cell survival rather than cell death . In our model , sequestration of TLR3 could be a way for the virus to prevent a TLR3-mediated pro-apoptotic response to infection , as neuronal integrity is required for RABV propagation though the CNS . Promotion of neuronal survival and virus replication are not exclusive and could be complementary strategies . Preliminary results indicate that RABV strains promoting either neuronal survival or death display different effects on NB size . Further experiments will be required to understand the role of NBs in the control of neuronal death . In conclusion , these findings describe a novel role for TLR3 and describe how viruses — in our case , RABV — hijack normal functions of neuronal proteins and exploit cell compartmentalisation to favour the progression of their life cycle .
Human Ntera-2clD/1 cells ( ATCC CRL , 1973 ) , human SK-N-SH cells ( ATCC HTB11 ) and Hek293A ( QBiogene ) were grown in Dulbecco's Modified Eagle Medium ( DMEM ) with Glutamax-I , high glucose and sodium pyruvate 100 mM ( Invitrogen , UK ) , supplemented with 10% FCS-N ( foetal calf serum for neuronal cultures , Invitrogen , U . K . ) , 100 µg/ml streptomycin and 100 U/ml penicillin ( Invitrogen ) . Human NT2-N cells [55] were differentiated from Ntera-2clD/1 cells by trans-retinoic acid and antimitotic treatment as previously described [56] , [57] . BSR cells ( a clone of BHK21 baby hamster kidney cells ) [58] were cultivated in DMEM supplemented with 8% FCS . Human grade III U373MG astrocytoma ( ATCC HTB 17 ) cells were propagated in Dulbecco's modified essential medium supplemented with 2 mM L-glutamine , 1 mM sodium pyruvate , 5% FCS , 2% sodium bicarbonate , 100 U/ml penicillin , and 100 µg/ml streptomycin at 37°C . The human microglial cell line CHME was cultivated as previously described [59] . The laboratory RABV strain CVS ( ATCC vr959 ) , a highly pathogenic strain causing fatal encephalomyelitis in mouse after intramuscular injection [60] , was propagated as previously described [61] . Cells were infected at a multiplicity of infection ( MOI ) of 3 and cultivated for 12 , 24 or 48 h at 37°C , in 5% CO2 . HSV-1 strain KOS [62] was propagated in U373MG cells . TLR3−/− mice were generated as described [2] . Six-week-old C57Bl6 from Janvier ( St . Berthevin , France ) or TLR3−/− female mice were inoculated intramuscularly in both hind legs , with 1×107 infectious particles of RABV . Disease progression was evaluated by scoring clinical signs and mortality . Mobility and mortality were scored as follows: 0 = normal mice , 1 = ruffled fur , 2 = one paralysed hind leg , 3 = two paralysed hind legs , 4 = total paralysis ( defined as the total loss of mobility ) and 5 = death . Daily clinical score was obtained by adding individual scores . Dead mice were reported in the clinical score of the day of the death and thereafter ( cumulative scores ) . Disease progression was presented by a curve of cumulative clinical scores . Mortality was scored daily . Dead mice were counted on the day of death and thereafter ( cumulative score ) . At day 11 after infection , groups of three mice were perfused with PBS . Brains were removed separately and stored at –80°C before being processed for RNA extraction . Animal housing and experimental protocols followed guidelines approved by the French Ministry of Agriculture and Ethical committee . Antibodies ( Ab ) to TLR3 ( Q18 , C20 , H125 ) and anti-IgG goat biotinylated Ab were from Santa Cruz Biotechnology ( Santa Cruz , CA , USA ) . Monoclonal antibodies ( mAb ) to TLR3 - IMG-315C ( FITC-conjugated , immunostaining: 1/50 ) , IMG-315A ( Immunoblot: 1/500 ) and anti-TRIF ( 1/100 ) mouse polyclonal were from Imgenex ( California , USA ) . Hoechst 33342 , RIPA buffer , glutaraldehyde ( G-5882 ) , 20× SSC buffer , poly-D lysine and Ab to vimentin ( Cy3 conjugate ) ( clone V9 ) and γ-tubulin ( clone GTU-88 ) were from Sigma ( Saint-Quentin Fallavier , France ) . Ab to Hsp70 ( SPA-810 , 1/200 ) was from Stressgen Bioreagents ( Victoria , BC , Canada ) . Ab directed against calnexin ( clone 37 , 1/500 ) and EEA1 ( 1/500 ) were purchased at BD Transduction Laboratories . Fc Block™ and CellFIX™ were from BD Pharmingen ( BD , Franklin Lakes , NJ , USA ) . KaryoMAX® Colcemid® Solution in PBS , Alexa Fluor® 488-conjugated anti-IgG rabbit and Alexa Fluor® 594-conjugated anti-IgG mouse were from Invitrogen ( Cergy-Pontoise , France ) ; ProLong® Gold Antifade reagent with or without DAPI and Alexa Fluor® 594-conjugated anti-IgG goat were purchased at Molecular Probes ( Eugene , Oregon , USA ) . R-phycoerythrin-conjugated streptavidin was purchased from Dako ( Trappes , France ) . Ab against CD63 is a kind gift from Eric Rubinstein ( Inserm 602 , Villejuif , France ) . Peroxidase-conjugated donkey anti-IgG mouse and AMCA-conjugated Streptavidin were from Jackson ImmunoResearch Laboratories ( Suffolk , UK ) . For hybridisation experiments: RNA storage solution , proteinase K solution , deionised formamide , ultra pure BSA and yeast tRNA were from Ambion . Mouse mAb directed against the RABV P protein ( 721 . 2 ) , RABV N protein ( PVA-3 ) or the RABV G protein ( PVE-12 ) have been previously described [63]–[65] . FITC-conjugated rabbit anti-RABV NC Ab and kaleidoscope prestained protein standard ( 161-0324 ) were obtained from Biorad ( Marnes-La-Coquette , France ) . Streptavidin peroxidase , hybond-P PVDF ( PolyVinylidine DiFluoride ) , biotinylated sheep anti-mouse IgG ( RPN1001 ) and ECL+ kit were from GE Healthcare ( UK ) . Micro BCA Protein Assay Reagent was from Pierce ( Rockford , IL , USA ) . Ab against α-tubulin ( clone DM1A , 1/1000 ) was from Oncogene Research Products , PhosphoSafe™ Extraction Buffer was from Novagen . Fluoromount-G was obtained from Southern Biotechnology ( Birmingham , AL , USA ) . IgG2a mAb J2 ( 1/1000 ) directed against dsRNA , of length less than or equal to 40 bp , were obtained from English and Scientific Consulting Bt , Szirak , Hungary . Total RNA was isolated from cells using an RNeasy kit ( Qiagen , Germany ) . cDNA synthesis was performed with 2 µg RNA using oligo ( dT ) primers ( 100 ng ) . Superscript II reverse transcriptase ( RT ) and Taq DNA polymerase ( QBiogene ) were used for RT-PCR in a Px2 thermal cycler ( Hybaid Corp . , USA ) . 35 cycles of amplification were performed as follows: 4 min at 94°C , 1 min at 55°C , 1 min at 72°C , 10 min at 72°C , and final cooling . Ribosomal 18S RNA was used as a housekeeping gene . Primers used for amplification of the TLR3 and 18S genes were synthesised by Eurogentec ( Belgium ) , with the following sequences ( 5′→3′ ) : forward-18S: CTTAGAGGGACAAGTGGCG; reverse-18S: ACGCTGAGCCAGTCAGTGTA; forward-TLR3: GAGGCGGGTGTTTTTGAACTAGAA; reverse-TLR3: AAGTCAATTGTCAAAAATAGGCCT . The primer pair used for TLR3 exon IV deletion is ( 5′→3′ ) : Forward-exon4-TLR3: CTCCAGGGTGTTTTCACGCAATTGG; Reverse-exon4-TLR3: TTCAGGTACCTCACATTGAAAAGCC . For quantitative PCR , specific primers for TLR3 were from QIAGEN ( QuantiTect Primer #QT00007714 ) . For amplification of RABV genome the following primers were used ( 5′→3′ ) : F = GGAATTCTCCGGAAGACTGGACCAGCTATGG;R = AGAATTCCCACTCAAGCCTAGTGAACGG . Real-time RT-PCR analysis was performed with an ABI Prism 77700 sequence detection system . Methods and relative quantification of gene expressions were carried out using the comparative method according to the manufacturer's instructions . Sequence of neuronal TLR3 was determined and assigned the GenBank accession number DQ445682 . RABV-infected and mock-infected NTera2clD/1 cells were washed once with phosphate-buffered saline ( PBS ) containing Ca2+Mg2+ ( PBS Ca2+Mg2+ ) , scraped ( Cell Scraper , Corning ) and pelleted in staining buffer ( SB ) ( PBS , 1% inactivated FCS , 0 . 1% sodium azide , pH 7 . 5 ) . Cells were fixed in 4% paraformaldehyde ( PFA ) /PBS for 30 min at 4°C , and resuspended in permeabilisation buffer ( PB ) ( PBS , 1% FCS , 0 . 1% sodium azide , 0 . 1% saponin ) . Cells were then incubated with FITC-conjugated anti RABV NC Ab for detection of viral proteins and/or successively incubated with goat anti-TLR3 Ab ( sc-Q18 ) followed by biotinylated anti-goat IgG Ab and finally with R-phycoerythrin-conjugated streptavidin . Cells were then washed with PB . For staining of non-fixed cells , cells were washed with SB instead of PB and fixed in CellFIX™ ( BD Biosciences , USA ) . Cytofluorimetry was performed with a FACSCalibur™ ( BD Biosciences ) . Results were analysed using the CellQuest™ Pro ( BD Biosciences ) software . Cells were mock- or RABV-infected ( at MOI 3 ) and treated or not at 24 h pi with colcemid ( 0 . 4 µg . ml−1 ) or vehicle alone ( PBS ) . Cells were fixed at 48 h pi and immunostained for NC and tubulin proteins . Widefield observation was performed on Zeiss Apotome and Zeiss Axiovision 4 . 2 software for image acquisition . Nuclei were detected by DAPI staining and NB by anti-NC FITC Ab . Acapella software ( PerkinElmer Acapella 2 . 0 ( TM ) ) was used to delineate inclusions and measure of size of viral inclusions ( area determined by number of pixels ) . The attribution of NB in each cell was computed by defining a region of interest around each nucleus . A set of negative controls has been used to fix the Acapella parameters ( i . e . threshold or contrast adjustment ) . The different characteristics ( area , position and number of NB for each cell ) were saved and exported to Excel ( Microsoft ) . RABV- and mock-infected cells were washed once with PBS Ca2+Mg2+ , fixed with 4% PFA for 30 min at room temperature ( rT ) , washed again , and treated with gelatin ( 1% in water ) for 5 min at 4°C . The samples were then incubated for 20 min at rT in 0 . 3% Triton X-100-PBS and surface IgG receptors were blocked with a saturating buffer ( ST ) ( 2% bovine serum albumin and 5% FCS in PBS ) for 30 min at rT followed by a 10 min incubation at 4°C with Fc-block ( 1/50 ) . Viral NC were detected by incubation with a FITC-conjugated rabbit Ab for 2 h at rT . Primary and secondary Abs were diluted in ST . For immunostaining , cells were incubated with secondary Ab , biotin/ streptavidin , followed by incubation with DAB , resulting in a brown coloration . Nuclei were stained with Hoechst 33342 for immunofluorescence analysis or haematoxylin for transmitted light acquisition . Slides were washed with PBS Ca2+Mg2+ , using water for the last wash . Coverslips were mounted in Fluoromount-G™ or ProLong Gold Antifade reagent ( +/− DAPI if Hoechst previously used ) . Widefield observation was performed on a Leica DM 5000B UV microscope equipped with a DC 300FX camera ( ×40 and ×63 objectives ) . Leica FW 4000 software was used for image acquisition and processing . Confocal images were acquired using a Zeiss LSM 510 META ( version 3 . 2 ) on Axiovert 200 M with a Plan Apochromat 63× and N . A . 1 . 4 objective . Confocal stacks were deconvoluted on Huygens® ( Scientific Volume , Imaging , Netherlands ) and 3D rendering was carried out using Imaris® ( Bitplane AG , Switzerland ) software . Confocal images were compiled using serial Z-stacks 0 . 3 µm apart , representing at least two experiments . Co-localisation of TLR3 and organelle markers was analysed using the colocalisation finder and threshold plugins of WCIF Image J . This allows quantification of the colocalisation ignoring pixels below the automatic thresholds . RABV- and mock-infected cells were washed once with PBS Ca2+Mg2+ . Cells were lysed with PhosphoSafe™ Extraction or RIPA buffer . Soluble and insoluble cellular protein fractions were separated using previously described protocols [33] , [42] . Lysate protein was quantified using the Micro BCA Protein Assay Reagent . Proteins ( 20 µg ) were loaded into a 10% Tris/Glycine SDS polyacrylamide gel with Kaleidoscope pre-stained standards ( 161-0324 , Biorad ) . Proteins were separated and transferred to a Hybond-P PVDF membrane for 2 h ( 150 mA ) , followed by saturation for 2 h at rT in saturating buffer ( PBS-Tween 0 . 1%-Milk 5% ) . Membranes were incubated with primary Ab overnight at 4°C , washed in PBS-Tween 0 . 1% , then incubated with secondary Ab coupled to horseradish peroxidase ( 1 h at rT ) . Membranes were washed again and analysed by chemoluminescence using the ECL+ kit . Signals are acquired with a GBOX ( Syngene ) monitored by the Gene Snap ( Syngene ) software . Confluent RABV-infected or mock-infected SK-N-SH cells were cultured for 48 h at 37°C , in 5% CO2 on untreated 4 mm2 square glass coverslips . Cells were fixed for 30 min at 4°C in a PFA/glutaraldehyde solution . Culture medium was removed and immediately replaced by fixative solution with 2% freshly depolymerised PFA + 2% Glutaraldehyde in 0 . 1 M PBS . Subsequent treatments were performed at 4°C . Cells were rinsed three times in PBS and post-fixed for 30 min in 1% osmium tetroxide in milliQ water and rinsed . They were dehydrated in graded concentrations of ethanol followed by pure acetone before being infiltrated with Araldite . The resin was cured for 3 h at 37°C , then 36 h at 61°C . Ultrathin sections were obtained with a Reichert Ultracut . Sections were counterstained ( or not ) with uranyl acetate and lead citrate and observed on a Jeol 1010 electron microscope . Electron microscopy was also performed on cells fixed in 3% PFA , permeabilised with 0 . 1% Triton ×100 , incubated with anti-RABV NC or anti-TLR3 ( sc-H125 ) antibodies and revealed with DAB . Under these conditions , Abs can access intracellular targets , but cell structures are poorly conserved . Probe was designed to target the leader sequence genome of the RABV strain SAD B19 ( 5′ ACCAGATCAAAGAAAAAACAGACATTGTCAATTG 3′ ) . RNA probe was synthesised by Dharmacon and a fluorescein molecule was added at the 3′ end . RNA probes were prepared according to Dharmacon's protocol and kept in the RNA storage solution . BSR cells were grown on poly-D lysine-coated slides and infected or not with RABV . Medium was removed at 36 h pi and cells were fixed for 30 min at 4°C with 4% PFA in PBS . All steps were carried out in RNAse-free conditions , except for the last washes . Cells were incubated for 5 min in 0 . 2 N HCl , rinsed in 2× SSC at rT and incubated for 10 min at 37°C with Proteinase K ( 20 µg/ml ) in PBS . Slides were subjected to two series of washes in PBS and RNAse free-water , re-incubated for 10 min at 4°C with 4% PFA , dehydrated in graded ethanol solutions ( 70 , 80 , 90 , 95 and 100% ) for 1 min each and air dried . Cells were overlaid for 1 h at rT with pre-hybridisation solution ( 4 mM EDTA , 5× SSC , 50% deionised formamide , 100 µg/ml yeast tRNA , 0 . 5 mg/ml ultrapure BSA ) . Slides were then rinsed in 2× SSC for 1 min , drained and dried with a paper towel . Cells were incubated for 3 h at 55°C with fluorescein-conjugated RNA probe ( 20 ng/µl ) diluted in hybridisation solution ( 4× SSC , 50% deionised formamide , 0 , 1% SDS , 8% dextran sulphate , 50 mM DTT ) or with hybridisation solution only . Following hybridisation , cells were washed twice at RT in 2× SSC and 0 . 1× SSC for 10 min . Slides were then air-dried and mounted using ProLong® Antifade ( Invitrogen ) . Samples were viewed using a Leica DM 5000B UV microscope and Leica TCS SP5 for confocal analysis . TLR3-specific miRNA sequences cloned into pcDNA™6 . 2-GW/EmGFP-miR were purchased from Invitrogen ( U . K . ) . High purity plasmid DNA was purified over JetStar columns ( Genomed , Bad Oeyenhausen ) . For transient transfections , cells were grown to 80%–90% confluence and nucleofected using the Amaxa system as follows: HEK293A cells ( Qbiogene ) ( 106 ) in 100 µl Nucleofector solution L ( Amaxa ) were mixed with 5 µg of the TLR3-specific miRNA or control ( pcDNA™6 . 2-GW/EmGFP-miR-NEG ) plasmid in a cuvette and subjected to Amaxa program Q-001 conditions . Nucleofected cells were grown in supplemented complete DMEM medium . 24 h post-nucleofection cells were incubated with blasticidin ( 5 µg/mL ) and infected with RABV ( MOI 3 ) . 48 h pi cells were fixed and stained with the different Ab for further flow cytometry analysis . Total RNA extraction ( RNeasy Kit , Qiagen , Germany ) was also performed to determine the efficiency of silencing . Cells grown on slides were fixed with PFA 4% for further staining of TLR3 and viral NC proteins . Calculation of silencing efficiency was done as follows ( cf TechNotes Volume 15 ( 2 ) , Applied Biosystems ) : the mean CT were calculated for each condition and for both the reference gene ( 18S ) and the gene of interest ( TLR3 ) . 2 values are calculated: ΔCT ( = mean CT ( experimental ie TLR3 ) - mean CT ( endogenous ie 18S ) ) and ΔΔCT ( = ΔCT ( experimental ) −ΔCT ( negative control ) ) . The percent knockdown is obtained as follows: %KD = ( [1–2−ΔΔCT]*100 ) . pUno-mcs and pZERO-hTLR3- ( deltaTIR ) plasmids ( Invivogen ) were amplified according to Invivogen protocol and purified using JetStar kit Plasmid purification MAXI kit ( Genomed ) . DNA was quantified and plasmids used for nucleofection of Hek293 cells ( Amaxa program Q-001 ) ( 5 µg plasmid for 1×106 cells ) . 48 h post-nucleofection cells were infected with RABV and 24 h post-infection cells were harvested for total RNA extraction . Q-PCR was carried out as described above . Stable cell lines were established by addition of selective antibiotics ( blasticidin or puromycin ) to culture medium; cells were passaged over several weeks ( ≥5 weeks ) . Graphs ( Figures 1C , 4D , 7B , 7C , 8A , 8B , 9 , S4 ) and Student's T tests ( Figures 4D left , 7B , 7C , 9 , S4 ) were generated and performed using GraphPad Prism version 5 . 00 for Windows ( GraphPad Software , San Diego California USA , ) . For mice experiments , collected data were plotted for comparison of Kaplan-Meier survival curves and statistical analysis was performed with MedCalc software . | Viruses are obligate parasites . The progression of their life cycle depends on their hijacking the cellular metabolism and machinery . Human neurons produce TLR3 , a protein involved in early host defence mechanisms and the modulation of neuronal survival . Rabies virus is a neurotropic virus , infecting mainly neurons . In this study , we showed that rabies virus exploits TLR3 function to store viral proteins and viral genomic material in particular areas of the cell where virus multiplication occurs . We found that , during the course of infection , large ( 1–3 µm ) spherical inclusions were formed within the region around the nucleus . These inclusions were composed of an inner core of aggregated TLR3 surrounded by a coat of viral proteins and genomic material . These inclusions were revealed to be the previously described Negri Bodies ( NBs ) . In absence of TLR3 , NBs were no longer formed and virus multiplication rate decreased . Mice deficient in TLR3 were more resistant to rabies and had lower levels of infection in their brains . This study shows how neurotropic viruses , such as rabies virus , hijack normal functions of neuronal proteins and use cell compartmentalisation to promote viral multiplication . | [
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] | 2009 | Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies |
Leptospirosis is an epidemic-prone neglected disease that affects humans and animals , mostly in vulnerable populations . The One Health approach is a recommended strategy to identify drivers of the disease and plan for its prevention and control . In that context , the aim of this study was to analyze the distribution of human cases of leptospirosis in the State of Rio Grande do Sul , Brazil , and to explore possible drivers . Additionally , it sought to provide further evidence to support interventions and to identify hypotheses for new research at the human-animal-ecosystem interface . The risk for human infection was described in relation to environmental , socioeconomic , and livestock variables . This ecological study used aggregated data by municipality ( all 496 ) . Data were extracted from secondary , publicly available sources . Thematic maps were constructed and univariate analysis performed for all variables . Negative binomial regression was used for multivariable statistical analysis of leptospirosis cases . An annual average of 428 human cases of leptospirosis was reported in the state from 2008 to 2012 . The cumulative incidence in rural populations was eight times higher than in urban populations . Variables significantly associated with leptospirosis cases in the final model were: Parana/Paraiba ecoregion ( RR: 2 . 25; CI95%: 2 . 03–2 . 49 ) ; Neossolo Litolítico soil ( RR: 1 . 93; CI95%: 1 . 26–2 . 96 ) ; and , to a lesser extent , the production of tobacco ( RR: 1 . 10; CI95%: 1 . 09–1 . 11 ) and rice ( RR: 1 . 003; CI95%: 1 . 002–1 . 04 ) . Urban cases were concentrated in the capital and rural cases in a specific ecoregion . The major drivers identified in this study were related to environmental and production processes that are permanent features of the state . This study contributes to the basic knowledge on leptospirosis distribution and drivers in the state and encourages a comprehensive approach to address the disease in the animal-human-ecosystem interface .
Even though leptospirosis is now recognized as a disease of epidemic potential with a significant health impact in many parts of the world , it remains a neglected disease . Its burden is estimated at 500 , 000 severe human cases per year worldwide , but a WHO expert group recently put its annual global incidence at 1 . 03 million people with 58 , 900 deaths [1 , 2] . Nevertheless , leptospirosis continues to be a silent disease [3] , mainly due to the paucity of data in many countries , including of the Americas [4 , 5] . The Leptospira bacteria may also affect a wide variety of animal species , both wild and domestic , which serve as reservoirs for human infection [6] . The diversity of animal carriers represents a significant challenge for prevention and control [7] . Exposure to water and soil contaminated by the urine of infected animals is the most common route of transmission to people and domestic animals [6] . Leptospirosis is an excellent example for the “One Health” approach , where the relationship between humans , animals and ecosystems is studied to improve knowledge on a disease and to enhance collaborative intersectoral and multidisciplinary control strategies [8] . The One Health working definition states that “it is feasible to integrate human , animal , and environmental health efforts to predict and control certain diseases at the human–animal–ecosystem interface; integrated approaches that consider human , animal , and environmental health components can improve prediction and control of certain diseases” [9] . Yet this approach is rarely used to advance knowledge on leptospirosis transmission , develop evidence-based policies , and create tools to save human lives and reduce the impact on domestic animals . Leptospirosis is one of the major neglected diseases in Latin America [10] . It has been reported in a variety of settings , from large urban centers , to remote rural areas [11 , 12 , 13 , 14 , 15] . Socioeconomic drivers include living in dense urban or peri-urban areas with inadequate waste collection and sanitation , which is often associated with vulnerable populations [16] . Leptospirosis has been linked to poverty , lack of water and sanitation , and poor housing conditions [12 , 17] . Environmental drivers have been identified in previous studies . Heavy rains or floods have been linked to a higher number of cases of leptospirosis [12 , 16 , 18 , 19 , 20 , 21] . Alkaline and neutral soil are suspected of promoting a longer survival of the bacteria , especially in young , not-yet-structured soils like those of volcanic origin [12 , 22] . In addition , soil temperature and proximity to water bodies were also reported as potential enablers for bacterial survival [23] . Leptospirosis is also considered an occupational disease , affecting rice laborers , sewer workers , animal handlers and gold miners [6 , 24 , 25] . A better understanding of the drivers for leptospirosis would provide crucial information for decision-makers to be able to target risk areas for priority interventions . Indeed , the current gaps in scientific and technological knowledge hinder the detection of cases and limit surveillance and control programs . Finally , because the instruments needed for control and elimination–such as broad rapid tests and vaccines–are not available at this time , leptospirosis is not considered to be “tool ready” and as a result , it is not targeted through large-scale global initiatives . Brazil is the fifth most populous country in the world ( approximately 200 million people ) and has the seventh highest gross domestic product ( USD 2 , 250 , 673 ) . Even though the economy has been growing steadily , there are still around 20 million people ( 10% of the population ) living in poverty in the country [26] . This population group is the most vulnerable to neglected diseases and other poverty-related infections . Previous studies have demonstrated the impact of neglected tropical diseases in Brazil and the need to develop new tools and technologies to fight them [27 , 28] . There is no accurate surveillance system in place for leptospirosis globally or in the Americas; however , some countries’ surveillance systems include the disease and estimates of its public health burden are available [5 , 29] . Notification of human leptospirosis is mandatory in Brazil and an annual average of 3 , 888 cases with 9 . 48% fatality are officially reported by the country’s surveillance system [30] . The Brazilian state of Rio Grande do Sul ranks fifth in the incidence rate ( 4 . 7 cases per 10 , 000 population ) and presents around 15% of the total number of cases in the country [31] . In a previous study conducted to identify high transmission areas and possible ecological components of leptospirosis transmission in Rio Grande do Sul , the highest incidence rates were found in the coastal sedimentary areas with low altitude and predominantly agricultural land use in the central valley [32] . The state economy is based on agribusiness , including cattle and rice paddies , with an associated increased risk of leptospirosis in some areas that needs to be evaluated . The objective of this study is to analyze the distribution of human cases of leptospirosis in the state of Rio Grande do Sul and explore possible drivers using the One Health approach . This analysis may orient further studies of the disease in the human-animal-ecosystem interface and the results of this study may be used as evidence to inform decision makers in the state .
An ecological study was carried out using aggregated data by municipality to analyze the situation of leptospirosis in all 496 municipalities ( corresponding to the second subnational administrative level ) in the state of Rio Grande do Sul , Brazil , between 2008 and 2012 . A geo-coded database was created using different sources . Variables were either downloaded or created from original sources . The data source used for each variable is described in the S1 Supporting Information . Human leptospirosis case data , de-identified and aggregated at the municipality level , were obtained from the Ministry of Health of Brazil national surveillance system database ( acronym in Portuguese SINAN ) [33] . All case data were publicly available by open consultation on the government website . Seven environmental variables ( ecoregion , type of soil , temperature , precipitation of the wettest month , altitude , slope of the land ( hill incline ) , and drainage ) were gathered from diverse “open-access” data sources and used in the study . When disaggregated , the ecoregion and type of soil variables turned into six and twelve variables , respectively . Altitude and hydrology information used in the background were obtained from the USGS-EROS , HYDRO1k Elevation Derivative Database [34] . Bioclimatic variables were calculated from monthly temperatures and rainfall values [35] . Geo-processing techniques were applied to assign and measure environmental variables for each municipality . Data for the socioeconomic variables ( gross domestic product per capita , Gini coefficient , illiteracy rate ) were gathered from the Brazilian Institute of Geography and Statistics ( acronym in Portuguese IBGE ) [36] . As the economy of Rio Grande do Sul is based on agribusiness , variables related to rice and tobacco production were collected from the IBGE database and data on bovine raising were provided by the Department of Agriculture of the state of Rio Grande do Sul and the Federal University of Rio Grande do Sul [37] . Leptospirosis cases: According to the Ministry of Health of Brazil , human cases of leptospirosis are those presenting clinical symptoms consistent with the disease and confirmed by laboratory diagnosis either with ELISA–IgM or MAT [38] . These laboratory confirmation techniques are available at the state level at the Central Public Health Laboratory , which is part of the National Public Health Laboratory Network . In Brazil , a case could also be confirmed by clinical-epidemiological criteria ( selected symptoms with epidemiological history ) [39] . All cases in the SINAN database were considered confirmed . In the original database , the cases were classified according to area of residence ( urban , peri-urban or rural ) [39] . Cumulative incidence: the number or proportion of a group ( cohort ) of people who experience the onset of a health-related event during a specific time interval [40] . In this study , it was estimated per 10 , 000 inhabitants . Criteria for risk stratification: The criteria used were based on a previous risk stratification study conducted in Nicaragua [12] . In summary , geographical areas were classified into two categories: i ) Silent Area ( no cases were reported during the study period ) and ii ) Productive Area ( active transmission was reported during the study period ) that could be an endemic or critical area ( higher quintile ) . Rio Grande do Sul is the southernmost state in Brazil bordering Argentina and Uruguay . The state covers an area of 281 , 731 , 445 km² , divided into 496 municipalities . In 2010 , the population was 10 , 693 , 929 inhabitants , 85 . 1% of which lived in urban areas , among them the Porto Alegre metropolitan area where 15% of the state population resides ( 1 , 472 , 482 inhabitants ) [41] . In 2010 , the state had the 4th highest gross domestic product per capita in the country and the Gini index ( 0 . 5472 ) was lower than the national level [36] . There are three major economic regions: 1 ) the south with greater land concentration , large cattle raising farms , and mechanized plantation of rice , soybean and wheat . This area also presents higher income inequality; 2 ) the northeast region , which includes the state capital , with more industries and predominantly small properties; and 3 ) the northern region , mostly colonized by European immigrants , with higher forest coverage , valleys , and plain areas with small agricultural lands . The most common agrarian structure in the state ( 90% of properties ) is a small family farm covering an area smaller than 100 hectares [42] . The hydrology of the state is basically divided into two main areas: the La Plata and Atlantic East Coast watersheds . The La Plata watershed is located in the northern area bordering with Argentina , and comprises the Uruguay River and mayor tributaries ( Caboa , Pelotas , Ibicui and Mirinay ) . The Atlantic East Coast watershed is mainly shaped by the Guaiba and Litoranea basins . The Guaiba basin includes tributaries of the Dos Patos coastal lagoon , the Jacui and Tacuari rivers , which run in the central area of the state and wash its most populated areas; other tributaries include the Sinos and Cai rivers , which also flow into the Dos Patos lagoon . ( S2 Supporting Information ) . The Litoranea basin encloses the Camaqua and Piratini rivers also flowing into the lagoon . The city of Porto Alegre is located in the Atlantic East Coast watershed . Two types of analyses were carried out to investigate associations between the risk of human leptospirosis and 26 variables selected as possible environmental , socioeconomic or livestock drivers: 1 ) a spatial analysis and thematic mapping of possible drivers , showing the municipalities in the higher quintiles of rates over the distribution of the variable also divided by quintiles ( range cuts ) and 2 ) a statistical analysis with univariate and multivariable regressions as described below . The spatial analysis consisted in computing zonal statistics and surface for the environmental variables , in addition to the geocoding of health and socioeconomic data . ArcGIS zonal statistics by municipality ( min , mean , max , standard deviation , range ) were calculated for the altitude , slope , temperature , and rain variables . Geo-processing geometric intersection of environmental features shaped the municipal surface of ecoregions and soils . Quintile thematic mapping was conducted once the municipal statistics of environmental , health and socioeconomic variables were geo-processed . For the multivariable regression , the dependent variable was the case count per municipality and the independent variables all 26 possible drivers . The ecoregion and soil variables were dichotomized as follows: they were coded 0 when they were a minority ( when land coverage or proportion was less than or equal to 50% of the municipality ) and they were coded 1 when they represented a majority ( when land coverage or proportion was above 50 . 01% of the municipality . ) The proportion of farms with up to ten animals per farm ( small farms ) was dichotomized in the same fashion . In addition , two bovine-related variables were created and used as continuous variable: the proportion of farms per km2 and the proportion of bovines per km2 . Productive processes such as the cultivation of tobacco and rice were analyzed on a unit of 10 , 000 tons . Predictors were analyzed with negative binomial regression ( NB ) and robust variance was used to estimate the relative risk ( RR ) and 95% confidence interval ( CI ) of the estimates [43] . The link function used was the default logit-function and the offset was the natural log-transformed number of population per municipality . Univariate analyses were first run for each of all 26 variables and 14 were preselected due to P ≤ 0 . 15 . Variance inflation factors ( VIF ) were estimated to verify the relations among all selected independent variables and check for potential collinearity . When a high VIF was found ( VIF>4 ) , the variable with a lower P-value was eliminated and the process was reiterated until only variables with a VIF<4 were left . Confounding effects were investigated by checking changes in the point estimates of the variables that remained in the model . Parameters with changes in their estimates > 25% were considered confounders and were retained during the variable selection process . Finally , two-way interaction terms between environmental variables with biological plausibility were investigated ( slope and altitude , altitude and drainage , altitude and precipitation of the wettest month ( mm ) ) . We used deviance performance as a goodness of fit test for the overall model . Results of the variables with P value > 0 . 15 are included in the S3 Supporting Information . Statistical tests were performed for over-dispersion evaluation and model fit comparison using literature-recommended approaches [44] . For the negative binomial model , the dispersion parameters were tested for difference with chi-squared statistics . To compare goodness of fit between pairs of the proposed regression models , Vuong statistics were calculated [45] . The following models were compared: Poisson regression , zero-inflated negative binomial ( ZINB ) and a negative binomial regression ( NB ) [46] . The differences in AIC and Vuong statistics were computed for all pairs of non-nested models ( i . e . , Poisson vs . ZINB , Poisson vs . NB , NB vs . ZINB ) . Based on the AIC and Vuong tests , the negative binomial regression model fit the data better in comparison with others . More details about the procedures carried out in order to compare models can be found in the S4 Supporting Information .
During the study period , 2 , 141 confirmed cases of leptospirosis were reported with an average of 428 cases annually . The yearly incidence remained similar over the period ( 412–543 cases ) , except for a lower than average number of cases ( 277 ) in 2012 ( Table 1 ) . A total of 233 municipalities out of 496 reported at least one case ( range 1–208 ) during the study period . Among the 263 municipalities not reporting cases , most had small populations ( less than 10 , 000 people ) . On average , there were 4 . 32 cases per municipality . A high number of urban cases were clustered in the Metropolitan Region of Porto Alegre ( 50 . 52% of all urban cases ) and groups of predominantly rural cases were located in the Center Oriental Region along the Jacui and Tacuari rivers and in the Southeast Region , near the great lagoons ( Fig 1 ) . Forty-seven municipalities concentrate 79 . 40% of the cases . Most of these municipalities are located in the Atlantic East Coast watershed , specifically in the Jacui-Tacuari and Cai basins ( 1 , 116 cases ) . The cumulative incidence for the entire state was 2 cases per 10 , 000 inhabitants ( range: 0–56 . 56 ) . The spatial distribution of the municipalities’ cumulative incidence by quintiles , as well as of those not reporting cases , are presented in Fig 2 . The cumulative incidence for rural areas ( 6 . 16 per 10 , 000 people ) was eight times higher than for urban areas ( 0 . 74 per 10 , 000 people ) . Risk stratification was carried out for the 496 municipalities in Rio Grande do Sul to inform planning of leptospirosis prevention and control activities . It shows that out of the 496 municipalities in the state , 263 ( 53 . 02% ) can be considered silent areas . Among the 233 productive areas ( 46 . 98% ) , 58 were found to be endemic and 75 municipalities were considered critical areas ( S5 Supporting Information ) . These 75 municipalities reported 1 , 766 cases ( 82 . 48% ) out of the total of 2 , 141 cases in the entire state . The negative binomial regression modeling identified fourteen variables that suggested some relation with the number of cases per municipalities in the univariate analysis ( P≤0 . 15 ) ( Table 2 ) . The 12 variables that were analyzed as possible drivers ( P>0 . 15 ) are presented in the S3 Supporting Information . The final model identified four variables as significantly associated with the leptospirosis case count ( P ≤ 0 . 05 ) ( Table 2 ) : the Parana/Paraiba interior forest ecoregion ( RR = 2 . 25; IC95% = 2 . 03–2 . 49; P < 0 . 001 ) ; Neossolo Litolítico soil ( RR = 1 . 93; IC95% = 1 . 26–2 . 96; P = 0 . 006 ) ; to a weaker extent , production of tobacco per 10 , 000 tons ( RR = 1 . 10; IC95% = 1 . 09–1 . 11; P <0 . 001 ) ; and finally , a borderline association with production of rice per 10 , 000 tons ( RR = 1 . 003; IC95% = 1 . 002–1 . 04; P <0 . 001 ) . The goodness-of-fit model was tested by a deviance chi-squared test and was found not to be significant ( P > 0 . 05 ) .
Leptospirosis is an important public health problem for the state of Rio Grande do Sul , with an average of 428 cases reported annually . The actual incidence is probably much higher , particularly in vulnerable populations , because leptospirosis is commonly misdiagnosed and unreported . The study results show that rural populations of the state have an approximately eight times higher risk of contracting leptospirosis than their urban counterparts , even though the number of reported cases was high in both areas . Rural cases are concentrated mostly in two zones , characterized by higher tobacco production ( central state ) and higher rice production ( south ) . This result suggests that preventive strategies in these regions require strong collaboration between the Health and the Agriculture sectors . Preventing cases among rural workers will be the main goal for these areas . In order to contribute to the One Health discussion and support possible intersectoral collaboration , suggestions for components of a leptospirosis plan and activities in the state of Rio Grande do Sul are presented in the S6 Supporting Information; using both the working definition of One Health and the results of this study as a basic scenario [9] . The highest concentration of urban cases was found in the metropolitan areas of cities with low altitude , including the state capital , which also suffer from frequent flooding . At the center of the state , high rate areas were found within the Atlantic East Coast watershed , which receive water from the Jacui , Taquari , Cai , Sinos and others rivers as mayor tributaries , all well known for their periodic flooding [48] . This suggests that leptospirosis prevention should also be considered in natural disaster plans and that the main goals for these areas should be to reduce the number of severe cases and save lives during an outbreak . Other possible drivers for leptospirosis identified in the final regression model were the Parana-Paraiba ecoregion , Neossolo Litolítico soil , and the production of tobacco . The Parana/Paraiba interior forest ecoregion showed an RR of 2 . 25 when compared to other ecoregions . This ecoregion is part of the Atlantic semi-deciduous forest biome that covers central and northern areas of the state and extends over numerous areas of Brazil and neighboring countries . It ranges from river plains to middle-level highlands . The climate is subtropical [49] so humidity may act as an important factor in the leptospirosis bacteria survival . The Parana-Paraiba ecoregion includes a diversity of soil types that vary from very fertile to impoverished . Neossolo Litolítico , a very young and not yet structured type of soil , is one of those that correlate geographically with high rates of human leptospirosis . Its RR is 1 . 93 when compared with municipalities with other predominant soil type . It has been suggested that its pH [50] and/or its low drainage capacity [51] may favor the bacteria survival . With each additional 10 . 000 tons of tobacco produced by the municipality , there was an increased RR in the leptospirosis cases of 1 . 10 . There is spatial overlapping of municipalities with high production of tobacco and higher rates of the disease in the Jacui-Tacuari valley . In the central area , several conditions concur with higher leptospirosis cases , in particular tobacco ( and rice ) production in a subtropical forest environment , over slope and inclined terrain with shallow weathered soils . We did not find information in the literature suggesting an association between tobacco plantation and leptospirosis cases . However , tobacco growth requires a pH of 5–6 . 5 [52] , which matches the leptospirosis bacterial soil requirement of 6 . 2 for survival over 7 weeks in the environment [22 , 53] . PH survival requirements vary among serovars , and as an example L . interrogans serovar hardjo prefers a pH of 6 . 5–6 . 8 [54] . Further studies analyzing which serovars are circulating in humans and in animals in these high risk areas will provide additional knowledge on the cycle of leptospirosis transmission . The agro-industrial production and export of tobacco are concentrated in three states in the south of Brazil . Rio Grande do Sul and the neighboring state of Santa Catarina are responsible for 80% of the production . Brazilian tobacco is grown by approximately 186 , 000 farming families in properties with an average area of 16 hectares [55] . Every step in the plantation of tobacco in Southern Brazil is carried out manually , mostly by family members from several generations of small farmers descended from Europeans immigrants [55] . Most of the farms have a small number of bovines , mostly dairy cows , although swine are also very frequent [37] . In this region , the most common feed for livestock is corn , mostly stocked on the farm where it attracts rodents . Insects and rodents are responsible for an estimated loss of 15% of all corn storage on small farm barns [47] . This region also includes large areas of preserved forests , which probably increases contact with wild rodents . Further studies about the presence of rodents and programs for rodent control would provide important information on their true impact . In the final model , the association with rice production was weak ( around 1 but statically significant ) , but there was some spatial overlapping of municipalities with high leptospirosis cumulative incidence and rice plantations in the Lagoa dos Patos shoreline . The state of Rio Grande do Sul produces 63% of all rice in Brazil [56] . The association between rice paddy workers and leptospirosis has already been described in the literature [6 , 57] . Working conditions in rice fields have been studied in Peru and results highlight occupational hazards linked to leptospirosis infection , such as long period of exposure to water , lack of use of any personal protection equipment , and presence of skin wounds [57] . It is very important to raise awareness about the risk of this disease in critical areas and promote personal protection equipment ( PPE ) . In the state of Rio Grande do Sul , where temperatures exceed 24°Celsius ( 75°Fahrenheit ) for half of the year , educational campaigns for the adequate use of PPE ( such as boots ) among workers are unlikely to be effective , especially during long shifts . The most important prevention tool for leptospirosis in high risk areas and especially under these conditions is vaccination , however , a human vaccine is not available in the majority of affected countries . In the central areas of the state , smaller livestock producers ( less than 10 bovines by property ) are predominant [37] , but this variable only presented relevance in the univariate analysis . Similarly , the proportion of bovine farms per km2 has shown some relation with the number of leptospirosis cases . The proximity with animals such as dogs increases the risk for leptospirosis [58] . A previous study found that leptospirosis titers were present in 39% of bovines in the state [59] . Further studies are required to better understand the epidemiological situation in the animal sector . This study provides evidence of the importance of the environmental component and the One Health approach for leptospirosis . As highlighted here , where the ecoregions were suitable for specific plantations ( such as tobacco in Parana-Paraiba ) human occupation of these territories has led to transformations of the natural environment and increased the use of natural resources for economic purposes [60] . Further research is required , using primary data , to further explore this finding , as well as the potential relationship between the type of ecoregion and economic processes , and their relationship with the animal-human interface . For other diseases , such as rabies transmitted by vampire bats , outbreaks have been reported rapidly in the Amazon region due to some productive processes such as gold prospection [61] . Rio Grande do Sul has one of the highest GDP per capita in the country [62] . For this reason , the study includes the Gini index that reflects possible inequalities related to the distribution of income among the inhabitants of the municipalities . However , the Gini index presented relevance only in the univariate analysis and no association was found in the final model . This results of this study provide potentially useful information for government decision-makers in the elaboration of a possible intersectoral plan . Evidence-based policy-making requires advanced social planning capacity , including the capacity to monitor and evaluate programs in the different ministries and agencies that provide services to the most vulnerable populations . Risk stratification generates evidence for decision-making and the critical areas identified by this study could be used as pilots for future local intersectoral studies . In the same way , areas identified as silent require attention from the health authorities to confirm the absence of disease in humans or the need to improve surveillance . The prediction , detection , prevention , and response to outbreaks of leptospirosis will be improved and better defined through knowledge generated by an integrated approach within the animal-human-ecosystem interface . This study clearly highlights the importance of a multidisciplinary and intersectoral approach , which was at the root of the creation of initiatives such as the Global Leptospirosis Environmental Action Network ( GLEAN ) . GLEAN brings together experts from different disciplines and sectors with the objective of orienting knowledge regarding the prediction , prevention , detection , and response to leptospirosis; and transforming relevant research findings into operational tools for affected communities and countries , as well as for organizations [63] . Although the use of aggregated data by municipality is a possible limitation of this study , the geographical approach helped to gather , unify and shape all factors ( environmental , socioeconomic , and health ) into the same area and detect spatial patterns . Ecological studies are an inexpensive way to use secondary available data; however they are commonly associated with the ecological fallacy [21] . Another limitation possibly resides in the large number of municipalities with small populations ( under 10 , 000 people ) . However , the possible effect of this data characteristic was considered in the statistical analysis through the use of negative binomial regression , by testing the effects using strata with and without small population . Also , no significant difference was found by analyzing the same approach using only municipalities with at least one positive case . This study contributes to the basic knowledge on leptospirosis distribution and drivers in the state of Rio Grande do Sul and points to the direction of tool-readiness for the disease . It also encourages a holistic approach for the disease , taking into account human , animal , and ecosystem interactions and supporting research in the development and validation of a rapid diagnostic test for humans and animals , and eventually production of a vaccine that is still desperately needed . | Leptospirosis is a bacterial disease affecting humans and several animal species , which serve as reservoirs for infection . Contamination happens through exposure to urine of infected animals in water and soil . A better understanding of the factors that affect the transmission of the disease , incorporating the relationship between humans , animals , and ecosystems within the One Health approach , would allow for tailored prevention and control measures at the local level . The aims of this study were to analyze the distribution of leptospirosis human cases and the possible factors that influence the transmission of the disease in the state of Rio Grande do Sul , as well as provide evidence to support the development of interventions and guide new studies in the region . Leptospirosis cases and possible environmental , socioeconomic , and livestock factors were analyzed . The study used data by municipality ( all 496 ) obtained via an open access database . Georeferenced maps were constructed and statistical analysis performed for 26 variables . A multivariable regression analysis was carried out . An annual average of 428 human cases of leptospirosis was reported in the state from 2008 to 2012 . The cumulative incidence in rural populations was eight times higher than in urban populations . Variables significantly associated with leptospirosis cases in the final model were: ecoregion , type of soil and to a lesser extent tobacco and rice production . Results showed that leptospirosis cases were concentrated in the state capital and in specific ecoregions . Environmental and agricultural production processes were identified as possible risk factors and these conditions are probably permanent characteristics of the state . This study contributes to the knowledge on leptospirosis distribution and risk factors in the state , and also highlights the importance of a holistic view and intersectoral approach in preventing the disease . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [] | 2015 | Leptospirosis in Rio Grande do Sul, Brazil: An Ecosystem Approach in the Animal-Human Interface |
Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira . Infection can lead to illness ranging from mild and non-specific to severe , with jaundice , kidney and liver dysfunction , and widespread endothelial damage . The adhesion of pathogenic Leptospira species ( spp . ) , the causative agent of leptospirosis , to host tissue components is necessary for infection and pathogenesis . While it is well-established that extracellular matrix ( ECM ) components play a role in the interaction of the pathogen with host molecules , we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components . Using in vitro phage display to select for phage clones that bind to EA . hy926 endothelial cells , we identified the putative lipoproteins LIC10508 and LIC13411 , and the conserved hypothetical proteins LIC12341 and LIC11574 , as candidate L . interrogans sv . Copenhageni st . Fiocruz L1–130 adhesins . Recombinant LIC11574 , but not its L . biflexa homologue LBF1629 , exhibited dose-dependent binding to both endothelial and epithelial cells . In addition , LIC11574 and LIC13411 bind to VE-cadherin , an endothelial cell receptor for L . interrogans . Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction , a likely indication that these proteins are outer membrane localized . All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot . This work has identified bacterial adhesins that are potentially involved in L . interrogans infection of the mammalian host , and through cadherin binding , may contribute to dissemination and vascular damage . Our findings may be of value in leptospirosis control and prevention , with the bacterial adhesins potentially serving as targets for development of diagnostics , therapeutics , and vaccines .
Leptospirosis , caused by pathogenic species of the genus Leptospira , is the most widespread zoonosis and has emerged as a major public health problem worldwide , particularly in the humid tropical and subtropical areas in developing countries . The clinical manifestations of human leptospirosis are diverse , ranging from mild illness to a severe disease form known as Weil's syndrome , characterized by jaundice , acute renal and hepatic failure , pulmonary distress , and hemorrhage . Specific groups at risk for infection include farmers , sewer workers , slaughterhouse workers , and veterinarians [1] . Additionally , outbreaks of this disease are associated with severe flooding events particularly in urban slum areas [2]–[4] . During infection , Leptospira spp . can rapidly disseminate from the portal of entry to any tissue of the host , and persist in the proximal tubules of the kidney of reservoir hosts such as rodents . Leptospirosis is spread through the contact of mucous membranes , cuts , or abrasions with fresh water contaminated by the urine of infected animals [5]–[7] . The mechanisms involved in leptospiral pathogenesis are not well elucidated . However , it is generally thought that the adhesion of Leptospira spp . to host tissue components is a necessary step for infection and pathogenesis . Attachment to host cells , and extracellular matrix ( ECM ) components , is likely to be necessary for the ability of leptospires to penetrate , disseminate and persist in mammalian host tissues . It has been demonstrated that pathogenic Leptospira binds to a variety of cell lines including fibroblasts , epithelial , endothelial , microglial and monocyte/macrophage [8]–[16] , and ECM components [17]–[25] in vitro . Many pathogenic bacteria express adhesive molecules ( adhesins ) on their surfaces that promote interaction with host cell receptors or with other macromolecules . Adhesins are surface-exposed proteins , examples of which include intimin in Escherichia coli [26] and invasin in Yersinia pseudotuberculosis [27] . A number of L . interrogans proteins have been shown to bind different ECM components in vitro , including leptospiral immunoglobulin ( Ig ) -like repeat proteins LigA and LigB [20] , [22] , [24] , [28]–[31] , LenA [21] , and OmpL1 [25] . OmpL1 , a 31-kDa transmembrane outer membrane protein ( OMP ) , binds to laminin , plasma fibronectin , and plasminogen [25] , [32] , [33] . Although it is well established that ECM components play a role in the interaction of the pathogen with host molecules [17] , [20] , [21] , [23]–[25] , [30] , [34] , recent data generated in our laboratory show that pathogenic Leptospira bind host cells more efficiently in vitro [8] , [35] . Attachment of L . interrogans to endothelial cell monolayers is mediated at least in part by the host cell surface protein VE-cadherin [35] , although the leptospiral adhesin ( s ) involved in this interaction remained unknown . In this study , we identified L . interrogans proteins involved in interactions with host cells using phage display . Phage display has been successful in identifying adhesins such as P66 in another spirochete , Borrelia burgdorferi [36] , [37] . This work also describes the characterization of the binding properties of the candidate adhesins , and provides evidence that these proteins are expressed during Leptospira infection .
All work with animals was performed under guidelines of the Public Health Services Policy on Humane Care and Use of Laboratory Animals . Mice were euthanized by controlled CO2 administration followed by cardiac puncture . The Institutional Animal Care and Use Committee of the Medical College of Wisconsin approved all work with animals . The protocol number is AUA2391 . BALB/c mice were purchased at the age of 3 weeks from Charles River Laboratories ( Wilmington , MA ) . Mice were fed and watered ad libitum throughout the course of the experiment . Leptospira interrogans serovar Copenhageni ( pathogenic , strain Fiocruz L1–130 ) was reisolated by infection of hamsters , and then stored at passage 1 and 2 in liquid nitrogen . Frozen aliquots were thawed and passaged in liquid Ellinghausen-McCullough-Johnson-Harris ( EMJH ) medium [6] supplemented with 5-fluorouracil ( Sigma-Aldrich , St . Louis , MO ) and 10% heat-inactivated rabbit serum ( Gibco , Grand Island , NY ) at 30°C . This strain has a 50% lethal dose range of 37–104 in hamsters [38]–[40] and the genome sequence was previously reported [41] . L . biflexa serovar Patoc ( avirulent; strain 23582 ) was obtained from the American Type Culture Collection ( ATCC ) ( Manassas , VA ) and grown in EMJH medium . Genomic DNA used to construct the phage library was obtained from growing 1 liter of L . interrogans . L . interrogans DNA was phenol- and chloroform-extracted , ethanol-precipitated and resuspended in 100 µl Tris-EDTA pH 8 . 0 as previously described [42] . Escherichia coli strain MC1061 ( F− ) was used as the host for the generation of the phage display library while E . coli strain TG1 ( F+ ) was used to recover selected phage clones through infection . E . coli strain Top10 was utilized for gene cloning , and E . coli strain KS330 was used for protein expression and purification . E . coli were grown in Luria-Bertani ( LB ) or 2x YT medium ( BD , Sparks , MD ) supplemented with 0 . 2% v/v dextrose at 30°C with shaking . For plating , the media were supplemented with 15 g/l agar . Antibiotics ( tetracycline , 12 . 5 µg/ml , ampicillin , 100 µg/ml and chloramphenicol , 30 µg/ml ) ( Sigma-Aldrich ) were added as appropriate . Human epithelial and endothelial cell lines were used in this study , as these represent cell types that are relevant to Leptospira spp . infection . The human laryngeal epithelial cell line HEp-2 was purchased from the ATCC , and grown in Eagle's Minimum Essential Medium ( ATCC ) supplemented with 10% fetal bovine serum ( FBS ) ( Gibco , Grand Island , NY ) at 37°C under 5% CO2 . The human macrovascular endothelial cell line EA . hy926 , a kind gift from Dr . C . J . Edgell ( University of North Carolina , Chapel Hill , NC ) [43] , [44] , was grown at 37°C under 5% CO2 in Dulbecco's modified Eagle medium ( DMEM ) ( Gibco ) with 4 . 5 g/l glucose , supplemented with 10% FBS , 100 µM hypoxanthine-0 . 4 µM aminopterin-16 µM thymidine ( Sigma-Aldrich ) , and 2 mM L-glutamine ( Gibco ) . The human microvascular endothelial cell line HMEC-1 [45] obtained from the Centers for Disease Control and Prevention ( CDC ) , was grown in MCDB 131 medium ( Gibco ) and supplemented with 15% heat-inactivated FBS ( Hyclone , Logan , UT ) , 2 mM L-glutamine ( Gibco ) , 10 ng/ml epidermal growth factor ( EGF ) ( BD Biosciences , Bedford , MA ) , 1 µg/ml hydrocortisone ( Sigma ) and 25 mM HEPES ( Gibco ) . All cell lines were grown in the presence of 100 U/ml penicillin and 100 µg/ml streptomycin ( Gibco ) . The display system used in this study is based on in-frame fusions to the phage minor coat protein encoded by gene III . The vector used to construct the filamentous phage library is derived from fdDOG [46] inserted with a stuffer region containing stop codons in all three reading frames upstream of gene III and flanked by SfiI restriction sites [37] . The resultant vector , fdBUG , cannot express the minor coat protein which is necessary for infecting naïve F+ E . coli cells , causing a reduction in infectious phage particle production [37] . The phage vector fdBUG was digested with SfiI ( New England Biolabs , Ipswich , MA ) for 4 hr at 50°C , which removes the stuffer region and leaves 3′-GTG overhangs . To prevent self-ligation , the digested vector was treated with alkaline phosphatase ( calf intestinal , New England Biolabs ) for 1 hr at 37°C . L . interrogans genomic DNA was partially digested for 1 hr at 37°C using MseI ( New England Biolabs ) , which leaves a 5′-TA overhang . The genomic DNA fragments were ligated with pre-annealed synthetic oligonucleotides ( Table 1 ) at a molar ratio of 2 equimolar mixture of adaptors to 1 genomic DNA using T4 ligase ( New England Biolabs ) . The three adaptors each have 5′-TA overhangs cohesive with the partially digested L . interrogans DNA at one end , and 3′-CAC overhangs cohesive with the ends left by SfiI digestion of the vector at the other end as previously described [37] . The adapted DNA was fractionated by agarose gel electrophoresis and fragments between 0 . 2 and 1 kilo base pairs ( kbp ) were extracted using a commercially available gel extraction kit ( QIAquick Gel Extraction kit , QIAGEN , Germantown , MD ) . The size-selected L . interrogans DNA fragments were ligated with SfiI-digested , dephosphorylated fdBUG vector ( 20 insert: 1 vector molar ratio ) using T4 ligase . The resulting ligation mixture was electroporated into E . coli MC1061 cells and selected on LB agar supplemented with 12 . 5 µg/ml tetracycline . As a control , fdBUG was transformed into E . coli MC1061 . The presence of L . interrogans DNA inserts in transformants were assessed by performing colony PCR as previously described [37] on twenty-four randomly picked colonies per transformation using primers oDOG1 and oDOG5 ( Table 1 ) , which amplify the region flanked by the two SfiI restriction sites . Three rounds of selection for phage that bind to EA . hy926 cells were performed using five independent pools of the phage library . Phage particles from 100 ml overnight cultures were prepared as described previously [36] , [37] with the exception that the phage were resuspended in DMEM supplemented with 1% BSA and protease inhibitors 1 mM benzamidine and 0 . 01 trypsin inhibitory units ( TIU ) /ml aprotinin ( 2PI ) ( Sigma-Aldrich ) . To eliminate clones that bind to ECM , the phage library was initially pre-adsorbed on ECM . ECM was obtained from EA . hy926 cells grown in a 6-well plate ( BD Falcon , Bedford , MA ) . Once the monolayers reached confluence , medium was removed and cells were washed with PBS . Two milliliters of 10 mM EDTA were added to each well and incubated for 10 min at RT on a rocker to lift the cells . The cells were removed by aspiration and the plates were visually assessed under a microscope to ensure complete removal of cells . The ECM left on the plate was washed three times with HBSC buffer ( 25 mM HEPES , pH 7 . 8 , 150 mM NaCl , 1 mM MnCl2 , 1 ( mM MgCl2 , 250 ( ( M CaCl2 ) prior to pre-adsorption of phage . Six ml of a 1 ( 100 dilution of the phage suspension ( approximately 1013 phage particles ) were added to ECM and incubated for 3 hr at 37°C under 5% CO2 . The unbound phage suspension was collected and added to 6-well plates containing confluent EA . hy926 monolayers pre-washed three times with HBSC . After 2 hr of incubation at 37°C under 5% CO2 , the unbound phage particles were removed and the wells were washed 15x with HBSC . The phage clones that specifically bound to intact monolayers were eluted using 3 ml of glycine pH 2 . 2 , and pH was then adjusted to pH 7 . 4 with 1 M Tris pH 10 . The eluted phage particles were used to infect E . coli strain TG1 . The infected clones were selected by overnight growth at 30°C on LB plates supplemented with 12 . 5 µg/ml tetracycline . All colonies obtained from each elution step were pooled and used to make new phage stocks for the second round of selection , performed as described . The same steps were repeated for the third round of selection , after which the selected pools were kept at −80°C until further use . As a control , phage particles were also prepared from vector fdDOG , which has an intact gene III , and therefore can produce the minor coat protein and so is capable of infecting bacterial cells . Three rounds of selection for fdDOG were performed in parallel with the phage library . After 3 rounds of selection , individual colonies of phage clones in E . coli TG1 were purified by plating pooled phage clones , then restreaking on LB plates supplemented with tetracycline . Individual colonies were picked and colony PCR was performed using primers oDOG1 and oDOG5 ( Table 1 ) . The amplified products were sent for sequencing ( McLab , South San Francisco , CA ) . The sequences obtained were compared by Basic Local Alignment Search Tool ( BLAST ) with the complete genome of L . interrogans sv . Copenhageni st . Fiocruz F1-130 at the NCBI site ( http://blast . ncbi . nlm . nih . gov ) to identify open reading frames to which the selected DNA fragment corresponded . The L . interrogans DNA inserted in the phage genome was also checked for correct gene orientation and intact reading frame . The genes identified were analyzed for the presence of signal peptide using online versions of prediction programs SignalP 3 . 0 ( http://www . cbs . dtu . dk/services/SignalP-3 . 0 ) and LipoP 1 . 0 ( http://www . cbs . dtu . dk/services/LipoP ) . The β-barrel prediction program PRED-TMBB ( http://biophysics . biol . uoa . gr/PRED-TMBB ) was utilized to determine if any of the gene hits might encode transmembrane OMPs . The predicted protein sequences were also manually analyzed for the presence signal peptides recognized by the spirochaetal lipoprotein signal peptidase ( Lsp ) or leader peptidase ( Lep ) [47] . Selected phage clones containing fragments of genes that are predicted to encode proteins that contain secretion signal sequences were further assessed based on the following criteria: ( a ) their known or putative surface localization ( predicted or shown experimentally ) , ( b ) the presence of a predicted transmembrane β-barrel motif , and ( c ) whether they have homologues in pathogenic and/or saprophytic strains . The genes encoding the Leptospira candidate adhesins LIC10508 , LIC11574 , LIC12341 , and LIC13411 identified by phage display , LBF1629 , which encodes the LIC11574 homologue in L . biflexa , and a known L . interrogans ECM adhesin OmpL1 , were cloned in the expression vector pMalC2 ( New England Biolabs ) and expressed as fusions to maltose binding protein ( MBP ) . The Leptospira genes without the sequences encoding the signal peptides were amplified by PCR using L . interrogans sv . Copenhageni st . Fiocruz F1–130 genomic DNA as template . The oligonucleotide primers containing restriction sites used in this study are listed in ( Table 1 ) . The amplified products were cleaved with appropriate restriction enzymes to generate non-compatible cohesive overhangs to facilitate directional cloning when ligated to pMalC2 digested with the same enzymes . The ligation products were transformed into E . coli strain Top10 by electroporation . The transformants were selected on LB plates supplemented with 100 µg/ml ampicillin . Plasmids from selected colonies were isolated using a commercially available kit ( Plasmid Mini Kit , QIAGEN ) and the presence of insert DNA was verified by PCR using vector specific primers oMal01 and oMal02 ( Table 1 ) , which hybridize to vector DNA sequences flanking the insertion site . Plasmid DNA was also sequenced in a commercial facility ( McLab , San Francisco , CA ) using oMal01 , oMal02 and custom-designed internal gene primers ( Table 1 ) . The plasmids with appropriate inserts were transformed into E . coli expression strain KS330 . The production of MBP-fusions from 1 liter E . coli cultures in 2x YT was induced with 1 mM isopropyl-β-D-thiogalactopyranoside ( IPTG ) ( Sigma-Aldrich ) for 2 hr at 30°C as previously described [48] , and assessed by silver staining and by immunoblot analysis using an α-MBP antibody . Bacterial cells were harvested by centrifugation and lysates were prepared by pressing the cells at 10 , 000 psi . The recombinant proteins were purified from the supernatant by amylose affinity chromatography . The protein concentrations of MBP fusions were determined using a Bradford method-based protein assay kit ( Bio-Rad , Hercules , CA ) . To test the binding of purified phage particles or recombinant MBP fusions to cell monolayers , the epithelial cell line HEp-2 , and endothelial cell lines EA . hy926 or HMEC-1 , were seeded in 96-well tissue culture plates ( BD Falcon ) at 50% confluence and incubated at 37°C , 5% CO2 for 2 days until cells were confluent . In some cases , the cells were incubated for an additional 1–2 days to reach post-confluence , while cell viability was maintained . Controls for binding included incubation of phage particles or recombinant proteins in medium alone ( binding to plastic ) or in ECM prepared from the cell lines as described above . To test the ability of the phage particles to bind to cell monolayers , purified phage particles were prepared from individual selected clones as previously described [37] . Approximately 1010 phage particles resuspended in 50 µl DMEM supplemented with 1% BSA and protease inhibitors benzamidine and aprotinin were added to confluent monolayers in quadruplicate on a 96-well tissue culture plate . The plates were incubated for 2 hr at 37°C under 5% CO2 to allow phage attachment . The unbound phages were removed by washing with HBSC , while the bound phages were fixed with 3% PFA in PBS . After washing with Tris-buffered saline ( TBS ) , the phage particles bound to monolayers or ECM were detected using an enzyme linked immunosorbent assay ( ELISA ) -based assay . Briefly , wells were blocked for 1 hr with 1% BSA in HBSC . The plates were washed 3x with TBS , then incubated with rabbit anti-M13+ fd bacteriophage antibody ( diluted 1∶10 , 000; Abcam , Cambridge , MA ) . After 1 hr , the plates were washed 3x with TBS , followed by incubation with anti-rabbit IgG horseradish peroxidase ( HRP ) -conjugated antibody ( diluted 1∶10 , 000; Promega ) for 40 min at RT . The blocking step and incubations with antibodies were performed at RT on a rocker . Plates were developed for 30 min with 3 , 3′ , 5 , 5′-Tetramethylbenzidine ( TMB , Sigma-Aldrich ) and 0 . 006% hydrogen peroxide in phosphate-citrate buffer ( 0 . 05 M Na2HPO4 , 0 . 02 M citric acid , pH 5 . 0 ) for 30 min . The absorbance at 655 nm was determined using an automated plate reader ( SpectraMax M5 , Molecular Devices ) . Results were expressed as fold difference in the absorbance readings in the presence of cells over no cells . To test the binding of recombinant Leptospira proteins to cells in vitro , cell monolayers were incubated with various concentrations ( ranging from 0 . 1–3 . 0 µM ) of MBP fusion proteins in DMEM supplemented with 1% BSA , 25 mM HEPES and 2PI for 1 hr at 37°C under 5% CO2 . The addition of recombinant proteins was done in quadruplicate wells . The plates were washed with 200 µl HBSC to remove the unbound proteins and fixed with 3% PFA in PBS . The wells were washed once with TBS , then the recombinant proteins bound to host cells were measured using an ELISA format . The wells were blocked with 1% BSA in TBS with 3% goat serum , washed , and then incubated with rabbit anti-MBP antibody ( diluted 1∶10 , 000; New England Biolabs , Beverly , MA ) for 1 hr at RT . After washing , HRP-conjugated anti-rabbit antibody ( diluted 1∶10 , 000; Promega ) was added to each well and incubated for 40 min at RT under constant shaking . Wells were developed for 25 min with TMB and 0 . 006% hydrogen peroxide in 0 . 05 M phosphate-citrate buffer ( pH 5 . 0 ) and absorbance at 655 nm was read as described previously . The cell-specific attachment was determined by subtracting the absorbance signal of recombinant proteins in empty wells/plastic from the absorbance in wells containing cells , with each assay done in quadruplicate . The attachment of recombinant proteins to purified receptors was also tested using the ELISA described above . The wells of pre-chilled 96-well Linbro plates ( MP Biomedicals , OH ) were coated with 50 µl/well 0 . 01 µM purified VE-cadherin ( Human CD-144 , cadherin-5 ) obtained from Bioclone Inc . ( San Diego , CA ) and diluted in HBSC supplemented with 2PI in quadruplicate wells . The amount of receptor used to coat wells was optimized and described previously by our group [35] . The plates were incubated overnight at 4°C with shaking . The controls used in the assay included HBSC buffer alone or BSA , coated at the same concentration as VE-cadherin . The plates were washed with HBSC and blocked with 1% BSA in DMEM for 1 h at 4°C , prior to the addition of 0 . 001–1 µM recombinant adhesins . The addition and detection of MBP fusion proteins on plates were carried out as described above . Receptor binding was expressed as the difference in the absorbance ( OD655 ) readings between empty and VE-cadherin-coated wells . Sera used for this evaluation were from de-identified banked samples obtained from subjects enrolled in protocols approved by the Institutional Review Board ( IRB ) committees of Yale University and Oswaldo Cruz Foundation . Convalescent-phase sera were obtained from Brazilian patients with confirmed leptospirosis [49] . Sera from control subjects were obtained from individuals residing in urban slum communities from the same site [4] . The serum from each individual at 1∶500 dilution was pre-cleared by incubation with a PVDF membrane blotted with whole cell lysate of E . coli expressing MBP-β-galactosidase . The purified recombinant proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes for western blotting . The membranes were blocked with TBS containing 5% nonfat dry milk overnight at 4°C . After 3 washes with TBS , the membranes were incubated in pre-cleared human serum for 3 hr at RT on a rocker . The membranes were washed 3x with 1x TBS before probing with anti-human IgG AP-conjugated antibodies ( diluted 1∶10 , 000; Promega ) . Proteins reacting with the antisera were visualized by use of the chromogenic substrates BCIP and NBT . Five 5-week old female BALB/c mice were immunized intraperitoneally with 100 µg ( in 100 µl volume ) of each recombinant MBP fusions to L . interrogans adhesins ( LIC10508 , LIC11574 , LIC12341 , LIC13411 , and control β-galactosidase ) with 100 µl Alum adjuvant ( Thermo Scientific , Rockford , Il ) . The mice were boosted 3 times at 14 day intervals with the same amount of protein in Alum . At day 56 , the mice were bled by cardiac puncture and sera were prepared from the collected blood . Sera obtained from control groups inoculated with PBS alone or PBS with Alum , showed no reactivity against any of the candidate adhesins or L . interrogans lysate . The cellular localization of L . interrogans proteins was determined using fractionation and phase separation assays adapted from [50] . L . interrogans was grown in 800 ml EMJH medium at 30°C until late log phase ( ∼108–109/ml ) . Bacteria were harvested by centrifugation for 30 min at 2 , 683 x g . Bacterial pellets were washed 3x with PBS pH 7 . 4 supplemented with 5 mM MgCl2 at 4°C , then resuspended in ice cold 10 mM Tris-HCl , 2 mM EDTA ( TE ) , pH 7 . 4 supplemented with protease inhibitors 0 . 01 TIU/ml aprotinin , 1 mM benzamidine and 10 µM pepstatin . L . interrogans concentration was adjusted to 5×109 bacteria/ml in a total volume of 15 ml . Ice-cold 10% TX-114 ( Sigma-Aldrich ) in TE supplemented with protease inhibitors was added to the bacteria to a final concentration of 1% . The solution was gently mixed and incubated at 4°C for 45 min on a rocker . Following incubation , the bacterial suspension was centrifuged for 15 min at 17 , 000 x g at 4°C . The supernatant was transferred to a sterile tube and CaCl2 was added to the supernatant to a final concentration of 20 mM . The supernatant fraction was kept at 4°C until use for phase separation steps . The pellet was resuspended in 1 ml of final sample buffer ( FSB; 62 . 5 mM Tris-HCl pH 6 . 8; 10% glycerol; 2% SDS supplemented with aprotinin , benzamidine and pepstatin ) . This is the detergent-insoluble fraction containing the protoplasmic cylinder ( PC ) . Ice cold 10% TX-114 was added to the supernatant ( detergent soluble fraction ) to a final concentration of 2% . Phase separation was performed by warming the detergent-soluble supernatant to 37°C for 10 min followed by centrifugation for 10 min at 1 , 000 x g . Centrifugation allows the separation of the detergent-poor aqueous phase ( AQ ) found at top from the detergent-rich hydrophobic phase ( DET ) observed as an “oily droplet” at the bottom of the tube . The AQ fraction is made up mostly of periplasmic proteins , while outer membrane lipoproteins and transmembrane proteins are found in the DET phase . The DET and AQ were then washed four times in the following manner . The aqueous phase was chilled in an ice bath , and 10% TX-114 was added to a final concentration of 2% , followed by warming to 37°C , then centrifugation at 2 , 000 x g at RT . The detergent phase was chilled in an ice bath , diluted with nine parts TE buffer , warmed to 37°C , and centrifuged as described above . The AQ and DET were precipitated with acetone and resuspended in FSB . L . interrogans whole cell lysates ( WCL ) were prepared by harvesting cultures , washing twice with PBS-MgCl , resuspended in PBS supplemented with protease inhibitors aprotinin and benzamidine , followed by sonication . L . interrogans WCL , PC , DET and AQ fractions were run on a 12 . 5 or 15% SDS-PAGE gel , loading equivalents of 5×108 or 2 . 5×109 bacteria per lane . One gel was silver-stained and a duplicate gel was transferred to a PVDF membrane and blocked with 5% milk , 3% goat serum ( GS ) in TBS for 1 hr at RT . Immunoblots were incubated with antisera against control periplasmic protein flagellin A1 ( FlaA1 ) ( 1∶5 , 000 ) which would be attached to the inner membrane , cytoplasmic membrane lipoprotein LipL31 ( 1∶5 , 000 ) , outer membrane proteins OmpL47 ( 1∶5 , 000 ) , OmpL1 ( 1∶5 , 000 ) , and LipL21 ( 1∶5 , 000 ) , and candidate L . interrogans adhesins LIC10508 ( 1∶100 ) , LIC11574 ( 1∶100 ) , LIC12341 ( 1∶100 ) , and LIC13411 ( 1∶500 ) in blocking buffer overnight at 4°C . After washing 3x with TBS , the membranes were probed with 1∶10 , 000 goat anti-rabbit antibody conjugated to AP or anti-mouse antibody conjugated to HRP for 40 min at RT . The membranes were washed with TBS 3x followed by visualization of protein bands using chromogenic substrates NBT and BCIP or by enhanced chemilumescent ( ECL ) substrate ( Thermo Scientific ) . Analyses were performed using Prism 6 ( GraphPad Software , Inc ) utilizing two-tailed Student's t-test . P values lower than 0 . 05 were considered statistically significant .
To identify bacterial cell surface proteins that mediate attachment to host cells , a L . interrogans filamentous phage display library was constructed . The vector used in generating the library , fdBUG , cannot express the phage coat protein III because of the stop codons in all three reading frames near the 5′ end of gene III [37] . Cloning of L . interrogans DNA fragments that encode an open reading frame continuous with that of the phage gene III allows expression of leptospiral peptides as fusions to the minor coat protein on the surface of the phage . Based on the average L . interrogans DNA fragment size of 500 bp ( inserted in fdBUG vector ) and its genome size of 4 . 5 Mbp , the number of clones necessary to represent the entire genome is ∼41 , 800 . The number of transformants recovered was 1 . 2×106 , with approximately 82% of the clones containing L . interrogans DNA inserted in the phage vector . It should be noted that the leptospiral DNA inserted in the phage vector was ligated with 3 pairs of adaptors , so one of the nine possible combinations will result in production of the protein encoded by the inserted DNA in frame with phage protein III . Therefore , the total number of suitable clones obtained is 1 . 1×105 , enough to represent the genome with approximately 2 . 6-fold redundancy . To select for phage clones that bind specifically to cells , five independent pools of phage were pre-adsorbed on ECM and the unbound phage particles were subjected to three rounds of selection for phage that bind to EA . hy926 endothelial cells in vitro . The endothelial cell line EA . hy926 was used in this study because it represents a cell type that is relevant to Leptospira infection and because of its relative ease in cultivation . Additionally , L . interrogans exhibited attachment to EA . hy926 monolayers in vitro [8] , [11] , [35] , and binding to the cells was stronger than to the ECM [35] . As a control , a parallel selection was also performed using fdDOG to assess non-specific binding of phage to cells . The number of clones eluted after each round of selection against endothelial cells increased as selection progressed . After the third round of selection , the number of colonies recovered from the phage library was on average 70-fold higher compared to the control fdDOG . A total of 931 clones from the three rounds of selection for binding to endothelial cells were sequenced ( Figure 1 ) . To identify the L . interrogans gene fragments inserted in the selected phage , the sequences obtained were compared by BLAST searches with the complete genome of L . interrogans serovar Copenhageni strain Fiocruz L1–130 . Out of the 931 sequences analyzed , 152 clones have inserts of L . interrogans DNA outside of annotated genes while 779 clones have DNA inserts corresponding to open reading frames , representing 185 unique genes ( Figure 1 ) . Many of the top gene hits in the selection encode proteins that do not possess a signal peptide ( Table S1 ) . Because the proteins encoded by these genes are not predicted to be present on the bacterial cell surface and there is no literature suggesting their surface localization , these gene hits were not further pursued . Although the number of hits may suggest strong selection to bind to endothelial cells , these interactions may not be biologically relevant if these proteins are not found on the surface of the bacterial cell . A similar phenomenon was previously observed using this system [37] . Since our aim was to identify bacterial proteins that interact with the host , L . interrogans genes encoding proteins that are predicted to be surface-localized or found extracellularly were of interest and priority . The gene hits were analyzed using programs SignalP 3 . 0 and LipoP 1 . 0 for the presence of a signal peptide at the N-terminus of the predicted protein product . All spirochetal OMPs studied have signal peptides that initiate export of the protein and are subsequently cleaved following translocation [47] . Out of the 185 genes analyzed , 37 were predicted to encode proteins that contain secretion signal sequences ( Table S2 ) , and therefore at least may be exported out of the cytoplasm . Four genes were prioritized for further screening based on additional criteria described in the Materials and Methods . These include L . interrogans genes predicted to express putative lipoproteins LIC10508 and LIC13411 , and conserved hypothetical proteins LIC11574 and LIC12341 ( Table 2 ) . Alignment of the L . interrogans peptides encoded in the selected phage clones revealed diverse sequences without any obvious consensus ( Table S3 ) . This is not surprising given that we selected phage clones using intact cells . It should be noted that each cell type expresses a number of different receptors that can potentially interact with clones in a phage display library [8] , [35] . This accounts for why we obtained a number of peptides with no clear consensus . To confirm cell-specific attachment of the selected phage clones , purified phage particles were prepared from individual phage clones and were tested for binding to cell monolayers . Phage suspensions were incubated with confluent monolayers of EA . hy926 or HEp-2 cells . The unbound phages were washed off and the phages bound to cells were fixed and quantified in an ELISA-based assay using anti-M13 ( fd phage coat ) antibodies . Cell monolayers were also incubated with fdDOG as control . Results were expressed as fold difference in the absorbance readings in the presence of cells over no cells . All phage clones carrying L . interrogans gene fragments showed binding to EA . hy926 endothelial cells ( ≥4 fold compared to vector control ) ( Figure 2 ) . Phage clones expressing fragments of the LIC10508 and LIC11574 genes also adhered to the epithelial cell line HEp-2 . Collectively , the selected phage clones bind more efficiently to endothelial as compared to epithelial monolayers , demonstrating that the selection process was successful in identifying clones that bind directly to this endothelial cell line . The observed disparity in the binding of phage clones between the two cell lines can be attributed to the likelihood that the receptors recognized by these phage clones are differentially expressed by the cell lines used . To determine whether the L . interrogans proteins encoded by genes selected through phage display bind to host cells beyond the context of the phage particle , recombinant forms of LIC10508 , LIC11574 , LIC12341 and LIC13411 were generated . The genes were cloned in pMalC2 expression vector to generate the mature protein ( without the signal peptide ) fused to maltose binding protein ( MBP ) ( Figure S1 ) . The 43 kDa MBP was chosen as tag as it increases the solubility of many fusion proteins , and the recombinant proteins can easily be purified by amylose affinity chromatography . Recombinant forms of other known adhesins such as invasin from Y . pseudotuberculosis and P66 of Borrelia burgdorferi have been successfully generated using this vector [37] , [48] , [51] . The relative binding capacities of the MBP fusions to endothelial and epithelial cells were determined using an ELISA format . For the binding assays , we chose to use the microvascular endothelial cell line HMEC-1 because of the physiological relevance of this cell type in the widespread hemorrhage observed in patients with leptospirosis . Increasing concentrations of recombinant proteins were incubated with confluent cell monolayers for 1 hr . The protein bound to cells was quantified and expressed as the absorbance reading of proteins in wells containing cells minus background signal or plastic binding . MBP-β-galactosidase and MBP-invasin served as negative and positive controls , respectively . The recombinant form of the conserved hypothetical protein LIC11574 demonstrated binding to HMEC-1 cell monolayers in a dose-dependent and saturable manner . The putative lipoprotein LIC13411 also binds HMEC-1 cells ( Figure 3A ) . Attachment of LIC11574 to epithelial HEp-2 cell monolayers was significantly higher as compared to control protein ( Figure 3B ) , but was not saturable ( Figure 3C ) . Both endothelial and epithelial cells bound the control protein , invasin efficiently ( not shown ) . LIC11574 has a homologue in the saprophytic L . biflexa sv . Patoc st . Patoc I ( Ames ) annotated as LBF1629 ( Accession number YP_001962715 ) . The 24 . 3 kDa protein product is annotated as conserved hypothetical protein containing a possible Type IV pilin N-terminal methylation site [52] . LBF1629 has a 33% identity and 55% similarity to LIC11574 . To assess the cell-binding activity of the protein encoded by the saprophytic strain , a recombinant form of LBF1629 was tested in adhesion assays and its binding activity was compared to LIC11574 . Results show that LBF1629 did not bind to either HMEC-1 endothelial or HEp-2 epithelial cells after 1 hr of incubation ( Figure 4 ) . This may suggest that despite some similarity in the amino acid sequences of these two proteins , there is a difference in the function of the proteins encoded by the pathogenic and saprophytic strains . Our group has previously identified the cadherins as receptors for L . interrogans [35] . To determine whether the candidate adhesins facilitate bacterial attachment to vascular endothelial ( VE ) -cadherin , we incubated MBP fusions of LIC10508 , LIC11574 , LIC12341 , and LIC13411 with immobilized VE-cadherin . The bound adhesins were quantified using the ELISA approach described above . Both LIC11574 and LIC13411 exhibited higher adherence to VE-cadherin than did the negative control β-gal . Interestingly , both of these proteins also exhibited attachment to HMEC-1 cells , which have been previously shown to express VE-cadherin [35] . This result suggests possible roles of these adhesins in mediating bacterial attachment to endothelial cells through the cadherin receptors . A dose response assay was performed to quantify the binding of LIC11574 and LIC13411 to VE-cadherin vs . control BSA immobilized on wells ( Figure 5 ) . The results show that LIC11574 binds efficiently to VE-cadherin with an estimated KD = 7 . 6±2 . 5 nM . The binding of LIC13411 to VE-cadherin was also efficient , KD = 0 . 4±0 . 2 nM . The binding of both adhesins to VE-cadherin was dose-dependent and saturable . LIC13411 also showed binding to BSA at high recombinant protein concentrations , but this was not saturable . To determine if our candidate adhesins are expressed during Leptospira infection , we screened sera from confirmed leptospirosis patients in the convalescent phase and from control individuals for reactivity to the adhesin proteins by immunoblot analysis . For this assay , sera were initially pre-cleared by incubation with membranes blotted with whole cell lysate of E . coli expressing MBP-β-galactosidase . MBP is expressed by E . coli , which is part of the gut flora of both humans and animals [53] . This step is to ensure that anti-MBP- or other anti-E . coli-specific antibodies are removed from the sera , and thus reduces non-specific signals . None of the sera from healthy individuals recognize recombinant forms of LIC12341 , LIC13411 , LIC11574 or LIC10508 ( Table 3 ) . In contrast , all candidate adhesins were recognized by at least one of the five sera from patients with leptospirosis . All patient sera recognized OmpL1 , a known OMP [54] , [55] that was used as the positive control for the test . β-galactosidase was used as negative control and did not react with any of the pre-cleared sera tested . Cellular fractionation of L . interrogans using Triton X-114 followed by phase partitioning experiments was performed to determine cellular localization of candidate cell-binding adhesins . This non-ionic detergent allows solubilization of the outer membrane but keeps the cytoplasmic membrane intact . Shifting the temperature from 4°C to 37°C allows separation of hydrophilic and hydrophobic fractions . TX-114 has been extensively used in the study of spirochete OMPs [32] , [50] , [56]–[60] . L . interrogans Copenhageni was treated with TX-114 followed by centrifugation to separate the detergent-insoluble material or protoplasmic cylinder fraction ( PC ) , made up of the inner membrane , bacterial components attached to the inner membrane , and cytoplasmic contents . Phase separation was performed by warming the detergent-soluble supernatant to 37°C followed by centrifugation to separate the detergent-rich hydrophobic phase ( DET ) from the detergent-poor aqueous phase ( AQ ) . The AQ fraction is made up mostly of periplasmic proteins while outer membrane lipoproteins and transmembrane proteins are found in the DET phase . As a control , whole cell lysates ( WCL ) of leptospires were prepared by sonication . Different fractions were assessed by western blot using antisera against L . interrogans proteins with known cellular locations such as inner membrane-associated FlaA1 and LipL31 , and outer membrane proteins OmpL47 and OmpL1 . The TX-114 fractionation analysis ( Figure 6 ) showed that the periplasmic protein FlaA1 and inner membrane protein LipL31 remain in the protoplasmic cylinder fraction ( PC ) , as expected . The transmembrane OMP OmpL47 was solubilized by the detergent but fractionates in the AQ phase . A similar result was observed by Pinne and Haake [50] which was unanticipated by the authors . However , additional assays such as surface proteolysis , immunofluorescence , membrane affinity and surface biotinylation confirmed that OmpL47 is indeed surface-exposed and membrane-integrated . OmpL1 is another transmembrane protein but behaves differently from OmpL47 . Both OMPs LipL21 and OmpL1 are partially solubilized by TX-114 extraction but partition exclusively to the DET phase [32] . All candidate adhesins were observed in the DET fraction . LIC10508 and LIC11574 behaved similarly to LipL21 and OmpL1 , with both proteins also detected in the protoplasmic cylinder after TX-114 treatment . LIC10508 has previously been reported to fractionate in the detergent phase , and to induce production of ICAM-1 and E-selectin by HUVEC cells , although direct adhesion to the cells or to extracellular matrix molecules was not demonstrated [61] . LIC10508 migrated at 23 kDa , slightly larger than the predicted 21 kDa mature protein product . Interestingly , two protein bands for LIC10508 , 21 kDa and 23 kDa were observed at the DET phase . LIC11574 also showed two different-sized species in different fractions; a band in detergent migrated faster ( 20 kDa ) than bands in detergent-insoluble fraction and in the whole cell lysate ( 25 kDa ) . Similarly , LIC12341 also showed two distinct species in different fractions . The different apparent molecular weights of LIC10508 , LIC11574 , and LIC12341 in different fractions , with the lower apparent Mr forms appearing in the detergent-soluble phase , suggests that the proteins may be undergoing processing . While differences in Mr between the protoplasmic cylinder fraction , which might include protein molecules that have not yet had secretion signal sequences removed , and the outer membrane fraction would not be surprising , the differences seen between the aqueous and detergent-soluble portions of the outer membrane fraction suggest that additional post-translational modifications might occur for these proteins . LIC11574 was observed in the AQ fraction when we increased the bacterial load to equivalents of 5×109 per lane . LIC12341 and LIC13411 were both found to be completely TX-114 soluble but found in DET and AQ phases . Lower dilutions of the antisera against the cell-binding adhesins described here ( 1∶100 or 1∶500 ) were used to detect the L . interrogans candidate adhesins , in comparison to the sera used to detect other proteins used as markers of cellular fractionation . This low reactivity is likely due to the low abundance of proteins expressed by L . interrogans because all antisera recognized the recombinant candidate adhesins at 1∶20 , 000 dilution; this is consistent with low transcript abundance for these adhesins [62] .
The binding of Leptospira spp . to ECM components of the host has long been established [17] , [20] , [21] , [23]–[25] , [30] , [34] . However , our group has shown that L . interrogans binds more efficiently to cells than to ECM in vitro [8] , [35] , and that the host cell surface protein VE-cadherin contributes to bacterial attachment to endothelial cells [35] . With the limitations in the available genetic tools in Leptospira spp . , phage display represents an alternative but powerful tool to identify Leptospira proteins with cell-binding activities , as well as the specific portions of the proteins with adhesin activities . This approach has also been employed in identifying Leptospira proteins that bind to ECM components [63] . Our experimental strategy eliminated adhesins that might bind to host molecules that might be deposited as extracellular matrix , or remain cell-associated . Our focus , however , was on cell-surface receptors due to the potential for trans-membrane signaling that could allow the bacteria to manipulate host cell biology to their own advantage . The putative lipoproteins LIC10508 and LIC13411 , and conserved hypothetical proteins LIC12341 and LIC11574 were identified using phage display and were prioritized for further screening . Studies focusing on these four candidate adhesins are sparse in the literature . Pinne et al . [64] described all four proteins as probable OMPs using a set of algorithms and criteria to identify candidate lipoproteins and transmembrane OMPs in L . interrogans sv . Copenhageni st . Fiocruz L1–130 . Using a protein microarray screening assay , LIC13411 , LIC11574 and LIC10508 all exhibited low binding activities to fibronectin [64] . Gamberini and colleagues also identified LIC10508 ( putative lipoprotein; LipL23 ) and LIC11574 ( conserved hypothetical protein; OmpL22 ) as surface proteins using in silico analysis [65] . Both proteins reacted with Leptospira-infected human sera suggesting that these proteins are expressed in the course of human infection . A serological cross reactivity assay performed by the group also demonstrated that LIC10508 is highly conserved among pathogenic strains . The cell-binding properties of these proteins are first described in this work . One advantage of phage display is that it allows the mapping of the binding domains , providing information on the peptide sequence sufficient to establish interactions with the substrate . In this study , the alignment of the L . interrogans peptide fragments inserted in the cell-binding phage clones did not reveal any discernable pattern . This was not surprising as the selection of the phage library was carried out using intact monolayers rather than a homogenous substrate , suggesting that the different selected phages may have recognized and bound various components of the monolayer . It should be noted that this study started prior to the identification of cadherins and other proteins as L . interrogans receptors on host cell surfaces [35] . In addition , our finding that phage clones carrying L . interrogans gene fragments bind more efficiently to endothelial cells than epithelial monolayers was not surprising since selection was carried out using an endothelial cell line . This disparity in the binding activities may be attributed to differences in the level or type of receptors expressed between the two cell types . We speculate that L interrogans adhesins recognize and bind specific receptors that are differentially expressed in different cell types . We have previously demonstrated that the production of host receptor proteins VE-cadherin , E-cadherin and ICAM-2 varies among the various endothelial and epithelial cell lines tested [35] . This finding suggests that pathogenic Leptospira spp . have evolved mechanisms to adhere to a variety of cell types through expression of numerous adhesins . The attachment of L . interrogans to host molecules is thought to be important to establish bacterial infection and dissemination resulting in leptospirosis . Like any experimental approach , the use of phage display has its caveats . One limitation of this approach is that peptides displayed on the surface of the phage may be folded differently from the purified recombinant fusion forms or on the surface of the leptospires ( in vivo ) . Phage display is limited to protein fragments that some host cell receptors may not bind if three-dimensional structures are required . This may explain the differences in the binding activities of the phage clones expressing adhesin peptide versus purified recombinant forms of the intact candidate adhesins . Both LIC11574 and LIC13411 displayed high affinity binding to VE-cadherin , a recently identified host protein receptor for L . interrogans [35] . However , whether the abilities of LIC11574 and LIC13411 to bind to purified VE-cadherin contribute to the ability of pathogenic Leptospira spp . to attach to endothelial cells , and whether multivalent interactions are required , need to be further explored . The interactions of pathogenic leptospires with VE-cadherin , in particular , is of interest due to the critical importance of VE-cadherin in maintenance of endothelial integrity [66] , [67] and the disruption thereof by pathogenic Leptospira species . The roles of LIC11574 and LIC13411 in pathogenesis as potential virulence factors also need to be established , and the potential for functional redundancy requires further exploration . Generation of targeted knockout mutants in each of these genes will be required to address these questions , and it is possible that inactivation of both genes will be required to detect a phenotype in vivo . It is thought that the ability of the pathogenic Leptospira spp . to recognize and bind host surface components is an essential step in the pathogenesis of leptospirosis . Outer membrane proteins are likely mediators of these interactions , leading to the entry and dissemination of the pathogen . This has led to several studies identifying OMPs and evaluating their roles in virulence . OMPs that are unique to pathogenic strains are of interest to a number of investigators , as their presence exclusively in infectious strains may support their role in virulence . One of our candidate adhesins , LIC11574 , has a homologue in the saprophytic strain L . biflexa . Only the OMP encoded by the pathogen demonstrated binding to cell monolayers . This suggests either there are distinct functions between protein homologues or adhesion is an additional role acquired during evolution in pathogenic strains to survive in hosts . Most studies focus on the roles of proteins unique to infectious strains but proteins shared by all Leptospira species should not be overlooked . Our findings may suggest that small differences in primary , secondary , or tertiary structures may bring about significant disparities in the roles of the proteins . Development of classic genetic knock-outs to assess roles in virulence may not be ideal in this system because of the potential for redundancy in the functions of adhesins . Loss of a gene ( inactivation ) may not result in obvious phenotypic change if the function is redundant to that of another protein with the same activity . For example , site-directed mutagenesis through allelic exchange was successful in the inactivation of ligB [68] , but the loss of this ECM-binding adhesin did not affect the ability of the pathogenic L . interrogans to infect hamsters . The expression of pathogen-specific LigB on saprophytic L . biflexa resulted in enhanced bacterial attachment to ECM and cells in culture [24] . Therefore , gain-of-function approaches by heterologous expression of adhesins from pathogens in saprophytic L . biflexa at least allowed confirmation of the role of this protein in adhesion to host cells in vitro . Interestingly , we recovered a fragment of LigB that did not contain any of the known ECM-binding domains [28] , [31] in our selection using endothelial cells ( Table S2 ) . This result will be pursued in the future . The mechanism of host resistance to pathogenic Leptospira spp . is mediated largely by humoral responses [7] , [69] , [70] . The candidate adhesins identified by phage display , LIC12341 , LIC13411 , LIC11574 and LIC10508 , are recognized by sera from convalescent leptospirosis patients but not by sera from control individuals . This implies expression of these proteins during infection . Because of their accessibility to host immune responses during infection , a number of surface-exposed Leptospira OMPs are considered as candidate vaccinogens . Leptospiral OMPs OmpL1 and LipL41 were protective immunogens when used in combination [71] , which is promising in the development of subunit vaccines with greater protective effects and fewer side effects . Further tests are necessary to determine if the adhesins identified in this work have potential immunoprotective properties . Bacterial adhesive molecules have surface-exposed moieties that allow them to interact with host cells , and may play important roles in establishing bacterial attachment and dissemination in hosts . Therefore , determining surface localization is vital to support the role of pathogenic Leptospira OMPs in virulence . In silico approaches can help predict protein export from cytoplasm to periplasm [47] and lipidation of lipoproteins [72] however , direct examination is necessary to validate their cellular localization . We used a non-ionic detergent Triton X-114 as other groups were successful in removing spirochete outer membrane using this detergent [50] , [73] . Our data reveal that all candidate proteins are found in the DET phase suggesting possible surface localization . Although the majority of LIC10508 and LIC11574 partition in the DET , these adhesins were also observed in the protoplasmic cylinder . Similar results were observed with the transmembrane porin OmpL1 [32] , LipL21 [58] , and other known Leptospira OMPs . Several groups have suggested that the incomplete detergent solubilization of these proteins may be attributed to amphipathic properties of transmembrane OMPs [32] , [58] . The candidate adhesins LIC12341 and LIC13411 were completely solubilized in TX-114 but fractionated to both AQ and DET . Although the results were unexpected , similar findings were observed with the identified outer membrane protein OmpL54 [50] . We also observed low expression of the candidate adhesins , which corresponds to the paucity of their transcript levels [62] . This precluded the use other approaches such as immunofluorescence and surface proteolysis . However , multiple approaches to determining whether spirochetal proteins are localized on the outer surface of the bacterial cell , as outlined by Pinne and Haake [50] , [74] , will be prioritized in further studies of our candidate adhesins . Prior to this work , the attachment of pathogenic L . interrogans to host molecules was mainly attributed to the ability of bacterial OMPs to recognize and bind to ECM components . The study presented here provides evidence of direct binding of L . interrogans to host cells , and identifies bacterial components potentially involved in this interaction . Identification of molecular components necessary for the initial interaction between host and pathogen will greatly improve our understanding of Leptospira spp . pathogenesis . Surface-exposed OMPs that are recognized by the host immune response early during infection and play a role in bacterial virulence are ideal targets for diagnostic screening and development of vaccines . With the current robust leptospiral research output , in the near future we may see the development of simple and inexpensive diagnostic systems appropriate for highly endemic , resource-poor areas , as well as the application of state-of-the-art technologies to vaccine development . | Leptospirosis , caused by pathogenic species of the genus Leptospira , is an infectious disease that has emerged as a globally important health problem . Infection can either lead to mild illness or can progress to a severe disease form manifested by jaundice , kidney and liver dysfunction , and widespread blood vessel damage . It is thought that the ability of the bacteria to recognize and bind to human and animal cells is important for Leptospira spp . to cause the disease . Using phage display , we were able to identify bacterial proteins that mediate the binding of the bacteria to host cells . One of the identified proteins , LIC11574 , attaches to different types of host cells , and to VE-cadherin , a cell surface protein previously identified as receptor for disease-causing L . interrogans . All bacterial proteins identified were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals . Our findings may be of value in leptospirosis control and prevention , with these bacterial surface proteins as new targets for serodiagnosis and vaccine development . | [
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] | 2014 | Identification of Cell-Binding Adhesins of Leptospira interrogans |
The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones , growth factors , and biogenic amines . Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells , but it is unclear how this specialized organelle is formed and acquires its specific cargos . To identify proteins that act in dense-core vesicle biogenesis , we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function . We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting . Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein ( EARP ) complex . By assaying behavior and the trafficking of dense-core vesicle cargos , we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway . Genetic epistasis data indicate that RAB-2 , EIPR-1 and EARP function in a common pathway . In addition , using a proteomic approach in rat insulinoma cells , we show that EIPR-1 physically interacts with the EARP complex . Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment .
The dense-core vesicle is a specialized secretory organelle found in neurons and endocrine cells . In endocrine cells , these vesicles are often called secretory granules; for simplicity , we will here use the term ‘dense-core vesicles’ to refer to both the neuronal and endocrine carriers . Dense-core vesicles package several classes of cargo , including peptide hormones , growth factors , and biogenic amines . In response to elevated calcium , dense-core vesicles release their cargos to modulate a variety of biological processes ranging from blood glucose homeostasis to development , function and plasticity of the nervous system . Despite the importance of the signaling molecules released by dense-core vesicles , surprisingly little is known about how dense-core vesicles are generated and acquire their proper cargos [1–4] . Dense-core vesicles originate at the trans-Golgi network , but it is unclear how their compartmental identity is established and how cargos are correctly sorted into nascent dense-core vesicles . After budding from the trans-Golgi , immature dense-core vesicles undergo a poorly defined maturation process that involves homotypic vesicle fusion , peptide processing , and the removal of some soluble and transmembrane cargos [5–11] . Some studies suggest that luminal dense-core vesicle cargos sort purely through an intrinsic ability to aggregate in the low pH/high Ca2+ milieu of the trans-Golgi [12] , while other studies suggest that aggregation is not sufficient and that sorting relies on poorly defined structural motifs in cargo proteins that may interact with multiple sorting receptors [13 , 14] . Two general models of dense-core vesicle cargo sorting have been debated for more than twenty years: the ‘sorting by entry’ and ‘sorting by retention’ models that propose that sorting occurs in the trans-Golgi as dense-core vesicles bud off ( sorting by entry ) , or in a post-Golgi step where non-dense-core vesicle cargos are removed ( sorting by retention ) [15 , 16] . Experimental evidence supports both models , which are not mutually exclusive , suggesting that both mechanisms contribute to dense-core vesicle biogenesis . Molecular mechanisms of dense-core vesicle biogenesis are poorly understood largely because few proteins have been identified that function in this process . In recent years , the small GTPase Rab2 has emerged as a major regulator of dense-core vesicle cargo trafficking . RAB-2 , its effector ICA69/RIC-19 , and the putative RAB-2 GTPase-activating protein ( GAP ) TBC-8 have all been shown to be important for early steps in dense-core vesicle cargo sorting at or near the trans-Golgi [17–22] . To identify additional factors in this pathway , we used a forward genetic screen in the nematode C . elegans to isolate mutants that affect dense-core vesicle function . We identified RUND-1 , a RUN domain protein , and CCCP-1 , a coiled-coil protein , that act as RAB-2 effectors to mediate normal dense-core vesicle cargo sorting [23] . In mutants of rab-2 , rund-1 , or cccp-1 , morphologically normal dense-core vesicles are generated and transported to their axonal release sites , but these dense-core vesicles have reduced levels of both luminal and transmembrane cargos . RAB-2 and its effectors colocalize at or near the trans-Golgi , suggesting that they act in the neuron cell body to mediate proper cargo-sorting as dense-core vesicles bud off from the trans-Golgi or undergo post-Golgi maturation . Here we investigate the function of several additional molecules identified in our genetic screen for dense-core vesicle mutants: the conserved WD40 domain protein EIPR-1 and the VPS-52 and VPS-53 subunits shared by the Golgi-associated retrograde protein ( GARP ) and endosome-associated recycling protein ( EARP ) complexes . We show that EIPR-1 physically and genetically interacts with the EARP complex . Mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants that lack RAB-2 , RUND-1 or CCCP-1 , and genetic epistasis data indicate that they function in the same pathway . The requirement of EARP for proper sorting of cargos to dense-core vesicles suggests that dense-core vesicle cargo sorting is achieved in part through the trafficking of dense-core vesicle cargos or cargo receptors through an endosomal sorting compartment . While there is ample prior evidence that cargos are removed during dense-core vesicle maturation and trafficked to endosomes , our experiments suggest that there is also trafficking from endosomes to maturing dense-core vesicles .
To identify proteins required for dense-core vesicle biogenesis , we performed a forward genetic screen in the nematode C . elegans based on suppression of the hyperactive locomotion of an activated Gq mutant [23] ( S1A Fig ) . We previously characterized the function of two novel conserved proteins identified from this screen , RUND-1 and CCCP-1 . Both RUND-1 and CCCP-1 interact with the small GTP-binding protein RAB-2 when it is in its activated GTP-bound state [23] . Mutants in rab-2 , rund-1 and cccp-1 have a stereotypical defect in locomotion behavior characterized by little spontaneous movement on food , but coordinated slow locomotion when stimulated . One additional uncloned mutant from our screen ( ox316 ) had defects in locomotion and egg-laying very similar to rab-2 , rund-1 , and cccp-1 mutants . We cloned this mutant by single nucleotide polymorphism ( SNP ) mapping and transgenic rescue experiments ( Materials and Methods ) . We named the gene eipr-1 ( EARP-interacting-protein , see below ) . The eipr-1 mutant phenotype is fully rescued by microinjection of the single gene Y87G2A . 11 , and the ox316 mutation leads to a premature stop codon in this gene ( Fig 1A–1C ) . We subsequently obtained a deletion allele ( tm4790 ) that causes identical locomotion and egg-laying phenotypes ( Fig 1C and S1B Fig ) . eipr-1 encodes a novel WD40 domain protein of 362 amino acids ( Fig 1B ) . Like RUND-1 and CCCP-1 , EIPR-1 has a single conserved ortholog in other metazoans . However , unlike RUND-1 and CCCP-1 , EIPR-1 is also found outside metazoans in plants and some fungi and protozoa ( S2 Fig ) . Interestingly , EIPR-1 is not present in all protozoa and fungi , suggesting an ancestral origin and loss in specific lineages including the budding yeast Saccahromyces cerevisiae . The mouse Eipr1 cDNA could also rescue the worm eipr-1 mutant ( Fig 1C and S1B Fig ) , demonstrating functional conservation of the mammalian ortholog . The phenotypic similarities between eipr-1 and rab-2 , rund-1 , and cccp-1 mutants suggests that these genes act in a common pathway . To test this , we built double mutants between eipr-1 and either rab-2 or rund-1 and assayed locomotion and egg-laying behavior . In both cases , the double mutants do not show enhanced locomotion or egg-laying defects compared to the single mutants ( Fig 1D and S1C Fig ) . Thus , eipr-1 acts in the rab-2 pathway to control locomotion and egg-laying behaviors . Mutants in rab-2 , rund-1 and cccp-1 have reduced axonal levels of dense-core vesicle cargos [19 , 22 , 23] . To test whether eipr-1 also acts in dense-core vesicle cargo trafficking , we examined trafficking of both luminal and membrane cargos of dense-core vesicles . For luminal cargos , we assayed the Venus-tagged peptides NLP-21 , FLP-3 , and INS-22 . NLP-21 and FLP-3 are endogenous neuropeptides that undergo proteolytic processing [25 , 26] , while INS-22 is an insulin-family peptide that does not undergo proteolytic processing [27] . For a membrane cargo , we assayed GFP tagged IDA-1 , the worm ortholog of the dense-core vesicle transmembrane protein phogrin [28] . Like the rab-2 pathway mutants , we found that eipr-1 mutants also had reduced axonal levels of all luminal cargos and the transmembrane cargo IDA-1::GFP ( Fig 2A–2D and S3A–S3C Fig ) . Double mutants of eipr-1 with either rab-2 or rund-1 did not show an enhanced defect compared to the single mutants ( Fig 2A ) , again suggesting that eipr-1 acts in the rab-2 pathway . To determine whether the eipr-1 defect is specific to dense-core vesicle cargos , we examined trafficking of the synaptic vesicle cargo UNC-17 , the vesicular acetylcholine transporter . eipr-1 mutants showed no reduction in axonal levels of UNC-17 ( Fig 2E ) . eipr-1 mutants also showed no reduction in axonal levels of the synaptic vesicle SNARE protein synaptobrevin ( SNB-1 ) ( S3D Fig ) . We conclude that EIPR-1 acts in the RAB-2 pathway to specifically mediate trafficking of dense-core vesicle cargos . A mutation in the egl-3 proprotein convertase blocks neuropeptide processing and leads to an increase in axonal levels of luminal dense-core vesicle cargos [19 , 22] . As with rab-2 and rund-1 , there is increased axonal fluorescence of Venus-tagged NLP-21 in eipr-1; egl-3 double mutants as compared to the eipr-1 single mutant ( Fig 2F ) . However , the egl-3 mutant on its own has increased axonal fluorescence compared to wild-type , and the eipr-1 mutant decreases axonal fluorescence about 50% in either wild-type or egl-3 backgrounds ( Fig 2F ) , indicating that eipr-1 , like rab-2 and rund-1 , acts in parallel to egl-3 . Thus , eipr-1 mutants have reduced axonal levels of both processed and unprocessed cargos . The behavioral and dense-core vesicle phenotypes of eipr-1 mutants suggest that eipr-1 acts in neurons . Given that eipr-1 is the fourth gene in an operon , it has been difficult to determine the endogenous expression pattern of eipr-1 ( see Materials and Methods for details and S4A Fig ) . Thus , we instead expressed the eipr-1 cDNA under the control of neuronal-specific promoters to determine whether the gene functions in neurons . Expression of eipr-1 ( + ) under the neuronal specific rab-3 promoter fully rescued eipr-1 mutant locomotion , dense-core vesicle trafficking , and egg-laying ( Figs 3A , 3B and S1B ) . Expression of eipr-1 ( + ) under the cholinergic unc-17 promoter also rescued the locomotion defect . Expression in cholinergic motor neurons ( using the acr-2 promoter ) did not rescue the locomotion phenotype , but expression driven by a head-specific derivative of the unc-17 promoter ( unc-17Hp ) rescued the locomotion phenotype , indicating that eipr-1 acts mainly in head cholinergic neurons to regulate locomotion behavior ( Fig 3A ) . The dense-core vesicle trafficking defect of eipr-1 mutants is cell autonomous , as expression of eipr-1 ( + ) driven by the unc-129 promoter fully rescued the axonal trafficking defect of NLP-21::Venus expressed by the same unc-129 promoter ( Fig 3B ) . Thus , EIPR-1 acts in neurons to control dense-core vesicle trafficking at the cellular level and locomotion behavior at the organismal level . The cell-specific constructs described above had EIPR-1 C-terminally tagged with GFP . Because these constructs were able to rescue the mutant phenotype , the EIPR-1::GFP protein must be functional; thus we examined these strains to determine the subcellular localization of EIPR-1 . Unlike RAB-2 , RUND-1 and CCCP-1 which are localized predominantly to the trans-Golgi [19 , 22 , 23] , EIPR-1::GFP was expressed diffusely throughout the cytoplasm when expressed under such promoters as rab-3 or unc-17 , even when coexpressed with a GTP-locked mutant of RAB-2 ( S4B and S4C Fig ) . However , given that endogenously tagged EIPR-1 is undetectable ( see Materials and Methods: Expression of eipr-1 , vps-52 , and vps-53 ) , these EIPR-1::GFP constructs are almost certainly overexpressed even though they are single-copy insertions , and the lack of localization could be an artifact of this overexpression . Thus , although the genetic interactions indicate that EIPR-1 acts in the RAB-2 pathway , it is unclear whether EIPR-1 colocalizes with RAB-2 and its interactors . WD40 domain proteins typically act as scaffolds that mediate multiple protein-protein interactions [30] . Given the genetic interactions seen between eipr-1 and members of the rab-2 pathway , we asked whether the EIPR-1 protein shows physical interactions with any of the RAB-2 pathway proteins . Using yeast two-hybrid assays , we did not detect interactions between EIPR-1 and RAB-2 , RUND-1 or CCCP-1 . RAB-2 and RUND-1 were also localized normally in eipr-1 mutants . Thus , neither cellular localization nor physical interactions connect EIPR-1 directly to RAB-2 or its interactors , suggesting that EIPR-1 may act at a distinct step in this trafficking pathway . To identify interactors of EIPR-1 , we expressed GFP-tagged rat EIPR1 in the rat insulinoma cell line 832/13 and performed mass spectrometry on GFP pulldowns . 832/13 cells package insulin into dense-core secretory granules that closely resemble neuronal dense-core vesicles and share similar mechanisms of biogenesis , maturation , and release [31 , 32] . Mammalian orthologs of RAB-2 , RUND-1 , CCCP-1 and EIPR-1 are all expressed endogenously in 832/13 cells ( Fig 4A ) , so these cells are a reasonable place to look for interactors of these proteins . We performed two independent pulldowns of GFP-tagged rat EIPR1 . The top four hits identify the proteins VPS51 , VPS50 ( also known as syndetin or VPS54L ) , VPS52 , and VPS53 ( Fig 4B and S5A Fig ) . Also high on the list is VPS54 . These five proteins define the related GARP and EARP trafficking complexes that share the VPS51 , VPS52 , and VPS53 proteins , but use either VPS54 or VPS50 as a fourth subunit [33–35] . Thus , EIPR1 is likely tied closely to one or both of these complexes , neither of which has been previously linked to dense-core vesicle biogenesis . These proteomic results are especially striking because , as described below , we had independently identified the vps-52 and vps-53 genes in our forward genetic screen for dense-core vesicle mutants . To confirm the interactions detected by mass spectrometry , we performed coimmunoprecipitation experiments . GFP-tagged EIPR1 expressed in 832/13 cells coimmunoprecipitated with either mCherry-tagged VPS51 or with endogenous VPS50 ( Fig 4C ) , validating the interactions . Since no other proteins showed as strong an interaction by mass spectrometry as the GARP/EARP subunits , EIPR1 is a specific interactor of the GARP/EARP complexes . Further supporting the model that EIPR-1 functions with either the GARP or EARP complex , we isolated mutants in the worm orthologs of vps-52 and vps-53 in our original forward genetic screen . We cloned these mutants by mapping , whole-genome sequencing , and transgenic rescue ( see Materials and Methods ) . Both the vps-52 ( ox345 ) and vps-53 ( ox339 ) mutants are predicted to be molecular nulls , an early stop and deletion respectively , and both mutants phenocopy other deletion mutants in the same genes . These mutants also resemble a deletion mutant in the vps-51 gene . Neuronal roles of vps-51 , vps-52 , and vps-53 have not been previously described in C . elegans . We tested whether vps-51 , vps-52 and vps-53 mutants have behavioral and cellular phenotypes similar to those of eipr-1 and rab-2 . Like eipr-1 and members of the rab-2 pathway , vps-51 , vps-52 and vps-53 mutants have slow but coordinated locomotion ( Fig 5A ) and an egg-laying defect . A double mutant between rab-2 and vps-53 did not have a stronger locomotion phenotype than the rab-2 single mutant ( Fig 5B ) , indicating that rab-2 and vps-53 act in the same pathway to control locomotion . These mutants also exhibit defects in trafficking dense-core vesicle cargos . Like rab-2 and eipr-1 , vps-51 , vps-52 and vps-53 mutants all show reduced axonal levels of Venus-tagged NLP-21 or FLP-3 peptides ( Figs 5C , 5D and S6C ) . A rab-2; vps-53 double mutant did not show an enhanced NLP-21::Venus trafficking defect compared to the single mutants ( Fig 5E ) , also suggesting that rab-2 and vps-53 act in the same pathway . However , unlike the rab-2 and eipr-1 mutants which show reduced axonal accumulation of the dense-core vesicle transmembrane protein IDA-1 , mutants in vps-51 , vps-52 and vps-53 all show increased axonal accumulation of IDA-1 ( Fig 5F ) , indicating that these genes have at least some function distinct from rab-2 or eipr-1 . Since VPS-51 , VPS-52 , and VPS-53 are common to both GARP and EARP , these data suggest that GARP , EARP , or both are required for normal dense-core vesicle biogenesis and regulation of worm locomotion . To determine the expression pattern of vps-52 and vps-53 , we made transgenic worms expressing the YFP-derivative Citrine under either the vps-52 or vps-53 promoter . Both vps-52 and vps-53 were expressed strongly and widely in neurons and were also expressed in several other tissues ( Fig 6A and 6B , and Materials and Methods for details ) . These data are consistent with the reported expression patterns of vps-51 , vps-52 , and vps-53 [36] . To determine where VPS-52 and VPS-53 localize in neurons , we tagged them with tagRFP at their C-termini and expressed them under their own promoters as single-copy transgenes to achieve endogenous expression levels . Both VPS-52 and VPS-53 localized mainly to two or three perinuclear puncta in neuronal cell bodies ( Fig 6C and 6D ) , reminiscent of the localization of RAB-2 , RUND-1 and CCCP-1 to the trans-Golgi [23] . We examined colocalization of VPS-52::tagRFP or VPS-53::tagRFP with GFP-tagged RAB-2 and found that they are partially overlapping in worm neurons ( Fig 6C and 6D ) , consistent with the reported localization of GARP to the trans-Golgi in other cell types and other systems [36–39] . To determine whether it is GARP or EARP ( or both ) that is required for dense-core vesicle biogenesis , we obtained deletion mutations in the GARP-specific vps-54 and EARP-specific vps-50 subunits . Unlike the other GARP mutants , vps-54 mutants did not have defects in locomotion or axonal accumulation of the luminal dense-core vesicle cargos NLP-21 and FLP-3 ( Figs 7A , 7C , S6A and S6D ) . In contrast , vps-50 mutants had defects in locomotion and reduced axonal levels of luminal dense-core vesicle cargos very similar to those of mutants in eipr-1 , vps-51 , vps-52 and vps-53 ( Figs 7A–7C and S6A ) . An eipr-1; vps-50 double mutant did not have an enhanced defect in locomotion or dense-core vesicle cargo trafficking when compared to the vps-50 single mutant ( Figs 7A , 7B and S6A ) , indicating that eipr-1 and vps-50 act in the same pathway . Moreover , like eipr-1 , mutations in vps-50 did not affect axonal levels of the synaptic vesicle protein synaptobrevin ( S6E Fig ) , indicating that VPS-50 is not required for the trafficking of synaptic vesicle proteins . In 832/13 cells , endogenous VPS50 showed a perinuclear localization that partially overlapped GFP-tagged RAB2A and CCCP1 ( S4D and S4E Fig ) . Providing additional evidence that vps-50 acts in the dense-core vesicle pathway regulating worm locomotion , we found that a vps-50 mutation suppressed the activated Gq mutant egl-30 ( tg26 ) that was used in the genetic screen for dense-core vesicle mutants , similar to the suppression seen by eipr-1 , vps-52 and vps-53 ( S1A Fig ) . However , a vps-54 mutation did not suppress this activated Gq mutant . Taken together , these data support a role for EARP , but not GARP , in dense-core vesicle biogenesis and regulation of worm locomotion . The decreased axonal levels of luminal dense-core vesicle cargos like NLP-21::Venus could be due either to decreased levels of the cargo in dense-core vesicles , or to increased release of the cargo . Exocytosis of dense-core vesicles and release of their cargos is reflected by accumulation of NLP-21::Venus in scavenger cells called coelomocytes that reside in the body cavity of the worm [40 , 41] . Thus , we measured coelomocyte fluorescence as an indirect measure of dense-core vesicle release . Like rab-2 mutants , eipr-1 , vps-50 , and vps-52 mutants all showed reductions in the accumulation of Venus fluorescence in coelomocytes that is approximately proportional to the decrease in axonal fluorescence seen in these mutants ( S6B Fig ) . This suggests that these mutants are not defective in dense-core vesicle release per se , but release reduced amounts of dense-core vesicle cargo because the vesicles contain less cargo . Additionally , double mutants between eipr-1 and rab-2 or vps-50 did not have stronger coelomocyte uptake phenotypes than the single mutants , providing further evidence that these genes all act in the same dense-core vesicle cargo sorting pathway . Two independent vps-50 mutants exhibited either a slight decrease or no decrease in axonal levels of the dense-core vesicle transmembrane cargo IDA-1 ( Fig 7D ) . Thus , vps-50 and eipr-1 have a similar IDA-1 trafficking phenotype that is distinct from the increased axonal accumulation of IDA-1 seen in vps-51 , vps-52 and vps-53 mutants . Interestingly , vps-54 mutants showed no change in axonal accumulation of IDA-1 ( Fig 7D ) . These results suggest that vps-51 , vps-52 , and vps-53 may have a function in IDA-1 trafficking independent of vps-50 and vps-54 . Previously , C . elegans GARP mutants , including vps-54 , were shown to have enlarged lysosomes in coelomocytes [36] . We examined eipr-1 and vps-50 mutants for this phenotype using the lysosomal marker LMP-1::GFP expressed in coelomocytes . Unlike GARP mutants , neither eipr-1 nor vps-50 had enlarged lysosomes ( S3E Fig ) , indicating that EARP is not required for normal lysosomal morphology . Thus , GARP is required for normal lysosomal morphology and EARP is required for dense-core vesicle biogenesis .
Through a convergence of worm genetics and proteomics in rat 832/13 cells , we identified the WD40 domain protein EIPR1 as a direct physical and functional interactor of the EARP complex . The EARP complex was recently identified as a new complex structurally related to the GARP complex [34 , 35] . EARP shares the VPS51 , VPS52 and VPS53 subunits with the GARP complex , but uses VPS50 instead of VPS54 as a fourth subunit . VPS50 was also recently shown to interact with GARP subunits in three large-scale mass spectrometry interactome data sets , demonstrating that the EARP complex is present in various cell types in several species [42–44] . In two of these data sets , EIPR1 was shown to interact with EARP subunits in human HEK293T or HeLa cells [42 , 43] , providing support for our results and indicating that the EIPR1 interaction with EARP is robust and conserved . In fact , using VPS50 as a bait protein , VPS51 , VPS52 , VPS53 and EIPR1 were pulled down as a stoichiometric complex [42] , indicating that EIPR1 may form part of a stable complex with EARP . No VPS54 was pulled down . This is consistent with our genetic experiments showing that eipr-1 and vps-50 mutants have similar locomotion and dense-core vesicle cargo trafficking phenotypes that are shared by vps-51 , vps-52 , and vps-53 mutants , but not vps-54 . Thus , we propose that EIPR1 is a new interactor of the EARP complex . The human Eipr1 gene was originally named Tssc1 for “tumor-suppressing subtransferable candidate 1” because it was first identified as one of several genes thought to be candidates for a tumor-suppressing activity on chromosome 11 [45] . However , when the human genome was completed , Tssc1 was located on chromosome 2 , and thus could not be the putative tumor suppressor . Because the TSSC1 protein appears to be both physically and functionally connected to the EARP complex , we propose that it be renamed EIPR1 ( EARP-Interacting PRotein ) . WD40 domain proteins typically act as scaffolds that provide several interaction surfaces for the assembly of larger protein complexes [30] . Though EIPR1 clearly interacts with EARP , it is unclear whether EIPR-1 is required for the localization or stability of the EARP complex . In an eipr-1 mutant , VPS-52 still showed a punctate localization ( S4F Fig ) , though this could also reflect its participation in the GARP complex . In C . elegans ( unlike yeast ) , the remaining members of the GARP complex were shown to be stable and localized in the absence of one of the subunits [36] , so the same may hold true for EARP . Surprisingly , GFP-tagged EIPR-1 was found throughout the cytoplasm when overexpressed in C . elegans neurons and not associated with a membrane-bound compartment . However , overexpression of membrane-localized proteins can sometimes lead to a diffuse cytoplasmic appearance [46] . Because we could not detect an endogenously tagged EIPR-1::GFP fusion protein , we presume that eipr-1 is normally expressed at low levels . This conclusion is supported by the few cDNAs or RNAseq reads reported for eipr-1 on Wormbase ( http://www . wormbase . org/species/c_elegans/gene/WBGene00013599#0df9-gec6-2 ) . One other hit found in both of our EIPR1 pulldowns is SNAP29 , a late Golgi/endosomal SNARE protein that has been shown to function in endocytic recycling and autophagosome fusion [47–50] . SNAP29 was also identified as an interactor of EIPR1 or EARP subunits ( but not VPS54 ) in other recent mass spectrometry studies [35 , 42 , 43] , and we confirmed that SNAP29 interacts with EIPR1 by a coimmunoprecipitation experiment ( S5B Fig ) . GARP has been shown to interact with several late-Golgi SNARE proteins and has been proposed to both tether vesicles and promote SNARE assembly that leads to fusion [36 , 51–54] . We propose that SNAP29 is part of a SNARE complex that interacts with EARP . EARP was originally shown to be localized to a Rab4 positive endosomal compartment in both fly S2 cells and rat hippocampal neurons [34 , 35] , and depletion of the EARP-specific VPS50 subunit led to a modest defect in the endocytic recycling of the transferrin receptor back to the cell surface [35] . Here we identify a new neuronal role of this complex in the sorting of cargos to dense-core vesicles . The GARP complex was originally characterized as an endosome to Golgi retrograde trafficking complex that may tether endosome-derived vesicles at the trans-Golgi [37 , 51 , 53 , 55] . Loss or depletion of GARP subunits leads to a variety of phenotypes that are presumed to be secondary to a primary defect in retrograde trafficking , including missorting of lysosomal proteins , abnormal lysosomal morphology , defective autophagy , defective lipid and sterol transport , defective anterograde transport of glycosyl phosphatidylinositol ( GPI ) -anchored and transmembrane proteins , and defects in sphingolipid homeostasis [33 , 36 , 37 , 52 , 55–57] . GARP and EARP subunits are expressed especially strongly in the nervous system in both mice and worms [35 , 36 , 58 , 59] , but until now , neuronal phenotypes of GARP/EARP mutants have not been carefully characterized . However , several mutations in GARP/EARP subunits have been connected to neurological diseases . Mutations in VPS54 and VPS52 lead to neurological disorders in mice that have been studied as models of the human diseases amyotrophic lateral sclerosis ( ALS ) and the seizure disorder high-pressure nervous syndrome , respectively [60 , 61] . Also , mutations in the human VPS53 gene cause progressive cerebello-cerebral atrophy type 2 ( PCCA2 ) , a neurodegenerative disease characterized by severe mental retardation and early-onset epilepsy [62] . It will be interesting to determine whether the GARP/EARP mutations cause these diseases due to one of their previously characterized roles such as sorting lysosomal cargos [37 , 55] , or due to their newly identified role in dense-core vesicle biogenesis . It will also be interesting to determine whether neurological diseases associated with VPS52 and VPS53 are caused by dysfunction of GARP or EARP or both . The overt behavioral phenotypes of C . elegans mutants affecting the GARP/EARP common subunits ( vps-51 , vps-52 and vps-53 ) are very similar to those of vps-50 and eipr-1 , suggesting that EARP is more important than GARP for neuronal function in C . elegans . Our identification of EIPR-1 and EARP as important for dense-core vesicle cargo sorting supports a recent study that showed that C . elegans vps-50 mutants have locomotion and dense-core vesicle cargo trafficking defects [59] . Like eipr-1 , the slow locomotion of vps-50 mutants on food was partially rescued by expression in cholinergic neurons [59] , suggesting that eipr-1 and vps-50 function together in the same neurons to control locomotion behavior . VPS-50 was also shown to physically interact with the vacuolar-ATPase ( v-ATPase ) complex that acidifies dense-core vesicles , and vps-50 mutants had defects in vesicle acidification and assembly of the v-ATPase complex [59] . These defects may be indirectly caused by a primary defect of EARP mutants in missorting dense-core vesicle cargos , possibly including subunits of the v-ATPase . One discrepancy between the two studies is that we found a modest reduction in axonal accumulation of the dense-core vesicle transmembrane cargo IDA-1 in the vps-50 ( ok2627 ) mutant and no change in vps-50 ( n4022 ) ( Fig 7D ) . In contrast , Paquin et al . reported a modest increase in axonal accumulation of IDA-1 in vps-50 ( n4022 ) [59] . Though we do not know the basis of this discrepancy , one difference between the two studies is that Paquin et al . imaged a more posterior section of the dorsal nerve cord [59] . How does EARP contribute to the sorting of dense-core vesicle cargos ? EARP has been reported to localize to endosomal compartments [34 , 35] , suggesting that dense-core vesicle cargo sorting may involve trafficking through endosomes . We propose a speculative model based on the idea that the RAB-2/EARP pathway proteins may contribute to dense-core vesicle cargo sorting by a passive mechanism that does not involve direct interactions with cargo ( Fig 8 ) . We propose that EARP and the RAB-2 pathway may control the trafficking of cargo into or out of the endosomal compartment , perhaps as a way to remove non-aggregated cargo from the dense-core vesicle . In mutants in this pathway , increased trafficking of cargos to endosomes or reduced retrieval of cargos from endosomes leads to their ultimate loss to lysosomes and hence reduced cargo levels in mature dense-core vesicles . In this model , EARP and the RAB-2 pathway do not act instructively to sort cargos in the way a sorting receptor would , but instead have a permissive role . Such a permissive role for EARP and the RAB-2 pathway would be consistent with the fact that none of these mutants has a complete loss of cargo sorted to the mature dense-core vesicles that accumulate in axons . Several previous studies are consistent with our model . Following dense-core vesicle exocytosis , the dense-core vesicle transmembrane protein peptidylglycine α-amidating monooxygenase ( PAM ) has been shown to be recycled back to dense-core vesicles via endosomal compartments [63] , demonstrating that there must be trafficking of cargos from endosomes to dense-core vesicles . Our model suggests that such endosomal trafficking occurs not only following exocytosis , but also during the biogenesis of dense-core vesicles . Another study demonstrated that soluble dense-core vesicle cargos are segregated from self-aggregating cargos in immature dense-core vesicles near the trans-Golgi [64] , consistent with the idea that aggregation of cargos at this point in vesicle maturation can be sufficient to drive sorting into mature dense-core vesicles . The identification of EIPR-1 and EARP adds to a growing list of proteins that have been recently identified as being involved in mediating aspects of dense-core vesicle biogenesis . In C . elegans , we and others previously identified the small GTPase RAB-2; the putative RAB-2 GAP TBC-8; the RAB-2 effectors RIC-19 , RUND-1 and CCCP-1; and the membrane-associated Golgi-localized protein HID-1 [19 , 20 , 22 , 23 , 65 , 66] . Mutations in all of these proteins result in similar defects in dense-core vesicle cargo trafficking , and genetic epistasis data support the idea that they function in a common pathway . In other systems , a number of factors have been recently identified as having roles in dense-core vesicle biogenesis . These include factors that belong to protein families implicated in various aspects of membrane trafficking such as BAR domain proteins [18 , 21 , 67 , 68] , members of the Arf family of small GTPases [69] , the clathrin adaptor complexes AP-1 and AP-3 [70–73] , members of the BLOC-1 and HOPS complexes that are important for the biogenesis of lysosome-related organelles [74–76] , and members of the sortilin family of lysosomal sorting receptors [77 , 78] . Among these , there are a few possible connections to the RAB-2/EARP pathway we have described in C . elegans . AP-1 is involved in trafficking between various cellular compartments , most notably between the trans-Golgi and endosomes [79] , and has been shown to be associated with immature dense-core vesicles [6 , 8] , so it may be involved in the endosomal recycling pathway proposed by our model . The BLOC-1 subunit dysbindin was identified as an interactor of EIPR1 , VPS51 and VPS53 by mass spectrometry [43] , and the possible dense-core vesicle coat protein VPS41 ( along with other HOPS subunits ) was identified as an interactor of Rab2 [34] . One final factor shown to be important for dense-core vesicle biogenesis in mouse chromaffin cells is the SNARE protein Vti1a [80] . Interestingly , Vti1a is one of the components of a trans-Golgi SNARE complex that binds GARP [53] , and it also localizes to immature dense-core vesicles near the trans-Golgi [80] . Syntaxin 6 , another member of this SNARE complex , has also been shown to be associated with immature dense-core vesicles and small vesicles near the trans-Golgi or endosomes [8 , 11 , 81] . Blocking antibodies to syntaxin 6 were shown to inhibit the homotypic fusion of immature dense-core vesicles in an in vitro fusion assay [11] , but the physiological importance of syntaxin 6 for dense-core vesicle biogenesis in vivo remains unclear . Interestingly , the RAB-2 effector RUND-1 is tightly colocalized with syntaxin 6 in C . elegans neurons [23] and the BAR domain protein PICK1 tightly colocalized with syntaxin 6 in mouse pituitary cells [21] . Also , syntaxin 6 physically interacts with SNAP29 [11] , a SNARE protein that we and others identified as an EIPR1 or EARP interactor [35 , 42 , 43] . It will be interesting to determine whether SNAP29 , Vti1a , and syntaxin 6 are members of a SNARE complex that interacts with EARP , and to identify the compartment where they function .
Worm strains were cultured and maintained using standard methods [82] . A complete list of strains and mutations used is provided in the Strain List ( S1 Table ) . The eipr-1 ( ox316 ) , vps-52 ( ox345 ) and vps-53 ( ox339 ) mutants were isolated in an ENU screen for suppressors of the hyperactive locomotion of an activated Gq mutant , egl-30 ( tg26 ) [23] ( S1A Fig ) . When outcrossed away from the egl-30 ( tg26 ) mutation , all three of these mutants had sluggish unmotivated locomotion and defects in egg-laying . The phenotypes of all three of these mutants are partially maternally rescued; for example , the phenotype of an eipr-1 mutant is weaker if its parent is heterozygous ( eipr-1/+ ) than if its parent is homozygous ( eipr-1/eipr-1 ) . The basis of this maternal rescue is not clear , but indicates the likely maternal deposition of these proteins in the egg . We mapped these mutations using single nucleotide polymorphisms ( SNPs ) in the Hawaiian strain CB4856 as described [83] . The eipr-1 ( ox316 ) mutation was mapped to a 205 kb region on the right arm of chromosome I with 31 predicted protein-coding genes . Because there was no coverage of this region by C . elegans cosmids , we obtained C . briggsae BACs ( from CHORI ) carrying orthologs of the genes in this region and injected them into eipr-1 ( ox316 ) mutants . The BAC RPCI94_01L06 rescued the eipr-1 locomotion defect , while the BAC RPCI94_13K10 did not . RPCI94_01L06 carries orthologs of six C . elegans genes in the mapped region . We sequenced the best two candidate genes in the ox316 mutant and found a C to T transition mutation in exon 4 of Y87G2A . 11 , leading to a premature stop codon at Q310 . We obtained and sequenced the eipr-1 cDNA from the ORFeome library , confirming the gene structure predicted on Wormbase . The clone carried a single mutation that we fixed by Quick Change mutagenesis . We then cloned the mutation-free cDNA into a Gateway entry vector for use in rescue experiments . We rescued the ox316 mutant with a transgene carrying only Y87G2A . 11 , confirming the gene identification . The vps-52 ( ox345 ) mutation was mapped to a 720 kb region in the middle of the X chromosome with 122 genes . The vps-53 ( ox339 ) mutation was mapped to a 92 kb region in the middle of chromosome III with 23 genes . We constructed a double mutant strain carrying both the ox339 and ox345 mutations and performed Illumina whole-genome sequencing . Within the region where ox339 mapped , we found no SNPs predicted to alter protein function , but did identify a 687 bp deletion within the T05G5 . 8 ( vps-53 ) ORF . Within the region where ox345 mapped , we found only a single SNP predicted to alter protein function , a C to T transition mutation in the F08C6 . 3 ( vps-52 ) ORF that created a premature stop codon at S119 . The mutations in the vps-53 and vps-52 genes were confirmed by Sanger sequencing . We confirmed the gene identifications by performing complementation tests with other deletion mutations in each gene and by performing single-gene rescue experiments . The vps-50 ( ox476 ) mutation was isolated as a background mutation in a strain carrying the linked ced-6 ( n2095 ) mutation . It was shown to be an allele of vps-50 by a complementation test with vps-50 ( ok2627 ) , and by sequencing vps-50 to identify the molecular lesion [59] . vps-50 ( ox476 ) was separated from the ced-6 mutation before further analysis . A complete list of constructs is provided in the Plasmid List ( S2 Table ) . Most of the constructs were made using the three slot multisite Gateway system ( Invitrogen ) . Typically , a promoter , a coding sequence ( genomic DNA or cDNA ) , and an N- or C-terminal fluorescent tag ( GFP or tagRFP-T ) were cloned along with a 3’UTR into the pCFJ150 destination vector used for Mos1-mediated single copy insertion ( MosSCI ) on chromosome II at ttTi5605 [84 , 85] . All insertions were made by the direct injection MosSCI method . For most constructs , we isolated multiple independent insertions that behaved similarly . Extrachromosomal arrays were made by standard transformation methods [86] . eipr-1 is the fourth gene in an operon about 30 kb downstream from the start of the first gene in the operon , so it is not clear what promoter regulates eipr-1 . We made a construct driving expression of GFP by the promoter region upstream of the first gene of the operon ( 1348 bp upstream of the W09C5 . 1 start codon ) . Extrachromosomal arrays of this construct showed expression in several tissues including the intestine , pharynx and hypodermis ( S4A Fig ) , but not neurons , suggesting that this promoter fusion does not represent the endogenous expression pattern of eipr-1 . We also tagged the chromosomal eipr-1 gene with GFP using the CRISPR/Cas9 method [87] . However , neither N-terminal nor C-terminal GFP-tagged eipr-1 was bright enough to detect , even with the use of an anti-GFP antibody to amplify the signal , indicating that eipr-1 is likely to be expressed endogenously at low levels . We made constructs driving expression of the yellow-fluorescent protein Citrine under the vps-52 and vps-53 promoters and generated worms with extrachromosomal arrays . For vps-52 , 822 bp upstream of the start codon was used as a promoter . vps-53 is the second gene in an operon downstream of the gene gcc-2 . For vps-53 , 4502 bp upstream of the gcc-2 start codon was used as the promoter . vps-52 showed strong expression in neurons in the head , ventral cord , and tail ( Fig 6A ) , as well as expression in the pharynx , intestine , uterus , spermatheca , skin , and several muscle types ( body wall , vulval ) . Other than the skin and muscle expression , this expression pattern matches that of rund-1 [23] . vps-53 was not as strongly expressed , but was clearly visible in many head neurons ( though possibly not all ) , ventral cord neurons , tail neurons , the pharynx , and weakly in the body wall muscles ( Fig 6B ) . To measure locomotion , first-day adults were picked to thin lawns of OP50 bacteria , allowed to rest for 30 seconds , and then body bends were counted for one minute . A body bend was defined as the movement of the worm from maximum to minimum amplitude of the sine wave [88] . To measure egg-laying , L4 larvae were placed on plates with OP50 and allowed to mature at 25°C overnight . The next day , five adult animals were moved to a fresh plate and allowed to lay eggs at 25°C for 2 . 5 hours . The number of eggs present on the plate was then counted . The INS-1-derived 832/13 cell line [89] was obtained from Dr . Christopher Newgard ( Duke University School of Medicine ) via Dr . Ian Sweet and Dr . Duk-Su Koh ( University of Washington ) . 832/13 cells were routinely grown at 5% CO2 at 37°C in RPMI-1640 , GlutaMAX™ ( GIBCO ) , supplemented with 10% FBS , 1 mM Sodium pyruvate , 10 mM HEPES , 0 . 0005% 2-beta-mercaptoethanol , and 1X Pen/Strep ( GIBCO ) . Cells were passaged biweekly after trypsin-EDTA detachment . All studies were performed on 832/13 passages between 70 and 90 . 832/13 cells were transfected using Lipofectamine 2000 ( ThermoFisher ) according to the manufacturer’s instructions . The anti-GFP nanobody was expressed and purified as previously described [90] with few modifications . In brief , bacterial expression vector pLaG16 containing the anti-GFP nanobody was transformed in Arctic Express ( DE3 ) cells ( Agilent ) . Cells were induced with 0 . 1 mM IPTG for 16 h at 8°C and centrifuged at 5 , 000g for 10-min at 4°C . To isolate the periplasmic fraction by osmotic shock [91] , cells were incubated for 1h in TES buffer ( 0 . 2 M Tris-HCl pH 8 , 0 . 2 mM EDTA and 0 . 5 M sucrose ) . Cells were spun down for 10 min at 6 , 000g at 4°C and the pellet was suspended in TE buffer ( 0 . 2 M Tris-HCl pH 8 and 0 . 5 mM EDTA ) following a 45 min incubation . Cells were pelleted by a 30 minutes spin at 30 , 000g at 4°C . The supernatant ( periplasmic fraction ) was bound to PerfectPro Ni-NTA Agarose affinity resin ( 5 Prime ) for 1h at 4°C . The resin was washed once with wash buffer I ( 20 mM sodium phosphate pH 8 . 0 , 0 . 9 M NaCl ) and twice with wash buffer II ( 20 mM sodium phosphate pH 8 . 0 , 150 mM NaCl and 10 mM imidazole ) and eluted with His elution buffer ( 20 mM sodium phosphate pH 8 . 0 , 150 mM NaCl , 250 mM imidazole ) . The eluent was then dialyzed with PBS . Recombinant anti-GFP nanobody was conjugated to epoxy-activated magnetic Dynabeads M270 ( Life Technologies ) using 10 μg recombinant protein per 1 mg of Dynabeads , with conjugations carried out in 0 . 1 M sodium phosphate pH 8 . 0 and 1 M ammonium sulfate , with an 18 h incubation at 30°C . Beads were washed once with 100 mM Glycine pH 2 . 5 , once with 10 mM Tris pH 8 . 8 , 4 times with PBS and twice with PBS plus 0 . 5% Triton X-100 . Beads were stored at -20°C in PBS with 50% glycerol . For immunoprecipitation , 4x106 832/13 cells were plated onto 10 cm petri dishes . Twenty-four hours later , cells were transfected with either EIPR1::GFP or GFP . After 24 hours , cells were washed with PBS and harvested in lysis buffer [50 mM Tris pH 7 . 5 , 150 mM NaCl , 1% NP-40 , Protease inhibitor cocktail ( Pierce ) ] . Lysates were passed 10 times through a 20G needle and incubated for 30 min at 4°C . Lysates were pre-cleared by a 20 , 000 g centrifugation at 4°C for 15 minutes and the supernatant was incubated with 30 μg of anti-GFP nanobody bound to magnetic beads , for two hours at 4°C . The beads were washed three times with 250 μL wash buffer ( 50 mM Tris pH 7 . 5 , 300 mM NaCl , 1% NP-40 ) . Samples were eluted in two steps with ( 1 ) 0 . 2 M glycine pH 2 . 5 and ( 2 ) 1 M NH4OH . Eluents were neutralized with 0 . 33 M Tris-HCl pH 7 . 5 and stored at -80°C . Samples were processed using modified FASP ( filter aided sample preparation ) to remove mass spec incompatible substances [92] . Samples were filtered with 10K Amicon Ultra 0 . 5 ml filters ( Millipore ) . Samples were buffer exchanged with 8 M urea in 50 mM ammonium bicarbonate pH 7 . 8 , reduced with TCEP ( Pierce ) , alkylated with IAA ( Sigma ) and digested with trypsin ( Pierce ) . Samples were concentrated by speed vacuum . A total of 0 . 15 μg of peptides was chromatographically separated onto a Thermo LTQ-Velos Pro mass spectrometer coupled with a Waters nanoACQUITY liquid chromatography system . A 75 μm fused silica column ( Polymicro Technologies ) was loaded with 30 cm C12 Jupiter ( Phenomenex ) , 4 μm reverse-phase beads , and a 100 μm fused silica Kasil frit trap ( PQ Corporation ) loaded with 4 cm of C12 Jupiter . Peptides were separated by a 110 minute gradient consisting of buffer A ( 95% water , 5% acetonitrile and 0 . 1% formic acid ) and buffer B ( 95% acetonitrile , 5% water and 0 . 1% formic acid ) . Two analytical replicates were run for each sample with each set of replicates randomized . Quality control samples were run every sixth sample and at the beginning of the runs to assess column chromatography stability . Data were searched with SEQUEST [93] against a Rattus norvegicus FASTA database containing contaminants . False discovery rates were determined via a decoy database using Percolator [94] at a q-value threshold of 0 . 01 and peptides were assembled into protein identifications using ID Picker [95] . Lists of EIPR1::GFP hits were assembled after first subtracting hits found in a GFP control pulldown ( S5A Fig , Left ) or subtracting hits found in the pulldowns of two other proteins , CCCP1::GFP and RUNDC1::GFP ( S5A Fig , Right ) , performed in parallel . For co-immunoprecipitation , 832/13 cells were cultured and lysed as described above ( Mass spectrometry ) . The lysates were incubated with 20 μg anti-GFP nanobody bound to magnetic beads , the beads were washed three times with lysis buffer and resuspended in Laemmli loading buffer . Samples were resolved on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose or PVDF membranes . To detect co-precipitated proteins , we added the following primary antibodies: mouse monoclonal anti-GFP ( 1:1000 , Santa Cruz #sc-9996 ) , monoclonal anti-mCherry ( 1:50 , a gift from Jihong Bai and the Fred Hutchinson Cancer Research Center antibody development shared resource center ) , monoclonal anti-VPS50 ( 1:1000 , FLJ20097 monoclonal antibody , clone 2D11 , Abnova #H00055610-M01 ) , rabbit polyclonal anti-SNAP29 ( 1:1000 , Sigma #S2069 ) , or monoclonal anti-beta-tubulin as a loading control ( 1:1000 , ThermoFisher , BT7R , #MA5-16308 ) . The secondary antibody was an Alexa Fluor 680-conjugated goat anti-mouse antibody ( 1:20 , 000 , Jackson Laboratory #115-625-166 ) or Alexa Fluor 680-conjugated goat anti-rabbit antibody ( 1:20 , 000 , Jackson Laboratory #115-625-144 ) . A LI-COR processor was used to develop images . Worms were mounted on 2% agarose pads and anesthetized with 50 mM sodium azide . Images were obtained using a Nikon 80i wide-field compound microscope except for the images shown in Fig 6A and 6B which were obtained on an Olympus confocal microscope , and the images in Fig 6C and 6D which were obtained on a Deltavision deconvolution microscope . To image the dorsal nerve cords , young adult animals were oriented with dorsal side up by exposure to the anesthetic for ten minutes on the slide before placing the cover slip . For quantitative imaging of dorsal cord fluorescence , all strains in a given experiment were imaged on the same days and all microscope settings were kept constant . The same section of the dorsal cord around the vulva was imaged in all worms . Maximum intensity projections were quantified using ImageJ software , measuring the total fluorescence in a region of interest encapsulating the cord and subtracting the background fluorescence of a region of identical size adjacent to the cord . 2x105 832/13 cells per well were seeded onto sterilized cover slips placed in 12 well cell culture plates . The next day , cells were rinsed twice with ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature . The cells were rinsed twice with PBS and permeabilized with 0 . 5% Triton X-100 in PBS for 5 minutes at room temperature . The cells were again washed twice with PBS and then placed in 5% milk in PBS for 1 hour at room temperature , followed by immunofluorescence staining with rabbit polyclonal anti-CCDC132 ( 1:50 , Sigma #HPA026679 ) and mouse monoclonal anti-GFP ( 1:350 , Santa Cruz #sc-9996 ) in 0 . 5% milk in PBS at room temperature for 1 hour . The cells were then washed with PBS three times for 5 minutes each , and incubated with Rhodamine anti-rabbit secondary antibody ( 1:1000 , Jackson Immunoresearch #111-025-144 ) and Alexa Fluor 488 anti-mouse secondary antibody ( 1:1000 , Jackson Immunoresearch #115-545-146 ) at room temperature for 1 hour . The cells were washed with PBS three times for 5 min each and examined by fluorescence microscopy ( Nikon 80i wide-field compound microscope ) . The anti-CCDC132 antibody recognizes VPS50 . P values were determined using GraphPad Prism 5 . 0d ( GraphPad Software ) . Data sets with multiple comparisons were analyzed by a one-way ANOVA followed by a Bonferroni posthoc test to examine selected comparisons or by Dunnett’s test if all comparisons were to the wild type control . Pairwise data comparisons were analyzed by two-tailed unpaired t tests . | Animal cells package and store many important signaling molecules in specialized compartments called dense-core vesicles . Molecules stored in dense-core vesicles include peptide hormones like insulin and small molecule neurotransmitters like dopamine . Defects in the release of these compounds can lead to a wide range of metabolic and mental disorders in humans , including diabetes , depression , and drug addiction . However , it is not well understood how dense-core vesicles are formed in cells and package the appropriate molecules . Here we use a genetic screen in the microscopic worm C . elegans to identify proteins that are important for early steps in the generation of dense-core vesicles , such as packaging the correct molecular cargos in the vesicles . We identify several factors that are conserved between worms and humans and point to a new role for a protein complex that had previously been shown to be important for controlling trafficking in other cellular compartments . The identification of this complex suggests new cellular trafficking events that may be important for the generation of dense-core vesicles . | [
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"organisms... | 2016 | The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles |
Snake bite is one of the most neglected public health issues in poor rural communities worldwide . In addition to the clinical effects of envenoming , treatment with antivenom frequently causes serious adverse reactions , including hypersensitivity reactions ( including anaphylaxis ) and pyrogenic reactions . We aimed to investigate the immune responses to Sri Lankan snake envenoming ( predominantly by Russell's viper ) and antivenom treatment . Plasma concentrations of Interleukin ( IL ) -6 , IL-10 , tumor necrosis factor α ( TNFα ) , soluble TNF receptor I ( sTNFRI ) , anaphylatoxins ( C3a , C4a , C5a; markers of complement activation ) , mast cell tryptase ( MCT ) , and histamine were measured in 120 Sri Lankan snakebite victims , both before and after treatment with antivenom . Immune mediator concentrations were correlated with envenoming features and the severity of antivenom-induced reactions including anaphylaxis . Envenoming was associated with complement activation and increased cytokine concentrations prior to antivenom administration , which correlated with non-specific systemic symptoms of envenoming but not with coagulopathy or neurotoxicity . Typical hypersensitivity reactions to antivenom occurred in 77/120 patients ( 64% ) , satisfying criteria for a diagnosis of anaphylaxis in 57/120 ( 48% ) . Pyrogenic reactions were observed in 32/120 patients ( 27% ) . All patients had further elevations in cytokine concentrations , but not complement activation , after the administration of antivenom , whether a reaction was noted to occur or not . Patients with anaphylaxis had significantly elevated concentrations of MCT and histamine . We have demonstrated that Sri Lankan snake envenoming is characterized by significant complement activation and release of inflammatory mediators . Antivenom treatment further enhances the release of inflammatory mediators in all patients , with anaphylactic reactions characterised by high levels of mast cell degranulation but not further complement activation . Anaphylaxis is probably triggered by non allergen-specific activation of mast cells and may be related to the quality of available antivenom preparations , as well as a priming effect from the immune response to the venom itself .
Snake envenoming is a significant medical issue worldwide [1]–[4] . It is a particular problem in South and Southeast Asia , including Sri Lanka , where bites occur from a number of snakes , most importantly Russell's viper ( Daboia russelii ) [5]–[7] . Snake venoms contain an array of proteins , toxins and enzymes that may cause coagulopathy , neurotoxicity , myotoxicity , hypotension and tissue necrosis [1] . In addition to direct toxic effects , in vitro studies involving the addition of snake venom to human plasma have shown activation of the complement cascade , with the generation of anaphylatoxins ( C3a , C4a , C5a ) , but these results have not been confirmed in vivo in envenomed snakebite victims [8] , [9] . Studies of mice injected with various snake venoms have demonstrated release of Interleukin-6 ( IL-6 ) , nitric oxide ( NO ) , IL-5 , tumor necrosis factor-α ( TNFα ) , IL-4 , IL-10 , prostaglandins and leukotrienes , with distinct time courses in production post venom exposure for individual mediators [10]–[13] . A small number of studies investigating plasma concentrations of proinflammatory cytokines in envenomed humans have shown elevated concentrations of IL-6 , IL-8 and TNFα [14]–[16] . However , these studies were performed on relatively small numbers of patients ( n = 14–26 ) and it remains unknown whether the release of immune mediators contributes to the manifestations of envenoming or simply reflects the degree of tissue damage . Early systemic reactions to lyophilized equine polyvalent antivenoms , such as those used in Sri Lanka and many other tropical countries , have been reported to occur in up to 75% of patients , with severe reactions ( anaphylaxis ) in up to 50% of those treated [5]–[7] , [17] , [18] . Anaphylaxis is classified as either immune-mediated or non-immune-mediated , with immune-mediated further classified as IgE-dependent or non-IgE dependent [19] . IgE-dependent anaphylaxis , where allergen exposure results in crosslinking of allergen-specific IgE bound to the surface of mast cells which subsequently degranulate and release mast cell tryptase ( MCT ) and histamine , requires prior exposure to the allergen . However , this is unlikely to occur with antivenom because the majority of snakebite victims have not been previously treated . Complement activation by aggregates of immunoglobulins and/or other proteins has therefore been proposed as the principal mechanism underlying early reactions , largely on the basis of in vitro studies [1] , [20] , [21] . The complement system may be activated by envenoming , but no evidence of further complement activation after antivenom treatment has been found in clinical studies [22] , [23] , and purification to reduce anti-complementary activity does not appear to lower reaction rates [24] . Unlike IgE-dependent anaphylaxis , rigors and fevers are prominent during early antivenom reactions [18] , with pyrogens ( endotoxins ) introduced by poor production processes implicated in these reactions . We have previously reported elevated serum/plasma concentrations of MCT , histamine , IL-6 , IL-10 and the soluble TNF receptor I ( sTNFRI ) in patients presenting to the Emergency Department with anaphylaxis triggered by foods , insect stings or drugs ( predominantly antibiotics and non-steroidal anti-inflammatory drugs ( NSAIDs ) ) [25] . We therefore aimed to investigate both the immune response to envenoming and the mechanisms of adverse reactions to antivenom by measuring markers of complement activation , mast cell degranulation and inflammation .
The study was approved by the Ethical Review Committee , Faculty of Medicine , University of Colombo and was registered with the Sri Lanka Clinical Trials Registry , SLCTR/2007/005 . All patients gave written and informed consent to the study . For patients between 14–18 years , written informed consent was obtained from the patient's parents/guardians as well as from the patients themselves . Patients were recruited from a secondary referral hospital in Chilaw , Sri Lanka between 21st January 2007 and 31st July 2009 . Patients ≥14 years requiring treatment with snake antivenom were eligible for inclusion . Patients administered antivenom prior to recruitment to the study were excluded . After commencing antivenom ( 10 vials of polyvalent antivenom ( Bharat Serums and Vaccines Limited , India ) in 500 ml normal saline ) , patients were observed for four hours for evidence of a systemic hypersensitivity reaction . Blood was collected into EDTA tubes prior to antivenom administration and then 15 minutes , 2 hours , 6 hours , 12 hours and 24 hours after antivenom administration . Samples were immediately centrifuged and the plasma aliquoted and frozen . Demographic information , details of the bite , information on snake identification , clinical features of envenoming , treatments and outcomes were recorded . Baseline observations were recorded prior to antivenom administration . The clinical features of early reactions to antivenom were recorded along with blood pressure , heart rate , respiratory rate and pulse oximetry readings . Clinical envenoming syndrome ( s ) were defined in each patient as venom induced consumption coagulopathy ( INR>1 . 5 ) , neurotoxicity ( ptosis , diplopia , bulbar paralysis , limb weakness and/or respiratory muscle paralysis ) or non-specific systemic symptoms ( nausea , vomiting , headache , abdominal pain , diarrhoea ) . The type of snake was based on identification of the snake by the patient or clinician , and confirmed by enzyme immunoassay ( EIA ) for Russell's viper cases [26] . Adverse reactions to antivenom were classified as hypersensitivity reactions as we observed “objectively reproducible symptoms or signs , initiated by exposure to a defined stimulus at a dose tolerated by normal subjects” [27] . The use of the term hypersensitivity does not imply previous exposure to antivenom . Systemic hypersensitivity reactions were defined by typical skin manifestations ( urticaria/erythema ) or National Institute of Allergy and Infectious Diseases/Food Allergy and Anaphylaxis Network ( NIAID-FAAN ) diagnostic criteria for anaphylaxis [28] , which recognise that some reactions may occur in the absence of typical skin features . Our adaptation of this system is presented in Table 1 . Because gastrointestinal symptoms were a common effect of envenoming , only new and persistent gastrointestinal symptoms were included in the NIAID-FAAN Definition 2 for the purposes of this study . We also added a further classification of respiratory reactions ( Definition 4 ) to account for the severe respiratory reactions sometimes noted to occur without typical skin features . These reactions were distinguished from those arising from respiratory paralysis because they were not preceded by a descending paralysis and result in primary hypoxia rather than hypoxia secondary to ventilator failure . Reactions were graded as skin only ( which does not correspond with a diagnosis of anaphylaxis ) , moderate anaphylaxis or severe anaphylaxis if hypotension or hypoxia was observed during the course of a hypersensitivity reaction [29] . Pyrogenic reactions ( rigors and/or fever ) were coded separately and not included in the hypersensitivity reaction grading system . Treatment of anaphylactic reactions was directed by the attending physician and most commonly involved a combination of parenteral adrenaline ( 0 . 25 to 1 mg ) , corticosteroids ( hydrocortisone ) and H1-antagonists . Plasma concentrations of MCT , histamine , IL-6 , IL-10 , sTNFRI and TNFα , were measured as described previously [25] . MCT concentrations in plasma were analyzed using the Phadia ImmunoCAP system . MCT results greater than 11 . 4 ng/ml are considered positive by this system , however we also assessed change in MCT ( ΔMCT ) with a positive MCT result regarded as a deviation of 2 . 0 µg/L ( ng/ml ) between high and low values for each case as measured over time [30] . Histamine concentrations were determined using IBL Histamine ELISA Kits according to the manufacturer's instructions ( IBL , Hamburg , Germany ) . Plasma samples were analyzed for IL-6 , IL-10 , sTNFRI and TNFα using Flex Sets ( BD Biosciences , San Jose , USA ) . Plasma levels of C3a , C4a and C5a were determined using the Anaphylatoxin CBA Kit from BD Biosciences . We defined a positive result ( >upper limit ) as a concentration greater than the 99th centile of healthy control samples ( n = 34 ) in our laboratory . Data are presented as median ( interquartile range; IQR ) . Because of the skewed distributions of mediator concentrations , non-parametric tests were applied to non-transformed ( raw ) data . The measured baseline and peak mediator concentrations were compared across all groups using the Kruskal-Wallis test and between two groups using the Wilcoxon rank-sum ( Mann-Whitney ) test . Matched data pairs ( i . e . baseline versus peak ) were compared using the Wilcoxon signed-rank test . Correlations between baseline and peak mediator concentrations were assessed with Spearman rank correlation and Bonferroni adjustments were applied for multiple simultaneous testing . The semi-parametric lowess smoothing procedure was used to illustrate the relationship between time from antivenom and the concentration of each mediator . Statistical analysis was performed with Stata version 10 . 1 ( StataCorp , College Station , Texas ) .
Of 1004 patients presenting with suspected snakebites during the study period , 247 patients received antivenom . One hundred and ninety eight were randomised to receive antivenom by their allocated infusion rate , and of these , 120 patients had blood samples collected for this study . Baseline characteristics for these patients are presented in Table 2 . INR results were available for 112/120 patients , with 111/112 patients presenting with coagulopathy ( INR>1 . 5 ) . The median value for the maximum INR measured was 7 . 0 ( IQR 3 . 7–13 ) . Signs of neurotoxicity were present in 72/120 ( 60% ) patients and non-specific systemic symptoms were observed in 91/120 ( 76% ) patients . Eighty-six patients bitten by Russell's viper had pre-antivenom venom concentrations measured , with a median of 171 ( IQR 85–399 ) ng/ml . Thirty-three patients received a second dose of antivenom a median of 8 ( IQR 7–13 ) hours after the first dose , and six of these patients received a third dose of antivenom . Hypersensitivity reactions after the initial dose of antivenom occurred in 77/120 ( 64% ) patients , including 20/120 ( 17% ) with skin-only reactions and 57/120 ( 48% ) with anaphylaxis . All reactions occurred within the first four hours of starting antivenom treatment . Cases of anaphylaxis satisfied Definition 1 in 32/57 ( 56% ) , Definition 2 in 11/57 ( 19% ) , Definition 3 in 6/57 ( 11% ) and Definition 4 in 8/57 ( 14% ) . Severe anaphylaxis was observed in 51/57 ( 89% ) anaphylaxis cases , of which 21/51 ( 41% ) were hypotensive , 18/51 ( 35% ) were hypoxemic and 12/51 ( 24% ) were both hypotensive and hypoxaemic . As reported previously , there was no difference in hypersensitivity reaction rates between the two study arms ( i . e . 20 minute vs 2 hour antivenom infusion rate ) [5] . Pyrogenic reactions were observed in 32/120 patients ( 27% ) , 10/32 ( 31% ) reactions were pyrogenic alone and 22/32 ( 69% ) had a pyrogenic reaction in addition to a hypersensitivity reaction ( 1 skin-only , 21 anaphylaxis ) . Pyrogenic reactions were seen more often in patients with anaphylaxis ( 21/57 , 41% ) compared to those without ( 11/63 , 17% ) . A comparison between pre-antivenom mediator concentrations in envenomed patients and healthy controls is shown in Table 3 . Almost all patients with snake envenoming had evidence of complement activation before receiving antivenom , with plasma concentrations of C3a elevated above the 99th centile of normal in 91% of patients . Plasma concentrations of IL-6 , IL-10 , C4a and C5a were also significantly higher in envenomed patients before they received antivenom . Although plasma concentrations of MCT were greater than the 99th centile of our healthy control group in 31% of envenomed patients , only 13/120 ( 11% ) patients had MCT concentrations above 11 . 4 ng/ml , the upper limit of normal defined by the assay ( median 15 ( IQR 13 . 9–28 . 6 ) . Pre-antivenom concentrations of histamine and sTNFRI were not significantly elevated in envenomed patients compared to healthy controls . The median and interquartile ranges for TNFα were zero for both groups . Pre-antivenom mediator concentrations did not correlate with maximum INR or pre-antivenom serum venom concentrations ( p>0 . 5 for all comparisons , Spearman's ) . Patients with signs of neurotoxicity had similar pre-antivenom plasma concentrations of all mediators when compared to patients without signs of neurotoxicity ( p>0 . 2 for all comparisons , Mann Whitney ) . Patients with non-specific systemic symptoms of envenoming had significantly higher pre-antivenom levels of IL-6 , IL-10 and sTNFRI compared to those without ( median serum concentrations of IL-6 [12 . 6 ( IQR 0–31 . 9 ) versus 6 . 2 ( IQR 0–19 . 5 ) , p = 0 . 044] , IL-10 [29 . 6 ( IQR 10 . 7–68 . 7 ) versus 7 . 6 ( IQR 0–16 . 5 ) , p<0 . 001] and sTNFRI [2043 ( IQR 1131–3257 ) versus 962 ( IQR 603–1546 ) , p<0 . 001] ) . There was no significant difference in pre-antivenom mediator levels between patients subsequently having no reaction to antivenom and patients experiencing pyrogenic reactions and/or anaphylaxis . Changes in serum concentrations of all immune mediators measured before and after treatment with antivenom are illustrated in Figure 1 ( MCT and histamine ) , Figure 2 ( IL-6 , IL-10 , TNFα and sTNFRI ) and Figure 3 ( C3a , C4a and C5a ) . As all adverse reactions to the initial dose of antivenom occurred during the first four hours of antivenom treatment , and second and third doses of antivenom were administered at least four hours after the initial dose for all patients , only patients with samples available from the first four hours of antivenom treatment were used for comparisons of baseline ( pre-antivenom ) and peak mediator concentrations ( post-antivenom ) by Wilcoxon Signed Rank Test ( n = 98 ) . There were significant increases ( baseline versus peak ) in MCT in all patient groups , whereas histamine only increased significantly during skin-only and anaphylactic reactions ( Figure 1 ) . Following antivenom administration , peak levels of MCT and histamine were significantly higher in anaphylaxis cases than patients with no reaction and patients with skin-only reactions ( Table 4 ) . ΔMCT was positive in 77% of anaphylaxis cases , 56% of skin-only reactions and 31% of patients with no reaction . The proportion of anaphylaxis patients with a positive ΔMCT was significantly higher than patients with no reaction ( p<0 . 001 , Fisher's Exact Test ) . In patients who had anaphylaxis , concentrations of histamine returned to baseline levels between 5–10 hours after the initial dose of antivenom whereas concentrations of MCT remained elevated in some patients for up to 24 hours ( Figure 1 ) . Plasma concentrations of IL-6 , IL-10 , TNFα and sTNFRI all increased significantly after antivenom compared with baseline in all patient groups with the exception of TNFα and sTNFRI in skin-only reactions ( Figure 2 ) . However , peak mediator concentrations were not different between patients with no reaction , skin-only reactions and anaphylaxis ( Table 4 ) . In patients who had anaphylaxis , concentrations of IL-6 and IL-10 remained elevated for up to 24 hours after the initial dose of antivenom , whereas concentrations of TNFα and sTNFRI returned to baseline levels between 10–15 hours after the initial dose of antivenom ( Figure 2 ) . Figure 3 demonstrates that plasma concentrations of C3a , C4a and C5a were significantly elevated above healthy control levels prior to antivenom , but did not increase significantly in any patient group after administration of antivenom . There were no significant differences in peak concentrations of C3a , C4a and C5a between groups ( Table 4 ) . In agreement with our finding that there was no difference in hypersensitivity reaction rates between the two study arms ( i . e . 20 minute vs 2 hour antivenom infusion rate ) , there was also no significant difference in serum concentrations for any immune mediator measured between the different infusion rates . When all patients were considered together , patients who had pyrogenic reactions to the antivenom had significantly higher peak concentrations of IL-6 , IL-10 , TNFα and sTNFRI than patients without pyrogenic reactions ( data not shown ) . A confounding factor was that patients with pyrogenic reactions were also more likely to have anaphylaxis . Patients with pyrogenic reactions alone had significantly higher peak levels of IL-6 , IL-10 and sTNFRI compared to patients with no reaction ( pyrogenic or hypersensitivity ) ( Table S1 ) . Similarly , patients with both pyrogenic reactions and anaphylaxis had significantly higher peak concentrations of IL-6 , IL-10 , TNFα and sTNFRI compared to patients with severe anaphylaxis alone .
We found that envenoming by Sri Lankan snakes ( predominantly D . russelii , Russell's viper ) causes significant immune activation with elevated concentrations of MCT , IL-6 , IL-10 , C3a , C4a and C5a compared to healthy controls , but without clinical evidence of anaphylaxis to the venom . The pre-antivenom concentrations of IL-6 , IL-10 and sTNFRI correlated with the non-specific systemic symptoms of envenoming which included nausea , vomiting , headache , abdominal pain and diarrhoea . After treatment with antivenom there were no further changes in C3a , C4a and C5a , but there were rapid increases in IL-6 , IL-10 , TNFα and sTNFRI concentrations , even when no hypersensitivity reaction to antivenom occurred . These increases were highest in patients who also had pyrogenic reactions . Patients experiencing anaphylaxis also had rapid increases in plasma concentrations of MCT and histamine , with significantly higher increases compared to patients with no reaction to antivenom . As it would be unethical to have an envenomed control group that did not receive antivenom , an on-going effect of the venom cannot be ruled out as a cause of the increase in cytokine levels after treatment . However , the dramatic nature of the increase , timed with antivenom administration rather than time from bite , and correlations with the severity of hypersensitivity reactions both strongly suggest that the increase reflects an immune response to antivenom in addition to that caused by the envenoming . Treatment with antivenom is well known to trigger hypersensitivity reactions . Almost half of the patients in this study experienced severe anaphylaxis , characterised by hypoxia and/or hypotension . We observed significant increases in both MCT and histamine concentrations , correlating with the severity of the hypersensitivity reaction . This confirms a role for mast cells and/or basophils in the development of anaphylaxis after antivenom treatment , however , the mechanism of activation by antivenom remains to be elucidated . We observed no increase in C5a , C4a or C3a after treatment with antivenom , with plasma levels remaining steady or declining in the majority of patients post-antivenom . This contradicts previous theories , based on in vitro studies , of mast cell degranulation during anaphylaxis to snake antivenoms being caused by complement activation and subsequent binding of anaphylatoxins to their receptors on mast cells [1] , [20] , [21] . Therefore , the mechanism of mast cell activation during hypersensitivity reactions to antivenom remains to be elucidated . It is highly unlikely that the reactions are allergen-specific IgG or IgE-mediated reactions , because patients rarely have the prior exposure to antivenom that is required to generate allergen-specific antibodies . Repeat exposure to antivenom is very rare in most parts of the world where snake bites are due to unintentional encounters with snakes , unlike developed countries where snake handlers often have prior exposure [31] . Interestingly , reactions to snake antivenom are not more common in snake handlers , again supporting that reactions are not allergen-specific IgG or IgE-mediated . However , anaphylaxis may be triggered by non-allergen specific cross-linking of IgE bound to the surface of mast cells and basophils . For examples , potato lectin has been shown to activate and degranulate both mast cells and basophils by cross-linking cell bound IgE , which contains 10–12% carbohydrates , through interactions with IgE glycans [32] . A similar mechanism of non-allergen specific IgE-triggered anaphylaxis has been proposed for X-ray contrast media reactions , with the hypothesis that contrast media may bridge adjacent IgE molecules via non-specific attachment to their Fc segments [33] . Similarly , it is possible that antivenoms may contain aggregates of immunoglobulins and/or other proteins that have the ability to crosslink IgE receptors on the surface of mast cells resulting in non-allergen specific mast cell degranulation . Activation of mast cells via IgG receptors has long been proposed as a mechanism for antivenom-induced anaphylaxis . Studies of cultured human mast cells have demonstrated that IgG aggregates can activate mast cells [34] , and histamine is released after IgG-receptor FcγRIIA aggregation [35] . Most of these models have required an antigen-specific response and such a mechanism is unlikely in this setting for the reasons outlined above . However , heat-aggregated IgG1 ( i . e . without the presence of specific antigen ) can activate IFNγ-treated human mast cells and this effect is enhanced in the presence of C3a [36] . It is therefore plausible that the immune response to snake venom , including elevated levels of C3a observed in this study , primes cells to degranulate in response to microaggregates of IgG that may be present in antivenom preparations . This may be more likely in preparations that are reconstituted after lyophilisation , if the antivenom ( IgG ) does not completely dissolve . Mast cell activation can also occur via a number of other mechanisms , including engagement of receptors for ligands such as chemokines and cytokines , sphingosine 1-phosphate ( S1P ) and platelet activating factor ( PAF ) [37]–[40] . An interesting observation in this study was that prior to administration of antivenom there was already evidence of immune activation , including high concentrations of cytokines that are produced during anaphylaxis [25] . A proportion of envenomed patients also had clear evidence of mast cell degranulation prior to treatment with antivenom , with elevated baseline concentrations of MCT above normal range in 31% . Histamine concentrations were increased above normal range in some cases but this did not reach statistical significance , which may reflect poorer sensitivity of this assay for low levels of mast cell activation . In a mouse model , Metz et al found that mast cells significantly reduce snake-venom-induced pathology , by releasing carboxypeptidase A and possibly other proteases , which can degrade venom components [41] . This probably reflects an innate response to “dangerous” molecular patterns , reflecting the evolution of mast cells to provide a protective role against a range of noxious environmental toxins including venoms [42] . Thus , snake venom-induced activation of mast cells and the release of potent biologically active mediators has been proposed to contribute to the symptoms of envenoming , including local inflammation , vascular permeability ( oedema ) and shock [12] . It is possible that mast cell activation and the induction of cytokine and anaphylatoxin production by venom also primes cells to react to antivenom . Pyrogenic reactions , including shivering and sweating , occurred in 32 patients , 10 with no other features of immediate hypersensitivity reaction based on our criteria ( Table 1 ) . Pyrogenic reactions to antivenom are thought to be caused by contamination of the antivenom preparation by endotoxin or lipopolysaccharide ( LPS ) . Bacterial contamination of antivenom may also contribute to the development of anaphylaxis as LPS can directly activate mast cells [43] . Therefore , ensuring sterile manufacturing practises and filtering or heat-treating antivenom preparations may reduce both hypersensitivity and pyrogenic reactions [44] . We have demonstrated that Sri Lankan snake envenoming is characterized by significant complement activation and release of inflammatory mediators . Antivenom treatment then intensifies this immune response , resulting in anaphylaxis characterized by high levels of mast cell degranulation in almost half of cases . It is unlikely antivenom reactions are triggered by allergen-specific IgG or IgE . However , we did not observe an increase in C3a , C4a or C5a after administration of antivenom , suggesting anaphylaxis to antivenom is also not triggered by anaphylatoxins binding to their receptors on mast cells and triggering degranulation as previously proposed . Antivenom reactions may be triggered by immunogolublin or proteins complexes present in the antivenom binding to IgG receptors or non-specifically crosslinking IgE on the surface of mast cells . This effect may be related to the quality of available antivenom preparations , as well as a priming effect from immune responses to the venom itself , including venom-induced mast cell degranulation in some patients . | Snakebites cause life-threatening symptoms including uncontrolled bleeding and paralysis . The body's immune responses to snake venom may contribute to the severity of these symptoms but have not been well characterized in humans . Treatment with antivenom is potentially lifesaving , but also carries risk , as severe allergic reactions ( anaphylaxis ) are common . Anaphylaxis occurs when mast cells , triggered by either allergen-specific antibodies , other immunological mechanisms , or non-immune mechanisms , release mediators that cause skin rashes , shortness of breath and , in severe cases , life-threatening hypotension and/or hypoxia . We have studied 120 snakebite victims in Sri Lanka , both before and after treatment with antivenom . Our results have shown snakebite triggers activation of the complement cascade ( an important part of the body's innate immune defence ) and production of proinflammatory mediators . In addition , we have demonstrated a quite astonishing level of immune activation after antivenom treatment in virtually every person treated , regardless of whether they had a reaction to the antivenom . Half of the patients treated experienced anaphylaxis , with clear evidence of mast cell activation . Anaphylaxis to antivenom is unlikely to be triggered by allergen-specific antibodies , as patients had not been previously exposed to antivenom , but may be related to the quality of available antivenom preparations , as well as a priming effect from the immune response to the venom itself . | [
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] | 2013 | Immune Response to Snake Envenoming and Treatment with Antivenom; Complement Activation, Cytokine Production and Mast Cell Degranulation |
Group B Streptococcus ( GBS ) , a common commensal of the female genital tract , is the leading cause of invasive infections in neonates . Expression of major GBS virulence factors , such as the hemolysin operon cyl , is regulated directly at the transcriptional level by the CovSR two-component system . Using a random genetic approach , we identified a multi-spanning transmembrane protein , Abx1 , essential for the production of the GBS hemolysin . Despite its similarity to eukaryotic CaaX proteases , the Abx1 function is not involved in a post-translational modification of the GBS hemolysin . Instead , we demonstrate that Abx1 regulates transcription of several virulence genes , including those comprising the hemolysin operon , by a CovSR-dependent mechanism . By combining genetic analyses , transcriptome profiling , and site-directed mutagenesis , we showed that Abx1 is a regulator of the histidine kinase CovS . Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS , overcoming the need for an additional signal . Conversely , the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state . Using a bacterial two-hybrid system , direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing . We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1 . In conclusion , our study reports a regulatory function for Abx1 , a member of a large protein family with a characteristic Abi-domain , which forms a signaling complex with the histidine kinase CovS in GBS .
Some commensal microorganisms are also opportunistic pathogens . Harmless , and potentially beneficial , they may become causative agents of local or systemic infections [1] , [2] . To date , the signals dictating the switch from commensalism to virulence are mainly unknown . The complex set of genetic and environmental factors thought to be involved would affect the equilibrium between the host and the microbes . Deciphering the molecular events that govern the transition between commensalism and virulence will contribute to understanding and controlling infections due to opportunistic pathogens . Streptococcus agalactiae ( Group B Streptococcus , GBS ) is a commensal bacterium of the adult gastro-intestinal tract and is present asymptomatically in the vaginal flora of 10–30% of healthy women [3] . However , GBS is the leading cause of invasive infections in neonates ( pneumonia , septicaemia , and meningitis ) and a serious cause of mortality or morbidity in adults with underlying diseases [4]–[6] . As with most streptococci , the ability of GBS to cause infections is multifactorial [7] . The main virulence-associated GBS proteins identified to date are secreted or surface components , including the ß-hemolysin/cytolysin and specific adhesins [7]–[10] . Expression of several major GBS virulence genes is regulated at the transcriptional level by a two-component system ( TCS ) called CovSR ( Control of virulence Sensor and Regulator; also known as CsrSR ) [10]–[13] . TCS is the main signaling mechanism used by bacteria to respond to their changing environments [14] . CovSR controls the GBS response to acid stress in vitro and is necessary in vivo at several steps of the infectious process , such as resistance to macrophage killing and penetration of the blood-brain barrier [15]–[18] . Depending on the model of infection , inactivation of the CovSR system leads to a decrease or an increase of virulence [11]–[13] , [15] , suggesting that this system is tightly regulated in space and time to specifically adapt the bacterial virulence capacities to the infected compartments of the host . CovSR orthologs are present in several streptococcal species , including Streptococcus pyogenes ( Group A Streptococcus , GAS ) , where their function as a master regulator of virulence gene expression is conserved [19]–[23] . The CovSR system belongs to the EnvZ/OmpR family of TCS , where CovS is the membrane-bound histidine protein kinase ( HK ) and CovR is the cytosolic response regulator ( RR ) . Typically , HK is the sensor of an environmental stimulus that induces its autophosphorylation and , subsequently , phosphorylation of its cognate RR [14] . Phosphorylation of the RR alters its function , generally by modifying its binding affinity for target promoter regions and thereby changing expression of specific genes or operons . In contrast to the majority of TCS , the streptococcal CovSR regulatory pathway is a combination of two antagonistic negative regulators [24] . Indeed , in both GBS and GAS , the phosphorylated CovR acts mainly as a transcriptional inhibitor by direct binding to target gene promoters [11] , [13] , [25]–[27] . It was therefore suggested , but not formally demonstrated in vitro , that CovS acts mainly as a phosphatase on CovR to de-repress virulence gene expression during infection [13] , [24] , [28] . Currently , it is not known whether CovS is activated by general stresses [16] , [28] and/or is a direct sensor of specific ligands [22] . The latter hypothesis is supported in GAS by the identification of extracellular Mg2+ and sub-inhibitory concentrations of LL-37 , an antimicrobial peptide secreted by innate immune cells , as specific ligands that respectively activate and inhibit CovS activity [22] , [29] , [30] . Additional levels of TCS regulation exist that do not depend directly on environmental signal sensing by the HK sensor [31]–[33] . These cellular regulators target either the HK or the RR and were named “third components” , “auxiliary proteins” , “adaptors” or “TCS connectors” . While keeping the specific molecular links between an HK and its cognate RR , the additional cellular regulator allows to coordinate the cellular response and/or to integrate multiple signals [33] . This is the case in GBS where CovR activity is modulated by a second signaling pathway mediated by the serine/threonine kinase Stk1 [15] , [25] , [34] . Direct phosphorylation of the CovR T65 threonyl residue by Stk1 decreases CovR activity and interferes negatively with CovS-dependent phosphorylation of the D53 aspartyl residue [25] . These CovS- and Stk1- signaling pathways converge on CovR and are both necessary for GBS virulence [15] . In this study , we identified and characterized a transmembrane protein , Abx1 , belonging to a large family of bacterial proteins of unknown function that have a conserved domain called Abi [35] , [36] . We provide evidence that the putative protease activity of Abx1 , inferred from its similarity to eukaryotic CaaX proteases [35] , [36] , is not necessary for its function . Instead , we demonstrate that Abx1 is an additional partner of the CovSR system that is necessary to regulate CovS activity by a protein-protein interaction . We show that the Abx1-CovS signaling complex and the Stk1-dependent pathway are both necessary to control CovR activity . In addition to defining the genetic network controlling virulence gene expression in GBS , we provide the first report of a cellular regulator of HK activity belonging to the large family of bacterial Abi-domain proteins .
The ß-hemolysin/cytolysin expressed by GBS is an important virulence factor encoded within a cluster of twelve genes forming the cyl operon [8] , [37] , [38] . Hemolytic activity of GBS is always associated with the synthesis of an orange pigment . Among the 12 genes of the cyl operon ( gbs0644-gbs0655 genes ) , the CylE protein ( gbs0651 ) is the critical determinant for the dual hemolytic/pigmentation phenotypes [8] , [38]–[40] . Inactivation of cylE abolishes hemolytic activity and pigmentation , and the mutant is less virulent in animal models of systemic infections [39]–[41] . Screening of a collection of random Himar1 insertional mutants derived from the NEM316 wild-type ( WT ) strain allowed us to identify mutants that mapped in the cyl operon [40] . We extended this approach with approx . 2 , 500 new Himar1 mutants screened for hemolytic activity on blood agar plates and for pigmentation on Granada agar , a specific medium that stabilizes the GBS pigment [42] . We focused our analysis on four mutants displaying a strong decrease in pigmentation and hemolytic activity on Granada and Blood agar plates ( see Figure 1A for two of these four phenotypically identical mutants ) . The four mutants have unique and independent Himar1 insertions in the uncharacterized gene gbs1532 as revealed by direct sequencing on chromosomal DNAs . This 921-bp gene encodes a 306 amino acid protein annotated as a hypothetical protein or putative protease ( NCBI ref . seq . NP_735969 ) . Transposon insertions of the four Himar1 mutants were in positions 85 , 347 , 625 , and 632 in the DNA coding sequence , leading to truncated proteins at codons 29 , 116 , 209 , and 211 , respectively . Blast similarity searches , domain analyses , and topology prediction ( Figure S1 ) revealed that Gbs1532 belongs to a conserved superfamily of putative membrane-bound metalloproteases related to eukaryotic CaaX proteases [35] , [36] . The characteristic domain of this superfamily of more than 5 , 500 proteins is the Abi-domain ( Pfam02517 ) . To our knowledge , none of the bacterial Abi-domain proteins are functionally characterized to date [36] . We therefore named the translated gbs1532 gene product Abx1 , for Abi-domain protein related to CaaX protease , and renamed the corresponding gene accordingly ( i . e . , abx1 ) . This abx1 gene belongs to the core genome of all sequenced GBS strains [43] , [44] and 6 other genes coding for Abi-domain proteins similar to Abx1 are present in their genomes ( Figure S1 ) . To confirm that abx1 is required for pigmentation and hemolysis , we constructed in-frame deletion mutant ( Δabx1 ) in the NEM316 WT strain . The Δabx1 mutant displayed the same phenotypes as the Himar1 mutants ( Figure 1B ) . Furthermore , pigment production and hemolysis were restored upon ectopic expression of abx1 transcribed from its own promoter ( Figure 1A and 1B: pTCVΩabx1 complementing vector ) confirming that abx1 was responsible for the observed phenotypes . Wild-type or Δabx1 mutant strains expressing Abx1 from the complementing plasmid pTCVΩabx1 were more pigmented and more hemolytic than the control strains ( Figure 1B ) . This suggests that the expression level of Abx1 might be limiting for pigment production and hemolysis . To test this hypothesis , we cloned abx1 downstream of two constitutive promoters with different strength ( Ptet and Pcyl+ ) to generate the overexpressing vectors pTCVΩPtet_abx1 , and pTCVΩPcyl+_abx1 , respectively . Furthermore , we engineered the WT strain to replace the chromosomal Pabx1 promoter by the Pcyl+ promoter ( ΔPabx1::Pcyl+ ) . All recombinant strains carrying the different overexpression forms of abx1 displayed hyper-hemolysis on blood agar and hyper-pigmentation on Granada agar ( Figure 1C ) . Determination of the hemolytic titer of the different mutants and quantification of the corresponding abx1 transcription levels further confirmed the link between hemolysis and abx1 expression ( Table 1 ) . Remarkably , the pigmentation level also correlated with the strength of the abx1 upstream promoter ( Ptet>Pcyl+>Pabx1 ) on TH agar , a medium where pigmentation is usually not observed ( Figure 1C ) . Overexpression of Gbs1037 , the closest Abx1 homolog in GBS ( 40% identity over 222 residues , E-value = e-11; Figure S1 ) and of the unrelated EFGP ( Enhanced Green Fluorescence Protein ) did not modify pigmentation and hemolysis of NEM316 , underlining the specific activity of Abx1 ( Figure 1C ) . The activity of Abx1 is a conserved feature within the GBS species since abx1 deletion or overexpression in several clinical strains ( serotypes Ia , 515; Ib , H36B; II , 18RS21; III , BM110; and V , 2603V/R; [44] ) has a strong effect on their hemolytic activities and pigmentation ( Figure S2 ) . In eukaryotes , Abi-domain-containing proteins ( Pfam02517 ) are CaaX prenyl proteases involved in protein prenylation [35] , [36] , [45] , [46] . These transmembrane proteases remove the carboxy-terminal aaX tripeptide of a protein after the addition of an isoprenyl group on the cysteyl residue of the CaaX motif . One of the main features of the Abi-domain is the presence of four predicted core transmembrane helical segments ( labeled TMH1–4 ) containing conserved active-site residues ( Figure S1D ) . The overall topology and the critical residues for protease activity are conserved in Abx1 ( Figure 2A and S1 ) . To date , prenylation was never described in prokaryotic cells but , intriguingly , the CylE hemolysin contains a cysteyl residue at the fourth position from its carboxylic end ( i . e . , the critical residue of the CaaX motif [46] ) . We tested whether CylE could be post-translationally modified by a prenylation-like mechanism involving Abx1 . Overexpression of Abx1 using pTCVΩPcyl+_abx1 in the non-hemolytic and non-pigmented ΔcylE mutant did not increase hemolysis or pigmentation ( Figure 2B ) , confirming that Abx1 function is strictly dependent on the presence of a functional CylE . We then substituted the cysteyl residue of CylE located at the fourth position from its carboxy-terminal end by an alanyl ( CylE C664A ) . This mutation did not alter pigmentation and hemolysis significantly ( Figure 2B ) , which suggests that CylE is not prenylated on this cysteyl , and consequently that Abx1 might not be a CaaX protease . To further test whether Abx1 has protease activity , we replaced the conserved residues ( glutamyl at positions 164 and 165 and histidyl at positions 197 and 235 ) described as critical for this enzymatic activity by alanyl residues ( Figure 2A; [47] ) . All alleles obtained for Abx1 ( E164A , E165A , EE164/165AA , H197A , H235A ) restored hemolysis and pigment production when expressed in a Δabx1 mutant strain ( Figure 2C ) . Hence , the putative protease activity of Abx1 is not necessary for the CylE dependent phenotypes . Abx1 activity is dependent of CylE but appears to be independent of a CylE post-translational modification . To decipher the relationship between Abx1 and cyl-dependent hemolysis and pigment production , we took advantage of the previously described ΔCBSCyl mutant where the CovR binding sites ( CBS ) in the promoter of the cyl operon were deleted [40] . Since CovR acts as a transcriptional repressor of the cyl operon , deletion of CBSCyl leads to strong constitutive expression of the cyl operon . Consequently , the ΔCBSCyl mutant is hyperhemolytic and hyperpigmented in all tested conditions ( Figure 3A ) . Whereas abx1 deletion in a WT background abolishes pigmentation and hemolysis , deletion of abx1 in a ΔCBSCyl background ( Δabx1ΔCBSCyl ) did not affect the hyper-hemolytic and hyper-pigmented phenotypes of the parental ΔCBSCyl strain ( Figure 3A ) . This result suggests that Abx1 acts upstream of CovR-mediated transcriptional inhibition of the cyl operon . To confirm this epistatic interaction , we inactivated covR ( gbs1672 ) and the complete covSR two-component system ( gbs1671–1672 ) in the WT and Δabx1 backgrounds . As expected , inactivation of covR or covSR in the WT strain led to hyper-pigmentation and hyper-hemolysis ( Figure 3A ) . Similarly , inactivation of CovR or CovSR in the Δabx1 background restored pigmentation and hemolysis to levels similar to those observed in the ΔcovR and ΔcovSR mutants ( Figure 3A ) . This suggests that Abx1 is an inhibitor of CovR activity . Consistently , Western blot analysis of the adhesin BibA and CAMP factor whose expression are negatively and positively regulated by CovR [11] , [13] , respectively , revealed similar expression patterns in the ΔcovR and abx1 overexpression mutants: inactivation of CovR or overexpression of abx1 dramatically increased BibA production and abolished secretion of the CAMP factor ( Figure 3B ) . These results demonstrate that Abx1 activity is not restricted to pigmentation and hemolysin production , and suggest an antagonist function on CovR activity . To decipher the relationship between Abx1 and the CovSR two-component system , we compared the transcriptomes of ΔcovS , ΔcovR and ΔcovSR mutants with the Δabx1 deletion mutant and two abx1 overexpression mutants ( v_Oe_abx1 = vector based abx1 overexpression with the pTCVΩPcyl+_abx1 plasmid; and K_Oe_abx1 = chromosomal abx1 overexpressing strain ΔPabx1::Pcyl+ ) . Pairwise comparisons of Log2 expression ratios ( Table S1 , N = 1 , 905 genes ) revealed highly similar expression changes between the strain carrying the chromosomal overexpression of abx1 ( K_Oe_abx1 ) and the ΔcovS and ΔcovR deletion mutants ( Figure 4A: Pearson correlation = 0 . 704 and 0 . 727 , respectively ) . Strikingly , the transcriptome profiles of the abx1 overexpression and ΔcovS deletion mutants form a distinct cluster as revealed by hierarchical clustering analysis ( Figure 4B and S3A ) . Detailed analyses revealed a cluster of genes containing the direct and conserved targets that are negatively regulated by CovR ( Figure 4C and S3B: N = 29 and 40 genes with expression change log2>2 and >1 in at least one strain , respectively ) . Expression of nearly all of these genes , including the cyl operon ( gbs0644–gbs0655 ) and the bibA gene ( gbs2018 ) , were dependent on the expression level of abx1 . Moderate overexpression of abx1 in the chromosome ( ΔPabx1::Pcyl+; 3 . 7 fold change by microarrays ) induced a transcriptome response highly similar to that of the ΔcovS mutant ( Figure 4C ) . Greater overexpression of abx1 ( pTCVΩPcyl+_abx1; 9 . 1 fold change by microarrays ) further increased expression of the direct-CovR regulated gene set to a level close to that observed in a ΔcovR mutant ( Figure 4C ) . In contrast , downregulation below the significant threshold is observed in the Δabx1 mutant ( Figure 4C ) . qRT-PCR analysis on a set of 6 genes regulated by the CovSR system confirmed gene expression changes identified by microarray analysis ( Figure 4D ) . Moreover , bibA and CAMP factor transcription levels in the ΔcovR deletion and abx1 overexpression mutants are in accordance with the corresponding protein expression levels as observed by Western analysis ( Figure 3B ) . Taken together , the similarities between abx1 overexpression mutants and cov deletion mutants suggest that Abx1 exerts a specific effect on the CovSR two-component system , most likely by acting as a CovS antagonist . Our results suggest an inhibitory function of Abx1 on CovS activity or that Abx1 protects or sequesters CovR , impeding its phosphorylation by CovS either directly or indirectly . To discriminate between these possibilities , we used a bacterial two-hybrid system [48] to test the physical interactions of Abx1 with different putative partners . This showed that Abx1 is able to form homodimers and that it interacts indeed with CovS but not with CovR ( Figure 5A and 5B ) . The Abx1-CovS interaction is specific , as Abx1 did not interact with two other GBS HKs that are similar to CovS ( Figure 5A: Gbs2082 and Gbs0430 ) . To define the regions involved in the Abx1-CovS interaction , we tested the interaction of Abx1 with different domains of CovS ( Figure 5C ) . These experiments showed that Abx1 interacts specifically with the amino-terminal part of CovS containing its extracytoplasmic and transmembrane domains ( Figure 5D ) . Deletion of the extracytoplasmic loop of CovS did not impede interaction with Abx1 , suggesting that the loop is not directly required for Abx1-CovS interaction ( Figure 5D: CovS form VI ) . No interaction was detected either with the carboxy-terminal part of CovS containing the catalytic domains or with CovS truncated forms containing only one transmembrane domain ( Figure 5D ) . These results suggest that Abx1 interacts with the two CovS transmembrane domains involved in signal processing . In agreement with the proposed inhibitory function of Abx1 on CovS , deletion of covS in a Δabx1 background ( Δabx1ΔcovS ) restores pigment production and hemolysis of the non-pigmented and non-hemolytic Δabx1 mutant to levels similar to those of the ΔcovS mutant ( Figure 6A ) . However , as seen previously by transcriptome analysis , the ΔcovS mutant is not identical to the ΔcovR mutant . In particular , the ΔcovR mutant is hyper-pigmented and hyper-hemolytic ( see Figure 3A ) while the ΔcovS mutant is only slightly affected in pigmentation and hemolysis compared to the WT strain ( Figure 6A ) . To explain these observations , we must consider the three common activities of histidine kinases ( HK ) : auto-phosphorylation of the kinase on a histidyl residue , phospho-transfer on an aspartyl residue of the cognate regulator , and phosphatase activity of the HK on its cognate regulator [14] . Deletion of covS abolishes the three activities . In this case , cross-phosphorylation of the regulator by small metabolites or by a non-cognate HK can be observed due to the absence of the phosphatase activity of the cognate HK [49] , [50] . Thus , in the ΔcovS mutant , cross-phosphorylation of CovR might enable its partial activation . To gain experimental evidence , we constructed CovS and CovR alanyl substitution mutants in conserved residues critical for each of the three activities associated with HK [14] . First , we targeted the phospho-acceptor histidyl residue H278 to obtain an auto-phosphorylation deficient allele of CovS . Second , we replaced the aspartyl residue D53 of CovR normally phosphorylated by CovS to abolish phospho-transfer between CovS and CovR [25] , [26] . Third , we replaced the CovS threonyl residue T282 that was proposed to be specifically involved in HK phosphatase activity [50] , [51] . We then used pigmentation and hemolysis production as reporters of CovSR activity ( Figure 6B ) . The CovR D53A mutant displayed a hyper-hemolytic and hyper-pigmented phenotype like the ΔcovR mutant ( Figure 6B ) , as also observed previously in another GBS strain [25] , supporting the model of CovR as a transcriptional repressor under its D53P phosphorylated form . Analyses of the CovS H278A mutant strain showed phenotypes similar to the ΔcovS mutant ( Figure 6B ) , suggesting that the H278 residue is essential for all three activities , as assumed by a mechanistic model of HK activity [50] , [51] . We identified in CovS the conserved threonyl residues specifically required for the phosphatase reaction of several HK [50] , [51] . Interestingly , the CovS T282A mutant is non-hemolytic and non-pigmented ( Figure 6B ) indicating that CovR is locked in an active form that fully inhibits the expression of the cyl operon . Overexpression of abx1 in the ΔcovS , CovS H278A or CovS T282A mutants did not modify their phenotypes , indicating that Abx1 activity is dependent on CovS kinase/phosphatase activities ( Figure 6C ) . The fact that overexpression of abx1 in the WT context led to hyper-pigmented and hyper-hemolytic phenotypes , as seen for the ΔcovR and CovR D53A mutants ( Figure 6 ) , suggests that Abx1 actively stimulates CovS-dependent dephosphorylation of CovR . Expression level measurements of selected CovR-regulated genes ( cylE , cylJ , bibA , gbs0791 , gbs1037 , and cfb coding the CAMP factor ) in CovS and CovR alanyl substitution mutants confirm that the broad effect of abx1 overexpression depends upon a functional CovS histidine kinase ( Figure 6D ) . Overall , our results support a model where Abx1 inhibits the kinase-competent form and/or stabilizes the phosphatase-competent form of CovS . An additional regulator of the CovSR system is the eukaryotic-like serine/threonine kinase Stk1 . It has been shown in vitro that Stk1 regulates CovR negatively by direct phosphorylation of the threonyl residue at position 65 ( T65 ) [25] , [34] . The CovR T65 phosphorylation by Stk1 has been proposed to be mutually exclusive of CovR D53 phosphorylation by CovS [25] . Consistently , the Δstk1 ( Δgbs0307 ) mutant is strongly and negatively affected in pigment production and hemolytic activity ( Figure 7 ) , in agreement with an inhibitory role of Stk1 on CovR activity . To learn whether Abx1 and Stk1 function together , we tested if Abx1 and Stk1 interact using the bacterial two-hybrid system . No interaction was observed ( data not shown ) . Furthermore , pigmentation and hemolysis were restored in the non-pigmented and non-hemolytic Δstk1 strain upon increasing abx1 copy number with plasmid pTCVΩabx1 ( Figure 7 ) . This indicates that Abx1 is able to bypass CovR activation observed in the absence of Stk1 . However , pigmentation and hemolysis levels of the Δstk1 strain carrying the pTCVΩabx1 vector are below those of the WT strain ( Figure 7 ) . This result suggests that abx1 overexpression needs a functional Stk1 to fully inhibit CovR activity . Finally , a condition-dependent toxicity was observed when the overexpressing pTCVΩPcyl+_abx1 vector was introduced into the Δstk1 mutant . Specifically , strong overexpression of abx1 in Δstk1 is toxic on Granada agar ( Figure 7 ) . This phenotype is CovS-dependent as toxicity is alleviated in a Δstk1ΔcovS double mutant , which is unresponsive to abx1 overexpression ( Figure 7 ) . This result reinforces the link between Abx1 and a functional CovS . In addition , the Δstk1ΔcovS mutant is locked in an intermediate phenotype ( Figure 7 ) , revealing a partial inhibition of CovR in the absence of CovS . Thus , CovS appears necessary for complete activation of CovR in the absence of Stk1 , most likely by an active CovR D53 phosphorylation . As shown in the model depicted Figure 8 , we propose that the regulatory function of Abx1 is absolutely dependent on CovS but also depends on the serine/threonine kinase Stk1 to control CovR activity by a convergent signaling pathway . The CovSR two-component system is a master regulator of GBS virulence gene expression . In order to assess the role of Abx1 in GBS virulence , neonatal rat pups were infected by intraperitoneal injection of 5×106 bacteria of either the Δabx1 deletion or abx1 overexpressing mutants . In this model , both mutants exhibited reduced virulence compared to the parental WT strain ( Figure 9 ) . It is noteworthy that the abx1 overexpression mutant is totally avirulent ( 100% survival ) and that neonates did not display any sign of illness during the course of two independent experiments ( data not shown ) . These results show that abx1 expression at an appropriate level is required to develop GBS infection .
We first observed that Abx1 is required for hemolysin and pigment production in GBS and that abx1 overexpression in a WT background increases hemolysin and pigment production in a dose-dependent manner . These abx1 effects depend upon a functional CovSR pathway , which directly controls expression of the cyl operon . In agreement with a regulatory activity of Abx1 on the CovSR system , modulation of abx1 expression affected not only hemolysin/pigment production but also production of the adhesin BibA and the CAMP factor , two direct targets of CovR [11] , [13] . Indeed , genome-wide transcriptome profiling suggested a specific and antagonistic activity of Abx1 on the CovSR system . By combining genetic analysis and protein-protein interactions , we show that Abx1 acts through the HK CovS to control the activity of CovR . The transmembrane protein CovS is the cognate HK of CovR . However , in contrast to the majority of TCS , CovR is mainly a transcriptional inhibitor when phosphorylated on the conserved D53 residue and CovS is assumed to be primarily a phosphatase necessary to relieve repression of virulence genes during infection [11]–[13] , [24] . Our data support this regulatory logic ( i . e . , the inactivation by CovS of the CovR inhibitor ) but show in addition that Abx1 exerts a cellular control on the dual kinase/phosphatase activity of CovS ( Figure 8 ) . By using the cyl operon as reporter of CovR activity , we observed constitutive CovR activation with a CovS allele ( CovS T282A ) mutated in the conserved threonyl residue specifically involved in the phosphatase activity of several other HKs [50] , [51] or by deletion of Abx1 ( Figure 8B ) . Conversely , CovR can be inactivated by a D53A substitution , mimicking a non-phosphorylated allele , or by overexpression of Abx1 ( Figure 8C ) . Furthermore , all of these Abx1 effects are dependent on a functional CovS . Taken together , these results suggest that the level of Abx1 is critical to control the equilibrium between the kinase and phosphatase activities of CovS . It is noteworthy that inactivation of both CovS kinase and phosphatase activities , either with a CovS H278A allele or by deleting covS , led to intermediate phenotypes compared to those obtained by CovR inactivation . This pattern has been observed previously in GBS [13] and GAS for the CovSR ortholog [24] , [52] , [53] . In this condition , it is likely that CovR is partially activated by cross-phosphorylation , either by small metabolites like acetyl-phosphate or by a non-cognate HK . The specific phosphatase activity of CovS avoids inappropriate activation of CovR by other pathways , a general mechanism to insulate TCS signaling pathways [49] . Importantly , HK phosphatase activity is not simply the reverse reaction of the kinase activity [50] , [51] . The two activities involve different , but potentially overlapping , catalytic residues [50] and are mutually exclusive since they depend on different conformations of HK homodimers [14] . Both Abx1 and CovS are transmembrane proteins . We found that Abx1 interacts directly with CovS input domains ( i . e . , the extracellular and transmembrane domain ) , and ompacts CovS output domains ( i . e . , the catalytic cytoplasmic domain ) . Current models of HK activity regulation are based on different conformational states of HK homodimers [14] . It is therefore most likely that Abx1 interferes with the dynamic process of CovS conformational changes propagated along the whole proteins to control the kinase/phosphatase switch [14] . In the absence of Abx1 , CovS appears to be locked into a kinase-competent conformation while overexpression of Abx1 will result in stabilization of the phosphatase-competent conformation ( or destabilization of the kinase-competent form ) . Of note , the CovS extracellular loop is not necessary for the physical interaction with Abx1 and it is therefore likely that the main domains of CovS interacting with Abx1 are the transmembrane domains flanking this loop . These HK transmembrane domains control the conformational rotation of the cytoplasmic subunits of HK homodimer by a rotation or a piston mechanism [54] , [55] . Thus , intra- and inter-molecular interactions interfering with this dynamic process will impact the catalytic activity by blocking HKs in a kinase or a phosphatase form [56] , [57] . Positive and negative regulation of HK activity by physical interactions with transmembrane regulators has been described in both gram-negative and gram-positive bacteria [31] , [32] . Members of this category are mainly small hydrophobic peptides , like the PhoQ-B1500/SafA [58] , PhoQ-MgrB [59] and EnvZ-MzrA [60] pairs in enteric bacteria , or proteins with a single transmembrane domain , like the YycH and YycI proteins regulating the essential HK YyfG in Bacillus subtilis [61] , [62] . In particular , structural modeling suggests that the YyfG HK is inactivated by its interactions with YyfH and YyfI in the membrane environment [62] . In analogy , this suggests a mechanism of CovS inactivation by its interaction with the eight predicted transmembrane spanning domains of Abx1 ( Figure S1 ) . Abx1 controls GBS virulence gene expression by regulating CovS activity , via a mechanism independent of the direct sensing of an environmental signal by CovS . This cellular regulation of TCS by a “third” component raises the question of how this additional regulator is itself regulated [32] , [33] . The HK regulator may set up a feedback loop when located in the same operon as the HK , as for B . subtilis YycHI [61] , [62] , or it may act as a TCS connector when its transcription is controlled directly by another TCS [31] as for E . coli B1500/SafA , MgrB , and MzrA peptides mentioned above [58]–[60] . We did not find evidence that abx1 transcription is directly controlled by CovR ( data not shown ) but cannot exclude transcriptional regulation by an as yet unidentified RR . An alternative hypothesis would be an allosteric regulation and/or a scaffolding function of Abx1 [33] . Regulation of HK activity by signal ( s ) not directly recognized by the HK but sensed and/or transmitted by its interactor ( s ) might be a common mechanism , as already described for antimicrobial cationic peptide resistance modules in Firmicutes [63] , [64] and for HK-HK complex formation in Pseudomonas aeruginosa [65] . Until now , it is not known how CovS activity is regulated in GBS . In GAS , the CovS ortholog ( displaying 50 . 3% identity with the GBS CovS ) is thought to be a direct sensor of magnesium and of the human antimicrobial peptide LL-37 [22] , [29] , [30] . However , only two Abi-domain proteins are present in GAS genomes and neither is an Abx1 ortholog ( Figure S1 ) . Although the biological function of the CovS orthologs in virulence is conserved [19] , [20] , GAS and GBS inhabit different ecological niches ( nasopharynx and skin for GAS; gut and vagina for GBS ) , suggesting a species-specific response by CovS . The identification of conditions bypassing the Abx1 effects in GBS will be valuable for characterizing the putative signal ( s ) directly sensed by CovS . We found that activity of the Abx1-CovS module depends upon a second signaling pathway mediated by the serine/threonine kinase Stk1 [25] , [34] . The two pathways converge on CovR to regulate virulence gene expression ( Figure 8 ) . In vitro , direct phosphorylation of the CovR T65 threonyl residue by Stk1 interferes negatively with phosphorylation of the conserved D53 aspartyl residue and decreases CovR affinity for its target promoters [25] , [34] . By genetic analysis , we found that the Abx1-CovS and the Stk1 signaling pathways are dependent on each other to fulfill complete activation or inhibition of CovR . CovR phosphorylation on T65 and D53 are mutually exclusive [25] , strongly suggesting that CovR has three possible states ( Figure 8D: D53P T65 = active; D53 T65 = inactive I dephosphorylated; D53 T65P = inactive II ) . It should be noted that Stk1 has additional direct targets beyond CovR and that its activity depends upon its associated phosphatase Stp1 [66]–[69] . Interestingly , a Δstk1 mutant is affected in cell cycle progression ( [66] and data not shown ) , as observed in S . pneumoniae and other bacteria [70] , [71] , and Stk1 might respond to the release of cell wall components into the extracellular environment by growing bacteria [72] . Therefore , it appears that CovR integrates at least two types of signals to coordinate virulence gene expression ( via Abx1-CovS ) with the control of bacterial division ( through Stk1 ) . This integrated system might be necessary to accurately control GBS lifestyle from commensalism to invasive infection [15] . The attenuated virulence of both deletion and overexpression abx1 mutants supports this dynamic view of the infectious process . The survival of animals challenged intraperitoneally with the overexpressing abx1 mutant is reminiscent of the decreased virulence of ΔcovR mutants previously described [11]–[13] . Several virulence genes controlled by CovR are involved at several steps of the infectious process , ranging from stress resistance to adherence to host cell [10] , [11] , [13] , [16] , [18] , [73] . Among the genes directly regulated by CovR , the hemolysin/cytotoxin CylE has key functions [39] . At sub-lytic concentrations , CylE induces an anti-inflammatory response [74] , while high CylE expression induces a pro-inflammatory response [41] . The inappropriate activation of a strong pro-inflammatory response during initial steps of the infectious process might explain , at least in part , the loss of virulence of mutants overexpressing abx1 or inactivated for covR . In contrario , at a latter stage of infection , these mutants can be more virulent due to the cytotoxic effect of CylE on blood cells , as seen with a ΔcovR mutant injected intravenously in mice [15] . Overall , these results indicate that the Abx1-CovS-CovR-Stk1 regulatory network controls the fine-tuning of virulence gene expression which is critical for GBS disease progression . The demonstration that Abx1 is a cellular regulator of CovS represents a new function for a bacterial Abi-domain protein . The Abi-domain ( Pfam02517 ) of Abx1 is the characteristic domain of a large transmembrane protein family ( >5 , 000 sequences ) with up to 15 members per bacterial species [35] , [36] . This family is mostly uncharacterized to date in prokaryotes but includes the eukaryotic type II CaaX proteases involved in protein prenylation [36] , [45] . However , prenylation appears restricted to eukaryotes and the two examples of prenylated bacterial proteins , SifA of Salmonella typhimurium and AnkB of Legionella pneumophila , are bacterial effectors modified by the eukaryotic enzymes after their injection into the host cell [75]–[77] . Our characterization of targeted mutations ( Figure 2 ) did not sustain our initial hypothesis of a prenylation-like modification of the CylE hemolysin . Moreover , the putative catalytic residues inferred from similarities with CaaX proteases are not necessary for Abx1 activity . Given the high conservation of these residues ( Figure S1 ) , we cannot rule out that Abx proteins have protease activity . However , the main function of Abx1 as a regulator of the CovS histidine kinase does not require this putative protease activity . . Strikingly , the absence of a protease activity was already suggested for a Staphylococcus aureus Abi-protein involved in lysostaphin resistance [78] . Interestingly , three of the four S . aureus Abi-domain genes were identified recently in a genome-wide screen for altered targeting of cell-surface proteins [79] . These defects are mainly due to decreased transcription of the corresponding genes in the absence of the Abi-domain proteins [79] , suggesting a conserved mechanism in gene transcriptional regulation . Finally , it should be mentioned that about one-fifth ( >1 , 000 ) of bacterial Abi-domain proteins are annotated as “abortive infection protein” due to a historical miss-annotation [36] leading to confusion between Abi phenotypes [80] and Abi-domain proteins [36] . It was also suggested that Abi proteins are involved in self-immunity against bacteriocins [81] . However , only a small subset of Abi-proteins ( less than 1% ) is localized in putative bacteriocin-encoding loci [81] or in operons encoding small toxins [82] , [83] . In particular , the gene encoding the Abi-domain protein , SagE is located in the operon directing synthesis of the S . pyogenes ß-hemolysin known as streptolysin S or SLS [8] , [84] . However , genetic analysis of this locus did not provide any conclusive clues about the SagE function [84]–[86] . Abi-domain proteins localized in toxin operons remain to be experimentally characterized for their involvement in toxin production and/or regulation . Studies of TCS signaling in cellular context have identified a great variety of additional TCS partners , targeting either the HK or the RR [31] , [32] . In this study , we demonstrated that the Abi-domain protein Abx1 is a regulator of the HK CovS in GBS . The CovSR system was presumed to be a linear signaling pathway . We show here that the CovSR TCS is the core of a signaling network and propose a new regulatory model , depicted in Figure 8 , which includes Abx1-dependent CovS regulation and Stk1-dependent CovR regulation [25] , [34] . Deciphering the additional CovSR input/output associated with these cellular regulators might prove useful for understanding the GBS transition from commensalism to virulence . Strikingly , Abx1 belongs to a very large family of multi-spanning transmembrane proteins that remain mostly uncharacterized to date [35] , [36] and are highly conserved at the species level . It is therefore tempting to extend our findings to other members of this family . The characterization of other Abx-like proteins in GBS ( Figure S1 ) and in other bacteria will reveal whether they belong to a functionally conserved family of HK regulators .
Animal experiments were performed at the Institut Pasteur ( Paris , France ) animal husbandries in accordance with the policies of the European Union guidelines for the handling of laboratory animals ( http://ec . europa . eu/environment/chemicals/lab_animals/home_en . htm ) and were approved by the Institut Pasteur animal care and use committee ( N°04 . 118 ) . The reference WT GBS strain used in this study is NEM316 , a ST-23 and serotype III clinical isolate responsible for a fatal case of septicaemia , whose sequenced genome [43] is accessible ( NCBI RefSeq NC_004368 . 1 ) . The relevant characteristics of the bacterial strains and plasmids used in this study are summarized in Table S2 . GBS was cultured in Todd Hewitt ( TH ) broth ( Difco Laboratories ) at 37°C without agitation . Escherichia coli DH5α ( Invitrogen ) and XL1 Blue ( Stratagene ) were grown in Luriani Broth ( LB ) medium . When specified , antibiotics were used at the following concentrations: for E . coli: ticarcillin , 100 µg/ml; erythromycin , 150 µg/ml; kanamycin , 25 µg/ml; for GBS: erythromycin , 10 µg/ml; kanamycin , 1 , 000 µg/ml . Pigmentation and ß-hemolytic activity were detected on Granada agar and Columbia agar supplemented with 5% horse blood , respectively ( BioMérieux , France ) . Overnight GBS cultures in TH were washed , adjusted to 108 CFU/ml , serially diluted ( dilution factor 10 ) in microplates and spotted on appropriate agar plates . Horse blood plates were incubated at 37°C for 16–24 h , followed by an additional 16–24 h incubation at room temperature or 4°C depending on the hyper- or hypo-hemolytic mutants being tested . To highlight the halo of lysis due to the ß-hemolysin , photographic acquisitions of blood agar plates were taken with light from below . Whole images were further converted to gray scale and processed ( Photoshop CS4 , Adobe , US ) to adjust contrast and brightness . Granada plates were incubated 20–36 h at 37°C in anaerobic condition ( AnaeroGen , Oxoid , UK ) . Photographic acquisitions of TH and Granada plates were done with side lighting from above and whole images were processed for contrast and brightness . GBS hemolytic titers were determined by a semi-quantitative method as described previously , with slight modifications [11] . Briefly , culture adjusted to 2 . 108 CFU/ml in Phosphate Buffer Saline ( PBS ) solution supplemented with 0 . 2% glucose were serially diluted ( dilution factor = 2 ) in microplates . One volume ( 100 µl ) of 1% defibrinated horse blood ( Oxoid , UK ) in 0 . 2% glucose PBS was added in each well and plates were incubated 1 h at 37°C . After gentle centrifugation ( 5 min . at 1000 rpm ) to pellet unlysed cells , the amount of hemoglobin in 100 µl of supernatants was quantified by optical absorbance at 420 nm . Hemolytic activity of each strain was defined as the minimum dilution that lysed at least 10% of red blood cells . Hemolytic titers were defined by the ratio between hemolytic activities of each strain against the hemolytic activity of the WT strain ( titer X = activity X/activity WT ) . Negative and positive controls were PBS ( = 0% lysed cells ) and 0 . 1% SDS ( = 100% lysed cells ) instead of bacterial cells , respectively . All assays were performed in triplicates with independent cultures . Purification of GBS genomic DNA and E . coli plasmids DNA was done on columns following manufacturer instructions ( DNeasy Blood and Tissue kit and Quiaprep Spin Minipreps kit , respectively , Qiagen ) . Oligonucleotides ( MWG and Sigma ) used in this study are listed in Table S3 . Analytical PCR used standard Taq polymerase ( Invitrogen , Life technologies ) and preparative PCR for cloning and sequencing was carried out with a high fidelity polymerase ( Phusion , Finnzymes ) . Sanger sequencing was done at the Institut Pasteur sequencing core facility ( Paris , France ) using the ABI PRISM 3 . 1 dye terminator cycle sequencing kit ( Applied Biosystems ) or outsourced ( Beckman Coulter Genomic , UK ) . Plasmids for overexpression and complementation ( pTCV backbone , Table S2 ) , deletion ( pG+host5 backbone , Table S2 ) and for bacterial double hybrid ( pUT18 and pKNT25 backbones , Table S2 ) were constructed by standard cloning procedures and all inserts were fully sequenced . Primer pairs , DNA matrix and restriction enzymes used for each vector are detailed in Table S4 . For several constructs , we used a splicing by overlap-extension method [87] to generate in-frame deletion cassettes , site-directed mutagenesis inserts or translational fusions ( Table S4 ) . For instance , deletion cassettes for chromosomal in-frame deletions were generated by combining two 500 bp PCR products , corresponding to genomic sequences flanking the region to be deleted , that were designed to have 25–50 bp of homology with each other at one end [87] . The 1 kb amplification products obtained with external primers were subsequently cloned into the thermosensitive shuttle plasmid pG+host5 backbone and sequenced ( Table S4 ) . Plasmids were introduced in GBS by electroporation . For chromosomal gene inactivation/modification , allelic exchanges were selected as described [88] , [89] . Deletions and site-directed substitutions were confirmed by sequencing PCR products obtained with primers designed outside the genomic region used for the construction of the corresponding cassette ( Table S3 and S4 ) . Secreted proteins were purified from 40 ml TH broth cultures collected at mid-exponential phase ( DO600 nm = 0 . 5 ) . Supernatants were filter sterilized and concentrated 50-fold using Vivaspin 20 columns ( Sartorius ) . Cell-wall proteins were prepared from overnight cultures at 37°C in TH by mutanolysin ( Sigma-Aldrich ) digestion in osmo-protective buffer as described [73] , [89] . Following SDS-PAGE electrophoresis , proteins were transferred onto a nitrocellulose membrane ( GE Healthcare ) and detected using rabbit specific polyclonal antibodies [73] and horseradish peroxidase ( HRP ) -coupled anti-rabbit secondary antibodies . Signals were detected by chemiluminescence ( ECL , GE Healthcare ) . Total RNAs were extracted from exponentially growing cells ( OD600 = 0 . 4–0 . 5 ) in TH at 37°C with a phenol/Trizol-based purification method as previously described [11] . Reverse transcription was done with Superscript indirect cDNA kit ( Invitrogen , Life technologies ) and qPCR was carried out with SYBR Green PCR kits ( Applied Biosystems , Life technologies ) . Relative quantification of specific gene expression was calculated with the 2−ΔΔCt method , with gyrA as the housekeeping reference , and normalized against the NEM316 wild-type . Each assay was performed at least in triplicate on three independent cultures . For microarray analysis , identical culture conditions and RNA purification procedures were used . The three independent cDNA preparations of each strain were labeled with Cy5 or Cy3 ( Amersham Biosciences ) for dye swap hybridizations against NEM316 wild-type strain RNA prepared in parallel . The 15K custom microarray ( Agilent Technologies ) contains 8 , 691 60-mer oligonucleotides specific for the 2134 predicted genes and long intergenic region of the NEM316 strain [90] . Arrays were scanned in an Axon 4000B dual laser scanner and treated as described [90] . Raw data have been submitted to the ArrayExpress database under the Accession number E-MEXP-3703 ( http://www . ebi . ac . uk/arrayexpress/ ) . Hierarchical clustering were done with Cluster 3 . 0 and visualized with Java TreeView . For BACTH assays [48] , chimeric GBS proteins fused to either T18 or T25 fragments of adenylate cyclase ( CyaA ) were constructed by conventional cloning in pUT18 and pKNT25 vectors for T18/25 C-terminal tagging or in pUT18C and pKT25 for T18/25 N-terminal tagging ( Table S2 and S4 ) . Physical interactions were assayed in E . coli DHT1 cells ( Table S2 ) co-transformed with recombinant pKNT25/pKT25 and pUT18/pUT18C vectors . Interaction efficiencies between hybrid proteins were quantified by measuring ß-galactosidase activity in 96-well plates assays . Bacteria were grown overnight at 30°C in 1 ml LB broth in the presence of 0 . 5 mM IPTG and appropriate antibiotics in 2 . 2 ml 96-deepwell plates ( Thermo Scientific ) . To permeabilize cells , 100 µl of bacterial cultures in 96 deepwell polypropylene plates were treated with 500 µl of buffer containing ß-mercaptoethanol ( 50 mM ) , SDS ( 0 . 2% v/v ) and chloroform ( 3% v/v ) and vortex two times 1 min ( Mixmate , Eppendorf ) . For the enzymatic reaction , 40 µl of ONPG ( 4 mg/ml ) was added to 120 µl of the permeabilized cells solution in a new microplate . Reaction kinetics at 28°C were followed by recording the OD420 every 4 min for 60–90 min in a microplate reader ( Synergy , BioTek ) . Slopes ( min−1 ) with a correlation coefficient r2≥0 . 98 were used to calculated enzymatic activities relative to internal negative ( pUT18 and pKNT25 empty vectors; activity = O arbitrary units ) and positive controls ( pUT18-zip and pKNT25-zip vectors , [48]; activity = 1 , 000 arbitrary units ) incorporated in each microplate . Slopes420 was divided by the OD600 of each culture to normalize for initial cell density . At least four independent cultures were done for each plasmid combination . All recombinant pKNT25/pKT25 and pUT18/pUT18C vectors were tested against the corresponding empty vector and gave background level of ß-galactosidase activity ( not depicted in Figure 5 ) . Two-days old neonatal Sprague-Dawley rat pups ( Janvier , Le Genest Saint Isle , France ) were used for mortality curve experiments . Randomized groups of 10 neonatal rat pups were infected by intraperitoneal ( i . p . ) injection with 5×106 bacteria in 100 µl PBS . Survivals were monitored for five days after injection and two independent experiments were carried out . | The gram-positive Streptococcus genus includes three major human pathogens that are members of the normal microflora: Streptococcus pneumoniae ( also known as the pneumococcus ) , Streptococcus pyogenes ( Group A Streptococcus ) , and Streptococcus agalactiae ( Group B Streptococcus ) . Their carriage in the population is highly dynamic and mostly asymptomatic . However , each of these species can cause a wide spectrum of diseases , from local infections to systemic and fatal infections including septicemia and meningitis . Expression of streptococcal virulence-associated genes is tightly regulated at the transcriptional level . However , the signal ( s ) and the precise molecular events controlling the switch from commensalism to virulence are not yet understood . In this study , we identified and characterized a bacterial protein essential for virulence gene expression in Group B Streptococcus , the main pathogen of neonates . We show that this transmembrane protein , named Abx1 , interacts with the histidine kinase CovS to modulate the activity of the major regulator of virulence CovR . We define how a core set of four proteins , Abx1 , CovS , CovR , and the serine/threonine kinase Stk1 , interact to control the expression of virulence genes in S . agalactiae . We propose that Abx1-like proteins , that are widespread in bacteria , might be part of a conserved mechanism of two-component system regulation . | [
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"m... | 2013 | The Abi-domain Protein Abx1 Interacts with the CovS Histidine Kinase to Control Virulence Gene Expression in Group B Streptococcus |
We report that natural killer T ( NKT ) cells play only a minor physiological role in protection from Leishmania donovani infection in C57BL/6 mice . Furthermore , attempts at therapeutic activation of invariant NKT ( iNKT ) cells with α-galactosylceramide ( α-GalCer ) during L . donovani infection exacerbated , rather than ameliorated , experimental visceral leishmaniasis . The inability of α-GalCer to promote anti-parasitic immunity did not result from inefficient antigen presentation caused by infection because α-GalCer–loaded bone marrow–derived dendritic cells were also unable to improve disease resolution . The immune-dampening affect of α-GalCer correlated with a bias towards increased IL-4 production by iNKT cells following α-GalCer stimulation in infected mice compared to naïve controls . However , studies in IL-4–deficient mice , and IL-4 neutralisation in cytokine-sufficient mice revealed that α-GalCer–induced IL-4 production during infection had only a minor role in impaired parasite control . Analysis of liver cell composition following α-GalCer stimulation during an established L . donovani infection revealed important differences , predominantly a decrease in IFNγ+ CD8+ T cells , compared with control-treated mice . Our data clearly illustrate the double-edged sword of NKT cell–based therapy , showing that in some circumstances , such as when sub-clinical or chronic infections exist , iNKT cell activation can have adverse outcomes .
Natural killer T ( NKT ) cells are a unique subset of CD1d-restricted T cells that provide a link between innate and adaptive immune responses . In mice , invariant NKT ( iNKT ) cells express a semi-invariant TCR , consisting of a Vα14Jα18 TCR α-chain and a TCR β-chain biased towards Vβ8 . 2 , Vβ2 , and Vβ7 expression ( reviewed in [1] ) . Type II NKT cells are another cell subset in mice with more diverse TCR expression [1]–[3] . Upon stimulation , iNKT cells rapidly produce large quantities of pro- and anti-inflammatory cytokines , resulting in activation of other immune cells such as NK cells and conventional T cells [4]–[7] ( also reviewed in [8] ) . NKT cells recognise and respond to glycolipid antigens presented on CD1d molecules . The most well-defined antigen for iNKT cells is α-galactosylceramide ( α-GalCer ) , a marine sponge-derived glycolipid that specifically targets iNKT cells and no other lymphocyte populations directly [9] . The activation of iNKT cells by α-GalCer can enhance resistance in several infectious disease models , including viral , bacterial and parasitic infections ( reviewed in [10]–[12] ) . Among parasitic infections studied , α-GalCer has been shown to enhance resistance to malaria [13] , trypanosomiasis [14] and toxoplasmosis [15] . The ability of iNKT cells to produce IFNγ following stimulation with α-GalCer is important for this therapeutic effect and host protection during infection , although the robust induction of TNF , IL-4 and IL-13 by iNKT cells also occurs ( reviewed in [8] , [16] , [17] . However , the connection between therapeutically induced NKT cell responses and physiological NKT cell responses is not always clear . Nevertheless , there are parallels between physiological and therapeutic NKT cell responses in some disease models . For example , in experimental tumour models , the growth of methylcholanthrene ( MCA ) -induced sarcoma cell lines is restricted by physiological IFNγ produced by endogenous NKT cells [18] , while many other experimental cell lines , including the B16-F10 melanoma are only controlled by NKT cells following therapeutic activation with α-GalCer , again in an IFNγ-dependent manner [7] . It is also possible that following pathogen challenge , NKT cells will be exposed to foreign glycolipids , or self glycolipids , that , in the inflammatory environment , can trigger NKT cell responses that are similar to those induced by α-GalCer [19]–[22] ( reviewed in [23] ) . Visceral leishmaniasis ( VL ) is a potentially fatal human disease caused by infection with Leishmania donovani or L . infantum ( chagasi ) [24] , [25] . Infection of genetically susceptible mice with L . donovani results in an acute but ultimately resolving infection in the liver associated with the development of granulomas around infected Kupffer cells [26] . In contrast , a chronic infection develops in the spleen , associated with severe immunopathology [27] , [28] . Parasite numbers increase more slowly in the spleen , and total splenic parasite burdens usually only reach 5–10% of maximum levels in the liver , often with greater variation between individual mice than in the livers of the same animals [28] , [29] . NKT cells have been shown to regulate CXCL10 expression in the livers of C57BL/6 mice in the early stages of L . donovani infection , an event thought to be important for hepatic granuloma development [30] . A subset of mouse NKT cells has also been reported to be activated by L . donovani lipophosphoglycan presented by host CD1d [31] . Studies in CD1d-deficient BALB/c mice have also suggested that NKT cells are important for the efficient control of L . donovani growth [31] . In addition , infection of immature human DC with L . infantum results in increased CD1d cell surface expression , and subsequent increased recognition and killing by IFNγ-producing iNKT cells [32] . CD4+ NKT cells have also been implicated in protection against the early stages of cutaneous leishmaniasis caused by L . major infection in genetically resistant mouse strains [33] . However , a more recent study has shown that clinical recovery from L . major infection can occur in NKT cell-deficient mice in the same time period as wild type mice , despite this early NKT cell role in protection [34] . Here we investigated the role of NKT cells during experimental VL in C57BL/6 mice , and tested whether stimulation of iNKT cells with α-GalCer could enhance anti-parasitic activity . Our data indicate that NKT cells are neither required for the development of immunity in the liver , nor for the control of parasite burden in the spleen following L . donovani infection . Furthermore , iNKT cell activation by α-GalCer hinders disease resolution in the liver . These results have important implications for the modulation of iNKT cell function during established disease .
To determine the relative roles of iNKT cells and type II NKT cells during experimental VL , we infected C57BL/6 mice deficient in iNKT cells only ( B6 . Jα18−/− ) and all CD1d-restricted NKT cells ( B6 . CD1d−/− ) with L . donovani and compared their course of infection to C57BL/6 controls . Only minor differences in parasite burdens were found between NKT cell-deficient mice and C57BL/6 controls . B6 . Jα18−/− mice had significantly higher hepatic parasite burdens than C57BL/6 mice at day 56 post-infection ( p . i . ) ( p<0 . 05; Figure 1A ) , suggesting that iNKT cells may be required for optimal control of parasite growth at later stages of infection . However , C57BL/6 and B6 . Jα18−/− mice had resolved hepatic infection to a similar extent by day 90 and 180 p . i . ( liver parasite burdens were 93±42 Leishman-Donovan units ( LDU ) versus 119±36 LDU in C57BL/6 versus B6 . Jα18−/− mice at day 90 p . i . , and 0 . 4±0 . 5 LDU versus 6 . 2±3 . 0 LDU in C57BL/6 versus B6 . Jα18−/− mice at day 180 p . i . ) . A small but significant difference ( p<0 . 05 ) in hepatic parasite burdens was also observed between B6 . CD1d−/− and C57BL/6 mice at day 7 p . i . ( Figure 1A ) suggesting a minor role for type II NKT cells in the early control of L . donovani growth . Alternatively , the CD1d molecule itself may signal to antigen presenting cells independently of NKT cells [35] , thereby contributing to anti-parasitic immunity . B6 . CD1d−/− mice had resolved hepatic L . donovani infection to the same extent as C57BL/6 control mice by day 56 p . i . ( Figure 1A ) , and were not examined after this time point . There was no decrease in the total numbers of IFNγ- or TNF-producing cells in the liver measured by ELISPOT at any time point in any group of mice ( Figure S1 ) . In addition , parasite burdens in the spleen were similar in all three groups of mice throughout the first 56 d of infection ( Figure 1B ) and until day 180 p . i . in C57BL/6 and B6 . Jα18−/− mice ( splenic parasite burdens were 24±17 LDU versus 18±5 LDU in C57BL/6 versus B6 . Jα18−/− mice at day 90 p . i . , and 5±3 LDU versus 10±3 LDU in C57BL/6 versus B6 . Jα18−/− mice at day 180 p . i . ) . Therefore , control of parasite growth in the liver was achieved in the absence of both iNKT and type II NKT cells , with only minor roles for these cells at different times following infection . The links between physiological NKT cell responses and iNKT responses induced by ligands such as α-GalCer are not always apparent [8] , and even though we were unable to detect a clear physiological role for NKT cells in protection against L . donovani , it was important to determine whether therapeutic iNKT cell activation with α-GalCer could improve disease resolution . Therefore , we next tested whether iNKT cells could be activated by α-GalCer at the time of infection to improve control of L . donovani growth . We focused these studies on the liver because NKT cells comprise a major cell population in this tissue site and parasite burdens are significantly higher and more consistent in the liver than in the spleen . The activity of α-GalCer was confirmed prior to all experiments by measuring IFNγ production by hepatic iNKT cells 2 h following α-GalCer administration ( data not shown ) . A single injection of α-GalCer was administered so that the iNKT cell response was not tolerised or biased towards a Th2 response , as has previously been reported [12] , [36]–[38] . First , α-GalCer was administered the day prior to infection , and no differences in hepatic parasite burdens were observed in treated mice , compared with controls on day 14 p . i . ( Figure 2A ) . The same result was observed when α-GalCer was administered to mice 2 h prior to infection ( Figure 2B ) . Stimulation with α-GalCer–loaded bone marrow–derived dendritic cells ( BMDC ) results in more prolonged cytokine responses than with soluble α-GalCer in mice due to selective targeting of the glycolipid to DC and improved presentation of the antigen to iNKT cells [37] . L . donovani has also been shown to interfere with antigen processing pathways [39] . Although there is no direct evidence for CD1d antigen presentation being compromised during experimental VL , we sought to eliminate this possibility by delivering α-GalCer–loaded BMDC to mice 2 h prior to infection . However , despite the stimulation of efficient iNKT cell activation by α-GalCer–loaded BMDC , demonstrated by increased serum cytokine levels ( data not shown ) , and as previously reported [37] this mode of α-GalCer delivery also had no impact on hepatic parasite burdens relative to control mice that had received vehicle-loaded BMDC ( Figure 2B ) . We next determined whether and when iNKT cells were first activated in response to L . donovani infection , so that we could test whether α-GalCer stimulation at this time was effective at activating these cells for anti-leishmanial effects . Although the percentage of iNKT cells in the liver was reduced at day 14 p . i . ( Figure 3A ) , due to the hepatic recruitment of other leukocytes during granuloma formation ( reviewed in [26] ) , total iNKT cell numbers doubled by day 7 p . i . , and this increase was sustained at day 14 p . i . ( Figure 3B ) due to an approximate 10-fold increase in total liver mononuclear cell ( MNC ) number . There was also evidence of TCR down-regulation by iNKT cells at day 7 p . i . ( data not shown ) and day 14 p . i . ( Figure 3A ) , but not earlier , suggesting TCR-dependent NKT cell activation , as seen following α-GalCer stimulation [40]–[42] . Furthermore , NK1 . 1 expression on iNKT cells was also reduced at these times , consistent with activation of these cells ( Figure 3C ) . The expression of CD69 on iNKT cells also increased above normal intermediate levels following L . donovani infection from day 7 p . i . onwards ( Figure 3C and 3D ) . Together , these data suggested that day 7 p . i . was an appropriate time to stimulate iNKT cells with α-GalCer to coincide with the time when hepatic iNKT cells were naturally activated following L . donovani infection . However , when C57BL/6 mice were given α-GalCer at this time , there was a significant increase in hepatic parasite burdens on day 14 p . i . ( p<0 . 05 ) , relative to controls ( Figure 3E ) . Thus , α-GalCer–stimulation of iNKT cells at the time when activation occurred naturally during infection suppressed , rather than improved , control of parasite growth in the liver . To test whether the activation of iNKT cells during an established L . donovani infection had any therapeutic potential , α-GalCer was administered on day 14 p . i . , which is around the time of peak hepatic parasite burden in C57BL/6 mice ( Figure 1A ) . Mice were sacrificed 1 wk later , and again , there were significant increases ( p<0 . 05 ) in hepatic parasite burdens in mice that received α-GalCer , compared to controls ( Figure 4A ) . When α-GalCer–loaded BMDC were used instead of soluble α-GalCer , there were no significant changes in hepatic parasite burdens , compared with controls that received vehicle-loaded BMDC ( Figure 4A ) . Serum TNF levels were reduced in α-GalCer-treated mice on day 21 p . i . compared to controls ( p<0 . 05 ) , but this did not occur in mice that received α-GalCer-loaded BMDC compared to controls that received vehicle-loaded BMDC ( Figure 4B ) . A similar trend was observed for reductions in serum IFNγ levels in mice that received either soluble α-GalCer or α-GalCer–loaded BMDC compared to controls , but these differences were not statistically significant ( Figure 4C ) . It should also be noted that vehicle-pulsed BMDC caused small reductions in serum TNF and IFNγ levels , relative to mice receiving vehicle alone ( Figure 4B and 4C ) , suggesting that a DC-derived mediator might contribute to anti-protective effects seen following α-GalCer treatment . Thus , stimulation of iNKT cells with α-GalCer during an established infection , either in soluble form or loaded onto DC , did not improve disease control , and furthermore , soluble α-GalCer enhanced parasite growth in mice with an established infection . To determine whether α-GalCer–treated mice recovered at later time points , α-GalCer was administered on day 14 p . i . , and mice were sacrificed on both day 21 p . i . and day 70 p . i . ( Figure S2 ) . At day 70 p . i . , hepatic parasite burdens had diminished significantly in mice receiving either vehicle or α-GalCer , and there was no significant difference between the groups . Therefore , the anti-protective effects of a single dose of α-GalCer during an established infection were transient , and anti-parasitic mechanisms were ultimately re-established in treated mice . L . donovani infection results in the generation of immune regulatory mechanisms that prevent efficient disease resolution . In particular , IL-10 suppresses pro-inflammatory responses and parasite clearance during experimental VL [27] , [43]–[45] . Previous work has reported an expansion of IL-10–producing CD4+ T cells in mice following L . donovani infection [46] , and more recently , high levels of IL-10 mRNA accumulation were found in T cells isolated from splenic aspirates taken from Indian VL patients [47] . Furthermore , activated NKT cells are capable of IL-10 production [48] . Therefore , mice were treated with either anti–IL-10 receptor ( IL-10R ) mAb or control rat IgG on day 12 p . i . , 2 d prior to the administration of α-GalCer . Liver parasite burdens were measured 1 wk following α-GalCer treatment . Again , the administration of α-GalCer to control mice on day 14 p . i . resulted in increased hepatic ( p<0 . 05 ) parasite burdens compared to vehicle-treated controls ( Figure 5A ) . IL-10R blockade was extremely potent in reducing parasite burdens ( Figure 5A ) , as previously reported [43] . Stimulation of iNKT cells with α-GalCer in the absence of IL-10 signalling did not have an additive effect on the control of parasite growth , compared to vehicle-treated controls ( Figure 5A ) , but instead resulted in a small , but significant ( p<0 . 01 ) , increase in hepatic parasite burdens ( Figure 5A ) . IL-10 blockade led to significantly elevated levels of serum TNF in both vehicle and α-GalCer treated mice ( p<0 . 01; Figure 5B ) , and a significant increase in serum IFNγ in mice that received α-GalCer ( p<0 . 05; Figure 5C ) . However , α-GalCer treatment resulted in increased hepatic parasite burdens despite these increased levels of inflammatory cytokines . α-GalCer treatment reduced the levels of serum TNF but not IFNγ relative to vehicle treatment in mice that received IL-10R blockade . Thus , stimulation of iNKT cells with α-GalCer in the absence of IL-10 signalling during an established L . donovani infection did not reduce parasite burdens in the liver , thereby indicating that this mechanism of immune regulation was not responsible for the failure of α-GalCer to improve control of parasite growth . We next examined whether iNKT cells responded differently to α-GalCer stimulation during an established L . donovani infection . Naïve and infected mice ( day 14 p . i . ) were administered α-GalCer and sacrificed 2 h later . The down-regulation of TCR on iNKT cells following α-GalCer stimulation was noted in naïve mice ( Figure 6A ) , as previously described [40] , as was down-regulation of TCR on iNKT cells during experimental VL ( Figure 6A ) . L . donovani infection did not alter the ratio of CD4+ to CD4− iNKT cells in the liver , and therefore did not appear to selectively favour the expansion of a particular subset of iNKT cells ( data not shown ) . IFNγ was readily detected in hepatic iNKT cells ( Figure 6B ) , NK cells and conventional T cells ( Figure S3 ) by day 14 p . i . . Following α-GalCer stimulation , the frequency of hepatic iNKT cells producing IFNγ and TNF was significantly reduced in infected animals relative to naïve animals ( p<0 . 05 ) , while the frequency of hepatic iNKT cells producing IL-4 was significantly increased ( p<0 . 05; Figure 6B–6D ) . However , due to the increase in liver MNC number that had occurred by day 14 p . i . ( 3 . 3×106±1 . 8×105 versus 3 . 5×107±7 . 9×106 in naïve versus day 14 p . i . , respectively ) , there was an approximate 3-fold increase in the absolute number of IL-4–producing hepatic iNKT cells , but little difference in the absolute numbers of IFNγ-producing and TNF-producing iNKT cells following α-GalCer stimulation in infected mice relative to α-GalCer–treated naïve controls . These data suggest an iNKT cell bias towards Th2 cytokine production in the liver following α-GalCer stimulation during L . donovani infection . Importantly , there was also an increase in the number of hepatic NK cells producing IFNγ following α-GalCer stimulation in infected animals relative to α-GalCer–treated naïve animals ( p<0 . 05; 8 . 2×104±5 . 3×103 versus 6 . 7×105±1 . 7×105 in naïve versus day 14 p . i . , respectively ) , indicating that L . donovani infection does not interfere with this important α-GalCer–mediated effector pathway . To determine whether there was a general enhancement in Th2 and/or regulatory cytokine production in response to α-GalCer treatment in L . donovani–infected mice , we next measured hepatic IL-10 , TGFβ , IL-13 and IL-4 mRNA accumulation at day 21 p . i . in mice that had received either vehicle or α-GalCer at day 14 p . i . ( Figure 7A–7D ) . We found no significant differences in hepatic IL-10 ( Figure 7A ) or TGFβ ( Figure 7B ) mRNA accumulation between vehicle- and α-GalCer–treated mice . Similar to the increased iNKT cell IL-4 production observed 2 h following α-GalCer administration shown above , we found a significant ( p<0 . 05 ) increase in hepatic IL-4 mRNA accumulation ( Figure 7C ) 1 wk following α-GalCer administration , relative to vehicle-treated controls . Interestingly , we also found a similar increase ( p<0 . 01 ) in hepatic IL-13 mRNA accumulation ( Figure 7D ) at the same time . Therefore , α-GalCer treatment of L . donovani–infected mice did not cause a general increase in Th2 and/or regulatory cytokine production , but instead , resulted in a selective increase in IL-4 and IL-13 production . We next tested whether increased IL-4 production following α-GalCer stimulation during an established L . donovani infection was responsible for impaired disease resolution . C57BL/6 and B6 . IL-4−/− mice were infected and administered α-GalCer or vehicle control 14 days later . At this time , B6 . IL-4−/− mice had similar hepatic parasite burdens to C57BL/6 mice ( Figure 8A ) . α-GalCer treatment resulted in increased hepatic parasite burdens ( p<0 . 05 ) in both C57BL/6 and B6 . IL-4−/− mice , compared to vehicle-treated controls ( Figure 8A ) . To ensure that redundancies in IL-4 deficient mice were not influencing the outcome of these experiments , we also neutralised IL-4 activity in C57BL/6 mice using a mAb at a dose previously shown to improve control of L . donovani growth in a Th2 environment [49] ( Figure 8B ) . Again , we found that L . donovani-infected mice treated with α-GalCer had significantly higher ( p<0 . 01 ) hepatic parasite burdens , compared with vehicle controls , regardless of whether they received anti–IL-4 mAb or control rat IgG . Together , these data suggest that increased IL-4 production in response to α-GalCer treatment in L . donovani–infected mice , either by iNKT cells or another cell type following iNKT cell activation , could not alone explain the impaired clearance of parasites . To test whether the activation of iNKT cells during an established L . donovani infection had any impact on the cellular composition of the liver that could explain the impaired parasite clearance , α-GalCer was administered on day 14 p . i . , and the numbers of different hepatic mononuclear cell populations were assessed 7 d later . We found no significant differences in hepatic B cell , NKT cell , neutrophil , monocyte/macrophage or DC numbers in mice that had received α-GalCer , relative to vehicle-treated control animals ( Figure S4 ) . However , we did observe a small decrease in the number of hepatic CD4+ T cells ( Figure 9A ) and a larger , significant ( p<0 . 05 ) decrease in the number of hepatic CD8+ T cells 7 d after α-GalCer-treatment ( Figure 9B ) relative to controls . We also found a small , but significant ( p<0 . 05 ) increase in hepatic NK cell numbers at the same time ( Figure 9C ) . We next examined whether the activation status of various cell populations had been altered by α-GalCer administration at day 14 p . i . . We first focused on both hepatic DC and monocytes/macrophages and found no alterations in the expression of MHC-II , CD40 , CD80 or CD86 on either population at 1 , 2 , 3 , or 7 d following α-GalCer treatment , compared with vehicle-treated controls ( data not shown ) . In addition , TNF production by monocytes/macrophages was not different between treatment groups at these times after infection , and no TNF was detected in DC ( data not shown ) . Therefore , the activation status of the major hepatic antigen-presenting cell populations did not appear to be modulated by α-GalCer . To investigate whether lymphocyte activation had been altered , we examined IFNγ production following α-GalCer or vehicle treatment and found no difference in the number of hepatic IFNγ+ NK cells ( Figure 9C ) or IFNγ+ NKT cells ( data not shown ) by 7 d post-treatment . However , we did observe a small decrease in the number of IFNγ+ CD4+ T cells ( Figure 9A ) and a larger , significant ( p<0 . 05 ) decrease in the number of IFNγ+ CD8+ T cells 7 d after α-GalCer-treatment ( Figure 9B ) . Given the importance of IFNγ [50] and CD8+ T cells [51] for the effective resolution of hepatic L . donovani infection , this loss of IFNγ+ CD8+ T cells after α-GalCer treatment is a likely explanation for the enhanced parasite growth observed in our studies .
We report that CD1d-restricted NKT cells have only a minor physiological role in experimental VL in C57BL/6 mice . More importantly , our results indicate that intentional stimulation of iNKT cells with the glycolipid α-GalCer during L . donovani infection can exacerbate disease . This suggests that the therapeutic use of NKT cell activators , such as α-GalCer , may not always provide beneficial effects , particularly in situations where sub-clinical or chronic infection may be present . Previous studies in CD1d-deficient BALB/c mice suggested that NKT cells were required for efficient control of hepatic L . donovani infection [31] . The most likely explanation for this discrepancy with our data is a different requirement for NKT cells in C57BL/6 and BALB/c mice , possibly reflecting different kinetics of parasite control between these mouse strains [28] . Alternatively , NKT cell responses may be influenced by host genetic background , as previously reported in experimental cerebral malaria [52] . Another study in B6 . Jα18−/− mice suggested that NKT cells were required for the efficient induction of CXCL10 production during the early stages of L . donovani infection in the liver [30] . However , given the limited differences observed in hepatic parasite burdens between B6 . Jα18−/− mice and control mice at early time points ( Figure 1A ) , our data suggest that NKT-independent chemokine signals can compensate for this lack of early CXCL10 production . Although only a limited physiological role was found for CD1d-restricted NKT cells during experimental VL , the activation of these cells has been shown to improve disease outcome in many infections , often via the increased production of IFNγ [11]–[15] , [53] . This can occur even when there appears to be little requirement for NKT cells during natural infection , and is often due to downstream activation of NK cells . For example , although iNKT cells have only a minor role in the clearance of murine cytomegalovirus , the activation of iNKT cells by α-GalCer during infection resulted in reduced viral replication in visceral organs [53] . However , despite iNKT cell IFNγ and TNF production , and enhanced NK cell IFNγ production in the liver following α-GalCer administration during infection , the control of L . donovani was not improved . In fact , α-GalCer treatment during an established infection suppresses parasite clearance from the liver . In contrast , the administration of another strong inducer of a Th1 response , recombinant IL-12 , does enhance disease resolution in experimental VL [54] , indicating that α-GalCer has a different qualitative effect despite its ability to induce a strong Th1 response in L . donovani-infected mice . This may be related to the ability of α-GalCer to stimulate both Th1 and Th2 cytokine production . The reason for the different effects of α-GalCer stimulation on parasite growth before and after infection is unclear , but it seems likely that changes in local tissue environments during infection may alter the outcome of iNKT cell activation with an impact on anti-leishmanial immunity . This explanation is supported by the finding that the timing of α-GalCer administration was also critical to the effect on murine experimental conjunctivitis [55] . The effect of other regimes of α-GalCer therapy , other sites of administration and/or other analogues such as α-C-GalCer [56] , [57] have not yet been tested in experimental VL , and it remains possible that these other strategies might represent a therapeutic regime by which iNKT cell activation may improve disease resolution in this model . However , our data clearly show that α-GalCer therapy can exacerbate disease in L . donovani-infected mice . A major functional change following α-GalCer-stimulation during L . donovani infection in the current study was rapidly increased numbers of IL-4 producing hepatic iNKT cells and a sustained increase in hepatic IL-4 mRNA accumulation 1 wk later . However , when L . donovani–infected B6 . IL-4−/− mice were treated with α-GalCer , hepatic parasite burdens were still increased compared to vehicle-treated B6 . IL-4−/− mice . A similar failure to reverse the immune-dampening effect of α-GalCer was also found when mice were treated with anti–IL-4 mAb before and after α-GalCer treatment . Therefore , the increased α-GalCer induced IL-4 production by iNKT cells or another hepatic cell population during L . donovani infection could not alone explain the enhanced hepatic parasite growth . It is possible that the induction of other Th2 and/or regulatory cytokines by α-GalCer might contribute to the increased parasite burden observed . Although no α-GalCer–related increase in hepatic IL-10 or TGFβ mRNA accumulation was found , we did observe a significant increase in IL-13 mRNA accumulation . Studies in IL-13–deficient mice indicate that IL-13 itself does not influence resistance to L . donovani infection [58] . However , the enhanced production of both IL-4 and IL-13 may work together to exacerbate parasite growth . This possibility is currently being examined . Other major changes observed in α-GalCer–treated infected mice were alterations to liver cell composition . Total hepatic NK cell number increased in mice that received α-GalCer , yet the number of IFNγ+ NK cells remained similar to vehicle-treated mice . NK cells can produce regulatory cytokines and modulate antigen presenting cell function ( reviewed in [59] ) and in this way NK cells may have contributed to the enhanced parasite growth seen following α-GalCer treatment . Studies to further elucidate the impact of NK cells on the immune dampening effect of α-GalCer in L . donovani-infected mice are underway . The other significant change in liver cell composition that occurred in α-GalCer–treated mice was a decrease in the number of CD8+ T cells , and importantly , a decrease in the number of IFNγ+ CD8+ T cells . Previous studies by Murray and colleagues have demonstrated a critical role for both CD8+ T cells [51] and IFNγ [50] for the effective resolution of hepatic L . donovani infection . Thus , it is likely that such a change to CD8+ T cell effector numbers will have a major impact on the ability of an animal to control parasite growth . The reason for this reduction in CD8+ T cell number is currently unknown , but could involve enhanced activation-induced cell death ( AICD ) and/or failure of expansion in this cell population . These possibilities are being investigated . In summary , our data indicate that although NKT cells have a relatively minor physiological role in experimental VL , intentional stimulation of iNKT cells with α-GalCer can exacerbate infection . This finding is significant because it highlights the fact that therapies aimed at modulating NKT cell function are not always beneficial to the host , and it is important to take into account the possibility of adverse consequences , such as may occur in the presence of pre-existing infections .
Female C57BL/6 and BALB/c mice were purchased from the Australian Resource Centre ( Canning Vale , Western Australia ) , and maintained under conventional conditions . B6 . RAG1/J−/− mice were bred at the Queensland Institute of Medical Research . B6 . Jα18−/− mice [60] were bred at the Peter MacCallum Cancer Centre and the Queensland Institute of Medical Research . B6 . CD1d−/− mice [61] and B6 . IL-4−/− mice ( B6 Il4tm1Nnt , The Jackson Laboratory , Bar Harbor , ME ) [62] were bred at the Peter MacCallum Cancer Centre . These mouse strains were originally generated on either the C57BL/6 background or the 129sv background and backcrossed 10 or more generations to C57BL/6 mice before use . Mice used were sex- and age-matched ( 6–10 wk ) , and all animal procedures were approved by the Queensland Institute of Medical Research Animal Ethics Committee . L . donovani ( LV9 ) was maintained by passage in BALB/c or B6 . RAG1/J−/− mice , and amastigotes were isolated from the spleens of chronically infected mice . Mice were infected by injecting 2×107 amastigotes i . v . via the lateral tail vein , killed at the times indicated in the text by CO2 asphyxiation and bled via cardiac puncture . Spleens and perfused livers were removed , and parasite burdens were determined from Diff-Quick-stained impression smears ( Lab Aids , Narrabeen , Australia ) and expressed in Leishman-Donovan units ( LDU ) ( the number of amastigotes per 1 , 000 host nuclei multiplied by the organ weight in grams ) [63] . Hepatic mononuclear cells ( MNC ) and splenocytes were isolated immediately following death as previously described [64] , [65] . Allophycocyanin ( APC ) -conjugated anti-TCRβ chain , phycoerythrin ( PE ) -conjugated anti-CD8α , PE-Cy5-conjugated anti-CD4 , PE-conjugated anti-Ly6G , fluorescein isothiocyanate ( FITC ) -conjugated anti-Ly6C , APC-conjugated anti-B220 , FITC-conjugated anti-CD19 , APC-conjugated anti-CD11c , FITC-conjugated anti–I-A/I-E ( MHC-II ) , PE-Cy5-conjugated anti-CD11b , PE-conjugated anti-CD69 , PE-conjugated anti-IFNγ , PE-conjugated anti-TNF , PE-conjugated anti–IL-4 , PE-conjugated rat IgG1 , PE-conjugated rat IgG2a , and biotinylated anti-NK1 . 1 mAbs were purchased from Biolegend ( San Diego , CA ) or BD Biosciences ( Franklin Lakes , NJ ) . PE-Cy5-conjugated α-GalCer–loaded or vehicle-loaded mouse CD1d tetramers were produced as previously described [66] . Biotinylated antibodies were detected using Alexa Fluor 488–conjugated streptavidin ( Invitrogen Life Technologies , Eugene , OR ) . Anti–IL-10R ( 1B1 . 3a; rat IgG1 ) was purchased from BD Biosciences . Purified control rat IgG was purchased from Sigma-Aldrich ( Castle Hill , Australia ) . The labelling of hepatic MNC for cell surface antigens and intracellular cytokines for FACS analysis was performed as previously described [40] , [65] . Flow cytometric analysis was performed on a FACScalibur flow cytometer and analysed using Cell Quest Pro ( BD Biosciences ) and FlowJo Software ( Tree Star , Ashland , OR ) . Cell populations in the liver were defined as follows: CD4+ T cells ( CD4+ TCRβ+ ) , CD8+ T cells ( CD8α+ TCRβ+ ) , B cells ( B220+ CD19+ ) , neutrophils ( CD11b+ Ly6G+ ) , macrophages/monocytes ( CD11b+ Ly6C+ ) , dendritic cells ( DC; CD11chi MHCIIhi ) , NK cells ( NK1 . 1+ TCRβ− ) , NKT cells ( CD1d α-GalCer tetramer+ TCRβ+ or NK1 . 1+ TCRβ+ , as indicated in the text ) . α-GalCer ( KRN7000 ) was provided by the Kirin Pharmaceutical Research laboratories ( Kirin Brewery , Gumna , Japan ) and was prepared in saline/0 . 5% ( w/v ) polysorbate-20 . No endotoxin contamination was detected in the supplied material ( Tomoaki Kuwaki , Kirin Brewery , personal communication ) . Mice received a single i . p . injection of 2 µg α-GalCer or vehicle control ( saline/0 . 5% ( w/v ) polysorbate-20 ) at the times indicated in the text . Bone marrow cells were isolated from the femurs of C57BL/6 mice and cultured in 20 ng/ml recombinant mouse GM-CSF and 20 ng/ml recombinant mouse IL-4 ( R&D Systems , Minneapolis , MN ) , as previously described [37] . BMDC were supplemented with fresh media and 100 ng/ml α-GalCer or vehicle control on days 6–8 of culture . BMDC ( 5×105 ) were injected i . v . at the times indicated in the text . Mice were injected i . p . with 1 mg of anti-IL-10R mAb ( 1B1 . 3a ) or purified control rat IgG ( Sigma-Aldrich ) at day 12 p . i . . Mice were injected i . p . with 1 mg of anti–IL-4 mAb ( 11B11 ) or purified control rat IgG on days 14 , 16 and 18 p . i . . Serum samples were analysed for IFNγ and TNF using a cytometric bead array ( mouse inflammation kit , BD Biosciences ) on a FACScan cytometer equipped with Cell Quest Pro and CBA Software ( BD Biosciences ) . The frequency of liver MNC producing IFNγ or TNF was determined by direct ex vivo ELISPOT assay , as previously described [67] using mAbs R46A2 and TN3-19 . 12 , respectively , as capture antibodies and polyclonal anti-murine IFNγ or TNF as detecting antibodies ( BD Biosciences ) . Total RNA was extracted from liver tissue using TRIzol reagent ( Invitrogen Life Technologies ) , and an RNeasy Mini Kit with on-column DNase digestion ( Qiagen , Valencia , CA ) . RNA samples were reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit ( Applied Biosystems , Foster City , CA ) according to the manufacturer's instructions . The number of IL-10 , TGFβ , IL-4 , IL-13 and HPRT ( house-keeping gene ) cDNA molecules in each sample were calculated using Taqman Gene Expression Assays ( Applied Biosystems ) . All real-time RT-PCR were performed on a Corbett Research RG-3000 Rotor Gene ( Corbett Life Sciences , Sydney , Australia ) . Standard curves were generated with known amounts of cDNA for each gene , and the number of cytokine molecules per 1 , 000 HPRT molecules in each sample was calculated . Statistical significance of differences in parasite burdens over a time course of infection were determined using a one way analysis of variance ( ANOVA ) followed by a Tukey's Post-Test . Statistical significance of other parameters were determined using a Mann-Whitney U test or Student's t-test . All analysis was conducted using GraphPad Prism version 4 . 03 for Windows ( GraphPad Software , San Diego , CA ) and p<0 . 05 was considered statistically significant . | Natural killer T ( NKT ) cells are a unique subset of T cells that can produce large quantities of inflammatory cytokines very rapidly upon stimulation . They are known to be strongly stimulated by a molecule called α-galactosylceramide ( α-GalCer ) that is derived from a marine sponge , and in this way α-GalCer is hoped to provide effective immunotherapy for a wide range of diseases . We attempted to stimulate NKT cells with α-GalCer in mice infected with Leishmania donovani , a protozoan parasite that causes a chronic disease known as visceral leishmaniasis in humans . L . donovani characteristically causes an acute resolving infection in the liver where NKT cells are abundant . Therefore , we hypothesised that by stimulating these cells with α-GalCer we would improve the rate of hepatic disease resolution . However , while α-GalCer administered prior to infection had no effect on hepatic parasite burden , α-GalCer administered during an established infection exacerbated hepatic disease , associated with a decrease in IFNγ-producing CD8+ T cells . These results are important as they demonstrate that therapies aimed at modulating NKT cell function are not always beneficial , and adverse consequences may occur in certain circumstances , such as in the presence of persistent and/or sub-clinical infections . | [
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] | 2008 | Activation of Invariant NKT Cells Exacerbates Experimental Visceral Leishmaniasis |
After fertilization but prior to the onset of zygotic transcription , the C . elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres , each of which goes on to generate distinct cell lineages . To understand how patterns of RNA inheritance and abundance arise after this first asymmetric cell division , we pooled hand-dissected AB and P1 blastomeres and performed RNA-seq . Our approach identified over 200 asymmetrically abundant mRNA transcripts . We confirmed symmetric or asymmetric abundance patterns for a subset of these transcripts using smFISH . smFISH also revealed heterogeneous subcellular patterning of the P1-enriched transcripts chs-1 and bpl-1 . We screened transcripts enriched in a given blastomere for embryonic defects using RNAi . The gene neg-1 ( F32D1 . 6 ) encoded an AB-enriched ( anterior ) transcript and was required for proper morphology of anterior tissues . In addition , analysis of the asymmetric transcripts yielded clues regarding the post-transcriptional mechanisms that control cellular mRNA abundance during asymmetric cell divisions , which are common in developing organisms .
Asymmetric cell divisions produce daughter cells of different size , molecular content , or developmental potential . These events promote tissue-type diversity in developing embryos , specify terminal differentiation , and allow for the maintenance of adult tissues [1 , 2] . Asymmetric cell divisions trigger divergent cell fates through the unequal distribution of cell fate determinants or by moving daughter cells into different morphogen fields [3 , 4] . Searches to identify intrinsic cell fate determinants and the mechanisms that guide their asymmetric distribution have been difficult to adapt to high-throughput strategies . A key challenge is separating daughter cells with sufficient purity and yield for genome-wide and proteome-wide assays . We sought to overcome this challenge in Caenorhabditis elegans by coupling a low-input RNA-seq protocol [5 , 6] with hand-dissection of blastomeres , which ensures absolute purity in each pool of isolated cells ( Fig 1A ) . In C . elegans , an asymmetric cell division cleaves the recently-fertilized zygote into a larger anterior AB cell and a smaller posterior P1 cell , each of which has distinctive characteristics and fates ( Fig 1B ) [7] . The AB cell undergoes a series of rapid , symmetric cell divisions to eventually produce hypodermal , muscle , and neuronal tissue [8–10] . The smaller daughter cell , P1 , is rich in perinuclear bodies of ribonucleoparticles called P granules . Unlike the AB cell that undergoes symmetric cell division , the P1 cell initiates a series of successive asymmetric cell divisions in which the smaller cell progressively inherits P granules and primordial germ cell fate and the larger cell becomes somatic tissues such as hypodermal , intestine , neuronal , and muscle tissues [8 , 11 , 12] . The somatic branches of the AB- and P1-lineages are transcriptionally active but the germ cell branch of the P1 lineage remains largely transcriptionally quiescent with some notable exceptions [13–16] ( Fig 1B ) . Four features make the first division of C . elegans embryogenesis an excellent model for studying the apportionment of mRNA through asymmetric cell division . First , the cell divisions of the early embryo are invariant and cell fates are precisely mapped [8 , 13 , 15–19] , allowing one to connect any mRNA asymmetries to functional consequences later in embryogenesis . Second , many of the mechanisms and proteins responsible for polarity and asymmetry have been identified , ultimately allowing the mechanisms that drive mRNA partitioning to be placed into an established framework [20] . Third , zygotic transcription does not initiate until the 4-cell stage and becomes widespread at the 16-cell stage [13 , 15 , 21] , meaning that RNA segregation , stabilization , and degradation can be observed independent of de novo transcription . Fourth , a few transcripts have been previously identified as asymmetrically abundant at this stage using in situ hybridization ( mex-3 , pos-1 ) allowing for verification by independent methods [22–24] . A previous study [16] identified 14 AB-enriched mRNA transcripts and 4 P1-enriched transcripts using a single-cell RNA-seq approach , in which only one cell is measured in each sample . Because our goal was to obtain as comprehensive a set of asymmetrically patterned genes as possible and to guard against cell-to-cell variability and noise , each of our samples consisted of a pool of 20 individual hand-dissected cells . By pooling AB and P1 cells , we were able to achieve lower variance in our samples ( S2 Fig ) , allowing us to identify 80 AB-enriched and 201 P1 enriched transcripts . This is consistent with other studies showing that pooling cells to quantities of 20-cells or more buffers against cell-to-cell variation and yields more reliable and reproducible quantification [25] . The larger number of asymmetric transcripts we discovered allowed us to derive common properties of mRNAs that are asymmetrically partitioned in the early embryo . We complemented our transcriptome profiling approach with quantitative microscopy to verify our findings and further resolve patterns of mRNA distribution . We also used our dataset to identify neg-1 as a gene newly discovered to be important for anterior morphology .
We developed a low input RNA-seq protocol ( RNA-amp-seq ) that maintains relative transcript abundance and minimizes the potential for contamination . Total RNA was amplified by in vitro transcription ( Eberwine amplification ) of a low input cDNA pool [26] , and amplified RNA was subjected to RNA-seq . Transcript abundance determined by RNA-amp-seq had a high correlation to standard RNA-seq ( r2 = 0 . 84; S1A Fig ) and preserved calls of enriched and depleted transcripts ( S1B Fig ) . Although there is high concordance between amplified and unamplified samples , some transcripts may not be captured by the amplification procedure , resulting in false negatives . These results agree with previous studies demonstrating that in vitro transcription amplifies whole transcriptomes linearly , preserving relative transcript abundances [6 , 16 , 27] . To obtain AB and P1 specific transcriptomes , we isolated AB and P1 from 2-cell stage embryos . Two-cell stage embryos were removed from their eggshells with chitinase and chymotrypsin treatment and were extracted from the remaining envelope through mechanical sheering . AB and P1 cells were separated using a hollow-tipped glass needle attached to a mouth aspirator [28 , 29] . Once separated , the two blastomeres were clearly distinguishable by their relative sizes . Twenty matched AB and P1 cells were pooled for each of three replicates ( Fig 1A ) . We performed RNA-amp-seq and identified transcripts with statistically significant differential abundances [30–33] . Eighty of these RNAs were enriched in the anterior AB cell and 201 in the posterior P1 cell ( Fig 1C–1G ) with a Benjamani-Hochberg-adjusted P value of less than 0 . 10 . For some analyses , these were ranked by adjusted P value ( S1 Dataset , S1 Table ) . We used several independent methods to confirm subsets of our identified asymmetrically abundant mRNA transcripts . First , we compared asymmetric transcripts to an existing database of C . elegans RNA in situ hybridization images [34] . Note that the in situ database was created systematically and was not designed or optimized to detect transcript asymmetries at the two-cell stage . Therefore , a lack of a discernable positive signal was not evidence of a false positive in our dataset . We queried in situ hybridization entries for our 80 AB-enriched , 201 P1-enriched , and the 80 symmetric transcripts with the most uniform distribution ( of 7664 ) ( Fig 2A–2D , S2 Dataset ) . Many transcripts were absent from the online database , showed no staining , or were uninterpretable . Entries that had successful staining were scored in a blind survey that was used to generate a symmetry score . We found a strong association between entries that were identified as AB-enriched or P1-enriched by our RNA-seq analysis and those that yielded either high or low symmetry scores , respectively ( Fig 2A–2D , S2 Dataset ) . Rates among all in situ images with positive staining were less than 3% AB-enriched and less than 1% P1-enriched ( 97% symmetric ) when we queried a random set of 100 genes present at the 2-cell stage . Given that the hybridizations were not optimized for detecting cell-to-cell variation at the 2-cell stage of development , it is remarkable that so many of our transcripts were validated by this resource . Second , we performed qRT-PCR on pools of five blastomeres to measure the abundance of individual RNA transcripts . We selected transcripts to test from each set of genes: AB-enriched , P1-enriched , and symmetric . The ratio of transcript abundance ( P1/AB ) fold change determined by qRT-PCR was highly correlated with that determined by RNA-seq ( r2 = 0 . 86 ) ( Fig 2D ) . One caveat to this technique is that reliable quantification requires a linear standard dilution series for each primer set . Only transcripts with mean RNA-seq abundance values over 15 , 000 passed this requirement and were quantifiable by this method . We also compared our data to the few transcripts whose mRNA localization had been previously characterized at the 2-cell stage of development . The mex-3 mRNA transcript is preferentially abundant in the AB cell [23] , pos-1 is P1-enriched [24] , and cey-2 appears initially uniform in early 2-cell stage but becomes P1-enriched in late 2-cell stage [22] . Of these transcripts , mex-3 appeared in our set of statistically significant AB-enriched transcripts . However , neither cey-2 nor pos-1 appeared on our list of P1-enriched transcripts . Though cey-2 and pos-1 were enriched 1 . 4 fold and 1 . 2 fold in the P1 cell in our dataset , they did not meet the conservative FDR-adjusted P value threshold we used . The transcripts for ama-1 , eft-4 , dpy-3 , act-1 , and tba-1 were previously characterized as having uniform distribution at the 2-cell stage [22] and appear among our symmetrically patterned transcripts . Further , a recent study reported that a transcript we identified as AB-enriched , W02F12 . 3 , was quantifiable as AB-enriched by microscopy [35] . There was high concordance between the AB and P1-enriched transcripts reported in Hashimshony et al . [16] and those identified by our study ( S2B and S2C , Fig ) . Five of the 14 genes previously identified as AB-enriched were also classified as AB-enriched in our study , and 3 of 4 P1-enriched genes previously identified were also in our set of 201 P1-enriched genes ( S2B and S2C , Fig ) . Both Hashimshony et al . and our study failed to identify cey-2 and pos-1 transcripts as P1-enriched , which had been previously shown by in situ hybridization [22 , 24] . The limitations of qRT-PCR and the publicly available in situ hybridization datasets motivated us to employ an alternative method of measuring mRNA transcript abundance . We performed quantitative in situ hybridization , also known as single-molecule FISH ( smFISH ) . In separate experiments , we used probes designed to hybridize to three AB-enriched transcripts ( mex-3 , rank #4; neg-1 , rank #42; tes-1 , rank #77 of 80 total ) , three P1-enriched transcripts ( chs-1 , rank #1; pgl-3 , rank #32; bpl-1 , rank #170 of 201 total ) , and three symmetric transcripts ( gpd-2 , set-3 , and B0495 . 7 ) . We chose mex-3 because it has been previously reported to be AB-enriched and neg-1 because of our interest in this gene specifically . Other candidates were chosen to span a range of expression levels and to represent asymmetric transcripts at the top and bottom of our RNA-seq-based P value ranked lists . For each probe set , in each cell of the 2-cell embryo , both the number of fluorescent particles and the total fluorescence generated by the particles were quantified ( Fig 3A–3F , S4A and S4B Fig ) . We report the ratio of “particle density” , where “particle density” is the volume-normalized count of fluorescent particles in the P1 cell as compared to the AB cell ( Fig 3A–3F ) . In the case of mex-3 , which is known to be enriched in AB , and neg-1 , which was discovered to be AB-enriched by RNA-seq in this study , quantitative analysis confirmed AB enrichment ( Fig 3A and 3B ) . We next performed multiplex FISH with dual probe sets , which allowed us to compare an asymmetric transcript to a symmetric transcript within the same embryo . This approach provided an internal control and buffered against variations in hybridization efficiency . We tested for significant differences in the ratios of particle densities ( P1/AB ) of tes-1 ( AB-enriched ) and chs-1 , pgl-3 , and bpl-1 ( P1-enriched ) relative to gpd-2 , set-3 , or B0495 . 7 ( symmetric transcripts ) . The three symmetric transcripts were nearly always within a 2-fold range of abundance in the AB and P1 cells ( Fig 3C–3F ) . In contrast , tes-1 was up to 8-fold enriched in the AB cell relative to P1 ( Fig 3C ) , confirming AB enrichment . This is important because tes-1 is a low-abundance transcript that is not reliably detectable by qRT-PCR . It is the 77th of 80 AB-enriched asymmetric transcripts ranked by P value . Thus , we have observed that even transcripts near the bottom of our asymmetrically abundant lists can exhibit reproducible , quantitative , and microscopically verifiable asymmetric patterns . Three transcripts that were P1-enriched by RNA-seq were tested by smFISH ( chs-1 , pgl-3 , and bpl-1 ) . Of these , chs-1 and bpl-1 transcripts were P1-biased relative to internal controls ( Fig 3D–3F ) . chs-1 and bpl-1 are ranked at the top and the bottom of the RNA-seq P1-enriched list of transcripts respectively . pgl-3 was also tested but did not show statistically significant differences between an internal control ( Fig 3E ) . Instead , pgl-3 showed high variability in P1-enrichment ranging from marginal AB-enrichment to 4-fold higher P1–enriched particle density . We noticed that cells with higher ratios of P1 particle density were often later 2-cell stage embryos ( Fig 3H ) indicating that variability may be due to a greater degree of asymmetry in older 2-cell embryos , and a lesser degree in 1-cell embryos . We also noted a qualitative difference in the chs-1 hybridization signal . The fluorescence particles appeared much larger than signal produced from other transcripts . This granular pattern was suggestive of P granule localization ( Fig 3D–3I ) . Because of their granular nature , we were not able to quantitate these transcripts with reliable single molecule resolution . For this reason we also measured total fluorescence for all our experiments . However , even in the case of chs-1 , which had the most notable granular patterning , the ratios and statistics calculated from total fluorescence density measurements closely paralleled measurements for particle density ( S4 Fig ) . smFISH allowed us to observe RNA abundance patterns in embryos throughout development . The chs-1 , pgl-1 , and bpl-1 transcripts exhibited uniform transcript abundance in 1-cell stage embryos whereas transcripts in 2-cell stage embryos were localized asymmetrically ( Fig 3G and 3H , S5 Fig ) . This suggests that differential transcript degradation or stabilization may contribute to pattern these particular mRNAs . Cell-specific distributions continued for chs-1 and bpl-1 transcripts , with particles concentrated in one or two posterior cells over the course of several cell divisions ( Fig 3H ) . We hypothesized that genes encoding asymmetrically abundant transcripts might have lineage-specific roles in the development of the early embryo . At least one known example supports this idea . MEX-3 mRNA and protein are both distributed preferentially to the AB cell and mex-3 is required for proper AB lineage specification . We used RNAi to survey the knockdown phenotypes of 33 of our asymmetrically abundant transcripts ( Fig 4A ) . Though RNAi assessments have been performed in the early embryo for some of these genes as part of systematic studies [36–42] , we re-tested these 33 genes and scored and ranked relative embryonic lethality under uniform and controlled conditions . RNAi corresponding to 9 of the 33 genes tested yielded greater than 5% embryonic lethality in either wild type ( N2 ) or sensitized ( rrf-3 ) worms [40] ( Fig 4A ) . We next tested whether any of these 9 genes with embryonic lethality phenotypes were involved in lineage-specific functions by performing timecourse microscopy after dsRNA injection . RNAi to neg-1 yielded the most dramatic anatomical defects , so we selected this gene for further study ( Fig 4B–4D ) . neg-1 encodes a small protein of unknown function predicted to contain an unstructured N-terminus and a potentially structured and positively charged C-terminus [43] . The only known homologs to this protein occur in C . elegans and C . brenneri . F32D1 . 6 has recently been named neg-1 ( Negative Effect on Gut development 1 ) . We found that neg-1 RNA was enriched and highly abundant in the anterior AB cell ( Fig 1D , Fig 2E , Fig 3A ) . Disruption of neg-1 by RNAi led to partially-penetrant embryonic lethality , with a median lethality rate of 96% by injection at 15°C , 81% at 20°C and 30% at 25°C ( Fig 4B ) , and thus was cold-sensitive . Lethality was lower by RNAi feeding ( 30% at 20°C ) , which is consistent with previously published studies of neg-1 RNAi feeding , which reported 40% lethality [44] ( Fig 4A ) . To achieve a more detailed characterization of the cause of embryonic lethality , we performed time-lapse microscopy on embryos from mothers that either were or were not injected with neg-1 dsRNA ( Materials and Methods ) ( Fig 4C and 4D ) . These experiments were conducted at 20°C . Among neg-1 depleted embryos , 22 . 2% arrested without apparent signs of elongation , 50% arrested with partial elongation and 27 . 7% elongated . In embryos undergoing partial elongation , we noticed a failure of the cells in the anterior of the embryo to enclose , which likely caused the elongation defect . During this time , development of the posterior continued normally ( Fig 4C and 4D ) . This is reminiscent of hammerhead ( hmr-1 ) mutation in that the hypodermis failed to enclose the anteroventral regions of the embryo leading to a failure of elongation [45] . It is also similar to humpback ( hmp-1 , hmp-2 ) phenotypes although we did not observe the classic dorsal rippling or bulging that is typical of those mutants [8 , 45 , 46] . We suspected that the primary defect in neg-1 depleted embryos was a failure of the hypodermis to fully enclose . During elongation , hypodermal cells encircle the embryo by extending from the dorsal half of the embryo and expanding to the ventral side , meeting and forming junctions at the ventral midline [47] . Once the ventral enclosure is complete , circumferential constriction of seam cells ( a file of mid-body cells ) and hypodermal cells powers the extension of the body into an elongated shape [48] . To more precisely monitor hypodermal cell behavior in neg-1 knock-downs , we performed time-lapse microscopy in a dlg-1::GFP strain that marks apical adherens junctions of epithelial cells of the hypodermis and gut [49] . We found that in 55% of neg-1 RNAi-treated embryos ( 18/33 ) , hypodermal cells were misshapen prior to ventral enclosure ( Fig 4E and 4F ) . Within this group , 9 embryos failed to complete ventral enclosure and did not undergo elongation ( Fig 4E and 4F ) . The other 9 did not complete ventral enclosure on the anterior end and only partially underwent elongation . In these embryos , elongation led to an organized posterior region of the worm but forced internal cells out through the unenclosed anterior leading to the hammerhead-like phenotype . Interestingly , 18% ( 6/33 ) of embryos exhibited hypodermal cell enclosure that was largely normal , but mistimed . Typically , two actin-rich “leader” cells on flanking sides of the body are the first to meet at the ventral midline and are then followed by the more-posterior hypodermal cells . In the subset of embryos with defective timing , the leader cells and posterior cells met synchronously at the ventral midline . Of these 6 mistimed-enclosure embryos , 4 failed to complete elongation , whereas the other two hatched . The remainder of embryos ( 9/33 , 27% ) exhibited normal hypodermal morphogenesis , and of these , 7 of 9 hatched ( Fig 4E and 4F ) . While there was a strong relation between abnormal behavior of hypodermal cells in neg-1 compromised embryos and the likelihood of successful hatching ( Fig 4F ) , the correlation was not perfect , suggesting that processes other than hypodermal cell behavior are also defective when neg-1 is lacking . mRNA abundance usually , but not always , correlates with protein production . To test whether anterior neg-1 mRNA enrichment correlated with NEG-1 protein enrichment , we compared smFISH staining for neg-1 mRNA to fluorescence in worms harboring a NeonGreen::neg-1 protein fusion . As visualized by smFISH , neg-1 mRNA appeared uniform in the 1-cell fertilized embryo , became localized to the anterior in the AB cell and its daughters , and then declined dramatically in signal during the 6–10-cell stages ( Fig 4G ) . We observed NeonGreen fluorescence in the ABa and ABp nuclei at the 4-cell stage . NeonGreen persisted in the anterior nuclei of the 6-cell and 8-cell stage embryos , but then decreased by the 10–32-cell stages . Therefore , anterior enrichment of neg-1 mRNA preceded fluorescent protein accumulation by one cell cycle , and the pattern of anterior-localization between mRNA and protein was correlated . Having identified an anterior transcript required for embryogenesis and required for full function in many AB-derived hypodermal cells , we asked whether asymmetric transcripts had functions associated with the lineage in which they were enriched . Transcripts that accumulated in AB were enriched in GO biological process categories [50] that were distinct from the categories enriched in P1 transcripts ( Fig 5A ) . Categories associated with the AB transcripts included organelle organization , cellular organization , chromosome organization , and cell-cycle processes , locomotion , and morphogenesis ( Fig 5A , S5 Dataset ) . The AB cell lineage undergoes more rapid cell cycle progression than the P1 lineage [8 , 9 , 49 , 50] , which explains the enrichment for genes involved in cell cycle processes . Transcripts preferentially abundant in the posterior P1 cell were associated with translational control , embryo development , proton transport , and cell death ( Fig 5A , S5 Dataset ) . Although translational control is prevalent throughout C . elegans embryogenesis , germ line development is especially dependent on translational control due to the transcriptional quiescence of this lineage until later stages of embryogenesis [12 , 52 , 53] . Competition and cooperation among RNA-binding proteins , some of which are asymmetrically distributed in the AB and P1 lineages , control translational repression and activation of maternally inherited mRNAs [54] . For example , MEX-3 is required for full translational repression of PAL-1 , NOS-2 , GLP-1 , and ZIF-1 in anterior blastomeres [23 , 55–57] , and POS-1 and GLD-1 account for translational repression of key transcripts in posterior blastomeres [24 , 55 , 58] . Maternally loaded mRNA transcripts of the C . elegans early embryo have been previously categorized into classes according to the dynamics of their distribution . Whereas Class I mRNAs are maintained in the embryo over time , Class II mRNAs are progressively degraded in somatic blastomeres . Known Class II mRNAs are limited to a few examples such as nos-1 and nos-2 , which degrade in somatic cells beginning at the 4-cell stage but co-localize with the P granules in the P lineage [59] . Baugh et al . identified 1749 genes that produced “Maternal Degradation” [MD] transcripts by microarray time-course assays on whole embryos . MD transcripts are hypothesized to contain Class II mRNAs as well mRNAs that are degraded in other spatial patterns [60] . AB-enriched and P1-enriched transcripts were almost twice as likely to be categorized as MD maternal mRNAs when compared to symmetrically distributed transcripts ( 43 . 9% [26/66] of AB-enriched , 51 . 0% of P1 enriched [25/147] , and 26 . 1% [737/2815] of symmetric transcripts were MD; Fig 6A–6C ) . This suggests that asymmetrically distributed transcripts were more likely to decrease in relative abundance as embryogenesis progressed compared to symmetrically distributed transcripts . This decline in abundance could be due to a uniform degradation of the transcript across the whole embryo or a relative reduction in the amount of transcript as it becomes restricted to a specific cell lineage . Indeed , by in situ hybridization ( Fig 3H , S4 Fig ) transcripts asymmetrically abundant in the P1 cell often exhibited P2-specific localization at the 4-cell stage . Transcription is largely quiescent in the P1 cell and its descendants , so it is possible that maternal mRNAs are specifically sequestered in that lineage for later use in germ line development . The transcriptomes of the Z2/Z3 progenitor germ cells isolated from early embryos have been previously characterized by sorting them using Ppie-1::GFP::PGL-1 fluorescent markers [61] . We did not find a statistically significant over-representation of P1-enriched transcripts among Z2/Z3-enriched transcripts ( Fig 6B and 6C ) . This indicates that P1-enriched transcripts are not set aside for retention in the Z2/Z3 germ line precursors at a higher rate than those found in the AB-enriched or symmetric sets at the 2-cell stage . However , individual P1-enriched transcripts may be of particular interest in germ line precursor biology . Indeed , transcripts that are both enriched in P1 and in Z2/Z3 include pgl-3 , which encodes one of the two key scaffolding proteins of the P granules [65] ( Fig 6B ) , and plk-3 , a component whose presence is correlated with P granule development [66] . To determine whether asymmetric transcripts were associated later in development with a specific cell or tissue type , we asked whether AB- or P1-enriched transcripts were over-represented in any specific embryonic cell type [61] . We found significant associations between AB transcripts and hypodermal transcripts , AB transcripts and pharyngeal transcripts , and between P1 transcripts and pharyngeal transcripts ( Fig 6C , S6 Dataset ) . However , we saw no clear association between AB-enriched transcripts and cell types that were exclusive to the AB-lineage or between P1-enriched transcripts and cell types that were exclusive to the P1-lineage . This suggests that the primary purpose of asymmetric transcript abundance following the first embryonic division is not to retain transcripts for cells that arise later in each lineage . Together these results suggest that mRNAs asymmetrically abundant at the 2-cell stage tend to decline rapidly in relative abundance as embryogenesis progresses , but that only a small fraction of the corresponding transcripts are tissue-specific or lineage-specific later in development . These findings are consistent with the observation that the transcriptome undergoes waves of change throughout development [60] . These waves are shaped through RNA degradation and zygotic transcription [67 , 68] . RNA-binding proteins recognize sequences and topologies in their RNA targets to affect localization , stabilization , polyadenylation , sequestration or degradation . We asked whether transcripts enriched in the AB or P1 cells shared RNA sequence features that could account for their patterning . Though we found no associations between 5′ splice leader usage , 5′ UTR length , 5′ UTR nucleotide composition , 3′ UTR length , or 3′ UTR sequence composition and cell-specific categories ( S6A and S6B Fig ) , we found that short gene models ( both spliced and unspliced ) were associated with P1 enrichment , and longer gene models with AB-enrichment . The median AB-enriched transcript length was longer than the median length of symmetric transcripts , which in turn was longer than the median of P1-enriched transcripts ( Fig 6D , S6C Fig ) . A large number of P1-enriched transcripts were very short ( 250–750 bp ) , a length class that was virtually non-existent in the AB set . Of the 73 genes whose models yielded spliced lengths of less than 750 bp and were P1-enriched , 13 were neuropeptide-like proteins ( nlp and flp ) and 14 were ribosomal protein components ( rpl or rps ) . We speculated that asymmetrically abundant transcripts might contain different RNA-binding protein associated sequence motifs or miRNA target sites . We searched de novo for RNA sequences that distinguished the AB-enriched or P1-enriched 3′ UTRs from those of the symmetric set . We identified two motifs over-represented in the 3′ UTRs of P1-enriched genes: UUUAUUGCAU and polyC ( 10-mer C ) but none that were over-represented in the AB-enriched set of genes ( Fig 6E ) [62 , 69 , 70] . The UUUAUUGCAU motif is an Adenine and Uracil rich element ( ARE ) that contains features similar to known POS-1 and MEX-3 recognition sequences . The UAUU sequence is similar to the zinc-finger coordinated UAUU sequence found in the recognition sequence of the CCCH-type zinc finger protein POS-1 ( UA ( U2-3 ) RD ( N1-3 ) G ) and its human homolog , tristetraprolin ( TPP-1 , UAUUUAUU ) . The UUUAUUGCAU motif also resembles two stretches of UUUAUUGA within the nos-2 3′ UTR that are perfectly conserved in three Caenorhabditis species , are required for MEX-3 binding in vitro , and are required for preventing NOS-2 protein accumulation in anterior blastomeres [52 , 56] . POS-1 and MEX-3 are two RNA binding proteins that are asymmetrically distributed in the 2-cell stage embryo and whose target sequence motifs have been described ( Fig 6F ) . We noticed a striking similarity between the POS-1 target sequence and the P1-enriched sequence we identified ( Fig 6E ) suggesting that POS-1 or another CCCH-type zinc finger may function in the retention of P1-enriched transcripts .
We have expanded the knowledge of which mRNA molecules are asymmetrically distributed during the first cell division in C . elegans , a powerful model for asymmetric cell division and early development . Our approach led to the identification of neg-1 . a gene that produces anterior mRNA and protein and is important for ventral enclosure and elongation , processes that involve hypodermal cells derived from the AB-lineage . We identified a transcript , neg-1 , that is more abundant in the anterior cell following the first cell division of the C . elegans zygote . The protein product of this transcript is also preferentially localized to anterior nuclei , starting in the following cell division . Disruption of neg-1 by RNAi led to defective anterior morphogenesis due to failures in hypodermal cell ventral enclosure and elongation . The hypodermal leader cells ( ABpraappap , ABpraapppa , ABplaappap , and ABplaapppa ) , the ventral cells posterior to the leader cells ( ABpraapppp , ABplaapppp , and the P cells ) , and the seam cells ( H0 , H1 , V1–V6 , and T ) are all derived from the AB lineage and are all actively involved in hypodermal cell enclosure along the ventral midline . The C founder cell yields other hypodermal cells but these are posterior and dorsally located and do not play as large a role in ventral enclosure [8 , 71] . Thus , the data presented here show that the asymmetrically abundant neg-1 mRNA is associated with subsequent asymmetric protein abundance , and that loss of neg-1 function has consequences on anterior morphogenesis . It remains an open question as to how a protein whose anterior patterning is most striking at the 4-cell stage yields an RNAi phenotype 300 minutes later . NEG-1 may alter or regulate gene expression in the AB lineage , which would be consistent with its positive charge and concentration in the nucleus . The functional characterization of neg-1 in this manuscript indicates that cell-specific RNA profiling may be a fruitful way to identify transcripts and proteins that contribute to lineage-specific processes . We identified neg-1 based on its preferential abundance in the AB cell . With this in mind , other transcripts identified in our study warrant special attention with respect to their potential for lineage-specific functions . For example , cdc-25 . 3 was AB-enriched . Its homolog cdc-25 . 1 regulates relative cell-cycle rate in other lineages [9] . cdc-25 . 1 and cdc-25 . 3 may work together to regulate lineage-dependent cell-cycle rates . Many of the AB-enriched transcripts , such as erm-1 and hmp-2 , are associated with epithelial adherens junctions , possibly foreshadowing the important role of hypodermal cell function among the progenitors of the AB lineage . Cytoskeletal regulatory genes enriched in the AB lineage such as cyk-1 and dnc-1 may indicate the requirement for greater cytoskeletal control in the AB lineage . RNAs enriched in the P1 cell also function in processes known to be important to the P lineage . One such transcript encodes PGL-3 , the protein that comprises the scaffold for P granules . Further , several RNAs encoding ribosomal protein genes and RBPs important for translation were over-represented in the P1 cell . This is consistent with the differential translation of specific mRNAs ( glp-1 , pal-1 , and nos-2 ) in the anterior and posterior blastomeres that generates proteomic diversity prior to the onset of zygotic transcription [52 , 57 , 72] . Because key RBPs that regulate those mRNAs also bind other targets , cohorts of mRNAs may be coordinately regulated [73 , 74] . The process of translation in general may be distinct between the P lineage and somatic lineages because P granules and germ granules contain mRNAs and proteins , such as IFE-1 , that regulate general aspects of translational biology [75] . Future studies may address how P1-enriched transcripts associated with translation impact protein production in that lineage . The search for asymmetrically partitioned cell-fate determinants has traditionally concentrated on differential gene expression patterns , which can arise through both transcriptional differences and differential post-transcriptional regulation of mRNAs . For example , ASH1 mRNA localizes to Saccharomyces cerevisiae bud cells and marks bud-versus-mother cell identity [76 , 77] , and the mRNA transcripts of gurken , bicoid , oskar , and nanos distribute asymmetrically in the Drosophila melanogaster embryo and are required for embryonic patterning ( recently reviewed in [78] and [79] ) . The concentration of a particular mRNA transcript in a specific lineage can also be important for lineage-related functions even if it is not acting to determine the identity of that lineage directly . Our study identified an asymmetrically distributed mRNA transcript , neg-1 , which was important for morphogenesis . However , we have yet to establish whether neg-1 is a cell fate determinant or whether AB-enrichment of neg-1 mRNA is required for its function for embryogenesis . The AB-enrichment of neg-1 mRNA preceded an anterior enrichment of NEG-1 protein by one cell division , but it remains possible that the asymmetric RNA localization is not required for asymmetric NEG-1 protein localization . For example , translational control could be the primary mechanism shaping the proteomes of daughter cells . Translation of specific transcripts is regulated during oogenesis , early embryonic development , and in the germ cell lineage . It could be that the asymmetric mRNAs we observe arise as a downstream effect of translational control . Even if so , cataloging asymmetrically abundant mRNAs is likely to be an efficient way to identify genes that are spatially regulated and important for development . The initial symmetry of the round oocyte is broken by the ellipsoid shape of the fertilized egg and by the entry point of the sperm centrosome [7 , 80 , 81] . The PAR proteins transduce those polarity signals to create a polarized actinomyosin network , directional cytoplasmic flow , and a skewed arrangement of the mitotic spindle [82–86] . As a result , the division plane of first mitosis in the C . elegans zygote is situated closer to the posterior . This process involves progressive stages of polarity initiation , maintenance , and amplification . How is mRNA location and abundance regulated during this process ? While a variety of mechanisms , including differential diffusion , stability , degradation , localization , sequestration , or polyadenylation may be regulating the RNA abundance patterns we identified in our study , our data provided two clues regarding where to look next . First , we observed that AB-enriched transcripts were significantly longer than symmetric transcripts , and that these were , in turn , longer than P1-enriched transcripts . It is possible the shorter length of the P1-transcripts decreases the likelihood that they will contain miRNA binding sites or RNA-Binding Protein ( RBP ) target sequences , allowing for preservation of the shorter mRNAs in the P1 cell . Alternatively , the shorter 3′ UTRs may lack motifs that direct or stabilize transcripts in the AB cell . Finally , it is possible that bulk cytoplasmic flow within the zygote passively accumulates smaller transcripts in the posterior end of the cell , whereas longer transcripts are retained in the anterior side . Given that the actinomyosin network is known to adopt asymmetric qualities after fertilization [84] and given that poroelastic diffusion properties of the cytoplasm are highly dependent on actiomyosin [87] , passive diffusion may contribute to differential distribution of molecules in the early embryo . Second , we identified an AU-riche element ( UUUAUUGCAU ) that was over-represented among the P1-enriched transcripts . This motif bears striking resemblance to the recognition motif of POS-1 ( UA ( U2-3 ) RD ( N1-3 ) G ) but also bears similarity to the direct repeats ( called DR1 and DR2 ) recognized by MEX-3 within the nos-2 3′ UTR ( UUUAUUGA ) [63 , 74] . In general , AU-rich elements are associated with mRNA stability and decay via RBPs . Our identified sequence motif could be a MEX-3 recognition motif , a degenerate POS-1 motif , the binding motif for another RBP such as MEX5/6 , or a motif that is bound combinatorially by multiple RBPs . nos-2 is translationally repressed in anterior blastomeres in a MEX-3-dependent manner [56] , and nos-2 mRNA is degraded in anterior blastomeres in a MEX-5/MEX-6-dependent manner [52] . It is possible that multiple P1-enriched transcripts and their protein products are similarly restricted from anterior blastomeres . Alternatively , POS-1 or some other RBP may recognize this degenerate motif to sequester transcripts in the P1 cell . In addition to these two observations derived from the bulk behavior of asymmetric transcripts in our RNA-seq approach , our smFISH observations also provide some guidance regarding mechanism . The transcripts chs-1 and bpl-1 were uniformly distributed in 1-cell embryos just before division and apparently became asymmetrically abundant after division occurred . In addition , the asymmetry of the pgl-3 transcript seemed more dramatic in late 2-cell stage embryos compared to early 2-cell embryos . This suggests that mechanisms responsible for asymmetric abundance of some mRNA transcripts are not wholly dependent on differential trafficking prior to cell division . Differential transcript stabilization or degradation may be responsible for generating cell-specific mRNA patterns throughout the cell cycle . We identified a PolyC sequence over-represented in the 3′ UTRs of P1-enriched transcripts . Recently , PolyC motifs were shown to be required for a wave of post-fertilization RNA degradation during the oocyte-to-embryo transition [88] . It is possible that the P1-lineage may be more resistant to this degradation than the AB-lineage . The smFISH experiments also revealed that chs-1 and bpl-1 transcripts were present in larger granular particles . Despite the superficial resemblance to P granules , we were unable to determine whether these structures were P granules . Unlike P granules , the chs-1 and bpl-1 transcript granules did not appear to coalesce or posteriorly localize during prophase of the 1-cell stage embryo . It is possible that granular association promotes P1-enrichment of transcripts through sequestration or stabilization . Asymmetric cell divisions are commonplace in development and influence fate decisions in systems ranging from plant stomata to cancer stem cells . The C . elegans early embryo is unique in the reproducibility of division timings and planes , the highly deterministic nature of the resulting lineages , and the lack of zygotic transcription in the earliest divisions . This study determined the mRNA abundance in each of the two cells created after the first zygotic cell division . This led to the identification of a new gene important for lineage-specific functions and clues regarding the post-transcriptional mechanisms that control cellular mRNA abundance in early embryogenesis .
N2 ( wild type ) worm strains were obtained from the Caenorhabditis Genome Center and maintained at 20°C unless otherwise indicated using standard protocols [89] . dlg-1::GFP ( FT64 ) was obtained from the CGC . Blastomere isolations were performed by embryo isolation , eggshell degradation and micromanipulated separation as previously described [90] . We used chitinase from Streptomyces griseus ( sigma , C6137 ) at 10 U/ml due to the recent unavailability of Serratia marcascens chitinase . Isolated AB and P1 cells were dropped directly into Trizol solution and stored at -80°C if not immediately processed . Blastomere preparations were thawed and re-frozen up to three cycles to lyse them . RNA extraction was performed using a Trizol/chloroform extraction followed by RNeasy Micro ( Qiagen ) preparation using On Column DNaseI Digestion ( Qiagen ) . RNA amplification was performed by in vitro transcription of an oligo dT-primer fused T7 promoter producing up to a 10 , 000-fold increase in mRNA using one round of a TargetAmp aRNA Amplification Kit ( Epicentre ) . The resulting in vitro amplified RNA was used for a standard RNA-seq protocol ( Illumina mRNA Sequencing Sample Preparation Guide , September 2009 ) . Magna beads were used for purification steps and for the final purification . Gel extraction was not necessary due to the size of in vitro amplified RNA fragments . Samples were sequenced on either GA-2 or Hi-Seq Illumina sequencers with either 36 or 50 rounds of sequencing . Data from this study are available on NCBI GEO ( GSE59943 ) Reviewer access: http://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ? acc=GSE59943 We sequenced 5 ug of unamplified RNA by the standard Illumina RNA-seq methodology . The same RNA sample was diluted down to 500 pg and sequenced using RNAamplification and sequencing . Sequencing reads were filtered for primer and adapter sequences [91] and a minimal quality score of 20 using Tagdust ( Version 1 . 12 ) [91] , aligned to the C . elegans ce10 genome using Tophat ( 1 . 4 . 0 ) [30] . The resulting alignment files were quantified using HT-Seq ( 0 . 5 . 3 ) [33] and the RefSeq gene annotation for WS220 downloaded March 23 , 2012 and reads were assigned per gene model . The significance of differentially represented transcripts were quantified by DESeq ( 1 . 2 . 1 ) [32] in R 2 . 12 [92] using size estimation normalization . Reads were called as present if the number of mapped reads met a minimum 50 reads in the mean of all samples and at least 1 read in the mean of either AB or P1 samples . We identified 7945 present genes of 20 , 240 of the predicted gene models . Present genes were called as giving rise to significantly asymmetric transcripts if they met a P value < 0 . 1 after FDR adjustment using the Benjamani-Hochberg method within the DEseq package ( 1 . 2 . 1 ) [32] in R 2 . 12 [92] ( All other R analyses were performed in 3 . 0 ) . When necessary , genes were ranked by negative log P value . The 80 AB-enriched transcripts and 201 P1-enriched transcripts identified by RNA-seq were surveyed for entries in a publicly available database of RNA in situ hybridization images [34] . A subset of 80 ( of 7664 ) symmetric transcripts were selected as a comparison . These 80 were the middle-most symmetric transcripts as ranked by log2 P1/AB fold change . Many transcripts were absent from the online database , showed no staining , or were uninterpretable for other reasons . Entries that had successful staining were scored in a blind survey we constructed . Multiple reviewers categorized sets of images from each entry as either “AB” , “Marginal AB” , “Symmetric” , “Marginal P1-biased” , P1” , “No expression” , or “Ambiguous” . The rubric for these categories is included as S1 Text . The full dataset including each reviewer’s response , the combined overall score , and links to publicly available images [34 , 93] is included as S3 Dataset . Pools of five P0 , AB , and P1 blastomeres were isolated using dissection methods above and immediately processed using the CellsDirect cDNA kit ( Invitrogen ) . The abundance of mRNA transcripts was quantified using the SYBR Maxima qPCR system ( Thermo Scientific ) on an ABI 7900HT Fast Real Time PCR System using a dilution series to calibrate relative abundance of each transcript . Only transcripts with an abundance over 15 , 000 mean reads passed the requirement of generating a linear standard dilution curve over a 1–1/1000 fold dilution series . Primers were designed to lie within the 3′ most exon and within 200 bp of the terminal codon . Each gene reported represents at least 2 independent biological samples , each of which was measured in 3 technical replicates; means of sample means and standard error of the mean were calculated . Stellaris smFISH fluorescent probe sets ( Biosearch ) were generated to hybridize to mex-3 neg-1 , tes-1 , chs-1 , pgl-3 , bpl-1 , B0495 . 7 , set-3 , and gpd-2 ( S2 Text ) . N2 worms were grown at 20°C to gravidity on NGM plates , bleached for embryos , resuspended in -20°C methanol , freeze cracked in liquid nitrogen , and fixed at -20°C for 2–48 hours . A protocol that combined Shaffer et al . [94] and Ji et al . [95] was performed . Embryos were equilibrated in WB1 ( 100 mg/ml dextran sulfate , 10% formamide , 2 x SSC ) , hybridized in hybridization buffer ( 100 mg/ml dextran sulfate , 1 mg/ml E . Coli tRNA , 2 mM vanadyl ribonucleoside complex , 0 . 2 mg/ml BSA ) containing 50 pmoles of each primer set . Hybridization at 30°C overnight was followed by two WB1 washes , DAPI staining , and three 2 x SSC washes . Embryos were mounted as described in Ji et al . [95] using SlowFade ( Life Technologies ) to prevent photobleaching . All smFISH images were acquired using a Cool Snap HQ2 camera on a DeltaVision-modified inverted microscope ( IX71; Olympus ) , with a UPlanSApo 100 x ( 1 . 40 NA ) objective and SoftWorx software ( Applied Precision ) using fixed exposure and acquisition conditions ( 0 . 3 μm z-stacks , up to three wavelengths ) . Images were deconvoluted . Quantification was performed using specialized scripts in ImageJ and R to identify thresholded particles , their pixel intensities , and their cell boundaries . The number of discrete particles was quantified and normalized to cell area ( proportional to volume because cells are flattened ) . The total internal intensity of each particle was also measured , and the background-subtracted fluorescence was normalized to cell area . For statistical tests , two probes were multiplexed within the same embryo and Wilcoxon signed-rank tests were performed on the paired ratios . dsRNA feeding was carried out as previously described [96] by moving worms to RNAi feeding plates at L4 stages of development . 24 hours and 48 hours after feeding , worms were assessed for their ability to lay viable embryos by capturing all eggs laid within the next 24-hours and scoring for hatching and viability 48–72 hours later . For each strain queried , we performed 2–3 replicates in which 4–6 mothers were assayed at both the 24- and 48-hour time points . dsRNA was produced using the T7 RiboMAX Expression System ( Promega , P1700 ) . RNA was injected into L4 stage worms as previously described [96 , 97] . Images were acquired 24 hours post-injection for injected , uninjected , and mock-injected worms . Experiments were carried out at 20°C unless otherwise noted . RNAi knockdown and control worms were imaged on the same slide . Time-lapse images collected in Fig 5C , were acquired using Nomarski DIC optics on a Nikon Eclipse E800 using a Nikon 1 . 40 NA 60x objective as previously described in Dickinson et al . [98] . Images were collected every 2 minutes over 9 hours with z-stacks at 1 μm intervals for a total depth of 30 μm . For Fig 5D , images were captured on a spinning disc CSU-XI Yokogawa confocal system mounted on an inverted microscope ( Eclipse Ti , Nikon ) , 16-bit cooled CCD camera ( Image E , Hamamatsu ) , 50 mW air-cooled Arbon laser ( Laser Physics ) , Nikon 1 . 4 NA 60x objective , and Metamorph Software . Images were captured every 15 min over 9 hours with z-stacks at 3 μm intervals for a total depth of 22 μm . Embryos were illuminated at 488 nm at 25% power , 500 ms , 1x1 bin for GFP settings and 100 ms , 1 x 1 bin for DIC settings . We fused the mex-5 promoter driving mneongreen ( Allele Biotechnology ) in frame , to the neg-1 gene beginning at its start site and containing its full 3’ UTR plus 100 base pairs downstream . This was cloned into a hygromyocin selectable MOSSCI plasmid using a pCFJ150 backbone [99] constructed using Gibson Assembly ( New England BioLabs ) . The resulting mex-5promoter::neongreen::neg-1::neg-1-3’UTR plasmid was transformed into N2 worms , selected with hygromycin , and counter selected for single copy insertion using the peel-1 system . NEONGREEN::NEG-1 fluorescence was imaged on an inverted microscope ( IX71; Olympus ) , with an 100 x objective , and using 0 . 3 μm sections . Max intensity projections are shown . The list of present genes were separated by fold change and ranked by FDR adjusted P value as calculated in DESeq to produce a ranked list of AB-enriched genes and P1-enriched genes . Ranked lists were processed as input by the GOrilla algorithm and a false discovery rate-adjusted P value of 10-5 was set as a minimum cutoff [50] . GO terms and scores were summarized in REViGO to merge excessively overlapping terms [51] . Categories were filtered for those with frequencies of less that 5% of the genome and the REViGO adjusted log10 P values greater than 7 were plotted . The intersection between Maternal Degraded ( MD ) mRNAs [60] and AB-enriched , P1-enriched , and symmetric genes was calculated . Symmetric genes were defined as the 50% of present genes with the largest P values , as calculated in DEseq . The association between maternal mRNA category and differential abundance category was calculated using Fisher’s exact test . The intersection between tissue-specific transcriptomes [61] and AB-enriched , P1-enriched , and symmetric sets were also calculated . The association between representation and category was calculated using Fisher’s exact test between symmetric and AB-enriched or symmetric and P1-enriched categories . Splice leader ( SL ) sequences , 5′ UTR sequences , spliced gene models , unspliced gene models , and 3′ UTR sequences were obtained from Mangone et al . [100] and Wormbase ( November 27 , 2013 ) . To avoid redundancy in pools of sequences , only the longest 3′ UTR from the union set of multiple 3′ UTR models identified in both Mangone et . al . and Wormbase annotations were used . Association between cell-type category ( AB-enriched , P1-enriched , symmetric ) and gene feature were calculated using Fisher’s exact test for discrete data and Wilcoxon rank-sum ( Mann-Whitney U ) test for continuous data . De novo motif searching was carried out using MEME ( 4 . 9 . 0 ) . We sought discriminating motifs 5–12 nucleotides in length that distinguished 3′ UTR sequences in the AB-enriched or the P1-enriched set from the symmetric set focusing on the sense strand . 3′ UTR gene models were identified as the overlap between Mangone et al . [100] and Wormbase ( November 27 , 2013 ) . The search model [–mod zoops] was used . High throughput sequencing data was submitted to NCBI Geo as GSE59943 ( http://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ? acc=GSE59943 ) . Raw sequences are available as SRP/SRP045/SRP045110 . GSE59943_ABP1_Nishimura_raw_matrix . txt . gz reports the number of raw read counts for each gene model , tabulated for 20 , 240 genes . GSE59943_ABP1_Nishimura_normalized_matrix . txt . gz reports the number of size-normalized read counts for each gene model , tabulated for 20 , 240 genes . GSE59943_AB_average . wig . gz is a wig file that averages three scaled AB samples . This file is suitable for upload to UCSC genome browser . GSE59943_P1_average . wig . gz is a wig file that averages three scaled P1 samples . This file is suitable for upload to UCSC genome browser . | At key moments in development , asymmetric cell divisions give rise to daughter cells of differing characteristics , a process that promotes cell-type diversity in complex organisms . The first cell division of the C . elegans early embryo is a powerful model for understanding asymmetric cell division because the timing of divisions and the placement of their division planes are precise and reproducible . We surveyed the mRNA content of each daughter cell in the C . elegans 2-cell embryo using low-input RNA sequencing . We identified several hundred asymmetric transcripts and tested them for functions in development . We found that the gene neg-1 produced mRNA and protein preferentially on the anterior ( head-side ) of 2-cell and 4-cell stage embryos and that loss of neg-1 led to consequences in anterior morphogenesis later in development . We also analyzed the asymmetric transcripts using quantitative microscopy , bioinformatics comparisons with previously existing datasets , and RNA sequence motif discovery to gain insight to the mechanisms by which asymmetric abundance patterns arise . | [
"Abstract",
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] | [] | 2015 | Asymmetric Transcript Discovery by RNA-seq in C. elegans Blastomeres Identifies neg-1, a Gene Important for Anterior Morphogenesis |
Schmid metaphyseal chondrodysplasia ( MCDS ) involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen , Col10a1 . Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum ( ER ) in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS . The resultant unfolded protein response ( UPR ) in affected chondrocytes involved activation of canonical ER stress sensors , IRE1 , ATF6 , and PERK with the downstream effect of disrupted chondrocyte differentiation . Here , we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS . Mice with a MCDS collagen X p . N617K knock-in mutation ( ColXN617K ) were crossed with mice in which Xbp1 was inactivated specifically in cartilage ( Xbp1CartΔEx2 ) , generating the compound mutant , C/X . The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K , revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS . Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant ( XBP1-independent ) or pathologically redundant ( XBP1-dependent ) . XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes . Moreover , these changes were consistent with disruption of C/EBP-β , a master regulator of chondrocyte differentiation , by CHOP , a transcription factor downstream of PERK that inhibits C/EBP proteins , and down-regulation of C/EBP-β transcriptional co-factors , GADD45-β and RUNX2 . Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-β-mediated chondrocyte differentiation . Our data suggest that modulation of C/EBP-β activity in MCDS chondrocytes may offer therapeutic opportunities .
Longitudinal growth of endochondral bones occurs bi-directionally under the control of cartilaginous growth plates located at each end of growing bones . In the growth plate , the extracellular matrix of cartilage is synthesized and remodelled by chondrocytes undergoing differentiation involving proliferation and hypertrophy , before being remodelled , calcified , and vascularised to produce primary bone [1] . As with other professional secretory cells , chondrocytes rely on the maintenance of endoplasmic reticulum ( ER ) homeostasis through molecular pathways that regulate protein folding quality control [2] . Known collectively as the unfolded protein response ( UPR ) , these pathways alleviate ER stress by enhancing the protein folding capacity of the ER , by up-regulating protein degradation machinery such as the ER-associated degradation ( ERAD ) pathway , or by regulating the translation or half-life of transcripts encoding secreted proteins . The UPR is activated by ER membrane-spanning sensors , including activating transcription factor 6 ( ATF6 ) , PRKR-like endoplasmic reticulum kinase ( PERK ) , and inositol-requiring enzyme-1 ( IRE1 ) , which detect the accumulation of misfolded proteins in the ER via their lumenal domains , and transmit this information to activate downstream signalling pathways via their cytoplasmic domains [3–5] . Activated ATF6 translocates to the Golgi apparatus , where it is proteolytically cleaved by site 1 protease to produce a 50kDa transcription factor that targets genes harbouring ERSE elements in their promoters , such as the molecular chaperone BiP [6] . PERK is a kinase that catalyses the phosphorylation of eIF2α in response to ER stress , down-regulating overall translation by inhibiting assembly of the translational initiation complex [7] , and driving the increased translation of specific transcripts including activating transcription factor 4 ( ATF4 ) [8 , 9] . Activated IRE1 splices a 26nt fragment from the coding sequence of the X-box binding protein 1 ( XBP1 ) mRNA to encode a transcription factor responsible for the expression of multiple UPR target genes [10] . While the UPR exists primarily as an adaptive mechanism to accommodate relatively minor fluctuations in protein misfolding during normal cellular function , it may also have deleterious consequences when chronically activated , as occurs in pathologies characterized by constitutive expression of mutant , misfolding proteins . We demonstrated in two mouse models of Schmid metaphyseal chondrodysplasia ( MCDS ) , a condition involving dwarfism and growth plate hypertrophic zone expansion caused by autosomal dominant mutations in the hypertrophic zone marker collagen X ( Col10a1 ) , that hypertrophic zone ER stress per se is sufficient to phenocopy the disease [11] . Using transcriptional profiling approaches , we characterised the UPR deployed by chondrocytes under such stress in vivo [12] . Our work revealed that ATF6 , PERK , and IRE1 are each activated , that components of the ERAD pathway are up-regulated , and that although this response enables the chondrocytes to survive the stress , it also results in disrupted differentiation and developmental arrest in a proliferative chondrocyte-like state [12] . Since IRE1/XBP1 regulates the most highly conserved of the UPR pathways , present in all eukaryotes from yeast through to higher vertebrates [4] , and is a key factor in the pathology of numerous diseases involving ER stress [13] , we sought to address its influence on the pathology of MCDS . Thus , we crossed our collagen X p . Asn617Lys knock-in mouse model of MCDS ( ColXN617K ) [11] with our Xbp1CartΔEx2 mouse , in which XBP1 activity is ablated specifically from chondrocytes [14] , to generate the compound mutant , ColXN617K/Xbp1CartΔEx2 ( C/X ) . Here we demonstrate surprising redundancy for the IRE1/XBP1 pathway in the MCDS UPR by showing that the pathology of the ColXN617K mouse is not substantially altered by the inactivation of XBP1 in C/X . Through transcriptional profiling of mutant and wildtype hypertrophic zones , we separate genes whose expression is regulated in the chondrocyte UPR in an XBP1-dependent manner from those regulated independently of XBP1 . Since XBP1 is redundant in the pathology of MCDS , we focus on genes regulated independently of XBP1 in order to identify those that are central to the pathology of the disease . We demonstrate that for chondrocytes , the XBP1-independent components of the UPR up-regulate gene networks associated with the ER and translation of mRNA , and down-regulate genes encoding glycoproteins and components of the extracellular matrix , as well as those associated with angiogenesis and skeletal system development . Thus we suggest that the hypertrophic zone expansion and delayed ossification observed in MCDS results from XBP1-independent transcriptional suppression of genes involved in cartilage matrix turnover , vascular invasion , and growth plate ossification . We propose interaction between C/EBP homologous protein ( CHOP ) , a transcription factor up-regulated downstream of PERK in response to ER stress with known roles in regulating apoptosis and cell differentiation [15] and CCAAT/enhancer binding protein beta ( C/EBP-β ) , a transcription factor important for the transition from chondrocyte proliferation to hypertrophy [16–18] , as a key point at which the UPR and chondrocyte differentiation machinery intersect in MCDS . This is the first study to present evidence supporting a direct link between the UPR and a blockage in cell differentiation mediated by transcriptional suppression of C/EBP-β in a mouse model of human disease . Moreover , it establishes a rational foundation for future studies investigating both C/EBP-β as a potential therapeutic target in the treatment of MCDS , and the possible role of disruption to differentiation pathways controlled by C/EBP-β in other ER stress-associated human disease contexts .
We crossed our collagen X p . Asn617Lys knock-in mouse model of MCDS ( ColXN617K ) [11] with mice in which Col2a1-Cre/loxP-mediated deletion of Xbp1 exon 2 renders Xbp1 completely inactive specifically in chondrocytes ( Xbp1CartΔEx2 ) [14] , to generate ColXN617K/Xbp1CartΔEx2 ( C/X ) . C/X mice were viable , fertile , and bred normally . RT-PCR and sequencing analysis of cDNA derived from femoral head cartilage of 14 day old C/X and wildtype mice confirmed the complete inactivation of XBP1 by Cre/loxP-mediated deletion of Xbp1 exon 2 in the mutant ( Fig 1A ) . PCR on genomic DNA derived from C/X and wildtype tail lysates revealed the homozygous presence of the collagen X p . Asn617Lys allele in the mutant , identifiable due to the presence of a residual loxP site downstream of the Col10a1 coding sequence remaining from the gene targeting construct used to create the ColXN617K mouse from which C/X was derived ( Fig 1B ) . To determine the impact of XBP1-dependent UPR signaling in the pathology of MCDS , we used morphometric and histological approaches to compare the skeletal phenotypes of wildtype , ColXN617K , Xbp1CartΔEx2 , and C/X mice . Skeletal preparations of newborn , seven day old , and two week old mutant and wildtype mice were stained with Alcian blue and Alizarin red to visualize cartilage and bone . Although no overt phenotype was apparent by visual inspection ( Fig 1C ) quantitative analysis of individual skeletal elements from two week old animals indicated significant reductions in the length of endochondral bones ( tibiae and femora ) when ColXN617K was compared to wildtype , as previously reported [11] , and also when C/X was compared with Xbp1CartΔEx2 ( Fig 1D and 1E ) . When skeletal elements from C/X were compared with ColXN617K however , there was no significant difference in femoral length , while the tibial length was found to be only very modestly reduced in C/X compared with ColN617K . No difference was observed in intramembranous bone growth ( as approximated by intercanthal distance measurements ) between any of the mutants compared with wildtype ( Fig 1F ) . Growth plate sections from each strain were analyzed histologically by H&E staining ( Fig 2A ) , and by immunofluorescence with antibodies for cartilage-specific collagen II ( Fig 2B ) to visualize the organization and extent of the growth plate cartilage extracellular matrix , and collagen X to demarcate the hypertrophic zone of the growth plate ( Fig 2C ) . Using H&E-stained sections to perform quantitative analyses of growth plate zone lengths between our various mouse strains , we found there was no significant difference between the length of the pathologically expanded hypertrophic zones observed in ColXN617K [11] and C/X ( Fig 2D–2F ) . Consistently however , we observed a progressive increase in the severity of hypertrophic zone expansion in the C/X mice from the anterior to posterior margin of the growth plate , whereas the severity of hypertrophic zone expansion was unchanged across this gradient in ColXN617K ( Figs 2A and 3A ) . No obvious difference in the abundance and organization of collagen II in the extracellular matrix was apparent between each mutant and wildtype . Collagen X staining was reduced and largely intracellular in both ColXN617K and C/X hypertrophic zones reflecting previously described reduced secretion of the mutant misfolded collagen X and its increased intracellular degradation by the ER-associated proteasomal degradation pathway [11 , 12] . These morphometric and histological data indicate that the severity of the dwarfism caused by expression of the p . N617K collagen X in ColXN617K mice was not substantially altered by loss of XBP1 activity in C/X , revealing surprising redundancy for the IRE1/XBP1 pathway in the pathology of MCDS , and implying that XBP1-independent consequences of collagen X-induced ER stress must underpin the disease pathology . TUNEL analysis was conducted on 14 day old wildtype , ColXN617K , Xbp1CartΔEx2 , and C/X tibial growth plates to determine whether loss of XBP1 from chondrocytes would alter cell fate during ER stress ( Fig 3A ) . The rate of apoptosis in each mouse was quantified by determining the extent of apoptosis as a percentage of the total number of chondrocytes in the zones ( Fig 3B ) . As expected the percentage of apoptotic cells observed in the hypertrophic zones of wildtype and Xbp1CartΔEx2 growth plates was low , typically in the order of 1–2% as previously described [14] . Significantly more apoptotic cells were detected in ColXN617K compared with wildtype , and in C/X compared with Xbp1CartΔEx2 . While a trend towards increased apoptosis was observed in the hypertrophic zones of C/X versus ColXN617K , the difference was not statistically significant . Thus our data suggest that apoptosis is a feature of the pathology of MCDS , occurring in chondrocytes in the ColXN617K and C/X hypertrophic zones by two weeks of age , independently of XBP1 signaling . To explore whether the apparent antero-posterior gradient of hypertrophic zone expansion in C/X is related to cell death , we set out to quantify the lateral distribution of apoptotic cells in the hypertrophic zones of the collagen X mutant growth plates . Thus , we divided the hypertrophic zones of ColXN617K and C/X growth plates into 10 columns of arbitrary width and scored the number of TUNEL-positive chondrocytes in each column , again normalised against the total number of cells per column ( Fig 3C and 3D ) . The distribution of apoptotic cells was found to be asymmetrical across the width of the ColXN617K ( Fig 3C ) and C/X ( Fig 3D ) hypertrophic zones with the highest percentages of apoptotic chondrocytes generally found in the peripheral-most columns in both mutants , at both the anterior and posterior margins of the growth plate . Thus , the antero-posterior gradient of hypertrophic zone expansion observed in C/X growth plates does not correlate with the extent of apoptosis observed in these tissues . Since cartilage-specific inactivation of XBP1 was shown to have a negligible effect on the severity of the disease phenotype in our MCDS mouse models , it follows that genes important to the pathology of MCDS must be regulated by the UPR independently of XBP1 . Conversely , those genes whose expression is regulated in the chondrocyte UPR in an XBP1-dependent manner must not contribute to the overall disease pathology . To assign genes to XBP1-independent or XBP1-dependent cohorts , we conducted expression profiling of RNA derived from hypertrophic zones microdissected from our mutant and wildtype mice . Samples were initially validated by qPCR using Agc1 , Ctgf , and Matn1 as markers of cartilage extracellular matrix , and Creld2 , Derl3 , Ero1l , Fgf21 , Steap1 , and p58IPK as markers of the hypertrophic chondrocyte UPR [12] . Agc1 , Ctgf , and Matn1 were down-regulated ( Fig 4A–4C ) and Creld2 , Derl3 , Ero1l , Fgf21 and Steap1 were up-regulated ( Fig 4D–4H ) in ColXN617K and in C/X compared to their respective controls , suggesting that the downstream consequences of the collagen X-induced cartilage UPR are similar in the absence of an active XBP1 pathway . Creld2 and Derl3 were both significantly upregulated in ColXN617K compared with C/X , indicating that these genes are at least partially XBP1-dependent . A comparable pattern was observed for Fgf21 and Steap1 , however the difference between ColXN617K and C/X was not statistically significant . One UPR gene , p58IPK , was up-regulated in ColXN617K but not in C/X ( Fig 4I ) , consistent with its known role as a direct downstream target of XBP1 signalling [19] . This further confirms the lack of XBP1 signalling in the C/X cartilage . To explore XBP1-independent or XBP1-dependent dysregulated genes in more detail transcriptomic analysis was performed by interrogating these samples with whole genome microarrays . Initially , we performed separate comparisons of C/X , Xbp1CartΔEx2 , or ColXN617K with wildtype to identify microarray probes showing greater than two-fold differential expression and with an adjusted p value of ≤0 . 01 in each mutant compared to wildtype . By these criteria , differential expression was detected with 1337 probes for C/X versus wildtype ( S1 Table ) , 215 probes for Xbp1CartΔEx2 versus wildtype ( S2 Table ) , and 1633 probes for ColXN617K versus wildtype ( S3 Table ) . Subsequently each of these sets of differentially expressed genes were compared with one another as shown in the Venn diagram in Fig 5A . Of the 1337 probes with differential expression between C/X and wildtype , 688 were differentially expressed in C/X versus wildtype and ColXN617K versus wildtype but not in Xbp1CartΔEx2 versus wildtype ( Fig 5A , cohort i ) . These probes represent genes regulated in the chondrocyte UPR independently of XBP1 . Of the 1633 probes indicating differential expression between ColXN617K versus wildtype , 885 were differentially expressed in ColXN617K versus wildtype but not in Xbp1CartΔEx2 versus wildtype or C/X versus wildtype ( Fig 5A , cohort ii ) , representing genes regulated in the chondrocyte UPR in an XBP1-dependent manner . Ontological analysis revealed that the XBP1-independent cohort ( Fig 5A , cohort i ) was enriched with probes corresponding to genes associated with angiogenesis , glycoproteins , the extracellular matrix , the endoplasmic reticulum , and positive regulation of apoptosis ( Fig 5B ) . Dysregulated apoptosis was confirmed by TUNEL analysis ( Fig 3 ) . We next partitioned the 687 probes of cohort i into those representing up-regulated genes or down-regulated genes in the collagen X mutants versus wildtype . The sub-cohort of 340 probes up-regulated independently of XBP1 was enriched with the majority of probes from cohort i corresponding to the endoplasmic reticulum , oxidation and reduction , and mRNA translation ( tRNA metabolism and amino acid biosynthesis; Fig 5C ) . The sub-cohort of 348 probes down-regulated independently of XBP1 was enriched with the majority of probes from cohort i associated with angiogenesis , glycoproteins , the extracellular matrix , and skeletal system development ( Fig 5D ) , consistent with the qPCR validation data for markers of ER stress , cartilage ECM ( Fig 4 ) and angiogenesis , Vegfa and Hbb-b1 ( S2A and S2B Fig ) . Thus the XBP1-independent consequences of the chondrocyte UPR are two-fold , both designed to restore ER homeostasis . On the one hand , they include the transcriptional up-regulation of genes encoding components of the cell that support protein folding . On the other , they involve transcriptional down-regulation of genes encoding secreted proteins , many of which include glycoproteins , components of the cartilage extracellular matrix , and pathways involved in skeletal system development . The XBP1-dependent cohort ( Fig 5A , cohort ii ) was enriched with probes corresponding to genes associated with organelle lumen , mitochondria , RNA processing and the cytoskeleton ( S1A Fig ) . We partitioned the 885 probes of cohort ii into those representing genes up-regulated or down-regulated in response to ER stress in an XBP1-dependent manner . The sub-cohort of 479 probes up-regulated in ColXN617K versus wildtype in an XBP1-dependent manner was enriched with the majority of probes from cohort ii associated with organelle lumen ( which includes ER-related genes ) , mitochondria , the nucleolus , and non-coding RNA processing and metabolism ( S1B Fig ) . qPCR validation of the up-regulation of several ER genes ( Creld2 , Derl3 , Ero1l and p58IPK ) is shown in Fig 4 . The sub-cohort of 406 probes down-regulated in ColXN617K versus wildtype in an XBP1-dependent manner was enriched with the majority of probes from cohort ii corresponding to the cytoskeleton , extracellular matrix , vasculature development , and ossification ( S1C Fig ) . By qPCR we validated the down-regulation of several genes related to ossification , Pthr1 and Bmp8a ( S2C and S2D Fig ) . Thus as with the XBP1-independent consequences of the chondrocyte UPR , XBP1-dependent gene expression changes include the transcriptional up-regulation of genes encoding components of the cell that support ER homeostasis , and the transcriptional down-regulation of genes encoding membrane-bound and secreted proteins that are trafficked through the ER for post-translational modification and assembly . We have previously shown that ER stress in the growth plate hypertrophic zone disrupts chondrocyte differentiation such that there is a delay in both the down-regulation of the proliferative chondrocyte gene expression signature , and up-regulation of the hypertrophic chondrocyte gene expression signature in mouse models of MCDS , including ColXN617K [12] . To determine whether XBP1 contributes to this disruption , we performed gene set tests comparing the differential expression of probes representing previously established [12] proliferative zone signature genes ( Fig 6A ) or hypertrophic zone signature genes ( Fig 6B ) in ColXN617K hypertrophic zones versus wildtype , C/X hypertrophic zones versus Xbp1CartΔEx2 , and in Xbp1CartΔEx2 hypertrophic zones versus wildtype . Significantly elevated expression of the proliferative zone gene signature was observed in C/X versus Xbp1CartΔEx2 ( Fig 6Ai ) , as in ColXN617K versus wildtype ( Fig 6Aii ) , but not in Xbp1CartΔEx2 versus wildtype ( Fig 6Aiii ) . Likewise we demonstrated significantly reduced expression of the hypertrophic zone gene signature in C/X versus Xbp1CartΔEx2 ( Fig 6Biii ) , similar to ColXN617K versus wildtype ( Fig 6Bii ) , but not in Xbp1CartΔEx2 versus wildtype ( Fig 6Bi ) . These results indicate that the disrupted differentiation observed in chondrocytes expressing misfolding protein in the hypertrophic zone is caused by an XBP1-independent aspect of the chondrocyte UPR . Several recent studies have implicated C/EBP-β in regulating the transition of chondrocytes from proliferation to hypertrophy [16–18] . Moreover , GADD45-β [20 , 21] and RUNX2 [17] have been identified as transcriptional co-factors of C/EBP-β required for full induction of the hypertrophy program . It has also been established that the function of C/EBP transcription factors , including C/EBP-β , may be inhibited through interaction with the ER stress-responsive transcription factor , CHOP [22] . Therefore we investigated whether the blockage in chondrocyte differentiation observed in our ColXN617K and C/X mice could be caused by inhibition of the transcriptional activity of C/EBP-β ( Fig 7 ) . By immunofluorescent analysis of wildtype and mutant growth plates , we confirmed that ATF4 , a marker of PERK activation required for ER stress-responsive expression of CHOP [15] , was up-regulated in the ColXN617K hypertrophic zone compared with wildtype , and in the C/X hypertrophic zone compared with Xbp1CartΔEx2 ( Fig 7A ) . Accordingly , by qPCR analysis of microdissected mutant and wildtype hypertrophic zones , we also confirmed up-regulation of Chop in ColXN617K versus wildtype , and in C/X versus Xbp1CartΔEx2 ( Fig 7B ) . Further qPCR analysis of the same samples was performed to establish expression profiles for Cebpb , Gadd45b , and Runx2 , as well as C/EBP-β transcriptional targets , p57Kip2 , Col10a1 , and Mmp13 ( Fig 7C–7H ) . Cebpb expression was up-regulated in ColXN617K versus wildtype , but differential expression was not observed between C/X and Xbp1CartΔEx2 . p57Kip2 , Runx2 , Col10a1 , and Mmp13 were all down-regulated in both ColXN617K versus wildtype , and C/X versus Xbp1CartΔEx2 . Gadd45b was significantly downregulated in ColXN617K versus wildtype; in C/X versus Xbp1CartΔEx2 downregulation of Gadd45b did not reach statistical significance . The qPCR expression profiles were broadly consistent with our microarray data for the same genes ( S1 and S3 Tables ) . Overall our results support the hypothesis that C/EBP-β transcriptional activity is inhibited as a result of post-transcriptional inhibition of C/EBP-β , rather than reduced expression of Cebpb mRNA , coupled with ER stress-dependent down-regulation of C/EBP-β transcriptional co-factors , GADD45-β and RUNX2 .
We and others have previously demonstrated that ER stress induced by expression of misfolding proteins in the mouse growth plate hypertrophic zone is sufficient to phenocopy MCDS [11] . Characterization of the molecular pathology of these mouse models of MCDS demonstrated that a canonical UPR is initiated involving activation of each of the canonical ER stress sensors that ultimately impairs bone growth by disrupting chondrocyte differentiation [11 , 12] . Here we demonstrate surprising redundancy of the IRE1/XBP1 signaling pathway in the MCDS UPR by showing that ablation of XBP1 signaling from chondrocytes in a mouse model of MCDS has no effect on the overall severity of the disease phenotype . It has been reported previously that by comparison with ATF6 and PERK , the XBP1 pathway regulates the differential expression of only a small subset of ER stress-responsive genes in mammalian cells [23 , 24] . This raises the question of what purpose the IRE1/XBP1 pathway serves in the cartilage UPR , and what genes it controls . To assess the contribution of the IRE1/XBP1 pathway to the MCDS UPR , we used a transcriptional profiling approach to interrogate gene expression in hypertrophic zones microdissected from our MCDS mutant mouse models . We identified 886 probes indicating significant differential gene expression between ColXN617K and wildtype in an XBP1-dependent manner . Given the impact of XBP1 at the transcriptional level when activated in response to chondrocyte ER stress and the effects its activation can have on the secretory capacity of the cell , and considering that cartilage-specific inactivation of XBP1 leads to a mild dwarfism characterized by hypertrophic zone shortening [14] , it is surprising that XBP1 is pathologically redundant in MCDS . In addition to regulating the activity of XBP1 , IRE1 activated during ER stress may also influence gene expression by degrading transcripts encoding membrane-bound and secreted proteins through regulated IRE1 dependent decay ( RIDD ) [25] and promote apoptosis via phosphorylation of JNK [26] . Of the established mammalian targets of RIDD , only Scara3 and Sparc were downregulated in both ColXN617K versus wildtype ( S1 Table ) and C/X versus Xbp1CartΔEx2 ( S3 Table ) , suggesting that RIDD does not have a significant role in the pathology of MCDS . Whether or not JNK is phosphorylated during ER stress in ColXN617K or C/X chondrocytes is unknown . Nevertheless , our study is the first to reveal dysregulated cell death as a feature of the pathology of MCDS . Previously we [12] and others [27] reported that apoptosis was not increased above wildtype levels in the hypertrophic zones of mouse models of MCDS up to 10 days of age . It is uncertain why a significant increase in the rate of ER stress-induced apoptosis was apparent in ColXN617K and C/X by two weeks of age but not earlier . A steady trajectory in the post-natal growth rate of the ColXN617K mouse was observed until 3 weeks of age , where it increased markedly to reach its peak between three to four weeks [11] . Thus , it is unlikely that the delay in ER stress-induced apoptosis in these mice until two weeks of age is attributable to significant growth-related increases in ER protein load at this time . An alternative possibility is that the delay might coincide with increased physical activity of the mice , implying that the fate of ER-stressed chondrocytes in the ColXN617K and C/X hypertrophic zones may be influenced by biomechanical force . Favouring a mechanism involving the combined influence of ER stress and biomechanical strain , we observed skewing in the antero-posterior distribution of apoptotic chondrocytes in the hypertrophic zones of both ColXN617K and C/X such that significantly more apoptotic chondrocytes were present closer to the periosteum than in the central portion of the growth plate . Moreover the bowing of weight-bearing long bones and coxa vara observed in human MCDS and mouse models [28] also points towards biomechanical stresses influencing endochondral bone development in this disease , in addition to ER stress . The onset of hypertrophic zone expansion and ER stress well before the increase in apoptosis in MCDS [11 , 12 , 27 , 29] indicates that ER stress-induced cell death is not a central feature of the disease pathology . Moreover , our data suggest that the proposal that the UPR in MCDS is adaptive and permits cell survival by alleviating the stress [27] may be restricted to early stages of cartilage development and maturation . We and others have demonstrated previously that ATF6 is proteolytically cleaved [11] and eIF2α phosphorylated [12] in mouse models of MCDS , implying activation of ATF6 and PERK respectively . Our transcriptomic analyses ( S1 and S3 Tables ) corroborate these findings , with ATF6 targets including BiP , Creld2 , Derl3 , and Grp94 [30] , PERK/ATF4/CHOP targets including Aldh1l2 , Aldh18a1 , Angptl6 , Clcn3 , Cdsn , Cyb5r1 , Cxad2 , Erlin1 , Fads3 , Gpt2 , Hspa9 , Iars , Leprotl1 , Mthfd2 , Nars , Otub2 , Ppp1r15a , Sars , Slc7a11 , Steap1 , Vldlr , Wars , Xpot , and Zfp238 [8 , 31] , and Erp72 , Grp58 , Herpud1 , and Pig-A , which are recognised as targets of both ATF6 and PERK , all upregulated in ColXN617K and C/X versus wildtype . Thus , we cannot definitively discriminate the relative contributions of ATF6 versus PERK to the IRE1/XBP1-independent effects of ER stress in MCDS chondrocytes , since both pathways seem to be activated strongly . We identified a cohort of 688 probes indicating XBP1-independent differential gene expression between our collagen X mutant mice and their respective controls . As with the XBP1-dependent cohort , several subsets were associated with processes and machinery designed to improve protein folding . Conspicuously , we also identified large subsets associated with angiogenesis , glycoproteins and the extracellular matrix . Subsequent partitioning of the XBP1-independent probes into subsets corresponding to up-regulated genes or down-regulated genes revealed that most of the genes associated with organelles or functions predicted to enhance protein folding were up-regulated in ColXN617K and C/X compared with controls , while most of the genes encoding markers of angiogenesis , glycoproteins , extracellular matrix molecules , and proteins associated with skeletal system development were down-regulated in the collagen X mutants compared with their respective controls . As with previous studies proposing UPR-mediated transcriptional suppression of physiological gene networks as a critical consequence of ER stress in other disease contexts [32–34] , so too our results suggest a pattern of ER stress-induced disruption to growth plate maturation in MCDS through transcriptional suppression of genes encoding secreted proteins , including components of the cartilage extracellular matrix . Several lines of evidence point towards the UPR interfering with chondrocyte differentiation by post-translational interruption of C/EBP-β . Various studies have implicated C/EBP-β as a key regulator in the transition of chondrocytes from proliferation to hypertrophy . Cebpb-/- mice were characterized by dwarfism involving elongation of the growth plate proliferative zone and delayed chondrocyte hypertrophy [16] . Proliferative zone elongation in these mice was due to reduced expression in the pre-hypertrophic zone of p57Kip2 , a gene identified as a transcriptional target of C/EBP-β that encodes a cyclin-dependent kinase inhibitor important for the exit of chondrocytes from cell division [16 , 35] . In addition to driving the expression of p57Kip2 , C/EBP-β represses the expression of Sox9 and Col2a1 , both important markers of chondrocyte proliferation [18] . Thus , C/EBP-β appears to have dual roles as a transcription factor controlling chondrocyte proliferation , switching off the expression of genes involved in maintaining the proliferative phenotype and switching on the expression of genes involved in terminating chondrocyte proliferation . As well as promoting the exit of chondrocytes from their proliferative program , C/EBP-β also actively promotes the entry of chondrocytes into hypertrophy . It has been shown that C/EBP-β co-localizes in the growth plate hypertrophic zone with GADD45-β and collagen X [20] , and that it acts cooperatively with GADD45-β to regulate Col10a1 and Mmp13 expression [20 , 21] . MMP13 is critical for endochondral ossification , since Mmp13-null mice are characterized by hypertrophic zone expansion , reduced collagen turnover , and delayed ossification [36] . In addition to GADD45-β , RUNX2 has also been implicated as a transcriptional co-factor of C/EBP-β . The Cebpb-/- mouse dwarfism phenotype was significantly exacerbated when crossed with a heterozygous Runx2 knockout mouse to generate Cebpb-/-;Runx2+/- , in which impaired cartilage remodelling through loss of Mmp13 expression resulted in elongation of the hypertrophic zone , in addition to the elongated proliferative zone seen in Cebpb-/- [17] . Thus , C/EBP-β actively promotes chondrocyte hypertrophy and growth plate matrix remodelling and turnover by interacting cooperatively with GADD45-β and RUNX2 to drive the expression of key markers of terminal chondrocyte maturation including Col10a1 and Mmp13 . Histomorphometric and expression profiling data in this and previous studies [11 , 12 , 27] are consistent with inhibition of C/EBP-β activity in ColXN617K and C/X growth plates . The hypertrophic zone expansion we have observed in ColXN617K [11 , 12] and C/X , the manner in which growth plate zone gene signatures were dysregulated in ColXN617K and C/X , and the down-regulation of key C/EBP-β transcriptional targets , p57Kip2 , Col10a1 , and Mmp13 observed here and previously [27] are all highly reminiscent of the skeletal phenotypes reported for the Cebpb-/- and Cebpb-/-;Runx2+/- mice [16 , 17] . Moreover , the mis-expression of SOX9 and Col2a1 in the 13del collagen X transgenic mouse is consistent with suppressed C/EBP-β activity in the MCDS growth plate [27] . Crucially however , the expression of Cebpb itself was not significantly down-regulated in the hypertrophic zones of either ColXN617K or C/X , implying that disruption to C/EBP-β activity in these mice must have occurred post-transcriptionally . The down-regulation of Gadd45b and Runx2 that we observed in ColXN617K and C/X relative to their controls is expected to have depleted the availability of C/EBP-β transcriptional co-factors required to promote hypertrophy in these mutants , and may thus have contributed to the elongation of their hypertrophic zones , as well as the dwarfism characteristic of each model . Nevertheless , that neither Runx2+/- nor Cebpb+/-;Runx2+/- mice display a skeletal phenotype [17] suggests that partial depletion of hypertrophy-dependent C/EBP-β transcriptional co-factors alone is insufficient to disrupt bone growth in the manner we have observed in ColXN617K and C/X . Rather , it suggests that loss of C/EBP-β activity itself , is also required . Moreover , while the depletion of GADD45-β and RUNX2 coupled with inactivation of C/EBP-β may have contributed to the delayed onset of chondrocyte hypertrophy , there is no evidence to suggest that it is in any way connected with the reduction in p57Kip2 expression , and associated mis-expression of proliferative zone signature genes that we observed in the ColXN617K and C/X hypertrophic zones . Thus it would appear that besides the depletion of GADD45-β and RUNX2 , the UPR must interfere with C/EBP-β activity by other means as well . Another mechanism by which C/EBP-β activity might be impaired in the growth plates of ColXN617K and C/X mice is through interaction with CHOP , which was induced in both mutants as a consequence of increased ATF4 expression . It has been shown that the transcriptional activity of C/EBP transcription factors can be attenuated by CHOP [22] . Other work has demonstrated that a consequence of prolonged UPR activation resulting from chemically induced ER stress in mice compromised by genetic inactivation of either of various components of the UPR , is transcriptional suppression of metabolic gene expression networks in the liver leading to hepatic steatosis , at least partially through the inhibition of C/EBP-α by CHOP [33 , 34] . These studies provide a precedent for disrupted cell differentiation in vivo by inhibition of C/EBP proteins by CHOP following ER stress . Thus we expect that in addition to ER stress-induced down-regulation of Gadd45b and Runx2 , inhibition of C/EBP-β by CHOP probably contributes to the down-regulation of C/EBP-β transcriptional targets we observed in ColXN617K and C/X . Expression of CHOP is understood to be a later event in the kinetics of UPR activation [37] . Supporting this , in situ analysis of UPR protein expression in the 13del collagen X transgenic mouse indicated that CHOP is activated relatively late in the MCDS UPR , after the onset of stress indicated by expression of the spliced form of XBP1 at the top of the mutant hypertrophic zone [27] . The delayed inhibition of C/EBP-β following the onset of ER stress may explain the observation from several MCDS mouse models that hypertrophic differentiation begins to proceed briefly before being interrupted and appearing to revert in response to UPR induction [11 , 27] . In all , these findings have led us to propose a model to explain the pathology of MCDS as follows . The onset of chondrocyte hypertrophy in MCDS is marked by the expression of misfolding collagen X , leading to ER stress . Each of the canonical ER stress-sensing pathways is activated . Through the combined , XBP1-independent effects of ER stress-induced up-regulation of CHOP and down-regulation of Gadd45b and Runx2 , C/EBP-β transcriptional activity is inhibited . Consequently , the stressed chondrocytes maintain or reactivate the expression of proliferative chondrocyte markers , and fail to express several key markers of terminal chondrocyte maturation , leaving the cells in a proliferative chondrocyte-like state . The collective consequences of these inputs are disruption to cartilage remodelling , vascularisation , and mineralisation , leading to hypertrophic zone expansion and dwarfism . This is the first study to our knowledge to provide evidence consistent with disruption to C/EBP-β-mediated gene transcription by ER stress in the pathology of a model of human disease . Moreover , it adds to the growing body of evidence arguing for the importance of CHOP in modulating the expression of physiological gene networks regulated by C/EBP transcription factors during ER stress [32–34] .
Col10a1 p . Asn617Lys mice ( ColXN617K ) [11] were crossed with mice in which Xbp1 mRNA is inactivated by the Cre recombinase-mediated deletion of exon 2 in Col2a1-expressing cells ( Xbp1CartΔEx2 ) [14] to generate the compound mutant , Col10a1 p . Asn617Lys/Xbp1CartΔEx2 ( C/X ) . These mice were viable , fertile and bred normally and were housed under pathogen-free conditions . All animal studies were approved by the Murdoch Childrens Research Institute Animal Ethics Committee ( Approval numbers AEC#718 and #787 ) . Genotyping was performed as previously described [11 , 14] . As previously [14] , RT-PCR and sequencing were subsequently performed on cartilage RNA as described to confirm deletion of Xbp1 exon 2 in C/X chondrocytes . Morphometric analyses were performed on skeletal preparations following Alcian blue/ Alizarin red staining as described [14] . Histology was performed on 10μm neutral buffered formalin-fixed cryosections of proximal tibial epiphyses from two week old wildtype , ColXN617K , Xbp1CartΔEx2 , or C/X mice . Toluidine blue staining [12] and immunofluorescent analyses using antibodies specific for collagen II or collagen X [14] were performed as described . Immunofluorescent analysis of ATF4 expression was performed using 1:100 rabbit anti-human ATF4 antibody ( D4B8; Cell Signaling Technology ) and an appropriate fluorescent secondary antibody ( 10μg/ml; Molecular Probes , Life Technologies ) , as follows . Prior to antigen retrieval , all sections were incubated for 10 min at room temperature in PBS , 0 . 2% Triton X-100 ( Sigma-Aldrich ) . For ATF4 antigen retrieval , sections were incubated for 10 min at room temperature in 1% SDS ( Sigma Aldrich ) in PBS . All immunofluorescence sections were counterstained and mounted using VECTASHIELD Mounting Medium with DAPI ( Vector Laboratories , Inc ) , and visualized by fluorescent microscopy with an Axio Imager M1 fluorescent microscope ( Zeiss ) . TUNEL was performed with the In Situ Cell Death Detection Kit , Fluorescein ( Roche ) to detect DNA fragmentation in cells undergoing programmed cell death as described [12] . Hypertrophic zones were microdissected , and RNA isolated and amplified from one proximal tibial growth plate from each of three two week old wildtype , ColXN617K , Xbp1CartΔEx2 , and C/X as described [14] . The yield and integrity of all samples were validated as appropriate with a Qubit 2 . 0 fluorometer ( Invitrogen ) , Nanodrop 1000 spectrophotometer , or a 2200 TapeStation ( Agilent Technologies ) , using a High Sensitivity R6K Screen Tape Kit ( Agilent Technologies ) . Each RNA validation procedure was performed according to the relevant manufacturer’s specifications . Quantitative PCR ( qPCR ) was performed on equal quantities of cDNA derived from tibial growth plate hypertrophic zone aRNA from two week old wildtype , ColXN617K , Xbp1CartΔEx2 , and C/X mice using the LightCycler 480 Probes Master Kit ( Roche Applied Science ) on a LightCycler 480 II qPCR machine ( Roche Applied Science ) as described [12] . All aRNA samples were interrogated by microarray analysis using single-colour hybridisations to MouseWG-6_V2 whole genome microarrays according to the manufacturer’s specifications ( Illumina ) . Microarrays were analysed using the package Limma [38] from the statistical language R . Background correction and quantile normalization were performed using the necq function . Probes that did not have above background expression in at least three samples were removed from the analysis after normalisation . Probes that were classified as “bad” or “no match” from illuminaMousev2 . db [39] were also removed . Self-contained gene set testing was performed using the ROAST [40] from the Limma package . Heatmaps were generated using heatmap . 2 from the gplots R package . All quantitative data were generated as stated in figure legends . All analyses were conducted using GraphPad QuickCalcs software ( La Jolla , CA , USA ) . | A significant component of the molecular pathology of many inherited skeletal disorders caused by mutations that cause misfolding and intracellular retention of extracellular matrix proteins is the induction of a cellular response to endoplasmic reticulum stress called the unfolded protein response ( UPR ) . In the case of Schmid metaphyseal chondrodysplasia ( MCDS ) caused by collagen X misfolding mutations , the consequences of the UPR have been shown to be the central cause of the cartilage pathology . Thus understanding the involvement of canonical UPR sensors , IRE1 , ATF6 , and PERK and their downstream signalling effects on chondrocyte differentiation and function is important for defining disease mechanisms and devising new therapies . Using a mouse model expressing misfolding collagen X and lacking IRE1/XBP1 pathway activity in chondrocytes , we demonstrate that this highly conserved UPR pathway is redundant to the cartilage pathology thus implicating XBP1-independent UPR signalling pathways . Based on detailed analysis of gene expression patterns we propose that XBP1-independent UPR driven disruption of C/EBP-β , a master regulator of chondrocyte differentiation , is important for the pathophysiology . Strategies designed to modulate C/EBP-β activity may thus offer therapeutic opportunities . | [
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] | [] | 2015 | XBP1-Independent UPR Pathways Suppress C/EBP-β Mediated Chondrocyte Differentiation in ER-Stress Related Skeletal Disease |
The control and prevention of dengue largely depends on vector control measures , environmental management , and personal protection . Dengue control programmes are facing great challenges due to development of insecticide resistance among vector mosquitoes . Information on susceptibility status to different insecticides is important for national programmes to formulate vector control strategies . We have studied the larval susceptibility of Aedes albopictus to temephos and adult susceptibility to 4% DDT , 0 . 05% deltamethrin , and 5% malathion as per WHO protocols in the northern districts of West Bengal . Polymorphisms in the VGSC gene were studied by direct sequencing of PCR products . The Ae . albopictus larval population showed sensitive [Resistance Ratio ( RR99 ) <3] to moderate levels of resistance ( 5<RR99>10 ) to temephos at different study sites . Adult bioassay results revealed that Ae . albopictus was highly resistant to DDT [Corrected Mortality ( CM ) < 90%] in all the study sites and susceptible to deltamethrin and malathion ( CM > 98% ) , except in Dhupguri where a low level of resistance to deltamethrin ( CM = 96 . 25% ) was recorded . None of the six important kdr mutations ( S953P , I975M/V , L978 , V980G , F1474C , D1703Y ) were found in the VGSC of studied mosquitoes , but we identified 11 synonymous and 1 non-synonymous mutation in the VGSC gene . The higher susceptibility level to deltamethrin and malathion , along with the absence of important kdr mutations indicates that these two insecticides are still effective against Ae . albopictus in the study areas . The susceptibility status of temephos should be monitored closely as low to moderate levels of resistance were observed in few sites . A similar study is recommended for monitoring and early detection of insecticide resistance in other parts of the country .
Dengue is a mosquito-borne flavi-viral disease and a major public health problem in more than 120 countries [1 , 2] . In recent years , dengue transmission has increased predominantly in urban , semi-urban areas and has even extended to the rural areas , becoming a major public health concern globally . A recent estimate showed 390 million new dengue infections throughout the world , of which , 96 million cases manifested the severe form of the disease [2] and almost half of the world’s population are at risk of dengue infection [3] . In India , dengue is spreading into new areas and emerging as a major public health problem . In 2016 , a total of 129166 dengue cases and 245 deaths were reported from India , of which 22865 cases and 45 deaths were reported from West Bengal [4] . Aedes aegypti and Aedes albopictus are the vectors of dengue along with three other important human viral diseases: yellow fever , chikungunya , and Zika . No effective vaccine against dengue is available to date . Vector control and personal protection from mosquito bites are suggested to reduce its transmission . For proper formulation and implementation of vector control strategies , thorough information about vector species distribution and their susceptibility to available insecticidal agents are necessary [5] . Four different classes of insecticides are in use as adulticides against Aedes mosquitoes: organophosphates , pyrethroids , organochlorines , and carbamates [6 , 7] . Among these , pyrethroids and organophosphates are widely used throughout the world [8 , 9 , 10] . Pyrethroids are used as indoor residual treatment and impregnation of bed nets whereas organophosphates are used as larvicides and space treatments [6] . The National Vector Borne Disease Control Programme ( NVBDCP ) of India recommends different insecticides for vector management , such as temephos ( 50 EC ) as a larvicide , DDT and synthetic pyrethroids ( recently introduced ) for indoor residual spray ( IRS ) , deltamethrin ( pyrethroid ) for impregnation of bed nets , and malathion for ultra low volume ( ULV ) spray . In India , Aedes mosquito control is mainly based on anti-larval measures and the use of insecticides by space spraying of pyrethrum and fogging of malathion during a disease outbreak to kill adults . The development and spread of resistance by the vector mosquitoes against all available insecticides is a great challenge to prevent the transmission of mosquito-borne diseases . Ae . albopictus and Ae . aegypti showed resistance to DDT [11 , 12 , 13] , but were susceptible to malathion and deltamethrin [11 , 12 , 14 , 15] in different parts of India . Pyrethroids are synthetic analogues of naturally occurring pyrethrum from the extracts of the Chrysanthemum flower and represent the most widely used insecticide against insect vectors [16] . Unfortunately , pyrethroid efficacy is being threatened due to rapid development of resistance by the vector mosquitoes [8 , 17] . The World Health Organisation ( WHO ) formulated standard diagnostic bioassay test kits to monitor the susceptibility of mosquitoes against different insecticides [18] . Exposure to pyrethroids and DDT results in “knockdown” ( i . e . , rapid paralysis ) due to prolonged-activation of sodium channels . Pyrethroids and organochlorines cause overstimulation of the mosquito nervous system by repeated action potentials form the opening of the sodium channel [19 , 20 , 21] . Knockdown resistance ( kdr ) is the major mechanism of pyrethroid resistance , caused by mutations in the voltage-gated sodium channel gene ( VGSC gene ) [22 , 23] . In insects , the voltage-gated sodium channel is an integral transmembrane protein which is composed of four homologous domains ( I-IV ) . Each domain consists of six subunits ( S1-S6 ) which are connected by loops . The segments S5 , S6 , and the P-loop between them form a central aqueous pore , and the S1-S4 segments of each domain unite to form four independent voltage-sensitive domains [24 , 25] . Insects have only one functional sodium channel gene [19] . There are two receptor sites in the four-domain sodium channel for simultaneous binding of pyrethroids [26] . So far , ten different mutations at eight codons comprising fifteen haplotypes have been reported in Ae . aegypti . The frequency of these mutations varies geographically [27 , 28] but such reports from India are very rare . Periodical monitoring of insecticide resistance among the prevailing vector population in a given geographical region will be helpful to formulate vector control strategies by the NVBDCP . The present work was designed to study the susceptibility status of Ae . albopictus to temephos , DDT , deltamethrin , and malathion , as well as polymorphisms in the VGSC gene in dengue endemic areas of northern West Bengal .
This study was carried out in one municipality and two blocks of Darjeeling , two blocks of Jalpaiguri , and one block of Uttar Dinajpur districts of West Bengal during June 2016 to September 2016 . The study locations were Siliguri Municipal Corporation ( SMC ) , Matigara , and Khoribari of the Darjeeling district; Malbazar , Dhupguri of the Jalpaiguri district , and the Itahar block of Uttar Dinajpur . Most of the study sites were sub-urban except Siliguri Municipal Corporation ( urban ) and Khoribari ( rural ) ( Fig 1 ) . The aquatic stages ( larvae and pupae ) of Aedes sp . were collected from the seventeen localities of three districts . For each collection site , larvae and pupae were collected from domestic , peri-domestic and natural breeding places . The collected immature stages of mosquitoes were stored in plastic containers containing water from the same breeding habitat and transferred to the laboratory . In the laboratory , the wild caught mosquito larvae and pupae were transferred into a larvae rearing tray along with water collected from the field and supplied with food for ornamental fishes available in the local market along with yeast . The mosquito larvae and pupae were reared to the adult stages in the laboratory under controlled conditions ( temperature 25°C ± 2°C; relative humidity 80% ± 10% ) . After emergence , the adults were identified by using the standard identification keys of Barraud , 1934 [29] and Tyagi et al . , 2012 [30] . The identified Ae . albopictus were allowed to breed under laboratory conditions . The larvae and adults of the F1 generation were used for larval and adult insecticide bioassays . Susceptibility of larvae to temephos ( 50EC; Nitapol Industries Pvt Ltd . , Kolkata ) was estimated using the standard WHO bioassay protocol [31] . The stock temephos solution of 1 ppm concentration and other subsequent dilutions were prepared in 95% ethanol and stored at +4°C for use in the susceptibility bioassay . Bioassays were conducted using 20–25 third instar to early fourth instar larvae ( wild caught strain and laboratory strain ) in disposable paper cups filled with the required concentration of insecticide solution and double distilled water at room temperature ( 25°C ± 2°C ) . Eight different concentrations ( 0 . 0005 , 0 . 001 , 0 . 005 , 0 . 01 , 0 . 05 , 0 . 1 , 0 . 5 and 1 . 0 ppm ) were used as per WHO recommendation [32 , 33] and each experiment was replicated at least three times . Each set of the bioassay was accompanied by two sets of controls ( equal concentration of 95% ethanol ) . Larval mortality was recorded after 24 h of exposure . The larvae that were motionless or convulsive upon a sharp stimulation were counted as dead [31] . Larval mortality was determined by dividing the number of dead larvae by the total number tested . A test was considered as invalid if pupation rate was greater than 10% , or mortality rate in the control was greater than 20% [31] . The degree of resistance was determined by the resistance ratio ( RR99 ) , which is calculated by comparing the lethal concentration ( LC99 ) value for a population with the LC99 value for the insecticide for a laboratory colony . The RR99 ≤3 was considered as susceptible , and 3 < RR99 ≤ 5 as low resistance , 5 < RR99 ≤ 10 as moderate resistance , and RR99> 10 as high resistance [34] . Two to three day old laboratory emerged unfed female Ae . albopictus mosquitoes were used for the insecticide susceptibility bioassay as per WHO protocol [18] . The tested insecticides were 4% DDT , 0 . 05% deltamethrin , and 5% malathion . The insecticide-impregnated papers were procured from the Vector Control Research Unit ( VCRU ) , Universiti Sains Malaysia , Malaysia . Five different holding tubes were used for each set of the experiment of which four were a test and one was a control . In each holding tube , 15–20 adult female mosquitoes were kept for one hour . After one hour of holding , mosquitoes from four tubes marked as test were exposed to insecticide-impregnated papers . The control tests were performed using silicone oil , olive oil , and risella oil pre-impregnated papers for deltamethrin , malathion , and DDT , respectively . Mosquitoes were allowed in the exposure tube for one hour and cumulative knock down was recorded after 10 , 15 , 20 , 30 , 40 , 50 , and 60 minutes . After 60 minutes of exposure , the mosquitoes were transferred to holding tubes and fed on a 5% sucrose solution for the next 24 h . Mortality was scored after 24 h to determine the susceptibility status as per WHO recommendation [18] . Mosquitoes were considered dead if they were motionless , when they were mechanically stimulated , following the method of Gonzalez Audino [35] . The live and dead mosquitoes obtained from the adult bioassays were stored at -20°C and used for molecular biological assays . Genomic DNA was isolated from both live and dead mosquitoes ( individually ) by using the DNeasy Blood & Tissue Kit ( Qiagen , Germany ) , as per the manufacturer’s instructions . Before initiation of DNA isolation , the wings of the mosquitoes were removed and the remaining part of the mosquito was carefully homogenised by a Tissue Ruptor ( Qiagen , Germany ) . Extracted DNA was stored at -20°C until further study . PCR was done using three different primer pairs targeting six amino acid loci ( S953P , I975M/V , L978 , V980G of domain II , F1474C of domain III and D1703Y of domain IV ) of the voltage-gated sodium channel gene ( VGSC ) of Ae . albopictus , which is responsible for knockdown resistance ( kdr ) . The details of primers and PCR conditions are given in Table 1 as described earlier by Kasai et al . , 2011 [36] . The quality of PCR products was ascertained by 2% agarose gel electrophoresis following ethidium bromide stain . The PCR product was gel purified using the Qiagen gel extraction kit ( Qiagen , Germany ) and sequencing was outsourced to Chromous Biotech , Bangalore . Four different primers i . e . , aegSCF3 , aegSCR22 ( forward and reverse primer for domain II ) , aegSCR8 ( reverse primer for domain III ) , and albSCF7 ( forward primer for domain IV ) were used for sequencing of the PCR products . In the present study , we numbered the codons of the VGSC gene according to the sequence of Ae . albopictus . The sequences were analysed using the software BioEdit Sequence Alignment Editor version 7 . 0 . 9 . 0 . The sequences were aligned with the reference sequence for Ae . albopictus ( GenBank accession no . AY663384 . 1 ) , using an online multiple sequence alignment tool . Before initiation of the work , the objectives of the study were explained to the local population of each study site . Permission was taken from the owners of private houses/lands before collection of immature stages of mosquito . The study did not involve any endangered and protected species . Mosquitoes were maintained under optimal conditions such as temperature , humidity , and adequate food supply in the laboratory . The study protocol was approved by the Institutional Ethics Committee of Calcutta School of Tropical Medicine , Kolkata . Larval bioassay data were analyzed using Log dose probit ( Ldp ) Line computer software ( Ehabsoft , Cairo Egypt; available at: http://www . ehabsoft . com/ldpline ) according to the Finney’s method [37] . Chi-squared ( χ2 ) test was used to estimate the goodness of fit , while linear regression was used to evaluate the data linearity . Lethal concentrations ( LC10 , LC50 , and LC99 ) along with the slope were estimated at 95% confidence intervals ( CI ) . For adult bioassays , observed mortality was calculated by the formula: observed mortality ( % ) = ( Total no . of dead mosquitoes / Total mosquitoes exposed ) x 100 . The observed mortality was corrected using Abbott’s formula when the mortality rate of control was within 5% - 20% . Corrected Mortality ( CM ) ( % ) = [ ( % of observed mortality—% of control mortality ) / ( 100 - % of control mortality ) ] x 100 . For adult bioassays , resistant/susceptibility status was defined according to WHO recommendations [18] . Mosquitoes were considered susceptible ( S ) if the corrected mortality ( CM ) rate was greater than 98% and resistant ( R ) if mortality rate was less than 90% . Mortality rate between 90–98% was considered as possible resistance ( PR ) and needs verification by alternative methods like enzyme bioassay and molecular marker studies [18] . The cumulative knock down rates ( KDR ) were calculated by observing the number of knocked down mosquitoes after 10 , 15 , 20 , 30 , 40 , 50 and 60 minutes during the hour-long exposure period . Knockdown time ( KDT10 , KDT50 , and KDT95 ) is the time required for knockdown of a particular proportion of mosquitoes following exposure to any insecticide . KDTs were determined using Log dose probit ( Ldp ) Line computer software ( Ehabsoft , Cairo Egypt; available at: http://www . ehabsoft . com/ldpline ) programme according to the Finney’s method [37] .
The study was conducted in one municipality and 5 different blocks of 3 districts in the northern part of West Bengal during June 2016 –September 2016 . The study sites of Dhupguri and Itahar blocks were surrounded by paddy fields , whereas the presence of both paddy fields and tea gardens were characteristic of the remaining study sites except Siliguri Municipality Corporation ( SMC ) and Matigara . Most of the study sites were suburban in nature except the Siliguri municipality area ( Urban ) and Khoribari ( rural ) ( Fig 1 ) . Storage water tanks , discarded tyres , tree holes , construction sites , flower pots , plastic cups , coconut shells , and discarded containers were the different seasonal breeding sites found in the study area . The climatic conditions of all study sites were humid and sub-tropical in nature and the temperature varies from 8°C in winter to 40°C in summer . The summary of larval bioassay results is presented in Table 2 . The LC10 , LC50 , and LC99 values of different study sites did not follow a normal distribution for mortality to the log dose ( χ2 ≥ 16 . 08; p ≤ 0 . 01 ) . The LC50 values ranged from 0 . 0009 to 0 . 0015 mg/L and LC99 from 0 . 1565 to 0 . 3343 mg/L . The calculated RR50 and RR99 values in different study sites were ranged from 1 . 0 to 2 . 5 and 2 . 45 to 5 . 24 , respectively . The results of the adult susceptibility bioassay for Ae . albopictus are given in Table 3 . After 24 hours of exposure , the corrected mortality rates for 4% DDT were 23 . 75% to 85 . 53% in different study sites . The obtained mortality rates were well below the WHO recommended 90% mortality rate for resistance . So , results suggested that the Ae . albopictus population of the study areas was highly resistant to DDT . In all of the study sites , the corrected mortality rate for 0 . 05% deltamethrin ranged from 98 . 08% to 100% , except in Dhupguri where the corrected mortality was 96 . 25% . So , Ae . albopictus population of all the study sites was susceptible to deltamethrin except Dhupguri . The corrected mortality rate for 5% malathion was >98% in all the study sites indicating susceptibility to malathion . The knock down time ( KDT10 , KDT50 , KDT95 ) for DDT , deltamethrin , and malathion showed a linear probit for knock-down rates with time in most of the study sites ( Table 3 ) . The observed KDT50 values were 23 . 62 to 51 . 39 mins for DDT , 10 . 14 to 13 . 82 mins for deltamethrin , and 17 . 52 to 25 . 31 mins for malathion . The KDT95 values for DDT were 80 . 10 to 212 . 11 mins , for deltamethrin 22 . 85 to 43 . 28 mins and for malathion 32 . 39 to 77 . 04 mins . The survival rate of Ae . albopictus against DDT , deltamethrin , and malathion over an exposure time of 1 hour is given in Fig 2A–2C . During 1 hour of exposure , the knock down rate ( KDR ) varies from 68 . 75% - 93 . 75% for DDT , 100% for deltamethrin , and 95 . 00% - 100% for malathion . DNA was isolated from 30 dead and 10 alive , deltamethrin-exposed Ae . albopictus mosquitoes and used for PCR amplification . For detection of kdr mutations three DNA fragments of 480 bp , 740 bp , and 280 bp for domain II , III , and IV of VGSC gene were amplified , respectively . None of the six important kdr mutations ( i . e . , S953P , I975M/V , L978 , V980G , F1474C , D1703Y ) were found among the studied mosquitoes . We detected 3 synonymous mutations in domain II , 1 non-synonymous and 3 synonymous mutations in domain III , and 5 synonymous mutations in domain IV . The frequencies of observed mutations are presented in Table 4 . The DNA sequences have been submitted to GenBank under accession nos . MF776970 and MF774494 .
Emergence and spread of insecticide resistance is the biggest challenge to control vector-borne disease transmission [38] . In Aedes mosquitoes there are two major mechanisms for pyrethroid resistance: increased detoxification and mutation in the VGSC gene . To date more than 50 different VGSC mutations have been identified in different insect species [19] . The six non-synonymous amino acid substitutions: S989P , I1011M , L1014F , V1016G in domain II , F1534C in domain III and D1763Y in domain IV of house fly are found to be associated with pyrethroid resistance . These codons are orthologous to the codons 953 , 975 , 978 , 980 , 1474 and 1703 , respectively of Ae . albopictus . The involvement of other mutations in pyrethroid resistance remains to be investigated . The L1014F , at S6 subunit of domain II was the first pyrethroid-resistance-associated mutation identified in the house fly and German cockroach [39 , 40 , 41] . I1011M was identified in domestic house fly from Brazil , Guyana , whereas V1016G was identified from Indonesia and Thailand [42] . Later , different substitutions , I1011V and V1016I , were found in Ae . aegypti populations from Latin America [43] . The most significant F1534C , located in S6 subunit of domain III was discovered in DDT/permethrin-resistant Ae . aegypti in Thailand and Vietnam [44 , 45] . The adult insecticide susceptibility bioassay is applied to determine the lethal dose of different insecticides by direct exposure . Additional tests , such as polymorphisms in marker genes and biochemical assays of different enzymes are used as supplementary evidence to clarify the results of bioassays and potential mechanisms . In the present study , we determined the susceptibility status of Ae . albopictus against DDT , deltamethrin , and malathion . The results showed that Ae . albopictus is significantly resistant to DDT with a higher KDT and KDR and a lower mortality rate . Similar observations have also been reported from other parts of the country [11 , 12 , 14] . Though DDT is not in use against Aedes vector mosquitoes , this compound is still in use for control of malaria vectors . The present study areas have been highly endemic for malaria for a long time , with the exception of Itahar . Thus , Aedes mosquitoes have been exposed to DDT for many generations which might be the cause of the high level of resistance that has developed to DDT . Ae . albopictus from the present study areas were susceptible to deltamethrin and malathion . Pyrethroid resistance in adult Aedes sp . is a problem worldwide . The level of resistance varies from region to region . A lower level of resistance is found in Asian , African , and Northern American countries , [46 , 47 , 48 , 49] whereas higher levels of resistance are found in South American countries [50 , 51] . In the present study , lower values of knock down time and knock down rate were observed in Ae . albopictus against deltamethrin and malathion . The KDT values recorded in the present study did not follow a normal distribution pattern which indicates that the prevailing Ae . albopictus population is susceptible to these insecticides . In India , temephos is used as larvicidal agent . In contrast to adult susceptibility , higher levels of larval resistance have been found in Asian , African , and North American countries [49 , 52 , 53 , 54 , 55] . In the present study we found that the Ae . albopictus larvae were sensitive to temephos in Khoribari and Dhupguri ( RR99<3 ) ; showed a low level of resistance in Matigara , Malbazar , and Itahar ( 3<RR99>5 ) , and moderate resistance in Siliguri ( 5<RR99>10 ) [33] . A similar type of observation was also reported from the north eastern part of India [11 , 12] . The Siliguri Municipal Corporation is the only urban site in the present study , where temephos has been in use for a long time . A longer duration of exposure to temephos might be the cause of the observed moderate level of resistance against it in Ae . albopictus from Siliguri . In contrast , a recent report from the northern part of West Bengal showed susceptibility of Ae . albopictus larvae to temephos assessed by larval susceptibility and bioassay of detoxifying enzymes [56] . The KDR is a mechanism of DDT and pyrethroid resistance . Mutations at codons 953 , 975 , 978 , 980 , 1474 , 1703 of the VGSC gene of Ae . albopictus have been found to be associated with reduced susceptibility to both DDT and pyrethroids [22 , 23] . As per our present study , the only previous report from India did not reveal any mutation in the VGSC gene of Ae . albopictus [8] but two other reports reveal mutations at codon F1534C [57] and at codon T1520I + F1534C of the Ae . aegypti VGSC gene [13] . In the present study , we detected only one non-synonymous mutation at S1485L in three samples . Interestingly , all three mosquitoes were susceptible to deltamethrin . So , the role of this mutation in pyrethroid resistance cannot be explained . We also detected 11 synonymous mutations among both dead as well as live deltamethrin-exposed mosquitoes . We did not assess the detoxifying enzyme levels associated with DDT and deltamethrin resistance . The higher susceptibility level in deltamethrin with absence of important kdr mutations and higher susceptibility to malathion indicate that these two insecticides are still effective in the study areas . The susceptibility status of temephos as a larvicide should be monitored closely as moderate and lower levels of resistance were observed in mosquitoes from a few study sites . A similar study is highly recommended for monitoring and early detection of pyrethroid and malathion resistance in other parts of the country . | Dengue is one of the most important arboviral infections in India , and transmitted by Aedes mosquitoes . Control of dengue largely depends on effective vector control measures as no specific drugs or vaccines are available , to date . The knowledge of insecticide susceptibility status for local vector mosquitoes is essential for the formulation of effective vector control measures . Therefore , regular monitoring of insecticide susceptibility is essential for the early detection of resistance . In the present study , sensitive to moderate levels of resistance to temephos were recorded among the Aedes albopictus larval populations of different study sites . Adult Ae . albopictus were highly resistant to DDT and susceptible to deltamethrin and malathion in all the study sites , except one site where a low level of resistance to deltamethrin was observed . By sequencing the VGSC gene we observed that the studied Ae . albopictus population does not contain any of the important kdr mutations which are associated with DDT and pyrethroid resistance in other insects . We found only one non-synonymous mutation at codon S1485L , but its role in pyrethroid resistance is yet to be established . | [
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"inse... | 2018 | Polymorphisms in voltage-gated sodium channel gene and susceptibility of Aedes albopictus to insecticides in three districts of northern West Bengal, India |
High serum uric acid levels elevate pro-inflammatory–state gout crystal arthropathy and place individuals at high risk for cardiovascular morbidity and mortality . Genome-wide scans in the genetically isolated Sardinian population identified variants associated with serum uric acid levels as a quantitative trait . They mapped within GLUT9 , a Chromosome 4 glucose transporter gene predominantly expressed in liver and kidney . SNP rs6855911 showed the strongest association ( p = 1 . 84 × 10−16 ) , along with eight others ( p = 7 . 75 × 10−16 to 6 . 05 × 10−11 ) . Individuals homozygous for the rare allele of rs6855911 ( minor allele frequency = 0 . 26 ) had 0 . 6 mg/dl less uric acid than those homozygous for the common allele; the results were replicated in an unrelated cohort from Tuscany . Our results suggest that polymorphisms in GLUT9 could affect glucose metabolism and uric acid synthesis and/or renal reabsorption , influencing serum uric acid levels over a wide range of values .
Serum uric acid ( UA ) levels are frequently elevated in conjunction with several disorders , including obesity , hyperlipidemia , atherosclerosis , and hyperinsulinemia [1] . Most widely discussed have been findings of high serum UA in individuals at risk of cardiovascular disease [2 , 3] . High UA levels are causal in gout crystal arthropathy , and according to some authors , are associated with a negative cardiovascular risk profile . Elevated UA is a risk factor for all-cause and cardiovascular mortality in patients with prevalent cardiovascular disease , and an important prognostic factor in hypertension . Identifying factors that affect UA levels could help our understanding of their role in pathology , improve clinical decisions about whether to treat moderate hyperuricemia [4 , 5] , and help identify new targets for intervention aimed at managing undesirably high UA levels . UA levels in humans are generally higher than in other species because the gene for the primary enzyme that transforms UA in other species , uricase , is inactivated . Consequently , UA levels are determined by the balance between the production of UA through purine catabolism ( the two important contributors being dietary purine levels and purines released from the DNA and RNA of damaged cells ) and the rate of excretion/reabsorption in the kidney and intestine . Several metabolic defects that may increase serum UA levels have been described . Changes in the level of UA synthesis can result from mutations or variation in activity of enzymes involved in purine metabolism . In one relevant instance , a microRNA has been reported that seems to modulate the synthesis of UA through its repression of phosphoribosyl pyrophosphate synthetase 1 [6] . Tissue ischemia can also stimulate UA production by the upregulation of xanthine oxidase . The alternative route to increased UA levels is a decline in UA clearance in the kidney . The urate/anion transporter [7] is highly expressed in the proximal kidney tubule , and genetic variants in the genes that code for the transporter may contribute to hyperuricemia [8] . In an attempt to discriminate genetic factors affecting UA levels , we scanned the genome with a panel of 362 , 129 single nucleotide polymorphisms ( SNPs ) in 4 , 305 Sardinian individuals . Here , we report association between SNPs found in a glucose transporter gene , GLUT9 , and UA levels . We also show the association is confirmed in an independent population .
UA levels and genotypes were assessed for a cohort of individuals from Sardinia ( see Materials and Methods ) . Using a Bonferroni threshold of 1 . 3 × 10−7 ( 0 . 05/362 , 129 ) , we selected 38 SNPs potentially associated with UA levels . Unexpectedly , rather than identifying variants in purine catabolism or in the urate transporter , the scan pointed to GLUT9 [9] on Chromosome 4 ( Figure 1; Table S1 ) . All SNPs exceeding the Bonferroni significance threshold map to the region that encodes this Class II glucose transporter [9] . The rare allele “G” of the top ranked SNP rs6855911 has an effect size of −0 . 317 mg/dl , with a more marked effect in women than in men ( Table 1 ) . Figure 2 shows the contributions of the two alleles of the SNP in homozygotes and heterozygotes . Their effect is observed across the full range of UA values . The strongest observed association of SNP rs6855911 and distribution of the SNPs in linkage disequilibrium are shown in Figure 3 , along with the comparative linkage disequilibrium patterns in the HapMap Utah residents with ancestry from northern and western Europe ( CEU ) and Yoruba in Ibadan , Nigeria ( YRI ) samples . In a sequential analysis in which we selected the best SNP for each trait and then conditioned on it to successively select the next best SNP , a marker in the adjacent gene , WDR1 , was selected at the second round , meaning that its signal is—at least in part—statistically independent from rs6855911 ( Table 2 ) . These results can support either the presence of multiple variants in the same region or reflect different degrees of linkage disequilibrium with a causal SNP that remains to be identified . The only other gene showing a cluster of SNPs associated with UA levels—though at far less striking p-values ( on the order of 10−7 ) —is PJA2 , a ubiquitin ligase gene on Chromosome 5 [10]; its position marked in Figure 1 . This possible association has not been examined further . Based on the genetic results , we hypothesized that variants underlying the association with UA levels affect a critical step in UA production or elimination . We started to test this hypothesis by studying the relationship between UA and the two top strongly associated SNPs in GLUT9 in an independent population sample . We examined participants in the InCHIANTI study , an epidemiological study ongoing in two Italian towns in Chianti , Tuscany . A detailed description of the study design and data collection methods of the InCHIANTI study has been published [11 , 12] . The study population consists of 1 , 301 individuals ( 579 males and 722 females ) , comprising a random sample of the population aged 65 years and older supplemented with 30 men and 30 women randomly selected in each decade between ages 20 and 70 . Table 1 and Figure 2 show that the results in this cohort replicate the findings in the Sardinian group . For SNP rs6855911 , the allele G has a negative additive effect of 0 . 39 mg/dl ( p = 1 . 54 × 10−11 ) . Similarly , the rare allele G for rs7442295 has a negative additive effect of 0 . 42 mg/dl ( p = 2 . 57 × 10−12 ) . Again , GLUT9 variants significantly and comparably affect UA levels over the whole distribution of values , without a threshold ( Figure 2 ) . Interestingly , while none of the Sardinian participants had hyperuricemia ( UA > 7 . 5 mg/dl in men and >6 . 2 mg/dl in women ) , in the InCHIANTI study , participants with at least one G allele had a significantly lower prevalence of hyperuricemia ( AA: 11 . 1%; AG: 7 . 2%; GG: 3 . 5%; p = 0 . 009 ) . UA levels generally increase with age ( e . g . , [13] ) , and the InCHIANTI cohort is considerably older than the Sardinian sample , resulting in higher mean values for each genotype ( Figure 2 ) . However , the values for corresponding age groups in the two are very similar , as are the contribution of the GLUT9 alleles and the greater effect in females ( Figure 2; Table 1 ) . Because the Sardinian population , as a founder population , is relatively homogeneous ( reviewed in [13] ) , it can facilitate the detection of associated SNPs but might emphasize genes that are important only on the island . In this respect , mainland Italian residents are both genetically much more heterogeneous and distinct from Sardinians ( as distant from Sardinians as American Caucasians are [14] ) . Thus , the replication of allele effects in Chianti suggests that GLUT9 is involved in a general mechanism of UA production/elimination that may be biologically important in other populations as well . The initial SNPs tested and found associated with UA levels all fall in noncoding regions . To assess any candidate SNPs in the coding region and/or promoter of the gene , the exons and a 1 . 5 kb presumptive promoter region upstream of the transcription start site were amplified by PCR and sequenced in 30 individuals ( ten for each homozygote and ten heterozygotes for rs6855911 ) . The primers used to amplify the genes are listed in Table S2 . The sequencing found no new SNPs . However , we identified several SNPs in Sardinian DNA samples that were previously described in other populations . They include six SNPs in the promoter region and nine in exons ( results are in Table S3 ) . Among the other SNPs in the region , rs16890979 , a nonsynonymous SNP in exon 8 that produces a Val253Ile amino acid change , was in strong linkage disequilibrium with rs6855911 ( r2 = 0 . 74 in the HapMap CEU and r2 = 1 . 0 among the 30 resequenced individuals ) . However , genotyping of that coding SNP in a further 541 Sardinians suggests that rs6855911 and other noncoding SNPs in the region are more strongly associated with UA levels ( p < 0 . 0004 for rs737267 and other noncoding SNPs in strong linkage disequilibrium with rs6855911 , p = 0 . 02 for rs16890979 ) . Further analyses are necessary to identify the causal variant ( s ) in the region .
How might variants of GLUT9 differentially affect UA levels ? Studies have verified that GLUT9 transports glucose [15] , and both characterized isoforms , comprising 540 and 511 amino acids [15] , respectively , are highly expressed in liver and distal kidney tubules . In the liver , where much UA is synthesized , rare deficiency of glucose-6-phosphatase ( Glycogenosis Type I; Online Mendelian Inheritance in Man 232200 ) leads to increased levels of UA . Variations in the uptake of glucose via GLUT9 might also result in increases or decreases in glucose-6-phosphate and modulate metabolism through the pentose phosphate shunt . Augmented levels of phosphoribosyl pyrophosphate synthesis , for example , could then lead to increased hepatic production of UA . Alternatively , or in addition , the effect of GLUT9 on UA level may be exerted through effects on renal excretion . In the kidney , urate transport occurs in the proximal tubular epithelium , whereas GLUT9 is expressed in more distal nephron segments , perhaps the distal convoluted or connecting tubules [16] . However , those segments are relatively anaerobic , and one can speculate that the levels of lactate and other anions could be altered by metabolism from the glucose supplied by GLUT9 , with consequent changes in organic anion concentration in the interstitium and neighboring proximal tubule cells . Anions are exchanged for urate by the transporter URAT1 , with lactate as a preferred exchange ion [7] , affecting the coupled process whereby filtered urate is reabsorbed from the urine in exchange for cytosolic organic anions . Hence , GLUT9 variants in the kidney could change the ambient level of organic anions and thereby alter urate reabsorption and circulating urate levels . Effects could be more marked in individuals with purine-rich diets , or in hypertensive individuals in whom reabsorption is favored by reduced blood flow in the proximal tubule [17] and/or localized ischemia [2] . Regardless of underlying mechanism , GLUT9 variants clearly have a significant modulating effect on levels of UA . Homozygotes for the two allelic variants differ in serum UA by 0 . 6 mg/dl , or on the order of 10% of the mean levels . Based on the findings in an independent study [18] , such a difference in UA levels would change the rate of cardiovascular events by about 1% . In further assessing the impact of such variation , it is notable that many authors ( [19] and review in [4] ) have pointed out that UA can function as an antioxidant , and that at earlier times in evolution , this may have been beneficial to human populations , in fact leading to the inactivation of uricase . Consistent with a possible need for UA in past generations , we found that the observed allele associated with higher levels of UA , AA , is more common on a population level . Allele GG , which is associated with lower levels , occurs in only 6 . 9% of the Sardinian and 6 . 6 % of the InCHIANTI population sample ( Table S4 gives the absolute numbers ) . In those individuals with intermediate levels of UA , it remains unclear whether it functions simply as an antioxidant that tends to increase in conditions characterized by tissue damage or whether it has a deleterious effect; and at very high levels UA is patently pathological . However , in individuals with very high levels , UA is patently pathological and mortality increases [3] . In such individuals , the G allele may be partially protective , and intriguingly , in the InCHIANTI study , participants with at least one G allele had a significantly lower prevalence of hyperuricemia ( defined as UA >7 . 5 mg/dl in men and >6 . 2 mg/dl in women ) compared to those with only A alleles . It would be of interest to verify whether variants of associated SNPs in the GLUT9 gene are less represented in particular groups of individuals with different clinical forms of hyperuricemia and in sporadic gout patients . Such effects would support the suggestion that GLUT9 may eventually provide a target for clinical intervention .
We recruited and phenotyped 6 , 148 individuals , male and female , 14–102 y old , from a cluster of four towns in the Lanusei Valley [13] . Blood samples were collected in the morning after the participants had been fasting for at least 12 h and after sitting for 15 min , both for SardiNIA and InCHIANTI cohorts . Aliquots of serum were immediately obtained and stored at −80 °C , and were subsequently used for the assessment of UA . UA ( mg/dl ) was measured using enzymatic–colorimetric methods ( Bayer for SardiNIA; Roche Diagnostics for INCHIANTI ) . The lower limits of detection were 0 . 2 mg/dl , range 0 . 2–25 . 0 mg/dl , intra-assay and inter-assay coefficients of variation were equal to 0 . 5% and 1 . 7% , respectively . Hyperuricemia was defined as a serum urate concentration >7 . 5 mg/dl ( 450 μmol/l ) in men and >6 . 2 mg/dL ( 372 μmol/l ) in women , in agreement with clinical laboratory standards . In the Sardinian cohort , 3 , 329 and 1 , 412 individuals were genotyped with the Affymetrix 10K and Affymetrix 500K Mapping array set , respectively , with 436 individuals generating an overlapping dataset . We took advantage of the relatedness among individuals in our sample to reduce study costs . Using a modified Lander-Green algorithm , full genotypes on the 2 , 893 individuals typed with only the 10K panel were imputed based on stretches of shared haplotype , permitting analyses on 4 , 305 individuals [20] . For genotyping for replication in the InCHIANTI cohort , allelic discrimination assays with predeveloped assay reagents ( Applied Biosystems ) for SNPs rs6855911 and rs7442295 were carried out using an ABI 7900HT sequencer detector system . Each 10 μl reaction volume was prepared according to manufacturer's directions , and contained 5 μl of twice-concentrated TaqMan Universal PCR master mix , 0 . 25 μl of 40-fold concentrated SNP genotyping assay mix with amplifying primers and oligonucleotide labeled with VIC or FAM reporter dyes for the corresponding alleles , 1 μl containing 5 ng of template DNA from an InCHIANTI subject , and 3 . 75 μl of water . Assays were run in 96-well format . Controls included DNA with sequence-verified homozygous alleles , DNA with heterozygous alleles , or no template DNA , in each assay plate . Following allele discrimination assays , a total of 14 random samples representing each allelic state were also checked by direct sequencing , confirming allele calls in all cases . Analyses in the Sardinia cohort were carried out as in [20] . We focused on 362 , 129 SNPs that passed quality control checks . We used a linear regression model to estimate the association between each SNP and UA levels . To avoid inflated type I error rates due to the departure from normality of measured UA levels , we applied an inverse normal transformation to the phenotype prior to analysis . We used an additive model and included sex , age , and age2 as covariates . For individuals who had genotype data available at the SNP being tested , we coded genotypes as 0 , 1 , or 2 , depending on the number of copies of an arbitrary reference allele for each SNP . For individuals with missing genotype data , we used the Lander-Green algorithm to estimate the number of copies of the allele carried by each individual ( based on the genotypes of family members ) and assigned each individual a score ranging between 0 and 2 [21] . This estimate incorporates allele frequency information , the genotypes of relatives for the SNP of interest , and flanking marker data . For computational efficiency , the Lander-Green algorithm was applied to sub-pedigrees , each including no more than 20–25 individuals , resulting in a dataset where the average analysis unit consisted of a family with 12 . 3 members and 3 . 2 generations . Finally , we used a variance component approach to account for correlation between different observed phenotypes within each family [21] . To evaluate association on the X chromosome , we modeled a polygenic variance component shared according to an X-linked kinship coefficient in addition to the usual autosomal polygenic variance component [13 , 21] . Further , we assumed that average phenotypic values for hemizygous males would be the same as for homozygous females [13 , 21] . Analysis for replication in the InCHIANTI population was carried out by again coding genotypes as 0 , 1 , or 2 and fitting a linear regression model adjusting for sex , age , and age2 . For the sequential analysis , the best SNP for each trait was selected first , and the analysis was conditioned on it to select successively the next best SNP . To carry out this conditioning , we simply added the best SNP as a covariate to the model and repeated the genome-wide association scan for all other SNPs . The same process was repeated in successive rounds . This sequential analysis can help identify regions with multiple independent association signals . After five rounds , additional SNPs identified had p-values that were far less significant . Oligonucleotide primers to amplify putative promoter regions and exons , including the splice sites for two isoforms of the GLUT9 gene , were designed based on the exon–intron borders represented in the University of California Santa Cruz ( UCSC ) genome assembly [22] using the primer prediction program Primer3 [23] interfaced with the UCSC genome browser . In the putative promoter regions , repeats were masked by Repeat Masker [24] , and only primers outside the repeats were selected . A list of primers used to amplify the exons is given in Table S2 . The exons were amplified by PCR in 25 μl reactions containing 0 . 2 mM dNTPs , final concentration 1 μM each of forward and reverse primers , 2 mM MgCl2 , and 50 ng of genomic DNA as template , with 1 unit recombinant Taq DNA polymerase ( Invitrogen ) . The cycling conditions were as follows: 95 °C 5 min , followed by 95 °C for 30 sec , 60 °C for 30 sec , and 72 °C for 30 sec for 30 cycles . The PCR products were purified by using ExoSAP-IT ( USB Corporation ) and were sequenced bidirectionally using the ABI PRISM BigDye Terminator v3 . 1 Cycle Sequencing Kit according to the manufacturer's recommendations , on an ABI PRISM 3100 Sequencer , and visualized with DNA Genetic Analyzer software ( ABI PRISM 3100 Genetic Analyzer ) .
The National Center for Biotechnology Information ( NCBI ) Online Mendelian Inheritance in Man ( OMIM , http://www . ncbi . nlm . nih . gov/entrez/dispomim . cgi ? id=232200 ) accession number for Glycogen storage disease Ia , simulating primary gout and xanthomatosis , is MIM 232200 . The NCBI Entrez database ( http://www . ncbi . nlm . nih . gov/sites/gquery ) accession number for Solute carrier family 2 , member 9 protein isoform I , is NP 064425 . | High serum uric acid levels lead to gout and increase the risk of cardiovascular and kidney disease . To determine what genetic factors might contribute to uric acid levels , we conducted genome-wide scans of single nucleotide variations in DNA in population samples from Sardinia and Chianti . We report here that variants in the GLUT9 gene are associated with altered uric acid levels in both populations . Unexpectedly , rather than being directly involved in uric acid synthesis or secretion , the GLUT9 gene encodes a glucose transporter . It is of interest that the gene is predominantly expressed in liver , a major site of uric acid synthesis , and in kidney , where uric acid is excreted and reabsorbed . However , it now remains to be determined how altered glucose uptake can indirectly affect synthesis or excretion/reabsorption of uric acid , and whether GLUT9 may provide a target for the therapeutic modification of uric acid levels . | [
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"genomics"
] | 2007 | The GLUT9 Gene Is Associated with Serum Uric Acid Levels in Sardinia and Chianti Cohorts |
Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals . One critical virulence attribute is its morphogenetic plasticity . Hyphal development requires two temporally linked changes in promoter chromatin , which is sequentially regulated by temporarily clearing the transcription inhibitor Nrg1 upon activation of the cAMP/PKA pathway and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling . Molecular mechanisms for the temporal connection and the link to Tor1 signaling are not clear . Here , through a forward genetic screen , we report the identification of the GATA family transcription factor Brg1 as the factor that recruits Hda1 to promoters of hypha-specific genes during hyphal elongation . BRG1 expression requires both the removal of Nrg1 and a sub-growth inhibitory level of rapamycin; therefore , it is a sensitive readout of Tor1 signaling . Interestingly , promoters of hypha-specific genes are not accessible to Brg1 in yeast cells . Furthermore , ectopic expression of Brg1 cannot induce hyphae , but can sustain hyphal development . Nucleosome mapping of a hypha-specific promoter shows that Nrg1 binding sites are in nucleosome free regions in yeast cells , whereas Brg1 binding sites are occupied by nucleosomes . Nucleosome disassembly during hyphal initiation exposes the binding sites for both regulators . During hyphal elongation , Brg1-mediated Hda1 recruitment causes nucleosome repositioning and occlusion of Nrg1 binding sites . We suggest that nucleosome repositioning is the underlying mechanism for the yeast-hyphal transition . The hypha-specific regulator Ume6 is a key downstream target of Brg1 and functions after Brg1 as a built-in positive feedback regulator of the hyphal transcriptional program to sustain hyphal development . With the levels of Nrg1 and Brg1 dynamically and sensitively controlled by the two major cellular growth pathways , temporal changes in nucleosome positioning during the yeast-to-hypha transition provide a mechanism for signal integration and cell fate specification . This mechanism is likely used broadly in development .
Candida albicans is a major opportunistic fungal pathogen of humans [1] , [2] . In most healthy individuals C . albicans exists as a harmless commensal in the oral cavity and the gastrointestinal and urogenital tracts . But the fungus can cause mucosal infections and systemic disease in immunocompromised people . One critical virulence attribute of C . albicans is its ability to undergo hyphal development in response to environmental cues . Mutants that are defective in hyphal formation display much reduced virulence in animal models of systemic candidiasis [3] , [4] . Hyphal morphogenesis is coupled with virulence as genes that control hyphal morphology are co-regulated with genes encoding virulence factors such as proteases and adhesins [4] . For example , HWP1 , ALS3 , and RBT5 encode cell wall proteins that are important for adhesion to host cells and iron acquisition from the host [5]–[8] . The transcription factor Ume6 , specifically expressed during hyphal development , controls the level and duration of hypha-specific genes and is important for hyphal elongation [9]–[11] . Hyphal morphogenesis and cell chain formation are under the control of another hypha-specific gene that encodes the G1 cyclin-related protein Hgc1 [12]–[16] . Hyphal development is regulated by multiple signal transduction pathways . Among them , the cAMP-dependent protein kinase A ( PKA ) pathway is essential for morphogenesis and virulence [17] . We recently showed that activation of the hyphal transcriptional program involves two phases of temporal dynamic changes in promoter chromatin [18] . Initiation requires a rapid but temporary disappearance of Nrg1 , a major repressor of hyphal morphogenesis [19] , [20] , via activation of the cAMP-PKA pathway [18] . Nrg1 disappearance from the promoters of hypha-specific genes is correlated with dissociation of the Rpd3 histone deacetylase from the promoters , increase in H4 acetylation , nucleosome disassembly , and transcriptional activation . Maintenance requires the recruitment of the Hda1 histone deacetylase to promoters under reduced Tor1 ( target of rapamycin ) signalling [18] . Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module , leading to eviction of the NuA4 acetyltransferase module and chromatin remodelling that blocks Nrg1 access to the promoters of hypha-specific genes . Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone . Such temporally linked regulation of promoter chromatin provides a unique mechanism for integrating multiple signals in the regulation of gene expression and phenotypic plasticity during hyphal development . C . albicans contains a conserved Tor1 protein kinase [21] , [22] . Gln3 and Gat1 , two GATA family transcription activators for genes that are subject to nitrogen catabolite repression , are targets of the TOR kinases in Saccharomyces cerevisiae [23] . Their homologs in C . albicans [24] , [25] , however , are not important for hyphal maintenance . The transcription factor that recruits Hda1 to the promoters of hypha-specific genes has not been identified . Furthermore , the molecular mechanism for the temporal connection between hyphal initiation and elongation is not clear . Here , we report , through a forward genetic screen the identification of the GATA family transcription factor Gat2 , recently found as a biofilm regulator Brg1 [26] , as the one that recruits Hda1 to promoters of hypha-specific genes . BRG1 expression is a sensitive readout of Tor1 signalling level in C . albicans , and Brg1 expression during hyphal elongation leads to nucleosome repositioning . Different nucleosome positions at the promoters of hypha-specific genes lead to different accessibility of Nrg1 and Brg1 to their DNA-binding sites at the promoters . This provides molecular insights to the sequential and integrative nature of promoter chromatin regulation . We further show that Brg1 functions before Ume6 in a feed-forward loop that regulates the yeast-to-hypha transition in C . albicans .
To uncover how rapamycin regulates hyphal elongation , we used a forward genetic screen to identify transcription factors responsible for recruitment of Hda1 under reduced Tor1 signaling . Based on the phenotype of the hda1 mutant , we expected the mutants of interest to be capable of germ-tube formation , but defective in sustained hyphal growth . Furthermore , they were expected to block rapamycin-mediated hyphal elongation . Mutants defective in hyphal elongation , but with defects which could be suppressed by rapamycin , were not studied further as their gene products were expected to act upstream or parallel to Tor1 signaling . We screened a knockout library of 165 transcription factor genes in C . albicans [27] for mutants defective in hyphal elongation in the presence of rapamycin . Only 8 mutants showed defects to varying extents in hyphal elongation in the presence of rapamycin , and could form germ tubes during hyphal initiation ( Table 1 ) . Among the 8 mutants , we found that promoter recruitment of Hda1 in the presence of rapamycin was dramatically reduced in brg1 , rob1 , and ahr1 mutants compared to the wild-type strain by chromatin immunoprecipitation ( ChIP ) ( Figure 1A ) , and Hda1 protein levels were not reduced in those mutants ( data not shown ) . Because promoter recruitment of Hda1 could only be established within the time window of reduced Nrg1 [18] , any defects in Nrg1 down-regulation would also block Hda1 recruitment . To exclude this possibility , a Western of Nrg1-Myc was performed in these three mutants . Nrg1 protein level decreased sharply in the brg1 mutant at 30 min upon hyphal induction as in the wild-type strain . But no obvious change in Nrg1 level was observed in the rob1 or ahr1 mutant ( Figure 1B ) . This is consistent with the efficient germ tube formation ( ∼90% ) observed in brg1 cells ( Figure 1C ) . The brg1 mutant showed a similar defect in hyphal elongation and Hda1 promoter recruitment in response to serum ( Figure S1 ) . The Brg1-dependent Hda1 recruitment to the UAS region of HWP1 is shown in Figure 1A , and to the UAS regions of ALS3 and ECE1 shown in Figure S2 . Therefore , Brg1 is the only transcription factor specifically required for the recruitment of Hda1 to hypha-specific promoters , but not involved in Nrg1 down-regulation during hyphal initiation . Previous genome-wide mutant screens have identified Brg1 as a general positive regulator of morphogenesis [27] and a brg1 mutant carrying a transposon insertion proximal to the ORF was found to be hyperfilamentous [28] . Our results provide a molecular function of Brg1 in filamentous growth . C . albicans Brg1 is most homologous in protein sequence to S . cerevisiae Gat2 by BLAST analysis . Both proteins contain a GATA family zinc finger motif most similar to Gat3 , Gat4 , Gat1 and Gln3 of S . cerevisiae . Functions and regulations of Gat2 , Gat3 , and Gat4 in S . cerevisiae are not well studied . Gln3 and Gat1 are phosphorylated by the TOR kinases [23] , and sequestered in the cytoplasm . In response to rapamycin treatment , Gln3 and Gat1 increasingly accumulate in the nucleus , where they bind to DNA and activate transcription of nitrogen catabolite repression-sensitive genes [29] . To examine if Brg1 accumulates in the nucleus in response to reduced Tor1 signaling , we localized Brg1 by indirect immunofluorescence . Brg1 was tagged at its N terminus with 13 Myc , and its expression was under the control of the MAL2 promoter . Unlike Gln3 and Gat1 , Myc-Brg1 displayed nuclear localization under all conditions with or without rapamycin ( Figure S3 ) . To determine if Brg1 interacts with Hda1 , Hda1 was fused at its C-terminus with a TAP tag of two copies of protein A sequence followed by calmodulin binding protein [30] . Using strains carrying the Myc-Brg1 under the MAL2 promoter , we found that immunoprecipitation of Hda1 with IgG beads was able to pull down Myc-Brg1 ( Figure 2A ) . The interaction was not regulated by growth forms , and was independent of rapamycin and temperature ( Figure 2A ) . Since Brg1 is the transcription factor required for the promoter recruitment of Hda1 and it interacts with Hda1 in vivo , we anticipated that Brg1 directly binds to the promoters of hypha-specific genes . We tagged Brg1 at the C-terminus with 13 Myc under its endogenous promoter for Brg1 ChIP experiments and performed a time course ChIP during hyphal induction . Brg1-myc was not detected at the promoters of hypha-specific genes in yeast cells from the starting culture . During hyphal growth at 37°C , Brg1-myc became associated with the UAS regions of the hypha-specific promoters in a rapamycin-dependent manner ( Figure 2B ) , very similar to rapamycin-dependent promoter recruitment of Hda1 [18] . Our results suggest that Brg1 recruits Hda1 to hyphal gene promoters to sustain hyphal development . This Tor1-mediated regulation could be at the level of Brg1 expression , stability , and/or promoter binding . We next examined whether Brg1 protein levels changed in response to rapamycin . The protein level of Brg1-myc under its endogenous promoter was low , but detectable in cells from overnight cultures . It increased rapidly at 1 h upon hyphal induction at 37°C and stayed at a high level in rapamycin-containing medium during hyphal elongation , but returned to a low level in the absence of rapamycin ( Figure S4A ) , suggesting that reduced Tor1 signaling was required for a sustained high level of Brg1 expression . However , rapamycin alone was not sufficient for a high level of Brg1 , as Brg1 levels were low at 25°C even in the presence of rapamycin ( Figure S4A ) . Brg1 protein was unstable , but its stability was not regulated by temperature or rapamycin ( Figure S3B ) . This suggested that rapamycin-dependent accumulation of Brg1 protein likely reflected the transcriptional regulation of BRG1 by the Tor1 pathway . Similar to its protein levels , we found that BRG1 expression was activated by a shift in temperature to 37°C and maintained at a high level in the presence of rapamycin ( Figure 3A ) . In the absence of rapamycin , BRG1 expression level increased briefly when cells were inoculated to YPD at 37°C , but decreased afterwards ( Figure 3A ) . BRG1 expression level remained low at 25°C regardless of the presence of rapamycin . Since Nrg1 is temporarily cleared when cells are inoculated into fresh medium at 37°C [18] , a prerequisite condition for the induction in BRG1 expression , we examined whether the down-regulation of Nrg1 is essential for the activation of BRG1 expression . We found that ectopic expression of NRG1 under the MAL2 promoter completely blocked the induction of BRG1 in YPD medium with rapamycin at 37°C ( Figure S5 ) . Furthermore , rapamycin could induce BRG1 expression independent of temperature in an nrg1 mutant ( Figure 3B ) . These results suggest that Nrg1 removal is required for rapamycin-induced BRG1 expression during hyphal elongation . Unlike hyphal genes , BRG1 transcript or Brg1 protein is present in the starting yeast cell , although at a low level . Furthermore , removing Nrg1 inhibition is not sufficient for the induction of BRG1 expression , as BRG1 transcript levels at time 0 were similar between the nrg1 mutant and the wild-type strain ( Figure 3B vs . 3A . the value at time 0 in A was set to 1 and used for normalization in both A and B ) , and BRG1 cannot be induced in the nrg1 mutant in the absence of rapamycin ( Figure 3B ) . Therefore , relief of Nrg1 inhibition by the activation of the cAMP/PKA pathway and reduced Tor1 signaling are both required for BRG1 expression; neither one is sufficient . Similar to hypha-specific genes , we predicted that Nrg1 and Brg1 can bind to the BRG1 promoter , and Brg1 recruitment of Hda1 can remodel the promoter chromatin to block Nrg1 from binding onto the BRG1 promoter during hyphal elongation . Nrg1 has been reported to bind to [A/C][A/C/G]CCCT , CCCCT or CCCT/C containing sequences [20] and Brg1 binds to the DNA sequence [A/C]GGTA[C/A] [26] . We analyzed the intergenic region upstream of BRG1 ( ∼10 kb ) and found 3 regions of ∼250 bp long that contain binding motifs for both Nrg1 and Brg1 ( Figure S6 ) . The ChIP DNA of Nrg1-Myc at time 0 ( yeast cells ) and Brg1-Myc at 6 h in the presence of rapamycin at 37°C ( hyphal cells ) were analyzed by qPCR at these regions ( Figure S6 ) . Among them , the region −1959∼−1710 bp showed much higher ChIP signals for both Nrg1-Myc and Brg1-Myc than the other regions ( Figure S6 ) . This region was therefore considered as a potential UAS region for BRG1 , and was used for subsequent ChIP time course experiments . As expected , Nrg1 dissociated rapidly from the BRG1 promoter during hyphal initiation and remained unbound in a rapamycin-dependent manner; Brg1 bound to the promoter in the presence of rapamycin only during hyphal elongation ( Figure 3CD ) , similar to its temporal association with the promoters of hypha-specific genes . In strains carrying Brg1-myc under its endogenous promoter , we found that Brg1 binding to the promoters of hypha-specific genes and BRG1 is rapamycin-dependent ( Figures 2B and 3D ) . However , BRG1 expression also required rapamycin ( Figure 3AB ) , which could lead to the rapamycin-dependent promoter binding observed with Brg1-myc under its own promoter ( Figures 2B & 3D ) . Since BRG1 expression also requires the initial removal of Nrg1 and subsequent binding of Brg1 ( Figure 3B ) similar to hypha-specific promoters , we wanted to examine whether Brg1 DNA binding is under the control of Tor1 . Ectopically expressed Brg1-myc under the MAL2 promoter was used in ChIP experiments to circumvent the Tor1 effects on BRG1 expression . Unexpectedly , we found that Brg1 could bind to several promoters of hypha-specific genes at 37°C independent of rapamycin ( Figures 4A and S7 ) . Interestingly , Brg1 could not bind to its own promoter without rapamycin at 37°C ( Figure 4B ) . Nrg1 was not responsible for the Tor1-regulated Brg1 binding , as the association of Brg1 to its own promoter was still rapamycin dependent in the nrg1 mutant ( Figure 4B ) . Our data show that reduced Tor1 signaling is required for the positive feedback activation by Brg1 on its own promoter . Tor1 signaling does not regulate Brg1 nuclear localization , its interaction with Hda1 , or its binding to the UAS regions of hypha-specific genes . Nrg1 can bind to promoters of hypha-specific genes in yeast cells but not in hyphal cells [18] . Brg1 binds to the promoters in hyphal cells to sustain hyphal development through Hda1 recruitment ( Figure 2B ) . However , the lack of promoter-bound Brg1 in yeast cells could be due to low levels of Brg1 protein , as BRG1 expression is activated during hyphal elongation ( Figure 3 ) . To examine whether Brg1 can bind the promoters of hypha-specific genes in yeast cells , ChIP was performed in yeast cells carrying ectopically expressed Brg1 . As shown in Figure 4A , Brg1 could not bind to the UAS region at the HWP1 promoter in yeast cells at 25°C ( Figure 4A ) . In contrast , Brg1 could bind to the HWP1 promoter at 37°C ( Figure 4A ) , a condition where Nrg1 was temporarily removed [18] . Similarly , Brg1 could also bind to the UAS regions of ECE1 and ALS3 promoters at 37°C , but not at 25°C ( Figure S7 ) . Growth at 37°C was required to clear Nrg1 because Brg1 could bind to the promoters in the nrg1 mutant regardless of temperature ( Figure 4A ) . Therefore , promoters of hypha-specific genes are not accessible to Brg1 in yeast cells . Since reduced TOR signaling sustains hyphal growth through the activation of BRG1 expression , we examined whether overexpression of BRG1 could bypass the requirement of rapamycin in sustained hyphal development . Ectopic expression of BRG1 in a wild-type strain could sustain hyphal growth at 37°C independent of rapamycin ( Figure 4C ) . This functional assay further supports our conclusion that the Tor1 pathway does not directly regulate hypha-specific transcription . We also observed that overexpressing Brg1 at 25°C could not induce hyphal development ( Figure 4C ) , consistent with the result that Brg1 could not bind to promoters of hyphal genes and BRG1 in yeast phase ( Figure 4AB ) . Although ectopic expression of Brg1 did not induce robust hyphal development after 6 hours , a few cells became elongated . The low percentage of elongated cells from ectopic Brg1 expression in yeast condition was in contrast to its ability to sustain hyphal development in the absence of rapamycin . Therefore , Brg1's function is to sustain hyphal development and Tor1 signaling controls hyphal elongation through the regulation of BRG1 expression . Nucleosomes are generally inhibitory to transcription factor binding , and changes in nucleosome position are associated with changes in gene expression by affecting the accessibility of transcription factors to promoters [31] , [32] . We suspected that changes in nucleosome positioning during yeast-to-hypha development determined the differential promoter accessibility to Nrg1 and Brg1 in yeast and hyphal cells . To test this hypothesis , we mapped nucleosome positions around the UAS region of the HWP1 promoter with cells in yeast phase , during hyphal initiation , during hyphal elongation with and without rapamycin , or with ectopic expression of Brg1 ( Figure 5A ) . We observed a nucleosome free region ( NFR ) in the middle of the UAS region on the HWP1 promoter in yeast phase cells from the starting culture at 0 hours . After 0 . 5 hours of hyphal induction in YPD at 37°C , the two nucleosomes surrounding the NFR disappeared , leaving a large region without nucleosomes . Interestingly , the two nucleosomes stayed off in the hyphal cells after 4 hours of growth in YPD at 37°C , a condition in which BRG1 expression is low . During sustained hyphal growth in the presence of rapamycin for BRG1 expression , or when Brg1 was ectopically expressed , a new nucleosome appeared in the middle of the UAS region , leaving two NFRs around the nucleosome . Our nucleosome mapping experiments demonstrated changes in nucleosome positioning at the UAS region of the HWP1 promoter during yeast-to-hypha development: from two nucleosomes in yeast phase , to no nucleosomes in the initiation phase , to one nucleosome in the hyphal phase . The observed changes in nucleosome occupation at the UAS region are consistent with our previous findings . Removal of Nrg1 during hyphal induction leads to rapid histone disassembly on UAS regions of hyphal genes [18] , which correlates to the dissociation of the two nucleosomes around the NFR in the UAS of the HWP1 promoter . The formation of a new nucleosome during hyphal elongation requires Brg1 binding and the recruitment of Hda1 , consistent with the finding that Hda1 at the promoters leads to eviction of the NuA4 acetyltransferase module and chromatin remodeling [18] . DNA sequence analysis for potential Nrg1 and Brg1 binding motifs in the UAS region of the HWP1 promoter identified one Nrg1 binding site and one Brg1 binding site . Interestingly , the Nrg1 binding site is located in the NFR in yeast cells; therefore it should be accessible to Nrg1 binding . However , the Brg1 binding site is occupied by a nucleosome , making it not accessible to Brg1 ( Figure 5A , 0 hour ) . During sustained hyphal development , the Nrg1 binding site is occupied by a nucleosome and becomes inaccessible whereas the Brg1 binding site is exposed in NFRs ( Figure 5A , +Rapa or MAL2p-BRG1 ) . Both binding sites are accessible during the hyphal initiation phase ( Figure 5A , 0 . 5 hour ) . Our data suggest that changes in nucleosome positioning during the yeast-to-hypha development underlie the differences in promoter accessibility to Nrg1 and Brg1 . Consistent with the concept that nucleosomes are inhibitory to transcription factor binding , Nrg1 binding motifs are found in nucleosome free regions upstream of repressed genes by genome-wide nucleosome mapping of the yeast form of C . albicans [32] . Furthermore , DNA sequence analysis of the intergenic regions upstream of BRG1 , HWP1 , ALS3 and ECE1 identified many Brg1 and Nrg1 binding motifs , but only the UAS regions are bound by Nrg1 in yeast cells and by Brg1 in hyphal cells ( Figures S6 and S8ABC ) . Interestingly , all UAS regions of these promoters contain both Nrg1 and Brg1 binding motifs . In addition , Brg1 also binds to the promoters of two hypha-specific regulatory genes , UME6 and HGC1 , during hyphal elongation ( Figure S9AB ) . Consistent with other hyphal regulated genes , Brg1-bound regions on the two promoters contain both Nrg1 and Brg1 binding sites within the distance of a nucleosome , and regions with only Brg1 binding sequence are not bound by Brg1 ( Figure S9AB ) . All these support the model that dynamic changes in nucleosome positioning on promoters of hypha-specific genes during the yeast-to-hypha transition lead to different accessibilities of these promoters to Nrg1 and Brg1 . Our results infer that Brg1 mediates its effects by binding directly to its binding sites at nucleosome free regions on the promoters of many hypha-specific genes . To examine the functionality of the putative Brg1 binding site in the UAS region for HWP1 , we replaced the Brg1 binding sequence AGGTAA at −1130 to −1125 on the HWP1 promoter with CTAGCC . The mutated HWP1 promoter was introduced to a wild-type C . albicans strain , generating a strain that carries both the wild-type HWP1 promoter and a copy of the HWP1 promoter mutated at the putative Brg1 binding site . ChIP of Brg1 under a condition of hyphal elongation showed the mutated HWP1 UAS region was not associated with Brg1 in comparison to the wild type copy of the HWP1 promoter in the same cells ( Figure 5B ) . Therefore , the predicted Brg1 binding site in the UAS region ( −1130 to −1125 ) is directly responsible for Brg1 recruitment . Ume6 has been shown to control the level and duration of hypha-specific genes and is important for hyphal elongation [9]–[11] . To address the functional relationship between Ume6 and Hda1 recruitment by Brg1 in hyphal maintenance , we performed a time course ChIP experiment of Hda1-Myc in brg1 and ume6 mutants . Unlike Brg1 , Ume6 was not essential for promoter recruitment of Hda1 , although levels of promoter-associated Hda1 decreased in the ume6 mutant compared to that in the wild-type strain ( Figure 6A ) . Similar to other hypha-specific genes , UME6 expression is dependent on Brg1 and Hda1 ( Figure 6B ) . However , protein levels of Ume6 increased much slower and reached a much lower level during hyphal induction in the absence of rapamycin than expected ( Figure 6C ) . Unstable UME6 transcript or protein likely made Ume6 level sensitive to the level of UME6 expression and rendered Ume6 protein level more dependent on Brg1 function than other proteins specifically expressed in hyphal cells . We then examined whether overexpression of UME6 could bypass the requirement of Hda1 in hyphal elongation . As shown in Figure 6D , constitutively expressed Ume6 , but not Brg1 , restored the hyphal growth defect in hda1 mutant cells . This result strongly suggests that expressing UME6 is a major function of Brg1-mediated chromatin remodeling by Hda1 during hyphal elongation . We further showed that Ume6 binds directly to the UAS region of the HWP1 promoter ( Figure 6C ) . Interestingly , levels of promoter-bound Ume6 peaked later than Brg1 during hyphal induction ( Figure 6C vs . 2B ) . Based on our data , we suggest that Ume6 functions directly on the promoters of hypha-specific genes after Brg1 as a built-in positive feedback for sustained hyphal development .
Here we report the identification of Brg1 , by a forward genetic screen , as the transcription factor that recruits Hda1 to promoters of hypha-specific genes under reduced Tor1 signaling for sustained hyphal development . Many signaling pathways and transcription factors play a role in hyphal morphogenesis in C . albicans . This is evident from the vast literature on this topic and number of genes implicated in hyphal morphogenesis [33] . In addition , large-scale mutant screens have also identified many regulators of hyphal morphogenesis including Brg1 [27] , [28] . A challenge is how to dissect the complicated regulatory network and understand the underlying molecular mechanisms for each regulator or pathway in hyphal development . Our recent study suggests that hyphal development undergoes two phases of regulation in promoter chromatin: initiation and elongation [18] . Nrg1 is temporally removed upon activation of the cAMP pathway during initiation; Hda1 is recruited to the promoters of hypha-specific genes under reduced Tor1 signaling during elongation . With this as our framework , we find that , among 165 putative transcription factor mutants , 7 mutants are defective in germ tube formation , and 47 mutants in hyphal elongation to different extents . Among the 47 mutants , 8 are still defective in the presence of rapamycin . Some of the 8 regulators may act downstream of Tor1 signaling . Out of the 8 mutants , we further narrowed it down to brg1 as the only mutant defective in the promoter recruitment of Hda1 . Consistent with the mutant phenotype , we find that Brg1 interacts with Hda1 . Furthermore , it binds to the promoters of hypha-specific genes during hyphal elongation in a rapamycin-dependent manner . Therefore , by separating defects in hyphal initiation vs . elongation , and by examining whether rapamycin could bypass the defects , as well as by using Nrg1 protein level and Hda1 promoter recruitment as readouts , we are able to identify one , from many mutants defective in hyphal development , that is specifically involved in Hda1 promoter recruitment . We also find that ahr1 and rob1 mutants are partially defective in hyphal development due to impaired down-regulation of Nrg1 . Ahr1 has been previously shown to function in hyphal development [27] , [34] . It is a zinc-finger transcription factor that recruits Mcm1 to promoters of genes involved in biofilm formation , filamentous growth , etc . including EFG1 and NRG1 [34] . Based on our data and Ahr1 target genes [34] , Ahr1 likely affects hyphal development through the regulation of EFG1 and/or NRG1 expression . Our forward genetic screening demonstrates that this scheme of mutant analyses can provide a framework for further characterization of other mutants defective in hyphal development . BRG1 expression is a sensitive output of Tor1 signaling in C . albicans . Sustained hyphal development requires only a sub-growth inhibitory level of 5–10 nM rapamycin [18] . This is 10-fold lower than the concentration required for nuclear localization of the GATA transcription factors Gln3 and Gat1 in S . cerevisiae [23] . Similarly , we have observed nuclear localization of Gln3 in C . albicans only in the presence of 100 nM rapamycin , but not at 10 nM ( our unpublished data ) . In contrast to Gln3 , Brg1 nuclear localization is not regulated by rapamycin . Instead , BRG1 expression requires the presence of 5–10 nM rapamycin , making it a sensitive readout of Tor1 signaling . In addition to reduced Tor1 signaling , activation of BRG1 expression also requires the removal of Nrg1 inhibition . This is different from hypha-specific genes where lack of Nrg1 is sufficient for their expression . Therefore , BRG1 expression uses an AND gate logic gating for signal integration . This type of logic gating is also seen in other GATA factor regulated gene expression . For example , the expression of nitrate and nitrite reductase in Aspergillus nidulans requires both the presence of nitrate and the absence of ammonium and glutamine [35] . Nrg1 and Brg1 act directly on the BRG1 promoter . Similar to hyphal specific promoters , Nrg1 dissociates from the BRG1 promoter during hyphal initiation and subsequently , Brg1 binds to the promoters during hyphal elongation in a rapamycin-dependent manner . Brg1 protein is not a direct target of the Tor1 pathway because Brg1 association with hypha-specific promoters is independent of rapamycin and ectopic expression of Brg1 can sustain hyphal elongation in the absence of rapamycin . Therefore , Tor1 function in hyphal development is through the transcriptional regulation of BRG1 . Experiments are underway to identify the additional regulators of BRG1 expression . The position of nucleosomes in a gene promoter impacts the accessibility of transcription factors to their DNA-binding sites . Ectopically expressed Brg1 binds promoters of hypha-specific genes only in hyphal cells , but not in yeast cells . Conversely , Nrg1 binds to the promoters in yeast cells , but not in hyphal cells . By mapping nucleosome positions during yeast-to-hypha development at the UAS region of the HWP1 promoter , we find dynamic changes in nucleosome positioning at the UAS region . The observed change in nucleosome positioning provides a mechanism for differential accessibility to Nrg1 and Brg1 in yeast and hyphal cells . Other hypha-specific promoters , including UME6 and HGC1 as well as BRG1 , likely have different nucleosome positioning in yeast and hyphae similar to HWP1 as differential Brg1 and Nrg1 binding is observed for all hypha-specific promoters examined . Furthermore , Brg1 bound regions on the examined promoters all have both Brg1 and Nrg1 binding sites within the distance of one nucleosome . We propose that hyphal development undergoes sequential changes in nucleosome positioning from the yeast state , to a transition state , and then to the hyphal state ( Figure 7 ) . In the yeast state Nrg1 binding sites are in a nucleosome free region and accessible , whereas Brg1 bindings sites are occupied by nucleosomes and therefore not accessible . Upon activation of the cAMP/PKA pathway when cells are inoculated into fresh media at 37°C , Nrg1 is temporarily removed , leading to rapid nucleosome disassembly at the UAS regions [18] . During the transition state , binding sites for both Nrg1 and Brg1 are accessible , providing a time window for Brg1 accumulation and binding to promoters before Nrg1 returns . In the hyphal state , nucleosomes reposition through Brg1-mediated Hda1 recruitment and chromatin remodeling , thus the Nrg1 binding sites are not accessible . The precise positions of Brg1 binding sites relative to nucleosomes in the hyphal state may vary among the promoters as they differ in levels or extent of expression during hyphal development . This effect of nucleosome positions on transcription factor binding and transcription induction is convincingly demonstrated by the Pho5 system in S . cerevisiae [36] . On the other hand , nucleosome occupancy is dynamic and is determined by a balance of assembly and disassembly activities . When highly expressed , Brg1 can compete with nucleosomes and convert some cells from the yeast to hyphal state . This is a slow and occasional event , consistent with the existence of a few elongated cells in a culture of mostly yeast cells when Brg1 is expressed from the MAL2 promoter under yeast growth conditions . A change in nucleosome positioning during yeast-to-hypha development provides a molecular mechanism for temporally linked regulation of promoter chromatin . It also provides a mechanism for plasticity in hyphal development and explains cell-to-cell variation among a cell population , which is important for adaptation and survival of unicellular microorganisms in stress conditions . The function of Brg1 in hyphal development is to recruit Hda1 to the UAS regions of hypha-specific promoters . Hda1 in turn remodels promoter chromatin by deacetylating a subunit of the NuA4 histone acetyltransferase module , leading to the occlusion of Nrg1 binding sites [18] . We have also shown that hyphal induction either in the presence of rapamycin , a condition for BRG1 expression or with the ectopic expression of Brg1 , leads to nucleosome repositioning . This function of Brg1 in chromatin remodeling in likely common as other GATA factors have also been shown to play a role in nucleosome rearrangements [35] , [37]–[39] . The ultimate function of Brg1 in chromatin rearrangement during hyphal development is to remove Nrg1 inhibition . This provides another layer of Nrg1 regulation , in addition to regulating its expression and stability . The additional layer of regulation provides C . albicans with a flexible response to nutrition and various growth environments . One key downstream target of the Brg1-mediated chromatin regulation is UME6 . Brg1 binds upstream of UME6 , and UME6 expression depends on Brg1 and Hda1 . Furthermore , ectopic expression of Ume6 completely bypassed the requirement of Brg1 or Hda1 . In this sense , hypha-specific expression of some genes could be regulated indirectly by Brg1 through the control of the level of Ume6 . Our results demonstrate that Ume6 functions after Brg1 and Hda1 as a built-in positive feedback regulation of the hyphal transcriptional program to sustain hyphal development , consistent with its reported functions [9]–[11] . We suggest that the yeast-to-hypha transition is controlled by a feed-forward loop of regulations by Nrg1 , Brg1 , and Ume6 . This temporal regulatory model is in agreement with delayed hyphal development by ectopic expression of Ume6 in yeast growth conditions [10] . Brg1 plays a critical function as a mid-regulator in this feed-forward loop . It provides C . albicans with a means to sense and integrate multiple nutritional and environmental signals for control of its morphological program that is essential for its pathogenesis . Considering that misregulation of either Nrg1 or Ume6 level causes altered virulence [9] , [40] , our identification of Brg1 as an output of the Tor1 signaling that regulates Nrg1 binding and Ume6 expression should provide a better understanding of the molecular mechanisms that control the yeast-hyphal development in its hosts , and how C . albicans senses and integrates multiple nutritional and environmental signals in the host for its adaption to exist as both a commensal and pathogen .
165 putative transcription factor mutants [27] and the wild-type reference strain SN250 were grown overnight in liquid YPD at 30°C . 15 mutants grew as elongated pseudohyphae and they were excluded from further analysis . The remaining 150 mutants and wild type cells were diluted at 1∶100 to YPD+10% Serum at 37°C for 1 h for a germ tube formation screen . 14 mutants were defective in germ tube formation . Among them , 7 completely blocked germ tube formation and were excluded . For mutants defective hyphal elongation , the 143 remaining mutants were grown in Lee's medium for 6 h at 37°C , and 47 mutants were defective in hyphal elongation . Among these 47 mutants , 39 mutants could be restored in hyphal elongation by rapamycin in YPD at 37°C . Only 8 transcription factor mutants were defective in response to rapamycin induced hyphal maintenance . The C . albicans strains used in this study are listed in Table S1 . Primer sequences are listed in Table S2 . The 8 transcription mutants defective in rapamycin induced hyphal elongation and UZ43 [11] were streaked on 5-fluoro-orotic acid-containing medium to generate Ura– strains for these mutants . pPR673-HDA1 was digested with BamHI to target the integration of the plasmid into the HDA1 locus to express Hda1-13Myc in these mutants . A 1 . 2-kb PCR product ( primers 1 and 2 ) containing the C-terminal BRG1 coding region was inserted into the BamHI-MluI sites of pPR671 and pPR673 [30] . The resulting plasmids were digested with SpeI for target integration into the BRG1 loci to express Brg1-13Myc . The pMAL2-MYC-BRG1 plasmid was constructed by amplifying BRG1 ( primers 3 and 4 ) to replace HGC1 from the pMAL2-MYC-HGC1 plasmid [41] . The resulting plasmid was digested with AscI to target integration into the ADE2 locus to express Brg1-13Myc . A 1 . 3-kb PCR product ( primers 5 and 6 ) containing the C-terminal HDA1 coding region was digested with BglII and MluI to replace CPH1 from the pACT1-CPH1-TAP plasmid ( our unpublished data ) . The resulting plasmid was digested with BamHI to target the integration of the plasmid into the HDA1 locus in HLY3636 to express Hda1-TAP . The pMAL2-UME6-13MYC plasmid was constructed by amplifying UME6 ( primers 7 and 8 ) to replace NRG1 from the pMAL2-NRG1-MYC plasmid . The resulting plasmids were digested with AscI for target integration into the ADE2 locus in HLY4032 to express Ume6-13Myc . To construct a mutant HWP1 promoter with a site-specific mutation at the predicted Brg1 binding site in the UAS region , the AGGTAA sequence at −1130 to −1125 upstream of HWP1 was replaced by CTAGCC . Two pairs of primers ( primers 17 and 18 , 19 and 20 ) were used to PCR amplify overlapping HWP1 promoter fragments with the mutation in the overlapping region . The resulting PCR products were purified and mixed as templates for another round of PCR amplification using the primers 17 and 20 , which produced the full-length mutated HWP1 promoter sequence . The resulting PCR product was cloned into the KpnI-ClaI site of the plasmid pHL471 [42] to express GFP , and was confirmed by DNA sequencing . The resulting plasmid was digested with BglII within the HWP1 promoter region ( from −1465 to −1460 ) for integration into the endogenous HWP1 promoter locus . Primers 21 and 22 were used to quantitate the binding of Brg1 to the wild type HWP1 promoter , and primers 21 and 23 were used to quantitate the binding of Brg1 to the mutated HWP1 promoter . Chromatin immunoprecipitation was performed as described with modifications [30] . DNA was sheared by sonication six times for 20 seconds at high power on a Bioruptor ( diagenode ) with 40 second intervals on ice . 10 µl of anti-Myc ( SC-789 , Santa Cruz ) antibodies were used for ∼4 mg of chromatin proteins in an immunoprecipitation volume of 200 µl . Protein extraction and immunoprecipitation were performed as described previously ( Cao et al . , 2006 ) with modifications . Protein extract containing 10 mg protein were subjected to immunoprecipitation using 60 µl of immunoglobulin G ( IgG ) agarose bead slurry ( Sigma ) , which was preincubated once with 0 . 2 mg/ml sheared salmon sperm DNA , 0 . 5 mg/ml bovine serum albumin in phosphate-buffered saline . Proteins were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane ( Hybond; GE Healthcare ) . After blocking in 3% skim-milk powder in Tris-buffered saline/0 . 05% Tween 20 , a peroxidase-conjugated anti-c-Myc antibody ( Roche ) was used to probe for Myc-tagged proteins , which were then detected using the enhanced chemiluminescence system ( Pierce Chemical . Rockford , IL ) . Methods for RNA isolation were carried out as previously described [43] . 10 µg of total RNA was DNase-treated at 37°C for 1 h using the DNase-free kit ( Qiagen ) , cDNA was synthesized using the SuperScript II Reverse Transcriptase kit ( Invitrogen ) , and qPCR was done using the iQ SYBR Green Supermix ( Bio-Rad ) using the primers 9 and 10 for BRG1 , primers 11 and 12 for ACT1 and primers 13 and 14 for UME6 . For the MNase assay , we used the protocol described as previously described [44] , [45] . In brief , we harvested 5×107 cells of each sample , and washed in 1 ml water . Then we resuspended the cells in 1 ml of sphaeroplasting solution ( 1 M sorbitol , 0 . 5 mM 2-mercaptoethanol , 0 . 18 mg/ml zymolyase ) , and incubated at room temperature for 5 min with gentle stirring . We harvested the cells , washed it in 1 ml of 1 M sorbitol , then resuspended the pellet in 500 µl of digestion buffer ( 1 M sorbitol , 50 mM NaCl , 100 mM Tris-Cl [pH 7 . 4] , 5 mM MgCl2 , 1 mM CaCl2 , 1 mM 2-mercaptoethanol , 0 . 5 mM spermidine , 0 . 075% NP-40 , micrococcal nuclease with a final concentration 1–10 U/ml ) for 8 min at 37°C . After terminating the MNase digestion by adding 50 µl quench buffer ( 250 mM EDTA , 5% SDS ) , DNA was extracted with phenol/chloroform , and then we proceeded with the qPCR analysis . The PCR product was designed to be ∼100 bp , and the neighboring PCR primers are ∼40 bp apart . In the nucleosome map , the x-axis represents the midpoint of the PCR product . The signal of each primer pair obtained from genomic DNA from each sample was used for normalization . The values of nucleosome occupancy at −5188 on the WOR1 promoter , which is not regulated by the yeast-hypha transition , were set to be 1 . 00 . | Candida is part of the gut microflora in healthy individuals , but can disseminate and cause systemic disease when the host's immune system is suppressed . Its ability to grow as yeast and hyphae in response to environmental cues is a major virulence attribute . Hyphal development requires temporary clearing of the transcription inhibitor Nrg1 upon activation of cAMP/PKA for initiation and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling for maintenance . Here , we show that , during hyphal initiation when Nrg1 is gone , expression of the GATA family transcription factor Brg1 is activated under reduced Tor1 signaling . Accumulated Brg1 recruits Hda1 to hyphal promoters to reposition nucleosomes , leading to obstruction of Nrg1 binding sites and sustained hyphal development . The nucleosome repositioning during the yeast-hyphal transition provides a mechanism for temporal integration of extracellular signals and cell-fate specification . The hypha-specific transcription factor Ume6 functions after Brg1 in this succession of feed-forward regulation of hyphal development . Since misregulation of either Nrg1 or Ume6 causes altered virulence , and Brg1 regulates both Nrg1 accessibility and Ume6 transcription , our findings should provide a better understanding of how Candida controls its morphological program in different host niches to exist as a commensal and a pathogen . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"medicine",
"biology"
] | 2012 | A GATA Transcription Factor Recruits Hda1 in Response to Reduced Tor1 Signaling to Establish a Hyphal Chromatin State in Candida albicans |
The mechanisms controlling cell fate determination and reprogramming are fundamental for development . A profound reprogramming , allowing the production of pluripotent cells in early embryos , takes place during the oocyte-to-embryo transition . To understand how the oocyte reprogramming potential is controlled , we sought Caenorhabditis elegans mutants in which embryonic transcription is initiated precociously in germ cells . This screen identified LIN-41 , a TRIM-NHL protein and a component of the somatic heterochronic pathway , as a temporal regulator of pluripotency in the germline . We found that LIN-41 is expressed in the cytoplasm of developing oocytes , which , in lin-41 mutants , acquire pluripotent characteristics of embryonic cells and form teratomas . To understand LIN-41 function in the germline , we conducted structure-function studies . In contrast to other TRIM-NHL proteins , we found that LIN-41 is unlikely to function as an E3 ubiquitin ligase . Similar to other TRIM-NHL proteins , the somatic function of LIN-41 is thought to involve mRNA regulation . Surprisingly , we found that mutations predicted to disrupt the association of LIN-41 with mRNA , which otherwise compromise LIN-41 function in the heterochronic pathway in the soma , have only minor effects in the germline . Similarly , LIN-41-mediated repression of a key somatic mRNA target is dispensable for the germline function . Thus , LIN-41 appears to function in the germline and the soma via different molecular mechanisms . These studies provide the first insight into the mechanism inhibiting the onset of embryonic differentiation in developing oocytes , which is required to ensure a successful transition between generations .
There is a special relationship between germ cells and pluripotency , i . e , . the ability to adopt alternative cell fates . First , germ cells transmit the pluripotent potential to recreate all types of cells in a new individual . Second , germ cells give rise to pluripotent cell lines such as embryonic germ or carcinoma cells and oocyte cytoplasm has the capacity to reprogram somatic nuclei [1] , [2] . Finally , in disease , germ cells can abnormally differentiate into diverse somatic cell types , forming teratomas . However , during normal development , the ability to differentiate into all three embryonic germ layers is restricted to the cells of the early embryo . Combined , these observations suggest that the reprogramming potential of germ cells is kept at bay by repressive mechanisms . Depletion of several chromatin modifiers , either alone or combined with an ectopic overexpression of somatic cell fate-specifying transcription factors , can induce reprogramming of C . elegans germ cells into somatic cells [3]–[5] . The loss of these factors appears to primarily impact proliferating ( pre-meiotic ) germ cells and affects chromatin-based regulation . In contrast , our previous work in the same animal demonstrated that a conserved RNA-binding protein , GLD-1/Quaking , prevents teratomatous differentiation of post-mitotic germ cells [6] , [7] . Importantly , in gld-1 mutants , the germline-to-soma transition is accompanied by a precocious onset of embryonic ( or zygotic ) genome activation ( EGA ) , suggesting a causal connection between EGA and pluripotency . In other animals , the connection between EGA and pluripotency has been also postulated based on the temporal correlation between EGA and the acquisition of a pluripotent chromatin landscape [8] , [9] . These observations prompted us to examine whether new regulators of pluripotency can be identified based on a precocious onset of EGA in the germline . Here , we report the discovery of one such novel regulator of pluripotency , LIN-41/TRIM71 . LIN-41 belongs to the TRIM-NHL protein family [10] . These proteins contain a TRIpartite Motif ( TRIM ) consisting of a RING finger domain ( commonly endowing a protein with E3 ubiquitin ligase activity , for example [11]–[13] ) , two B-Box motifs and a coiled-coil domain . Additionally , they also carry six so-called NHL repeats ( named after NCL-1 , HT2A and LIN-41 ) and may contain a filamin domain , which have been implicated in both protein-protein and protein-RNA interactions [13]–[17] . Consistently , different molecular functions have been attributed to LIN-41-like proteins , but many questions remain open; for example , it is not clear whether all the domains function together and/or are used in a tissue context-dependent manner [11] , [14] , [18]–[20] . The TRIM-NHL family includes well-known regulators of self-renewal and differentiation . For example , in Drosophila melanogaster , Brat inhibits neuroblast self-renewal , cell growth and ribosome synthesis in the larval brain [21]–[24] and Mei-P26 restricts growth and proliferation in the ovarian stem cell lineage [25] . Defects in TRIM-NHL proteins have also been associated with human pathologies , for example TRIM32 has been implicated in the Bardet–Biedl Syndrome and the Limb-Girdle Muscular Dystrophy [12] , [26] , [27] . Recently , human LIN-41 has been shown to promote reprogramming of differentiated cells into induced pluripotent stem cells ( iPSCs ) [28] . Here , we demonstrate a role for LIN-41 in controlling pluripotency during development of an animal . In C . elegans , LIN-41 is a well-known component of the somatic heterochronic pathway , which temporally controls the transition from larval to adult cell fates [29] , [30] . The lin-41 germline phenotype described here indicates that , by preventing the onset of embryonic events in developing oocytes , LIN-41 also ensures a successful transition between generations . However , based on our analyses on both existing and newly created LIN-41 mutations , LIN-41 appears to function in the germline and the soma via two distinct molecular mechanisms . Our study identifies the first cytoplasmic “molecular roadblock” to reprogramming in developing oocytes and we propose it to be required to delay the onset of embryonic differentiation until after fertilization .
To understand how the onset of pluripotency is controlled during C . elegans development , we executed a genetic screen to identify factors that prevent EGA in the adult germline . To monitor EGA , we created a strain expressing GFP from an early embryonic promoter , vet-4 ( very early transcript 4 ) [31] , [32] . Thus , to identify novel regulators of developmental plasticity , we searched for mutants expressing the EGA-GFP in the adult germline ( Figure 1A ) . In addition to a new allele of gld-1 , this screen yielded two mutants that , in contrast to the embryo-specific EGA-GFP expression in wild-type animals , expressed EGA-GFP within the gonads ( Figure 1B ) . Several lines of evidence suggested that the phenotype of the two mutant strains was caused by alterations in the same gene , lin-41 ( Figures 1C and S1A–D ) . In these mutants ( alleles rrr3 and rrr4 , Figure 1C ) , the EGA-GFP expression was restricted to the proximal region of the oogenic germline ( Figure 1B ) . Consistent with this , RNAi-mediated depletion of lin-41 resulted in a similar expression of EGA-GFP in the gonad ( Figure S1C ) and a transgenic construct expressing LIN-41 fully rescued the germline defects of lin-41 ( rrr3 ) animals ( Figure S1D ) . To further examine the role of LIN-41 in controlling EGA , we verified that the endogenous vet-4 is also abnormally transcribed in lin-41 ( rrr3 ) gonads . Indeed , by in situ hybridization , we could detect vet-4 to be expressed in the proximal gonads of lin-41 ( rrr3 ) , but not wild-type animals ( Figure 1D ) . Next , to examine the extent of embryonic-like transcription in lin-41 ( rrr3 ) gonads , we monitored the levels of vet-4 and other additional early embryonic transcripts by reverse transcription and quantitative PCR ( RT-qPCR ) ( Text S1 ) . We found that these transcripts were expressed in mutant , but not wild-type gonads ( Figure 1E ) , further demonstrating that , in lin-41 mutants , embryonic transcription is prematurely activated in the germline . Importantly , we detected no obvious changes in levels or expression pattern of GLD-1 in lin-41 ( rrr3 ) gonads ( Figure S2 ) , suggesting that the gonadal phenotype of lin-41 mutants is not caused by defective expression of GLD-1 . In wild-type animals , Pol II-dependent transcription is repressed in oocytes , which is seemingly at odds with the embryonic-like transcription in the proximal gonads of lin-41 animals . To investigate this potential discrepancy , we examined the transcription-initiating phosphorylation of serine 5 ( Ser5P ) within the C-terminal domain ( CTD ) of Pol II [33] . In contrast to wild-type gonads , Ser5P was detected in the majority of the cells in the proximal gonads of lin-41 ( rrr3 ) animals ( Figure 2A ) , indicating ongoing Pol II-dependent transcription . Apart from EGA , the onset of embryonic development is marked by the degradation of germline mRNAs and proteins [34] . To examine this aspect of the germline-to-soma transition in lin-41 animals , we followed the expression levels of RME-2 , a yolk receptor present in oocytes [35] , and PGL-1 , a constitutive component of germ cell-specific RNA/protein granules [36] . In contrast to wild-type animals , which express RME-2 in developing oocytes and PGL-1 throughout the germline , we found that both proteins were absent from the proximal lin-41 ( rrr3 ) gonads ( Figure 2B–C ) , indicating that cells in this gonadal region lose germline identity . To test this further , we monitored expression of several transcripts that are normally expressed in somatic lineages . By RT-qPCR ( Text S1 ) , we found that several of these transcripts ( for example the myogenic hlh-1/MyoD ) were abnormally expressed in lin-41 ( rrr3 ) gonads ( Figure 2D ) . Additionally , we examined the expression of several hox genes , which control the positional identities of cells during animal body formation [37] . While the hox transcripts were not expressed in wild-type gonads , they were strongly expressed in lin-41 ( rrr3 ) gonads ( Figure 2D ) . Finally , we analyzed the expression of the muscle lineage markers UNC-120 and muscle myosin , the intestine lineage marker ELT-2 and a GFP reporter driven from a pan-neuronal unc-119 promoter ( nGFP ) . We observed that lin-41 ( rrr3 ) gonads contained numerous cells expressing muscle and neuronal markers ( Figures 2E and S3; 44/45 examined gonads contained cells expressing UNC-120 , 10/18 cells expressing muscle myosin , and 57/57 cells expressing the nGFP ) . Only few gonads contained ELT-2-expressing cells ( 3/35 gonads and only in few cells ) , which might reflect a competitive advantage of some differentiation programs in the lin-41 teratoma . During embryogenesis , most body-wall muscles of an adult animal are specified by the transcription factor PAL-1/CDX [38] . The PAL-1-dependent transcription is relatively well understood and involves the activation of its direct targets , such as HLH-1/MyoD and UNC-120/SRF [39] . In wild-type oocytes , expression of PAL-1 is insufficient for the induction of its target genes ( Figure S4A–B ) [40] . Nevertheless , we observed that the numbers of UNC-120-expressing cells in lin-41 ( rrr3 ) gonads were significantly reduced upon pal-1 RNAi ( Figure S4B ) . Thus , the differentiation into muscles in lin-41 gonads appears , at least partly , to mimic the pathway driving muscle formation in embryos . Together , these findings indicate that lin-41 germ cells in the proximal gonad undergo a dramatic reprogramming , which results in the acquisition of an embryonic-like state and teratomatous differentiation . To better understand the germline-to-soma transition in lin-41 animals , we examined cells in lin-41 ( rrr3 ) gonads in a time-course experiment ( Figure 3A ) . Until immediately after the end of spermatogenesis , the morphology and numbers of germ cells in lin-41 and wild-type gonads appeared similar . However , concomitantly with the onset of oogenesis , differences between the lin-41 and wild-type germlines began to emerge . The proximal region of wild-type gonads contained fully-grown oocytes harboring chromosomes arrested at the diakinesis stage of meiosis I . In stark contrast , the proximal region of lin-41 gonads contained oocyte-like cells that were about to divide , as evidenced by the presence of highly condensed chromosomes ( marked by the phosphorylation of histone H3 on serine 10 , Ser10P [41] , and microtubule spindles ( Figure 3A–B ) . Consistent with entering a mitotic cell cycle , cells in the proximal lin-41 ( rrr3 ) gonads did not express HIM-3 ( Figure 3C ) , a synaptonemal complex component [42] . Wild-type oocytes eliminate centrosomes , presumably to ensure the correct ploidy in embryos [31] , [43] , [44] . In contrast , by monitoring a constitutive centrosome component , SPD-2 [45] , we found that centrosomes were present in the proximal lin-41 ( rrr3 ) gonads ( Figure S5 ) . These centrosomes could duplicate ( Figures 3D and S5 ) and were able to nucleate microtubule spindles ( Figure 3D ) . Finally , in addition to the cell cycle markers , we monitored expression of an EGA reporter ( EGA-mCherry ) and the muscle-lineage marker UNC-120 and observed that their expression followed the onset of mitosis ( Figure 3A ) . Taken together , the absence of LIN-41 leads to the elimination of germline proteins , induction of EGA , a change from the meiotic to the mitotic cell cycle and somatic-like differentiation . Thus , rather than completing oogenesis , cells in the proximal lin-41 gonads execute events that , in wild-type development , only occur during embryogenesis . In addition to the germline defects , lin-41 ( rrr3 ) animals displayed somatic abnormalities: decreased size ( dumpy phenotype ) , appeared sick and occasionally bursted through the vulva . These phenotypes have been extensively described by Slack and colleagues and are caused , at least in part , by a precocious translation of the transcription factor LIN-29 [29] . To determine whether the gonadal phenotype reflects LIN-41 function in the germline , or it is indirectly caused by the loss of LIN-41 in the soma , we created a transgene driving lin-41 expression from a heat-shock promoter ( hsp-16 . 41 ) . Due to a general insensitivity of germ cells to the heat-shock promoter-driven expression [46] , this transgene was not expressed in the germline but , when crossed into the lin-41 ( rrr3 ) mutant background and cultivated at an elevated temperature ( 24–25°C , which is apparently enough to drive sufficient expression of lin-41 in the soma ) , it rescued the somatic lin-41 defects ( the transgenic animals no longer appeared sick or short; Figure 4A ) . Despite the somatic rescue , these animals still developed teratomas ( Figure 4B , 50/50 examined animals ) , suggesting that , in controlling the germline-to-soma transition , LIN-41 functions autonomously in the germline . To examine this further , we immunostained gonads using antibodies raised against LIN-41 and found that , indeed , LIN-41 was present in the cytoplasm of germ cells starting from the late pachytene stage and culminating in the fully-grown oocytes ( Figure 4C ) . Interestingly , LIN-41 was often absent from the most-proximal oocytes ( Figures S1D and S6 ) , suggesting a possible connection between oocyte maturation and/or ovulation and LIN-41 levels . LIN-41 expression was limited to the oogenic germline ( i . e . , it was absent in sperm , e . g . , S1D ) , suggesting that the germline-to-soma transition in lin-41 gonads is caused by the loss of LIN-41 function in the developing oocytes . In the soma , LIN-41 is thought to associate with and repress the mRNA encoding a transcription factor , LIN-29 , and LIN-29 depletion suppresses the somatic defects of lin-41 mutants [29] . In contrast to these observations in the soma , lin-29 mRNA appears to be either poorly or not at all expressed in the germline ( our unpublished results and [47] ) . Consistently , we found that RNAi-mediated depletion of LIN-29 did not suppress the germline defects of lin-41 ( rrr3 ) mutants ( though , it suppressed the somatic defects , as expected [29] ) . We obtained similar results in lin-41; lin-29 double mutants ( Figure S7 ) . Thus , LIN-41 may function in the germline and soma via distinct targets and/or mechanisms . The domain structure of LIN-41 reflects the diversity of functions that have been associated with TRIM-NHL proteins ( Figure 1C ) . Several of these proteins function as E3 ubiquitin ligases , which require a functional RING domain [10] , [30] . However , a sequence alignment of the C . elegans LIN-41 RING domain with those of other Caenorhabditis species indicates that a highly conserved proline , critical for canonical E3-E2 interactions [48] , is not found in the nematode LIN-41 RING domains ( Figure 5A ) . Moreover , mutating five cysteine residues that are critical for the RING domain zinc finger structure ( C114S , C117S , C130S , C151S , C154S; Figure 5A ) resulted in a protein that rescued both somatic and germline defects of lin-41 ( rrr3 ) animals ( Figure 5B ) . Although we cannot rule out the possibility that LIN-41 associates with additional factors to regulate ubiquitination , these results suggest that the nematode LIN-41 does not function as a direct E3 ubiquitin ligase . The coiled-coiled , filamin and NHL domains of the human homolog of LIN-41 , TRIM71 , constitute the minimal region responsible for binding mRNA and inhibiting translation [19] , which is the function attributed to the C . elegans LIN-41 in the soma [29] . One previously isolated lin-41 allele , ma104 , is a transposon insertion into the sequence coding for the filamin domain [29] . Intriguingly , lin-41 ( ma104 ) mutants display the somatic defects , but the animals are apparently fertile [29] . We confirmed this and , although the brood size in lin-41 ( ma104 ) animals is decreased [29] , we found no obvious differences in the levels or localization of the germline LIN-41ma104 ( Figure 6A ) and no evidence for a precocious EGA in the gonads ( 0/35 worms were expressing the EGA reporter in their gonads ) . To understand the effect of the ma104 mutation on the LIN-41 protein , we examined the cDNA product of the lin-41 ( ma104 ) allele . We found that the ma104 mutation resulted in an insertion of 16 amino acids into the filamin domain ( Figure 6B ) . To gain a mechanistic insight into the ma104 mutation , the filamin domain ( residues 691–821 ) was subcloned , overexpressed in bacteria , and the protein purified to homogeneity . The protein was crystallized and the structure determined at high resolution ( 1 . 68 Å; for data collection and refinement statistics , see Table S1 ) . The final crystallographic model encompasses residues 691–729 and 758–820 , whereas a long insert ( 730–757 ) that is only found in Caenorhabditis LIN-41 protein sequences could not be built due to high flexibility . We found that the LIN-41 filamin structure exhibits a classical immunoglobulin ( IG ) -like domain fold consisting of seven β-strands arranged in two antiparallel β-sheets ( Figure 6C ) [49] . A structural search with the LIN-41 filamin domain against the Protein Data Bank ( PDB ) , using DALI , identified the filamin domains most structurally similar to that of LIN-41 , which yielded the filamin domains from the Dictyostelium discoideum gelation factor , the human TRIM45 and Filamin-A ( PDB IDs 1QFH , 1WLH , 2DS4 and 3RGH , respectively ) with root-mean-square deviation values for Cα positions between 1 . 6 and 2 . 0 Å [50] . Both crystal packing analysis using PISA [51] and SEC MALS experiments of the protein in solution reveal the oligomeric state of this protein domain as monomeric . Importantly , the 16-residue insertion present in the ma104 allele maps to the mid-section of the second β-strand and is very likely perturbing the filamin IG-like fold ( Figure 6B–C ) . Specifically , the 16-residue insert will prevent completion of one of the β-sheets resulting in solvent access to the hydrophobic protein core of the IG-like β-sandwich and thereby severely destabilizing the fold . This makes it very unlikely that the filamin domain of the ma104 allele is properly folded to exert its biological function . In the fly Brat and the mammalian TRIM71/LIN-41 , the NHL domain is essential for mRNA regulation [14] , [19] , [52] . One of the lin-41 alleles reported here , rrr4 , introduces a premature stop codon within the first NHL repeat ( Figure 1C ) , potentially triggering mRNA degradation via nonsense-mediated mRNA decay ( NMD ) . Indeed , inhibiting NMD ( by depleting an NMD component , SMG-2 [53] ) , restored the wild-type expression pattern and levels of LIN-41rrr4 ( Figure 7A ) . LIN-41rrr4 is expected to lack the NHL domain and we found that the gonads expressing this LIN-41 variant displayed lin-41-like germline and somatic defects ( Figure 7A ) . Thus , the NHL domain appears to be essential for LIN-41 functions in both germ and somatic cells . The NHL domain structure of Brat forms a six-bladed β-propeller [52] . Several point mutations in Brat and TRIM71 that disrupt mRNA regulation affect residues on the electropositive side of the NHL domain [19] , [23] , [52] , [54] ( Figures S8 , S9 ) , highlighting the importance of this surface for mRNA regulation . Point mutations in the NHL domain have been also reported in LIN-41 ( Figures 7B and S9A–B ) [29] . Importantly , although these mutations display defects in the soma , the animals are fertile , suggesting that the mutant proteins fulfill the gonadal functions [29] . To better interpret these mutations , we initially attempted , unsuccessfully , to express the LIN-41 filamin-NHL or a NHL-only domain constructs for protein structure determination . Thus , we created a homology model of the LIN-41 NHL domain based on the crystal structure of the Brat NHL domain . Interestingly , we found that most of the existing point mutations in the LIN-41 NHL domain also affect amino acids residing on the electropositive surface of the NHL domain ( Figure 7B ) . These observations suggest that i ) the electropositive surface of the NHL domain plays a conserved function in mRNA regulation and ii ) the germline and somatic functions of the NHL domain involve different mechanisms . To explore this further , we introduced an additional mutation ( Y941A ) on the electropositive surface of the NHL domain ( Figures 7B and S9 ) . Potentially , this mutation is more informative than the other existing NHL mutations because mutation of the corresponding residue ( Y702A ) in TRIM71 is known to abolish mRNA regulation [19] . In contrast to the deletion of the whole NHL domain from otherwise rescuing ( FLAG- and GFP-tagged ) LIN-41 protein , which , as expected , caused defects in both the soma and the germline , we found that the LIN-41Y941A variant largely suppressed the germline defects and sterility of lin-41 animals ( Figure 7C ) , including the precocious expression of the endogenous vet-4 transcript ( Figure 7D ) , though it continued to display the somatic defects . Thus , if the same domains/residues determine mRNA regulation in LIN-41 as in TRIM71 , the germline function of LIN-41 might be independent from mRNA binding .
In contrast to the much-publicized regulation of pluripotency via DNA and chromatin modifications , the potential for cytoplasmic regulation has largely been neglected . Our findings suggest that proteins like LIN-41 and GLD-1 can function in the cytoplasm as molecular “roadblocks” to reprogramming , analogous to the nuclear factors . Similarly , components of P-granules ( germline-expressed RNPs ) have been recently reported to facilitate maintenance of germline identity in proliferating germ cells , i . e . , at the stage prior to GLD-1 expression [55] . Whether teratomatous differentiation in the absence of P-granules reflects precocious activation of embryonic transcription , or has a different etiology , remains to be determined . However , in contrast to P-granules , that impact multiple aspects of RNA metabolism , GLD-1 and LIN-41 are expected to have more specific functions . Intriguingly , the germline-to-soma transition in the absence of LIN-41 or GLD-1 involves similar events: loss of germline proteins , retention of centrosomes , execution of mitosis and activation of the embryonic genome . Two GLD-1 mRNA targets , important for the germline-to-soma transition , encode the CDK-2 partner protein CYE-1/cyclin E and the transcription factor PAL-1/Cdx [6] , [31] . However , cyclin E and PAL-1 are co-expressed with LIN-41 in the developing wild-type oocytes [56] , suggesting that their expression is not regulated by LIN-41 . Thus , GLD-1 and LIN-41 may regulate pluripotency via different targets and/or mechanisms . While GLD-1 directly binds and regulates the expression of its mRNA targets , the molecular function of LIN-41 remains elusive . Our analysis suggests that the germline function of LIN-41 may be independent from mRNA binding , though it does not exclude its role in posttranscriptional regulation , for example as a component of a regulatory RNP . In addition to binding RNA , several TRIM-NHL proteins have been shown to modulate functions of other proteins , for example , by their sequestration ( e . g . , TRIM3 appears to regulate p21 [13] , [17] ) or by linking structural proteins ( e . g . , Wech bridges Talin and ILK for proper embryonic muscle attachment [16] ) . These interactions depend , at least in part , on the NHL domains of both proteins . Thus , LIN-41 could regulate the germline-to-soma transition by associating , via its NHL domain , with another protein . LIN-41-mediated regulation of cell fate transition between generations is somewhat reminiscent of LIN-41 function in the hetrochronic pathway in the soma . However , specific mutations within the NHL and filamin domains of LIN-41 result mainly in somatic but not germline defects ( this study and [29] ) . In addition , LIN-41-dependent repression of LIN-29 appears to be restricted to the soma . Thus , although LIN-41 regulates developmental transitions in both germ- and somatic cells , it may do so through different molecular mechanisms and/or targets . In the soma , down-regulation of LIN-41 , which is mediated by the let-7 miRNA , allows terminal differentiation [57] , [58] . In the germline , LIN-41 levels decrease in the most-proximal oocytes , so that LIN-41 is absent from the early embryos . An interesting possibility is that the absence of LIN-41 is a trigger for the onset of embryonic differentiation . In order to test this hypothesis , we attempted to over-express LIN-41 from the heat shock promoter in very early embryos . However , LIN-41 was efficiently expressed only after gastrulation ( our unpublished observation ) , i . e . , several cell divisions after EGA , making the experiment inconclusive . The down-regulation of LIN-41 occurs while Pol II-dependent transcription is globally repressed in the oocytes , suggesting that the regulation occurs at the mRNA or the protein level . To test for possible regulation at the mRNA level , we expressed a rescuing LIN-41 under the control of a truncated 3′UTR missing most of the sequence , including the let-7 binding sites [58] . While the expression of this LIN-41 protein started earlier ( more distally ) in the germline , suggesting posttranscriptional regulation of lin-41 mRNA in this part of the gonad , LIN-41 was still down-regulated in the oocytes and embryos ( our unpublished observation ) , hinting at a possible regulation at the protein level . If so , testing the functional significance of LIN-41 degradation will require the dissection of regulatory motifs in the protein ( for example phosphorylation ) . TRIM-NHL proteins are known to control the proliferation versus differentiation decision in germ- and neuronal stem cell lineages [23] , [25] , [59] and , intriguingly , C . elegans LIN-41 has been recently reported to control the regenerative ability of neurons [60] . While these examples highlight the importance of TRIM-NHL proteins for maintaining homeostasis in self-renewing tissues , these proteins have not previously been implicated in controlling pluripotency during development . In our study , we describe LIN-41 as a critical component of the timing mechanism controlling the oocyte reprogramming capacity . To our knowledge , this is the first example of such a regulator in cells that are ready for embryonic development , providing the initial glimpse into a pathway controlling one of the most fundamental developmental transitions . While the in vivo roles of the mammalian LIN-41/TRIM71 are poorly understood , the murine TRIM71 is expressed and functions in developing embryos [61] , [62] . TRIM71 is also preferentially expressed in embryonic stem ( ES ) cells [18] , which are derived from pluripotent embryonic cells . In ES cells , TRIM71 represses the expression of Cdkn1 , an inhibitor of the cell cycle progression , thereby promoting proliferation [18] . While this role appears opposite to LIN-41 function in the C . elegans germline , TRIM71 presumably associates with many mRNAs , making additional roles likely . Intriguingly , the human LIN-41 has been recently shown to facilitate reprogramming of fibroblasts into iPSCs [28] . In this context , LIN-41 , combined with several “pluripotency” transcription factors , can circumvent the requirement for c-Myc in reprogramming [28] . c-Myc facilitates reprogramming in several ways , including by inhibiting differentiation [63] , and LIN-41 appears to play a similar role by repressing mRNAs encoding pro-differentiation factors [28] . Although the targets and , perhaps , the mechanisms may differ , it is striking that the C . elegans LIN-41 appears to fulfill an analogous function in the germline . Thus , dissecting LIN-41 targets and the mechanism are exciting objectives for the future research .
N2 animals were maintained as previously described [64] and were grown at 20°C unless stated otherwise . For alleles and transgenic lines , see Supplemental Material . For RNAi , L1 larvae ( L4 for fog-2 ) , grown at 25°C , were fed with bacteria expressing dsRNAs ( targeting lin-41 , pal-1 , fog-2 or smg-2 from the Open Biosystem library or lin-29 from the Ahringer library ) and screened one day after the L4-to-adult molt in the same ( lin-41 , pal-1 and lin-29 ) or in the second generation ( fog-2 and smg-2 ) . A bacterial strain carrying an “empty” vector was used as a negative control ( mock RNAi ) . EMS mutagenesis [64] was performed on a strain ( # 1284 , see Text S1 ) carrying the EGA-GFP ( integrated at two chromosomal locations to increase GFP fluorescence ) . F2 animals derived from ≈10 . 000 F1s were screened . Candidate mutations were identified as previously described [65] . Each mutant was back-crossed four times against the parental strain before genome sequencing . Genomic DNAs ( gDNAs ) were isolated using Gentra Puregene Tissue Kit 4 g ( Qiagen ) . DNA libraries were created from 50 ng of gDNA ( Nextera DNA kit from Illumina ) . The sequencing data were generated using Hi Seq 2000 ( Illumina ) . Sequence reads were aligned to the May 2008 C . elegans assembly ( obtained from http://hgdownload . soe . ucsc . edu/goldenPath/ce6/chromosomes/ ) using “bwa” [66]; version 0 . 6 . 1-r104 ) with default parameters , but only retaining single-hit alignments ( “bwa samse -n 1” and selecting alignments with “X0:i:1” ) . The resulting alignments were converted to BAM format , sorted and indexed using “samtools” [67]; version 0 . 1 . 18 ) . In order to quantify contamination by Escherichia coli , reads were similarly aligned to a collection of E . coli genomes ( NCBI accession numbers NC_008253 , NC_008563 , NC_010468 , NC_004431 , NC_009801 , NC_009800 , NC_002655 , NC_002695 , NC_010498 , NC_007946 , NC_010473 , NC_000913 and AC_000091 ) , which typically resulted in less than 1% aligned reads . Sequence variants were identified using GATK [68]; version 1 . 5 . 31 ) indel realignment and base quality score recalibration , followed by SNP and INDEL discovery and genotyping for each individual strain using standard hard filtering parameters , resulting in a total of six to eight thousand sequence variations in each strain compared to the reference genome . Finally , the number of high quality ( score > = 500 ) single nucleotide substitutions of EMS-type ( G/C→A/T transitions [69] , not found in other any other mutant strain or in the parent strain ( typically less than 1% of the total number of variants per strain ) were counted in sequential windows of 1 Mb to identify regions of increased variant density . RNA was isolated from gonads dissected from one day-old ( after the L4-to-adult molt ) animals . cDNA was synthesized with oligo ( dT ) primers using the ImProm II Reverse transcription system from Promega according to manufacturer's instructions . cDNA was used for qPCR with the Absolute QPCR SYBR green ROX mix ( AbGene ) on an ABI PRISM 7700 system ( Applied Biosystems ) . qPCR reactions were performed as previously described [31] . At least one primer in each pair is specific for an exon-exon junction . Human carrier RNA was added to each sample before RNA extraction , allowing normalization to hGAPDH . Standard curves for quantification were generated from a serial dilution of input cDNA for each primer pair . The amount of target present in each replicate was derived from a standard curve; an average was calculated for the triplicates . To compare total mRNA levels , the qPCR results were normalized to human GAPDH and to the wild-type values for each primer pair and fold enrichments were calculated . For primers used , see Text S1 . Immunostaining experiments were performed as previously described [70] with the following antibodies: PGL-1 [36] ( dilution 1∶1000 ) ; SPD-2 [45] ( “969LA” , 1∶800 ) ; GFP ( Roche , 1∶700 ) ; phospo-Histone H3 Ser10 ( “Ser10P” , Millipore , 1∶200 ) ; muscle myosin [71] ( “5–6” , 1∶2 . 500 ) ; and UNC-120 ( courtesy of Michael Krause , 1∶500 ) . Immunostainings against RME-2 [35] ( “INT” , dilution 1∶100 ) , GLD-1 [72] ( dilution 1∶5 ) and LIN-41 ( courtesy of Helge Grosshans , “4796” , 1∶2 . 000 ) were performed as previously described [73] and against the Ser5P of Pol II CTD [74] ( “3E8” , 1∶5 ) according to Seydoux and Dunn [33] . Immunostainings against HIM-3 [42] ( courtesy of Monique Zetka , dilution 1∶500 ) were performed as previously described [75] . Secondary antibodies used in this study: goat anti-mouse IgG alexa-488 ( Molecular Probes , 1∶600 , ) , goat anti-rabbit IgG alexa-568 ( Invitrogen , 1∶750 ) and goat anti-rat IgG alexa-568 ( Molecular Probes , 1∶500 ) . In situ hybridizations against the vet-4 mRNA were performed as previously described [31] . Unless indicated otherwise , the gonads were dissected from 1 day-old adults . Zeiss AxioImager Z1 microscope equipped with an Axiocam MRm REV 2 CCD camera was used for capturing pictures . Images were then exported into Adobe Photoshop CS4 and processed in an identical manner . A spinning disk multipoint confocal microscope equipped with an EM-CCD Cascade II camera ( Photometrics ) was used for capturing images for Figure 3D . Pictures were , then , deconvolved with the Huygens software and then processed in Imaris XP 7 . 1 . 1 . The affinity-purified ( ELISA ) rabbit anti-LIN-41 antibody ( “4796” ) was provided by Helge Grosshans ( Magdalene Rausch & Helge Grosshans , unpublished data ) and created against the VKNLKLSVLISQAESLQSKQIDLQQAIQTATKLMDSSDCDEMVLRQVFEKLASCQMGNEGTEPNNNILNVLMLACQVNEDDRLKFTAPQDGILLNKARQF sequence ( residues 587–686 ) . The rabbit was raised by SDIX in Newark , DE , USA . The LIN-41 point mutant transgene constructs “RING” and “Y941A” were created from the wild-type LIN-41 transgenic template by site-directed mutagenesis ( Stratagene QuikChange method ) , whereas the deletion construct “ΔNHL” was created via two-step PCR . In any case , Phusion High-Fidelity DNA Polymerase ( Fermentas ) was used . For primers used see Text S1 . The filamin domain ( residues 691–821 ) of C . elegans LIN-41 ( isoform B of Q9U489 ) was cloned into pOPINF [76] using In-Fusion ( Clontech Laboratories Inc ) . The resulting expression construct was transformed into BL21 DE3 cells and the protein expressed via auto-induction at 20°C for 20 hours . Cells were harvested , then resuspended in lysis buffer ( 50 mM Tris , pH 7 . 5 , 500 mM NaCl , 20 mM imidazole , 0 . 2% Tween-20 ) and frozen at −80°C . The cell suspension was thawed and freshly supplemented with Complete EDTA-free protease inhibitors ( Roche Diagnostics ) and 3 U/ml Benzonase ( Sigma ) before passing through an Avestin EmulsiFlex-C3 cell disruptor . The clarified lysate was incubated with NiNTA affinity resin ( Qiagen ) in batch mode and the bound protein eluted in 50 mM Tris , pH 7 . 5 , 500 mM NaCl , 125 mM imidazole . The protein was fractionated on a Superdex 75 HiLoad 16/60 ( GE Healthcare ) gel filtration column in GF buffer ( 20 mM Tris , pH 7 . 5 , 200 mM NaCl , 2 mM TCEP and 0 . 02% NaN3 ) . The single peak fraction was pooled and digested overnight at 4°C with 3C protease to remove the N-terminal histidine tag . The released protein tag and 3C protease were removed by a second nickel-affinity step and the untagged filamin domain was further purified over a Superdex 75 column in GF buffer and concentrated to 7 . 5 mg/ml . All crystallization experiments were performed at 20°C using the sitting-drop vapour diffusion method via a Phoenix robot ( Art Robbins ) dispensing 100 nl drops . Removal of the N-terminal histidine tag from the filamin domain was needed to obtain crystals . The untagged filamin domain readily crystallized in many conditions . Crystals grown in 1 . 1 M sodium malonate , 0 . 1 M HEPES , pH 7 . 0 , 0 . 5% v/v Jeffamine ED-2001 , were harvested and cryoprotected in mother liquor containing 25% ethylene glycol . These crystals diffracted to 1 . 68 Å resolution at the SLS PX-III beamline and belonged to space group C2221 with one molecule per asymmetric unit . Diffraction data were integrated and scaled using XDS [77] and the structure was solved by the molecular replacement method using PHASER [78] . Phases from this solution were calculated and used for automatic model building with BUCCANEER [79] . The LIN-41 filamin structure was further improved by the crystallographic simulated annealing routine followed by individual B-factor refinement in PHENIX [80] and several rounds of manual rebuilding in COOT [81] and refinement in BUSTER [82] . The final structure was validated using COOT . Structural images for figures were prepared with PyMOL ( http://pymol . sourceforge . net/ ) . Atomic coordinates and structure factors for the LIN-41 filamin domain have been deposited in the PDB with entry code 4UMG . Amino acid sequences of the C . elegans LIN-41 ( Uniprot Q9U489 , 830–1147 ) and Homo sapiens TRIM71 ( Uniprot Q2Q1W2 , 591–868 ) NHL domains were submitted to the HHPRED server for homology detection and structure prediction [83] . The structure of the D . melanogaster Brat NHL domain ( PDB 1Q7F ) was the top hit in both searches resulting in very high scores for the LIN-41 NHL domain ( Score = 241 . 22 , E-value = 1e-33 , 28% sequence identity ) and the TRIM71 NHL domain ( score = 22 . 54 , E-value = 4 . 5e-35 , 31% identity ) . The top alignments were edited for minimal local corrections and sent to the HHPRED MODELLER pipeline for modeling . Loops lacking template information for both the C . elegans and the human NHL domain models were removed in the final models . Structural figures were prepared using PyMOL ( www . pymol . org ) . | Reprogramming into a naïve , pluripotent state during the oocyte-to-embryo transition is directed by the oocyte cytoplasm . To understand how this reprogramming is controlled , we searched for C . elegans mutants in which the activation of embryonic genome , a landmark event demarcating the switch from a germline- to embryo-specific transcription , is initiated precociously in germ cells . This screen identified a novel function for LIN-41 , a member of the TRIM-NHL protein family , in preventing a premature onset of embryonic-like differentiation and teratoma formation in developing oocytes , thus ensuring a successful passage between generations . This is the first example of such a regulator in cells that are poised for embryonic development . Interestingly , the majority of molecular “roadblocks” to reprograming that have been identified so far are epigenetic regulators . However , we propose that , at least in germ cells , LIN-41-like regulators may fulfill an analogous role in the cytoplasm , which has possible implications for the generation of human pluripotent stem cells . | [
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"a... | 2014 | The TRIM-NHL Protein LIN-41 Controls the Onset of Developmental Plasticity in Caenorhabditis elegans |
Glycosylation plays crucial regulatory roles in various biological processes such as development , immunity , and neural functions . For example , α1 , 3-fucosylation , the addition of a fucose moiety abundant in Drosophila neural cells , is essential for neural development , function , and behavior . However , it remains largely unknown how neural-specific α1 , 3-fucosylation is regulated . In the present study , we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library . We obtained 109 genes affecting glycosylation that clustered into nine functional groups . Among them , members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1 , 3-fucosylation . Further analyses revealed that an RNA–binding protein , second mitotic wave missing ( Swm ) , upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus . This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells .
Neural cells require correct glycosylation patterns for their development , function , and viability . An example of this is the attachment of an α1 , 3-fucose moiety to an N-glycan core via α1 , 3-linkage [1] , a process which is prevalent in neural cells in Drosophila [2] . This α1 , 3-fucose moiety can be detected with an anti-horse radish peroxidase ( HRP ) antibody and therefore has also previously been referred to as an HRP epitope . The α1 , 3-fucose is thought to be essential for neural development , function , and behavior because a nac ( neurally altered carbohydrate ) Drosophila mutant that lacks this α1 , 3-fucose moiety exhibits deformation of the eyes [3] , the misrouting of wing sensory neurons [4] , and abnormal grooming behavior [5] . However , as it remains unclear that the nac mutation impairs only α1 , 3-fucosylation , the necessity of α1 , 3-fucosylation for neural development and/or function in Drosophila has not been conclusively demonstrated . The enzyme α1 , 3-fucosyltransferase ( FucTA ) [6] , which is mainly expressed in neural cells , directly catalyzes α1 , 3-fucosylation . In addition to FucTA , other glycosylation-related proteins such as UDP-GlcNAc: α-3-D-mannoside-β -1 , 2-N-acetylglucosaminyltransferase I ( Mgat1 ) [7] , GDP-mannose 4 , 6-dehydratase ( Gmd ) [8] , and a GDP-fucose transporter ( Gfr ) [9] , [10] are required for α1 , 3-fucosylation . Whereas Mgat1 provides a preferred substrate for FucTA by adding N-acetylglucosamine to the nonreducing end of an N-glycan , Gmd and Gfr are responsible for the synthesis and transport , respectively , of GDP-fucose , another substrate for FucTA . These genes , in contrast to the gene encoding FucTA , are widely expressed in various tissues and also utilized for other glycosylation processes such as O-fucosylation of Notch , α1 , 6-fucosylation of N-glycans , and formation of complex type N-glycans . Hence , the neural-specific expression of FucTA appears to account for the neural-specific regulation of α1 , 3-fucosylation . However , the mechanisms regulating FucTA expression have remained largely unknown . Forward genetic approaches have proven to be powerful methods of elucidating novel mechanisms . For example , the study of Drosophila genetics has yielded important contributions to our understanding of the developmental significance of proteoglycans [11] , [12] and Fringe-dependent Notch glycosylation [13] . Genetic screens for mutations affecting morphogenesis and growth factor signaling have now identified a number of genes involved in Notch glycosylation and/or proteoglycan formation . Most of these genes are conserved in mammals , suggesting that Drosophila is a useful model system for the study of glycosylation in metazoans . However , although previously performed screens of this nature have identified glycosyl enzymes and nucleotide sugar transporters , to date they have not been used to uncover regulators of these molecules . To elucidate novel regulatory mechanisms underlying neural-specific glycosylation , we performed a genetic screen in Drosophila and identified 109 genes required for glycosylation of a retinal neural cell-specific protein . These included 95 genes that are newly implicated in this process and 9 functional groups . Furthermore , 17 genes were identified to be specifically required for α1 , 3-fucosylation . Among these genes , we further analyzed the function of second mitotic wave missing ( Swm ) , which contains an RNA-binding motif . Here , we show that Swm directly binds to fucTA mRNA , upregulates fucTA mRNA and protein levels , and facilitates the nuclear export of fucTA mRNA in neural cells . These results indicate that Swm is involved in neural-specific glycosylation in addition to the cell cycle , in which its involvement has been previously reported [14] . This report , the first large-scale screen for glycosylation in a multicellular organism , has thus identified a number of new genes directly or indirectly involved in glycosylation and unveiled a novel regulatory mechanism of neural-specific glycosylation .
As α1 , 3-fucosylation has not proven to be essential for neural development and/or function in Drosophila , we first aimed to determine the neural function of α1 , 3-fucosylation . A phenotype of a piggyBac insertional mutant for the fucTA gene , fucTAf03774 , was examined . To validate this mutant , its central nervous system ( CNS ) was stained using anti-HRP antibody . The mutant CNS was negative for anti-HRP staining , suggesting that α1 , 3-fucosylation was compromised ( data not shown ) . CNS morphology was then compared between wild-type and fucTA mutant third instar larvae . The longitudinal length-to-width ratio of the ventral nerve cord ( VNC ) was significantly lower in fucTA mutants than in wild-type larvae ( Figure 1A and 1B ) , indicating that α1 , 3-fucosylation plays an important role in neural development . However , the staining patterns of antibodies such as 22C10 , BP102 , anti-FasI , anti-FasII , and anti-FasIII were not noticeably different in the fucTA mutant ( data not shown ) . To reveal novel regulatory mechanisms underlying neural-specific glycosylation , a genetic screen was performed in adult Drosophila using the RNAi-mediated tissue-specific gene knockdown method . The procedure is summarized in Figure 2A . We chose eye-specific knockdown because eyes bear the neural-specific glycan α1 , 3-fucose . Additionally , the ablation of genes essential for development and/or cell viability in eyes might not cause knockdown fly lethality , whereas their ablation in the central and/or peripheral nervous systems would be lethal . DsRNAs targeting different genes were expressed in Drosophila eyes by crossing the eye-specific Gal4 driver , GMR-Gal4 , with the corresponding strains . These strains harbored inverted repeats of a portion of the corresponding cDNA downstream of the yeast upstream activating sequence ( UAS ) , which is activated by the Gal4 protein ( http://www . shigen . nig . ac . jp/fly/nigfly/about/aboutRnai . jsp ) . In our screen , the glycosylation of a model eye-specific glycoprotein was examined , as this allowed us to accurately measure the number of glycans added . We chose Chaoptin ( Chp ) [15] as our model glycoprotein based on the following advantages: ( i ) Chp is modified by neural-specific α1 , 3-fucosylation; ( ii ) detailed information is available on both the structures of the N-glycans and their attachment sites on Chp ( described below in detail ) [16]; and ( iii ) simple protein affinity purification is possible using an anti-Chp antibody ( 24B10 ) . As shown in Figure 2B , Chp protein could be purified as a single band detected by Coomassie Brilliant Blue ( CBB ) staining . Chp is a Drosophila glycoprotein essential for rhabdomere formation in adult photoreceptor cells , which is mediated through cell adhesion activity [15] , [17] . N-glycosylation of Chp was recently shown to play a pivotal role in its stability , transport to the plasma membrane , and cell adhesion activity [18] . Chp N-glycan structures are classified as high-mannose type , pauci-mannose type , or complex type [16] . Glycan structures added to Chp are summarized in Figure S1 . Interestingly , α1 , 3-fucose is added only at N1012 , suggesting that Chp has a single difucosylated glycan at N1012 . To detect these glycan structures , the binding of glycan probes such as lectins and anti-HRP antibody was examined . Immunopurified Chp was subjected to lectin blot or immunoblot analysis using ABA , ConA , DBA , DSA , LCA , Lotus , PHA-L4 , PNA , SBA , UEA , and WGA lectins and anti-HRP antibody . The specificity of these lectins and anti-HRP antibody is summarized in Table S1 . The binding abilities of these probes were quantified relative to the amount of Chp protein ( Figure 2C ) . These analyses suggested that the WGA , ConA , LCA , and DSA lectins and anti-HRP antibody would be useful as glycan probes in our assay system . As represented in Figure S1 , WGA and ConA detect N-acetylglucosamine ( GlcNAc ) and mannose moieties of N-glycan core regions , respectively . LCA preferentially binds to N-glycans with α1 , 6-fucose and even weakly recognizes the core regions of N-glycans . Anti-HRP antibody binds to the α1 , 3-fucose moieties attached to the core [1] , [19] . DSA recognizes GlcNAc at the non-reducing end of N-glycans . Chp protein purified from knockdown flies was subjected to lectin and immuno-dot blot analyses using the same glycan probes described above and an anti-Chp antibody . An example of the dot blot analysis is shown in Figure S2 . The glycan moieties detected by these probes were quantified relative to Chp protein , and the z-scores for each knockdown experiment were thus calculated . For verification of this procedure , a dsRNA that targets FucTA was expressed . The resulting knockdown of fucTA decreased the affinity of Chp to the anti-HRP antibody ( 66 . 2±11 . 8% of the control , Figure 2D ) . Given the results of our previous mass spectrometry study showing that α1 , 3-fucose is added to the Chp N1012 site [16] , Chp was purified from fucTA knockdown eyes , and mass spectrometry analysis was performed . The glycan structures attached to N1012 are summarized in Figure S3 . The number of glycans with two fucoses present at N1012 ( M3F2Gn2 and M3F2Gn3 ) selectively decreased in fucTA knockdown eyes ( Figure 2E ) . This finding is consistent with those of previous studies reporting that most single- or double-fucosylated N-glycans bear α1 , 6-fucose alone , or both α1 , 6- and α1 , 3-fucose , respectively , in embryos [20] and adults [6] . A large-scale screen was performed in the current study using an RNAi library that was previously constructed in National Institute of Genetics , Japan ( NIG , http://www . shigen . nig . ac . jp/fly/nigfly/about/aboutRnai . jsp ) . With this library , the expression of 6923 Drosophila genes can be suppressed . From the primary screen followed by tests for reproducibility , 171 genes were identified as candidate genes , knockdown of which compromised glycosylation ( Table S2 ) . Since these candidate genes likely included false-positives due to the off-target effects of RNAi [21] , the results were validated by repeating the knockdown experiments using secondary sets of dsRNAs . During the construction of these secondary sets of dsRNAs , we ensured that the target regions did not overlap . Of the 171 primary candidate genes , only 80 could be tested because , in most cases , the induction of the secondary sets of dsRNA resulted in lethal or severe eye malformation or cDNAs were too short to permit the design of secondary target regions . Of the 80 genes tested successfully , 57 were verified to be involved in glycosylation . Sources of the RNAi strains used for the verification of the 57 genes are listed in Table S2 ( see “Line ID or transformant ID used for validation” ) . When the validation results were compared with the calculated off-target probability scores ( OTPS ) provided by dsCheck software [22] , 95 . 6% of the validated genes had scores of less than 3 , whereas the scores of 60 . 7% of the genes yet to be validated were greater than 2 . Thus , the genes with OTPS less than 3 were rescreened by testing whether knockdown of their suspected off-target genes led to glycosylation defects . When knockdown of the suspected off-target genes did not induce any glycosylation defects , the corresponding genes were classified as glycosylation-related genes ( rank 2 ) along with the genes that were validated by secondary dsRNAs ( rank 1 ) ( Table S4 ) . To successfully identify additional glycosylation genes , we searched for genes that interacted with our newly identified genes in the yeast two-hybrid database BIOGRID ( http://www . thebiogrid . org/ ) and in the genetic interaction data listed in FlyBase ( http://flybase . bio . indiana . edu/ ) . The RNAi fly strains harboring candidate genes that did not already exist in our library were obtained from the Vienna Drosophila RNAi Library Center ( VDRC ) [23] ( Table S3 ) . Among the 186 genes tested , 10 genes showed glycosylation defects . Further validation experiments identified 1 rank-1 and 5 rank-2 glycosylation-related genes among them . In total , 109 genes were eventually isolated and identified as glycosylation-related genes ( Table S4 ) . Significantly , although 14 of the 109 identified genes have already been shown to be involved in glycosylation , the remaining 95 genes were newly assigned to this category by our analysis . Glycosylation-related genes were classified based on their domain structures and presumptive functions using the InterPro ( http://www . ebi . ac . uk/interpro/ ) , FlyBase ( http://flybase . org/ ) , and Panther ( http://www . pantherdb . org/ ) databases . The gene functional groups obtained included glycosylation reactions , transcription , RNA regulation , translation , intracellular trafficking , cytoskeletal regulation , signal transduction , protein degradation , mitochondrial function , and other functions ( Figure 3A , Table S4 ) . To identify genes specifically involved in neural-specific glycosylation , we selected genes whose knockdown induced only abnormal anti-HRP binding activity from among the 109 genes listed in Table S4 . From this analysis , 17 genes showing α1 , 3-fucosylation-specific defects were identified belonging to seven functional groups ( Figure 3B ) . In the RNA regulation group , swm ( CG10084 ) [14] , Pabp2 ( CG2163 ) [24] , and Hel25E ( CG7269 ) are suggested to interact with each other in the Drosophila interaction database STRING ( http://string . embl . de/ ) and literature [25] . Since Swm and Pabp2 have been reported to physically interact with each other in BIOGRID , the roles of these genes were further analyzed in the context of glycosylation . To analyze the glycosylation defects induced by the knockdown of these genes in further detail , Chp glycans in the swm and Pabp2 knockdown lines were subjected to mass spectrometric analysis . The ratio of the α1 , 3-fucosylated form ( M3F2Gn2 ) at N1012 was markedly decreased in both knockdown lines , whereas the other form ( M3F2Gn3 ) was only slightly affected by swm or Pabp2 knockdown ( Figure 4A ) . These results suggest that Swm and Pabp2 are required for α1 , 3-fucosylation and that the M3F2Gn2 form might be more sensitive than the M3F2Gn3 form to decreases in Swm and Pabp2 . Since swm and Pabp2 mutants have been previously isolated [26] ( Flybase ) , glycosylation defects were also examined in the amorphic and hypomorphic alleles of swm , swmF14 and swmF15 , respectively , and the hypomorphic allele of Pabp2 , Pabp2KG02359 . All cells homozygous for swm mutations were generated in whole Drosophila eyes using a modified FLP/FRT system [27] . Chp was isolated from these mutant eyes and detected by immunoblotting with anti-Chp and anti-HRP antibodies to quantify the amount of α1 , 3-fucose added to Chp . The α1 , 3-fucose added in swmF14 , swmF15 , and Pabp2KG02359 cells decreased to 22 . 7% ± 4 . 8% , 28 . 3% ± 7 . 3% , and 59 . 5% ± 8 . 8% of wild-type levels , respectively ( Figure 4B ) . These results are further evidence from knockdown experiments that Swm and Pabp2 are somehow required for α1 , 3-fucosylation . Although Pabp2 is known to participate in polyA addition , the function of Swm is largely unknown . Thus , we focused on the role of Swm in glycosylation . Modification of α1 , 3-fucose is widely utilized for Drosophila neural proteins . Thus , we first examined whether swm depletion affects not only Chp but also a wide range of other proteins . Homogenates of adult eyes heterozygous or homozygous for swmF14 and swmF15 were analyzed by immunoblot analysis with an anti-HRP antibody ( Figure 5A ) . Several positive bands were detected , including a high-molecular-weight band corresponding to Chp ( ∼150 kDa ) . Quantitative comparison between heterozygotes and homozygotes of the same swm alleles revealed that anti-HRP antibody affinity was reduced in most bands in the homozygotes , with the exception of an approximately 30-kDa product ( Figure 5A and 5B ) . The decrease was much larger in swmF14 than in swmF15 mutants , consistent with the severity of these mutations . Taken together , our data indicate that Swm is required for the α1 , 3-fucosylation of most proteins that are usually modified by α1 , 3-fucose . The observed requirement of Swm for most α1 , 3-fucosylation was also supported by the immunostaining of eyes with anti-HRP antibody . Adult photoreceptor cells from swm knockdown and control flies were simultaneously stained with anti-HRP and anti-Chp antibodies ( Figure 5C ) . Control photoreceptor cells appeared yellow in color because the intensities of green signal ( anti-HRP ) and red signal ( anti-Chp ) were nearly equal in this condition . In contrast , swm knockdown cells appeared relatively red in color , suggesting that the intensity of green signal was reduced compared to the red signal . These data clearly indicate that the signal strength of anti-HRP relative to anti-Chp was reduced in the knockdown photoreceptor cells compared to control cells . Since α1 , 3-fucosylation is directly catalyzed by FucTA , the Swm-dependence of FucTA protein expression was examined . Because no anti-FucTA antibody was available , a knock-in fly was generated in which the fucTA locus was replaced by a Myc-tagged fucTA gene ( Figure S4A and S4B ) . When swm dsRNA was expressed by GMR-Gal4 , FucTA-Myc protein levels were reduced to 47 . 5±2 . 4% of control values as revealed by immunoblot analysis ( n = 2 ) ( Figure 6A and Figure S4C ) . The reduction of FucTA-Myc levels in swm mutant tissue was also examined . We costained the CNS of first instar larvae with anti-Myc and anti-GM130 , a Golgi marker . The number of Golgi harboring FucTA-Myc protein was markedly decreased in swmF14 mutants ( 2 . 0% of total Golgi ) compared to wild-type larvae ( 43 . 1% of total Golgi , Figure 6B ) . The different ratios of FucTA-Myc reduction observed in the biochemical and immunostaining experiments may be due to different levels of Swm depletion in knockdown and mutant flies . Although Swm has an RNA recognition motif ( RNP-1 ) that is present in a wide variety of other RNA binding proteins [26] , it remained undetermined whether Swm directly bound to mRNA . Binding of Swm to fucTA mRNA was thus examined by expressing Flag-tagged Swm in the Drosophila neural cell line BG2-c6 [28] and by performing immunoprecipitation with an anti-Flag antibody . The quantity of endogenous fucTA mRNA that coprecipitated with Swm-Flag was higher in Swm-Flag–expressing cells than in controls in which Swm-Flag was not expressed or immunoprecipitation was performed using a control IgG ( Figure 6C ) . These results strongly suggest that Swm binds to fucTA mRNA in vivo . The amount of fucTA mRNA under the control of Swm was next examined . dsRNA targeting swm was introduced into BG2-c6 cells , and the amount of endogenous fucTA mRNA was measured . In addition to a decrease in swm mRNA , to 27 . 3 ± 1 . 9% of the control level ( n = 3 ) , the amount of fucTA mRNA also decreased to 60 . 6 ± 10 . 5% of the control level ( Figure 6D ) . Furthermore , the stability of fucTA mRNA was examined in swm knockdown cells . The amount of fucTA mRNA was measured at 30 , 60 , and 90 min after total transcription was inhibited by addition of actinomycin D . Decay rates of fucTA were indistinguishable between swm knockdown and control cells ( swm , t1/2 = 97 . 72; control , t1/2 = 100 . 48 , Figure 6E ) . These data suggest that Swm is involved in the expression of fucTA mRNA but not in its stability . Swm was recently reported to incorporate into the complex that regulates nuclear export of mRNAs [29] . The necessity of Swm for the nuclear export of fucTA mRNA was therefore examined in BG2-c6 cells . Following separation of cell lysates into nuclear and cytoplasmic fractions , fucTA mRNA levels were measured in each fraction of swm knockdown and control cells . The amount of fucTA mRNA in the nucleus increased by 9 . 6-fold in swm knockdown cells compared to control gfp knockdown cells ( Figure 6F ) . The nuclear export of actin ( act5c ) mRNA was also examined in swm knockdown cells . The amount of actin mRNA in the nucleus increased by 1 . 7-fold in swm knockdown cells compared to control cells . However , the fold change in nuclear accumulation of actin mRNA was significantly lower than that of fucTA mRNA in swm knockdown cells ( Figure 6F ) . These results suggest that relatively more Swm is required for nuclear export of fucTA mRNA than for nuclear export of actin mRNA . Since nuclear export defects have been reported to result in an abnormally longer polyA length of mRNAs [29] , the polyA length of fucTA mRNA was examined in swm knockdown cells . The polyA length of fucTA mRNA was clearly longer in swm knockdown cells than in control cells ( Figure 6G ) . Collectively , these results indicate that Swm positively regulates α1 , 3-fucosylation by promoting nuclear export of fucTA mRNA in neural cells .
In the current study , the fucTA mutant showed an imbalanced VNC . The VNC longitudinal length-to-width ratio was significantly shorter in fucTA mutants compared to wild-type larvae . To our knowledge , this is the first report of such shortened VNC in the loss-of-function mutant , although elongated VNC has already been reported in mutants for integrin [30] , collagen IV [31] , DHR3 [32] , worniu [33] and C1GalTA [34] . Interestingly , C1GalTA encodes a core 1 galactosyltransferase that adds galactose to O-GalNAc attached to Ser or Thr protein residues . Taken together with our results , glycosylation patterns might affect the VNC shape . Although how α1 , 3-fucose affects VNC formation remains to be revealed , we think that two possible defects may result in a shortened VNC in fucTA mutants . ( 1 ) The aberrantly elongated VNC in the C1GalTA mutant is proposed to result from deglycosylation of laminin , a component of the extracellular matrix . In addition , overexpression of FucTA has recently been reported to enhance cell migration in the embryonic nervous system [35] . By analogy with the case of C1GalTA , cell migration in the larval VNC may be impaired in fucTA mutants because of aberrant interactions between migrating cells and extracellular matrix . ( 2 ) Another possibility is that neural activity may be impaired in fucTA mutants because neural activity has been reported to be necessary for VNC condensation in embryos [31] . This possible defect is consistent with the finding that one of the major α1 , 3-fucosylated proteins is a β-subunit of Na+/K+ ATPase essential for the maintenance of neural cell membrane potentials [36] . It will be interesting to examine whether neural activity is controlled by glycosylation in the future . Three types of genes were identified in this screen: ( 1 ) genes that specifically regulate glycosylation such as glycosyltransferases , ( 2 ) genes with multiple functions including regulation of expression , activity and/or localization of glycosyltransferases , and ( 3 ) genes with primary functions other than glycosylation that secondarily affect glycosylation . For example , knockdown of cytoskeletal regulation and mitochondrial function genes may secondarily affect glycosylation as a result of damage to cell structures and viability . For several reasons , swm might be the second type of gene . Although swm has been previously reported to show involvement in the cell cycle [14] , no glycosylation defects were observed with the knockdown of other known cell cycle regulators such as cdc2 ( CG5363 ) or cdk4 ( CG5072 ) ( Table S2 and S3 ) . In addition , no cell cycle defects could be detected in the fucTA mutant . These data suggest that cell cycle and glycosylation are regulated independently . Therefore , swm would play at least two independent roles in cell cycle and glycosylation . Traffic genes identified in this screen may be another example of the second type of genes . The conserved oligomeric Golgi ( COG ) complex has been reported to participate in the Golgi localization of glycosyltransferases and membrane traffic [37] , but no involvement of the COG complex in glycosylation has been reported in Drosophila . Our screen identified Cog3 as a gene whose knockdown impaired α1 , 3-fucosylation . Thus , localization of FucTA protein was examined in cog3 knockdown photoreceptor cells . Expressed FucTA was hardly localized to the Golgi complex in knockdown photoreceptor cells ( data not shown ) . Moreover , Golgi localization of FucTA was also preliminarily found compromised by knockdown of vps2 and vps20 , other traffic genes ( data not shown ) . These data raise the possibility that some traffic genes may be involved in glycosylation by localizing glycosyltransferases to appropriate Golgi and/or endoplasmic reticulum sites . Our detailed analysis of glycan structures using mass spectrometry revealed that one ( M3F2Gn2 ) of the two forms bearing an α1 , 3-fucose ( M3F2Gn3 and M3F2Gn2 ) is more sensitive to the decrease of swm and Pabp2 than the other ( M3F2Gn3 ) . Two reasons may account for this difference , which are not mutually exclusive . ( 1 ) FucTA productivity differs between these two glycoforms . The structural difference between the glycoforms consists of a GlcNAc moiety attached to the nonreducing end of N-glycans . Given that this GlcNAc moiety has been shown to increase the reactivity of FucTA to the core of N-glycans [6] , we assume that M3FGn3 is preferentially modified by FucTA to form M3F2Gn3 . Therefore , under the condition of reduction , not complete loss , of FucTA by swm and Pabp2 knockdown , M3F2Gn2 might be preferentially lost . ( 2 ) The other possibility is that Swm or Pabp2 regulate Mgat1 or Fused lobes ( Fdl ) [38] , the enzymes that add and remove the GlcNAc moiety , respectively , from the nonreducing end . In fact , the amount of mRNA encoding Mgat1 , but not fdl , was slightly but significantly increased in swm knockdown cells ( Figure S5 ) . This finding is consistent with the selective decrease of M3F2Gn2 induced by swm knockdown . However , it remains undetermined whether the preferential decrease of glycoforms is caused by an increase in Mgat1 , the preferential productivity of FucTA , or both . Expression of mRNA is generally regulated at transcription , splicing , 5′- or 3′-end processing , and nuclear export , and by the stability of the mRNA . The stability of fucTA mRNA was not affected by swm knockdown ( Figure 6E ) . Given that 5′-end processing is essential for mRNA stability , the 5′-end processing of fucTA mRNA is not regulated by Swm . In addition , splicing of fucTA mRNA does not appear to depend on Swm activity since the misspliced form of fucTA mRNA in the swm knockdown cells could not be detected when we amplified fucTA mRNA using PCR primers that targeted different exons ( data not shown ) . Furthermore , both transcription and nuclear export of mRNAs are regulated by the THO/TREX complex [39] and Sus1 [40]; thus , Swm may be a factor participating in both processes , with its depletion resulting in reduced expression and defective nuclear export of fucTA mRNA . We assume that abnormal extension of polyA in swm knockdown cells would be a secondary effect of nuclear export defects , as observed in mutants of genes required for nuclear export [29] . Moreover , Pabp2 , Hel25E/UAP56 [41] , and Nup358 [42] , which participate in the nuclear export of mRNA , were also identified in our screen ( Table S4 ) , suggesting that nuclear export might be essential for glycosylation regulation . Nuclear accumulation of polyA ( + ) RNA in swm knockdown S2 cells was confirmed in our in situ hybridization experiment , as previously reported [43] . However , in neural BG2-c6 cells , weak polyA ( + ) RNA signals in nuclei were comparable between control gfp knockdown and swm knockdown cells ( data not shown ) , suggesting that only a small portion of mRNA might be exported by Swm . In addition , the ratio of nuclear to cytoplasmic fucTA mRNA was relatively higher than that of actin mRNA in swm knockdown BG2-c6 cells ( Figure 6F ) . Collectively , Swm may have preferred mRNAs for nuclear export . Moreover , the export efficiency and/or target RNA preference of Swm may differ between S2 and BG2-c6 cells . Most of the genes identified in our present study have mammalian homologs ( Table S5 ) . Among these , Cog3 has been reported to be involved in glycosylation in organisms ranging from yeast to humans [37] . Interestingly , mutations of some COG components are causative for human diseases including congenital disorders of glycosylation ( CDG ) [44] . Further investigation of the genes identified here will likely provide additional insights into novel glycosylation regulatory mechanisms conserved in organisms ranging from Drosophila to humans and , possibly , into diseases involving glycosylation pathways .
The following fly strains were used: Canton-S as the wild-type strain , GMR-Gal4 ( Bloomington Drosophila Stock Center ( BDSC ) ) , fucTA mutant Pbac{WH}FucTAf03774 ( BDSC ) , swm mutants swmF14 and swmF15 ( BDSC ) , and Pabp2 mutant P{SUPor-P}Pabp2{KG02359} ( BDSC ) . RNAi lines were mainly supplied by the National Institute of Genetics ( Japan ) , with the remainder purchased from the Vienna Drosophila RNAi Center ( VDRC ) . The transgenic flies harboring UAS-dicer2 were also purchased from VDRC . The swm and Pabp2 mutant mosaic clones were generated using the FLP-FRT system [27] . Adult heads from a cross of w[*]; P{w[+t*] ry[+t*] = white-un1}30C swm[F14] P{ry[+t7 . 2] = neoFRT}40A/CyO , P{Wee-P . ph0}2 or w[*]; P{w[+t*] ry[+t*] = white-un1}30C swm[F15] P{ry[+t7 . 2] = neoFRT}40A/CyO , P{Wee-P . ph0}2 with y[1]w[*]; P{w[+mC] = GMR-hid}G1 P{ry[+t7 . 2] = neoFRT}40A , l ( 2 ) CL-L[1]/CyO; P{w[+m*] = GAL4-ey . H}SS5 , P{w[+mC] = UAS-FLP1 . D}JD2 ( BDSC ) , or y[1]; P{y[+mDint2] w[BR . E . BR] = SUPor-P}Pabp2[KG02359] P{neoFRT}42D/SM1 with y[1] , w[*]; P{ry[+t7 . 2] = neoFRT}42D P{y[+t7 . 7] ry[+t7 . 2] = Car20y}44B , P{w[+mC] = GMR-hid}SS2 , l ( 2 ) CL-R[1]/CyO; and P{w[+m*] = GAL4-ey . H}SS5 , P{w[+mC] = UAS-FLP1 . D}JD2 ( BDSC ) were dissected and used for glycosylation analyses as described below . To investigate the localization of FucTA in RNAi-mutant flies , inducible Myc-tagged FucTA-expressing flies ( UAS-fucTA-Myc ) were generated . fucTA cDNA was a gift from S . Nishihara . A DNA fragment encoding the 3Myc sequence was ligated to the 3′end of the coding region of fucTA and inserted into the pUAST vector . The vector was injected into w1118 embryos for the production of transgenic flies . To generate fucTA-Myc knock-in flies ( fucTA-MycKI ) , the procedure was adopted from the ends-out knock in method [45] . Details are shown in Figure S4 . Commercial sources provided biotin-conjugated Lotus; HRP-conjugated ConA , WGA , DSA , LCA , UEA , PHAL4 , PNA , SBA , and DBA lectins ( Seikagaku Kogyo ) ; and HRP-conjugated ABA ( Sigma ) . Anti-HRP antibody was obtained from Jackson Immunoresearch . In addition , the antibodies used in this study are as follows: streptavidin-POD ( Roche ) ; anti-Myc A14 and anti-Myc 9E10 ( Santa Cruz ) ; anti-Flag M2 ( Sigma ) ; HRP-conjugated anti-mouse IgG , HRP-conjugated anti-rabbit IgG , and normal mouse IgG ( Jackson Immunoresearch ) ; Alexa 488-conjugated anti-mouse IgG , Alexa 488-conjugated anti-rabbit IgG , and TOPRO3 ( Invitrogen ) ; and Cy3-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG ( Kirkegaard & Perry Laboratories , Inc . ) . Anti-Chp 24B10 , anti-α-tubulin , 22C10 , BP102 , anti-FasI , anti-FasII , and anti-FasIII were obtained from the Developmental Studies Hybridoma Bank . Anti-Chp antibody N8A [18] and anti-GM130 [46] were generated as described previously . The primer sets used for the generation of transgenic flies are listed in Table S6 . The primer sets used for analysis in cultured cells are listed in Table S7 . The central nervous systems were manually isolated from CS and fucTAf03774 third instar larvae and fixed in 4% paraformaldehyde/PBS at 4°C overnight . After several washes with PBS , brain hemispheres were manually removed and placed flatly onto slide glasses . The images were captured using an AxioCam-equipped Zeiss AxioImager M1 , and the length and width of VNCs were measured using AxioVision Rel . 4 . 7 software . The first screen was carried out using a collection of transgenic fly lines carrying inducible dsRNA constructs . To drive expression of dsRNA for each gene in the eye , males from each RNAi fly line were crossed with GMR-Gal4 virgin females . Mating to inducible RNAi males and egg laying were carried out at 25°C for two days , and the resulting embryos were then incubated at 28°C . After 2 weeks , the hatched knockdown flies were collected and cultured for an additional 14 days at 28°C to enhance the effects of dsRNAs . More than 100 heads per line were isolated and homogenized in lysis buffer consisting of 10 mM sodium phosphate ( pH 7 . 0 ) , 100 mM NaCl , 0 . 5% Triton X-100 , and complete protease inhibitor ( Roche ) . The lysate was incubated with anti-Chp ( 24B10 ) covalently coupled with protein G-Sepharose ( GE Healthcare ) for 2 h . The Chp-bound Sepharose was subsequently washed with lysis buffer , and Chp was eluted with 50 mM triethylamine-acetic acid ( pH 3 . 5 ) . Purified Chp was spotted onto Hybond ECL nitrocellulose membranes ( GE Healthcare ) using a Bio Dot slot format ( Bio-Rad ) . After the membrane was blocked with 2% bovine serum albumin , immuno- or lectin blot analysis was performed with anti-Chp ( N8A ) , anti-HRP , or HRP-conjugated ConA , LCA , DSA , and WGA . HRP-conjugated anti-mouse or HRP-conjugated anti-rabbit IgG was used as the secondary antibody . Detection was performed using Supersignal West Pico Chemiluminescent Substrate ( Thermo ) , and quantification performed using a LAS-1000 Luminescent Image Analysis System ( FujiFilm ) . An example of the dot blot is shown in Figure S2 . Lectin affinity values per Chp values were converted to log values for normalization . On the basis of standard deviations from the values of GMR>GFPRNAi ( as a control ) , genes with a z-score >3 were considered primary candidates . In the primary screen , we performed a single assay round . For the primary candidates , the lectin analysis was repeated ( n = 3 ) . Two-sided Student's t-tests were applied to the reproducibility test data between lectin values of knockdown and control flies . Genes with p<0 . 005 were considered to be reproducible findings . Collection of the second transgenic fly stock lines was designed in such a way that there was no overlap between the primary construct in the original library and the new dsRNA sequence , and that the new dsRNA sequence had a low off-target probability score calculated by dsCheck [22] . Using the second lines , lectin analysis was independently repeated ( n = 2 ) , and the values between the knockdown and control ( GMR>GFPRNAi ) were tested by two-sided Student's t-tests . Genes with p<0 . 05 were considered glycosylation-related genes ( Rank 1 ) . When no change in lectin values was observed solely using GMR-Gal4 , the examination was repeated using GMR-Gal4; UAS-dicer2 ( VDRC ) . RNAi flies from VDRC whose target sequence had no overlap with the primary construct were also used as second lines . For candidate genes that were not evaluated with the second lines , first the number of 19mers with a perfect match to each Drosophila transcript was calculated using dsCheck . The highest number of 19mers with a perfect match to the off-target gene was defined as the “off-target probability score” . The genes with a score below 3 were tested to determine whether knockdown of their suspected off-target genes led to glycosylation defects . When the knockdown of the suspected off-target genes did not lead to any evidence of glycosylation defects , the corresponding genes were added to the list of genes required for glycosylation ( Rank 2 ) . Our final glycosylation hit list was annotated to include information on protein domain , molecular function , and protein family by cross-referencing InterPro ( version 20 . 0 and 21 . 0 ) [47] , Flybase , and Panther [48] . On the basis of the resulting annotation , genes were manually classified into 9 categories . These annotations are shown in Table S4 . MS analysis of glycopeptides was performed as previously described [16] . Purified Chp was digested with modified trypsin ( Promega ) . Glycopeptides and peptide mixtures were separated by reversed-phase high-performance liquid chromatography ( RP-HPLC ) using an Inertsil C18 column ( GL Science ) . The matrices for peptide analysis by matrix-assisted laser desorption/ionization time-of-flight MS ( MALDI-TOF-MS ) spectra were acquired using a Voyager mass spectrometer ( Applied Biosystems ) . MS/MS measurements were performed using an Ultraflex TOF/TOF mass spectrometer with the LIFT-MS/MS facility ( Bruker Daltonics GmbH ) . To investigate the amount of α1 , 3-fucosylation in vivo , frozen sections of adult eye demonstrating swm or gfp knockdown were prepared . Immunostaining was performed as previously described [49] . Fluorescent images were acquired with a laser scanning confocal microscope , LSM700 ( Zeiss ) . BG2-c6 cells were cultured in M3 medium ( Sigma ) containing 10% FBS and 10 µg/mL insulin ( Invitrogen ) at 25°C . Transfection was performed using a calcium phosphate transfection kit ( Invitrogen ) . To construct an expression plasmid for C-terminal Flag-tagged Swm ( Swm-Flag ) , cDNA containing the swm coding region was obtained from Canton-S total RNA by RT-PCR . A DNA fragment encoding the 3Flag sequence was ligated to the 3′end of the swm coding region and inserted into the expression vector pRmHa . For the transient expression of Swm-Flag , 1 . 5 µg plasmid was added to each well of a 24-well plate , in which 5×105 BG2-c6 cells had been seeded . Plasmid expression was induced by incubating cells in the presence of 0 . 1–0 . 5 mM CuSO4 for periods from 3 h to overnight . For knockdown experiments , dsRNA for swm or gfp was produced using in vitro transcription T7 kit ( Takara ) . A 3-µg aliquot of dsRNA was added to each well of a 24-well plate in which 5×105 cells had been seeded . After 5 days , cells were harvested for the following assays . For protein analysis , equal amounts of protein extracted from fly eyes were subjected to SDS-PAGE . After transfer to PVDF membranes ( Millipore ) , membranes were blocked in PBS containing 0 . 05% Tween 20 and 5% skim milk and incubated overnight with the primary antibody , followed by the secondary antibody . A monoclonal anti-α-tubulin antibody was used for normalization . Detection was performed using the Supersignal West Pico Chemiluminescent Substrate . Immunoprecipitation of protein–RNA complexes was performed according to a protocol by Niranjanakumari et al . [50] . BG2-c6 cells transfected with swm-Flag were harvested and cross-linked in PBS containing 0 . 2% PFA . Following sonication in RIPA buffer ( 50 mM Tris-HCl pH 7 . 5 , 1% NP-40 , 0 . 05% SDS , 1 mM EDTA , 150 mM NaCl ) with protease inhibitor ( Nakarai ) , each supernatant was mixed with a monoclonal anti-Flag M2 antibody or normal mouse IgG and then incubated with protein-G beads ( GE Healthcare ) at 4°C for 6 h . Sepharose beads were washed 5 times with RIPA buffer and incubated in 50 mM Tris ( pH 7 . 0 ) , 5 mM EDTA , 10 mM DTT , and 1% SDS at 70°C for 45 min to reverse cross-linking . RNA was extracted from the immunoprecipitates using Sepasol reagent ( Nakarai ) followed by DNase I treatment . The extracted RNA was used as a template to synthesize cDNA using Superscript III ( Invitrogen ) according to the manufacturer's protocol . Total RNA was extracted using the RNeasy mini kit ( Qiagen ) or Sepasol reagent ( Nakarai ) . Complementary DNAs were synthesized using Superscript III according to the manufacturer's protocol . Real-time PCR was carried out using a 7900HT Fast Real Time PCR system ( Applied Biosystems ) with Power Syber Green . Samples were normalized with Drosophila rpl32 . BG2-c6 cells transfected with gfp or swm dsRNA were grown for 5 days , and then actinomycin D ( Nakarai ) was added to a final concentration of 5 µg/mL to arrest de novo RNA synthesis . At 30 , 60 , and 90 min after actinomycin D treatment , the cells were harvested , and fucTA and rpl32 ( control ) mRNA was quantified by real-time PCR as described above . PolyA measurement of fucTA mRNA was carried out as described [51] . PCR fragments from the PAT assay were separated on 2% agarose gels and visualized by Southern blot using DIG DNA Labeling and Detection kit ( Roche ) . Fractionation and RNA isolation were performed as described [52] . The cytoplasmic fraction was extracted by hypotonic buffer ( 10 mM Tris ( pH 7 . 5 ) , 10 mM NaCl , 3 mM MgCl2 , 0 . 5% ( v/v ) Nonident P-40 , 40 units/mL RNase inhibitor , 1 mM DTT ) and then extracted by EDTA buffer ( 10 mM Tris ( pH 7 . 5 ) , 10 mM NaCl , 40 mM EDTA , 0 . 5% ( v/v ) Nonident P-40 , 40 units/mL RNase inhibitor , 1 mM DTT ) . The remaining pellet was used as the nuclear fraction . Sepasol solution was used to purify mRNA from each fraction as described above . The InParanoid database ( version 7 . 0 ) was searched for mammalian homologs [53] . | Glycosylation plays crucial regulatory roles in various biological processes such as development , immunity , and neural functions . Accordingly , some glycans are generated in a stage- and tissue-specific manner . To address how such distinct glycosylation is regulated in different tissues , we performed a large-scale screen for genes involved in glycosylation of a neural-specific protein . We identified 109 genes , 95 of which are assigned for the first time as directly or indirectly involved in glycosylation . We further found that neural-specific glycosylation is regulated at the RNA level , which is a novel regulatory mechanism of tissue-specific glycosylation . | [
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] | 2010 | Identification of Genes Required for Neural-Specific Glycosylation Using Functional Genomics |
Norovirus infection is the leading cause of food-borne gastroenteritis worldwide , being responsible for over 200 , 000 deaths annually . Studies with murine norovirus ( MNV ) showed that protective STAT1 signaling controls viral replication and pathogenesis , but the immune mechanisms that noroviruses exploit to induce pathology are elusive . Here , we show that gastrointestinal MNV infection leads to widespread IL-1β maturation in MNV-susceptible STAT1-deficient mice . MNV activates the canonical Nlrp3 inflammasome in macrophages , leading to maturation of IL-1β and to Gasdermin D ( GSDMD ) -dependent pyroptosis . STAT1-deficient macrophages displayed increased MAVS-mediated expression of pro-IL-1β , facilitating elevated Nlrp3-dependent release of mature IL-1β upon MNV infection . Accordingly , MNV-infected Stat1-/- mice showed Nlrp3-dependent maturation of IL-1β as well as Nlrp3-dependent pyroptosis as assessed by in vivo cleavage of GSDMD to its active N-terminal fragment . While MNV-induced diarrheic responses were not affected , Stat1-/- mice additionally lacking either Nlrp3 or GSDMD displayed lower levels of the fecal inflammatory marker Lipocalin-2 as well as delayed lethality after gastrointestinal MNV infection . Together , these results uncover new insights into the mechanisms of norovirus-induced inflammation and cell death , thereby revealing Nlrp3 inflammasome activation and ensuing GSDMD-driven pyroptosis as contributors to MNV-induced immunopathology in susceptible STAT1-deficient mice .
Human norovirus infection is the most common cause of food-borne gastroenteritis worldwide , responsible for an estimated 684 million cases per year [1] . While norovirus gastroenteritis is self-limiting in immunocompetent individuals , it can evolve to a seriously debilitating and even life-threatening infection in conditions of genetic or acquired immunosuppression [2] . Moreover , norovirus infections also account for more than 200 , 000 deaths annually , mostly affecting children below five years of age in developing countries [1] . Despite this significant global socio-economic burden , the cellular processes that are induced upon gastrointestinal norovirus infection are poorly understood . Norovirus challenge studies in human volunteers showed that viral shedding lasts long after the acute vomiting and diarrhea symptoms have subsided , and also revealed that in some individuals the viral burden peaks after clinical symptoms had been resolved [3] . This poor correlation between viral titers and gastrointestinal manifestations suggests that in addition to norovirus replication , some of the host innate responses to infection may contribute to provoking pathology . Studies using the murine norovirus ( MNV ) model showed that Interferon ( IFN ) -induced STAT1-dependent responses are required for controlling viral replication and associated pathogenesis [4–6] , but the deleterious innate immune mediators that contribute to norovirus-induced intestinal and systemic inflammation remain to be elucidated . Several studies identified inflammasome activation as a crucial innate immune mechanism that protects the host from a wide variety of viral infections [7 , 8] . Inflammasomes are cytosolic multi-protein complexes in which caspase-1 and -11 proteolytic activities exert important functions in innate immunity by mediating maturation of the pro-inflammatory cytokines Interleukin ( IL ) -1β and IL-18 , as well as by initiating a lytic form of cell death termed pyroptosis through cleaving Gasdermin D ( GSDMD ) [9] . For instance , inflammasome activation was shown to preserve respiratory function and to prevent lethality upon Influenza infection [10–12] . Similar host protective roles for inflammasomes were reported in encephalitis caused by West Nile virus as well as in gastroenteritis induced by rotavirus [13–15] . However , the role of inflammasomes in norovirus-induced immunopathology and norovirus pathogenesis remains to be elucidated . Here , we show that inflammasome activation has a detrimental rather than a beneficial function during gastrointestinal norovirus infection . We show that MNV activates the canonical Nlrp3 inflammasome leading to IL-1β secretion as well as GSDMD-dependent pyroptosis in macrophages . In addition , we show that these Nlrp3- and GSDMD-mediated responses do not contribute to the diarrheic manifestations of gastroenteritis but do promote MNV-induced intestinal inflammation and lethality in STAT1-deficient mice . These data show that Nlrp3 inflammasome signaling and GSDMD-driven pyroptosis contribute to immunopathology in the setting of gastrointestinal norovirus infection .
As an in vivo model for norovirus-induced gastroenteritis with concomitant systemic inflammatory responses , we infected Stat1-/- mice via oral gavage with the non-persistent MNV-1 CW1 strain [16] ( hereafter referred to as MNV ) , which was lethal for infected Stat1-/- mice but not for their Stat1+/- littermates ( S1A Fig ) . In order to identify putative innate immune mediators contributing to MNV-induced intestinal inflammation and lethality in Stat1-/- mice , we first characterized in vivo MNV replication kinetics . To this end , cohorts of Stat1-/- and Stat1+/- littermate mice were orally infected with MNV and tissues were harvested at one , two or three days post-infection . Quantification of viral RNA levels showed that Stat1-/- mice displayed overall significantly higher viral burdens when compared with Stat1+/- littermates over the entire gastrointestinal tract , in Peyer’s patches ( PP ) and mesenteric lymph nodes ( MLN ) , as well as in the spleen and liver only at 3 days post-infection ( S1B–S1J Fig ) . Analysis of IFNβ expression levels at a time point when systemic viral spread had occurred , indicated that MNV infection of Stat1-/- mice caused robust anti-viral type I IFN responses in all of these tissues examined , with the exception of the distal colon ( S2 Fig ) . MNV genomes detected in the distal colon may therefore derive from non-replicating viral particles , or alternatively MNV-induced IFN responses in the distal colon may occur with different kinetics or may be less potent compared to other parts of the gastrointestinal tract . Nevertheless , the above analyses showed that Stat1-/- mice displayed intestinal , as well as systemic dissemination of MNV at three days post-infection , which was associated with anti-viral responses within the corresponding tissues . We next investigated how these viral replication kinetics correlated with MNV-induced diarrhea and intestinal inflammation . Consistent with the peak of viral titers in these mice , we observed that gastrointestinal MNV infection caused diarrhea only in Stat1-/- mice at three days post-infection ( S3A Fig ) . Gastrointestinal MNV infection is known to provoke only subtle inflammation in the intestine , even in highly susceptible Stat1-/- mice . For instance , a study performing detailed histological examinations could not reveal increased numbers of inflammatory cells in the intestinal lamina propria of MNV-infected Stat1-/- mice [17] . Therefore , to monitor intestinal inflammation in MNV-infected mice non-invasively over time in a sensitive manner we measured fecal Lipocalin-2 ( Lcn-2 ) levels that were shown to correlate closely with varying degrees of inflammation restricted to the intestine [18] . While fecal Lcn-2 levels did not increase in MNV-infected Stat1+/- mice , Stat1-/- littermates displayed a significant elevation of fecal Lcn-2 levels at two and three days after oral MNV infection ( S3B Fig ) . This observation validated fecal Lcn-2 as a sensitive marker of MNV-induced intestinal inflammation , and indicated that the kinetics of intestinal pathology correlated with previously observed viral replication and anti-viral responses . Therefore , three days post-infection was selected as an appropriate time point for screening innate immune responses potentially associated with MNV-induced intestinal and systemic pathology . For this purpose , we quantified the expression levels of a panel of inflammatory chemokines and cytokines at selected MNV replication sites in Stat1-/- and Stat1+/- littermates that had been infected for three days with either UV-inactivated or live MNV . Statistical log-linear regression analysis identified several chemokines and cytokines that were specifically associated with MNV-induced pathology , as defined by statistically significant higher expression levels in Stat1-/- mice infected with live MNV relative to Stat1-/- mice challenged with UV-inactivated MNV , as well as relative to live MNV-infected Stat1+/- littermate controls ( Fig 1A , S1 Table ) . The various inflammatory cytokines expressed at higher levels in live MNV-infected Stat1-/- mice indicated a complex immune response raised to MNV in these mice . For instance , live MNV-infected Stat1-/- mice displayed increased levels of IFNγ , IL-4 and IL-17A in MLN , suggestive of ongoing ILC1/Th1 , ILC2/Th2 as well as ILC3/Th17 innate and/or adaptive lymphocyte responses ( Fig 1A , S1 Table ) . Amongst the unambiguously innate immune mediators associated with severity of MNV infection in Stat1-/- mice , the cell death-associated alarmin IL-1α and the inflammasome-dependent cytokine IL-1β were prominent in both MLN and the spleen ( Fig 1A and 1B ) . Because IL-1β is produced as a cytosolic precursor protein that needs maturation by inflammasomes for exerting biological activity , we performed additional Western blotting analyses . Consistent with inflammasome activation occurring in MNV-infected mice , these analyses showed in vivo maturation of pro-IL-1β to its active IL-1β form in live MNV-infected Stat1-/- MLN and spleen ( Fig 1C and 1D ) . In addition , although overall IL-1β levels in liver were not significantly increased ( Fig 1A and 1B ) , liver tissue of a subset of live MNV-infected Stat1-/- mice contained detectable levels of mature IL-1β ( Fig 1E ) . Together , these results showed that Stat1-/- mice produced mature IL-1β three days after gastrointestinal MNV infection . In line with our observations showing that MNV-infected Stat1-/- mice displayed detectable intestinal inflammation as well as systemic viral spread only at three days post-infection , we could not detect increased IL-1β levels in MNV-infected Stat1-/- mice at earlier time points post-infection in any tissue examined ( S4 Fig ) . These correlating cytokine , intestinal inflammation and systemic viral dissemination kinetics suggested that IL-1β could contribute to local as well as systemic MNV-induced pathologies , prompting us to further investigate the mechanisms by which MNV induces IL-1β maturation and release . Inflammasomes are cytosolic multiprotein complexes that engage caspase-1 and/or -11 to exert both inflammatory and cytotoxic functions in response to infectious agents [7] . Since myeloid cells are major targets during MNV infection [16 , 19 , 20] , we next turned to primary bone marrow-derived macrophages ( BMDMs ) as a controlled ex vivo setting to dissect the mechanisms underlying inflammasome responses to MNV infection . MNV-infected macrophages that were primed with the TLR2 agonist Pam3CSK4 secreted IL-1β into the culture supernatant starting 12–16 hours post-infection ( Fig 2A ) . This IL-1β secretion was associated with proteolytic maturation of pro-IL-1β and caspase-1 ( Fig 2B ) , confirming MNV-induced inflammasome activation in these cells . In contrast , unprimed MNV-infected macrophages failed to secrete IL-1β , consistent with the need for prior transcriptional upregulation of pro-IL-1β ( Fig 2A ) . Nevertheless , these unprimed WT macrophages displayed caspase-1 processing starting 12 hours after MNV infection ( Fig 2C ) , demonstrating that MNV itself has the capacity to trigger inflammasome activation even in the absence of sufficient amounts of pro-IL-1β that would contribute to inflammatory responses . We next set out to dissect the composition of the MNV-activated inflammasome . Experiments in caspase-11-deficient primary macrophages showed that caspase-11 did not contribute to MNV-induced caspase-1 activation and that the latter was sufficient for mediating IL-1β maturation and secretion upon MNV infection ( Fig 2D–2F ) . Next , MNV infections in primary macrophages lacking both caspase-1 and -11 , or lacking the adaptor protein ASC demonstrated that MNV induced maturation and secretion of IL-1β in an ASC- and caspase-1-dependent manner ( Fig 2G–2I ) . While MNV infections in Aim2- , Nlrc4- and Nlrp6-deficient primary macrophages showed that these Pattern Recognition Receptors were dispensable for MNV-induced inflammasome activation ( S5A–S5D Fig ) , MNV did not provoke caspase-1 processing or downstream IL-1β maturation and secretion upon infecting primary or immortalized Nlrp3-/- macrophages ( Fig 2J–2L , S5A–S5E Fig ) . Importantly , despite abrogating MNV-induced inflammasome responses , the absence of Nlrp3 did not impair MNV replication rates or MNV-induced type I IFN responses in macrophages ( S6A and S6B Fig ) , showing that MNV-induced inflammasome activation does not interfere with the protective IFN responses that are triggered upon MNV dsRNA recognition , at least in vitro . Together , the above observations established activation of the canonical Nlrp3 inflammasome as an innate immune pathway responsible for IL-1β secretion from MNV-infected macrophages . We next investigated the cellular mechanisms by which MNV-induced inflammasome activation mediated secretion of IL-1β . Several MNV infection studies using RAW264 . 7 macrophages showed that MNV provokes apoptotic cell death [21–24] . However , analyzing the kinetics of apoptotic signaling and cellular membrane permeability by simultaneous real-time monitoring of cleavage of a fluorogenic caspase-3/7 substrate and of cell-impermeable Sytox Green ( SG ) uptake , respectively , showed that MNV-infected primary macrophages displayed SG uptake due to membrane permeability starting from 12 hours post-infection , while caspase-3/7 activity was detected only at later stages of MNV infection ( Fig 3A ) . Consistent with this observation arguing against an exclusive role for apoptosis in MNV-induced cell death , confocal live cell imaging of MNV-infected primary macrophages revealed cells undergoing an increase in membrane permeability followed by cellular swelling , characteristic of a necrotic cell death mode ( Fig 3B , S1 Movie ) . Importantly , the above real-time and imaging analyses detected MNV-induced membrane permeability and cellular swelling from 12 hours post-infection , similar as observed for caspase-1 maturation and IL-1β secretion ( see Fig 2A–2C ) . These concordant kinetics suggested a functional link between inflammasome activation and loss of membrane integrity . Inflammasomes are known to provoke a lytic cell death mode termed pyroptosis . The biochemical hallmark of inflammasome-induced pyroptosis is the proteolytic processing of Gasdermin D ( GSDMD ) , resulting in its N-terminal p30 fragment capable of generating pores in the cellular membrane that eventually lyse the cell due to osmotic pressure build-up [25–30] . We therefore performed Western blotting analyses showing that MNV-infected BMDMs displayed GSDMD processing to its p30 fragment starting at 12 hours post-infection ( Fig 3C ) , in line with previously observed kinetics of inflammasome activation . In contrast , proteolytic activation of caspase-3 indicative of apoptotic cell death was detected only at 24 hours after MNV infection ( Fig 3C ) . Together , the above real-time imaging and biochemical observations suggested that apoptosis is a late-stage event after infecting primary macrophages with MNV , whereas a population of MNV-infected macrophages undergo an earlier lytic cell death associated with GSDMD processing , indicative of pyroptosis . After observing MNV-induced GSDMD processing concordant with the appearance of lytic cell death , we next sought to validate the functional role of GSDMD in MNV-induced cell death and IL-1β release . Real-time SG uptake analyses showed that both unprimed and TLR2-primed primary Gsdmd-/- macrophages displayed a delayed membrane permeability increase upon MNV infection ( Fig 3D–3E ) . Furthermore , in accordance with our above observations identifying Nlrp3 and caspase-1 but not caspase-11 as inflammasome components engaged by MNV ( see Fig 2D–2L ) , Nlrp3-/- and Casp1-/- macrophages showed a delay in MNV-induced membrane permeability similar to Gsdmd-/- cells , while Casp11-/- macrophages responded like WT cells ( Fig 3D–3E ) . Further biochemical evidence for inflammasome-dependent pyroptosis in MNV-infected macrophages was obtained by Western blotting analyses showing that MNV-induced generation of the GSDMD p30 fragment did not require caspase-11 ( Fig 3F ) but was dependent on the presence of Nlrp3 ( Fig 3G ) , consistent with the opposing roles of these proteins in MNV-induced inflammasome activation . We next evaluated the role of GSDMD-mediated pyroptosis in MNV-induced release of IL-1β from macrophages . While GSDMD deficiency did not affect maturation of caspase-1 or pro-IL-1β ( Fig 3H ) –consistent with its role downstream of inflammasome activation–also MNV-induced secretion of IL-1β into the culture supernatant was not diminished in Gsdmd-/- macrophages ( Fig 3I ) . This persisting IL-1β secretion in MNV-infected Gsdmd-/- macrophages aligns with the incomplete blockade of SG uptake in these cells , and supports the existence of additional mechanisms contributing to cellular permeability during MNV infection . As IL-1β secretion triggered by canonical inflammasomes indeed was reported to involve also GSDMD-independent mechanisms [25] , we investigated whether necroptosis contributed to MNV-induced IL-1β release . Necroptosis is executed by the pseudokinase MLKL , which upon phosphorylation creates nanopores in the plasma membrane leading to cellular swelling and lysis [31 , 32] . This MLKL-induced membrane damage was suggested as a trigger for Nlrp3 inflammasome activation [33 , 34] , raising the possibility that upon MNV infection , MLKL could act upstream to activate the Nlrp3 inflammasome as well as downstream to help release mature IL-1β through necroptosis . However , real-time SG uptake measurements did not reveal delayed membrane permeability in MNV-infected Mlkl-/- macrophages ( S7A and S7B Fig ) . In accordance , the kinetics of MNV-induced inflammasome activation and release of mature IL-1β were unchanged in Mlkl-/- macrophages ( S7C–S7E Fig ) . Together , these results showed that MLKL was not required for MNV-induced inflammasome responses . In addition , these results dismissed a role for MLKL-mediated necroptosis as a GSDMD-independent lytic cell death contributing to IL-1β release from MNV-infected cells , leaving apoptotic cells as the remaining potential source of IL-1β secretion . Interestingly , recent studies showed that GSDMD-deficient macrophages could relay inflammasome signaling into the execution of apoptotic cell death [35 , 36] . In support of this idea , real time monitoring of a fluorogenic caspase-3/7 substrate in MNV-infected BMDMs showed increased apoptotic activity in the absence of GSDMD ( S8A Fig ) . Moreover , Gsdmd-/- BMDMs also showed earlier and stronger caspase-3 cleavage upon MNV infection compared to WT BMDMs ( S8B Fig ) . Together , these observations showing increased apoptotic signaling in Gsdmd-/- macrophages suggest that the residual IL-1β release that is detected in the absence of GSDMD-driven pyroptosis might derive from apoptotic cell bodies that lyse through secondary necrosis . As our above observations pointed to a dual cytokine maturation and cell death inducing role of the Nlrp3 inflammasome upon MNV infection , we next evaluated the role of Nlrp3 in MNV-susceptible Stat1-/- conditions . Interestingly , we noted pronounced IL-1β secretion from Stat1-/- macrophages without the need for prior TLR2 priming ( Fig 4A ) . Indeed , while caspase-1 activation and downstream GSDMD p30 generation were evident in both unprimed Stat1+/- and unprimed Stat1-/- macrophages , STAT1 deficiency was additionally associated with pro-IL-1β processing leading to secretion of its mature form ( Fig 4A and 4B ) . The observed effect of STAT1 deficiency allowing IL-1β maturation in unprimed macrophages was specific for MNV-induced Nlrp3 inflammasome activation , as unprimed Stat1-/- macrophages did not display IL-1β maturation upon triggering the Nlrp3 inflammasome with ATP , Nigericin or MSU ( S9 Fig ) . Closer Western blotting examination of unprimed MNV-infected Stat1-/- macrophages revealed that STAT1 deficiency was associated with enhanced expression of pro-IL-1β but not of the Nlrp3 or ASC inflammasome components ( Fig 4B ) . Since MNV-infected unprimed Stat1-/- macrophages displayed faster MNV replication rates compared to Stat1+/- cells ( Fig 4C ) , more pronounced pro-IL-1β expression could be caused by enhanced dsRNA-induced NF-κB signaling triggered by increased viral RNA . Therefore , because MNV-induced NF-κB responses rely on dsRNA triggering of the MDA5-MAVS pathway [37] , we examined the role of MAVS in MNV-induced inflammasome responses . Deleting MAVS in otherwise WT BMDMs abrogated MNV-induced IFNβ secretion as expected ( S10A Fig ) , but did not alter MNV-induced inflammasome responses as assessed by IL-1β secretion as well as by pro-IL-1β , caspase-1 and GSDMD processing ( S10B–S10D Fig ) , showing that MNV-induced activation of the Nlrp3 inflammasome does not rely on MAVS signaling . Interestingly however , unlike unprimed STAT1-deficient macrophages , unprimed MAVS-deficient macrophages did not display increased pro-IL-1β expression upon MNV infection and did not alter MNV replication rates in unprimed conditions ( S10D and S10E Fig ) . These pronounced differences between the effects of deleting either STAT1 or MAVS during MNV infection of unprimed BMDMs supported the idea that enhanced MNV-induced signaling associated with increased viral replication could explain the propensity of unprimed Stat1-/- macrophages to boost pro-IL-1β expression . Indeed , abrogating MAVS-mediated signaling in Stat1-/- macrophages did not only abolish MNV-induced IFNβ secretion as expected ( S11 Fig ) , but the resulting Stat1-/-Mavs-/- BMDMs also lost their ability to secrete IL-1β upon MNV infection in unprimed conditions ( Fig 4D ) . Additional MAVS deletion in Stat1-/- macrophages did not alter inflammasome responses in terms of caspase-1 cleavage ( Fig 4E ) , consistent with its dispensable role in MNV-induced inflammasome activation as observed on a WT background , but it abrogated the increase in pro-IL-1β levels observed in unprimed Stat1-/- BMDMs , resulting in the lack of IL-1β maturation in unprimed MNV-infected Stat1-/-Mavs-/- BMDMs ( Fig 4E ) . Together , our observations in MNV-infected Stat1-/- , Mavs-/- and Stat1-/-Mavs-/- macrophages showed that STAT1 controls MNV-induced inflammasome-mediated IL-1β responses by restricting MAVS-mediated signaling towards upregulation of pro-IL-1β levels . However , while being responsible for this MNV-induced priming of the inflammasome , MAVS signaling does not participate in the Nlrp3 activation step of MNV-induced inflammasome responses . The above ex vivo experiments indicated that STAT1 signaling prevents the pro-inflammatory effects of MNV-induced Nlrp3 inflammasome activation by suppressing expression of its pro-IL-1β substrate . As this could be one of the mechanisms by which STAT1 signaling controls MNV pathogenesis , we generated Stat1-/-Nlrp3-/- mice to investigate whether Nlrp3 inflammasome-mediated IL-1β secretion contributes to the MNV-susceptible phenotype conferred by STAT1 deficiency . Whereas both MNV-infected Stat1-/-Nlrp3-/- and Stat1-/-Nlrp3+/- macrophages produced pro-IL-1β in the absence of TLR priming , MNV only triggered caspase-1 activation , GSDMD cleavage , pro-IL-1β maturation as well as IL-1β secretion in Stat1-/-Nlrp3+/- cells ( Fig 5A–5B ) . This demonstrated that Nlrp3 deficiency prevents the enhanced secretion of bio-active IL-1β from Stat1-/- macrophages by blocking its maturation ( Fig 5B ) . To evaluate whether Nlrp3 was also responsible for inflammasome responses upon MNV infection in vivo , Stat1-/-Nlrp3-/- and Stat1-/-Nlrp3+/- littermates were infected with MNV through the oral route . Western blotting analysis showed that both spleen and MLN from MNV-infected Stat1-/-Nlrp3+/- mice displayed maturation of IL-1β , which was abrogated in MNV-infected Stat1-/-Nlrp3-/- organs ( Fig 5C–5D ) . Moreover , these analyses revealed that MLN from MNV-infected Stat1-/-Nlrp3+/- mice also displayed Nlrp3-dependent GSDMD cleavage after MNV infection , indicative of pyroptosis ( Fig 5D ) . Together , these observations demonstrated that gastrointestinal MNV infection in Stat1-/- mice elicits Nlrp3-dependent IL-1β responses in MLN and spleen , as well as Nlrp3-dependent pyroptosis in MLN based on the appearance of the N-terminal p30 fragment of GSDMD as its biochemical hallmark . We next evaluated the pathophysiological outcome of Nlrp3-mediated inflammasome responses by assessing MNV-induced lethality in Stat1-/-Nlrp3-/- mice . Gastrointestinal MNV infections revealed that Stat1-/-Nlrp3-/- mice survived statistically significantly longer than their Stat1-/-Nlrp3+/- littermates ( Fig 5E ) , clearly showing that abrogating Nlrp3-mediated IL-1β maturation has a beneficial effect in overall MNV-induced immunopathology in STAT1-deficient mice . Next , to examine whether GSDMD-driven pyroptosis takes part in the Nlrp3-mediated effects during MNV-induced immunopathology , we also generated Stat1-/-Gsdmd-/- mice . Interestingly , Stat1-/-Gsdmd-/- mice phenocopied the statistically significant survival advantage of Stat1-/-Nlrp3-/- mice upon gastrointestinal MNV infection when compared to Stat1-/- mice ( Fig 5F ) . This observation indicated that also GSDMD-driven pyroptosis is crucially involved in the deleterious systemic effects of MNV contributing to lethality in Stat1-/- mice . Finally , in order to evaluate whether Nlrp3 inflammasome activation and GSDMD-driven pyroptosis also exerted deleterious effects locally in the intestinal tract , we evaluated diarrhea responses and we measured fecal Lcn-2 levels in MNV-infected Stat1-/-Nlrp3-/- and Stat1-/-Gsdmd-/- mice . These analyses showed that gastrointestinal MNV infection provoked similar degrees of diarrhea in Stat1-/-Nlrp3-/- and Stat1-/-Gsdmd-/- mice when compared with Stat1-/- mice ( Fig 5G ) , indicating that inflammasome activation does not take part in MNV-induced diarrheic responses . In contrast , the induction of fecal Lcn-2 observed in MNV-infected Stat1-/- mice at three days post-infection was significantly diminished in both Stat1-/-Nlrp3-/- and Stat1-/-Gsdmd-/- mice ( Fig 5H ) . Taken together , while not overtly involved in the diarrhea responses that are most relevant to norovirus gastroenteritis in immunocompetent humans , the fecal Lcn-2 results show that Nlrp3 inflammasome and GSDMD pyroptosis responses do not only take part in systemic inflammatory responses that most likely underlie MNV-induced lethality , but also contribute to local MNV-induced inflammation in the intestine . Thus , our in vivo observations identify activation of the Nlrp3 inflammasome and ensuing GSDMD-driven pyroptosis as a critical innate immune response to MNV that–in contrast to the previously described MNV-induced signaling pathways leading to protective IFN responses [37 , 38]–exerts an immunopathologic role upon gastrointestinal MNV infection in Stat1-/- mice .
In this study , we reveal activation of the canonical Nlrp3 inflammasome leading to IL-1β secretion and GSDMD-dependent pyroptotic cell death as a detrimental innate immune response triggered by MNV . Given that several studies demonstrated protective roles for inflammasomes in various viral infections [10–15] , our observation that the Nlrp3 inflammasome contributes to MNV-induced lethality in Stat1-/- mice is remarkable . Interestingly however , Influenza infection studies have shown that the level of host IFN responsiveness determines the physiological outcome of inflammasome activation in viral infections . While inflammasome deficiency was detrimental during Influenza infection when compared with wild-type mice [10–12] , abrogating inflammasome signaling prevented Influenza-induced lethality in Tlr7-/-Mavs-/- mice that are impaired in mounting type I IFN responses to Influenza [39] . Our study showing that the Nlrp3 inflammasome contributes to lethality upon gastrointestinal MNV infection in IFN-unresponsive Stat1-/- mice is in line with this observation in Influenza-infected mice . Moreover , our observation that STAT1 signaling prevents MAVS-mediated upregulation of pro-IL-1β suggests that enhanced expression of this inflammasome substrate may be one of the reasons why inflammasome activation is detrimental rather than protective in the absence of IFN signaling . Although enhanced pro-IL-1β expression enables Stat1-/- cells to produce more mature IL-1β upon inflammasome activation , releasing this mature IL-1β across the plasma membrane is still needed to provoke the overwhelming inflammatory responses that may contribute to lethality in Stat1-/- mice . While necroptosis was suggested to perform such a role upon infection with Influenza [40 , 41] , MNV did not provoke MLKL-mediated membrane permeability but rather induced GSDMD-mediated pyroptosis . Although this contrasts with previous studies showing that MNV infection caused apoptotic cell death in RAW264 . 7 macrophages [21–24] , the latter cell line is known to lack ASC expression [42] . This implies that inflammasome activation and downstream pyroptosis induction were defective in these cells . As such , apoptotic cell death in MNV-infected RAW264 . 7 cells is in line with our observation that primary macrophages display Casp3/7 activity at late stages upon MNV infection , and this notion supports a balance between pyroptosis and apoptosis during MNV infection in macrophages . Accordingly , we observed that MNV infection provoked elevated Casp3/7 activity and more pronounced caspase-3 cleavage in the absence of GSDMD , suggesting that Gsdmd-/- cells can switch to apoptotic cell death upon MNV infection . This could explain our observation that MNV-infected GSDMD-deficient macrophages still released IL-1β , as secondary necrosis of apoptotic cell bodies may also contribute to IL-1β release in macrophage cultures . Despite this in vitro observation predicting that GSDMD deficiency would not be able to prevent MNV-induced inflammasome-mediated immunopathology , our in vivo experiments demonstrated that Stat1-/-Gsdmd-/- mice reproduced both the survival advantage and the diminished fecal Lcn-2 levels of Stat1-/-Nlrp3-/- mice upon gastrointestinal MNV infection . These concordant observations in Stat1-/-Gsdmd-/- and Stat1-/-Nlrp3-/- mice indicate that Nlrp3 inflammasome-induced pyroptosis represents the dominant cell death mode promoting MNV-induced immunopathology . While difficult to obtain experimental evidence explaining the discordant in vitro and in vivo observations , it is plausible that the IL-1β release deriving from post-apoptotic necrosis of Gsdmd-/- cells in vitro is efficiently prevented by timely efferocytosis of apoptotic cells in vivo . Further supporting our observations pointing to Nlrp3-mediated pyroptosis as the physiologically relevant cell death mode during gastrointestinal MNV infection , Van Winkle et al . recently showed that MNV induced a lytic cell death mode in bone-marrow derived dendritic cells as well as in the BV2 microglia cell line [20] . Moreover , the ability of different MNV strains to induce this lytic cell death correlated with their persistence in the host , as lytic cell death was accompanied with myeloid cell recruitment to the MLN and the PP that provided MNV with novel target cells [20] . Although future experiments will be required to validate this hypothesis , our observations suggest that this cell death mediated process supporting systemic persistence of acute MNV infection could involve Nlrp3-induced and GSDMD-dependent pyroptosis . In conclusion , this work contributed to a more detailed understanding of norovirus-induced inflammatory and cell death pathways in vitro and in vivo . We identify Nlrp3 inflammasome activation and downstream GSDMD-dependent pyroptosis as an MNV-induced innate immune and cell death response . We demonstrate that Nlrp3 inflammasome activation by MNV provokes both GSDMD-dependent pyroptosis as well as secretion of mature IL-1β in macrophages . We further show that these inflammasome responses contribute to lethality in MNV-susceptible Stat1-/- mice . In addition , Stat1-/-Gsdmd-/- and Stat1-/-Nlrp3-/- mice showed diminished fecal Lcn-2 levels upon gastrointestinal MNV infection . Although the latter observations show that inflammasome responses also act locally to promote MNV-induced intestinal inflammation , diarrheic reactions upon gastrointestinal MNV infection were not decreased in Stat1-/-Gsdmd-/- or Stat1-/-Nlrp3-/- mice . This indicates that the mechanisms regulating intestinal inflammation and diarrhea in MNV-infected Stat1-/- mice are uncoupled , and suggests that the immunopathologic role of inflammasome activation in Stat1-/- mice may not be extrapolated to human norovirus gastroenteritis as occurring in immunocompetent individuals . Instead , our observations showing inflammasome responses upon systemic MNV dissemination such as occurring in Stat1-/- mice suggest that Nlrp3 inflammasome activation and GSDMD-driven pyroptosis may represent an immunopathologic response that could be more relevant in life-threatening cases of human norovirus infections such as in immunocompromised individuals [2] .
All animal experiments were performed according to institutionally approved protocols according to national ( Belgian Laws 14/08/1986 and 22/12/2003 , Belgian Royal Decree 06/04/2010 ) and European ( EU Directives 2010/63/EU , 86/609/EEG ) animal regulations . Animal protocols were reviewed and approved by the Ethical Committee Animal Experimentation VIB site Ghent—Ghent University—Faculty of Sciences ( permit number LA1400091 ) with approval ID 2016–030 . All necessary efforts were made to minimize suffering of the animals . Stat1−/− , ASC−/− , Casp1/11−/− , Casp1−/− , Casp11−/− , Nlrp3−/− , Nlrp6-/- , Nlrc4−/− , Aim2−/− , Gsdmd−/− , Mlkl−/− and Mavs-/- mice , either generated on C57BL/6 background or backcrossed at least ten generations to C57BL/6J background , were described previously [25 , 43–52] . In all in vivo experiments as well as ex vivo primary cell experiments , controls were either in-house bred C57BL/6J mice ( WT ) or the +/+ or +/- littermates of the respective mice as indicated . All animal experiments were performed according to approved protocols according to institutional , national and European guidelines . All mice used in this study were bred and housed in individually ventilated cages ( IVC ) in the Specific Pathogen Free facility at Ghent University , were sex- and age-matched and were fed autoclaved standard rodent feed ( Ssniff , Soest , Germany ) at libitum with free access to drinking water . In all experiments , up to 5 mice were housed per cage in a 12-h light-12-h dark cycle . Mice were assigned to experimental groups according to genotype and treatment . Bone-marrow-derived macrophages ( BMDMs ) were differentiated in Iscove’s modified Dulbecco’s medium ( IMDM ) supplemented with 30% L929 cell conditioned medium , 10% heat-inactivated FBS , 1% Gibco non-essential amino acids and 1% penicillin/streptomycin at 37°C and 5% CO2 . After 6 days , the differentiation medium was aspirated and the cells were scraped in cold infection medium ( IMDM supplemented with 10% heat-inactivated FBS and 1% Gibco non-essential amino acids ) . Then , 8 . 5 x 105 cells per well were seeded in a 12-well plate and incubated overnight at 37°C and 5% CO2 . On day 7 the MNV infection experiments were initiated , all with the MNV-1 CW1 strain [53] . Macrophages derived from immortalized myeloid progenitors ( generated by Eicke Latz , Institute of Innate Immunity , Bonn , Germany; and kindly provided by Dr . Ashley Mansell , Centre for Innate Immunity and Infectious Diseases , Clayton , Australia ) were grown in DMEM ( Gibco ) supplemented with 10% heat-inactivated FBS and 2 mM glutamine . For MNV infection experiments , unprimed macrophages were used , or macrophages were first primed with TLR agonists . TLR2 priming in BMDMs was performed by aspirating the medium and adding 500 μl/well of culture medium ( DMEM + 10% FBS ) containing 500 ng/ml Pam3CSK4 for 5h . TLR4 priming of immortalized macrophage was performed by aspirating the medium and adding culture medium ( DMEM + 10% FBS ) containing 100 ng/ml LPS for 3h . MNV infections were performed using viral dilutions made from a stock solution stored at -80°C in cell culture medium . First , 250 μl of medium was aspirated and then either 250 μl of medium was added ( mock-infected controls ) , 250 μl of UV-inactivated MNV was added , or 250 μl of live MNV was added , the latter two both at a multiplicity of infection ( MOI ) 5 . Control UV-inactivated MNV was prepared by placing the virus under UV light for at least 1h . After the infection the cells were incubated for 1h at 37°C with regular shaking , after which the medium was removed , the cells were washed with PBS and 1 ml culture medium was added . The cells were then incubated for indicated time periods at 37°C and 5% CO2 before sample collection . For canonical Nlrp3 inflammasome triggering , BMDMs were stimulated with 1 μg/ml Ultrapure LPS from Salmonella Minnesota ( Invivogen ) for 3 hours , after which ATP ( 5 mM , Roche ) , Nigericin ( 20 μM , Sigma Aldrich ) or Silica ( 0 . 5 mg/ml , Min—U—Sil5 US Silica ) were applied for 45 min , 45 min and 6 hours , respectively , in 37°C and 5% CO2 cell culture conditions . Cellular membrane integrity and Caspase-3/7 enzymatic activity were determined with an IncuCyte FLR imaging system ( Essen Bioscience ) , using the non-cell-permeable SYTOX Green ( SG ) DNA staining agent ( 30 nM ) ( Invitrogen ) and the Casp-3/7 fluorogenic substrate ( 1:46 v/v ) ( Life technologies ) , respectively , according to the manufacturer’s protocol . The percentage of positive cells was calculated with the IncuCyte software package . These percentages were normalized to cell confluency as well as to a 100% cell count control achieved by SG-labelling of Triton X-100 treated wells , and were corrected for the number of positive cells observed at time point 0 . Age- and sex-matched mice were infected by oral gavage with the MNV-1 CW1 strain . The infectious dose used was 106 or 107 PFU per mouse , as indicated , administered in 100 μl PBS . Survival was monitored daily . Peyer`s patches , mesenteric lymph nodes , spleen , liver , ileum and stool were collected at indicated days post-infection for homogenization and further analysis . Tissue samples were weighed and were homogenized in 500 μl PBS , after which lysis was completed by addition of lysis buffer ( 20 mM Tris HCl ( pH 7 . 4 ) , 200 mM NaCl , 1% Nonidet P-40 ) and incubation for 10 minutes on ice . Full speed centrifugation for 30 minutes cleared the homogenate and supernatant was used for further analysis . Mouse cytokines in cell culture supernatants and tissue homogenates were determined by magnetic bead-based multiplex assay using Luminex technology ( Bio-Rad ) and type I IFN levels were analyzed using eBioscience IFNα/IFNβ Procartaplex , all according to the manufacturer’s protocols . In S5E Fig , IL-1β was detected by the BD OptEIA ELISA kit ( BD biosciences ) according to the manufacturer’s protocol . Cytokines from tissue homogenates were normalized to weight of tissue , while cytokines from cell culture supernatants were expressed as concentration per ml of cell culture medium . For measuring Lipocalin-2 levels , fecal pellets were weighed and were homogenized in 500μl PBS using sterile soil grinding SK38 2ml tubes ( Bertin Technologies ) . Stool homogenates were collected in 1 . 5 ml eppendorf tubes and cleared upon full speed centrifugation for 30 minutes . Lipocalin-2 levels in stool supernatants were then analyzed using the mouse Lipocalin-2/NGAL duoset ELISA ( R&D systems ) according to the manufacturer’s instructions , and were normalized per mg of stool . Tissue samples collected on indicated days were homogenized in 500 μl TRIsure ( Bioline ) . For RNA isolation from primary macrophages grown in monolayer , cells were directly lysed in the culture plate by adding 700 μl of TRIsure . Cell lysate was pipetted up and down to ensure sufficient cell disruption . Total RNA isolation was performed according the manufacturer’s protocol . cDNA was synthesized using the iScript gDNA clear cDNA Synthesis kit ( Biorad ) and quantitative PCR was performed using Taqman gene expression assays ( Life technologies ) . Ifnβ mRNA levels were normalized to the levels of the Tbp reference gene . To determine MNV genome copy numbers , cell culture supernatants were aspirated , after which adherent BMDMs were lysed in TRIsure . RNA was isolated and qRT-PCR was performed using the specific 5’-CCGCAGGAACGCTCAGCAG-3’ and 5’-GGCTGAATGGGGACGGCCTG-3’ primers together with the custom made Taqman probe 5’-ATGAGTGATGGCGCA-3’ [16] . Viral genome copies were quantified according to a serially diluted MNV DNA standard . To visualize cell morphology over time , 105 BMDMs per well were seeded onto a poly-L-lysine ( Sigma ) -coated eight-well slide chamber ( ibidi ) . The next day , the cells were infected with UV-inactivated or live MNV ( MOI 5 ) , followed by incubation at 37°C and 5% CO2 . After 1 hour of infection , the medium was removed , cells were washed with PBS and 1 ml medium with fluorescent dye was added . To visualize membrane integrity , the non-permeable DNA stain Propidium iodide ( BD Bioscience ) was added to the medium ( 50 ng/ml ) . Cells were imaged every 30 minutes over a time-period of 24 hours on a Zeiss Spinning Disk microscope using a 25x objective . Cells and culture supernatants , and tissue homogenates were incubated with cell lysis buffer ( 20 mM Tris HCl ( pH 7 . 4 ) , 200 mM NaCl , 1% Nonidet P-40 ) and denatured in Laemlli buffer by boiling for 10 min . Proteins were separated by SDS-PAGE electrophoresis ( Thermo Scientific ) after which proteins were transferred to nitrocellulose membranes ( Thermo Scientific ) using semi-dry ( 20 min ) or turbo ( 7 min ) blotting . Blocking and antibody incubation were performed in PBS or TBS supplemented with 0 . 05% Tween20 ( vol/vol ) and 3% or 5% ( wt/vol ) non-fat dry milk . The membranes were incubated overnight at 4°C with primary antibodies against Caspase-1 ( 1:1000; Adipogen ) , IL-1β ( 1:2000; GeneTex ) , Nlrp3 ( 1:1000; Adipogen ) , Caspase-11 ( 1:1000; Novus biologicals ) , GSDMD ( 1:1000 ) [29] , ASC ( 1:1000; ECM Biosciences ) , MLKL ( 1:1000; Millipore ) , or Caspase-3 ( 1:1000; Cell signaling ) . After washing , membranes were incubated with HRP-conjugated anti-mouse , anti-rabbit or anti-rat secondary antibodies ( 1:5000; Jackson ImmunoResearch Laboratories , 111-035-144 , 112-035-143 , 112-035-143 ) or were incubated with the directly labeled primary antibody β-actin-HRP ( 1:10000; Santa Cruz ) for up to 3 h . Proteins of interest were detected by the enhanced SuperSignal West Pico Chemiluminescent Substrate ( Thermo Scientific ) . For log-linear regression analysis of the cytokine and chemokine expressions shown in Fig 1A , a Generalized Linear Mixed Model ( GLMM ) ( fixed model: poisson distribution , log link; random model: gamma distribution , log link ) as implemented in Genstat v18 ( VSN International , Hemel Hempstead , UK; www . genstat . co . uk ) was fit to the data . The linear predictor vector of the values was written as follows: log ( μ ) = η = Xβ + Zν , where the matrix X is the design matrix for the fixed terms ( i . e . genotype , treatment and tissue ) and their interactions , β is their vector of regression coefficients , Z is the design matrix for the random term ( i . e . subject ) , and ν is the corresponding vector of random effect having a gamma distribution . The significance of the regression coefficients was assessed by a t-test . Estimated mean values and their standard errors were obtained as predictions from the GLMM , formed on the scale of the response variable . Other statistics used include two-sided Student’s t-test with unequal variance and nonparametric log-rank test , both analyzed with Prism 7 . 0 ( GraphPad Software , San Diego , CA ) . | Gastrointestinal norovirus infections form a serious socio-economic worldwide problem , with about 684 million cases annually worldwide and mortality occurring both in underdeveloped countries and in immunocompromised patients . Despite the urgent global need , no specific treatments are available for norovirus induced gastroenteritis , partly because innate immune responses upon gastrointestinal norovirus infection are largely unresolved . Using the murine norovirus ( MNV ) model we showed that MNV infection in macrophages leads to a lytic form of cell death termed pyroptosis as well as to the maturation and release of the pro-inflammatory cytokine IL-1β . Maturation of IL-1β was observed also in vivo , after gastrointestinal infection of MNV-susceptible Stat1 knockout mice . We found that these innate immune responses upon MNV infection crucially depended on activation of the Nlrp3 inflammasome leading to Gasdermin D-driven pyroptosis , and inactivating this signaling pathway delayed lethality of MNV-susceptible STAT1 knockout mice after gastrointestinal MNV infection . We thus identified Nlrp3 inflammasome activation and ensuing pyroptosis as a signaling pathway contributing to norovirus-induced immunopathology . Taken together , this study resulted in a more detailed understanding of MNV-induced inflammatory and cell death pathways and provided insights into how gastrointestinal viruses induce immunopathology . | [
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"gas... | 2019 | Nlrp3 inflammasome activation and Gasdermin D-driven pyroptosis are immunopathogenic upon gastrointestinal norovirus infection |
FACT , in addition to its role in transcription , is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair . Here , we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER . We found that , upon oxidative stress , FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family . We also showed the rapid recruitment of FACT to the site of damage , coincident with the glycosylase OGG1 , upon the local generation of oxidized DNA . Interestingly , FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC . This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers . Altogether , our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER .
DNA is packaged under the form of chromatin in the eukaryotic nucleus . The nucleosome , the repeating unit of chromatin , consists of an octamer of core histones ( two each of H2A , H2B , H3 and H4 ) , around which DNA is wrapped in two superhelical turns [1] . Linker DNA , connecting the consecutive nucleosomes , interacts with a fifth histone H1 , termed linker histone , [1] . Each core histone contains a structured domain , the histone fold , and an unstructured NH2-terminus [2–4] . Both the linker histone and the NH2-termini of core histones are involved in the assembly and maintenance of the 30 nm chromatin [5–7] fiber and the mitotic chromosome [8 , 9] . The nucleosome is a barrier for several processes requiring access to the naked DNA underlying sequences [10 , 11] . The three main strategies that the cell uses to overcome the nucleosome barrier are the posttranslational modifications of core histones [12] , the incorporation of histone variants in chromatin [13 , 14] and the chromatin remodelers [15 , 16] . The chromatin remodelers are very sophisticated nanomachines able to perturb the histone-DNA interactions and to mobilize the histone octamer along DNA by using the energy freed by the hydrolysis of ATP [15–17 , 18 , 19] . Depending on the type of ATPase present in the complex , they are classified in at least four main groups , namely the SWI/SNF , ISWI , CHD and INO80 families [20] . The chromatin remodeler RSC belongs to the SWI/SNF family and it is involved in the repair of damaged DNA [21 , 22] . RSC contains a central cavity sufficient for binding of a single nucleosome [23] . We have recently analyzed the mechanism of RSC-induced nucleosome mobilization and have shown that RSC generates initially an ensemble of particles with highly altered histone-DNA interactions , which are further mobilized by RSC [24] . Base Excision Repair ( BER ) is the major pathway to repair highly mutagenic lesions present in cellular DNA due to inherent instability of some nucleobases or induced upon oxidative stress ( e . g . uracil and 8-oxo-7 , 8-dihydroguanine ( 8-oxoG ) ) . The molecular mechanisms of BER on naked DNA are well understood , but how it functions on chromatin templates remains elusive . The reported data show that the presence of nucleosomes interferes strongly with BER , although the different enzymes are affected in a distinct manner [25–29] . For instance , within nucleosomal DNA , the activity of uracil DNA glycosylase ( UDG ) on uracil facing the solvent is only slightly reduced , while the removal of uracil facing the histone octamer is greatly ( up to 3–4 orders of magnitude ) inhibited [30] . In addition , the very strong inhibition of 8-oxoG removal by the glycosylase OGG1 was overcome in the presence of nucleosome remodeling complexes [31 , 32] . Human FACT ( Facilitates Chromatin Transcription ) protein consists of two subunits , hSpt16 and SSRP1 , which are both required for its functionality [33] . FACT exhibits a histone chaperone activity; it makes a complex with the H2A-H2B dimer and the ( H3-H4 ) 2 tetramer and is able to deposit them on DNA [33 , 34] . Few reports indicate that , in addition to its role in transcription , FACT might be implicated in the Transcription-Coupled Nucleotide Excision Repair ( TC- NER ) of UVC-damaged DNA or DNA Double Strand Break ( DSB ) repair [35–38] . FACT was found in a complex with casein kinase 2 ( CK2 ) , known to phosphorylate p53 in a DNA damage dependent manner resulting in an increase of p53 activity [35 , 36] . FACT appears also to be involved in both phosphorylation and exchange of histone variant H2A . X , two events related to the DSB repair [37] . In this work we show that upon oxidatively induction of DNA damage , FACT switches protein partners from transcription-associated factors to DNA repair protein . FACT together with chromatin remodelers from the SWI/SNF family is recruited to the sites of DNA damage . This DNA damage response strongly suggests an implication of FACT in BER . To shed light on the mechanism of FACT in this process , we have carried out a series of in vitro experiments using uracil containing nucleosomes . We reveal that FACT greatly facilitates the removal of uracil by UDG from the nucleosomal DNA via a novel mechanism , implicating boosting of the activity of the ATP-dependent chromatin remodeler RSC . Collectively , our data points towards a role of FACT in in vivo BER of DNA lesions though assisting the remodeling of chromatin at the sites of lesions .
Some available data indicate that FACT , in addition to transcription , is involved , as mentioned above , in both transcription-coupled nucleotide excision repair ( TC-NER ) and DNA double strand break ( DSB ) repair [35–38] . However , no data is available on the implication of FACT in BER . If FACT is directly implicated in the repair of oxidatively damaged DNA it should be recruited to the sites of induced lesions in DNA . To test if FACT is recruited at BER-lesions we used HeLa cells transiently expressing fluorescently labeled FACT , i . e . a fusion of DsRed with SSRP1 ( DsRed-SSRP1 ) , the smaller subunit of FACT . As controls , HeLa cells expressing fluorescently labeled either the glycosylase OGG1 ( EGFP-OGG1 ) or the DSB repair factor Ku80 ( Ku80-GFP ) were used . These cells were treated as in [39] with Ro 19–8022 , a specific type II photosensitizer generating exclusively 8-oxoG [40 , 41] in a region microirradiated with low intensity 405 nm laser light . Upon microirradiation , DsRed-SSRP1 , similarly to the repair fusion EGFP-OGG1 , was rapidly recruited to the site of 8-oxoG ( Fig 1A ) . As expected , no recruitment of the Ku-80 protein was detected ( Fig 1B ) , thus revealing the absence of DSB generation under our irradiation conditions . Note , that our control experiments with shRNA FACT KD cells showed that the absence of FACT does not abolish OGG1 recruitment to the micro-damaged foci , as the low signal-to-noise ratio and statistical signal fluctuations did not allowed a reliable quantitative comparison . Therefore , FACT co-localizes with BER protein suggesting it might be implicated in the repair of oxidatively generated DNA lesions . To further consolidate this hypothesis we have carried out a series of biochemical experiments . Briefly , we generated stable HeLa cell lines expressing HA-tagged SSRP1 ( HA-SSRP1 ) and treated them with 10 mM H2O2 for 5 min at 4°C to induce oxidative damage to DNA . Then we isolated the chromatin-associated HA-SSRP1 complex ( HA-SSRP1 . com ) from both control ( non-H2O2 treated ) and H2O2-treated cells by immuno-affinity using anti-HA antibody . Both complexes were next run on SDS gels and the associated polypeptides were identified by mass spectrometry and Western blotting ( Fig 1C and 1D ) . As seen ( Fig 1D ) , the HA-SSRP1 complex , isolated from H2O2-treated HeLa cells , contains many additional polypeptides compared to the one isolated from the control untreated cells . Mass spectrometry analysis shows that a large number of proteins involved in repair of damaged DNA ( DDB1 , DDB2 , XRCC1 , CUL1 , MRE11A , XRCC4 , PARP-1 , etc . ) as well as SMARCA1 , SMARCA4 and SMARCC2 proteins , subunits of the human SWI/SNF family remodelers , co-immunopurify with the HA-SSRP1 complex , isolated from H2O2 treated HeLa cells ( see Fig 1C and 1D and S1 Table ) . Note that these proteins were not found associated with the HA-SSRP1 complex purified from the control HeLa cells ( Fig 1C and 1D and S1 Table ) . While HA-SSRP1 , in control cells , interact with proteins involved in transcriptional control ( PolR2A , PolR2B , PAF1 , HIRIP3 , TRIM24 , TRIM33 ) , HA-SSRP1 complex , isolated from the DNA damaged cells , ( Fig 1B and 1C and S1 Table ) are devoid to these proteins . Western blotting analysis for some of the proteins provided further support to this finding ( Fig 1D , bottom panel ) . Thus , induction of DNA damages results in release of FACT associated with Pol II containing chromatin . This is accompanied with recruitment of both FACT and repair proteins to the damage sites . Note that chromatin remodelers of the SWI/SNF family are known to be implicated in DNA repair [31 , 32 , 42] . Taken as a whole , this set of data suggests that FACT participates directly in the repair of oxidative DNA lesions . How could FACT function in the repair of oxidatively damaged DNA ? To address this , we focused on the role of FACT in BER and analyzed the effect of FACT on UDG removal of uracil from nucleosomal DNA using in vitro reconstituted centrally positioned nucleosomes . FACT was purified to homogeneity from HeLa cells by double immuno-affinity procedure ( S1C Fig and Materials and Methods ) . The histone octamer was assembled with highly purified recombinant core histones ( S1A Fig ) and the nucleosome reconstitution was carried out by using the 601 nucleosome positioning sequence , containing randomly incorporated uracil [30] . The reconstitution conditions were optimized and essentially all DNA was assembled into nucleosomes ( S1B Fig ) . The •OH and DNase I footprintings ( Figs 2 and 3 ) showed clear 10 bp cleavage pattern further confirming the proper wrapping and the strong translational-rotational positioning of the nucleosomal DNA around the histone octamer in the reconstituted samples . The reconstituted nucleosome samples were then incubated with increasing amount of UDG ( Fig 2 , lanes 3–6 ) , DNA was isolated and , after APE1 treatment ( to cleave the DNA phosphodiester bond at the abasic sites generated upon removal of uracil by UDG ) , the cleaved DNA was run on a PAGE under denaturing conditions . The cleavage patterns of linker DNA within nucleosomal and naked DNA are identical ( Fig 2 , compare lanes 3–6 with lanes 21–24 ) . By contrast , the cleavage pattern of the histone interacting nucleosome core DNA strongly differed from the respective regions within naked DNA ( compare lanes 3–6 with lanes 21–24 ) . Cleavage in nucleosome core DNA is only observed at the sites facing the solution , which are also accessible to •OH radicals ( lanes 1 and 25 ) even at the highest concentration of UDG used ( lane 6 ) . This is in perfect agreement with the reported data [30 , 43] and illustrates the inability of UDG to remove uracil from sites facing the histone octamer . Noteworthy , the presence of 1 . 6 pmol FACT in the reaction mixture does affect neither the efficiency nor the pattern of removal of uracil by UDG ( compare lane 13 with lane 4; note that in both cases the same concentration of UDG ( 1 . 2x10-2 units ) was used ) . Pretreatment of the nucleosome samples with increasing amount of RSC changed completely the UDG cleavage pattern of nucleosomal DNA , i . e . it became qualitatively indistinguishable from that of naked DNA ( compare lanes 8–12 with lanes 21–24 ) . Thus , the RSC induced remodeling of the nucleosomes renders all uracil residues ( including the ones facing the histone octamer ) accessible to UDG . Interestingly , the same effect was observed when only 0 . 15 units of RSC ( an amount of RSC unable to change the UDG accessibility to uracil , see lane 8 ) and increasing amount of FACT was used for the pretreatment of nucleosomes ( compare lanes 14–19 with lanes 21–24 ) . This shows that in vitro FACT and RSC act in concert to facilitate the UDG removal of uracil from nucleosomal DNA and suggests that in vivo they could act similarly to assist BER . Bearing in mind the above described results , we hypothesized that FACT facilitates UDG accessibility to uracil in nucleosomal DNA by acting on the remodeling activity of RSC , as it was found for nucleolin [44] . To address this we have used DNase I footprinting . Briefly , we incubated 601 end-positioned nucleosomes with RSC either in the absence or presence of FACT and after arresting the remodeling reaction , the samples were treated in controlled manner with DNase I . The cleaved DNA was then purified and analyzed on a PAGE under denaturing conditions ( Fig 3A ) . The presence of FACT alone , as expected , did not change the clear 10 bp cleavage pattern of nucleosomal DNA ( Fig 3A , compare lane 1 with lane 2 ) . So , under the conditions of the experiment , FACT does not alter the structure of the nucleosome . When incubated with 0 . 2 units of RSC , the nucleosomes exhibited some alterations in the DNase I cleavage pattern , testifying for some relatively small perturbations in the histone-DNA interactions induced by RSC ( compare lane 3 with lanes 1 and 2 ) . Remarkably , the treatment of the nucleosomes with the same amount of RSC , but in the presence of 1 . 6 pmol of FACT , resulted in pronounced alterations in the DNase I digestion pattern ( lane 4 ) . The same altered DNase I digestion pattern was observed with 5-fold more RSC in the absence of FACT ( 1 unit , lane 5 ) . We concluded that FACT exhibits strong “co-remodeling” activity and is able to boost no less than 5-fold the remodeling activity of RSC . The effect of FACT on the nucleosome mobilization efficiency of RSC was studied by EMSA ( Fig 3B and 3C ) . Treatment with 0 . 2 units of RSC led to mobilization of a very small part of the nucleosomes ( not exceeding 15% , see Fig 3B and quantification on Fig 3C ) . The presence of increasing amount of FACT in the reaction mixture led to a strong increase in the amount of slid end–positioned nucleosomes and already at the highest concentration ( 1 . 6 pmol ) of FACT , the slid nucleosomes represent ∼60% of the overall nucleosome population ( Fig 3C ) . Similarly , the presence of FACT in the remodeling mixture led to strong increase in the time-course of the mobilization reaction ( S2 Fig ) . Therefore , FACT boosts the RSC ability to mobilize the nucleosomes . Our high-resolution microscopy and biochemical data show an intriguing behavior of RSC nucleosome remodeling . This consists of a formation of RSC released stable , non-mobilized particles , termed remosomes [24] , which contain ∼180–190 bp of DNA loosely attached to the histone octamer [45] . The remosomes are formed by RSC-pumping ∼15–20 bp from each end of the free DNA linkers of the nucleosome without repositioning of the histone octamer . Subsequently , the remosomes are mobilized by RSC within a second interaction step [24] . Note that these “remosomes” are quite different from the Kornberg’s “altered nucleosomes” [46] and Kingston’s “novel band” [47] induced at a very high enzyme/nucleosome ratio ~0 . 5–2 by RSC or SWI/SNF , respectively , and identified as nucleosomal dimers . Interestingly , under our experimental conditions of enzyme/nucleosome ratio ~1/20–1/100 , we did not observe any histone eviction from remodeled nucleosomes , by contrasts to reported excessive dimer ( octamer ) eviction after sliding ( see [48] and references therein ) . Most likely the later observations , occurring at very high enzyme/substrate ratio ~0 . 5–1 , represents nucleosomal DNA border ( limited DNA length ) artefacts of excessively “over-mobilized” mono- ( di- , tri- ) nucleosomes leading to unstable non-canonical ( dyadless ) nucleosomal species described in [49] , that can lose histones due to excessive binding-dissociation events of the remodeler and/or interaction with surrounding histone acceptors . The main characteristic of the remosome is the higher accessibility of its DNA to restriction enzymes . To test if FACT was able to modify distinct compartments of RSC induced nucleosome remodeling , we have used the recently developed “in gel one pot assay” [24] . This approach detects quantitatively the alterations in histone-DNA interactions with a 10 bp resolution all along the nucleosomal DNA ( Fig 4 and [24 , 50] ) . Briefly , eight mutated 32P-end labeled 255 bp 601 . 2 sequences were used to reconstitute centrally positioned nucleosomes . A single HaeIII restriction site ( designated as d0 to d7 , where the subscript refers to the number of helical turns from the nucleosome dyad ) was inserted within each of these sequences . An equimolar mixture of the eight reconstituted nucleosomes was incubated with appropriate amount of RSC either alone ( to produce 10–15% of slid nucleosomes ) or in the presence of increasing amount of FACT ( Fig 4A ) . A FACT-concentration dependent mobilization of the nucleosome is observed as judged by the EMSA ( Fig 4A ) . The upper electrophoretic band , containing the remosome fraction as well as the designated end-positioned slid nucleosome fraction were excised and in gel digested with HaeIII . The digested DNA was purified from the gel and run on an 8% PAGE under denaturing conditions . A similar experiment using either control nucleosomes or nucleosomes treated with FACT ( in the absence of RSC ) was also performed . As seen ( Fig 4B and 4C ) , and in agreement with the reported data [24] , the restriction enzyme HaeIII accessibility of the control particles at d6 to d0 is very low and practically unaffected by the presence of FACT . This agrees with the available data [50] and reveals that FACT alone , in the concentrations used , does not destabilize the histone-DNA interactions within the nucleosome . However , upon incubation with both RSC and FACT , DNA exhibited highly altered accessibility all along the nucleosome ( Fig 4B and 4C ) . The accessibility of d7 decreased relative to the control particles ( this effect is due to the “pumping” of linker DNA in the nucleosome ) , while that of the other positions strongly increased . This increase in the HaeIII accessibility profile is concomitant with the increase of FACT concentration used in the remodeling reaction . Since this altered HaeIII accessibility profile is a remosome specific structural “signature” [24] , we conclude that FACT assists RSC in perturbing the histone-DNA interactions in the nucleosome , while maintaining the distinct compartments of remodeling . Since FACT significantly increases the capacity of RSC to generate remosomes and slid nucleosomes , its involvement in repair might be mainly associated with this property . If this was the case , one should expect damaged remosomal DNA to be easily repaired . We have addressed this question by studying the ability of UDG to remove uracil from remosomes ( Fig 5 ) . In agreement with the data in Fig 2 , UDG was unable to excise histone octamer facing uracil from the control nucleosome ( Fig 5 , “nucleosomes” ) . In contrast , the histone octamer facing uracil was rather efficiently removed in remosomes , even at our lowest concentration of UDG ( 3 . 9x10-3 units ) , just like solution facing uracil ( Fig 5 , “remosomes” ) and is almost saturated at concentrations above ∼2 . 3x10-2 units of UDG ( Fig 5 , “remosomes” ) . Excision of uracil from end positioned slid nucleosomes exhibits essentially the same behavior ( Fig 5 , “slid” ) . These results reveal that the alterations of either the histone-DNA interactions in the remosomes or mobilization of the nucleosomes allow efficient repair . The above presented data reveals that FACT assists very efficiently RSC to both alter the histone-DNA interaction and to mobilize the nucleosome in an ATP-dependent manner . To achieve this , FACT could either act on the nucleosomal substrate or on RSC or on both of them . To differentiate between these possibilities we have carried out the RSC nucleosome mobilization assay at increasing concentration of FACT , but at low ATP concentration ( 120 μM ) . At this low concentration of ATP it is possible to precisely measure the amount of ATP hydrolyzed by RSC and thus , to precisely determine the percentage of nucleosomes mobilized by the hydrolysis of a “unit” of ATP . Under these conditions of the experiment , the increase of the FACT concentration results in a ~ 4 fold increase ( from 16% to 64% ) of the slid by RSC nucleosomes ( Fig 6A ) . Remarkably , under the same experimental conditions no change in the amount of hydrolyzed ATP was detected ( Fig 6B ) . Kinetic experiments confirmed that FACT does not affect the ATPase activity of RSC ( S3 Fig ) . This demonstrates that FACT boosts strongly the generation of slid nucleosomes ( ∼4-times more slid nucleosomes are generated per unit of hydrolyzed ATP in the presence of the highest amount of FACT ( 1 . 6 pmol ) used in the experiments ) without affecting the ATPase activity of RSC . In other words , the presence of FACT allows RSC to transform much more efficiently the energy freed by the ATP hydrolysis into mechanical work . Since FACT does not affect the ATPase activity of RSC , it should act on the nucleosomes making them more prone to remodeling .
Several reports clearly demonstrated the involvement of FACT in assisting the transcription of chromatin DNA [33 , 34 , 51 , 52] . The reported data on the potential implication of FACT in DNA repair are , however , scarce and no data on implication of FACT in oxidative damage repair are available [35–37] . Here we show that FACT is directly implicated in the repair of oxidatively generated DNA lesions and decipher how FACT may function in BER . Our in vivo data reveals that upon oxidative stress FACT is released from transcribed chromatin and it is relocated to chromatin loci associated with both repair proteins and chromatin remodelers involved in DNA repair . This intriguing behavior of FACT points to a possible direct function in BER . To address this we have analyzed in vitro the effect of FACT on BER initiation by using chromatinized templates containing uracil . Our data shows that FACT alone has no effect on the removal of uracil by UDG from nucleosomal DNA . Although , FACT exhibits a strong “co-remodeling” activity and it is able to increase many-fold the efficiency of the involved in DNA repair chromatin remodeler RSC to both remodel and relocate the nucleosomes . FACT does not affect the ATPase activity of RSC , but instead makes the nucleosomes easier to be remodeled and mobilized , i . e . it increases the efficiency of transformation of the energy freed by the RSC-induced hydrolysis of ATP into mechanical work . This allows , in turn , very low amount of RSC to be sufficient to strongly alter the histone-DNA interactions as well as to slide the nucleosomes and thus , the lesions in chromatin DNA to be efficiently repaired . Our data suggest that in vivo FACT acts in BER , via RSC ( or through other ATP-dependent remodeling factors ) , by increasing the efficiency of repair of DNA lesions . FACT ( at the concentrations used in the experiments ) does not affect nucleosome structure as judged by both DNase I footprinting and the very sensitive restriction enzyme accessibility assay ( one pot assay ) . In addition , no stable binding of FACT to the nucleosome was detected by EMSA . Then how does FACT act on the nucleosome substrate to make it more easily “remodelable” ? It is difficult to answer to this question . Obviously , some transient FACT-nucleosome interactions during the RSC remodeling process should be generated allowing the remodeler to function with much higher efficiency . Noteworthy , genetic analysis in yeast has revealed evidences for functional relationship between the N-terminal domain of Spt16 , one of the FACT subunits , and the docking domain of H2A [53] . The proper folding and integrity of the docking domain of H2A is , however , required for chromatin remodeling [54] . For example , nucleosome reconstituted with deleted docking domain H2A or with the histone variant H2A . Bbd ( which possesses defective docking domain ) cannot be both remodeled and mobilized by remodelers from both SWI/SNF and ISWI family [54 , 55] . Of note , Hondele et al . , have provided structural insights into Spt16 binding to H2A-H2B dimers [56] and suggested a possible mechanism of unraveling of the outer 30 base pairs of the nucleosome by invasion of FACT . Although , it has been reported that FACT alone is able to alter the overall nucleosome structure and destabilize both histone-DNA and histone-histone interactions without any histone eviction [53] . In these last experiments the molar ratio FACT/nucleosome exceeded , however , at least 50 fold the ratio of FACT/nucleosome used in our experiments ( see Materials and Methods ) . Thus , the reported FACT-induced destabilization of the nucleosome would reflect the very high concentrations of FACT in the earlier reported experiments [53] . Displacement or transfer of histone dimers as a result of nucleosome remodeling has been reported [57] indicative of destabilization of nucleosomes in the process . As mentioned above , H2A variants or experimental mutations which weaken the H3-H4 tetramer and H2A-H2B dimer interface result in unwrapping of DNA from the nucleosomal ends and associated loss of remodeling . An attractive possibility is that depending on the local concentration of FACT , the equilibrium can be shifted into a ‘dynamically stable’ conformation of nucleosomes , which facilitates efficient nucleosome remodeling . It has been recently shown that at the sites of UV induced damage , the histone pair H2A-H2B is exchanged and this exchange is assisted by FACT . This FACT-dependent chromatin dynamics is implicated in the promotion of transcriptional restart after DNA repair of the lesions blocking transcription [38] . Thus , in Transcription-Coupled–Nucleotide Excision Repair ( TC-NER ) , the histone chaperone activity of FACT appeared to be used . This might reflect the nature of lesions to be repaired requiring the removal of histones to freeing-up a relatively long stretch of free DNA for TC-NER to proceed efficiently . Of note , the observed co-remodeling activity of nucleolin , a protein displaying histone chaperone activity [44] , suggests that RSC boosting could be a common feature for histone chaperones . However , under our experimental conditions we did not observe any SWI/SNF and RSC remodeler activity boosting mediated by some “classical” histone chaperones we tested , namely NPM1 ( B23 ) , mNAP1 , and nucleoplasmin ( S4 Fig ) . Since both FACT [33] and nucleolin [58] contains an HMG box domain and HMGB1&2 proteins have also co-remodeling activity [59] , this suggests that the presence of HMG-box would be important for co-remodeling . Interestingly , FACT facilitates also very efficiently the nucleosome mobilization by ACF ( S5 Fig ) , a remodeler belonging to the ISWI family . FACT was also found stably recruited to the CENP-A nucleosomal complexes [60] and RSF , a chromatin remodeler , is also a member of this complex [61 , 62] . In addition , FACT was shown to physically interact with CHD1 , another chromatin remodeler [63] . Thus , the cell appears to use the FACT nucleosome reorganization ability to control the activity of distinct remodelers belonging to different families for different purposes in different processes . The transient interaction of FACT with H2A docking domain and with nucleosomal DNA would be determinant for this peculiar property of FACT and the cell may use it not only to modulate the remodeling capacity of chromatin remodeling machines but also to control other “machines” working on chromatin . A typical example is the activity of FACT during transcription , which requires a transient interaction with the H2A/H2B dimer [52] .
The 255 bp 601 DNA probe used for reconstitution of centrally positioned nucleosomes was PCR amplified from pGEM-3Z-601 . 1 plasmid ( kindly provided by J . Widom ) . 5’ end labeling was performed by using γ-32P-labeled primer in PCR . For ‘One Pot Restriction enzyme Assay’ a set of eight pGEM-3Z-601 . 2 mutants were utilized , each containing HaeIII site at a different superhelical location , as described before [50]; note that the “dyad 7” fragment contains an additional HaeIII site located at 4 bp away from the d7 site ) . Briefly , a 281 bp fragment was amplified using primers targeting the vector specific sequence flanking the 601 . 2 sequence . Labeling of the fragment was done as described above . The fragments were subsequently digested with SphI to get a fragment of 255 bp with 57 and 51 bp linker DNA on left and right side respectively . For repair assays uracil was randomly incorporated in the 601 sequence by PCR using a dNTP mix containing dUTP/dTTP in a 5/95% ratio [30] . Top strand was 5’ end labeled using γ-32P-labeled primer in PCR as described . For DNase I footprinting , a 200 bp fragment with 601 positioning sequence at one end was obtained by digestion of 255 bp 601 with NotI . Fragments were labeled by gap filling using Klenow enzyme with [α-32P]CTP in the presence of 50 μM dGTP . For end positioned nucleosomes used for ACF induced nucleosome sliding assays , a 241 bp fragment was PCR amplified from p199 . 1 plasmid to generate 601 DNA with the 601 sequence at the end and labeled using γ-32P-labeled primer in PCR . All the DNA fragments were purified on 6% native acrylamide gel prior to use for nucleosome reconstitutions . The chromatin associated FACT complex was affinity purified from HeLa cell extracts as described previously [64] . Recombinant histones H2A , H2B , H3 and H4 from Xenopus laevis were expressed in Echerichia coli strain BL21 ( DE3 ) and purified to homogeneity under denaturing conditions as described [65] . Saccharomyces cerevisiae RSC complex was purified using standard tandem affinity purification as described [24] . ACF remodelling complex was purified according the protocol described in [55] . APE1 is from NEB and UDG from Invitrogen . Oxidative DNA lesions were generated by HeLa cells incubation on ice for 5 min with 10 mM H2O2 . HeLa extracts were prepared using a modification of the Dignam protocol [66] . Briefly , cells were lysed in hypotonic buffer ( 10 mm Tris-HCl at pH 7 . 65 , 1 . 5 mm MgCl2 , 10 mm KCl ) and disrupted by Dounce homogenizer . The cytosolic fraction was separated from the pellet by centrifugation at 4°C . The nuclear-soluble fraction was removed by incubation of the pellet in high-salt buffer ( to get a final NaCl concentration of 300 mM ) . Nuclear pellets were recovered by centrifugation and resuspended in TGEN 50 buffer ( 20 mM Tris at pH 7 . 65 , 50 mM NaCl , 3 mM MgCl2 , 0 . 1 mM EDTA , 10% glycerol , 0 0 . 01% NP40 ) . Nuclei were digested by micrococcal nuclease to generate mainly mononucleosomes . Mononucleosomes containing e-SSRP-1 were purified from the resulting material by immunoprecipitation on anti-Flag antibody-conjugated agarose . After elution with the Flag peptide , the e-SSRP-1 bound nucleosomes were incubated with 1M NaCl and further fractionated on a 15–45% glycerol gradient to dissociate FACT from the nucleosome . The e-SSRP-1 complex was further affinity-purified by anti-HA antibody-conjugated agarose and eluted with the HA peptide . The HA and Flag peptides were first buffered with 50 mM Tris-Cl ( pH 8 . 5 ) , then diluted to 4 mg/mL in TGEN 150 buffer ( 20 mM Tris at pH 7 . 65 , 150 mM NaCl , 3 mM MgCl2 , 0 . 1 mM EDTA , 10% glycerol , 0 0 . 01% NP40 ) , and stored at −20°C until use . Anti-FLAG beads were washed in TGEN 150 buffer . Complexes were resolved by SDS-PAGE and stained using the Silver Quest kit ( Invitrogen ) . Identification of proteins was performed by the Taplin Biological Mass Spectrometry Facility ( Harvard Medical School , Boston , Massachusetts , USA ) . Damage to DNA was induced mainly as described in [67] . HeLa cells transfected with the indicated Fusions with either DsRed or EGFP were grown on coverslips in DMEM medium and were treated with Ro-198022 for 5 min before 405 nm laser irradiation . The irradiation was performed at low intensity generating no DSB and UV-lesions as described in [39] . Nucleosomes were reconstituted using the salt dialysis procedure ( Mutskov et al . , 1998 , MCB ) . Briefly , 5 μg of chicken erythrocyte carrier DNA and 250 ng of abovementioned labeled DNA probes were mixed with pre-reconstituted core histone octamers in an equimolar ratio . The nucleosome reconstitution buffer was composed of 10 mM Tris–HCl ( pH 7 . 4 ) , 1 mM EDTA , 5mM β- mercaptoethanol and 2 M NaCl . Reconstitution mixtures were dialyzed at 4°C against a smoothly decreasing NaCl concentration up to 10 mM final . Efficiency and quality of nucleosome reconstitutions were checked on a 5% native PAGE run with 0 . 25X TBE . Nucleosome sliding reactions were performed typically with ~75 ng of nucleosomes ( ~0 . 5 pmol ) and the indicated amount of RSC or ACF in remodeling buffer ( RB ) consisting of 10 mM Tris pH 7 . 4 , 5% glycerol , 1 mM ATP , 2 . 5 mM MgCl2 , 1 mM DTT , 100 μg/ml BSA , 50 mM NaCl , 0 . 01% NP40 in a volume of 7 . 5 μl at 29°C for 50 min . Reactions were stopped by addition of 0 . 1 units of apyrase . Sliding products were analysed , when indicated , on a 5% native PAGE by quantifying the exposed autoradiographs . Similar conditions were used for remodeling assays to be probed by DNase I or followed by repair assays if not mentioned otherwise . One unit of the remodeling complexes is defined as the amount ( typically ~10 fmol ) necessary to induce 50% relocation to the end position of ~0 . 5 pmol middle positioned 601 ~250 bp nucleosomes in 50 min under our standard reaction conditions [14 , 31 , 44] . One μg of plasmid DNA was added to the end positioned control or remodelled nucleosomes after stopping the reaction by addition of apyrase . Samples were digested for 2 min with 0 . 5 units of DNase I at room temperature . After stopping the reaction by addition of SDS and EDTA to 0 . 1% and 20 mM respectively , DNA was extracted with phenol-chloroform , ethanol precipitated and run on 8% denaturing PAGE . Dried gels were exposed and imaged on Fuji-FLA5100 phosphorimager . Nucleosomes ( 0 . 5 pmol ) assembled on the end labelled 255 bp 601 DNA were adjusted to 5 mM Tris ( pH 7 . 4 ) and 0 . 25 mM EDTA in a 7 . 5 μl volume . The hydroxyl radical •OH reaction was carried out by mixing 2 . 5 μl each of 2 mM FeAMSO₄ and 4 mM EDTA , 1M ascorbate and 0 . 12% H2O2 in a drop on the side of the tube cap before mixing it with the nucleosome solution . The reaction was stopped by adding 0 . 1% SDS , 20 mM EDTA , 1% glycerol and 100 mM Tris ( pH 7 . 4 ) . DNA was purified and analyzed as described above . For carrying out the repair assay the DNA or nucleosomes incorporated Uracil were incubated with indicated amount of Uracil DNA glycosylase ( UDG ) in the remodeling buffer at 30°C for one hour . DNA was phenol chloroform extracted and precipitated with ethanol . Abasic sites generated by UDG were cleaved by incubation with 1 unit of APE1 . DNA was extracted , processed and imaged as described above . A sliding reaction was carried out in the presence of increasing concentrations of FACT and the amounts of RSC as indicated . Prior to loading on 5% native polyacrylamide gel , 6 . 25 pmol of cold 255 bp 601 middle positioned nucleosomes were added to each reaction as a carrier in order to maintain stability during subsequent procedures . Remodeling products were resolved on 5% native polyacrylamide gel . Bands , corresponding to control unremodeled and unmobilized remodeled nucleosomes , were excised , collected in siliconized eppendorf tubes , crushed very gently and immersed with 50 μl restriction buffer ( 10 mM Tris pH 7 . 6 , 10 mM MgCl2 , 50 mM NaCl , 1 mM DTT and 100 μg/ml BSA ) containing 8 units/μl of HaeIII for 10 minutes at 30°C . The reaction was stopped by adding an equal volume ( 50 μl ) of 2x stop buffer containing 0 . 2% SDS and 40 mM EDTA . DNA was eluted from the gel slices , purified as described above , and run on 8% denaturing gel . The quantification of extent of accessibility at different superhelical locations in the nucleosome was performed using Multi-Gauge Software ( Fuji ) as described [24] . Centrally positioned 601 nucleosomes were incubated for 50 min with RSC in the remodeling reaction as described above to achieve ~50–60% siding [24] . Reaction products were resolved on 5% native polyacrylamide gel . Bands , corresponding , the un-mobilized RSC remodeled “remosomes” , and the mobilized end-positioned “slid” fractions , as well as control naked DNA and untreated nucleosomes were excised . Excised bands were eluted in 80 μl Elution Buffer ( EB ) containing Tris 10 mM pH 7 . 4 , 0 . 25 mM EDTA , 0 . 01% NP40 , 100 μg/ml BSA and 30 mM NaCl , at 4°C for 1 h with gentle shaking . 7 . 5 pmol of cold 255 bp 601 nucleosomes were added in the elution buffer to maintain the stability of eluted nucleosomes . Eluted nucleosomes were filtered through glass fibre filter under low speed centrifugation ( 200g ) to remove acrylamide particles . Eluted naked DNA , nucleosomes , remosomes and slid particles were divided into equal aliquots , and uracil cleavage efficiency was assessed by incubating the fractions with UDG in increasing concentrations . Samples were processed for further analysis as described above . The ATPase assays were carried out in the remodeling reaction buffer with ATP concentration at 120 μM . To the reaction mixture 0 . 1 μl ( 3 . 3 μM ) of the source 32P ATP was added . The remodelling reaction was carried out at 29°C for 50 minutes . Reaction aliquots were analyzed by both , native 5% PAGE ( sliding assay ) and 15% acrylamide-50% urea denaturing gel ( ATPase assay ) . The native PAGE was dried , exposed overnight on a phosphor imager screen and quantified by using the Multi Gauge V3 . 0 software . The wet denaturing gels ( ATPase assay ) were covered by saran , exposed for 30’ and the digital images quantified . Briefly , %ATPhyd = 100[P*]/ ( [P*]+[ATP*] ) ( where the values in brackets represents the background corrected integrated volumes of the corresponding radioactive bands on the digital gel image . The experimental error was typically ±10% from two independent replicas . | In the nucleus , DNA is packaged into chromatin . The repeating unit of chromatin , the nucleosome , consists of a histone octamer around which DNA is wrapped into two superhelical turns . The nucleosome is a barrier for various nuclear processes which require access to DNA . To repair lesions on DNA , this barrier has to be overcome by either nucleosome remodeling or by histone eviction . Here we present evidence that FACT , a protein known to be involved in transcription , is also involved in BER , by boosting nucleosome remodeling . Upon in vivo oxidized DNA lesion induction , FACT exhibits a BER-like protein behavior , and it is recruited to the sites of DNA damages . In vitro experiments show that FACT boosts the remodeling activity of the chromatin remodeler RSC and is implicated in DNA repair . The presence of FACT greatly facilitates the removal of uracil from nucleosomal , but not from naked DNA , in a RSC-mediated reaction . Taken collectively , our in vitro and in vivo data reveal a role of FACT in BER by helping the remodeling of chromatin at the sites of lesions . | [
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"denaturation... | 2016 | FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC |
The reconstruction and synthesis of ancestral RNAs is a feasible goal for paleogenetics . This will require new bioinformatics methods , including a robust statistical framework for reconstructing histories of substitutions , indels and structural changes . We describe a “transducer composition” algorithm for extending pairwise probabilistic models of RNA structural evolution to models of multiple sequences related by a phylogenetic tree . This algorithm draws on formal models of computational linguistics as well as the 1985 protosequence algorithm of David Sankoff . The output of the composition algorithm is a multiple-sequence stochastic context-free grammar . We describe dynamic programming algorithms , which are robust to null cycles and empty bifurcations , for parsing this grammar . Example applications include structural alignment of non-coding RNAs , propagation of structural information from an experimentally-characterized sequence to its homologs , and inference of the ancestral structure of a set of diverged RNAs . We implemented the above algorithms for a simple model of pairwise RNA structural evolution; in particular , the algorithms for maximum likelihood ( ML ) alignment of three known RNA structures and a known phylogeny and inference of the common ancestral structure . We compared this ML algorithm to a variety of related , but simpler , techniques , including ML alignment algorithms for simpler models that omitted various aspects of the full model and also a posterior-decoding alignment algorithm for one of the simpler models . In our tests , incorporation of basepair structure was the most important factor for accurate alignment inference; appropriate use of posterior-decoding was next; and fine details of the model were least important . Posterior-decoding heuristics can be substantially faster than exact phylogenetic inference , so this motivates the use of sum-over-pairs heuristics where possible ( and approximate sum-over-pairs ) . For more exact probabilistic inference , we discuss the use of transducer composition for ML ( or MCMC ) inference on phylogenies , including possible ways to make the core operations tractable .
In 1968 , Francis Crick hypothesized that the first ribosome consisted entirely of RNA , without any protein cofactors [1] . A domain structure for this primeval ribosome was recently proposed [2] . To synthesize such a reconstructed ribosome or reconstructions of other evolutionarily significant RNAs such as group II introns [3] or telomerase [4] , it will be necessary to develop methods that can predict the sequences and structures of ancient RNAs based on the divergent sequences of their many descendants . An inspection of RNA alignments , such as those in the RFAM database [5] , suggests that an evolutionary model for RNA structure must eventually include multiple layers of detail: point substitutions , covariant substitutions of base-pairs [6] , [7] , indels [8] , local changes in secondary structure such as helix slippage [9] , and changes in domain structure [2] . Stochastic context-free grammars ( SCFGs ) , which can efficiently detect the long-range correlations of RNA base-pairing structures , are natural probabilistic models of such phenomena and have been used for ncRNA homology detection [10]–[13] , gene prediction [14] , [15] , folding [16] , [17] and alignment [18]–[20] . By analogy with models of substitution processes , which are well-understood [21] , we may take the problem of building phylogenetic models of RNA evolution and split it into two halves . The first half is the development of a pairwise model , describing the probability distribution of a descendant ( ) conditional on its immediate ancestor ( ) . In substitution processes , the pairwise model is a conditional substitution matrix . Often ( but not always ) the pairwise model , representing a finite evolutionary time , is derived from an instantaneous model of change over an infinitesimal time interval , i . e . , a continuous-time Markov chain ( parametrized by a rate matrix ) . Obtaining the transition probabilities of this chain ( via exponentiation of the rate matrix ) yields a pairwise model whose parameters are smoothly-varying functions of . A pairwise model represents an individual branch of a phylogenetic tree , with representing the length of that branch . The second half of the phylogenetic modeling problem involves extending the model ( and related inference algorithms ) from a single branch to a complete phylogeny , i . e . , from a pairwise model of two sequences to a multiple-sequence model of many sequences . In a typical situation , the sequences at the leaves of the tree are observed but those at internal nodes are not . Questions of interest then include: For substitution models , there has been extensive work focused on answering each of these questions . Given a pairwise substitution model , questions A and B can be answered exactly by Felsenstein's pruning algorithm [22] and question C can be answered by the peeling algorithm ( first presented for pedigree analysis by Elston and Stewart [23] ) . The estimation of evolutionary histories ( question D ) has been addressed by exact summarization [24] and sampling [25] approaches . Another representation of answers A–C is that the pruning and peeling algorithms ( combined ) are just the sum-product algorithm on a directed graphical model [26] , yielding exact marginal distributions for unobserved variables . Graphical models also suggest general-purpose sampling approaches in addition to the exact sum-product algorithm . The two halves of the reconstruction problem — developing a pairwise model and then extending it to multiple sequences — are largely independent . Felsenstein's pruning algorithm , for example , is essentially blind to the parametric form of the pairwise substitution model; it just assumes that a substitution matrix is provided for every branch . Subsequent models developed by other researchers can be plugged into the pruning algorithm without modification [27] , [28] . We therefore addressed the problem of modeling the indel-evolution of multiple structured RNAs in a similarly-modular fashion by separating the creation of pairwise and multiple-sequence models . In previous work , we addressed the first ( pairwise ) part of the RNA reconstruction problem by describing a simple continuous-time model of RNA structural evolution [29] . This model corresponded to a Pair SCFG with a time-dependent parametrization which we used to simultaneously align and predict the structure of pairs of related RNAs . The focus of the present work is to solve the second ( multiple-sequence ) part of the RNA reconstruction problem by giving a general procedure for extending a pairwise model to multiple sequences related by a phylogenetic tree . This process yields a multiple-sequence SCFG , a natural model of the evolutionary relationships between multiple structured RNAs . The main contributions of this paper are ( 1 ) an algorithm that transforms a phylogenetic ensemble of pair grammars , representing models on branches of a phylogenetic tree , into a coherent , multiple-sequence SCFG , ( 2 ) dynamic programming ( DP ) algorithms for performing inference under this multiple-sequence SCFG , and ( 3 ) freely-available software implementing algorithms ( 1 ) and ( 2 ) for the simplified case of a three-taxon star-topology tree . While the idea of composing conditionally-normalized models on trees is intuitive , the resulting models can be very complex , even for simple models of RNA evolution , making ( 1 ) necessary . Studies of related indel models have suggested that an implementation of dynamic programming ( DP ) algorithms on a three-taxon tree is sufficient to draw samples from the posterior distribution of ancestral sequences on more complex tree topologies , using Markov Chain Monte Carlo or MCMC [30]–[32] , suggesting that ( 2 ) and ( 3 ) are , in principle , sufficient for analyzing trees relating many sequences . We show that our algorithm produces a multiple-sequence grammar which is much more compact than suggested by naive approaches to model construction . We provide analyses of the asymptotic complexities of models constructed using our procedure and provide estimates of the time and memory required to reconstruct the structures of several RNA families for the case of a three-taxon phylogeny , which we have implemented in the program Indiegram . While by these estimates only the smallest sequences currently fit into affordable memory , thereby preventing us from applying our method to many problems of interest , a simulation study suggests that we can hope to accurately reconstruct ancestral structures over long evolutionary time , even in the presence of structural divergence . In the Discussion , we speculate on algorithmic extensions that may reduce memory requirements , inspired by related work in reconstructing DNA and protein sequences .
Our problem statement is this: Given a phylogenetic tree relating several structured RNAs and a description of the evolution of a structured RNA along a single branch of the tree ( in the form of a Pair SCFG ) , ( 1 ) find the corresponding phylogenetic multiple-sequence grammar and ( 2 ) use that grammar to reconstruct , a posteriori , the evolutionary histories of the RNAs . We assume here that the phylogeny , including both the tree topology and branch lengths , is given . This paper focuses on model construction and inference algorithms rather than the heuristics which will be necessary to make these algorithms fast enough for analysis of many biological datasets . As discussed below , the complexity of general inference algorithms is prohibitively high for many problems of interest . However , this complexity can be significantly reduced by incorporating outside knowledge . For example , if we know the consensus structure of several sequences or their individual structures , then we can constrain our algorithms accordingly . Similarly , we might consider only ancestral structures which are compatible with a given multiple sequence alignment , or a relatively small set of candidate alignments ( as in the ORTHEUS program [33] ) . Such constraints are commonly used by programs for SCFG-based RNA sequence analysis such as QRNA [34] , Stemloc [18] and CONSAN [19] . Alignment and structural constraints can be combined [18] . In the following sections we introduce more precise definitions for two-sequence models of RNA structure and outline our algorithms for ( 1 ) combining these two-sequence models on a phylogenetic tree and ( 2 ) using the composite phylogenetic grammars for inference . We discuss the general problem of creating state-space models of the evolution of related sequences , beginning with models of substitution processes acting at independent sites ( as studied in likelihood phylogenetics ) and generalizing to models of indels , first in primary sequences and then in sequences with conserved secondary structure . A stochastic model for the evolution of one sequence ( the ancestor , ) into another ( the descendant , ) over an interval of time ( ) can be described by a joint distribution , . This joint distribution can be factored , , where is the marginal distribution over ancestral sequences and is the conditional distribution over descendant sequences given an ancestral sequence . In terms of phylogenetics , the conditional distribution describes the evolution along a branch of length . It is possible to “multiply” two such models together . More precisely , one multiplies two conditional distributions and sums out the intermediate sequence . Thus , successive evolution along two branches is modeled by the distributionand we can sum sequence out of this , obtaining the distributionfor the composite branch . This formalism underlies likelihood phylogenetics . Working under the independent-sites assumption , is the 'th element of the joint substitution matrix for a single site and is the corresponding element of the conditional matrix . The conditional matrix is in fact the matrix exponential , where is the substitution rate matrix [24] . Composition of two branches just amounts to a matrix multiplication . A similar formalism can be used to describe the evolution of whole sequences with indels . Suppose that the joint distribution is the distribution modeled by a pair hidden Markov model ( Pair HMM ) [11] , a probabilistic model of the evolution of two sequences under the approximation that only adjacent characters are directly correlated , and the marginal is the distribution of a single-sequence HMM , a probabilistic model of single sequences under the same approximation . The conditional distribution then corresponds to a conditional Pair HMM , a discrete-state machine which transforms one sequence ( the input , ) into another ( the output , ) . Following computational linguists , we call this conditionally-normalized state machine a string transducer or simply a transducer [35] . Because of its conditional normalization , this state machine is distinct from a standard Pair HMM . A Pair HMM has two outputs and and emits symbols to both of those outputs , while a transducer absorbs symbols from the input and emits symbols to the output . Despite this distinction , Pair HMMs and transducers share very similar inference algorithms; for example , is computed using a direct analogue of the Forward algorithm [11] . We extend this formalism to the case of structured RNA as follows . Let and now represent structured RNA sequences or , more precisely , parse trees . A single-sequence SCFG models the marginal ; a jointly-normalized Pair SCFG [11] models the the joint distribution . The conditional distribution is modeled by a conditionally-normalized Pair SCFG . Following terminology from computational linguistics [36] , we call this conditionally-normalized grammar a parse-tree transducer . String transducers are special cases of parse-tree transducers , just as HMMs are special cases of SCFGs . Henceforth , we will drop the distinction between strings and parse trees . We will also refer interchangeably to “states” ( in the state-machine representation ) and “nonterminals” ( in the grammar representation ) . Likewise , we will refer interchangeably to “state paths” ( machines ) and “parse trees” ( grammars ) . We can use the concepts of factoring probability distributions introduced in the two-sequence framework to model the common descent of many homologous sequences . Given a phylogenetic tree and a two-sequence model , we wish to obtain a multiple-sequence SCFG describing the common descent of the observed sequences . A singlet transducer ( which emits , but does not absorb , symbols ) lies at the root of the phylogeny and serves as a generative model of the ancestral sequence . To represent the evolution of an ancestral sequence into many descendant sequences , we place a branch transducer on each branch of the phylogeny . Throughout this paper we frequently refer to two and three-taxon ( star ) phylogenies . In all cases , the sequence is assumed to be the ( unobserved ) ancestral sequence and the sequences , , and the ( observed ) extant sequences . In this section , we describe dynamic programming ( DP ) algorithms for inferring the alignment , structure and evolutionary history of multiple related RNAs , using the multiple-sequence SCFG we have derived . The transducer composition algorithm described above constructs a phylogenetic SCFG for both ancestral and extant sequences . A parse tree for this SCFG represents a structural and evolutionary explanation of the extant sequences , including a complete ancestral reconstruction . Consequently , given a set of extant sequences , many of the questions of interest to us can be reduced to searches over , or summarizations of , the set of possible parse trees . Well-known algorithms already exist for maxing or summing over SCFG parse tree likelihoods . The Cocke-Younger-Kasami ( CYK ) algorithm performs maximum-likelihood ( ML ) inference; the Inside algorithm can be used to sum over parse trees or sample them a posteriori; and the Inside-Outside algorithm yields posterior probabilities for individual parse tree nodes [11] . All of these algorithms are , however , complicated ( at least in our models ) by the existence of “null cycles” in the grammar . A null cycle is a parse tree fragment that is redundant and could be removed , such as a detour through states ( ) that could be replaced by a direct transition ( ) . Biologically , null cycles correspond to fragments of ancestral sequence that were universally deleted and therefore are unobserved in any of the extant sequences . These unobserved fragments can be unbounded in length ( and so , therefore , can the parse tree ) . Within the CYK , Inside and Outside recursions , this causes cyclic dependencies which cannot be resolved . Below we describe a method to eliminate null cycles from the ensemble model by transforming any SCFG to an equivalent acyclic SCFG . We then present multiple-sequence versions of the CYK , Inside and Outside algorithms . While some sort of null-cycle elimination is often required in order to deal with cyclic dependencies , there are several ways to accomplish this other than the algorithm presented below . A simpler approach ( that only works for the CYK algorithm ) appears in the computational linguistics literature [37] . We have also developed a heuristic for CYK that simply ignores null cycles as well as an iterative approximation that loops several times over cyclically-dependent cells of the DP matrix until the estimate starts to converge . For conciseness , we have omitted descriptions of these methods , presenting only the exact elimination algorithm .
We implemented our model construction algorithm on the three-taxon star phylogeny . Given a singlet transducer modeling ancestral structures and a branch transducer modeling structural evolution , our Perl modules generate C++ code for the corresponding jointly-normalized three-sequence ( Triplet ) SCFG . Any model of structural evolution which can be represented as a Pair SCFG and factored into singlet and branch transducers is permitted as input to the packages , allowing for flexible , automated model design . The available software is described in Text S3 . We illustrated our method for building models of structured sequences using a model which was introduced in previous work , the TKF Structure Tree [29] , a simplified probabilistic model of the evolution of RNA structure . The TKF Structure Tree ( TKFST ) model is based on the Thorne-Kishino-Felsenstein ( TKF ) model of the stochastic evolution of primary sequences via indel events [44] . In the original TKF model , sequence evolves under a time-homogeneous linear birth-death-immigration process [45] . Single characters ( “links” ) are inserted with rate and deleted with rate . At equilibrium , sequences obey a geometric length distribution with parameter . Although this model has flaws ( e . g . , it lacks affine gap penalties , rate heterogeneity and context-dependent mutation rates ) , it illustrates many of the key ideas used by more sophisticated indel models , notably the possibility for systematic derivation of pairwise alignment automata from first principles via analysis of birth-death processes [44] , [46] . The TKF Structure Tree model is an extension of the TKF model to RNA structure . In this model , loop and stem regions are mutually nested ( Figure 8 ) : the parameter determines the proportion of links within loop sequences that are nested stems , and every stem sequence has a nested loop at the end . Single bases are inserted and deleted in loops with rates and ; similarly , base-pairs are inserted and deleted in stems with rates and . Both loops and stems have geometric length distributions with parameters and . Insertions of a new stem into an existing loop sequence ( or deletions of an existing stem ) occur at the same rate as single-base insertions ( or deletions ) and can model large-scale structural changes ( Figure 9 ) . We parametrized the singlet and branch transducers of the TKFST model using estimates reported by a phylo-grammar for RNA secondary structure prediction , PFOLD [16] , and an implementation of pairwise alignment for the TKF Structure Tree model , Evoldoer [29] . The equilibrium distributions of unpaired and paired nucleotides of the singlet and branch transducers , as well as the substitution models of unpaired and paired nucleotides of the branch transducers , were derived from the substitution rate matrices of the PFOLD program . These rate matrices , which have proven useful for RNA structure prediction [16] , [17] , [47] , were derived from the Bayreuth tRNA database [48] and the European large subunit rRNA database [49] . This continuous-time model corresponds to a Pair SCFG and as such fits neatly into our modeling framework once the probability distribution is appropriately factored into marginal and conditional distributions ( generated by singlet and branch transducers ) . Tables 1 and 2 show the states and transitions of the singlet transducer ( single-sequence SCFG ) which generates ancestral sequence under the Structure Tree model . Tables 3 and 4 show the states and transitions of the branch transducer ( conditionally-normalized Pair SCFG ) which evolves a sequence and structure along a branch of the phylogenetic tree . The equilibrium distribution and transition probabilities between states of the TKFST model can be expressed in terms of functions of the evolutionary time along a branch and the insertion and deletion rates and of the model . The length of ancestral sequences is geometric in ( Table 2 ) , defined as . The three functions , and which govern the transition probabilities in Table 4 are defined for loop sequences asand similarly for stem sequences [29] . The above-described TKFST SCFGs must be transformed slightly before they can be loaded into Indiegram . The grammars are presented in Indiegram format in Text S4 . A few other useful statistics for the TKFST model: the expected number of links in a loop sequence is and in a stem sequence . Since of the links in a loop sequence are nested stems , and since each stem has twice as many nucleotides as it has links ( since each link is a base pair ) , the expected number of bases in a loop sequence isThe expected number of bases in a stem sequence isThe expected number of bases that are created/removed when a loop-sequence link is inserted/deleted isThe expected number of stems directly rooted in a given loop sequence is and the expected number of stems directly rooted in , or indirectly descended from , a given loop sequence is ( note that this is also the expected total number of loop sequences indirectly descended from a given loop sequence ) . Therefore , in the equilibrium structure , the expected number of stems is ; of loops , ; of unpaired bases , ; and of base-pairs , . In a tree with total branch length , the expected number of single-base deletions is ; of base-pair deletions , ; and of substructure deletions , . The TKFST model , like the original TKF model , probably needs refinements in order to accurately model many structural RNAs . For example , it fails to model certain phenomena observed in natural RNA structures ( such as base-stacking or tetraloops ) and in alignments of those structures ( such as helix slippage ) . We assessed its appropriateness as a model of RNA structural evolution by conducting benchmarks of its capabilities for ( 1 ) multiple sequence alignment of structured RNAs , summing over all possible structures , and ( 2 ) structure prediction of homologous structured RNAs and comparing its performance to Stemloc ( one of the better-performing pairwise SCFGs used for RNA multiple alignment [20] ) . The results of these benchmarks , reported in Table 5 and Table 6 , suggest that TKFST is a useful guide for deriving more complicated models of RNA evolution: while it has relatively poor sensitivity ( but high positive predictive value ) as a base-pairing predictor , it is competitive with one of the most accurate RNA multiple sequence alignment programs [20] . TKFST's poorer performance at base-pairing prediction is likely due to its much-simpler model of RNA structure . The richer grammar , as described in [18] , is much more complex than TKFST: excluding the substitution model , it has 14 free parameters ( compared to TKFST's 4 ) , uses an affine gap penalty ( compared to TKFST's linear gap penalty ) , and explicitly models structural features such as multiple-branched loops , symmetric/asymmetric bulges , and minimum loop lengths . Unlike TKFST , the richer grammar is structurally unambiguous: a one-to-one mapping exists from structures to parse trees . Although we use the TKFST model as an illustrative example of a Pair SCFG that can be extended with our method , the model is not fundamental to our approach and can be replaced by a different and more realistic pairwise model , such as the Stemloc pairwise SCFG used in these comparisons [20] . We anticipate that further improvements should be possible by reviewing other comparisons of SCFGs at structure prediction , such as the study of [17] . We used our model-construction algorithm to build the grammar corresponding to the TKFST model acting on a star phylogeny with three ( extant ) leaf sequences and a single ( unobserved ) ancestral sequence . We chose this phylogeny for two reasons: ( 1 ) it is the simplest extension of the well-studied , standard two-sequence ( Pair SCFG ) model and ( 2 ) algorithms on a phylogeny with three leaves should be sufficient for ergodic sampling of reconstructions on any larger phylogeny , using , e . g . , a Gibbs-sampling MCMC kernel [31] or a progressive suboptimal-alignment sampling heuristic [33] . The statistics of the TKFST model on the three-taxon phylogeny illustrate the advantages of our procedure for model construction . While the singlet and branch transducers are relatively simple—the singlet transducer , shown in Table 2 , has 7 total states and 2 bifurcation states and the branch transducer , shown in Table 4 , has 21 total states and 6 bifurcation states—the ensemble model of three extant sequences is very complex . The naive exponential upper-bound gives a maximal state space of size states . Using our uninformed search algorithm , we determined that there are 287 accessible states and 686 possible transitions between these states ( compare with the transitions estimated with the exponential calculation ) . After performing the transformations described in “Exact elimination of null cycles in SCFGs” to eliminate useless windback states , the ensemble model has a reduced state space with 230 states , albeit at the cost of extra transitions , bring the total to 1 , 789 transitions ( here we are trading reduced memory complexity , which is linear in the number of states , for increased time complexity , which is linear in the number of transitions ) . Note that both before and after the reduction in complexity , the total number of states and transitions are less than the approximate bounds of states and transitions suggested in “The composition algorithm” . Nonetheless , the extreme complexity of the ensemble model , despite the simplicity of the underlying model of RNA structure , makes clear the necessity for automated procedures for model construction . Dataset S1 gives the state space of the ensemble model constructed by the search algorithm and Dataset S2 the reduced model after eliminating windback states; both are in Graphviz format for visualization and show the state of the singlet transducer generating ancestral sequence as well as the states of the branch transducers generating observed sequences . We implemented constrained maximum-likelihood inference of the structural alignment and ancestral structure of three extant sequences in a C++ program ( Indiegram ) . For tractability , Indiegram uses the concept of fold envelopes described earlier to limit the fold space considered by the CYK algorithm , permitting structural information for the three extant sequences to be ( optionally ) supplied as input . If no structural information is supplied , then Indiegram uses a single-sequence SCFG to estimate a set of plausible folds [18] , which are used to constrain the CYK algorithm . The inference algorithms in Indiegram could be further constrained to enforce , for example , a fixed multiple alignment or a consensus structure for extant sequences . While experimentally-determined structures of individual RNAs are relatively rare , curated deep sequence alignments , such as those constructed for ribosomal RNAs [50] , are frequently available for characterized RNA families . By constraining the inference algorithms with such sequence alignments , the memory and time complexity of the algorithms could be dramatically reduced . Such constraints can be naturally expressed with “alignment envelopes , ” the alignment-space analogue of fold envelopes [18] . However , in this paper we focus on model construction and inference algorithms and postpone exploration of heuristics and constraints of these algorithms for future work . While reconstructing large RNAs such as ribosomal subunits is currently computationally-inaccessible without further heuristics to constrain our algorithms , reconstructing small RNAs of biological interest will soon be feasible . Table 7 shows estimates of the memory and time required to reconstruct biologically-interesting subunits of the nanos 3′ translational control element and tRNAs , as well as two small RNAs which show significant structural divergence , the Y RNAs and Group II introns , and therefore promise to be interesting candidates for ancestral reconstruction . The reconstructed structures for three nanos 3′ translational control elements ( TCEs ) and three tRNAs , which could be analyzed given current computational limitations , can be found at http://biowiki . org/IndieGram; however , the phylogenetic trees relating the tested sequences have short branch lengths , making the reconstruction problem easy by forcing the reconstructed structures to be essentially-identical to those of one of the extant RNAs . Guided by our experience with the nanos 3′ TCE and tRNA , where the reconstruction problem was made easy by the presence of a close outgroup , we conducted a simulation study of the dependence of reconstruction accuracy on outgroup branch length , with the further goal of comparing the performance of our reconstruction method ( when simulating directly from the model ) to simpler reconstruction methods that ignore either structure or phylogeny . ( We here use the term “outgroup” loosely to denote the variable-length branch in our three-taxon study , where the other two branches are held at unit length . ) We simulated the evolution of RNAs under the TKFST model along three-taxon phylogenies ( with one internal node ) , where we kept the branch lengths of two sibling species constant and varied the branch length of the outgroup between at steps of size . Parameters used in the simulation were and for loop sequence and and for stem sequence; the probability of a stem insertion was . These yielded a mean loop length of 5 bp and a mean stem length of 2 . 33 bp , with substructure indels per alignment . We selected alignments to reconstruct by requiring that there be at least two ancestral stems , loops of and stems of ; to reduce the complexity of our algorithms we additionally required that the sequences have . We then attempted three-way multiple alignment ( and , in some cases , reconstruction of the ancestor ) using a variety of statistical inference algorithms . We sought insight as to the relative importance of the following factors in reconstructing ancestral RNA: ( i ) modeling the secondary structure; ( ii ) modeling the phylogenetic topology & branch lengths; ( iii ) using posterior-decoding algorithms to maximize the expected alignment accuracy , rather than picking the single most likely alignment [20] , [51] , [52] . The alignment programs we used in this benchmark were Indiegram ( exact ML inference of alignment and ancestral structure , given phylogeny , descendant structures and correct model ) ; Stemloc ( a greedy ML heuristic , ignoring phylogeny in favor of a single-linkage clustering of the descendant structures ) ; Stemloc-AMA ( a posterior-decoding heuristic , maximizing the alignment's expected accuracy rather than its likelihood ) ; and Handel ( ML alignment under various indel models that ignore secondary structure completely ) . In detail , the reconstruction methods were Stemloc : the Stemloc program was used to align the three sequences via single-linkage clustering with a Pair SCFG [18] . The structures of the leaf sequences were provided , but not the phylogenetic branch lengths . Instead of modeling a true phylogeny by introducing unobserved ancestral sequences , it just does single-linkage clustering of the observed sequences . Stemloc-AMA : the Stemloc program was used to align the three sequences in “sequence annealing” mode , a posterior decoding method that attempts to optimize AMA , a sum-over-pairs alignment accuracy metric [20] . The structures of the leaf sequences were provided , but not the phylogenetic branch lengths . This program uses the same underlying pair SCFG as Stemloc , but instead of maximizing likelihood , it attempts to maximize an alignment accuracy metric . TKF91: with the TKF91 model [44] , the Handel package [30] , [39] , [40] was used to align the three extant sequences and reconstruct the ancestor . The correct phylogenetic tree and branch lengths were supplied ( as they were for the Indiegram benchmark ) . The insertion , deletion and substitution rates for the TKF91 model were set equal to those of the loop submodel of TKFST . This may be understood as a naive sequence-only reconstruction that completely ignores basepair structure ( i . e . the stem sub-model of TKFST ) . Long Indel: with a single-event trajectory approximation to the long indel model [53] , the Handel package was used to align the three extant sequences and reconstruct the ancestor . The correct phylogenetic tree and branch lengths were supplied . The deletion and substitution rates were set equal to those of the loop submodel of TKFST . The mean indel length was set equal to , the mean number of bases that are created/removed by an insertion/deletion in the loop submodel of TKFST; the mean equilibrium sequence length ( and thereby the insertion rate ) was equal to , the mean number of bases in TKFST at equilibrium ( “A simple model of RNA structural evolution” has formulae for these quantities in terms of the TKFST rate parameters ) . This model improves on the previous model ( TKF91 ) by introducing affine gap penalties . We measured alignment accuracy , under the simplifying assumption that this correlates well with ancestral reconstruction accuracy . We first consider the perfect alignment rate; that is , the number of times each method gets the alignment exactly correct . Theory predicts that Maximum Likelihood inference , using the correct model and parameters , should be asymptotically optimal ( if one only counts perfect guesses ) . Inspecting Figure 10 , we find this to be almost the case; the exception is when the outgroup is very distant and the bins may be undersampled ( the departure from prediction that we observe for low-identity alignments is not statistically significant: when the optimal success rate drops below , then 125 trials are probably insufficient to compare two near-optimal methods ) . We also note that the ML version of Stemloc is near-optimal , despite the Stemloc pair-SCFG being slightly different from the TKFST pair-SCFG in parameterization and structure ( e . g . Stemloc 's grammar does not allow insertion/deletion of entire substructures ) . The ML version of Stemloc is also observed to have a slightly higher perfect alignment rate than the posterior-decoding version ( Stemloc -AMA ) . Finally , we note that the structure-blind models ( TKF91 and Long Indel ) perform consistently worse than the structure-aware methods; furthermore , both linear ( TKF91 ) and affine ( Long Indel ) gap-penalties perform equally bad in this test ( note that the TKFST model , from which the true alignments were simulated , does not allow long-indel events , which may partly explain why affine gap-penalties do not help in this benchmark ) . A subtly different ranking emerges from consideration of the alignment accuracy . In Figure 11 , we abandon the all-or-nothing metric of counting only perfect alignments , instead using a metric that shows what proportion of the alignment is correct . Specifically , we plot the Alignment Metric Accuracy ( AMA ) as a function of outgroup branch length . AMA measures the proportion of residues which are correctly aligned , averaged over all pairs of sequences [54] . Figure 4 reveals that Stemloc-AMA ( which attempts to find the alignment with the maximum expected AMA ) edges out both Indiegram and the ML version of Stemloc ( both of which attempt to find the alignment with the maximum likelihood ) . These results , compared to the subtly different story told by the perfect alignment rate , underscore the point that benchmark results for alignment methods can depend exquisitely on the choice of accuracy metric . The superiority of ML methods is only assured in terms of perfect alignment rate , and not necessarily other accuracy metrics . Taken together , these results suggest that the most important factor distinguishing the various models we have examined is the incorporation of some form of basepair structure: structure-blind Handel ( regardless of linear vs affine gap penalty ) performs much worse than the structure-aware SCFG methods . Intuitively , this is to be expected: whenever a basepair-aware method aligns one half of a basepair , it gets the other nucleotide correctly aligned for free . In benchmarks of RNA multiple alignment programs , structure-aware scoring schemes routinely outperform structure-blind scoring schemes [55] , [56] . Since we know that modeling structure is very important , it's not too surprising that it turns out to be the most important of the factors we considered . The second most important , amongst the factors we have considered in this experiment , is selection of the most appropriate objective function for the task at hand ( c . f . perfect alignment rate vs AMA ) , followed by use of the correct posterior-decoding algorithm for the chosen objective function ( c . f . Stemloc vs Stemloc-AMA ) . This is a subtle but important point: before deciding exactly what inference algorithm we're going to use to reconstruct ancestral sequences , we need to decide whether we want to maximize ( a ) the probability that our reconstructed sequence is 100% correct , ( b ) the expected number of nucleotides that are correctly reconstructed , ( c ) the expected number of base-pairs that are correctly reconstructed , ( d ) the expected number of stems that are correctly reconstructed , ( e ) some other metric . Each of these metrics would require a slightly different inference algorithm . Lastly , the fine details of the scoring scheme—including branch lengths , substitution scores , gap penalties and so forth—appear to be the least important of the factors we considered , yielding observable differences only when all other aspects of the inference procedure were more-or-less equal . While such details of the model may affect reconstruction quality , they appear to have very minor influence on alignment quality .
Following the conception of paleogenetics [57] , a large number of synthetic reconstructions of ancestral protein sequences have been reported in the literature [58]–[65] . There is also scientific interest in reconstructing DNA sequences [33] , [66]–[71] . Given the importance of the RNA world hypothesis to current discussions of the origin of life [72]–[78] , the many modern-day relics of this world [79]–[82] and the recent proposal of a structural model for the primordial ribosome [2] , we believe that phylogenetic reconstruction of ancient RNA is a significant problem , deserving of strong bioinformatics support . The work reported in this paper builds on extensive prior art in the areas of evolutionary modeling and ancestral reconstruction . Reviewing all of this would take several books , but we can note some key references . The reconstruction of ancient sequences was first proposed in 1963 by Pauling and Zuckerkandl [57]; current applications of this idea , mostly using substitution models , are surveyed in the book edited by Liberles [83] . Many algorithms in phylogenetics implicitly reconstruct substitution histories , whether by parsimony [84] , [85] or likelihood [22] . There is a substantial body of work to model indels on phylogenies [30] , [35] , [39] , [44] , [53] , [86]–[92] . Recent work has extended these ideas to the reconstruction of indel histories [93] , [94] , particularly at the genomic scale [33] , [95] . There is also prior work in computational linguistics on the theory of transducers for sequences [96] and parse trees [36] , [37] , [97] , [98] ( from which we take the terms “string transducer” and “parse-tree transducer” ) . We draw on the bioinformatics literature for SCFGs [10] , [11] , [99] , especially Pair SCFGs [14] , [18] , [19] and phylogenetic SCFGs [16] . In particular , an early example of a pairwise conditional model for structure-dependent RNA evolution was given by Eddy et al [12] . A conditional framework similar to ours in some respects is described by Sakakibara et al [100] . The dynamic programming inference algorithms for multiple-sequence SCFGs are closely related to the protosequence algorithm of Sankoff [42] . While we have focused on the TKF Structure Tree model in our Results , our model-construction algorithm is applicable to any model of the evolution of secondary structure which can be expressed as a Pair SCFG . Realistic structural and thermodynamic effects—such as base-stacking or loop length distributions—can , in principle , be incorporated . Other phenomena of RNA evolution may prove more difficult: modeling helix slippage with a branch transducer is awkward , let alone more radical changes in structure; pseudoknots , too , are impossible with the models we have described here . Even so , variants of our models could be used for proposing candidate alignments for more accurate scoring by such models . An implementation of inference algorithms for models on the three-taxon phylogeny is sufficient to construct a MCMC sampling algorithm over many sequences on an arbitrary phylogeny . A sketch of such a sampling algorithm is as follows: at each step of the sampling algorithm , we re-sample the sequence and structure of the ancestral node , conditioned on the sequences and structures of , and . The structural alignment of all four sequences can change at each step , providing for fast mixing and guaranteeing ergodicity . This move is similar to the sampler proposed by [31] for models with a HMM structure . Note that this , in principle , permits construction of a crude sampler to simultaneously infer phylogeny as well , by proposing and accepting or rejecting changes to the underlying tree as well as the implied structural alignment . Reconstructing structural changes of large RNAs using the three-way sampling kernel which we have described would require resources far in excess of those currently available; barring the availability of supercomputers with terabytes of memory , such algorithms will only be feasible for short RNAs ( Table 7 ) . A promising direction is to consider variations on the three-way sampling kernel , such as the importance-sampling approach described for the TKF model by [32] . This approach first proposes an ancestor by aligning extant sequence to ( ignoring ) ; then , in a second step , the proposed is independently aligned to . The proposed three-way alignment and reconstruction is then randomly accepted ( or rejected ) using a Hastings ratio based on the three-way transducer composition . The complexity of this kernel is the same as the pairwise case; with suitable constraints , this is feasible for RNA grammars on present hardware , at least for ribosomal domains ( if not yet whole subunits—although pairwise alignment of those should also be possible soon ) . The approach of Redelings and Suchard therefore merits future consideration in the context of modeling the evolution of RNAs on a tree . An alternative MCMC scheme for sampling RNA phylogeny , structure and alignment was developed for the SimulFold program [101] . SimulFold does not use a strictly normalized probabilistic model , resulting in some oddities in the ways that structure and indels interact ( for example , it does not penalize deletion of one half of a basepair ) . Currently , it is not clear how appropriate SimulFold would be for ancestral reconstruction , although it has several advantages ( e . g . , explicit treatment of pseudoknots ) . Of course , MCMC kernels are inherently adaptable to other purposes: the MCMC moves developed for SimulFold may be useful for inference under different models . This paper focuses on the case where the tree topology is known , but many of the methods which we have described can be extended to the more general case where none of the possible constraints ( phylogeny , structure or alignment ) are final . For example , the probabilistic framework readily allows us to compare likelihoods of two different phylogenetic trees by constructing a composite transducer for each tree . Thus , the MCMC samplers described above for alignments could , in principal , be extended to phylogenies ( albeit at a computational cost ) . While MCMC provides the most information about the posterior distribution of evolutionary histories , in practice a maximum likelihood inference may be adequate ( and typically much faster ) . The progressive profiling used by the Ortheus program for reconstructing ancestral genomes is promising [33] . This approach is similar to a progressive multiple alignment algorithm , in that it proceeds via a single postorder ( leaf-to-root ) traversal of the phylogeny . As each node is visited , a profile is generated for that node , by aligning the profiles of its children to a composite transducer using DP , then sampling a finite number of traceback paths through the DP matrix . The profile is not linear: the sampled paths instead form a reticulate network , a . k . a . a partial order graph [102] . An equivalent of Ortheus for RNA reconstruction should be possible , representing the intermediate profiles using transducers . Given the excellent performance of Stemloc-AMA 's sequence annealing , particularly when measured using its own scoring metric ( AMA ) , such posterior-decoding methods should also be considered for reconstruction . In summary , the evolutionary models and algorithms we have described form a systematic theoretical platform on which we can test different optimization and sampling strategies for studying the structural evolution of RNA gene families in detail . Stochastic grammars are powerful tools for this task , although they will not be the only tools we need , particularly as we move towards modeling RNA evolution in greater detail . Our hope is that these algorithms will allow us to test and refine our understanding of RNA evolution by computational reconstruction and ( eventually ) direct experimental investigation of early ribonucleic machines . | A number of leading methods for bioinformatics analysis of structural RNAs use probabilistic grammars as models for pairs of homologous RNAs . We show that any such pairwise grammar can be extended to an entire phylogeny by treating the pairwise grammar as a machine ( a “transducer” ) that models a single ancestor-descendant relationship in the tree , transforming one RNA structure into another . In addition to phylogenetic enhancement of current applications , such as RNA genefinding , homology detection , alignment and secondary structure prediction , this should enable probabilistic phylogenetic reconstruction of RNA sequences that are ancestral to present-day genes . We describe statistical inference algorithms , software implementations , and a simulation-based comparison of three-taxon maximum likelihood alignment to several other methods for aligning three sibling RNAs . In the Discussion we consider how the three-taxon RNA alignment-reconstruction-folding algorithm , which is currently very computationally-expensive , might be made more efficient so that larger phylogenies could be considered . | [
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] | [
"computational",
"biology/evolutionary",
"modeling"
] | 2009 | Evolutionary Triplet Models of Structured RNA |
T-box proteins are conserved transcription factors that play crucial roles in development of all metazoans; and , in humans , mutations affecting T-box genes are associated with a variety of congenital diseases and cancers . Despite the importance of this transcription factor family , very little is known regarding how T-box factors regulate gene expression . The Caenorhabditis elegans genome contains 21 T-box genes , and their characterized functions include cell fate specification in a variety of tissues . The C . elegans Tbx1 sub-family member MLS-1 functions during larval development to specify the fate of non-striated uterine muscles; and , in mls-1 mutants , uterine muscles are transformed to a vulval muscle fate . Here we demonstrate that MLS-1 function depends on binding to the Groucho-family co-repressor UNC-37 . MLS-1 interacts with UNC-37 via a conserved eh1 motif , and the MLS-1 eh1 motif is necessary for MLS-1 to specify uterine muscle fate . Moreover , unc-37 loss-of-function produces uterine muscle to vulval muscle fate transformation similar to those observed in mls-1 mutants . Based on these results , we conclude that MLS-1 specifies uterine muscle fate by repressing target gene expression , and this function depends on interaction with UNC-37 . Moreover , we suggest that MLS-1 shares a common mechanism for transcriptional repression with related T-box factors in other animal phyla .
T-box transcription factors play essential roles in the development of all multicellular organisms , where their functions include the specification of primary germ layers and the specification of cell fates during organogenesis [1] , [2] . In humans , both decreased and increased activity of these factors are associated with congenital disease ( Holt-Oram syndrome , Ulnar-Mammary syndrome , DiGeorge syndrome , etc ) [3] , auto immune disorders [4] , and cancers [5] , [6] . Despite this importance the mechanisms by which T-box factors regulate target gene expression are not well established . Groucho family ( Gro/TLE ) proteins are conserved transcriptional co-repressors that interact with distinct Engrailed homology 1 ( eh1 ) or WRPW/Y motifs in a wide variety of transcription factors and , in many cases , recruit histone deacetylases to target gene promoters [reviewed in [7]] . Gro/TLE factors have recently been implicated in the regulatory mechanism of several T-box factors . Xenopus Tbx6 and Tbx1 , and zebrafish Tbx24 and Ntl/Brachyury interact indirectly with Gro/TLE factors through Ripply/Bowline family proteins , and this interaction can convert these proteins from transcriptional activators to repressors [8]–[10] . Two closely related members of the mouse Tbx1 subfamily Tbx15 and Tbx18 interact directly with the Gro/TLE protein TLE3 via eh1 motifs to repress reporter gene expression in mammalian cells [11] . Additional T-box factors likely function with Gro/TLE proteins , as T-box factors in several species contain eh1 motifs , including the Caenorhabditis elegans T-box factors MLS-1 and MAB-9 [12] . While this accumulating evidence suggests a variety of T-box factors interact with Gro/TLE factors , the significance of these interactions has not been examined in vivo . In this report we investigate the interaction between MLS-1 and the C . elegans Gro/TLE protein UNC-37 . MLS-1 is a member of the Tbx1 subfamily that includes mouse Gro/TLE-interacting proteins Tbx15 and Tbx18 [1] , [13] . MLS-1 functions to specify uterine muscle fate in the mesodermal ( M ) lineage during hermaphrodite larval development [14] . In wild-type hermaphrodites , the M mesoblast produces all post-embryonic mesoderm cells , including two sex myoblasts ( SMs ) that divide during the late L3 and L4 stages to produce eight uterine muscles ( four um1 and four um2 uterine muscles ) and eight vulval muscles ( four vm1 and four vm2 vulval muscles ) [15] . mls-1 loss-of-function results in a transformation of uterine muscle precursors to a vulval muscle fate resulting in the loss of all um1 and um2 muscles and the formation of excess vm1 and vm2 muscles . In comparison , ectopic expression of mls-1 throughout the M lineage results in supernumerary uterine muscles [14] . Here we demonstrate that MLS-1 interacts with UNC-37 in both yeast two-hybrid and in C . elegans bimolecular fluorescence complementation ( BiFC ) assays . This interaction is mediated by an eh1 motif near the MLS-1 N-terminus , and mutation of this eh1 motif eliminates the ability of MLS-1 to specify uterine muscles . Furthermore , unc-37 loss-of-function results in a loss of uterine muscles and a corresponding gain of vulval muscles similar to mls-1 loss-of-function . Taken together , these results indicate MLS-1 functions as an UNC-37 dependent transcriptional repressor to specify uterine muscle fate , and they provide the first in vivo evidence that interaction with Gro/TLE factors is essential for T-box factor function .
C . elegans MLS-1 is a relatively small T-box protein ( 252 aa; Accession NP_498640 ) consisting of a 187 residue T-box DNA-binding domain flanked by short N-terminal and C-terminal amino acid stretches [14] . A bioinformatic screen of predicted transcription factors in C . elegans identified a high-scoring eh1 motif outside of the T-box near the MLS-1 N-terminus [12] . The MLS-1 eh1 motif is conserved in the N-terminus of MLS-1 proteins of several Caenorhabditis species ( Figure 1A ) suggesting it is biologically significant , and we hypothesize MLS-1 functions as a Groucho-dependent transcriptional repressor . To test this hypothesis , we first asked if MLS-1 can interact with the C . elegans Groucho protein UNC-37 using yeast two-hybrid assays . A full-length MLS-1 bait was tested for interaction with a prey encoding UNC-37 residues 70–612 , which consists of the entire WD repeat domain ( Accession O02482 ) . The WD repeat domain of Gro/TLE proteins mediates interaction with target proteins [7] , [16] , [17] . We found that MLS-1 interacts specifically with the UNC-37 prey , but does not interact with the empty prey vector . Mutating two consensus residues in the MLS-1 eh1 motif to alanines abolished this interaction ( Figure 1A ) . In subsequent experiments we refer to this mutation as MLS-1FSI→ASA . To further explore this physical interaction we asked if MLS-1 and UNC-37 are able to undergo bimolecular fluorescence complementation ( BiFC ) in C . elegans . In BiFC assays separate pieces of a fluorescent protein are attached to potential protein interactors , and if these proteins interact the fluorescent protein is able to reconstitute giving off a fluorescent signal [18] , [19] . We generated three independent transgenic lines expressing mls-1 fused to a Venus N-terminal fragment ( mls-1::vn173 ) and unc-37 fused to a Venus C-terminal fragment ( unc-37::vc155 ) under control of the heat shock promoter hsp-16 . 41 . Heat shocking each of these lines resulted in BiFC signal in the nuclei of several cell types in which the hsp-16 . 41 promoter is strongly active after heat shock , most noticeably in nuclei in embryos and the intestinal cells ( Figure 1B ) [20] . The nuclear BiFC signal in the intestine was distinct from autofluorescence from endogenous gut granules ( Figure 1B ) , and transgenic lines expressing only unfused Venus protein halves had no BiFC signal as previously reported [18] . To determine whether the interaction between MLS-1 and UNC-37 requires the MLS-1 eh1 motif , we asked whether co-expression of mls-1FSI→ASA::vn173 and unc-37::vc155 would present a BiFC signal . After a one hour heat shock , transgenic animals co-expressing these proteins displayed no BiFC signal , whereas animals co-expressing wild-type mls-1::vn173 with unc-37::vc155 exhibited abundant BiFC signal ( Figure 1B ) . Substantially longer heat shocks caused some nuclei co-expressing mls-1FSI→ASA::vn173 and unc-37::vn155 to exhibit low intensity BiFC signal , suggesting that the eh1 mutation reduced but did not completely eliminate interaction with UNC-37 , and that the MLS-1FSI→ASA protein is expressed . To further demonstrate that the MLS-1FSI→ASA protein is expressed , we examined strains containing an mls-1FSI→ASA::mCherry transgene regulated by the mls-1 promoter , and found that this mutant fusion protein is expressed in enteric muscles at a level comparable to wild-type mls-1::mCherry ( Figure 1C ) . Taken together , these results indicate that MLS-1 can interact with UNC-37 in yeast and in C . elegans , and that these interactions depend on the MLS-1 eh1 motif . Furthermore , in C . elegans this interaction was detected only in cell nuclei , consistent with the hypothesis that these proteins interact to repress target gene expression . MLS-1 and UNC-37 are capable of interacting in yeast and when expressed broadly in C . elegans using a heat shock promoter , but it is not clear from these results whether these proteins interact in any of the cells in which MLS-1 functions . UNC-37 is believed to be ubiquitously expressed throughout development [17] , but MLS-1 is expressed in a very restricted pattern in uterine muscles , type 2 vulval muscles ( after specification of these cells as vulval muscles ) , and three enteric muscles ( left and right intestinal muscles and anal depressor muscle ) [14] . Our analysis of mls-1 promoter activity confirms that mls-1 is expressed in the above mentioned enteric muscles throughout the life of animals post-hatching , but is only very transiently expressed in the uterine muscles and vm2s at L4 and young adult stages ( Figure S1 ) . To determine if MLS-1 and UNC-37 interact in cells normally expressing MLS-1 , we generated three independent strains co-expressing mls-1::vc155 and unc-37::vn155 under control of the mls-1 and unc-37 promoters , respectively , and found that each of these lines exhibited BiFC signal in most cells where mls-1 is normally expressed . BiFC signal was observed postembryonically in one or two cells in the tail , which we tentatively identify as enteric muscles ( Figure 2C ) . BiFC signal was also observed near the vulva in young adults , in cells that we identify as the vm2 vulval muscles and the um1 and um2 uterine muscles based on position and morphology ( Figure 2A , 2B ) . The um2 cells had BiFC signal localized in either nuclei or cytoplasm , and we believe these differences reflect the different morphology of type 1 and type 2 uterine muscles [21] . The signals in the vulval and uterine muscles were very transient and were not observed in L4s or in older adults . MLS-1 promotes uterine muscle fate , and we wanted to determine if this activity depends on the MLS-1 eh1 motif . In mls-1 mutants uterine muscles are transformed into vulval muscles . This phenotype can be easily scored using an egl-15::gfp reporter , which in wild-type animals is expressed in four vm1 cells that form a characteristic X-shape near the vulva [22]–[24] , but in mls-1 mutants egl-15::gfp expression can be seen in four additional cells [14] ( Figure 3 ) . mls-1 mutants can be efficiently rescued by transformation with a wild-type mls-1 genomic DNA fragment injected at 10 ng/µl [14] . We wanted to ask if MLS-1 requires an intact eh1 motif to specify uterine muscle fate , but we were unable to obtain transgenic lines containing the same genomic fragment bearing the mls-1FSI→ASA mutation when injected at this concentration . This result strongly suggests that the mls-1FSI→ASA is expressed , but is toxic to the animals . We were able to generate both wild-type mls-1 and mls-1FSI→ASA transgenic strains by reducing the DNA concentration to 2 . 5 ng/µl . Wild-type mls-1 DNA at this lower concentration was able to rescue mls-1 ( cc569 ) mutants in five independent lines , but at reduced percentages ( Figure 3 ) ( Table 1 ) . In contrast the mls-1FSI→ASA transgene did not rescue mls-1 ( cc569 ) in seven of seven lines ( Figure 3 ) ( Table 1 ) . These results indicate that the MLS-1 eh1 motif is required for MLS-1 to specify uterine muscle fate . Because the MLS-1 eh1 motif mediates interaction with UNC-37 and is necessary for rescue of mls-1 mutants , we hypothesize MLS-1 requires UNC-37 to specify uterine muscle fate . Therefore unc-37 loss-of-function animals should phenocopy mls-1 mutants . To test this hypothesis , we performed unc-37 ( RNAi ) on egl-15::gfp animals . These RNAi affected animals displayed egl-15::gfp expression in the four vm1 cells but also in up to four additional cells ( Figure 4A , Table 2 ) . To more directly examine the effect on uterine muscle specification we repeated unc-37 ( RNAi ) experiments on animals with an integrated rgs-2::gfp transgene . In wild-type adults rgs-2::gfp is expressed in the pharynx , the ventral nerve cord , lumbar ganglia , and uterine muscles [14] , [25] . unc-37 ( RNAi ) greatly reduced the number of and intensity of rgs-2::gfp expressing uterine muscles , while having no effect on rgs-2::gfp expression in other tissues ( Figure 4B , Table 2 ) . Note that the morphology of uterine muscles made it much more difficult to count rgs-2::gfp expressing cells than egl-15::gfp expressing vulval muscles , but nearly half the unc-37 ( RNAi ) animals we examined had no rgs-2::gfp expressing uterine muscles . We next examined egl-15::gfp expression in two unc-37 mutants: unc-37 ( e262 ) and unc-37 ( wd17wd21 ) . unc-37 ( e262 ) is a hypomorphic allele that can be maintained as a homozygous strain , and it encodes a missense mutation affecting a conserved residue in the WD repeat domain that specifically affects UNC-37 function in VA motor neurons [17] . In contrast unc-37 ( wd17wd21 ) is a putative null allele containing a splice acceptor site mutation upstream of the WD repeats [17] . unc-37 ( wd17wd21 ) mutants exhibit a maternal effect embryonic lethal phenotype , but unc-37 ( wd17wd21 ) homozygotes produced from heterozygous mutant hermaphrodites grow to adulthood and exhibit uncoordinated and protruding vulva phenotypes . Most unc-37 ( e262 ) mutants exhibited a wild-type pattern of egl-15::gfp expression , but approximately 5% of these animals expressed egl-15::gfp in extra cells . A much higher percentage of unc-37 ( wd17wd21 ) adult animals ( 81% , n = 59 ) contained supernumerary egl-15::gfp expressing vulval muscles . In both cases GFP-expressing cells were near the vulva similar to mls-1 ( cc569 ) animals ( Figure 4A ) , but in other cases they were located in the posterior body wall muscle quadrants ( Figure S2 ) . A similar localization of egl-15::gfp expressing cells has previously been observed in mls-2 mutants [26] . This phenotype may be significant as MLS-2 is predicted to have a high scoring eh1 motif [12] , and it suggests that unc-37 ( e262 ) may affect the activity of both MLS-1 and MLS-2 in specifying cell fates in the M lineage .
MLS-1 is a selector gene that is necessary and sufficient to specify uterine muscle development in the M lineage [14] . Here we show that MLS-1 interacts with the UNC-37/Groucho co-repressor , and that MLS-1 function in C . elegans depends on this interaction . These results indicate that MLS-1 specifies uterine muscle fate at least in part by repressing target gene transcription . What types of genes does MLS-1 regulate ? We suggest that MLS-1 represses expression of regulatory genes that themselves encode inhibitors of uterine muscle development and activators of vulval muscle development . This model is consistent with previous observations that mls-1 loss-of-function results in a transformation of uterine muscle to vulval muscle , whereas ectopic mls-1 expression results in production of ectopic uterine muscles [14] . Analogous negative regulatory pathways have been suggested for specification of body wall muscle and coelomocyte cell fates elsewhere in the M lineage [27] , [28] . The MLS-1 targets must be expressed in the M lineage outside of the descendants of the SMs , because widespread expression of mls-1 in the M lineage can convert many M lineage cells to a uterine muscle fate [14] . We do not yet know of any direct targets of MLS-1 . The egl-15 promoter is active in the supernumerary vulval muscles in mls-1 mutants , and we asked if it might be directly repressed by MLS-1 . However mutation of predicted T-box binding sites in this promoter did not lead to expanded expression egl-15::gfp reporter ( R . Miller and P . Okkema , unpublished ) , suggesting MLS-1 regulation of the egl-15 promoter is indirect . MLS-1 may also function with UNC-37/Groucho in other MLS-1 expressing cells . When expressed with its own promoter , MLS-1 interacts with UNC-37 in BiFC assays in the vm2 vulval muscles and one to two intestinal/enteric muscles . The function of mls-1 in these cell types is unknown [14] , so we cannot determine if interaction with UNC-37 is necessary for MLS-1 , but we suggest that MLS-1 may similarly function as a Groucho dependent repressor in these cells . In addition to MLS-1 , UNC-37/Groucho likely interacts with other factors that are involved in vulval development or that are expressed in the M-lineage . unc-37 ( RNAi ) and some mutant alleles produce a protruding vulva ( Pvl ) phenotype that is not observed in mls-1 mutants [29] . Likewise , we also found egl-15::gfp expressing cells that looked like body wall muscles in unc-37 ( e262 ) and more frequently in unc-37 ( wd17wd21 ) mutants , while we never observed this phenotype in mls-1 mutants . At least two other transcription factors expressed in the M lineage contain high scoring eh1 motifs ( CEH-24 and MLS-2 see [12] ) , and we suggest that these and other factors expressed in the M lineage function as Groucho-dependent repressors . Our results suggest that mutation of the MLS-1 eh1 motif creates a gain-of-function protein that is deleterious . It was much more difficult to generate transgenic lines expressing mls-1FSI→ASA than lines expressing wild-type mls-1 using either the mls-1 promoter or a heat-inducible promoter , and we were only able to generate transgenic lines expressing mls-1FSI→ASA with arrays containing a low concentration of the expression vector . MLS-1FSI→ASA contains an intact T-box , and , because many T-box factors bind similar sequences , MLS-1FSI→ASA could recognize binding sites for wild-type MLS-1 or for other T-box factors . It is unlikely that mls-1FSI→ASA is simply a dominant negative mutation that interferes with wild-type mls-1 function , because mls-1 null mutants are viable and healthy [14] . Instead , we suggest that MLS-1FSI→ASA interferes with function of other T-box factors that are required for viability . Alternatively , mutation of the eh1 motif may allow MLS-1 to function as a transcriptional activator and inappropriately activate T-box target genes . Groucho has been shown to convert a variety of transcriptional activators to repressors , including several T-box factors [7]–[10] . MLS-1 is a member of the Tbx1 sub-family , which includes the mammalian proteins Tbx1 , Tbx15 , Tbx18 , Tbx20 , and Tbx22 . Like MLS-1 , each of these proteins has been shown to repress transcription , and there is evidence that these proteins interact either directly or indirectly with Gro/TLE proteins [8]–[12] . Thus this T-box sub-family shares a common mechanism for transcriptional repression in different animal phyla . Among the mammalian T-box genes , MLS-1 is most closely related to Tbx15 , Tbx18 and Tbx22 . These genes have diverse functions in mesoderm development , but recently described functions for Tbx18 in smooth muscle development may be most closely related to the function of MLS-1 . Tbx18 is expressed in the developing urogenital ridge where it is necessary for development of the ureter smooth muscle [30] , [31] . Likewise Tbx18 is also expressed in endocardial cells that contribute to the coronary smooth muscles , although its function in this tissue has not yet been characterized [32] . The C . elegans muscle types expressing MLS-1 share both structural and functional similarities with mammalian smooth muscles . C . elegans uterine muscles are non-striated with loosely organized myofilaments arranged circumferentially around the uterus , and contractions of these muscles help move embryos through the uterus toward the vulva [14] , [15] . Ultrastructurally , the uterine muscles contain thin filaments attached to the uterine basal lamina at randomly arranged points , which is similar to the organization found in smooth muscles [33] . Together , these observations suggest the interesting possibility that Tbx18 and MLS-1 share conserved function in smooth muscle development .
C . elegans were grown under standard conditions and were raised at 20°C unless otherwise noted [34] . The following strains were used: NH2447 ayIs2 [egl-15::gfp] IV [35]; PD4285 mls-1 ( cc569 ) I; ayIs2 [egl-15::gfp] IV [14]; LX354 lin-15 ( n765ts ) ; vsIs5 [rgs-2::gfp; lin-15 ( + ) ] [25]; CB262 unc-37 ( e262 ) I [36] , NC93 unc-37 ( wd17wd21 ) /dpy-14 ( e188 ) I [17] , OK0675 unc-37 ( e262 ) I; ayIs2 [egl-15::gfp] IV , and OK0787 unc-37 ( wd17wd21 ) /dpy-14 ( e188 ) I; ayIs2 [egl-15::gfp] IV . The following transgenic strains were constructed for this work . mls-1 ( Open Biosystems ) and unc-37 ( yk727f10 provided by Y . Kohara , National Institute of Genetics , Japan ) cDNA were cloned into pCE-BiFC-VN173 and pCE-BiFC-VC155 plasmids ( provided by Chang-Deng Hu , Purdue ) to generate pOK257 . 05 ( mls-1::vn173 ) and pOK257 . 06 ( unc-37::vc155 ) . mls-1FSI→ASA cDNA was generated by site directed mutagenesis of mls-1 cDNA ( Quikchange II XL , Stratagene ) , and cloned into pCE-BiFC-VN173 producing pOK258 . 06 ( mls-1FSI→ASA::vn173 ) . Transgenic adults were picked to OP50 seeded plates , heat shocked for one hour at 33°C as described in Results , allowed to recover for one hour at 20°C , and examined for BiFC signal at 63× magnification . Genomic mls-1::vn173 and unc-37::vc155 constructs , with gene expression under the control of their respective endogenous promoters , were produced by PCR of promoter+gene fragments using pSAK244 . 13 and N2 genomic DNA templates , respectively . hsp-16 . 41 promoter fragments were removed from pCE-BiFC-VN173 and pCE-BiFC-VC155 and replaced with these mls-1 and unc-37 promoter+gene cassettes generating pOK263 . 03 and pOK266 . 02 . Transgenic animals from various stages ( L1 to Adult ) were examined at 63× for BiFC signal . Yeast two-hybrid assays were performed as previously described [38] . The mls-1 bait plasmid ( pOK248 . 03 ) was constructed by inserting the full-length mls-1 cDNA in to pLexA-NLS , and the unc-37 prey plasmid was previously isolated from the pACT-RB1 cDNA library ( provided by R . Barstead ) . mls-1 ( cc569 ) ; ayIs2 strains with mls-1 ( + ) or mls-1FSI→ASA extrachromosomal arrays were generated as above . Adult transgenic animals from these lines and animals from NH2447 and PD4285 were examined and scored at 40× and 63× magnification for the number of cells expressing egl-15::gfp . unc-37 cDNA was cloned into the L4440 ( double T7 promoter ) vector and the resulting plasmid ( pOK247 . 03 ) was used to transform HT115 ( DE3 ) E . coli cells . unc-37 “feeding” RNAi was performed as before with some modifications [39] , [40] . Plates with large numbers of ayIs2 [egl-15::gfp] IV ( NH2447 ) or lin-15 ( n765ts ) X; vsIs5 [rgs-2::gfp] ( LX354 ) ( provided by M . Koelle , Yale ) adults and embryos were bleached and embryos were transferred to unseeded NGM plates and placed at 25°C overnight to allow embryos to hatch and synchronize as L1s . The next day synchronized L1s were transferred to unc-37 ( RNAi ) seeded plates and placed at 20°C for 48 hours to 72 hours . Adult animals were examined and scored at 40× and 63× magnification for egl-15::gfp or rgs-2::gfp ( mis ) expression . Note that it is only possible to qualitatively evaluate rgs-2::gfp expression due to the unusual morphology of uterine muscles . | T-box proteins are a conserved family of transcription factors that play important roles in development of all animals . A variety of human congenital diseases and cancers have been associated with mutations that either decrease or increase T-box factor activity . By learning more about how T-box transcription factors function , we can better understand and treat these disorders . Here we use genetic and molecular techniques in the roundworm C . elegans to explore the mechanism ( s ) of the muscle-specifying T-box factor MLS-1 . We show that MLS-1 physically interacts with another conserved protein called Groucho to repress gene expression and that this interaction is necessary for normal MLS-1 function . C . elegans MLS-1 is highly related to T-box factors in humans and other organisms , and we suggest Groucho interaction may be a common mechanism for T-box factor function . These insights may lead to therapies that modulate the activity of T-box factors in disease . | [
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"transcr... | 2011 | The Caenorhabditis elegans T-Box Factor MLS-1 Requires Groucho Co-Repressor Interaction for Uterine Muscle Specification |
Individuals in the indeterminate phase of Chagas disease are considered to have mortality rates similar to those of the overall population . This study compares mortality rates among blood donors seropositive for Chagas disease and negative controls in the city of São Paulo , Brazil . This is a retrospective cohort study of blood donors from 1996 to 2000: 2842 seropositive and 5684 seronegative for Chagas disease . Death status was ascertained by performing probabilistic record linkage ( RL ) with the Brazil national mortality information system ( SIM ) . RL was assessed in a previous validation study . Cox Regression was used to derive hazard ratios ( HR ) , adjusting for confounders . RL identified 159 deaths among the 2842 seropositive blood donors ( 5 . 6% ) and 103 deaths among the 5684 seronegative ( 1 . 8% ) . Out of the 159 deaths among seropositive donors , 26 had the 10th International Statistical Classification of Diseases and Related Health Problems ( ICD-10 ) indicating Chagas disease as the underlying cause of death ( B57 . 0/B57 . 5 ) , 23 had ICD-10 codes ( I42 . 0/I42 . 2/I47 . 0/I47 . 2/I49 . 0/I50 . 0/I50 . 1/ I50 . 9/I51 . 7 ) indicating cardiac abnormalities possibly related to Chagas disease listed as an underlying or associated cause of death , with the others having no mention of Chagas disease in part I of the death certificate . Donors seropositive for Chagas disease had a 2 . 3 times higher risk of death due to all causes ( 95% Confidence Interval ( 95% CI ) , 1 . 8–3 . 0 ) than seronegative donors . When considering deaths due to Chagas disease or those that had underlying causes of cardiac abnormalities related to Chagas disease , seropositive donors had a risk of death 17 . 9 ( 95% CI , 6 . 3–50 . 8 ) times greater than seronegative donors . There is an excess risk of death in donors seropositive blood for Chagas disease compared to seronegative donors . Chagas disease is an under-reported cause of death in the Brazilian mortality database .
Chagas disease was first described in 1909 by the Brazilian scientist Carlos Ribeiro Justiniano Chagas ( 1879–1934 ) . The etiologic agent that causes Chagas disease is the protozoan parasite Trypanosoma cruzi ( T . cruzi ) . The parasite is transmitted by the reduviid bug , a member of the Triatomine subfamily , popularly known as the “kissing bug” . Transmission may also occur by blood or organ transfusion , contaminated food , from mother to child and due to laboratory accidents [1] . Classified as one of the 17 most important neglected diseases by the World Health Organization ( WHO ) , estimates of the number of infected individuals in the world have decreased from approximately 20 million in 1981 [1] , to 7–8 million in 2014 [2] . In Brazil , from 1980 to 2000 there was a great effort by health authorities to eradicate the insect and to stop transfusion transmission . The reduction in disease burden is predominantly a result of improved vector control . The main measures of disease control implemented were the elimination of Triatoma infestans peri and in-house habitat by the use of insecticides , housing stock improvement , sanitary education , and the adoption of screening tests among blood donors to detect the parasite and thus avoid transmission [3] . Donor screening for T cruzi is mandatory in all Latin American countries with more than 92% of screening coverage of the blood supply . [4] The prevalence of T cruzi has declined among first time blood donors in Brazil , from 0 . 52% in 1996 [5] to 0 . 14% in 2008 [6] . In humans , Chagas disease manifests in two phases , acute and chronic . The acute phase has mild symptoms that may last for approximately two months . In the chronic phase , the majority of cases are of the asymptomatic indeterminate form , which may last a lifetime . Individuals in the chronic phase of the disease with normal electrocardiogram ( EKG ) and normal chest and esophagus X-rays are considered individuals in the indeterminate form of the disease [7] . Whereas , clinically apparent chronic disease form is divided into a cardiac , gastrointestinal , or combined form [8] . The most important health consequence of Chagas disease is cardiomyopathy , which over a lifetime occurs in 20–40% of infected persons with an incidence rate of 1 . 85% per person-year [9] . In Brazil , it is estimated that in 2013 there were 2 to 3 million infected individuals [10] , of which 600 , 000 had cardiac and/or gastrointestinal complications . The disease is also responsible for approximately 6 , 000 deaths per year in Brazil , i . e . 0 . 6% of all deaths that occur in the country [11] . This estimate is based on analyses of data from the National Mortality Information System ( SIM ) from 1999 to 2007 , in which records with 10th International Statistical Classification of Diseases ( ICD-10 ) codes B57 and all subcategories ( B57 . 0 to B57 . 5 ) marked on the underlying and/or associated causes of death field were selected [11 , 12] . One of the critical challenges for Chagas disease control is that many individuals with the indeterminate form of Chagas disease are not aware that they are infected , so that these individuals are potential onward T . cruzi transmitters and may even die without being diagnosed with the disease . Mortality-associated with Chagas disease is still inadequately estimated , particularly for those with the indeterminate form of the disease . Important unanswered questions include: do persons with indeterminate disease have an increased risk of dying ? Studies that compare mortality among seropositive and seronegative individuals can help reduce this knowledge gap as described in a systematic review and meta-analysis published in 2016 [13] . The objective of the study was to compare death rates during an up to 14 year follow-up period among Chagas disease seropositive blood donors and seronegative blood donors identified in the period 1996–2000 in the city of São Paulo , Brazil .
FPS is a reference center in Latin America , and the proportion of the supply collected is approximately 33% of all donated blood used in the metropolitan region of São Paulo city . FPS is a reference center for the Pan-American Health Organization ( PAHO ) , World Health Organization ( WHO ) and the Brazilian Ministry of Health , it collects and processes 90 , 000 blood units annually that are used by more than 100 health institutions [14] . Blood donors constitute a particular population in which , if infected , seropositive individuals are mostly asymptomatic and therefore are more likely to be in the indeterminate form Chagas disease . Although donation eligibility assessment does not include EKG or X rays , blood donors go through an interview before donating and requirements to donate include being in good health with no evidence of or reported history of Chagas disease or cardiomyopathies . During the study period ( 1996–2000 ) FPS screened all donors with 3 serological assays ( indirect immunofluorescence ( IIF ) , indirect hemaglutination ( IHA ) , and enzyme-linked immunosorbent assay ( ELISA ) ) [15] . Donors reactive on all 3 assays were considered confirmed positive . Using blood bank computerized records , all confirmed seropositive donors for Chagas disease were included ( n = 2 , 842 ) and a random sample of blood donors seronegative for all screening tests performed at FPS during the same time period was selected , that is , 2 seronegative donors to each seropositive matched by year of donation ( n = 5 , 684 ) . During the study period , FPS routinely performed the following serology screening tests on all donations before releasing units for transfusion: syphilis , Human immunodeficiency virus ( HIV ) , Human T-cell lymphotropic viruses I and II ( HTLV ) , Hepatitis C Virus ( HCV ) , Hepatitis B Virus ( HBV ) , and Chagas disease . In order to ascertain the death status of the study population , a record linkage ( RL ) was performed to link records of the FPS database with the Brazil national mortality information system ( Sistema de Informações sobre Mortalidade—SIM ) records . As Brazil does not have a unique national identity number , a probabilistic RL was performed using open source software . Patients’ name , mother´s name and date of birth were the chosen linking variables . One of the advantages of the use of this software in our context is that it links names and other variables through the use of a Soundex system which is specific for Portuguese phonetics ( Reclink III , version 3 . 0 . 4 4005 , Rio de Janeiro , Brazil ) [16] . The RL procedure used here has been described in depth in a previous publication [17] . In this previous study , we assessed the RL methodology validity by assembling a list of blood donors with known vital status . Sensitivity and specificity of the RL method to detect mortality were 94% ( 95%CI , 90–97% ) and 100% ( 95%CI , 98%-100% ) , respectively . In the SIM database , records containing the variables patients’ name , mother´s name , date of birth and address of residence were only available for the years 2001 to 2009 ( SIM version extracted on 11/01/2010 , Brasilia , Brazil ) . FPS donors included in the study donated blood during the period of 1996 to 2000 , this means that even though we were able to retrospectively follow all donors for at least nine years , up to first five years of follow-up were not available in SIM . For the cohort that donated blood in 1996 , we missed the first five years , for the 1997 cohort , we missed the first four years and so forth . In other words , irrespective of their Chagas serology status , we do not know whether those who donated blood in 1996 , for example , died in the period of 1996 to 2000 . However , based on findings of seropositive and seronegative individuals who donated blood in 2000 and for whom we only missed deaths that might have happened during the year of their blood donation , we found that deaths were extremely rare in the first year after donation . Because of this finding and with a view of circumscribing this data limitation , we made the analytical assumption that nobody in our cohort died in the years 1996 to 2000 . For the descriptive analysis , the underlying causes of death of seropositive and seronegative donors were grouped by chapters in the ICD-10 . For the multivariate analysis , causes of death were analyzed according to three groups: A ) Confirmed deaths due to Chagas disease , B ) Confirmed and possible deaths due to Chagas disease , and C ) Death due to all causes . Group A is hierarchically a subgroup of group B , which is a subgroup of group C . “Confirmed deaths due to Chagas disease” refers to deaths recorded with ICD-10 codes B57 . 0—B57 . 5 listed on the underlying or the associated causes of death fields . “Confirmed and possible deaths due to Chagas disease” refer to deaths recorded with ICD-10 codes B57 . 0—B57 . 5 plus other codes indicating cardiac abnormalities that are possibly due to Chagas disease listed on the underlying or the associated causes of death fields: I42 . 0—Dilated cardiomyopathy , I42 . 2—Other hypertrophic cardiomyopathy , I47 . 0—Re-entry ventricular arrhythmia , I47 . 2—Ventricular tachycardia , I49 . 0—Ventricular fibrillation and flutter , I50 . 0—Congestive heart failure , I50 . 1—Left ventricular failure , I50 . 9—Heart failure , unspecified and I51 . 7—Cardiomegaly . Comparisons of characteristics of donors that were seropositive and seronegative or that were alive or dead at the end of the follow-up period were performed using two-tailed Fisher Exact tests or Chi-squared tests , as appropriate . Mortality rates were calculated by dividing the number of donors that died ( numerator ) by the total person-years at risk of dying during the follow-up period , i . e . from donation to death or to the end of the study period ( denominator ) . Survival of seropositive and seronegative donors over time was graphically assessed by Kaplan-Meier ( KM ) curves and statistical differences tested with the Logrank test . Age and sex were the only two demographic variables available for analysis in the FPS database that represented potential confounders of the association between the outcomes variables and the main explanatory variable under investigation , i . e . serological status . Age was analyzed a categorical variable ( 18–29 years , 30–39 years , 40–49 years and >50 years ) . Multiple Cox Regression models were used to derive hazard ratios ( HR ) for the outcomes studied , adjusting for confounders and testing for interactions , where appropriate . Two sets of analyses were performed one for the whole cohort and another excluding Chagas disease positive donors co-infected with one or more of the following infections: syphilis , HIV , HTLV , HCV and HBV . Regression models containing all potential predictors were estimated . The predictor variables available for the regression models were age group and sex . Stepwise backward selection procedures were used to remove variables . Variables were removed with Wald test p-values ≥0 . 2 . The remaining variables were then successively removed based on lack of evidence of confounding and their contribution to the models . Variables whose removal from the models caused substantial changes ( defined as >10% ) in the HRs were retained , as were variables whose removal incurred significantly reduced model fit based on likelihood ratio tests ( p-values <0 . 05 ) . For each outcome and potential risk factor , the proportional hazards assumption was tested statistically using Schoenfeld residuals and by examining for parallel curves in log-log plots . Schoenfeld residuals tests were not statistically significant and the curves were mostly parallel , indicating no evidence of violation of the proportional hazards assumption . In addition , the proportional hazard was assessed by including a time-dependent interaction term in the multivariable Cox models and by testing the statistical significance of this interaction using the likelihood ratio test . Consistently , results indicated no evidence of violation of the assumption . Statistical analyses were conducted using Stata version 13 . 1 ( StataCorp LP , College Station , TX , USA ) software [18] . Ethical approval for this study was obtained from the Ethics Research Committee of Clinical Hospital of the School of Medicine—University of Sao Paulo , HC-FMUSP ( CAAE:03882512 . 4 . 0000 . 0065 ) . HC-FMUSP Ethics Research Committee determined it unnecessary to obtain informed consent from each individual for reviewing medical records , given the difficulty of obtaining such consent and because the study was conducted using 2 existing databases . The identifiers were removed from database after the probabilistic RL was performed .
Out of 1 , 354 , 569 blood donors to FPS during the study time period , 2 , 842 confirmed positive cases and 5 , 684 controls ( matched by year of donation ) were assessed for death status and are described on Table 1 . The RL method identified a total of 262 deaths , 159 ( 5 . 6% ) among the seropositive blood donors and 103 ( 1 . 8% ) among the seronegative . Mortality was significantly higher in seropositive donors . Out of the 2842 Chagas disease seropositive donors , 128 ( 4 . 5% ) were also seropositive for at least one of the other tested infections . Syphilis ( 51 donors ) was the most common co-infection , followed by HBV ( 35 donors ) and HCV ( 26 donors ) , HTLV ( 21 donors ) and HIV ( 2 donors ) . A total of 12 donors were co-infected with Chagas disease plus two of the other infections . The distribution of the underlying causes of death of seropositive and seronegative donors , as classified by ICD-10 chapters , is shown in Table 2 . Distribution by subcategories of ICD-10 is available in a supplemental table ( S1 Table ) . Seropositive donors died more frequently of diseases classified with codes of Chapter IX—Diseases of circulatory system and Chapter I—Certain infectious and parasitic diseases as compared to seronegative donors , On the other hand , seronegative donors died more frequently of external causes ( Chapter XX ) . Out of the 262 donors that died , 26 had codes B57 listed as the underlying cause of death and were therefore classified as "Confirmed deaths due to Chagas disease" . All of them were seropositive for Chagas disease . There was no record with B57 listed as an associated cause of death , but there was one seropositive donor with code B57 listed on part II ( co-morbidities ) on the death certificate . A total of 49 donors were classified as "Confirmed and possible deaths due to Chagas disease" . This number includes all 26 donors classified as "Confirmed deaths due to Chagas disease" plus 23 donors that had codes indicating cardiac abnormalities possibly related to Chagas disease , listed as an underlying or associated cause of death . Codes indicating cardiac abnormalities possibly related to Chagas disease were listed as associated causes of death in 16 ( 61 . 5% ) of the 26 donors classified as "Confirmed deaths due to Chagas disease" . Out of the other 236 donors that were not classified as "Confirmed deaths due to Chagas disease" , only 23 ( 9 . 8% ) had any of these codes listed as an underlying or associated cause of death ( p<0 . 001 ) . Moreover , these codes were listed in 35 ( 22% ) of the 159 deaths of seropositive donors and in only 4 ( 3 . 9% ) of the 103 deaths of seronegative donors ( p<0 . 001 ) . The underlying causes of death of the 49 donors classified as "Confirmed and possible deaths due to Chagas disease" is shown in Table 3 . It was unexpected to find code B56 . 9 ( African trypanosomiasis ) in a donor seropositive for T . cruzi . While possible , it may also have been a coding mistake . Donors’ cumulative survival according to serology , without excluding co-infected individuals , is shown in Fig 1 and according to serology and age group in Fig 2 . Survival curves of seronegative donors were better than those of seropositive ones ( p<0 . 001 ) . The same pattern was observed for the KM curves by age group . For the age group 40–49 years the difference did not reach statistical significance ( p = 0 . 097 ) . In the multivariate analysis , estimated crude and adjusted hazard ratios were calculated for each of the three different case definitions: A ) Confirmed deaths due to Chagas disease B ) Confirmed and possible deaths due to Chagas disease and C ) Death due to all causes ( Table 4 ) . As there were no confirmed deaths due to Chagas disease in the seronegative group , HRs could not be estimated for this case definition . The age adjusted HR for mortality in seropositive donors was 2 . 3 ( 95%CI 1 . 8–3 . 0 ) times higher than seronegative ones when deaths due to all causes were considered and the age and sex adjusted HR for mortality was 17 . 9 ( 95%CI 6 . 3–50 . 8 ) times higher for confirmed and possible deaths due to Chagas disease . Excluding co-infected donors did not appreciably change the results . When multivariate analyses were performed excluding the 128 co-infected donors , seropositive donors had a HR 2 . 2 ( 95%CI = 1 . 7–2 . 9 , p-value<0 . 001 ) times higher than seronegative ones for all cause deaths and a HR 16 . 1 ( 95%CI = 5 . 6–45 . 9 , p-value<0 . 001 ) times higher when considering confirmed and possible deaths due to Chagas disease .
In this study , we compared the mortality rates of blood donors identified as seropositive for Chagas disease and seronegative for all screening tests based on donation testing in the 5-year time period , 1996–2000 . Our results show that seropositive blood donors had a risk of dying over the study time period 2 . 3 times greater than seronegative individuals . When considering only deaths due to Chagas disease or those that had underlying causes of cardiac abnormalities that could indicate Chagas disease , seropositive blood donors had a much higher risk of death than seronegative donors . In a recent systematic review , Cucunuba et al . analyzed 25 papers published on mortality of Chagas disease from 10 , 638 individuals and 53 , 346 persons-year of follow-up . Only six of those studies focused on asymptomatic patients [19–24] , following a total of 4 , 393 individuals for 34 , 217 person-years of observation . Crude relative risk of mortality from these studies varied from 0 . 81 to 3 . 59 . In this study , we have been able to follow 8 , 526 individuals for 100 , 467 person-years of observation , doubling the data available and contributing to a more precise evaluation of the impact of Chagas disease-associated mortality among asymptomatic seropositive individuals . Classifying mortality for persons who are initially identified as being in the indeterminate form is difficult because disease progression is slow , studies need large sample sizes and very long follow-up periods . Population studies are expensive , and in areas with low prevalence such as the city of São Paulo , with prevalence rates of less than 1% [5 , 6] , screening of a large number of individuals would be necessary to achieve a sufficient number of seropositive cases . We overcame this problem by focusing on blood donors that are routinely screened for Chagas disease and that data are available through computerized systems . In Brazil , the national mortality information system has a national coverage and the quality of its cause of death data has improved substantially over the last two decades [25–28] . A provocative finding is that cardiac causes of death with no mention of Chagas disease was significantly more common among seropositive donors . Indeed , of the 45 deaths due to cardiac disease among seropositive donors only 26 ( 58% ) had Chagas disease listed on their death certificate as an underlying cause of death . This indicates that the impact of Chagas disease mortality in Brazil is underestimated when analyses of only death records that specifically mention Chagas disease as the underlying cause of death are conducted [11] . The fact that Chagas disease was not reported as an underlying or associated cause of death on the death certificate of 42% of seropositive donors that died due to cardiac causes demonstrates under ascertainment of Chagas disease pathogenesis , highlighting its status as a neglected disease . Given the context for donation test result notification in Brazil and because the seropositive donors were asymptomatic at time of donation , only those that returned to the blood bank to get their screening tests results would have been notified of being seropositive for T . cruzi infection . The use of the probabilistic RL method implies we might have missed some deaths [17] . However , as reported previously by our group , the methodology used had a sensitivity of 94% and a specificity of 100% , making us confident that the deaths found do represent true matches . But the main limitation of our study was that death information recorded by name was not available in the mortality database for the years 1996 to 2000 , so that we were unable to ascertain whether or not donors in our cohort died in the early years of follow-up . In the analysis , we could have removed donors from this period and analyzed only those donating from 2001 onwards , but we faced the problem that during the study years , donors seropositive for Chagas disease were progressively less frequent in our database [5] . This decrease is probably due to two reasons: 1 ) vector transmission control activities in the country causing a decreased overall number of seropositive individuals in the population [5] , and 2 ) in order to increase blood donations , from 1995–2001 FPS conducted a campaign to recruit previous donors seronegative for all screening tests to donate again , so that the proportion of donors who had not donated before became increasingly smaller compared to repeated donors [29] . If we had used in our cohort blood donors only from 2001 onward , the number of seropositive cases would be substantially lower and the follow-up period would be shorter , with the potential to attenuate the results of the analyses of this slow progressing disease . However , early deaths after donation were not frequent , as observed for those who donated in 1999 and 2000 for whom we were able to ascertain whether or not they had died after their first or second year of follow-up , respectively ( see S2 Table ) . If we had had mortality data for the whole cohort period , we have no reason to suppose that there would be an excess of early deaths in seronegative donors that would counterbalance the excess of late deaths that we found in the seropositive ones . Moreover , because the proportion of ICD-10 chapter XX , external causes of death , were equivalent in the seropositive and seronegative groups , an additional level of assurance is provided that there was no clear differential bias in mortality ascertainment between these two groups . Even if the number of deaths has been underestimated by the RL method and available data in SIM , the impact would be expected to be non-differential indicating the findings of our study are robust . In summary , we do not believe that this limitation greatly affected our results . Another limitation with greater potential impact on our results is the fact that data regarding socioeconomic status was not available in the blood bank database , so we could not control for this effect in our multivariate models . In general , persons with T . cruzi infection are mostly poor , less educated , and from rural areas . This combination of factors could lead to increase mortality for reasons such as less healthy lifestyles or access to care . In turn , this could lead to increased mortality . Yet , these individuals presented to blood donate and at the time were eligible and donated following the extensive health history assessment covering a range of topics , including current physical health , substance abuse history , and sexual history . Our analysis might have generated increased accuracy in the HR results if we had been able to adjust for socioecomic status . Although our study is focused in the city of Sao Paulo , we believe it well represent Brazil . Internal migration was an important factor for population increase in the city of Sao Paulo [30] . Data regarding State of Birth is not available at FPS data system , nevertheless , in a previous study done by our group in the same center ( Retrovirus Epidemiological Donor Study ( REDS–II ) ) , seropositive and seronegative donors were interviewed and individuals born in the State of Sao Paulo represented 13% of the seropositive and 55% of the seronegative blood donors [9] . We believe that most Chagas disease seropositive donors were in the indeterminate form of the disease . Individuals who present to donate blood have to report if they do not have any current serious conditions in a comprehensive questionnaire about their health status . Questions such as “Do you have a severe heart problem ? ” and “Do you have arrhythmia and use medication for this problem ? ” are part of the screening questionnaire . However , we do not know if any of the donors had abnormal EKGs or abnormalities identifiable by X-ray , in which case they would be classified in the clinical form of the chronic phase of the disease . The increased mortality findings have high relevance for common assumptions about Chagas disease morbidity . It is not uncommon to find articles in the literature that consider the indeterminate form of the disease as a benign disease not requiring close medical attention [20 , 31 , 32] . However , such studies are generally based on following a small number of people and often without appropriate comparison groups [13] . When clinicians read these articles they may feel justified in not treating their patients with the limited number of therapeutic options that are available . Because available pharmacotherapy is not very effective and all of them have potential important side effects [32 , 33] , clinicians may not want to treat indeterminate Chagas . Our findings suggest that this belief may be erroneous and that the indeterminate form , even in a relatively young population , is associated with higher mortality rates . Earlier treatment to reduce morbidity and mortality may be important . Chagas is a neglected disease and research is urgently needed in order to test new therapeutic options with fewer side effects and to find better correlates of disease progression . In conclusion , this analysis shows that there is an excess risk of death in donors who test seropositive for Chagas disease compared to seronegative donors . Chagas disease is an under-estimated cause of death in the Brazilian National Mortality Information System . | Chagas disease is classified as one of the 17 most important neglected diseases by the World Health Organization ( WHO ) . The majority of infected individuals live in 21 countries of Central and South America . Estimates of mortality attributable to Chagas disease vary considerably ( between 0 . 2% and 19 . 2% annually ) . To improve the measurement of disease burden and policy decision-making in regard to treatment availability , more accurate estimates of mortality , especially among otherwise asymptomatic seropositive individuals are needed , . This study compares mortality rates and causes of death of asymptomatic blood donors who tested seropositive for Chagas disease and seronegative for all screening tests conducted in Brazil . Mortality status was ascertained by linking donor names with the Brazilian national mortality information system ( SIM ) . The study found that donors who tested Chagas disease seropositive had risk of death from all causes 2 . 3 ( 95% CI , 1 . 8–3 . 0 ) times greater than seronegative ones . The data also suggest that the SIM may underestimate the total number of deaths attributable to Chagas disease in Brazil . | [
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"protozoan... | 2017 | Mortality among blood donors seropositive and seronegative for Chagas disease (1996–2000) in São Paulo, Brazil: A death certificate linkage study |
Bromodomain and Extra-terminal motif ( BET ) proteins play a central role in transcription regulation and chromatin signalling pathways . They are present in unicellular eukaryotes and in this study , the role of the BET protein Bdf1 has been explored in Saccharomyces cerevisiae . Mutation of Bdf1 bromodomains revealed defects on both the formation of spores and the meiotic progression , blocking cells at the exit from prophase , before the first meiotic division . This phenotype is associated with a massive deregulation of the transcription of meiotic genes and Bdf1 bromodomains are required for appropriate expression of the key meiotic transcription factor NDT80 and almost all the Ndt80-inducible genes , including APC complex components . Bdf1 notably accumulates on the promoter of Ndt80 and its recruitment is dependent on Bdf1 bromodomains . In addition , the ectopic expression of NDT80 during meiosis partially bypasses this dependency . Finally , purification of Bdf1 partners identified two independent complexes with Bdf2 or the SWR complex , neither of which was required to complete sporulation . Taken together , our results unveil a new role for Bdf1 –working independently from its predominant protein partners Bdf2 and the SWR1 complex–as a regulator of meiosis-specific genes .
Protein members of the BET family share a conserved modular architecture , with two bromodomains in their N-terminal part , an extra-terminal recruitment ( ET ) domain and other conserved motifs . Bromodomain modules bind acetylated lysines in histones and other proteins , and BET bromodomains specifically recognise the acetylated lysines of core histones , in particular H3 and H4 [1] . BET proteins are present in unicellular eukaryotes such as the model organism Saccharomyces cerevisiae , where two homologous genes , Bdf1 and Bdf2 , are expressed . Bdf1 has a typical BET protein structure , with two bromodomains , one ET domain and other conserved motifs . Multiple functional roles have been proposed for this protein , which was first described as a regulator of snRNA [2] and then identified as part of the yeast’s general transcription factor , TFIID [3] . In yeast , unlike its human homologue TAF ( II ) 250 , the TAF ( II ) 145 protein lacks a module with bromodomains . Bdf1 could thus represent the missing piece of this yeast TAF complex [3 , 4] . Moreover , Bdf1 plays a role in regulating gene expression in response to various stresses [5–8] . Bdf1 has been shown to be part of the SWR complex , which is responsible for the incorporation of the histone variant H2A . Z into chromatin [9 , 10] . Bdf1 is not required for the enzymatic activity of Swr1 but facilitates H2A . Z incorporation in vivo [5 , 11 , 12] . Two studies demonstrated that Bdf1 bromodomains are functional and bind multi-acetylated histones , where they mediate an anti-silencing function and prevent the spreading of Sir proteins at heterochromatin boundaries [13 , 14] . Unexpectedly , a high throughput study also implicated Bdf1 in pre-mRNA splicing [15] . Thus , this protein could potentially link chromatin remodelling , transcription initiation and pre-mRNA splicing . Most of the data describing the functions of Bdf1 were obtained using vegetative S . cerevisiae cells . However , Bdf1 was described to play an essential role for sporulation when its gene was first identified in yeast [16] . In this process , nutrient starvation triggers a specific differentiation program in diploid S . cerevisiae , starting with meiosis and ending with the formation of four quiescent spores [17 , 18] . In their seminal work , Chua et al . [16] identified Bdf1 as a chromosomal protein with two bromodomains which was required for meiosis progression . We also found that spore chromatin is highly compacted , hyperacetylated , and enriched in Bdf1 [19] . Altogether , this information suggests that Bdf1 plays a functional role during the meiotic and post-meiotic stages of sporulation . In mammals , Brd2 , Brd3 and Brdt play specific roles in cellular differentiation in a range of tissues [20–22] . Thus , Brd2 is involved in neuronal development and differentiation [23 , 24] , while Brd3 is essential to erythropoiesis [25] . Brdt is only expressed in the testis and regulates the expression program of meiotic and post-meiotic genes , driving the transcription program throughout sperm differentiation [26] . In addition , it is a key player in the final chromatin reorganisation step found in the final spermatic structure [27] . Brdt preferentially binds to hyperacetylated histones and promotes a drastic chromatin reorganisation during sperm differentiation [28 , 29] . Brd4 is also involved in regulating transcription during post-meiotic sperm differentiation and final chromatin reorganisation [30] . Altogether , BET proteins are important for gametogenesis . The role of Bdf1 during sporulation was the focus of the study presented here . Our results indicate that , although Bdf1 bromodomains are not required for growth on non-fermentable carbon sources , they are essential for sporulation to complete . Transcriptomic analysis during sporulation revealed that Bdf1 bromodomains play an essential coordinating role in the transcriptional program of meiosis leading to the formation of spores . In particular , Bdf1 was found to accumulate on the promoter of the master regulator NDT80 before meiosis . This recruitment requires functional Bdf1 bromodomains and is essential for a normal expression of NDT80 . Thus the ectopic expression of Ndt80 can partially overcome the defects observed when Bdf1 bromodomains are mutated . Finally , Bdf1 forms two exclusive complexes , neither of which was required for meiotic gene regulation or to complete sporulation .
Deregulation of some master regulators of sporulation could be an obvious explanation for failure to induce this differentiation program . The expression of early genes IME1 and IME2 , which is required for progression through meiotic S and G2 phases , was not significantly affected by mutation of the Bdf1 bromodomains ( S2A Fig ) . Middle and late sporulation genes are essential for meiotic divisions and post-meiotic spore differentiation . The expression of a selection of these genes was tested by RT-qPCR and found to be defective when Bdf1 bromodomains are mutated ( S2B and S2C Fig ) . This result is consistent with the sporulation phenotype of the strain , which is blocked at the transition between the pachytene phase and meiotic divisions . RNA-seq experiments provided a general view of the transcriptional defects caused by mutation of both Bdf1 bromodomains . The expression of BDF1 was monitored in triplicate experiments performed on the WT and in bdf1-Y187F-Y354F strains at three time-points ( before sporulation induction ( 0 h ) , 4 and 8 hours after induction , Fig 3A ) . The bioinformatics pipeline used in the analysis of the results of these experiments is presented in S3A Fig . Increased BDF1 transcript and protein levels were detected when its bromodomains were mutated , possibly as part of a compensatory response by the cell ( S3B Fig ) . Furthermore , our results confirmed that Bdf1 regulates BDF2 mRNA levels , particularly during sporulation when the mutation of Bdf1 bromodomains may induce a compensatory overexpression of BDF2 ( S3B Fig , [34] ) . The distance between each replicate was analysed and , as expected , biological replicates appeared clustered by strain and sporulation time-point ( Fig 3B ) . Thus , before sporulation was induced , the transcription program was similar in the WT and BDF1 bromodomain mutant strains . Defects started to accumulate from 4 h and culminated at 8 h when the BDF1 bromodomain mutant strain stopped progressing through the sporulation program ( Fig 3B and 3C ) . The same observation was made based on an independent assessment using principal component analysis ( S3C Fig ) . A total of 833 genes were differentially downregulated when combined between the 4h and 8h time points . The examination of their promoters revealed that binding sequences of the transcription factors NDT80 , CUP9 , CUP2 , FKH1 and ROX1 were statistically enriched ( p-value <0 . 001 , Fig 4A ) . Of these , only Ndt80 has been described as essential for sporulation and is known to be a key meiotic regulator [38–41] . Indeed , Ndt80 is induced during meiosis where it is expressed at much higher levels than Cup9 , Cup2 , Fkh1 and Rox1 ( Fig 4A ) . Defects in Ndt80 expression also explain the non-induction of middle sporulation genes , such as SMK1 and SSP1 , as expression of these genes is controlled by Ndt80; both were downregulated in the bromodomain mutant strain ( S2B Fig , [35 , 42] ) . Ndt80 is the key transcription factor that activates the promoters of middle sporulation genes and is expressed just before the middle genes are induced [41] . The examination of the promoters of the 833 differentially downregulated genes identified 531 genes with putative NDT80 binding sites ( Fig 4B , S5 Table ) . In addition , the probability that an Ndt80 binding sequence is present within the promoter of Bdf1-regulated genes was calculated based on the ROC-AUC value produced by the MORPHEUS webtool [43] ( Fig 4C ) . The ROC-AUC value of 0 . 63 , which is above 0 . 5 , indicates an enrichment of the Ndt80 binding sequence in the genes regulated by Bdf1 bromodomains when compared to a list of unrelated genes . Finally , a list of genes potentially regulated by Ndt80 was obtained from Chu et al [35] . These genes were statistically over-represented in the list of genes downregulated in the bdf1-Y187F-Y354F mutant ( Fig 4D , p-value = 10−48 with a hypergeometric test ) . In conclusion , a significant proportion of the genes which fail to induce when Bdf1 bromodomains are mutated are regulated by Ndt80 . An oestradiol-inducible form of NDT80 was used to test whether a reduction in Ndt80 levels could explain the phenotype observed when Bdf1 bromodomains are mutated [44 , 45] . This system was introduced into the bdf1-Y187F-Y354F mutant and oestradiol was added 6 hours after sporulation induction . Induction of NDT80 expression at this stage partially alleviated the defects observed when Bdf1 bromodomains are mutated , with 40% of spores formed in this mutant strain ( Fig 4E ) . This result indicates that mutation of Bdf1 bromodomains affects the expression of the master regulator NDT80 alters the expression of middle sporulation genes and that these sporulation defects can be partly rescued by ectopic overexpression of NDT80 . We next wondered whether Bdf1 interactants could contribute to the regulation of the meiotic transcription program and mediate the transcriptional activation of NDT80 . The interactome of Bdf1 during vegetative growth was determined using mass spectrometry-based proteomic analysis of affinity purified samples ( Fig 5 ) . The relative abundances of Bdf1 partners were determined using the mass spectrometry-based iBAQ metrics [49 , 50] . Interestingly , the most abundant Bdf1 partner was Bdf2 . The 12 subunits of the SWR complex , involved in the incorporation of the yeast histone variant H2A . Z , were also identified [9] . Both of these interactions with Bdf1 were confirmed by western blot ( S4B Fig ) . The relative stoichiometry of the SWR subunits found to interact with Bdf1-TAP was compatible with the recently published structure of the complex , where the subunits Rvb1/2 are 2 . 5 times more abundant than the other subunits ( Fig 5A bottom , [51] ) . Finally , the kinase CK2 was also detected ( Fig 5A and S6 Table ) . This protein had previously been associated with Bdf1 [9] . Additional purifications were then performed to refine our knowledge of how the Bdf1 interactome is organised . Bdf2-TAP purifications were performed and identified Bdf1 as the major partner ( Fig 5B ) . None of the SWR subunits was associated with Bdf2 . The Swr1 protein was then TAP-tagged and its complex purified . This analysis identified all the subunits of the SWR complex ( Fig 5C ) . Bdf1 was identified as a minor interacting partner of Swr1 compared to the well-established SWR complex subunits . In these experiments , a 9 fold difference in relative abundance was detected for Rvb1/2 and the other SWR subunits . This difference with the data obtained in the Bdf1 complex and the published structure of the SWR complex could be explained if the SWR complex is present in vivo in several forms . This hypothesis is strengthened by results from structural studies using cosslinking reagents to obtain a homogeneous population of the complex [51] . Interestingly , Bdf2 was never identified in SWR complex . This result suggests that Bdf1 could form two exclusive complexes , one with the SWR complex and another one with Bdf2 . Finally , Bdf1-TAP was purified from a strain lacking YAF9 , the subunit of the SWR complex responsible for recruiting Bdf1 [52] . In the yaf9Δ strain , Bdf1 still associated with Bdf2 , but its interaction with the SWR complex was completely abolished ( Fig 5D ) . Taken together , these results suggest that Bdf1 can be part of two exclusive complexes: one with the SWR complex or a Bdf1 / Bdf2 complex whose function remains to be characterised ( Fig 5E ) . The sporulation phenotypes induced by the mutations of Bdf1 bromodomains could be functionally explained by its interactions with the SWR complex or with Bdf2 . To investigate this hypothesis , YAF9 and SWR1 were mutated in the SK1 genetic background and compared to bdf1 mutant strains . Deletion of YAF9 or SWR1 had no effect on growth in any of these conditions ( Fig 6A ) , nor did YAF9 and SWR1 deletions affect sporulation efficiency ( Fig 6B ) . Therefore , the sporulation defects observed when Bdf1 bromodomains are mutated are not related to the function of the SWR complex . In the absence of a role for the SWR complex in sporulation , we hypothesised that the Bdf1 / Bdf2 complex could play a role during sporulation which may be related to the defects observed when Bdf1 bromodomains are mutated . As previously published [2 , 8 , 16 , 32] , strains deleted for BDF1 and BDF2 exhibit a slow growth phenotype on fermentable carbon sources , and fail to grow on non-fermentable carbon sources such as acetate or glycerol ( Fig 6A ) . Different regions of Bdf2 were deleted to map the regions required for its interaction with Bdf1 ( Fig 6C ) . This analysis indicated that Bdf2 bromodomains are essential for its interaction with Bdf1 ( Fig 6D ) . Interestingly , deletion of the two Bdf2 bromodomains had no phenotypic effects either on non-fermentable carbon sources or during sporulation ( Fig 6E , left ) . Finally , we studied the functional role of the Extra-Terminal domain ( ET ) , which is a conserved protein–protein interacting domain that regulates transcriptional activity [53 , 54] . Deleting this domain in BDF2 disrupted its interaction with Bdf1 ( Fig 6D ) but had no effect on sporulation ( Fig 6E , left ) . Deletion of the same domain in Bdf1 did not affect growth on non-fermentable carbon sources at 30°C but revealed some defects at 37°C ( S5B Fig ) . In contrast to Bdf2 , the ET deletion in Bdf1 affects sporulation to a similar extent to the mutation of its bromodomains ( Fig 6E , right ) . These last results underline the fact that even though Bdf1 and Bdf2 are highly similar in their modular organisation , they play different roles during sporulation . Thus , only Bdf1 bromodomains , and not Bdf2 bromodomains , are essential for the formation of spores . Moreover , the sporulation defects observed in strains with mutated Bdf1 bromodomains are not mediated by any of its partners . Indeed , the disruption of the interaction of Bdf1 with Bdf2 or with the SWR complex generated no sporulation defects . Finally , we examined whether deletion of BDF2 impacted Bdf1 recruitment to chromatin . ChIP experiments suggest that Bdf1 does not require Bdf2 to bind to its chromatin targets ( Fig 6F ) . In addition , we were unable to detect any recruitment of Bdf2 to these genes , which raises the issue of the recruitment of Bdf2 to chromatin [55] . The fact that Bdf1 loading onto chromatin does not depend on Bdf2 reinforces the idea that Bdf2 does not contribute to the transcriptional regulation of middle sporulation genes .
Here , and in previous studies , deletion of BDF1 or BDF2 was found to lead to severe growth defects in the absence of a fermentable carbon source [2 , 16 , 32] . Indeed , Bdf1 was shown to preserve mitochondrial activity in various metabolic conditions [6 , 56] . However , a putative role for Bdf2 has yet to be investigated . Our results indicated that this phenotype is not controlled by Bdf1 or Bdf2 bromodomains , thus possibly revealing a new function of BET proteins in cell metabolism , which is independent of their bromodomains and remains to be characterised . Bdf1 was first linked to sporulation when its gene was cloned , its bromodomains were identified and the growth and sporulation defects induced by its deletion were analysed [16] . Furthermore , a transposon-based screen indicated that the second Bdf1 bromodomain , but not the first one , was essential for the completion of meiosis and spore formation [16] . Here , we found that both bromodomains of Bdf1 have to be mutated to impair the formation of spores and cause an arrest during meiosis . These apparent discrepancies compared to the results presented in Chua et al . [16] could be explained by the facts that ( i ) they used a transposon mutational screen which did not precisely disrupt Bdf1 bromodomains and ( ii ) a transposon insertion in the second bromodomain may have also disrupted the subsequent ET domain , which we also found to be essential for sporulation ( Fig 6E , right ) . When Bdf1 bromodomains are mutated , early inducers of the transcription program for sporulation are correctly expressed but transcriptional defects accumulate as sporulation progresses ( Fig 3D , S2A Fig ) . In particular , expression of the middle sporulation genes and of the master regulator NDT80 failed when Bdf1 bromodomains were not functional ( Figs 4B and S2B ) . Interestingly , 65% of the genes downregulated in the bdf1 bromodomain mutant were also predicted targets of Ndt80 ( Fig 4B ) . Several studies identified Bdf1 as important for gene splicing [15 , 34] . This functional role appears to be independent of its bromodomains as no splicing defects were observed in the bdf1-Y187F-Y354F mutant ( S3D and S3E Fig ) . Quantitative proteomics analysis of the Bdf1 interactome revealed that Bdf1’s main partners are Bdf2 and the SWR complex . These interactions occur as part of two different , mutually exclusive , complexes ( Fig 5E ) . It is interesting to note that Bdf1 , Bdf2 and Yaf9 , the subunit of Swr1 which interacts with Bdf1 , share similarities: first , they all contain domains which recognise acetylated lysines: bromodomains for Bdf1 and Bdf2 and a YEATS domain for Yaf9 [57] . The Yaf9 YEATS domain was recently described as a reader of lysine crotonylation and to promote active transcription [58] . Second , they all possess a coiled-coil domain , well known to mediate protein dimerization ( S6 Fig , [59] ) . The B motif present in all the members of the BET family is also a coiled-coil domain and could mediate the dimerization of BET proteins [60] . Therefore , we hypothesised that coiled-coil domains could contribute to the formation of the two complexes the Bdf1 protein is involved in; Bdf1’s coiled-coil domain could interact exclusively either with the coiled-coil domain of Bdf2 or of Yaf9 . This would explain how Bdf1 forms exclusive complexes with these two proteins . In the SWR complex , Bdf1 promotes the incorporation of H2A . Z into acetylated regions [10 , 61 , 62] . The function of the interaction between Bdf1 and Bdf2 remains to be characterised . This interaction seems to be tight because deletion of any of the Bdf2 domains -bromodomains or ET- disrupts its interaction with Bdf1 . However , none of these BDF2 mutations induces any sporulation defects . Although Bdf1 and Bdf2 are functionally redundant—as only one is needed to maintain cell viability—they could play different roles in controlling chromatin dynamics . Indeed , they occupy distinct locations in the genome and larger amounts of Bdf1 bind to chromatin compared to Bdf2 [55] . Deletion of BDF2 in vegetative cells only affects a few transcripts , whereas the deletion of BDF1 provokes major transcriptomic changes [14] . The situation is similar during sporulation: Bdf1 is loaded on various loci , and especially middle sporulation genes , whereas Bdf2 does not seem to be strongly attached to chromatin ( Fig 4F and ref [19] ) . The timing of the recruitment of Bdf1 onto chromatin is particularly intriguing , with Bdf1 detected on the promoters of middle sporulation genes before their transcriptional activation . This recruitment is essential for the activation of these genes and involves Bdf1 bromodomains . When these domains are mutated , Bdf1 is no longer recruited to these promoters and their genes fail to induce . The non-induction of NDT80 causes the major defects observed when Bdf1 bromodomains are mutated . Indeed , this Bdf1 mutation phenocopies deletion of NDT80 , which also induces stalling in the pachytene phase and failure to accumulate middle-meiotic mRNA . Moreover , the ectopic expression of NDT80 can partially rescue the sporulation defects caused by mutation of Bdf1 bromodomains . However , the mechanisms through which Bdf1 specifically regulates the expression of NDT80 and middle sporulation genes remain unclear . Bdf1 was originally described as a missing piece of the TFIID complex , although as a loosely interacting protein [63] , which could partially explain why no TFIID proteins were identified in our purifications . Perhaps a minor fraction of Bdf1 interacts with the transcription machinery . Bdf1 alone could promote a chromatin state favourable for the expression of middle sporulation genes . Thus , Bdf1 could promote the initial low-level pool of Ndt80 in meiotic G2 and also promote the feed-forward autoregulatory loop required to trigger high expression levels of middle genes [64] . Bdf1 is a member of the BET family , which also includes the mammalian proteins Brd2 , Brd3 , Brd4 and Brdt . Brd4 has been shown to facilitate transcriptional activation and was recently found to have an intrinsic HAT activity [48] . However , the residues controlling this activity are not conserved in Bdf1 . Finally , H4 acetylation and H4K5 butyrylation have been shown to regulate the binding of Brdt to chromatin , resulting in highly active transcription [65] . Similarly , different acyl marks could regulate Bdf1 binding to chromatin and may explain why the expression of middle genes specifically requires Bdf1 bromodomains . This novel role for Bdf1 during yeast meiosis could be shared by mammalian BET proteins during spermatogenesis . Indeed , Brd2 and Brd4 are known to associate with mitotic chromosomes and Brd4 binds post-mitotic genes to promote their transcriptional reactivation after the M phase [66–68] . During male meiosis in mouse , Brdt is essential for the gene expression program , and its deletion affects the expression of testis-specific cyclin genes such as Ccna1 , which are required for meiotic division [26] . In yeast , the results presented here revealed that mutation of Bdf1 bromodomains abolished the expression of almost all the subunits of the APC complex , which is required for meiotic progression [69] . Interestingly , in mice , the absence of Brdt leads to a loss of FZR1 expression—an essential activator of the APC complex during meiosis—and subsequent deregulation of the APC complex ( S7 Fig and reference [70] ) . In conclusion , Bdf1 appears to be a key chromatin protein for the meiotic transcription program , regulating the expression of middle sporulation genes and promoting their rapid activation . Its role during meiosis appears to be evolutionarily conserved from yeast to mouse , and BET proteins are essential for the functionality of the APC complex and its regulation of meiotic progression . Our results suggest that the control of gene activation by Bdf1 does not rely on any of its major protein partners . Further studies will be required to explore its precise mode of action and whether these molecular mechanisms are conserved during the mammalian cell cycle in somatic cells and during meiosis and gamete differentiation .
GST , TAP and HA antibodies were obtained from Dutscher ( Ref . 27-4577-01 ) , Fisher ( Ref . 10506450 ) and Roche ( Ref . 11867423001 ) , respectively . Anti-Bdf1 antibody was developed in-house by injecting recombinant protein into rabbits ( Covalab ) . The use of bdf1Δ and Bdf1-TAP strains validated its specificity ( S4A Fig ) . Yeast Bdf1 bromodomain 1 ( residues 132–263 ) or 2 ( residues 317–430 ) and human Brd4 bromodomain 1 ( residues 22–204 ) were cloned into pGEX-4T1 as GST-tagged proteins . Expression was induced in E . coli strain BL21 ( DE3 ) grown in LB medium with kanamycin ( 50 μg/mL ) at 37°C by adding 0 . 5 mM IPTG at OD600 of 1 and then incubated for 16 h at 16°C . Cells were lysed by sonication in Tris-HCl 50 mM pH 7 . 5 , NaCl 150 mM and protease inhibitors . Clarified lysate was incubated with glutathione sepharose ( Fisher , ref . W7349W ) and washed in Tris-HCl 50 mM pH 7 . 5 , NaCl 500 mM , NP-40 1% . Bound proteins were eluted in glutathione 10 mM . For peptide pull-downs , 0 . 5 nmol of biotinylated H4 ( Millipore , ref . 12–405 ) and H4K5ac K8ac K12ac K16ac peptides ( H4ac4 , H4 ( Millipore , ref . 12–379 ) ) were bound to Streptavidin magnetic beads ( Thermo Dynabeads MyOne Streptavidin , ref . 65001 ) and used to pull-down 1 . 25 μg of GST-tagged Bdf1 bromodomain 1 and 2 ( wild-type and mutants , bdf1-Y187F and bdf1-Y354F ) and GST-tagged Brd4 bromodomain 1 of Brd4 in Tris-HCl pH 7 . 5 50 mM , NaCl 150 mM , NP-40 0 . 1% , Glycerol 10% and DTT 1 mM . Washes were optimized using 500 mM of NaCl . Bound proteins were eluted by boiling in Leammli buffer and proteins analysed by western blot using an anti-GST antibody . Histone peptide array were purchased from Active Motif ( ref . 13005 ) and used in accordance with the supplier’s recommendations . Briefly , arrays were blocked and then incubated with 1 μM of purified GST-tagged Bdf1 bromodomain 1 and 2 . Incubation was allowed to proceed for 2 h at 4°C in binding buffer ( Tris-HCl 50 mM pH 7 . 4 , NP-40 0 . 1% , NaCl 150 mM , glycerol 10% ) . The membrane was washed in PBS Tween 0 . 1% and detected with an anti-GST antibody mixed with an anti-myc antibody used as a positive control and for normalisation . The membrane was washed and incubated with horseradish-peroxidase-conjugated anti-Goat antibody ( Jackson , ref . 705 . 035 . 147 ) . The membrane was submerged in Clarity Western ECL Substrate ( Bio-Rad , ref . 170–5060 ) , imaged ( Bio-Rad ChemiDoc XRS Imaging ) and data were quantified using an array analyser software provided by Active Motif . Signal intensities are presented in S1 Table . Yeast strains , plasmids and primers are listed in S2 , S3 and S4 Tables respectively . The genotypes/expression profiles of all deletion mutants and tagged strains were confirmed by PCR analysis , sequencing and/or western blot analysis . Frogging assays on agar plates used 10-fold serial dilutions . Diploid yeast in the SK1 background was grown to an OD600 of 0 . 5 in YPD . Cells were washed and resuspended in YPA at an OD600 of 0 . 03 . After 12 h cells were washed and transferred into sporulation media ( K acetate 2% ) supplemented with auxotrophic amino acids . Six hours after sporulation induction , GAL-NDT80 GAL4 . ER strain was released from the meiotic arrest by the addition of 1 μM oestradiol ( 5 mM stock in ethanol , Sigma E2758-1G ) . Sporulation efficiency was assessed after 24 h of induction and is defined by the number of cells which formed tetrads . Meiotic progression was monitored by fixing 500 μl of cells in EtOH 70% and staining the nuclei with 0 . 5 μg/ml of DAPI . Fluorescence microscopy was used to determine the proportion of mono- , bi- , tri- and tetra-nucleated cells . The frequency of meiotic recombination between the heteroalleles his4-N/his4-G was quantified by plating sporulated yeast cultures on YPD and SC-HIS plates . The number of yeasts growing on SC-HIS plates was counted and normalised to the total number of cells growing on YPD . Samples were collected from sporulating cells ( wild-type and Bdf1 double point mutant bdf1-Y187F-Y354F ) at 0 , 4 and 8 h . Cell pellets were washed and stored at -80°C . Total RNA was extracted with phenol:chloroform ( Sigma Ref . 2190191 ) from three independent biological replicates . DNaseI treatment ( Thermo , ref . AM2222 ) was performed using 1 μg of total RNA subsequently purified by phenol:chloroform extraction . Intact poly ( A ) + RNA was isolated by using the NEB Next Poly ( A ) + mRNA Magnetic Isolation kit ( E7490S ) . cDNA libraries were constructed from 10 ng of total RNA using the NEB Next Ultra RNA Library Prep Kit for Illumina ( New England Biolabs , ref . E7530S ) . Fragments were enriched by 15 cycles of PCR amplification . Agilent 2100 Bioanalyzer ( 2100 , Agilent Technologies , CA ) was used to assess the quality and quantity of each library . Eighteen barcoded cDNA libraries were pooled and sequenced on an Illumina HiSeqTM 2000 , generating 5–8 million single reads ( SR50 ) for each sample . The bioinformatic analysis pipeline , presented S3A Fig can be summarized as follows: reads were quality-checked using FastQC before and after cleaning by Trimmomatic applying default parameters [71] . Reads were then aligned to S . cerevisiae S288c genome build R64-1-1 . 82 using Bowtie2 [72] . The attribute file was downloaded from Ensembl website and modified to include yeast intronic sequences , obtained from Yeastmine . Raw read counts for each gene were calculated using HTSeqCount using defaults parameters [73] . Read count data was normalised using the DESeq2 R-package ( S5 Table , [74] ) . DESeq2 normalised read counts were used to identify differentially expressed genes with an adjusted p-value < 0 . 05 and a fold change below -2 or above 2 ( S5 Table ) . Gene ontology analysis was performed using the generic GO-term mapper developed by the Max Planck Institute ( http://cpdb . molgen . mpg . de/YCPDB ) . Pscan tool was used to explore the promoter of the genes which are downregulated in bdf1-Y187F-Y354F 4 and 8h after sporulation induction ( http://159 . 149 . 160 . 88/pscan/ , [75] ) . Binding motifs for yeast transcription factors were obtained from the JASPAR 2016 database [76] and researched in the promoter of each gene ( -500 to 0 bp upstream each transcription start side ) . A binding motif was considered significantly over-represented when the p-value was inferior to 0 . 001 ( Fig 4A ) . The probability that an Ndt80 binding sequence was present in the promoter of bdf1-Y187F-Y354F deregulated genes was calculated using the Morpheus ROC tool [43] . This tool computes the ROC-AUC value as a measure of the enrichment of the binding sequence of a transcription factor in a list of candidate genes compared to a mock list of genes ( Fig 4C ) . Venn diagram was generated using Venny 2 . 1–0 ( http://bioinfogp . cnb . csic . es/tools/venny/index . html ) . A list of genes potentially regulated by Ndt80 was obtained from Chu et al [35] . The statistic representation of these genes in the list of genes downregulated in the bdf1-Y187F-Y354F mutant was tested using a hypergeometric test ( Fig 4D ) . ChIP analyses were performed as described [19] with minor changes . Crosslinking was done with 1% EGS for 15 min and then 1% formaldehyde for 10 min before quenching for 5 min with Glycine 125 mM . Cells were lysed in lysis buffer ( Hepes 50 mM pH 7 . 5 , NaCl 140 mM , EDTA 1mM , Triton X-100 0 . 1% , PMSF 0 . 5 mM , cOmplete , TSA 100 mg/l , phosphatase inhibitor cocktail ( Sigma , ref . P2850 ) ) in a Fastprep ( MP Biologicals ) for three periods of 45 s . Extracts were sonicated in cycles of 20 s with intermediate incubation for 40 s over a total of 30 min ( EpiShear , Active Motif ) . Clarified extracts were immunoprecipitated by incubating with Pan-Mouse IgG Dynabeads ( LifeTechnologies , ref . 11041 ) or with Protein G Dynabeads ( Thermo , ref . 10004D ) conjugated with anti-HA antibody ( Roche , ref . 11867423001 ) for TAP or HA tagged strains , respectively . ChIP data are presented as percent of input . In all cases , at least three biological replicates were performed to determine the standard errors in each experiment . TAP purifications were performed as described previously [77 , 78] . Calmodulin eluates from the TAP-purified complexes were analysed by SDS-PAGE by using Novex 4–12% gradient gels ( Invitrogen ) and visualised by staining with SilverQuest Silver Staining Kit ( Thermo , LC6070 ) . Western blot analysis of the calmodulin eluates was performed using the corresponding antibodies . Protein preparation and mass spectrometry-based proteomic analyses were carried out as described in [79] . Briefly , eluted proteins were stacked as a single band in a SDS-PAGE gel ( NuPAGE 4–12% , Invitrogen ) and submitted to in-gel digestion using trypsin ( Promega , sequencing grade ) . Resulting peptides were analysed by online nanoLC-MS/MS ( UltiMate 3000 and Q-Exactive Plus or LTQ-Orbitrap Velos Pro , Thermo Scientific ) using a 120-min gradient . Peptides and proteins were identified and quantified using MaxQuant ( version 1 . 5 . 3 . 30 , [80] ) and the SGD database ( November 2015 release ) . Proteins were quantified based on the iBAQ value [81] calculated by MaxQuant . Only proteins identified with a minimum of two unique + razor peptides were taken into account . For statistical analysis of results obtained with Bdf1-TAP triplicates , we used ProStaR [82]: proteins identified in the reverse and contaminant databases and proteins exhibiting fewer than 6 iBAQ values in a single condition ( 2 conditions and 2 analytical replicates per biological replicate ) were discarded from the list . After log2 transformation , iBAQ values were normalised by condition-wise median centring before imputing missing values using the QRILC algorithm ( missing not at random-devoted imputation method ) ; statistical testing was conducted using limma . For a protein to be considered as a potential binding partner of Bdf1-TAP , it had to exhibit a p-value < 0 . 001 and a log2 ( fold change ) ≥ 7 . Coimmunoprecipitation was performed using 50 mL of cells grown on YPD to an OD600 of 0 . 5 . Cells were harvested , washed with water , and resuspended in lysis buffer ( HEPES 50 mM pH 7 . 5 , NaCl 140 mM , EDTA 1 mM , glycerol 10% , NP-40 0 . 5% , PMSF 1 mM and protease inhibitors ) . An equal volume of glass beads was added . Cells were lysed in a Fastprep homogenizer ( MP Biomedicals ) for 45 s . The clarified extracts were immunoprecipitated during 2 h at 4°C using Dynabeads Pan Mouse IgG ( LifeTechnologies ) . The immunoprecipitates were washed and resuspended in 50 μl of SDS-PAGE sample buffer for subsequent western blot analysis . Reverse transcription was performed by following manufacturer instructions using kits iScript RT Supermix and Universal SYBR Green Supermix ( Bio-Rad ) on a CFX384 Touch qPCR machine ( Bio-Rad ) . Primers are presented ( S4 Table ) . At least three biological replicates were performed to determine the standard errors for each experiment . RT-qPCR data were then normalised relative to the reference gene , NUP85 [19] . RNA-seq data obtained in this study are available from the GEO repository ( GSE89530 ) . Mouse transcriptomic data was derived from RNA-seq dataset GEO GSE39909 as described in ( S7 Fig , [26] ) . Proteomic data produced in this study have been deposited on ProteomeXchange under identifier PXD005227 . | Chromatin modifying proteins play a central role in transcription regulation and chromatin signalling . In this study we investigated the functional role of the bromodomains of the chromatin protein Bdf1 during yeast gametogenesis . Our results show that the bromodomains of Bdf1 are essential for meiotic progression and the formation of mature spores . Bdf1 bromodomains are required for the expression of key meiotic genes and the master regulator NDT80 . Forced expression of NDT80 can partially rescue the formation of spores when Bdf1 bromodomains are mutated . The results presented here indicate that Bdf1 forms two exclusive complexes , with Bdf2 or with the SWR complex . However , none of these complexes are required for sporulation progression . To conclude , our findings suggest that Bdf1 is a new regulator of the meiotic transcription program and of the expression of the master regulator NDT80 . | [
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"saccharomy... | 2017 | Bdf1 Bromodomains Are Essential for Meiosis and the Expression of Meiotic-Specific Genes |
Genome-wide studies of post-transcriptional mRNA regulation in model organisms indicate a “post-transcriptional RNA regulon” model , in which a set of functionally related genes is regulated by mRNA–binding RNAs or proteins . One well-studied post-transcriptional regulon by Puf3p functions in mitochondrial biogenesis in budding yeast . The evolution of the Puf3p regulon remains unclear because previous studies have shown functional divergence of Puf3p regulon targets among yeast , fruit fly , and humans . By analyzing evolutionary patterns of Puf3p and its targeted genes in forty-two sequenced fungi , we demonstrated that , although the Puf3p regulon is conserved among all of the studied fungi , the dedicated regulation of mitochondrial biogenesis by Puf3p emerged only in the Saccharomycotina clade . Moreover , the evolution of the Puf3p regulon was coupled with evolution of codon usage bias in down-regulating expression of genes that function in mitochondria in yeast species after genome duplication . Our results provide a scenario for how evolution like a tinker exploits pre-existing materials of a conserved post-transcriptional regulon to regulate gene expression for novel functional roles .
Evolution of gene expression may account for significant phenotypic diversity among species [1]–[6] . Gene expression is regulated at various levels . Many studies have demonstrated gene expression changes caused by mutations in transcription [7] , [8] . Post-transcriptional regulation is also crucial for splicing , translation , localization and degeneration of mRNAs in eukaryotes , and thus is important in determining the abundance of gene expression [9] , [10] . It is likely that changes in post-transcriptional regulons are also important in the evolution of gene expression [11] . The control of post-transcriptional regulation is mediated by regulons such as mRNA-binding proteins ( RBP ) or RNAs ( e . g . , microRNAs ) which usually bind to elements in the 3′ untranslated regions ( UTR ) and determine the fate of their targeted mRNAs . Evolution of microRNA post-transcriptional regulons has been well studied due to recent improvement in understanding their functions . It was shown that novel microRNAs can turn over rapidly during evolution [12] , and for those that are highly conserved over long evolutionary distances , their targets can change dramatically even within populations [13] . Studies on microRNAs have revealed interesting information on evolution of this particular type of post-transcriptional regulon , whereas evolution of RBP regulons remains poorly understood . Furthermore , RBP regulons play major post-transcriptional roles in budding yeast because the species lost the microRNA regulatory machine [14] . One of the best-characterized RBP families is PUF ( Pumilio and FBF , FBF represents for fem-3 binding factor ) , which is conserved in a wide variety of eukaryotes from yeast to humans [15]–[19] . The PUF post-transcriptional regulon regulates diverse gene sets in various model organisms . For example , in the budding yeast , Saccharomyces cerevisiae , genes most commonly targeted by Puf3p are in the mitochondria and play essential roles in mitochondrial biogenesis [17] , [20] . In the fruit fly , Drosophila melanogaster , Pumilio ( a PUF protein ) , which binds with the same element as Puf3p in the budding yeast , is necessary for early embryogenesis and development of primordial germ cells [21] , . Genome-wide identification of the Pumilio targets in fruit flies uncovered genes involved particularly in nucleotide metabolism , transcriptional regulation and synthesis of membrane proteins [18] . In humans , two paralogous PUF proteins ( Pum1p and Pum2p ) , which interact with the microRNA system in post-transcriptional regulation , share the same binding-element with yeast Puf3p and bind to mRNAs from genes that function in transcriptional regulation and cell proliferation [19] , [23] . Previous studies have reported that the binding site of Puf3p is highly conserved in sensu stricto yeasts [24]–[26] . Taking advantage of a large number of genomic sequences , in this study we investigated the evolution of the Puf3p post-transcriptional regulon in fungi . Our results show continuous steps of functional innovation in the Puf3p regulon despite its ultra conservation in these fungal species . First , the regulation of mitochondrial biogenesis by the Puf3p regulon originated in the Saccharomycotina subdivision; second , the Puf3p regulon was coupled with codon usage bias to modulate expression of genes that function in mitochondria in yeasts after whole genome duplication ( WGD ) . Our work and reports from other labs show that mitochondria underwent significant functional changes during the origin of an efficient aerobic fermentation system in the yeast species that went through WGD [27]–[30] . This current report provides evidence suggesting that the Puf3p post-transcriptional regulon was involved in the evolution of this novel life history in yeasts .
The RNA-binding domain of Puf3p , called the PUF homology domain ( PUF-HD ) , consists of eight repeated peptide motifs [31]–[33] . In order to study the evolution of the RNA-binding domain , orthologous genes of PUF3 were identified from the forty-two sequenced fungal species ( Table S1 ) . Domain alignments in SMART [34] , [35] indicate that almost all Puf3p orthologs contain the eight repeated motifs even though some repeats are not highly conserved ( Figure 1A ) . Puf3p in Lodderomyces elongisporus and Rhizopus oryzae lost one repeat which may have resulted from insufficient genome sequencing or assembly because both orthologs are located at the end of the assembled contigs . This result demonstrates that the binding domain of Puf3p is conserved among all studied fungi . The evolutionary trajectory of the Puf3p-binding element in its target genes was further investigated . Because the 8nt-core motif of P3E is conserved from yeast to human , we used the 8nt-core P3E profiling in the budding yeast as a reference to identify all possible puf3p targeted genes that contain at least one P3E at their 3′ downstream sequences ( Figure 1B ) [19] . Because GC content is very low in the P3E , the genomic GC content would inevitably affect the frequency of P3E in each species . Indeed , as shown in Figure 1C , at the genome level , the number of genes with P3E is negatively correlated with the genomic GC content among the studied species . In order to exclude the impact of genomic GC content on our results , we generated 10 , 000 random sequences for each species based on the average GC content of 1 , 000 bp downstream sequences of all the annotated genes in this species . Using the occurrence of P3E in these random sequences as background in each species , we estimated the probability of having the observed P3E frequency in the 3′ downstream sequences of all the annotated genes in the same species . The probability was calculated in each sliding window of 50 bp in the 3′ downstream sequences of each species . As shown in Figure 1D , all studied species exhibited significant enrichment of P3E in the first several sliding windows in the 3′ downstream regions . As the 3′ UTR in yeast is usually shorter than 250 bp [36] , our results indicate that the enrichment of P3E in the studied fungi results from P3E conservation in the 3′ UTR sequences . When we used the GC contents in the 250 bp regions after the stop codon or in each 50 bp sliding window to calculate the background P3E motif distribution , similar enrichment of P3E motif in the 3′UTR regions are still observed in most fungi species ( Figure S1 ) . Previous reports showed that Puf3p plays an essential role in mitochondrial biogenesis in S . cerevisiae [17] , [37] , [38] . This observation prompted us to investigate whether the functional profile of the Puf3p regulon is also conserved among fungi species . Accordingly , we identified all of orthologous genes between each studied fungal species and budding yeast . Genes in each species are classified into categories based on the sub-cellular localization of their orthologs in the budding yeast [39] . We then estimated the enrichment of genes with P3E in each localization category . As shown in Figure 2A , we discovered that all of the studied species in the Saccharomycotina subdivision show significant enrichment of P3E in genes that function in mitochondria . Other clades of the studied fungi did not have this pattern . Indeed , close to 50% of genes that function in mitochondria have P3E in the Saccharomycotina subdivision , which is significantly higher than that of species in other clades ( Figure 2B , P-value = 6×10−22 ) . A similar pattern was observed when a slightly different P3E motif profile [17] was used to calculate the motif frequency ( Figure S2 ) . Because regulation of the mitochondrial translational machine is essential for mitochondrial biogenesis [40] , we further investigated conservation of P3E among the orthologous genes with this particular function in the Saccharomycotina subdivision . Our results showed that ∼80% of genes with highly conserved P3E were involved in mitochondrial translation , whereas this number is only ∼4% for genes with little P3E conservation , indicating that the Puf3p regulation of genes that are involved in the mitochondrial translational machine is highly conserved in these Saccharomycotina species ( Figure 2C ) . Gene expression is regulated at multiple levels . Biased usage of preferred codons can result in enhanced accuracy and speed of protein synthesis in highly expressed genes [41] , [42] . Previous studies reported that codon usage bias in mitochondrial genes is relaxed , possibly due to a relaxed function of the organelle with the origin of an efficient aerobic fermentation system in the fungal lineage with WGD [27] , [28] . We predicted that Puf3p-regulated mitochondrial genes , due to their importance in mitochondrial biogenesis and functions , would experience more relaxation of codon usage bias than other mitochondrial genes in the post-WGD yeast species . In order to test this , we calculated the average codon bias adaptation index ( CAI ) for the mitochondrial genes , with and without P3E , for each species . As shown in Figure 3A , the mitochondrial genes with P3E in the post-WGD species show significantly smaller CAI than those genes in the fungal species that diverged from the common ancestor before the WGD event ( student t-test , P = 3×10−8 ) , whereas mitochondrial genes without P3E did not show such a pattern ( Figure 3B , student t-test , P = 0 . 1 ) . Due to the importance of Puf3p regulation in mitochondrial gene degradation , we further investigated expression of its target mitochondrial genes under the fermentative condition . Using gene-expression profiling measured by microarray data [43] , we discovered that significantly more mitochondrial genes with P3E were down-regulated in the fermentative medium ( YPD ) than those without P3E ( Figure 4A , Fisher's exact test , P = 1 . 2×10−4 ) . Furthermore , we found that mitochondrial genes with P3E tend to be co-regulated because the average correlation coefficients of gene expression among mitochondrial genes with P3E in different conditions is significantly higher than that of genes without P3E ( Figure 4B , student t test , P = 0 ) . Therefore Puf3p regulon plays an important role in regulating mitochondrial genes in different conditions .
PUF protein was first characterized in Drosophila as an mRNA-binding factor that recruits other proteins to inhibit the translation of the bound mRNA [21] . Subsequently , many studies in yeast revealed that the PUF family regulates specific mRNA degeneration by their RNA-binding domains [17] , [31] , [44] , [45] . It was shown that the function of targeting mRNA for degeneration by Puf3p is much more efficient in 2% glucose ( YPD , fermentative ) than in 3% ethanol ( YPE , non-fermentative ) [17] , [46] . Furthermore , it was shown that Puf3p is crucial for mitochondrial biogenesis and motility under non-fermentative conditions in budding yeast [43] . Saint-Georges and his colleagues reported that Puf3p can transfer its target mRNAs to the peripheral mitochondria in the non-fermentative growth medium [37] . The expression of PUF3 gene is significantly higher in yeast growing in YPE than in YPD ( Figure 5A , P-value<0 . 05 ) . We speculate that this is true because the positive regulation of mitochondrial biogenesis might not be as important for Puf3p in fermentative conditions as that in respiratory conditions: First , based on gene deletion data , the mitochondrial genes with P3E are significantly more important ( having more severe growth defects after gene deletion ) than those genes without P3E ( P = 4×10−6 ) under non-fermentative conditions , but these two gene groups do not show obvious difference in deletion phenotype under fermentative conditions ( Figure 5B ) . Second , severe growth defect after PUF3 gene deletion was observed in YPE , but not in YPD ( Figure 5C ) . Therefore in non-fermentative condition Puf3p regulates both mitochondrial biogenesis and mRNA degradation , but in fermentative condition , it might only regulate mRNA degradation , albeit more efficiently in this condition . The expression difference of PUF3 in two growth conditions can also be explained by the fact that mitochondrial biogenesis in non-fermentative conditions is extremely important for yeast because the organism relies on respiration , and therefore mitochondria , to generate cellular energy in these conditions . In contrast , the function of mRNA degradation might not be as essential to the organism under fermentative conditions . Although loss of a functional PUF3 gene shows a negligible effect on organism growth in YPD ( Figure 5C ) , our results in this study indicate that Puf3p regulation of mitochondrial gene degradation might be important for yeast fermentative growth during evolution . After whole genome duplication , the post-WGD species ( including budding yeast ) evolved efficient fermentative ability [47] . Mitochondrial function became relaxed in these species [27] , [28] . Most post-WGD yeast species can live even without a functional mitochondrial genome [47] . A large number of mitochondrial genes are down-regulated in yeast fermentative growth [48] . Degradation of mRNA by Puf3p may accelerate this gene-expression regulation process during environmental switches . Interestingly , our results showed that mitochondrial genes having P3E had significantly relaxed codon usage bias in the post-WGD species , which is not true for other mitochondrial genes . Understanding the origin of genetic novelties is a challenging issue in evolutionary biology . One of the prominent models proposed by Francois Jacob for evolution of genetic novelties in gene regulatory network is tinkering evolution , in which evolution reorganizes pre-existing networks to produce novelties [6] , [49]–[52] . Our results provide an interesting paradigm for the evolution of post-transcriptional regulons that is consistent with this model . The Puf3p post-transcriptional regulon might have changed significantly at least twice in fungal evolution . First , although it is conserved in all the studied fungal species , the dedicated function of the Puf3p post-transcriptional regulon in mitochondrial biogenesis independently evolved in the Saccharomycotina subdivision . Second , although the regulation of mitochondrial mRNAs by Puf3p is conserved across the Saccharomycotina subdivision , the Puf3p target genes evolved a reduced codon usage bias in the post-WGD species , which might be consistent with the functional relaxation of mitochondrial genes in the post-WGD species due to the emergence of their fermentative life-style during evolution . It was shown that although some microRNAs are highly conserved , their target networks can change dramatically during evolution [13] . Our results , together with those from previous studies , indicate that post-transcriptional regulons by RBP and microRNAs might share similar evolutionary patterns: i . e . , the interaction mechanisms between the regulators and their target genes are conserved , whereas the target network is plastic during evolution . As post-transcriptional regulation plays an important role in regulating gene expression , this evolutionary scenario involving post-transcriptional regulons could lead to significant gene-expression divergence among species .
Sequences for the forty-two sequenced fungal species were downloaded from the Fungal Comparative Genomics database [53] and the National Center for Biotechnology Information ( http://www . ncbi . nlm . nih . gov/ ) . Using the InParanoid software package [54] , orthologs between budding yeast and other fungal species were identified . The PUF3 orthologs in other species of fungi were identified manually based on the best alignment . Eight repeated motifs of PUF protein were detected by the SMART sequence analysis ( http://smart . embl-heidelberg . de/ ) . Because the Puf3p binding motif in its target genes ( P3E ) is conserved between yeast and humans , based on the profile of P3E in budding yeast , we used a Perl script to detect the target locus of Puf3p by fixing all seven invariable sites and allowing flexibility in the fifth site ( Figure 1B ) . For each species , we scanned 1 , 000 bp of DNA sequence downstream of all annotated genes to discern the occurrence of the P3E motif . The percentage of motif occurrences in each 50-bp window among all genes in each species was calculated . Multiple occurrences of the motif in the same sliding window were regarded as independent events . To see whether the motif occurrences in a species are different from random expectation , we calculated GC content of the 1 , 000bp downstream sequences for all genes in each species . Based on the observed GC content , 10 , 000 random sequences of 1 , 000bp were generated by a perl script and the occurrences of PUF3 motif were calculated . The significance of PUF3 motif occurrences in each sliding window was calculated by comparing its motif frequency against the frequencies of P3E in these random sequences by Fisher's exact test ( more focused tests were also conducted in Figure S1 ) . The Bonferroni correction was used to correct for multiple comparisons . Genes with P3E in their 250bp 3′ downstream regions were defined as the target of Puf3p . The sub-localization information for genes in budding yeast was downloaded from the Saccharomyces Genome Database ( http://www . yeastgenome . org/ ) [39] . Based on the identified orthologous genes between each studied fungal species and budding yeast , genes in each species are classified into different localization categories based on the sub-cellular localization of their orthologs in the budding yeast . The hypergeometric test was used to test the enrichment of genes with P3E in each localization category . The Bonferroni correction was used to correct for multiple comparisons . According to the InParanoid results , we identified all the orthologous clusters that contained the known cytoplasmic ribosomal protein genes in S . cerevisiae , regardless of gene copy number in each species . The ribosomal protein genes in each species were used as references to calculate the codon adaptation index ( CAI ) value for each individual gene in the same species by CodonW ( http://codonw . sourceforge . net/ ) [55] . In order to compare codon usage bias between different species , CAI values in each species were standardized so that the mean and standard deviation were 0 and 1 , respectively . Cells were grown in the YPD and YPE media until optical densities at 600 nm reaches 1 . Total RNA was extracted using the Trizol protocol [56] and cDNA was synthesized using an Invitrogen kit ( Cat . No . 18080-051 ) . Using the ACT1 gene as reference , the expressional levels of PUF3 in fermentative and non-fermentative conditions were measured by quantitative real-time PCR . The rate of growth for deletion mutants were downloaded from [57] . Two-tails student t test was used to compare the average fitness contribution of mitochondrial genes with and without P3E in fermentative and non-fermentative growth conditions . The deletion of the PUF3 gene was conducted in the BY4741 strain ( Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ) by homologous recombination and ura- was used as the selection marker ( the primer sequences for gene deletion are available upon request ) . The mutant and the wild type strains were grown overnight in YPD ( 2% glucose ) and YPE ( 3% ethanol ) media . Cells were then transferred into fresh media and grown until the optical density ( 600 nm ) reached 0 . 2 . Then 4ul of growth media were dotted onto the YPD and YPE plates with ten-fold dilutions . YPD and YPE plates were incubated at 30°C for 48 and 72h , respectively . | It is well known that the evolution of gene expression can account for significant phenotypic diversity among species . Gene expression is regulated at various levels . Many studies have demonstrated gene expression changes caused by mutations in transcription . Post-transcriptional regulation is also crucial for splicing , translation , localization , and degeneration of mRNAs in eukaryotes and , thus , is important in determining the abundance of gene expression . Changes in post-transcriptional regulons are also important in the evolution of gene expression . In this study we investigated the evolution of a particular post-transcriptional regulon , Puf3p , in fungal species . Our results illustrated an important evolutionary mode for evolution of post-transcriptional regulon , i . e . , pre-existing materials of conserved post-transcriptional regulons can be recruited to regulate gene expression for novel functional roles . | [
"Abstract",
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] | [
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] | 2010 | Tinkering Evolution of Post-Transcriptional RNA Regulons: Puf3p in Fungi as an Example |
Rickettsia africae , the etiological agent of African tick bite fever , is widely distributed in sub-Saharan Africa . Contrary to reports of its homogeneity , a localized study in Asembo , Kenya recently reported high genetic diversity . The present study aims to elucidate the extent of this heterogeneity by examining archived Rickettsia africae DNA samples collected from different eco-regions of Kenya . To evaluate their phylogenetic relationships , archived genomic DNA obtained from 57 ticks a priori identified to contain R . africae by comparison to ompA , ompB and gltA genes was used to amplify five rickettsial genes i . e . gltA , ompA , ompB , 17kDa and sca4 . The resulting amplicons were sequenced . Translated amino acid alignments were used to guide the nucleotide alignments . Single gene and concatenated alignments were used to infer phylogenetic relationships . Out of the 57 DNA samples , three were determined to be R . aeschlimanii and not R . africae . One sample turned out to be a novel rickettsiae and an interim name of “Candidatus Rickettsia moyalensis” is proposed . The bonafide R . africae formed two distinct clades . Clade I contained 9% of the samples and branched with the validated R . africae str ESF-5 , while clade II ( two samples ) formed a distinct sub-lineage . This data supports the use of multiple genes for phylogenetic inferences . It is determined that , despite its recent emergence , the R . africae lineage is diverse . This data also provides evidence of a novel Rickettsia species , Candidatus Rickettsia moyalensis .
Rickettsiae are obligate intracellular gram negative bacteria , belonging to the class alpha-proteobacteria . They are found in a diverse array of hosts ranging from vertebrates , arthropods , annelids , amoeba and plants . Based on a host perspective , the non-vertebrate-associated Rickettsia remain understudied and poorly characterized [1] . In contrast , the vertebrate-associated Rickettsia that are vectored by hematophagous arthropods such as ticks , fleas , lice and mites are better studied , are responsible for rickettsial diseases that are important cause of illness and death worldwide [2] . To date , this genus consists of 29 validated species and numerous partially characterized species [3 , 4] , thus illustrating the difficulties of unravelling the composition of this seemingly homogeneous group of bacteria . Phenotypic characters such as pathogenicity , growth temperature requirements , ability to polymerize host cell actin , and cross reactivity to somatic antigens of Proteus vulgaris strains ( OX19 and OX2 ) and P . mirabilis OXK have been used to infer evolutionary relationships amongst rickettsiae [5] . From these criteria , the genus rickettsia was organized into three bio-types , namely , spotted fever group ( SFG ) , typhus group ( TG ) and scrub typhus group ( STG ) . The phenotypic characters have been found to be unreliable estimators of their phylogeny [6] . The advent of molecular tools brought major reorganizations in rickettsia taxonomy . For instance , by analysis of Rickettsia 16S rRNA ( rrs ) , STG was removed from the genus Rickettsia and placed into its own genus , Orientia . This genus currently has only two species , O . tsutsugamushi [7] and a recently described O . chuto [8] . Several genes have been used for Rickettsia phylogenetic systematics: the rrs [5] , gltA [9] , 17kDa [10] , ompA [11] , ompB [12] , sca4 [13] , sca2 [14] , and more recently , complete genomic sequences [15 , 16] . Currently , the genus delineates into four clades [17–19]: ( i ) The SFG which is the most derived , and consists of 23 validated species including R . africae , and numerous partially characterized species . Using whole genome approach , it has been realized that some of the members of rickettsiae such as R . helvetica do not fit in the SFG [16 , 20] ) . ( ii ) The transitional group ( TRG ) whose members are R . akari and R . felis . ( iii ) The TG which has only two members , namely , R . typhi and R . prowazekii; and ( iv ) , the ancestral group ( AG ) , whose members consists of R . bellii and R . canadensis . The use of the name TRG has been challenged [15 , 21] ) . In addition to the systemic of vertebrate-associated Rickettsia , clades associated with non-vertebrate Rickettsia have been described [16 , 19] . Of the rickettsiae , R . africae is probably the most important in Africa . It is the aetiological agent of African tick bite fever [22] and the most reported [23] . It has been reported in 22 sub-Saharan countries [3] , the West Indies [24] and Oceania [25] . In Kenya , a recent surveillance study of rickettsiae in ticks identified 104 rickettsiae , of which 93% were R . africae , clearly , demonstrating its dominance [26] . R . africae is vectored by Amblyomma ticks , primarily A . variegatum and A . hebraeum . Infections have also been detected in many other ticks species [3 , 26–28] by PCR and not by competency studies . In general , Rickettsia species are very closely related . Within the SFG where R . africae belongs , the mean nucleotide homogeneity with rrs , gltA , ompA , ompB and sca4 genes ranges from 82 . 2% to 99 . 8% [29] . Considering this high interspecies homogeneity , intraspecies differences are even smaller . For example , using the more variable multi spacer typing that combined dksA-xerC , mppA-purC , and rpmE-tRNAfMet spacer sequences , it was impossible to discriminate R . africae strains [30] . Using ompA and ompB genes , many groups have however reported heterogeneity [25 , 31 , 32] . A recent study reported an even higher heterogeneity of R . africae samples collected from a localized area in Western Kenya [33] . The study reported here sought to determine how widespread the heterogeneity of Kenya’s R . africae is by examining DNA samples collected from different eco-regions of Kenya .
This study was carried out using ticks collected from domestic animals presented for slaughter . The tick samples were collected under protocol SSC#1248 that was reviewed and approved by the Animal Use Committee of the Kenya Medical Research Institute . Details of the areas the tick were collected from , method of collection , DNA extraction and preliminary genotyping have been published before [26] and are summarized in S1 Table . Sequence data for 57 tick-extracted DNA samples that had been identified as R . africae by comparison to 385 bp citrate synthase ( gltA ) gene , 530 bp outer membrane protein A gene ( ompA ) and 444 bp outer membrane protein B gene ( ompB ) were obtained from our laboratory's database from a previous study [26] . The sequences of both strands were re-checked for correctness and errors cleaned . Samples with short or missing sequences were re-amplified and re-sequenced . Two additional genes: the 450 bp 17kDa and 2700 bp sca4 genes were also amplified and sequenced . Primers used to amplify target genes are listed in Table 1 and are previously described [34] . PCR reagents were obtained from Applied Biosystems ( CA , USA ) and reactions performed in a 25 μL reaction volume containing 10 μM of each primer , 200 μM of dNTP mix , 1 . 5U Taq polymerase and 2 mM MgCl2 . Amplification was carried out in a DNA thermal cycler ( HID Veriti ) from Applied Biosystems ( CA , USA ) . The following conditions were used for amplification: For ompA and gltA genes: 3 min of initial denaturation at 94°C , then 40 cycles at 94°C for 30 sec , 53°C for 30 sec , 68°C for 1 min . For the ompB gene: 3 min of initial denaturation at 95°C , then 40 cycles at 95°C for 30 sec , 50°C for 30 sec , 68°C for 1 min 30 sec . For the 17kDa gene: 5 min of initial denaturation at 94°C , then 35 cycles at 94°C for 30 sec , 50°C for 1 min , 68°C for 1 min . For sca4: 5 min of initial denaturation at 95°C , then 40 cycles at 95°C for 45 sec , 60°C for 30 sec and 68°C for 3 min . All the amplifications were then completed by holding for 7 min at 72°C . To ascertain correct product sizes , a portion of the amplicons ( 5μL ) was run on a 1% ( w/v ) agarose gel containing ethidium bromide . Product sizes were estimated by comparing with a molecular mass standard ( 1kb plus ladder , Invitrogen , ( CA , USA ) . The PCR products were purified using Isolate II PCR and Gel Kit ( Bioline , UK ) as recommended by the manufacturer . The purified PCR products were sequenced in both directions using the Big Dye Terminator Cycle Sequencing Kit v 3 . 1 ( Applied Biosystems , CA , USA ) and the sequences analyzed by capillary electrophoresis in a 3130 Genetic Analyzer ( Applied Biosystems ) . The sequences were proofread , edited and assembled into consensus sequences using CLC Main Workbench v 7 ( CLC Inc , Aarhus , Denmark ) , and used to query GenBank using the nucleotide Basic Local Alignment Search Tool ( BLAST ) [35] . Six different alignments were generated: Five of them corresponded to sequences of each target gene ( gltA , ompA , ompB , 17kDa and sca4 ) , and one corresponding to the concatenated sequence of all the five genes as well as the validated rickettsia strains derived from GenBank ( see S2 Table for names of reference strains and their accession numbers ) . To ensure the accuracy of these alignments , nucleotide sequences were translated to their respective amino acids using the translate tools in the CLC Main Workbench v7 . Amino acid alignments were made using Muscle v 3 . 8 software [36] . The protein alignments were then used to guide the corresponding nucleotide alignments using TOPALi V2 software [37] . For phylogenetic inference , MEGA v7 software [38] was used to estimate the best substitution model as well as estimate for the Maximum Likelihood ( ML ) trees for the individual genes . The concatenated alignment tree was also estimated with the ML method using a General Time Reversal ( GTR ) nucleotide substitution model with a gamma distribution ( GTR+G ) . For bayesian probability analysis , jModeltest v 2 . 1 [39] was used to determine the GTR+G as the best fit model for gltA , ompA , ompB , 17kDa and sca4 gene alignments . A partitioned analysis was then performed on the concatenated dataset using a bayesian Markov Chain Monte Carlo ( MCMC ) method implemented with MrBayes software v 3 . 2 [40] . The cluster confidence was given as posterior probabilities .
In total , 57 tick DNA samples were available for analysis . From these and as shown in supplementary information ( S3 Table ) , 45 sequences were obtained for gltA ( Genbank accession KX368721-KX368765 ) , 57 for ompA ( Accession: KX368868-KX368924 ) , 44 for ompB ( Accession: KX368823- KX368867 ) , 57 for 17kDa ( Accession: KX368766-KX368822 ) and 40 for sca4 ( Accession: KX368925-KX368964 ) . The gene sequences were subjected to BLAST analysis for a preliminary verification of their identity . BLAST results are shown in S1 Table . The topology of the gltA gene tree inferred with the ML method is shown in Fig 1 , panel A . The tree shows unresolved evolutionary relationships ( nodes with <50% bootstrap support values ) . Overlooking these clade credibility values , a major cluster ( clade I ) consisting of 89% of the sequences was observed in the more derived parts of the tree . The other two distinct clades were samples from the North Eastern part of Kenya . Clade II consisted of samples ( 044 and 045 from Wajir ) that diverged as sister operational taxonomic units ( OTUs ) . Clade III samples clustered at the basal part of the tree and came from Moyale ( 139 , 135 , 136 and 176 ) and Wajir ( 577 ) . In comparison to gltA , the ompA tree resolved majority of the nodes into three well supported groups ( Fig 1 , panel B ) . As in gltA , clade I had the majority of OTUs ( 52/57 , 91% ) , and was the most derived . Majority of the nodes were unresolved , and the members clustered in a polytomy . As in gltA , clade II formed a dichotomy made of samples 044 and 045 from Wajir . Clade III constituted a cluster of 176_Moyale , 164_Wajir and 195_Machakos in the basal parts of the tree . In this clade , only 176_Moyale was shared with the gltA gene tree . As shown in Fig 1 , panel C , ompB also resolved the study OTUs into three groups . Clade I contained 40/44 ( 91% ) members in a polytomy . Clade II contained the same sister OTUs ( 044 and 045 ) from Wajir . Clade III had the same samples as in the ompA gene tree . As with gltA , ompA and ompB genes , the topology of the 17kDa gene tree was consistent in delineating three clades ( Fig 1 , panel D ) . Clade I contained 52/57 ( 91% ) of unresolved OTUs . Clade II consisted of sister OTUs ( 044 and 045 ) from Wajir , while clade III consisted of 164_Wajir , 195_Machakos and 176_Moyale Compared to gltA , ompA , ompB and 17kDa , the sca4 gene tree was better resolved especially in delineation of clade I ( Fig 1 , panel E ) which consisted of 35/40 ( 86% ) OTUs . Clade II consisted of sister OTUs ( 044 and 045 ) from Wajir . Clade III consisted of 176_Moyale , 164_Wajir , and an additional member 293_Migori . In order to improve the phylogenetic resolution of individual genes , the five genes were concatenated . Out of the 57 samples initially available for analysis , only 39 were included in the concatenation . The choice of the 39 was influenced by availability of the limiting gene ( 39 sca4 samples ) , and not missing more than one of the other four gene sequences . The concatenated sequences also included validated Rickettsia species available in GenBank . The concatenated tree constructed with Bayesian method is shown in Fig 2 . The validated Rickettsia sequences delineated into the three known Rickettsia clades: TG , TRG and SFG . All the 39 study OTUs lay within the SFG clade , of which 33/39 ( 84% ) clustered with the validated R . africae str ESF-5 shown as clade I . The two sister OTUs ( 044 and 045 ) from Wajir formed a distinct clade ( clade II ) that shared the most recent common ancestor with clade I . Two other samples ( 164_Wajir and 195_Machakos ) previously identified as R . africae [26] delineated with the validated R . aeschlimanii . The positions of these OTUs held when tested by ML method ( S1 Fig ) . The position of 293_Migori was however tenuous , as it branched with R . aeschlimanii on ML method , but as a lone taxon between R . heilongjiangensis and R . slovaca on Bayesian analysis . Another interesting sample 176_Moyale branched as a lone taxon with total statistical support ( a posterior probability value of 1 ) , thus raising questions concerning its taxonomic status . We consider this sample as a novel rickettsiae and an interim name of “Candidatus Rickettsia Moyalensis” is proposed . The ompA gene sequences obtained from our study samples and those published previously collected in Kenya [23 , 31] were used to generate a phylogenetic tree ( Fig 3 ) . With this tree , three clades were discernible . Clade I contained majority of the OTUs 61/66 ( 92 . 4% ) and a cluster of other 7 that formed a subclade within clade I , that consisted of OTUs published in previous studies . Clade II consisted of sister OTUs ( 044 and 045 ) from Wajir , and a basal clade III that consisted of members 176_Moyale , 164_Wajir , and 195_Machakos . Clearly , five of our sequences ( 044 , 045 and 164 from Wajir , 176 Moyale and 195 Machakos ) are distinct from those described previously .
This study provides new information regarding the phylogenetic relationships of the R . africae lineage . This lineage , though only recently emerged [30] is clearly undergoing diversification , as observed in the branching order of the samples studied . A definite sub-lineage of R . africae ( samples 044 and 045 from Wajir ) was identified . It was impossible to confirm the placement of sample 293_Migori . Additional sequence data will be required to resolve the ambiguity . Lastly , a putative novel rickettsiae ( sample 176_Moyale ) with a proposed name of “Candidatus Rickettsia moyalensis” was identified . Further work will be needed to determine its prevalence in Kenya and its implications to human and/or animal disease . | Rickettsia africae is a bacterium mainly vectored by Amblyomma and Rhipicephalus species of ticks . It is the etiological agent of African tick bite fever ( ATBF ) , a spotted fever rickettsiosis that presents as an acute febrile illness characterized by petecheal skin hemorrhages , from which the name is derived . This bacterium is probably the most important in sub-Saharan Africa , including Kenya , in terms of incidence and prevalence . This notwithstanding , the disease is poorly understood and is often mistreated as malaria , and therefore qualifies as a highly neglected disease . This study examined the genetic relationships of R . africae collected from diverse eco-regions of Kenya . We present data that indicate high genetic diversity in Kenya’s R . africae and corroborate a recent study that reported similar genetic diversity in R . africae samples collected from a localized area in western Kenya . Importantly , we describe a divergent lineage and propose the name Candidatus Rickettsia moyalensis . | [
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"symptoms... | 2016 | Phylogenetic Variants of Rickettsia africae, and Incidental Identification of "Candidatus Rickettsia Moyalensis" in Kenya |
Although the causes of Parkinson's disease ( PD ) are thought to be primarily environmental , recent studies suggest that a number of genes influence susceptibility . Using targeted case recruitment and online survey instruments , we conducted the largest case-control genome-wide association study ( GWAS ) of PD based on a single collection of individuals to date ( 3 , 426 cases and 29 , 624 controls ) . We discovered two novel , genome-wide significant associations with PD–rs6812193 near SCARB2 ( , ) and rs11868035 near SREBF1/RAI1 ( , ) —both replicated in an independent cohort . We also replicated 20 previously discovered genetic associations ( including LRRK2 , GBA , SNCA , MAPT , GAK , and the HLA region ) , providing support for our novel study design . Relying on a recently proposed method based on genome-wide sharing estimates between distantly related individuals , we estimated the heritability of PD to be at least 0 . 27 . Finally , using sparse regression techniques , we constructed predictive models that account for 6%–7% of the total variance in liability and that suggest the presence of true associations just beyond genome-wide significance , as confirmed through both internal and external cross-validation . These results indicate a substantial , but by no means total , contribution of genetics underlying susceptibility to both early-onset and late-onset PD , suggesting that , despite the novel associations discovered here and elsewhere , the majority of the genetic component for Parkinson's disease remains to be discovered .
To date , a number of different genetic susceptibility factors have been identified for Parkinson's disease . Autosomal dominant factors involved in PD include mutations in the SNCA ( -synuclein ) [1] , [2] and LRRK2 ( leucine-rich repeat kinase 2 ) [3] , [4] genes . Autosomal recessive factors include mutations in the PARK2 ( parkin ) [5] , PINK1 ( PTEN induced putative kinase 1 ) [6] , PARK7 ( DJ1 ) [7] , and ATP13A2 ( ATPase type 13A2 ) genes [8] , [9] . Parkinson's disease is sometimes thought of as consisting of an early-onset form , characterized by familial clustering and high penetrance mutations , and a late-onset form , which occurs more sporadically . In contrast with the evidence for Mendelian inheritance of early-onset PD , studies comparing rates of concordance in monozygotic and dizygotic twins [10]–[12] have traditionally yielded low estimates of heritability for late-onset PD . Evidence of familial aggregation in late-onset PD , when present , is thus sometimes attributed to shared environmental effects or ascertainment bias ( see [13] , but cf . , [14] ) . Nonetheless , genome-wide association studies have uncovered a few new genes involved in late-onset PD in European [15]–[19] and Japanese [20] populations . These studies have shown repeatedly that common variation in SNCA and an inversion of the region containing the MAPT ( microtubule-associated protein tau ) gene are associated with PD . In addition , NUCKS1 ( nuclear casein kinase and cyclin-dependent kinase substrate 1 ) [16] , [20] , the HLA ( human leukocyte antigen ) region [18] , GAK ( cyclin G associated kinase ) [15] , [19] , BST1 ( bone marrow stromal cell antigen 1 ) [20] , [21] , and five additional loci [22] have recently been associated with PD . Here , we present a genome-wide association study of PD with a number of distinguishing features . The recruitment of PD cases took place primarily through targeted emails to PD foundations and support groups over the course of 18 months . The large set of controls was recruited through the 23andMe customer database . Determination of case status was conducted through a set of online questionnaires . We present three main scientific results . First , we identify two novel loci associated with PD and replicate many more . Second , we present lower-bound estimates of PD heritability based on observed genome-wide sharing that imply a large fraction of the genetic component underlying the etiology of PD has not yet been discovered . Third , we use sparse regression techniques to construct risk estimation algorithms that account for roughly 6–7% of the total variance in liability to PD and that suggest the existence of true associations lying just beyond genome-wide significance .
We performed a GWAS using the 23andMe dataset consisting of 3 , 426 cases and 29 , 624 controls , controlling for age , sex , genotyping platform , and five principal components . All 23andMe samples were genotyped using a custom Illumina HumanHap 550+ panel , with 522 , 782 markers passing quality control ( see Materials and Methods ) . Manhattan and q-q plots can be found in Figures S1 and S2 . All SNPs with -values under in the 23andMe cohort are shown in Table 2 . Summary data for the SNPs in Table 2 can be found in Table S1 . All SNPs with -values under can be found in Table S2 . Using a cutoff of for significance based on a Bonferroni correction across all markers , we identified two novel regions–SCARB2 and SREBF1/RAI1–and replicated six previously reported regions–LRRK2 , SNCA , GBA , MAPT , MCCC1/LAMP3 , and GAK . A seventh replication ( SLC41A1/PARK16 ) and two other potentially novel regions ( RIT2/SYT4 and USP25 ) appear nearly genome-wide significant as well . The first novel association is rs6812193 , with an odds ratio ( OR ) of and -value of , in an intron of FAM47E ( see Figure 1 ) , upstream of SCARB2 . This SNP replicates in the IPDGC cohort with an OR of and -value of . The second novel association is rs11868035 , with an OR of and -value of , located in an intron of SREBF1 ( see Figure 2 ) . This association also replicates in the IPDGC cohort with an OR of and -value of 0 . 03 . Of potential interest is a non-synonymous variant ( proline to threonine change ) , with a -value of , in RAI1 , rs11649804 , in tight linkage disequilibrium ( LD ) with rs11868035 ( ) . Among replications of previously reported associations , rs34637584 is the non-synonymous G2019S mutation in the LRRK2 gene , well known to be associated with PD [24] . GBA N370S is one of the mutations causing Gaucher's disease and has recently been associated with PD [25] , [26] . These two SNPs , both rare variants , were included as part of the custom set of variants used to genotype the 23andMe cohort . The associations with SNCA , MAPT , GAK , and SLC41A1 have been reported multiple times . Here we provide the first independent confirmation of the association of rs10513789 in the MCCC1/LAMP3 region with PD , as first reported in [22] . Of the three suggestive associations that do not reach genome-wide significance , rs823156 near SLC41A1/PARK16 has been previously reported [20] . The association with rs4130047 ( in an intron of RIT2 , with an OR of 1 . 16 and -value of ) is not quite significant , though it independently appears in the IPDGC cohort with a replication -value of , and also is included in the supplement of [15] as a suggestive association under a recessive model . The last suggestive SNP , rs2823357 , lies 170 kb upstream of USP25 , a ubiquitin-specific protease . This association , however , fails to replicate in the IPDGC cohort , with a -value of . See Figure 5 and Figure 6 in [15] for plots of the RIT2/SYT4 and USP25 regions . On the basis of candidate gene studies or modest significance levels in previous GWASs , researchers have proposed associations for many genes with PD to date . We used the set of “Top Results” from the meta-analysis at http://www . pdgene . org/ [27] as well as all SNPs appearing in a PD GWAS with -values under from [28] . After removing SNPs for which we did not have a good proxy , and omitting highly correlated or duplicate SNPs , we were left with 42 potential replications . In addition to LRRK2 G2019S , 19 other previously reported associations replicated with the correct directionality in the 23andMe cohort using a significance threshold of 0 . 05 ( see Table 3 ) . Of these , 17 of our confidence intervals include the published OR . Of the two that did not , one was using a proxy SNP with a rather weak of 0 . 16 , so it is not surprising that our OR is weaker in this case . We had good power ( over 86% in all but one case ) to replicate all 42 of these associations , assuming the reported odds ratio ( from the meta-analysis in the case of associations from [27] , from the original paper otherwise ) was correct . Thus the failure to replicate many of the reported associations ( including two with “A” or “B” meta-analysis grades ) is likely partially due to inflation of the odds ratios in those reports , although a few of the associations that were discovered in Asian populations , such as rs1994090 , may simply not have the same effect in European populations . Finally , we note that in all analyses above , we restricted consideration to a log-additive model of association . Allowance for dominant and recessive models might lead to increased power for detection in cases where a log-additive model is not appropriate , and may also affect the success rates in our replication experiment . To characterize the extent to which genetic factors tagged on genotyping panels play a role in PD susceptibility , we applied a recently proposed approach for estimating heritability based on genome-wide sharing between distantly related individuals [29] , [30] . Using the 23andMe cohort , we estimated the heritability of PD to be 0 . 272 with 95% CI of 0 . 229 to 0 . 315 . This estimate refers only to the proportion of phenotypic variance arising from causal variants that are in LD with the SNPs on our genotyping platform , which may be less than the corresponding proportion for all causal variants . This estimate has only a mild dependence on the assumed prevalence: using prevalences of or gives estimates of or , respectively . Thus , our estimates generally suggest a lower-bound on the actual heritability in the 0 . 25 to 0 . 3 range . Table 4 compares our heritability estimates using the 23andMe cohort with an estimate computed for the NINDS cohort based on the same analytic methods , and with numbers obtained from the literature . Where only relative recurrence risk ratios were provided , we inferred the corresponding heritability of liability under an assumption of no shared environmental covariance [31]; in practice , such an assumption is unlikely to hold for close relatives , and as such , the estimates of heritability we have inferred from those studies are likely to be upwardly biased . We note that our estimates of heritability for PD are most consistent with estimates from prior twin studies , though with substantially tighter confidence intervals even after accounting for the uncertainty in prevalence . Given our estimates of the genetic contribution to PD , we then sought to determine the proportion of this contribution that we could attribute to specific genetic factors on our genotyping panel by constructing risk prediction models for PD . We considered two settings: an internal five-fold cross-validation experiment , where we divided the 23andMe cohort into five matched sets of cases and controls and computed predictions for each set using models trained using the other four sets; and an external cross-validation experiment , where we trained risk prediction models using the entire 23andMe cohort and tested them on the NINDS cohort after restricting both datasets to the set of common SNPs passing quality control for each dataset individually . In both the internal and external validation experiments , we measured the discriminative accuracy of the risk prediction algorithm using the area under the receiver operating characteristic curve ( AUC ) . The AUC for a model can be interpreted as the probability that a randomly selected case will have a higher estimated risk of developing PD than a randomly selected control . An AUC of 1 implies that a model discriminates perfectly between cases and controls , whereas an AUC of 0 . 5 corresponds to a model based on random guessing . To measure predictive accuracy , we used a covariate-adjusted measure of AUC that removed the effect of potential confounding by sex , age , population structure , and , where appropriate , cross-validation fold or genotyping platform ( see Materials and Methods ) . To avoid making manual decisions in the choice of SNPs to include , we used a sparse logistic regression algorithm for building risk prediction models , and varied the strength of the sparsity-inducing prior ( see Materials and Methods ) . For a given training set , this procedure generated a series of risk prediction models of differing size , each of which we characterized using an approximate theoretical upper bound on the expected number of false positive SNPs , ; here , corresponds to a model containing only genome-wide significant associations . In the internal five-fold cross-validation experiment ( see Table 5 ) , the differences in AUCs between each of the largest three models ( i . e . , , , or ) and each of the smallest two models ( i . e . , or ) were significant ( e . g . , comparing the largest and smallest models , one-sided ; see Table S7 ) . In the external cross-validation results , the four largest models were significantly better than the genome-wide significant model ( e . g . , comparing the largest and smallest models , one-sided ) . Taken at face value , these results seem to suggest that the larger models , which include many more genomic regions than those deemed genome-wide significant , may harbor associations that account for their increased predictive accuracy . An alternative possibility , however , is that the differences in performance between models are actually just a consequence of differing levels of bias arising from the use of sparsity-inducing regularization . In Text S1 and Table S4 , we present an argument using a bias-corrected version of our external cross-validation experiment that the above caveats do not explain the improved accuracy for the and models , thus suggesting that these models are likely to harbor additional important loci for PD; see Tables S5 and S6 for the SNPs included in these models . We note that this argument may not be the only way of demonstrating the existence of meaningful associations beyond the genome-wide significance threshold; closely related arguments based on the sparse regression methods [32] or genetic profile scores have also been previously proposed [33] . Regardless , our heritability estimates imply an upper bound on AUC for a genetic risk prediction model of roughly 0 . 83 to 0 . 88 based on the method of [31] , though this would rise if the actual heritability were higher . Based on these numbers , the genetic risk prediction models detailed previously , which attain an AUC of roughly 0 . 6 in our cross-validated tests , account for approximately – of the total genetic variance in liability ( see Materials and Methods ) .
We found two novel associations at a genome-wide level of significance near SCARB2 ( rs6812193 ) and SREBF1/RAI1 ( rs11868035 ) , both of which were replicated in data from [23] . We also report two novel associations ( near RIT2 and USP25 ) just under the level of significance , one of which ( RIT2 ) was also replicated . While it is difficult to pinpoint any causal genes from a GWAS , there are a few biologically plausible candidates worthy of discussion . The PD-associated SNP rs6812193 lies in an intron of the FAM47E gene , which gives rise to multiple alternatively spliced transcripts , many of which are protein-coding; the functions of these hypothetical proteins are unknown . A more attractive candidate , located kb centromeric to the SNP , is SCARB2 ( scavenger receptor class B , member 2 ) , which encodes the lysosomal integral membrane protein type 2 ( LIMP-2 ) . LIMP-2 deficiency causes the autosomal-recessive disorder Action Myoclonus-Renal Failure syndrome ( AMRF ) , which combines renal glomerulosclerosis with progressive myoclonus epilepsy associated with storage material in the brain [34] . LIMP-2 is involved in directing -glucocerebrosidase to the lysosome where it hydrolyzes the -glycosyl linkage of glucosylceramide [35] . Deficiency of this enzyme due to mutations in its gene ( GBA ) causes the most common lysosomal storage disorder , Gaucher's disease . Recently , mutations in GBA have also been identified in PD [36] , pointing to a possible functional link between the newly identified candidate gene SCARB2 and PD . rs11868035 appears in an intron of the alternatively spliced gene , SREBF1 ( sterol regulatory element-binding transcription factor 1 ) , within the Smith-Magenis syndrome ( SMS ) deletion region on 17p11 . 2 . SREBF1 encodes SREBP-1 ( sterol regulatory element-binding protein 1 ) , a transcriptional activator required for lipid homeostasis , which regulates cholesterol synthesis and its cellular uptake from plasma LDL [37] . Studies of neuronal cell cultures have implicated SREBP-1 as a mediator of NMDA-induced excitotoxicity [38] . rs11868035 is directly adjacent to the acceptor splice site for the C-terminal exon of the SREBP-1c isoform of the protein [39] , suggesting that the effect of the polymorphism may be specifically related to the splicing machinery for this protein . The mutation is also in strong LD with rs11649804 , a nonsynonymous variant in the nearby gene RAI1 ( retinoic acid-induced protein 1 ) , which regulates transcription by remodeling chromatin and interacting with the basic transcriptional machinery . Heterozygous mutations in RAI1 reproduce the major symptoms of SMS , such as developmental and growth delay , self-injurious behaviors , sleep disturbance , and distinct craniofacial and skeletal anomalies [40] . Future work is needed to identify the functionally important variant ( s ) responsible for this association . The SNP rs4130047 , slightly below the genome-wide significance threshold , lies in an intron of the RIT2 ( Ras-like without CAAX 2 ) gene that encodes Rit2 , a member of the Ras superfamily of small GTPases . Though we do not claim this SNP as a confirmed replication , there are a number of reasons to suspect that this association may also be real . Rit2 binds calmodulin in a calcium-dependent manner , and is thought to regulate signaling pathways and cellular processes distinct from those controlled by Ras [41] . It localizes to both the nucleus and the cytoplasm . Independent of our study , RIT2 was previously proposed as a candidate gene for PD , based on the possibility that dopaminergic neurons may be especially vulnerable to high intracellular calcium levels , perhaps through an interaction with -synuclein [42] . The PD-associated region contains another biologically plausible candidate gene , SYT4 ( synaptotagmin IV ) , which encodes synaptotagmin-4 , an integral membrane protein of synaptic vesicles thought to serve as sensor in the process of vesicular trafficking and exocytosis . It is expressed widely in the brain but not in extraneural tissues [43] . Homozygous Syt4−/− mouse mutants have impaired motor coordination [44] . SYT4 is particularly interesting as a SNP near SYT11 ( synaptotagmin XI ) has been associated with PD in [22] , and the encoded protein , synaptotagmin-11 , is known to interact with parkin [45] . The suggestively associated SNP rs28233572 lies in a gene-poor region with only one candidate gene downstream , USP25 , encoding ubiquitin specific peptidase 25 , which regulates intracellular protein breakdown by disassembly of the polyubiquitin chains . Other ubiquitin-specific proteases ( USP24 , USP40 ) have been proposed as candidate genes for PD [46] ( although USP24 fails to replicate here , see Table 3 ) . Our heritability estimates , which suggest that genetic factors account for at least one-fourth of the total variation in liability to PD , represent the tightest confidence bounds determined for the heritability of PD to date . These estimates , which rely on observed genetic sharing rather than predicted relationship coefficients , avoid confounding from shared environmental covariance by restricting attention to very distantly related individuals . Furthermore , they complement estimates of heritability from twin studies by considering large numbers of individuals with low amounts of genetic sharing , rather than small numbers of twin pairs with large amounts of genetic sharing . These estimates should only be interpreted as lower bounds on the actual heritability of liability of PD for two reasons . First , they only reflect phenotypic variation due to causal variants in LD with SNPs on the genotyping platform . Second , they only capture the contribution to additive variance that arises from a polygenic model of many SNPs of small effect , but do not include the variance arising from known specific associations . This limitation is most apparent in our estimate of heritability based on only early-onset cases ( ) , which is considerably lower than reported in prior twin studies ( e . g . , in [10] ) . In early-onset PD , mutations in six specific genes ( SNCA , PRKN , PINK1 , DJ1 , LRRK2 , and GBA ) have been reported to account for 16% of cases [47]; these specific mutations are not directly accounted for in our estimate , which is based on a polygenic model . We note that a similar effect may explain the low heritability estimate for early-onset PD in [48] . Thus , the actual heritability of PD , and the corresponding true upper bound on discriminative accuracy achievable through genetic factors , may be even higher than the estimates we provide . Our estimates also indicate a substantial genetic component for late-onset PD ( ) , for which previous estimates of heritability have been inconclusive due to the lack of statistical power ( e . g . , 0 . 068 in [10] and 0 . 453 in [48] ) . One might ask , if late-onset PD is indeed so heritable , why do cases frequently appear sporadically in the general population ? Following the analysis of [49] , if one were to assume a heritability of and an average of three children per family , then the proportion of sporadic cases ( i . e . , no parent , child , sibling , grandparent , aunt or uncle , or first cousin with PD ) among all PD cases would be 64% for a prevalence of ; in the 23andMe cohort , 69% of PD cases would be considered sporadic by this definition based on self-reported family history . Similarly , the expected proportion of PD cases with no affected parent or sibling would be 88% under the same assumptions , compared with 84% as reported in [50] , or 89% based on the cohort in [51] . These examples illustrate the fact that the presence or absence of a familial pattern cannot always be used to determine pathogenesis , especially for diseases that are rare and have a complex etiology . Overall , our risk prediction results are consistent with a measured AUC of roughly 0 . 6 . The cross-validated AUCs presented here should be distinguished from more usual measurements of AUC in genome-wide association studies , which are typically only estimated on the development set , and which rely on weighted combinations of SNPs with independently estimated odds ratios . In some cases , the bias resulting from lack of proper external validation can be quite large . For example , a simple genetic profile score based on multiplying together odds ratios for the SNPs in Table 2 appears to achieve an AUC of in the 23andMe data ( or if no covariate adjustment is performed ) making it appear competitive with some of the best models described in Table 5 . However , when the same model is evaluated in the NINDS data , the AUC drops to , exhibiting a drop in performance characteristic of models that have been overfit to their training data . In contrast , the consistency between the internal and external validation results in the models shown in Table 5 demonstrate not only the predictiveness of our models within the 23andMe cohort but also their ability to generalize to other populations . Our empirical demonstration that including SNPs beyond the genome-wide significant level provides improved discriminative power mirrors the recent results of [32] , which also studied the performance of sparse regression methods in a risk prediction setting . In an applied setting where the goal is to achieve the best predictive accuracy rather than to isolate the contribution of individual genetic factors , however , even higher discriminative accuracies may be possible if one were to incorporate these covariates as part of the predictive models . Even without these , however , significant improvements in risk prediction are likely still possible , with our heritability analyses indicating asymptotic target AUCs above 0 . 8 . Our AUCs are generally conservative for a number of reasons . In the internal experiments , they were obtained by training on only 80% of the data . In the external experiments , the models included only the SNPs in common between the 23andMe and NINDS datasets and thus excluded several SNPs with large effects in LRRK2 and GBA that may add a percent or more to the AUC if included . Furthermore , our analyses adjusted for confounding from population structure and other covariates so as to ensure that the discriminative accuracies we reported were specifically due to genetic effects . Finally , we note that data for the 23andMe cohort used in this study were acquired in a novel manner , using genotype and survey data acquired through a commercial online personal genetic testing service . The use of self-reported phenotype data raised some unique challenges . For example , our cohort was not a true population sample for a number of reasons , such as the general bias toward higher socioeconomic status , as typical of 23andMe customers . In general , however , we would not expect these ascertainment biases to substantially affect our conclusions unless their effects varied differentially between the case and control sets . As another example , in compiling the cohort , we used participants with varying levels of completeness in their self-reported data ( see Materials and Methods ) . Out of the 3 , 426 cases in the 23andMe cohort , though most cases reported having PD in a questionnaire , 482 affirmatively stated they had PD upon entry to the research study but did not fill out any PD-related questionnaire during the study . However , we did not see a large difference between those answering questions and not . Among the 11 associations presented in Table 2 , only the association with MAPT showed a significant difference between the cohort who answered a questionnaire and those who did not ( see Table S7 ) . Also , approximately 84% of the cases filled out a questionnaire , and of them , over 96% reported a PD diagnosis . Even if a larger fraction ( say 10–15% ) of those who did not take a questionnaire did not have PD , the gain in power from the additional cases would more than offset the loss of power from having some 50 more false positive cases . Despite the challenges associated with using self-reported data collected through online surveys , ultimately , our results lend credibility to the accuracy of this novel research design . For example , the agreement between our study and previous studies in terms of the ORs estimated for the 19 associations replicated in Table 3 strongly suggests that our cohort is similar to those used in other PD studies . Similarly , the consistency of AUCs and heritability estimates across our cohort and the NINDS cohort both suggest a limited role of bias in our study . Importantly , our mode of data collection also provided a number of clear benefits . The use of internet-based techniques enabled rapid recruitment of a large patient community . The 3 , 426 cases in this study were enrolled in about 18 months , with over half joining in the first month of the study . Also adding significantly to the power and robustness of this study was the availability of a large cohort of controls derived from the 23andMe customer base . By using a non-traditional recruitment approach , we thus were able to attain good power for our study through large sample sizes . To our knowledge , this study represents the largest genome-wide association study of Parkinson's disease conducted on a single cohort to date , with only a recent meta-analysis achieving a larger number of cases [22] . We suggest that this methodology for study design may prove advantageous for other conditions where the advantage of having a large cohort is paramount for detecting subtle genetic effects . In summary , we have for the first time used a rapid , web-based enrollment method to assemble a large population for a genome-wide association study of PD . We have replicated results from numerous previous studies , providing support for the utility of our study design . We have also identified two new associations , both in genes related to pathways that have been previously implicated in the pathogenesis of PD . Using cross-validation , we have provided evidence that many suggestive associations in our data may also play an important role . Using recently developed analytic approaches developed for GWAS that take into account the ascertainment bias inherent in a case-control population , we have estimated the genetic contribution to PD in this sample . These findings confirm the hypothesis that PD is a complex disorder , with both genetic and environmental determinants . Future investigations , expanded to include environmental as well as genetic factors , will likely further refine our understanding of the pathogensis of PD , and , ultimately , lead to new approaches to treatment .
The 23andMe cohort consisted of customers of 23andMe , Inc . , a personal genetics company . Patients with PD were recruited to join this cohort through a targeted email campaign in conjunction with the Michael J . Fox Foundation , The Parkinson's Institute and Clinical Center , and many other PD patient groups and clinics . Emails or hard copy mailings were sent to all individuals who had registered with these groups as PD patients . A limited number of patients were also recruited in person at PD workshops and conferences . Family members of individuals with the LRRK2 G2019S were also recruited to participate in the general Parkinson's disease research at 23andMe , without regard to Parkinson's disease status; however , most of these individuals were not included in the cohort used for this particular study , due to our restriction of the dataset to unrelated individuals ( see below ) . Patients were invited to fill out a screening questionnaire asking if they had been diagnosed with PD and their physician's name , phone number , and institution . Patients who stated they had been diagnosed with PD and who gave complete , non-suspicious answers to the other questions were offered the 23andMe Personal Genome Service for a nominal fee of $25 . Individuals included in the 23andMe cohort were selected for being of primarily European ancestry , as determined through an analysis of local ancestry via comparison to the three HapMap 2 populations , using an unpublished method substantially similar to [52] . A maximal set of unrelated individuals in the 23andMe cohort was chosen for the analysis using a segmental identity-by-descent ( IBD ) estimation algorithm ( as used in [53] ) . Individuals were defined as related if they shared more than 700 cM IBD , including both regions where the two individuals share either one or both genomic segments identical-by-descent . This level of relatedness ( roughly 20% of the genome ) corresponds approximately to the minimal expected sharing between first-cousins in an outbred population . We determined that 29 individuals were included in both the 23andMe and NINDS cohorts and hence were removed from the latter cohort for all analyses . Genotype and phenotype data for the National Institute of Neurological Disease and Stroke ( NINDS ) cohort were obtained from the NINDS Database found at http://www . ncbi . nlm . nih . gov/gap through dbGaP accession number phs000089 . v3 . p2 , supplemented with individual-level data for 200 subjects from phs000089 . v2 . p2 who were left out of the later version as of December 12 , 2010 . Cases in the NINDS cohort consisted of North American Caucasians with Parkinson's disease , as assessed by a neurologist . Controls consisted of neurologically normal , unrelated , white individuals with no family history for a number of neurological conditions , including Parkinson's disease . A complete description of the inclusion and exclusion criteria can be found directly at the NINDS Database website as referenced above , and in related studies using the data from this cohort [16] , [54] , [55] . This study was conducted according to the principles expressed in the Declaration of Helsinki . The 23andMe study protocol and consent were approved by the external AAHRPP-accredited IRB , Ethical and Independent Review Services ( E&I Review ) . Our consent and privacy statement preclude the sharing of individual-level data without explicit consent . We have , however , shared summary statistics for all SNPs with -values under ( Table S2 ) . We also hope to further collaborate with the scientific community using this data . The NINDS dataset was analyzed anonymously . Patients recruited through the PD outreach initiative as well as individuals from the general 23andMe customer base were asked to take online questionnaires , including a general medical questionnaire and a detailed questionnaire specifically on PD ( covering disease onset , diagnosis , and symptoms ) . Both of these questionnaires asked the subject if he or she had ever received a PD diagnosis from a physician and if so , the age of onset . The detailed questionnaire also asked for much more specific information regarding the symptoms , clinical history , and family history of the patient . We selected as cases all participants who provided an affirmative diagnosis of PD from a physician ( on the initial screening form or on either of the two questionnaires ) and who did not provide any potentially contradictory information , defined here as: Due to the low prevalence of PD , we used controls taken from general 23andMe customer base in the analysis . Some of the controls filled out no questionnaires; however , others answered questions about possible PD-like symptoms or filled out a general medical history . In order to maximize our power to detect genetic associations with PD , we excluded some putative controls who might be at higher probability for developing PD in the future . Thus , the controls consisted of all consented European 23andMe customers who met all of the following criteria: Approximately 1 , 430 individuals in total were excluded from the control set due to these filters . We note that as a consequence of both our exclusion criteria and other recruitment biases associated with the 23andMe customer base , our controls are unlikely to be exactly representative of the general population . For the 23andMe cohort , DNA extraction and genotyping were performed on saliva samples by National Genetics Institute ( NGI ) , a CLIA-certified clinical laboratory and subsidiary of Laboratory Corporation of America . Samples were genotyped on the Illumina HumanHap550+ BeadChip platform , which included SNPs from the standard HumanHap550 panel augmented with a custom set of approximately 25 , 000 SNPs selected by 23andMe . Every sample that failed to reach 98 . 5% call rate was re-analyzed . Individuals whose analyses failed repeatedly were re-contacted by 23andMe customer service to provide additional samples , as is done for all 23andMe customers . Two slightly different versions of the genotyping platform were used in this study . See [53] for further details on the genotyping and sample quality controls . The NINDS dataset consisted of 519 samples genotyped on a combination of an Illumina HumanHap 250 K and Illumina HumanHap 300 K chip , and 1 , 183 samples genotyped on an Illumina HumanHap 550 K chip . As the proportion of cases genotyped on each platform in the NINDS dataset differed between the two platforms , any marker with differing frequencies across the two platforms ( due to problems with clustering or other genotyping error ) would show up as associated with PD status in the NINDS dataset . To account for potential stratification arising from this , we defined a binary covariate to indicate genotyping platform , which we used for covariate adjustment during analyses involving the NINDS dataset . In all analyses , SNPs with a call rate or minor allele frequency were excluded from analysis . Additionally , SNPs with Hardy-Weinberg -values or were excluded from the 23andMe and NINDS datasets , respectively [56] . For analyses involving external validation of risk prediction models trained on the 23andMe dataset against the NINDS dataset , only SNPs common to both datasets were used in both model development and testing . Altogether , 522 , 782 SNPs were retained for the 23andMe dataset with an average call rate of 99 . 8% , 514 , 362 SNPs were retained for the NINDS dataset with an average call rate of 99 . 8% , and 492 , 136 SNPS were common to both datasets . For the association analysis , all -values were calculated using a likelihood ratio test for the logistic regression model , adjusting for sex , age , and the first five principal components ( chosen based on an examination of the eigenspectrum of our data ) :Here , the phenotypic status of each individual was coded as for unaffected individuals and 1 for affected individuals . Genotypes were coded to indicate the number of minor alleles present for tested SNP ( corresponding to a log-additive model of association ) , and was the projection of the individual onto the th principal component of the genotype data matrix . Reported odds ratios for each SNP relative to the minor allele were defined as , and the alleles used throughout refer to the plus strand of NCBI build 36 . 3 of the human genome . Principal components were computed using multi-dimensional scaling over the allele-sharing distance matrix as in [53] . The SNPs used for the replication analysis ( Table 3 ) were taken from http://www . pdgene . org/ [27] and http://www . genome . gov/gwastudies/ [57] on November 18 , 2010 . We added the SNPs reported as genome-wide significant from [22] to this list . For the power calculations , we used the model from [58] . To estimate heritability of liability , we used the GCTA package ( v0 . 90 . 3 ) [29] for genome-wide complex trait analysis . Previously , this approach was used to estimate the proportion of the heritability in height that could be explained by common variation on a genomic panel [69] . Here , we used a recent adaptation of this method to case-control studies [30] to estimate the heritability of PD in both the NINDS and 23andMe cohorts . We analyzed the NINDS cohort data by using GCTA to remove individuals with genetic relationship greater than 0 . 025 , and estimating heritability of liability using 20 principal components as covariates and assuming a disease prevalence of 0 . 01 . For the 23andMe cohort , we adopted the same procedure but pre-filtered the data by stratifying the dataset on sex , and matching on age and five principal components in order to obtain a reduced size dataset with one case per four controls . For Table 4 , we converted relative recurrence risk ratios to heritability of liability estimates using a modification of the analysis described in [31] . Formulas for estimating the maximum AUC achievable for a given heritability , and for computing the proportion of variance in liability explained were also based on [31] . Details are provided in Text S1 . We note that in Table 4 , the heritability estimates shown were based on the authors' criteria for “broad-definition PD” as no heritability estimates could be provided for the strict PD definition due to the lack of concordant monozygotic twins present in the dataset . For [10] , 95% confidence intervals were obtained through a reanalysis of the original data using 100 , 000 bootstrap samples for the counts of doubly-ascertained concordant , singly-ascertained concordant , and discordant monozygotic and dizygotic twin pairs . Heritability of liability was estimated as twice the difference in tetrachoric correlations for monozygotic and dizygotic twins , using a specialized numerical integration procedure for multivariate normal densities [70] . We performed risk prediction experiments using a sparse logistic regression solver based on the “elastic net” regularization penalty [59] . In this approach , one solves the convex optimization problem , for fixed constants , where and , and where . Sparse regression methods , which tend to estimate solution vectors with very few non-zero components , have enjoyed increased popularity in recent years due to their effectiveness in picking out relevant features from extremely high-dimensional data . In the context of genetic association analysis , sparse regression methods can be used to identify the set of SNPs that are most relevant to prediction of a given phenotype . The “elastic net” approach we used is a particular sparse regression variant based on combining / regularization penalties that has the advantage of grouping together correlated features while maintaining sparsity . We note that elastic net regularization has also previously been applied in the context of GWAS and SNP-based risk prediction in a number of recent papers [32] , [60]–[62] . For all experiments , we used a default value of ( to ensure uniqueness of the solution to the optimization problem ) and evaluated the performance of the risk prediction algorithm in two different ways . We varied the hyperparameter to obtain different bounds on the expected number of false positive associations ( i . e . , genotype features with non-zero coefficients corresponding to SNPs that are not truly associated with the phenotype ) , using an adaptation of the analysis from [63] . We performed two types of cross-validation experiments: an internal cross-validation analysis using only the 23andMe cohort , and an external cross-validation analysis testing the predictive accuracy of a model trained using the 23andMe cohort on the NINDS cohort . In both cases , all aspects of the analysis following QC were included as part of the cross-validation . For the internal cross-validation experiment , we generated a matched dataset using a portion of the 23andMe cohort . More specifically , we separated the 23andMe dataset into 20 partitions based on sex and age decile . Next , all partitions were then balanced to contain roughly the same ratio of cases to controls . Finally , five equally-sized cross-validation folds were formed , containing the same amount of representation from each of the partitions . In total 3 , 380 cases and 21 , 640 controls were used across the five cross-validation folds . For the external cross-validation experiment , the entire 23andMe cohort was used as a training set , and evaluation was performed on the NINDS dataset . When estimating AUC using a test set , stratification biases can arise when the apparent discriminative accuracy of the model can be attributed to one or more covariates . This could occur , for example , if the prevalence of the disease were to vary by population , provided that the SNPs included in the risk prediction model were informative of ancestry . To control for confounding from covariate imbalance , we used a stratified variant of the AUC known as the “covariate-adjusted AUC , ” defined as the probability that a randomly selected case will have a higher estimated risk of developing PD than a randomly selected matched control . More details on our procedure for covariate adjustment are provided in Text S1 . The use of AUC-based statistics for risk prediction is not without controversy . In the setting of a case-control study , the AUC has the advantage of being neither dependent on an arbitrarily set threshold for risk ( which would be needed when computing sensitivity or specificity ) or the relative proportion of cases and controls in the study . Some authors have contended that the AUC is not a clinically relevant measurement of performance and may be insensitive to changes that would otherwise be considered important in a diagnostic setting [64]–[67] , while others have argued that changes in AUC are nonetheless meaningful in assessing discriminative performance [68] . Here , we have chosen to rely on AUC not as a summary of the clinical performance of a classifier , but rather as a mechanism for studying the genetic etiology of a disease , and for estimating the proportion of genetic variance captured by the SNPs used in our models . Were one specifically interested in developing a clinically useful classifier , other measures of accuracy may be more appropriate . Since the goal of our experiments was to measure the predictive capacity that could be attributed to SNPs in our model , rather than covariates such as sex , age , or ancestry , we intentionally excluded covariates when fitting our predictive models . We note that because of our use of covariate-adjusted AUCs , the decision to exclude covariates had little impact on our results since changes in predictive performance arising from the inclusion of covariates would have been “factored out” by the stratification procedure anyway . For example , the covariate-adjusted external validation accuracies of the smallest and largest models in Table 5 were 0 . 550 and 0 . 605 , respectively; the analogous risk prediction model including covariates would have achieved accuracies of 0 . 557 and 0 . 603 . | We conducted a large genome-wide association study ( GWAS ) of Parkinson's disease ( PD ) with over 3 , 400 cases and 29 , 000 controls ( the largest single PD GWAS cohort to date ) . We report two novel genetic associations and replicate a total of twenty previously described associations , showing that there are now many solid genetic factors underlying PD . We also estimate that genetic factors explain at least one-fourth of the variation in PD liability , of which currently discovered factors only explain a small fraction ( 6%–7% ) . Together , these results expand the set of genetic factors discovered to date and imply that many more associations remain to be found . Unlike traditional studies , participation in this study took place completely online , using a collection of cases recruited primarily via PD mailing lists and controls derived from the customer base of the personal genetics company 23andMe . Our study thus illustrates the ability of web-based methods for enrollment and data collection to yield new scientific insights into the etiology of disease , and it demonstrates the power and reliability of self-reported data for studying the genetics of Parkinson's disease . | [
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] | 2011 | Web-Based Genome-Wide Association Study Identifies Two Novel Loci and a Substantial Genetic Component for Parkinson's Disease |
Long interspersed elements ( LINEs ) are transposable elements that proliferate within eukaryotic genomes , having a large impact on eukaryotic genome evolution . LINEs mobilize via a process called retrotransposition . Although the role of the LINE-encoded protein ( s ) in retrotransposition has been extensively investigated , the participation of host-encoded factors in retrotransposition remains unclear . To address this issue , we examined retrotransposition frequencies of two structurally different LINEs—zebrafish ZfL2-2 and human L1—in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining ( NHEJ ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ . Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells , suggesting that NHEJ is involved in LINE retrotransposition . More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition , namely to ensure efficient integration of LINEs and to restrict their full-length formation .
Long interspersed elements ( LINEs ) and short interspersed elements ( SINEs ) are transposable elements widely distributed in eukaryotic genomes [1] , [2]; as such , they substantially affect genome complexity and evolution [3] , [4] . These elements mobilize and amplify their own sequences by a mechanism called retrotransposition . LINEs are 4–7 kbp in length and typically encode two open reading frames ( ORFs ) , ORF1 and ORF2 , both of which are essential for LINE retrotransposition [5] , [6] . During retrotransposition , LINEs are first transcribed into messenger RNA ( mRNA ) from which the LINE-encoded proteins are translated ( Figure S1A ) . Next , the LINE mRNA and proteins form a complex [7] , [8] and move to target sites on a host chromosome where the LINE-encoded endonuclease ( EN ) nicks a strand on the DNA duplex . The LINE-encoded reverse transcriptase ( RT ) then reverse transcribes the LINE mRNA using the 3′ hydroxyl group generated by the nick as a primer; this reaction is called target-primed reverse transcription [5] , [9] , [10] . Thereafter , the newly synthesized LINE is integrated into the host chromosome , at which time sequence alterations are generated at the target site . The position of the second strand cleavage is considered to define which kind of target site alteration is generated ( Figure S1B ) [11] . In the model , second-strand cleavage downstream of the initial first-strand nick generates target site duplication ( TSD ) , cleavage at the same site generates blunt end joining ( BEJ ) , and cleavage upstream generates target site truncation ( TST ) . However , the precise mechanism of the integration remains unclear ( Figure S1A ) . A DNA double-strand break ( DSB ) would necessarily need to be generated at the target site to integrate the newly synthesized LINE element . In fact , overexpression of human LINE L1 in mammalian cultured cells induces DSBs in the host chromosomal DNA [12] . Accumulating evidence has revealed that several host-encoded DNA repair proteins are involved in the mobility reactions of retrotransposons , such as yeast LTR retrotransposons and bacterial group II introns [for review , 13] . However , the roles of host factors in LINE retrotransposition remain unclear . Only a few genetic studies have identified host proteins that are involved in LINE retrotransposition: the ataxia-telangiectasia mutated ( ATM ) protein—a protein kinase involved in cellular responses to DSBs—is suggested to participate in L1 retrotransposition [12] , and the ERCC1/XPF endonuclease—which functions in nucleotide excision repair—is involved in limiting L1 retrotransposition [14] . It is conceivable that the LINE retrotransposition reactions involve other host factors , such as proteins of the non-homologous end-joining ( NHEJ ) pathway , that predominate in DSB repair in vertebrate cells [15] . The core components involved in vertebrate NHEJ are the Ku70 and Ku80 heterodimer ( Ku70/80 ) , the catalytic subunit of DNA protein kinase ( DNA-PKcs ) , DNA ligase IV ( LigIV ) , and Xrcc4 . Initially , Ku70/80 binds to the broken DNA ends . DNA-PKcs is recruited to the ends by Ku70/80 , with which it maintains the broken ends in proximity and provides a platform for the recruitment of other enzymes [16] . The kinase activity of DNA-PKcs—which is activated upon recruitment to the broken ends—is considered to enhance the DSB signal via phosphorylation of many downstream targets , although physiological targets of the phosphorylation remain obscure [for example , 17] . LigIV , which forms a tight complex with Xrcc4 , is responsible for ligation of the broken DNA ends [18] , [19] . There are other proteins implicated in NHEJ . During NHEJ , a pair of broken ends that are incompatible for ligation is processed into compatible ends by a nuclease ( s ) , such as Artemis , and/or a polymerase ( s ) , although their significance in NHEJ is less clear . Interestingly , broken ends are still repaired by NHEJ in cells deficient in the core NHEJ components such as Ku proteins or LigIV , suggesting that NHEJ can be achieved by at least two distinct pathways [20] , [21] . To distinguish these two processes , the NHEJ pathway that depends on the core components is denoted ‘classical’ , and the other pathway , which can occur without the core components , is called ‘alternative’ . In contrast to the classical NHEJ , the enzymes responsible for the alternative NHEJ remain uncharacterized . Recently , we established a new system to detect LINE retrotransposition in the chicken B lymphocyte cell line , DT40 , using two different kinds of LINEs , zebrafish ZfL2-2 and human L1 [22] ( Figure S2 ) . Here , we applied this system to Ku70−/− , Artemis−/− , and LigIV−/− DT40 cell lines to determine the effect of these knockouts on the retrotransposition frequencies ( RFs ) of ZfL2-2 and L1 . We then characterized ZfL2-2 insertions retrotransposed in the chromosomal DNA of DT40 cells to obtain evidence for the involvement of NHEJ factors in the LINE integration reaction . In addition , we examined the possible involvement of DNA-PKcs in LINE integration in human HeLa cells using NU7026 , an inhibitor of DNA-PKcs activity .
To investigate whether host factors participating in NHEJ are involved in LINE retrotransposition , we examined RFs of two types of LINEs that have different structural characteristics—zebrafish ZfL2-2 and human L1—using wild-type ( WT ) and five knockout DT40 cell lines . The knockout DT40 cell lines were deficient in the genes encoding Ku70 , Artemis , LigIV , SHIP1 , or Rad18; the first three cell lines are related to NHEJ , and the others are not ( Figure 1 , Tables S1 , S2 ) . Because the intrinsic colony-forming capacities varied among these cell lines , we compensated for this aspect by including the plating efficiency in the RF calculation ( see Materials and Methods ) . The RF of ZfL2-2 decreased by about 2- to 8-fold relative to the WT DT40 in all NHEJ-deficient cell lines examined here ( Figure 1B; Ku70−/− , Art−/− and LigIV−/− ) . On the other hand , knockout of the Rad18 or SHIP1 gene , neither of which is related to the NHEJ pathway , did not affect the RF ( Figure 1B; Rad18−/−and SHIP1−/− ) . These results suggest that the NHEJ pathway plays a role in ZfL2-2 retrotransposition in these chicken cells . Similar retrotransposition results were obtained using L1 , although the decrease in the L1 RF in Ku70−/− and LigIV−/− was smaller than that for ZfL2-2 ( Figure 1C; see also Table S1 , S2 ) . To confirm that the RF decrease in the Ku70-defective cells was caused by Ku70 disruption , ZfL2-2 retrotransposition was assessed in three DT40 cell lines , WT , Ku70−/− and LigIV−/− , with transient expression of a cloned chicken Ku70 gene ( Figure 1D , Table S3 ) . Transcription of the cloned and/or endogenous Ku70 genes in each sample was verified by RT-PCR . Expression of the cloned Ku70 in WT and LigIV−/− cells did not significantly alter the ZfL2-2 RF . In contrast , exogenous Ku70 expression in Ku70−/− cells dramatically increased the ZfL2-2 RF to a level comparable to WT cells . These results indicate that the decrease of ZfL2-2 RF in Ku70−/− cells was indeed caused by Ku70 disruption . The NHEJ-defective DT40 cell lines are sensitive to intense ionizing radiation [23] , [24] , indicating that the cells cannot efficiently repair radiation-induced DSBs , causing cell death . If the expression of ZfL2-2 or L1 in DT40 cells induces DSBs in chromosomal DNA as in the case of the L1 expression in HeLa cells [12] , the NHEJ-defective DT40 cells may be more sensitive to such LINE-induced DSBs than WT cells . If this is the case , it is possible that the decrease of ZfL2-2 and L1 RF observed in the NHEJ-defective DT40 cells only reflects cell death caused by the LINE-induced DSBs , which cannot be compensated for by the plating efficiency in our assay ( see Figure S3 ) . To examine this possibility , we monitored the viability of WT and mutant DT40 cells transfected with the LINE expression vector . As shown in Figure S4 , when two different fluorescence protein expression vectors ( enhanced green fluorescence protein ( EGFP ) and DsRed-Express ) were mixed and co-electroporated into DT40 cells , most transfected ( fluorescence-positive ) cells ( >80% ) express both of the two fluorescent proteins , and the amounts of proteins expressed from the co-transfected vectors were roughly proportional to each other ( Figure S4D and S4E ) . Hence , to trace the LINE-expressing cells , an EGFP expression vector was electroporated together with the LINE expression vector into DT40 cells , and the EGFP expression and its intensity were monitored as shown in Figures S5 , S6 , S7 , S8 , S9 , S10 , S11 , and S12 . This EGFP monitoring was conducted from 3 to 8 days after electroporation , during which cell division occurred at least eight times ( data not shown ) . EGFP expression observed on the eighth day was minimal , showing the detection limit . As shown in Figure 2 , the relative ratio of the amount of EGFP-expressing cells at each time point to that of the third day was similar between WT and Ku70−/− DT40 cells up to the end of the monitoring . In addition , the time course of the relative ratio of the geometric mean and the median of the EGFP intensity was similar between the WT and Ku70−/− DT40 cells ( Figure 2 ) . Moreover , the time course of the values did not change when a point mutation that abolishes LINE EN activity was introduced in the ZfL2-2 and L1 elements . Similar results were obtained from Artemis−/− and LigIV−/− cell lines ( Figure S13 , S14 ) . These results indicate that LINE EN expression does not influence the viability of WT and NHEJ-defective cell lines . Thus , the decrease in LINE RF in the NHEJ-defective cell lines is likely to be related to the involvement of NHEJ in LINE retrotransposition in DT40 cells . LINE retrotransposition typically depends on the LINE's own EN [5] , [25] , [26] . In contrast , a previous study reported that a fraction of human L1 retrotransposition in Chinese hamster ovary ( CHO ) cells was not dependent on L1 EN [27] , [28] . This L1 EN-independent retrotransposition was enhanced in mutant CHO cells defective in a gene involved in NHEJ [27] . This report prompted us to consider that ZfL2-2 and L1 might atypically retrotranspose in NHEJ-defective DT40 cells through an EN-independent manner . To examine this possibility , we examined RFs of the EN-defective ZfL2-2 and L1 elements in the NHEJ-defective DT40 cell lines ( Table S1 , S2 ) . In both NHEJ-defective and WT cell lines , no G418-resistant colonies were formed with these EN mutants , indicating that mobilization of ZfL2-2 and L1 in the DT40 cell lines examined here is dependent on their own ENs . Although we have not resolved the reason why the dependence of LINE retrotransposition on EN differs in chicken and hamster cells , this may reflect the differences in DNA repair that exist between these cells as discussed by Morrish et al . [27] . To determine in which step of the retrotransposition reaction each NHEJ factor is involved , we determined and analyzed the 5′ and 3′ junction sequences of 102 ZfL2-2 inserts in chromosomal DNA of WT , Ku70−/− , Artemis−/− and LigIV−/− DT40 cells ( 26 , 25 , 24 and 27 insertions , respectively; Table S4 ) . We previously showed that ∼40% of ZfL2-2 elements in the zebrafish genome had extra nucleotides at the 5′ junction , whereas ∼50% had microhomologies [29] . At the 3′ junction , on the other hand , ∼80% of these elements had microhomologies [29] . Similar tendencies were observed at both junctions of ZfL2-2 insertions in DT40 cells , and these tendencies were not altered by NHEJ defects ( Table S5 ) . Also , the length distribution of the 5′ and 3′ microhomologies did not differ between the WT and NHEJ-deficient DT40 cells ( Figure S15 ) . However , the ZfL2-2 insertions in Ku70−/− and Artemis−/− cells were significantly longer than those in WT cells ( Figure 3A and 3B; P = 0 . 008 and 0 . 036 , respectively ) . In particular , full-length elements were recovered only from NHEJ-deficient cells ( Figure 3A , 3C ) . Indeed , the fraction of full-length insertions differed significantly between WT and Ku70−/− cells and between WT and Artemis−/− cells ( P = 0 . 010 and 0 . 046 , respectively ) . These results indicate that Ku70 and Artemis inhibit the generation of longer inserts in DT40 cells , suggesting that these NHEJ factors , at least in part , participate in LINE 5′ truncation . We classified the target site alterations of the ZfL2-2 insertions in DT40 cells into five categories: long TST ( L-TST , >20 bp ) , short TST ( S-TST , ≤20 bp ) , BEJ , short TSD ( S-TSD , ≤20 bp ) and long TSD ( L-TSD , >20 bp ) ( Figure 3D , Table S5 ) . Consistent with our previous data regarding ZfL2-2 elements present in the zebrafish genome [29] , a large fraction of ZfL2-2 insertions ( 20 of 25 , 80% ) in WT DT40 had a TSD , and the rest of them had a TST ( Figure 3D ) . Most insertions ( 24 of 25 ) in the WT cells had short target site alterations ( ≤20 bp ) , and only one had a L-TSD ( 1228 bp ) , indicating that long target site alterations are relatively rare in WT cells . On the other hand , L-TST ( 343–50187 bp ) insertions were frequently observed in Ku70−/− cells ( Figure 3D; 5 of 25 ) , suggesting that Ku70 prevents the generation of L-TST . We next focused on insertions with short target site alterations ( Figure 3E ) . Insertions with S-TSD predominated in all cell lines . Still , 5 of 25 insertions ( 20% ) in WT cells and 3 of 20 insertions ( 15% ) in Ku70−/− cells had an S-TST . In contrast , only one of 23 insertions ( 4% ) in Artemis−/− cells had an S-TST , and no S-TSTs were observed in LigIV−/− cells . The difference in occurrence of S-TSTs between the WT and LigIV−/− cells is statistically significant ( Figure 3E; P = 0 . 016 ) . These results indicate that LigIV ( and possibly Artemis ) plays an important role in generating S-TSTs . To examine whether NHEJ is also involved in LINE retrotransposition in cells other than chicken DT40 , we performed the retrotransposition assay in human HeLa cells ( Figure 4 ) . No knockout HeLa cell line is available , but DNA-PKcs kinase activity can be specifically inhibited by NU7026 [30] . We first confirmed that NU7026 kills HeLa cells in a dose-dependent manner only in the presence of a DSB inducer , etoposide [31] . HeLa cells treated with NU7026 became more sensitive to etoposide , indicating that the NHEJ repair capacity is suppressed by NU7026 ( Figure 4A ) . The RFs of both ZfL2-2 and L1 decreased with increasing concentrations of NU7026 , suggesting that the NHEJ pathway is also involved in LINE retrotransposition in HeLa cells ( Figure 4B , Table S6 , S7 ) . Consistent with the results using DT40 cells , ZfL2-2 retrotransposition was more sensitive than L1 retrotransposition to NU7026 .
Host repair systems are likely to be involved in the later stages of LINE retrotransposition [12] , [13] , [29] , [32] , [33] . For example , bioinformatic studies have suggested that the ‘alternative’ NHEJ pathway is involved in LINE retrotransposition [33] ( see below ) . There is , however , no bioinformatic evidence for such involvement of the ‘classical’ NHEJ or experimental evidence for a role in LINE retrotransposition of any host repair system except for the ATM kinase [12] . Here , we studied the effects of defects in ‘classical’ NHEJ on ZfL2-2 retrotransposition and found that such defects considerably decrease the ZfL2-2 RF , suggesting that a large fraction of ZfL2-2 insertion events in DT40 cells utilizes these classical NHEJ factors . In addition , the characterization of ZfL2-2 insertions revealed that disruption of the genes encoding NHEJ components extended the length of inserted ZfL2-2 elements , allowing more full-length insertions ( Ku70−/− and Artemis−/− ) ; frequently generated L-TSTs ( Ku70−/− ) ; and diminished the generation of S-TSTs ( LigIV−/− ) . These results suggest that NHEJ proteins are involved in the 5′ joining of ZfL2-2 insertions during retrotransposition , as detailed below . During retrotransposition ( Figure S1A ) , the ZfL2-2 RNA-protein complex chooses a target site , at which the ZfL2-2 EN nicks the first strand of the host DNA . The ZfL2-2 RT then initiates reverse transcription of the ZfL2-2 RNA from the nick . Most ZfL2-2 elements in DT40 cells as well as those in the zebrafish genome have a certain length of truncation at the 5′ end ( 5′ truncation ) , which is a characteristic of a typical LINE element . The mechanism by which the 5′ truncation is generated is , however , unclear . Our data provide a possible mechanism for the 5′ truncation . The Ku70 defect produced longer insertions ( Figure 3A , 3B ) , implying that the Ku70/80 complex can obstruct the progression of the ZfL2-2 RT . For instance , transient dissociation of the RT from the template RNA could allow Ku70/80 to associate with the end of the newly synthesized ZfL2-2 DNA ( Figure 5 ) because Ku70/80 is able to interact with a single-to-double-strand transition of DNA [34] . The Ku70/80 association may interfere with further reverse transcription and initiate a joining reaction between the premature ZfL2-2 cDNA and upstream target DNA , resulting in a 5′ truncation . Because deficiencies of Artemis and LigIV—which act downstream of Ku70 in NHEJ—also caused longer insertions ( Figure 3A , 3B ) , the progression of the NHEJ pathway might be related to the switching of reaction modes from reverse transcription to 5′ joining . Ku70/80 protects DNA ends from exonucleolytic degradation [35] . Consistently , Ku70−/− cells frequently produced ZfL2-2 insertions containing long chromosomal DNA deletions ( Figure 3D; L-TST , 343–50187 bp ) . This suggests that Ku70/80 is associated with the end of the upstream target DNA as well as the end of the ZfL2-2 element during integration , and protects the chromosomal DNA from degradation ( Figure 5 ) . In the case of TST generation , genomic information is altered not only by inserting the ZfL2-2 sequence but also by deleting the pre-existing sequence . Thus , Ku70/80 may also serve as a barrier against the loss of genomic information caused by ZfL2-2 retrotransposition . The variability of target site alteration has been accounted for by the difference in the position of the second strand cleavage [11] ( Figure S1B ) ; however , it remains unclear what other factor or factors are involved in this variation . We found that LigIV−/− cells did not produce S-TSTs , whereas ∼20% of insertions in WT cells had an S-TST ( P = 0 . 016 ) , indicating that S-TST generation is dependent on LigIV activity . Moreover , the S-TST frequency was also decreased in Artemis−/− cells . Therefore , the 5′ overhang ( generated by the second-strand cleavage upstream of the first nick; see Figure S1B ) at the chromosomal end may be processed predominantly by Artemis and then ligated to the ZfL2-2 5′ end by LigIV ( Figure 5 ) . Hence , our results suggest that these NHEJ factors contribute to variation among target site alterations . Taken together , our data suggest the possibility that NHEJ proteins , originally recruited for the repair of chromosomal breaks generated by the ZfL2-2 EN , are necessarily utilized for ZfL2-2 integration . Deficiencies of NHEJ proteins remarkably decreased the ZfL2-2 RF , indicating that NHEJ proteins are required for efficient retrotransposition . On the other hand , these NHEJ factors restricted the generation of full-length ZfL2-2 copies by diverting initiated retrotransposition reactions toward the generation of truncated ZfL2-2 copies . The restriction of full-length copies that have the potential to undergo subsequent retrotransposition limits the amplification of ZfL2-2 copies in the next generation . Interestingly , Deininger's group showed that many more DSBs than retrotransposition events are generated by L1 EN expression in HeLa cells [12] , suggesting that a considerable fraction of L1-induced DSBs are repaired without L1 insertions . Because DSBs are predominantly repaired by NHEJ in vertebrate cells , it is plausible that these L1-induced DSBs are fixed by NHEJ . Thus , the NHEJ pathway probably limits retrotransposition at two different phases: 1 ) inhibition of LINE cDNA integration itself by immediate repair of DSBs , resulting in direct limitation of retrotransposition and 2 ) production of truncated insertions , resulting in limitation of retrotransposition in the next generation . Because more active L1 elements produce longer insertions [36] , the rapid or efficient progression of reverse transcription may counteract both of these NHEJ limitations . Thus , rapid cDNA synthesis prior to the operation of the NHEJ pathway may be vital for successful LINE amplification . Taken together , these observations indicate that there is opposition between DNA repair and LINE retrotransposition . Similarly , a retrotransposon conflict hypothesis has been proposed by Sawyer and Malik , in which NHEJ proteins are proposed to be hijacked for mobilization of Ty LTR retrotransposons or recruited to defend against them [37] . Our data show that disruption of the NHEJ pathway in DT40 cells did not completely suppress ZfL2-2 retrotransposition ( Figure 1 ) . Therefore , a pathway ( s ) other than classical NHEJ may exist to connect the ZfL2-2 integrants and the end of the target DNA at the 5′ junction . As proposed by Zingler et al . [33] , one possibility is the ‘alternative’ NHEJ pathway , which joins two DNA ends via microhomology in a manner independent of NHEJ factors such as Ku70/80 and LigIV . However , the fraction of insertions having 5′ microhomology was not elevated in Ku70−/− or LigIV−/− cells ( Table S5 ) , and thus this putative mechanism cannot fully account for the observed residual Ku70- or LigIV-independent retrotransposition activity . Rather , the template-jump model [33] , [38]–[43] seems more likely for the 5′ joining process , although the DNA ligase responsible remains unidentified . Indeed , a large proportion of extra nucleotides found at the 5′ junction of ZfL2-2 insertions in DT40 cells appears to be synthesized by the template jump reaction ( unpublished data ) . Cell death caused by LINE EN expression was not detected in chicken DT40 cells in our experimental system ( Figure 2 , S13 and S14 ) , although it causes considerable cell death in human HeLa cells [12] ( data not shown ) . Neither DT40 nor HeLa cells have detectable levels of p53 , a tumor suppressor that induces apoptosis or cell cycle arrest against DNA damage [44] , [45] . Thus , although a lack of p53 appears to confer tolerance to chromosomal instability in DT40 cells [44] , it cannot explain the observed differential sensitivity to LINE EN between DT40 and HeLa cells . We thus speculate that this differential sensitivity may reflect the presence of an intrinsic LINE retrotransposition mechanism in each cell line . The fact that LINE retrotransposition in chicken DT40 and hamster CHO cells is differentially dependent on EN supports this idea . Hence , comparative analysis of the mechanism of LINE retrotransposition in different cell lines and organisms is indispensable for understanding the generality and specificity of the LINE amplification mechanism . Deficiencies of NHEJ proteins in DT40 and HeLa cells also decreased the L1 RF , suggesting that NHEJ factors participate in retrotransposition of human L1 as well as zebrafish ZfL2-2 in these vertebrate cells . The degree of decrease in the L1 RF was , however , smaller than that for ZfL2-2 ( Figure 1 , 4 ) , indicating that NHEJ is not much involved in retrotransposition of human L1 . This implies that each LINE has its own dependency on NHEJ and probably other repair system ( s ) ; in other words , that the mechanism of LINE retrotransposition is considerably distinct between each LINE in the light of the participation of host repair systems . A major structural difference between these LINEs is the absence ( ZfL2-2 ) or presence ( L1 ) of ORF1p . Because L1 ORF1p has been suggested to be involved in the 5′ joining [46] , ORF1p might make L1 more independent of the host NHEJ system . Our study indicates that the factors of classical NHEJ are involved in the repair of breaks generated by LINEs during retrotransposition . Our results also indicate that the NHEJ pathway is not the only mechanism by which such breaks can be repaired . Elucidation of the entire ensemble of host factors involved in LINE mobilization will help us understand the interaction between hosts and molecular parasites during evolution .
pBZ2-5 expresses the WT zebrafish LINE ZfL2-2 containing the neomycin resistance gene that is disrupted by an intron in the antisense orientation ( mneoI ) [25] . p131 . 11 expresses the mneoI-marked ZfL2-2 element containing a point mutation ( E72A ) in the EN sequence [22] . pAZ2-2 , which expresses the WT ZfL2-2 element marked by mneoI400/ColE1 [11] , was constructed as follows . The mneoI400/ColE1 cassette was amplified from pCEP4/L1 . 3mneoI400/ColE1 [11] by PCR using primers Neo-NotF-1 ( 5′-TGTGTGTGGCGGCCGCGCACAAACGACCCAACACCC-3′ ) and Neo-BamR-1 ( 5′-CACACGGATCCGCTGCAGCATAGCCTCAGG-3′ ) . The PCR fragment of mneoI400/ColE1 was digested with NotI and BamHI . Using the mneoI400/ColE1 fragment , the NotI and BamHI fragment of pBB4 [25] , which contains mneoI , was replaced , resulting in pBB5-9 . The 3′ tail of ZfL2-2 was amplified from pBZ2-5 by PCR using primers Z2-3′F1 ( 5′-ATATGGATCCTGAAACTTGCCTTTAGTACTTATTCATTGTTGC-3′ ) and Z2-3′R1 ( 5′-ATATGGATCCTTTACATTTACATTTACATTTAGTCATTTAGCAGACGC-3′ ) . The PCR fragment of the 3′ tail was digested with BamHI and inserted in the BamHI site of pBB5-9 , resulting in pAZ2-2 . pJM102/L1 . 3 expresses the WT L1 ( L1 . 3 ) containing the marker mneoI [27] . pJM102/L1 . 3 H230A expresses the mneoI-marked L1 ( L1 . 3 ) containing a point mutation ( H230A ) in the EN sequence [27] . pEGFP-FLAG-1 expresses an enhanced green fluorescence protein ( EGFP ) [22] . Chicken Ku70/pAneo was constructed by cloning the chicken Ku70 gene into the expression vector pAneo [23] . The expression vectors were all purified using the QIAfilter Plasmid Midi or Mega kit ( Qiagen ) . WT DT40 and its SHIP1−/− and IP3R−/− derivatives were purchased from RIKEN Bioresource Center ( cell numbers RCB1464 , 1465 , and 1467 ) . Ku70−/− , DNA ligase IV−/− and Rad18−/− DT40 cell lines were established previously [23] , [24] , [47] . The Artemis−/− DT40 cell line was kindly provided by Dr . Minoru Takata [48] . These DT40 cells were cultured in RPMI medium 1640 ( Invitrogen ) supplemented with 10% fetal bovine serum , 1% chicken serum , 20 U/ml penicillin , 20 µg/ml streptomycin , and 10 µM β-mercaptoethanol , in a humidified atmosphere with 5% CO2 at 37°C or at the temperatures indicated . The retrotransposition assay procedure in DT40 cells has been described [22] . Briefly , DT40 cells were cotransfected with pEGFP-FLAG-1 ( 15 µg ) and one of the LINE expression vectors ( 15 µg ) , pBZ2-2 , p131 . 11 , pJM102/L1 . 3 , or pJM102/L1 . 3 H230A [22] , [27] . Transfection was carried out by electroporation at 250 V and 960 µF for ZfL2-2 expression vectors , and at 200 V and 960 µF for L1 expression vectors using the GENE Pulser ( Bio-Rad ) . After the transfected cells were incubated at 33°C for 3 days , the number of EGFP-positive and EGFP-negative cells were counted by flow cytometry to measure the transfection efficiency . To detect retrotransposition , the electroporated cells ( ∼1×106 cells per dish ) were plated in soft agarose medium containing G418 ( 1 . 6 mg/ml ) . In parallel , to determine the plating efficiency , the electroporated cells ( 200 cells per dish ) were plated in soft agarose medium without G418 . After an 11-day incubation at 37°C , visible colonies were counted . Plating efficiency was calculated as the number of visible colonies on the plate ( without G418 ) as a percentage of the 200 cells plated . RF was calculated as: RF = G/ ( E×P/100 ) , where G represents the number of G418-resistant colonies , E represents the number of EGFP-positive cells , and P represents the plating efficiency . DT40 cells ( WT , Ku70−/− or LigIV−/− ) were cotransfected with pEGFPFLAG-1 ( 10 µg ) , pBZ2-2 ( 10 µg ) , and one of two expression vectors ( 10 µg ) , chicken Ku70/pAneo or pAneo . Transfection was carried out by electroporation at 250 V and 960 µF using a GENE Pulser . After transfection , cells were processed by the same procedure described above , and the RF was calculated . To detect transcription of Ku70 from the exogenous and/or endogenous gene , cells transfected with the three plasmid DNAs were harvested 3 days after transfection . Total RNA was extracted from the cells using the RNeasy Mini kit ( Qiagen ) . The column-based preparation was repeated to avoid any DNA contamination . RT-PCR was performed using the total RNA ( 1 µg ) as the template with primers cKu70R1 ( 5′-CAGAGACAGTGAGCTTGCCC-3′ ) and cKu70F2 ( 5′-CGCTGGATATGCTGGAACCA-3′ ) . As a control , transcription of the chicken β-actin gene was detected by RT-PCR using primers cActinF1 ( 5′-GGTCAGGTCATCACCATTGG-3′ ) and cActinR1 ( 5′-TGCATCCTGTCAGCAATGCC-3′ ) . DT40 cells were cotransfected with pEGFP-FLAG-1 ( 15 µg ) and one of the LINE expression vectors ( 15 µg ) , pBZ2-2 , p131 . 11 , pJM102/L1 . 3 , or pJM102/L1 . 3 H230A [22] , [27] . Transfection was carried out by electroporation at 200 V and 950 µF using the GENE Pulser Xcell ( Bio-Rad ) . After electroporation , the cells were incubated at 33°C for 3 days . Then the cells were subcultured at 37°C . The percentage of EGFP-positive cells and their EGFP fluorescence intensity were monitored by flow cytometry at intervals of 12 h from 3 to 8 days after electroporation . Ten thousand cells were counted for each measurement by flow cytometry . Circular DNA containing ZfL2-2 insertions was isolated using the procedure developed by Gilbert et al . [11] . Briefly , DT40 cell clones derived from each G418-resistant colony produced by pAZ2-2 were cultured separately until the total number of cells reached ∼1×107 per clone . Genomic DNA was isolated from each clone using the GenElute mammalian genomic DNA miniprep kit ( Sigma ) . Genomic DNA ( ∼20 µg per clone ) was digested with 75 U of HindIII for 6 h at 37°C . The digested DNA ( ∼20 µg ) was then self-ligated overnight by T4 DNA ligase ( 350 U ) in 500 µl solution at 16°C . Ninety percent of the circular DNA was incorporated in E . coli DH10BT1R ( Invitrogen ) by electroporation with the GENE Pulser Xcell ( Bio-Rad ) under conditions of 2 , 500 V , 25 µF and 100 Ω , and the electroporated cells were plated on kanamycin-containing ( 70 µg/ml ) plates . Circular DNA containing a mneoI400/ColE1-marked ZfL2-2 insertion ( with its flanking chicken genomic DNA ) was isolated from the kanamycin-resistant cells . The 5′ and 3′ junctions of each isolated ZfL2-2 insertion were sequenced using the appropriate primers . Sequences flanking each ZfL2-2 insertion were used as probes in BLAT searches to identify the preintegration site in the chicken genome database ( http://genome . ucsc . edu; the May 2006 chicken ( Gallus gallus ) v2 . 1 draft assembly ) . Exponentially growing HeLa-RC cells [25] were exposed to increasing concentrations of NU7026 with or without etoposide ( 1 µM ) for 2 h . After treatment , the cells were trypsinized and reseeded into new 100-mm dishes at densities of 350 or 3 , 500 cells/dish and grown in fresh medium containing no drug . After 10 days , colonies were fixed with 100% ethanol and stained with 2% Giemsa solution . The survival rate was calculated as the number of colonies as a percentage of the reseeded cells . HeLa-RC cells ( 2×105 cells/well ) were seeded in 6-well dishes [25] . NU7026 of the indicated concentration was added to the medium 1 day after seeding . One hour after the addition of NU7026 , the cells were transfected with 1 µg plasmid DNA ( pBZ2-5 or pJM102/L1 . 3 ) . The cells containing the plasmid were selected with hygromycin ( 200 µg/ml ) for 6 days . NU7026 treatment was continued during the hygromycin selection . The hygromycin-resistant ( HygR ) cells were trypsinized and reseeded into new 100-mm dishes ( at the density of 100 , 000 cells/dish for pBZ2-5 and 5 , 000 cells/dish for pJM102/L1 . 3 ) and grown in medium with 400 µg/ml G418 . In parallel , 10 , 000 HygR cells for pBZ2-5 or 2 , 000 HygR cells for pJM102/L1 . 3 were also reseeded in a 100-mm dish and grown in medium without G418 to measure the plating efficiency . After a 12-day incubation , cell colonies were fixed by 100% ethanol and stained with 2% Giemsa solution . The plating efficiency was calculated as the number of visible colonies on the plate ( without G418 ) as a percentage of the number of cells plated . RF was calculated as the number of G418-resistant colonies per HygR cell , compensating for the plating efficiency . | Long interspersed elements ( LINEs ) are transposable elements that mobilize and amplify their own copies within eukaryotic genomes . Although LINEs had been considered as “junk” DNA , recent studies have suggested that the LINE-induced alterations of host chromosomes are a major driving force for eukaryotic genome evolution . LINEs mobilize via a mechanism called retrotransposition , in which transcribed LINE RNA is reverse transcribed into DNA that is then integrated into the host chromosome . Although the role of LINE-encoded proteins in retrotransposition has been revealed , the participation of host-encoded proteins has not been well investigated . Here , using knockout chicken DT40 cell lines , we present genetic evidence that the host-encoded proteins involved in repair of DNA double-strand breaks participate in LINE retrotransposition . More precise characterization of LINE insertions in DT40 cells suggested dual roles for these host DNA repair proteins in LINE retrotransposition; one function is required for efficient integration of LINEs and the other restricts their full-length formation . | [
"Abstract",
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] | [
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] | 2009 | Genetic Evidence That the Non-Homologous End-Joining Repair Pathway Is Involved in LINE Retrotransposition |
Simian-human immunodeficiency virus ( SHIV ) challenge stocks are critical for preclinical testing of vaccines , antibodies , and other interventions aimed to prevent HIV-1 . A major unmet need for the field has been the lack of a SHIV challenge stock expressing circulating recombinant form 01_AE ( CRF01_AE ) env sequences . We therefore sought to develop mucosally transmissible SHIV challenge stocks containing HIV-1 CRF01_AE env derived from acutely HIV-1 infected individuals from Thailand . SHIV-AE6 , SHIV-AE6RM , and SHIV-AE16 contained env sequences that were >99% identical to the original HIV-1 isolate and did not require in vivo passaging . These viruses exhibited CCR5 tropism and displayed a tier 2 neutralization phenotype . These challenge stocks efficiently infected rhesus monkeys by the intrarectal route , replicated to high levels during acute infection , and established chronic viremia in a subset of animals . SHIV-AE16 was titrated for use in single , high dose as well as repetitive , low dose intrarectal challenge studies . These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines , monoclonal antibodies , and other interventions targeted at preventing HIV-1 CRF01_AE infection .
The generation and evaluation of simian-human immunodeficiency virus ( SHIV ) challenge stocks are important for preclinical testing of vaccines , microbicides , monoclonal antibodies , and other interventions aimed at preventing HIV-1 infection . SHIVs are typically constructed with an SIV backbone and HIV-1 env , tat , rev , and vpu [1–3] . The predominant HIV-1 subtype in the United States and Europe is subtype B [4] . In contrast , circulating recombinant form 01_AE ( CRF01_AE ) viruses are predominant in Southeast Asia , including Thailand [5] . The RV144 study in Thailand has been the only partially successful vaccine clinical efficacy trial to date [6] . A SHIV challenge stock expressing CRF01_AE env would therefore be critical for testing concepts related to the protection observed in RV144 . Studies involving correlates of risk and molecular sieve analyses have raised important hypotheses for how the RV144 vaccine might have worked [7 , 8] , but mechanistic studies have been limited by the lack of an available CRF01_AE SHIV challenge stock . We therefore sought to produce mucosally transmissible SHIVs containing early CRF01_AE env sequences . HIV-1 transmission across a mucosal barrier is a relatively inefficient process and typically results in propagation of only a single transmitted/founder ( T/F ) virus [9] . A single T/F virus has been deemed responsible for propagation of HIV-1 in 60–80% of mucosal infections [10 , 11] . SHIVs encoding early or acute HIV-1 envs might therefore optimally model acute infection and thus be relevant for testing vaccines and other interventions aimed at preventing mucosal infection . Three challenge stocks , SHIV-AE6 , SHIV-AE6RM , and SHIV-AE16 , were generated in human or rhesus PBMC without in vivo passaging . These viruses displayed CCR5 tropism and a neutralization phenotype consistent with tier 2 primary isolates . They established mucosal infection in rhesus monkeys , exhibited robust acute viral replication , and maintained chronic viremia in a subset of animals challenged by the intrarectal ( i . r . ) route . These novel CRF01_AE SHIV challenge stocks should facilitate preclinical mechanistic and efficacy studies involving interventions aimed at preventing infection with CRF01_AE HIV-1 .
Indian origin rhesus monkeys ( Macaca mulatta ) were used in the current study . Eight Mamu-A*01-negative adult male and female animals were housed at Bioqual ( Rockville , MD ) , and 16 Mamu-A*01-positive adult male and female animals were housed at the New England Primate Research Center ( Southborough , MA ) . All animals were maintained in accordance with the Association for Assessment and Accreditation of Laboratory Animals with approvals from the relevant Institutional Animal Care and Use Committees ( IACUC ) . The Harvard Medical School Standing Committee on Animals ( #03970 ) and the Bioqual IACUC ( 14-3131-62P ) approved these studies . The animal protocols adhered to NIH standards set forth in the "Policy on Humane Care of Vertebrate Animals Used in Testing , Research , and Training" and the "Guidelines for the Care and Use of Laboratory Animals" ( DHHS publication #NIH 85–23 ) . Animal welfare was maintained by the NEPRC and Bioqual animal management programs , which are accredited by the American Association for the Accreditation of Laboratory Animal Care ( AAALAC ) and meet all applicable federal and institutional standards for standard housing , standard monkey diet , water ad libitum , social enrichments , and steps intended to minimize suffering , such as the use of anesthesia for all procedures and analgesia for invasive procedures such as biopsies . Animals were euthanized by ketamine followed by sodium pentobarbital , which is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association . Human and rhesus monkey peripheral blood mononuclear cells ( PBMC ) were isolated by Ficoll-Hypaque gradient purification , followed by stimulation with concanavalin A ( Con A; 6 . 25 μg/ml ) and human interleukin-2 ( 20 units/ml IL-2; AIDS Research and Reference Reagent Program ) overnight . Human PBMCs were purchased commercially ( Research Blood Components ) . Cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum , 2 mM glutamine , 100 units/ml penicillin , 100 μg/ml streptomycin , and 20 units/ml of IL-2 . TZM-bl cells ( JC53-bl clone 13 cells ) are derived from a HeLa cell line ( JC . 53 ) that stably expressed CD4 and HIV coreceptors , as well as luciferase and β-galactosidase under the control of the HIV-1 long terminal repeat . TZM-bl cells and GHOST cells expressed CD4 alone or with different chemokine receptors and were obtained from the NIH AIDS Research and Reference Reagent Program . Lymph node and colorectal biopsies were collected at week 12 and incubated in RPMI 1640 containing 10% FBS supplemented with 200 U/ml of type IV collagenase ( Sigma-Aldrich ) and 30 U/ml of DNase I ( Sigma-Aldrich ) at 37°C with rocking for 30 min . The digested biopsy tissues were then homogenized , and the solution was strained with a 70-μm ( pore size ) cell strainer ( BD Falcon ) . The cell suspension was then centrifuged at 695 × g for 25 min on a 35% Percoll ( Sigma-Aldrich ) gradient . Pellets containing lymphocytes were collected and processed for assays . Proviral DNA assays were conducted using the QIAcube HT ( Qiagen ) performed as previously described [12] . Twenty-nine HIV-1 CRF01_AE early env sequences ( AE1 –AE29 ) derived from acutely HIV-1-infected participants in either the RV217 Early Capture HIV Cohort Study in East Africa and Thailand ( Merlin Robb , AIDS Vaccine 2012 ) or the RV254/SEARCH study in Thailand [13] were used to generate SHIV molecular clones ( Table 1 ) . The env sequences were synthesized by GeneArt ( GeneArt , Invitrogen ) and inserted into the KB9-AC plasmid as described [14] . Plasmids were sequenced and transfected into 293T cells using LipoD293 ( SignaGen Laboratories ) . Cell culture supernatants were collected after 48 h and clarified through a 0 . 45 μm filter . Eight infectious molecular clones ( AE3 , AE4 , AE6 , AE15 , AE16 , AE17 , AE21 , AE22 ) that replicated to high levels in human PBMC were selected to generate large-scale challenge stocks . Each virus stock was produced from PBMC isolated from 120 ml of human or rhesus monkey whole blood . Cell culture supernatants harvested from transiently transfected 293T cells were used to inoculate ConA-stimulated PBMC in the presence of 20 U/ml human IL-2 ( AIDS Research and Reference Reagent Program ) . SHIV-AE6RM was prepared by propagation of the human SHIV-AE6 stock in rhesus monkey PBMC . The media was replaced and collected in 3-day increments over the course of 12–15 days . Virus was quantified by SIV p27 enzyme-linked immunosorbent assay ( ELISA; Zeptometrix ) , and the 50% tissue culture infectious does ( TCID50 ) was determined in TZM-bl cells . Virus stocks were assessed for infectivity by inoculation of TZM-bl cells using 5-fold serial dilutions in the presence of 11 μg/ml of DEAE-dextran hydrochloride ( Sigma , St . Louis , MO ) in quadruplicate wells . Virus infectivity was determined 48 h post infection by measuring the level of luciferase activity expressed in infected cells . The TCID50 was calculated as the dilution point at which 50% of the cultures were infected . The inhibitors TAK-779 and AMD-3100 were obtained from the AIDS Research and Reference Reagent Program and were diluted per manufacturer instructions . TZM-bl cells were seeded at 3 X 104 cells/well in flat-bottom plates . The cells were incubated for 1 h in 10-fold dilutions of TAK-779 ranging from 5 to 500 , 000 ng/ml or 10-fold dilutions of AMD-3100 ranging from 5 to 50 , 000 ng/ml or mock infected with supernatant from cultured PBMC without viral infection . After 1 h , 100 TCID50 of the SHIV stocks were added to the wells , and 48 h later the cells were lysed and analyzed by Steady-Glo ( Promega ) luciferase assay with luminescence measured on a Victor 3 luminometer ( Perkin-Elmer ) . GHOST cells expressing coreceptors CXCR4 , CCR5 , and CCR5/CXCR4 and the parental strain were obtained from the AIDS Research and Reference Reagent Program . These were maintained in Dulbecco modified Eagle medium with 10% fetal calf serum and penicillin-streptomycin and supplemented with 500 μg/ml G418 , 100 μg/ml hygromycin , and 1 μg/ml puromycin . Puromycin was excluded for culture of the parental cell line . Cells were plated at 104 cells per ml in 96-well round-bottom wells in medium supplemented with 20 μg/ml Polybrene . One hundred TCID50 of each virus stock was added to each well , followed by incubation overnight . After being washed with phosphate-buffered saline ( PBS ) and replacement with fresh culture medium , cell culture supernatants were harvested 4 days after incubation and assessed for SIV p27 ELISA ( Zeptometrix ) . Neutralizing antibody titers against SHIV isolates were determined using a luciferase-based assay in TZM . bl cells as previously described [15 , 16] . This assay measures a decrease in luciferase reporter gene expression following single-round viral infection of TZM . bl cells . Briefly , 3-fold serial dilutions mAb reagents were performed in duplicate and incubated with primary SHIV isolates for 1 hour at 37°C . TZM . bl cells were then added in growth media containing DEAE-dextran at a final concentration of 11 μg/ml , and assay plates incubated for 48 hours at 37°C , 5% CO2 . Luciferase reporter gene expression was measured using Bright-Glo luciferase reagent ( Promega ) and a Victor 3 luminometer ( Perkin Elmer ) . Neutralization titers ( 50% inhibitory concentrations , IC50 ) were calculated as the sample dilution at which the relative luciferase units ( RLU ) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells . MAbs 4E10 , 2G12 , 2F5 , b12 , PG9 , PG16 , and VRC01 ( Polymun Scientific ) and soluble human CD4 protein ( Progenics ) were obtained commercially . MAbs 3BNC117 , 8ANC195 , 10–1074 , 3BC176 , and 45-46W were provided by Michel Nussenzweig ( The Rockefeller University , New York , NY ) . MAbs PGT121 , PGT126 , and PGT128 were provided by Dennis Burton ( The Scripps Research Institute , La Jolla , CA ) . Animals received a single intrarectal ( i . r . ) inoculation with 1 ml of undiluted SHIV-AE6 , SHIV-AE6RM , or SHIVAE-16 stocks . For the SHIV-AE16 in vivo titration , six i . r . challenges with 1 ml of 1:1 or 1:10 diluted virus stock were performed with two week intervals from week 0 to week 10 . All animals were monitored for viral loads and CD4+ T cell counts . EDTA-anticoagulated whole blood , PBMC , and lymph node mononuclear cells ( LNMC ) were stained using anti-CD3-APC , anti-CD4-PE , and anti-CD8-FITC MAbs and analyzed using a FACSCalibur flow cytometer ( BD Biosciences , San Jose , Ca ) . Cell counts were determined using the BD True Count tubes according to the manufacturer’s instructions ( BD Biosciences ) . Viral RNA was isolated from cell-free plasma using the QIAamp viral RNA isolation kit ( Qiagen ) and quantified as previously described [4 , 14 , 17] . Quantitative PCR and viral load assays were performed as previously described [6 , 12 , 14] . All viral loads were run at the end of each study to maintain objectivity and ensure that all animals were handled identically . Viral RNA was extracted using a QIAamp viral RNA minikit ( Qiagen ) . RNA was eluted and immediately subjected to cDNA synthesis . Reverse transcription of RNA to single-stranded cDNA was performed using SuperScript III reverse transcriptase ( Invitrogen ) as described [9 , 14] . The env sequences of the amplicons were deposited in GenBank under accession numbers KT581024—KT581117 as detailed in Table 1 .
Twenty-nine CRF01_AE early HIV-1 env sequences from acutely HIV-1 infected individuals from Thailand ( Table 1 ) were cloned into the KB9-AC backbone , essentially as described [14] . Plasmids were used for transfections in 293T cells to generate virus , and virus infectivity titers were determined ( Table 2 ) . Culture supernatants were used to assess viral growth in human and rhesus PBMC . As is the case with other SHIVs [10 , 11 , 14] , these viruses replicated well in human PBMC but inefficiently in rhesus PBMC ( Table 2 ) . Interestingly , SHIV-AE22 was identified as the most infectious virus in this set of SHIVs , but this virus was confirmed to be subtype B rather than CRF01_AE ( Table 1 ) . We selected eight viruses ( AE3 , AE4 , AE6 , AE15 , AE16 , AE17 , AE21 , AE22 ) that replicated to high levels in human PBMC for the generation of large-scale challenge stocks by inoculation of the 293T transfection-derived supernatants into primary human PBMC . The large-scale challenge stocks ranged in infectivity from 6 . 4 X 102 to 7 . 0 X 108 TCID50/ml , and viral loads ranged from 3 . 2 X 108 to 2 . 1 X 109 RNA copies/ml ( Table 3 ) . The challenge stocks were then assessed for their ability to replicate in rhesus PBMC . Only two stocks , SHIV-AE6 and SHIV-AE16 , replicated at detectable levels in rhesus PBMC , and SHIV-AE6 exhibited the highest SIV p27 level as determined by ELISA ( Table 3 ) . This stock was therefore chosen to produce a large-scale challenge stock in primary rhesus PBMC , which was designated SHIV-AE6RM . The rhesus-derived SHIV-AE6RM stock replicated 114-fold more efficiently than did the human-derived SHIV-AE6 stock in rhesus PBMC ( Table 3 ) . We next performed sequencing by single genome amplification ( SGA ) [11] to analyze the env sequences in the SHIV-AE6 , SHIV-AE6RM , and SHIV-AE16 challenge stocks as compared to the original HIV-1 env sequences from the RV217 and the RV254/SEARCH studies in Thailand . We evaluated 31 SGA-derived sequences obtained for each challenge stock ( Fig 1 ) . The SHIV-AE6 stock had one to five random amino acid differences in 10 amplicons ( Fig 1A ) , while the SHIV-AE16 stock had one to three intermittent amino acid differences in 16 amplicons ( Fig 1B ) . The rest of the amplicons for both stocks was identical to the parent sequence . For the rhesus PBMC-derived SHIV-AE6RM challenge stock , 30/31 amplicons contained at least one amino acid difference as compared to the original HIV-1 env sequence , and 20/31 amplicons contained a consistent D168H mutation in the V2 loop ( Fig 1C ) . These data show that these SHIV stocks expressed envs that were >99% identical to the parental HIV-1 env sequences and only contained rare and sporadic amino acid changes , except for the D168H mutation in SHIV-AE6RM . We examined coreceptor usage of the early CRF01_AE SHIV stocks using TZM-bl cells in the presence of the coreceptor inhibitors TAK779 ( CCR5 ) and AMD3100 ( CXCR4 ) . All of the SHIV stocks were potently inhibited by the CCR5 TAK-779 inhibitor ( Fig 2A ) but were not inhibited by the CXCR4 inhibitor AMD3100 ( Fig 2B ) . We then tested the replication ability of SHIV-AE6 , SHIV-AE6RM , and SHIV-AE16 stocks in GHOST cells expressing alternate coreceptors and then measured the SIV p27 production of each SHIV stock in the culture supernatant ( Fig 2C ) . The assay verified that the SHIV stocks replicated in the R5 or dual X4/R5 expressing cells , but did not utilize X4 alone as a coreceptor . The results indicate that these SHIV stocks all utilized the CCR5 coreceptor for cellular entry . We next examined the neutralization sensitivity of each SHIV stock in TZM-bl cells with a panel of broadly reactive mAbs ( Table 4 ) targeting the major neutralization epitopes . The SHIV stocks all displayed neutralization profiles consistent with tier 2 neutralization resistant phenotypes , although SHIV-AE6RM showed sensitivity to soluble CD4 . We next assessed the capacity of SHIV-AE6 , SHIV-AE6RM , and SHIV-AE16 stocks that displayed detectable levels of replication in rhesus PBMC for their ability to infect rhesus monkeys in vivo by the i . r . route . Twelve adult rhesus monkeys were challenged with a single i . r . inoculation of 1 ml of undiluted virus for each stock ( n = 4/group ) . For the SHIV-AE6 stock , 3/4 animals exhibited peak viral loads ranging from 6 . 9 to 7 . 3 log RNA copies/ml at 2 to 3 weeks post inoculation , but minimal CD4+ T cell decline ranging from 10 to 14% during acute infection ( Fig 3A ) , and 2/4 animals were still viremic at week 10 . All 4/4 animals challenged with the SHIV-AE6RM stock became infected , and 2/4 animals were viremic at week 10 ( Fig 3B ) . For the SHIV-AE16 challenge , 2/4 animals displayed robust peak viral loads up to 7 . 2 log RNA copies/ml and detectable viremia at week 10 , while the other two animals had low or undetectable levels of viremia ( Fig 3C ) . SHIV-AE16 contained V2 sequences that match target epitopes of V2-specific mAbs isolated from the RV144 study [14 , 18] . Thus , SHIV-AE16 was chosen for further study . We conducted an in vivo titration experiment to determine the infectivity of SHIV-AE16 in a larger group of animals , as well as its infectivity following repetitive lower dose inoculations . Twelve adult rhesus monkeys received six repetitive i . r . challenges with either a 1:1 dilution ( n = 6 ) or a 1:10 dilution ( n = 6 ) of the SHIV-AE16 stock . 6/6 animals that received the 1:1 dilution were productively infected after the first challenge . These data suggest that this dose may be useful for single , high-dose challenge studies . Peak viral loads ranged between 6 . 1 to 7 . 3 log RNA copies/ml by week 2–6 and resulted in modest 6–14% declines in CD4+ T cell levels during acute infection ( Fig 4A ) . Viral loads declined but were still detectable in 6/6 animals at week 10 and in 3/6 animals by week 20 . In the 1:10 dilution group , 3/6 animals became infected after the first challenge , while the remaining three animals became infected after the second or third challenge ( Fig 4B ) . Animals established peak viral loads ranging from 5 . 8 to 7 . 4 log RNA copies/ml , and viral loads were detectable in 3/6 animals by week 20 . These data suggest that the 1:10 dilution may be useful for repetitive , low-dose challenge studies . In addition to viral RNA , we also confirmed the presence of viral DNA in peripheral blood mononuclear cells ( PBMC ) , lymph node mononuclear cells ( LNMC ) , and colorectal cells from these animals at week 12 ( Fig 5 ) , indicating viral infection in tissues . Taken together , these data show that the 1:1 and 1:10 dilutions of SHIV-AE16 stock led to productive infection in rhesus monkeys .
The development of CRF01_AE SHIV challenge stocks has to date remained elusive . This has been an unmet need for the HIV-1 research field , as such viruses are critical for preclinical testing of vaccines , antibodies , and other interventions aimed at preventing CRF01_AE HIV-1 infection . In this manuscript , we describe the generation of mucosally transmissible CRF01_AE SHIV challenge stocks . These challenge stocks will likely prove useful for preclinical testing of vaccines and mAbs related to the RV144 vaccine study , which showed a modest level of protection in Thailand [6] . Our goal was to generate mucosally transmissible CRF01_AE SHIV challenge stocks expressing early HIV-1 env CRF01_AE sequences . SHIV-AE6 , SHIV-AE6RM , and SHIV-AE16 encoded env sequences that closely matched the original sequences ( >99% ) from early HIV-1 CRF01_AE strains from Thailand . The SHIVs were highly infectious by the i . r . route in rhesus monkeys ( Table 5 ) , displayed robust acute viral replication , modest CD4+ T cell declines , and chronic viremia in 17/24 ( 71% ) animals at week 10 and in 6/12 ( 50% ) animals at week 20 ( Figs 3 and 4 ) . Previous research has shown that in vivo passaged SHIVs accumulate mutations in HIV-1 env that subsequently increase the pathogenicity of passaged viruses [14 , 19] . In an effort to prevent mutations and divergence from the parental HIV-1 env sequences , we generated our challenge stocks without in vivo passaging . Despite these attempts , SHIV-AE6RM developed a single D168H mutation in the V2 loop in 20/31 amplicons ( Fig 1 ) . Mutations in the V2 loop are common in the generation of SHIVs [3] , and other studies have suggested that laboratory adapted HIV-1 strains acquire positively charged amino acid mutations in variable loops that confer increased sensitivity to sCD4 ( Table 4 ) [20] . Further studies will be needed to determine if the D168H mutation is critical for conferring adaptation in rhesus PBMC . It is desirable for a SHIV challenge stock to be titrated for both single , high-dose challenges as well as repetitive , low-dose challenges . Vaccine studies in rhesus monkeys are often assessed by repetitive , low-dose challenges , whereas passive protection studies with monoclonal antibodies typically utilize single , high-dose challenges [21] . To develop a CRF01_AE SHIV stock that would be broadly useful , we titrated the SHIV-AE16 challenge stock in vivo ( Fig 4 ) . These data suggest the potential utility of SHIV-AE16 at 1:1 dilution for single , high-dose challenges and at 1:10 dilution for repetitive , low-dose challenges . In particular , this CRF01_AE SHIV challenge stock may prove useful for preclinical evaluations of vaccine regimens or V2-specific mAbs [7] related to the RV144 study to evaluate proposed mechanistic immune correlates of protection . The infectivity and pathogenicity of our CRF01_AE SHIVs appear lower than our previously published clade B and C SHIVs [14 , 22] . These CRF01_AE SHIV stocks will therefore likely prove useful for challenge studies in which protection from acquisition of infection is the endpoint , but for studies of chronic virus pathogenesis , in vivo adapted virus stocks will likely be required . We note that there was higher infectivity of the SHIV-AE16 challenge stock at 1:1 dilution ( 6/6 animals; Fig 4A ) as compared with undiluted virus ( 2/4 animals; Fig 3C ) , but the implications of this observation remain unclear given the small numbers of animals utilized in the initial study . In summary , we report the generation of novel CRF01_AE SHIV challenge stocks . These viruses did not require in vivo passaging and were titrated for use in rhesus monkeys . These CRF01_AE SHIV challenge stocks should prove useful to test vaccines , antibodies , and other interventions targeted to CRF01_AE HIV-1 . | In this study , we generated and evaluated novel simian-human immunodeficiency virus ( SHIV ) challenge stocks expressing env sequences from HIV-1 strains from Thailand . The lack of such SHIV challenge stocks has been a major unmet need for the field and has hindered progress to evaluate strategies aimed at preventing HIV-1 infection for this region of the world . The challenge stocks described in this manuscript should prove useful for studying the preclinical efficacy and mechanisms of vaccines , antibodies , and other interventions . | [
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"immunodefi... | 2016 | Production of Mucosally Transmissible SHIV Challenge Stocks from HIV-1 Circulating Recombinant Form 01_AE env Sequences |
The wMel strain of Wolbachia bacteria is known to prevent dengue and Zika virus transmission in the mosquito vector Aedes aegypti . Accordingly , the release of wMel-infected A . aegypti in endemic regions has been recommended by the World Health Organization as a potential strategy for controlling dengue and Zika outbreaks . However , the utility of this approach could be limited if high temperatures in the aquatic habitats where A . aegypti develop are detrimental to Wolbachia . We exposed wMel-infected A . aegypti eggs and larvae to fluctuating daily temperatures of 30–40°C for three , five , or seven days during their development . We found that Wolbachia levels in females emerging from heat treatments were significantly lower than in the controls that had developed at 20–30°C . Notably , seven days of high temperatures starting at the egg stage reduced Wolbachia levels in emerging females to less than 0 . 1% of the wMel control levels . However , after adult females returned to 20–30°C for 4–7 days , they experienced differing degrees of Wolbachia recovery . Our findings suggest that the spread of Wolbachia in wild A . aegypti populations and any consequent protection from dengue and Zika viruses might be limited in ecosystems that experience periods of extreme heat , but Wolbachia levels recover partially after temperatures return to normal .
Mosquito-borne arboviruses are a growing public health threat . The alarming geographic spread and costly health burden of dengue fever have led the World Health Organization ( WHO ) to deem it “the most important mosquito-borne viral disease in the world . ” Over the last 50 years , the incidence of dengue cases has increased 30-fold [1] . Now more than 100 countries have endemic dengue and over 40% of the world's population is at risk [2] . Zika virus , which recently caused a surge of children born with microcephaly and other neurological disorders , was declared a Public Health Emergency of International Concern ( PHEIC ) by the WHO after it spread from Brazil to 26 other countries or territories in the Americas within one year [3 , 4] . With no effective antiviral treatments in the arsenal and just one licensed dengue vaccine that is 65 . 6% effective for those 9 years or older , control of the mosquito vectors , Aedes aegypti and Aedes albopictus , is the most viable option for curbing transmission of these arboviruses [5–7] . However , in resource-limited cities with endemic dengue , vector control efforts are often only pursued in response to explosive epidemics [8 , 9] . Failure to control these vectors in tropical urban environments is one of the major drivers of the growing incidence and geographic expansion of dengue and other mosquito-borne arboviruses [10] . Alarmingly , existing control options for A . aegypti are of little use in urban areas [9 , 11] . Space spraying with ultra-low volume insecticides , including organophosphates and pyrethroids , has been used by many countries in the face of dengue outbreaks for the past 40 years despite limited evidence of its epidemiological benefits [10 , 12 , 13] . Vector densities inevitably recover after space spraying because ideal larval habitats for A . aegypti abound in cities—exposed water sources for drinking or washing and non-biodegradable trash that collects water [8] . Targeted spraying of potential larval development containers with residual insecticides [14 , 15] and indoor residual spraying targeting adult mosquitoes [16] in combination can substantially reduce local dengue incidence , but only if high coverage is achieved [17 , 18] . For countries faced with nearly ubiquitous breeding of A . aegypti in their sprawling cities , the comprehensive spraying required to stop transmission is unrealistic [19] . Consequently , there is no urban setting in which vector control has completely eliminated dengue virus ( DENV ) transmission or prevented dengue epidemics [13 , 20] . A potential solution for DENV and Zika virus ( ZIKV ) transmission involves releasing A . aegypti infected with Wolbachia , a common bacterium infecting the reproductive systems of many insects [21–24] . The fitness effects of Wolbachia on insect hosts are strain specific , ranging from life-shortening to pathogen-blocking phenotypes [25 , 26] . The pathogen-blocking properties of some strains of Wolbachia have led researchers to characterize them further and transfer them into vector species for potential use in vector-borne disease control . The wMel strain of Wolbachia shows the most promise currently , as it blocks DENV and ZIKV transmission by the mosquito , raising the possibility of disrupting dengue and Zika transmission cycles [27–30] . The wMel strain was transinfected from Drosophila melanogaster into A . aegypti , and wMel-infected A . aegypti have been released at sites in Australia , Vietnam , Brazil , Indonesia , and Colombia [31] . The success of each Wolbachia strain in invading insect populations is determined by the net fitness effect of the strain coupled with the extent to which it manipulates host reproduction [32] . One key mechanism of reproductive manipulation is cytoplasmic incompatibility ( CI ) . When a strain causes complete CI , Wolbachia-infected females can mate successfully with Wolbachia-infected males , while uninfected females cannot [32] . The wMel Wolbachia strain causes complete CI and has had some success in invading wild A . aegypti populations [28 , 33] . However , the prevalence of wMel Wolbachia must remain high in the A . aegypti population in order for wMel to reliably and substantially reduce the capacity of the mosquito population to transmit pathogens [32 , 34] . Protection against DENV in field-collected wMel A . aegypti is similar to that observed in the original transinfected wMel line [35] , indicating that this strategy might be used to reduce dengue transmission in endemic areas [28 , 29 , 36] . Recently the WHO recommended the use of Wolbachia for dengue and Zika control [4] , although there is currently insufficient epidemiological evidence to know if the approach is effective . It is also unknown whether the prevalence of wMel-infected A . aegypti and the wMel Wolbachia levels within individual mosquitoes will remain high enough to prevent DENV and ZIKV transmission in all environments . The levels of wMel Wolbachia load throughout the various stages of the A . aegypti lifespan have not been described , as most studies have focused on population dynamics and fitness effects of wMel Wolbachia after adult emergence [28 , 33 , 35 , 37–40] . The early stages of development comprise a sensitive period during the A . aegypti lifespan; immature forms are confined to their aquatic habitats , whereas adults can seek out favorable microclimates to increase their chances of survival [41–43] . Immature A . aegypti develop in containers in the domestic environment that hold water , including flower pots , tanks , and drums as well as bottles , cans , and automobile tires [8 , 44] . These containers sometimes hold as little as 5 mL of water [45] . Female A . aegypti preferentially lay their eggs in shaded containers , but it is not uncommon to find immatures in containers fully exposed to the sun [46 , 47] . Although comprehensive temperature measurements in sun-exposed containers have not been carried out , lab-reared A . aegypti larvae can tolerate aquatic temperatures as high as 43°C if they are pre-exposed to high but sublethal temperatures [48] . The ability of wMel Wolbachia to tolerate the same elevated temperatures as immature A . aegypti has not been investigated . The heat sensitivity of Wolbachia with respect to its hosts has been characterized in other arthropods . Exposure to high temperatures during development cured the Wolbachia infections of two-spotted spider mites Tetranychus urticae [49] , Tribolium flour beetles [50] , and Drosophila spp . [51–54] . In the mosquito Aedes scutellaris , the reproductive effect of CI caused by Wolbachia was lost when larvae were reared at 32 . 5°C , but it was unknown whether the loss of Wolbachia or host expression of heat-shock proteins was responsible [55–57] . In A . albopictus all life stages maintained at 37°C had a lower levels of Wolbachia than those reared at 25°C , indicating that high temperatures may reduce Wolbachia levels in mosquito hosts [58] . Reduced Wolbachia levels in response to high temperatures during larval development could represent a barrier to the spread of wMel Wolbachia in A . aegypti populations if fundamental drive mechanisms such as maternal transmission and CI are affected . Because only Wolbachia-infected females produce viable offspring with Wolbachia-infected males , CI creates a selective pressure for the spread of Wolbachia [32] . The spread of Wolbachia in mosquito populations is crucial , because incomplete wMel Wolbachia coverage in the A . aegypti population leaves the potential for DENV and ZIKV transmission . A recent study found geographical clusters of uninfected mosquitoes in a wMel-infected A . aegypti release suburb of Cairns , Far North Queensland , Australia [59] . The incomplete Wolbachia coverage was suggested to be due to immigration of uninfected mosquitoes from outside the release area , cryptic breeding sites , or other environmental phenomena such as “larval curing” ( loss of Wolbachia infection during larval development ) [59] . However , the occurrence of larval curing in mosquitoes has been poorly defined to date . Specifically , little is known about the temperature thresholds for Wolbachia during mosquito development or whether any potential curing persists after temperatures return to normal . Understanding larval curing in wMel-infected A . aegypti has important applications , as lower Wolbachia levels in adults might have downstream impacts on cytoplasmic incompatibility [60–67] ( although in D . simulans between-strain differences in CI are not explained by Wolbachia density [68] ) , maternal transmission [69 , 70] , and pathogen inhibition [29 , 68 , 71–73] . We investigated the effects of high temperatures during egg and larval development on laboratory-reared wMel-infected A . aegypti using fluctuating daily temperatures that simulate the real-world conditions of a heatwave in Cairns , Australia . Our results have implications for the projected spread of wMel Wolbachia through A . aegypti populations and for the vector competence of wMel-infected A . aegypti under different environmental conditions .
Blood feeding of mosquito colonies using human volunteers was performed in accordance to the QIMR Berghofer Human Research Ethics Committee permit QIMR HREC361 . Written informed consent was obtained from all volunteers who participated in the study . Mosquitoes were taken from a Wolbachia-free A . aegypti colony ( “Cairns” line ) started from eggs collected in Cairns , Australia , in January 2015 and from a colony of wMel-infected A . aegypti ( “wMel” line ) started from eggs collected in suburbs of Cairns in April 2015 . The colonies were maintained in separate , identical climate-controlled rooms at 27 ± 1°C and 70 ± 10% relative humidity with a 12:12 hour light:dark cycle and crepuscular periods . Eggs were flooded in aged ( ≥ 48 h ) tap water and allowed to hatch naturally . Larval stages were reared under a controlled density ( < 200 larvae per tray ) in trays with 3 L of aged tap water . Larvae were fed on ground TetraMin tropical fish food ( Tetra , Germany ) . Pupae were transferred into cages measuring 40 × 40 × 30 cm for adult emergence . Colonies were maintained with a population size of > 500 individuals per generation . Adult mosquitoes received 10% sucrose solution ad libitum , and females were blood-fed on a human volunteer for 15 min every 7 d . The wMel-infected A . aegypti colony was regularly screened for Wolbachia using PCR of the wsp gene from the time of establishment [74] . Prior to the start of the experiments , our screening showed that the colony was completely infected with Wolbachia . For the experiments , eggs were collected from A . aegypti wMel ( F16 and F17 generations used ) and A . aegypti Cairns ( F18 and F19 generations ) colonies at 8:30 A . M . following the first night of oviposition . Eggs were counted under a stereomicroscope at 23°C and were separated into batches of approximately 600 eggs . Each batch was placed inside a dry paper towel , which was folded and placed next to a damp paper towel inside an open plastic bag . Egg bags were placed inside their corresponding environmental chambers at the coldest point of the temperature cycles , which was 20°C for the control condition and 30°C for the treatment condition . Eggs were left to mature for 48 h , and then batches of approximately 150 eggs were flooded in 500 mL aged tap water in plastic trays ( 183 × 152 × 65 mm ) . Four replicate trays were used per treatment group . From the day of hatching until pupation , ground TetraMin tropical fish food ( Tetra , Germany ) was administered daily at the coldest point of the temperature cycles using the “medium” diet described by Hugo et al . [75] . Pupae were transferred into 1-L plastic containers with mesh tops , and emerging adults were given 10% sucrose solution ad libitum . Adult females were aspirated out at 0–2 days post-emergence and at 4–7 days post-emergence . They were frozen at -20°C until processing . We tested the effect of high temperatures during egg and larval development on Wolbachia levels in A . aegypti wMel adult females in two replicate experiments: Each replicate experiment compared various heatwave temperature regimes applied during particular periods of immature mosquito development that varied in duration and stage of onset . The temperature profiles we used simulated observed temperatures during average and extreme conditions in Cairns , Queensland . The Australian Bureau of Meteorology defines a heatwave as “a period of at least three days where the combined effect of excess heat and heat stress is unusual with respect to the local climate” [76] . We designed our treatment temperature profile to surpass the severe daily mean temperature threshold of 30 . 4°C for Cairns , which is based on temperature data from 1958 to 2011 [76] . Both treatment and control temperature profiles followed a truncated sinusoidal progression during the day and exponential decrease at night , representing a profile of daily temperature variation [77] . The shapes of the profiles were the same for each condition , but the profile was raised or lowered to adjust the mean temperature ( S1 Fig ) . Experiments were conducted in two environmental chambers ( 294-L Panasonic MLR-352H-PE and MLR-351H , Gunma , Japan ) . Nine treatment groups were exposed to fluctuating heatwave temperatures between 30°C and 40°C for varying durations beginning at various life stages . Controls consisted of wMel A . aegypti and wildtype Cairns A . aegypti exposed to diurnal temperature fluctuations between 20°C and 30°C . Transfers between environmental chambers were made at the coldest point of the temperature cycles ( 20°C for the control condition and 30°C for the treatment condition ) in order to minimize the likelihood of heat shock . As illustrated in Fig 1 , treatment groups exposed to high temperatures beginning from early embryogenesis ( eggs at ≤ 15 hours post-oviposition ) lasting three , five , or seven days are denoted by “E3 , ” “E5 , ” and “E7 . ” Groups exposed to high temperatures beginning at the immature larval stages ( 1st/2nd instars ) lasting three , five , or seven days are denoted by “I3 , ” “I5 , ” and “I7 . ” Groups exposed to high temperatures beginning at more mature larval stages ( 3rd/4th instars ) lasting three , five , or seven days are denoted by “M3 , ” “M5 , ” and “M7 . ” Prior to the two studies , a pilot study was conducted to determine differences in means for a range of onsets and durations ( S2 Fig ) . Data loggers , both factory installed and independent HOBO data loggers ( Onset , Cape Cod , MA ) , recorded light intensity and temperature variation . Actual water temperatures in the control chamber were within 1 . 00°C of the programmed air temperature throughout the duration of the experiments . This was also the case in the treatment chamber , except during the coldest periods , when water temperature was as much as 2 . 93°C lower than the programmed air temperature . Wolbachia densities within individual adult females were determined by quantitative PCR . The head was removed from each frozen adult female before DNA extraction . Genomic DNA was extracted using QuickExtract DNA Extraction Solution ( Epicentre Technologies Corporation ) as per the manufacturer’s instructions and was diluted 1:10 in purified water . Multiplex qPCR was performed , amplifying the target Wolbachia-specific wsp gene and the somatic Actin5c gene , which acted as a reference gene to standardize for mosquito body size ( wsp F: 5´–CATTGGTGTTGGTGTTGGTG–3´ , R: 5´–ACACCAGCTTTTACTTGACCAG–3´ , Actin5c F: 5´–GACGAAGAAGTTGCTGCTCTGGTTG–3´ , R: 5´–TGAGGATACCACGCTTGCTCTGC–3´ ) ( full methods in S1 Appendix ) [73 , 78 , 79] . Quantification cycles ( Cq ) were normalized by taking into consideration the different amplification efficiencies of the wsp and Actin5c genes , and Wolbachia to host genome ratios were calculated using Q-Gene [80] . Fluorescence in situ hybridization ( FISH ) was carried out using a Wolbachia-specific 16S rRNA probe [29] . Three freshly collected adult females ( legs and wings removed ) from each treatment group were fixed in 4% paraformaldehyde in 0 . 1 M phosphate buffer overnight and were transferred to 70% ethanol . Bodies were embedded in paraffin wax and sectioned with a microtome . Slides were dewaxed with two successive xylene washes for 10 min , two successive 5-min washes with 100% ethanol , and two successive 5-min washes in 95% ethanol . Slides were hybridized with the Wolbachia-specific W2 probe ( 5´–CTTCTGTGAGTACCGTCATTATC–3´ ) [29] conjugated on the 5´ end to the fluorescent probe Alexa Fluor 488 ( Molecular Probes , Inc ) . Slides were left in a dark humidity chamber at 37°C overnight and washed briefly in 1× saline sodium citrate ( SSC ) buffer + 10 mM dithiothreitol ( DTT ) at room temperature , then two 15-min washes in 1× SSC + 10 mM DTT at 55°C , two 15-min washes in 0 . 5× SSC at 55°C , a 10-min wash in 0 . 5× SSC + 10 mM DTT + 4' , 6-Diamidino-2-phenylindole ( DAPI ) ( 0 . 01 mg/50 mL ) at room temperature , and then a final 10-min wash in 0 . 5× SSC + 10mM DTT at room temperature . Slides were washed briefly with distilled water and mounted with Vectashield Hard Set mounting medium ( Vector Laboratories , Burlingame , CA ) . Slides were allowed to dry in a refrigerator overnight . Images from all sections were captured with a DeltaVision Core Deconvolution Microscope ( GE ) using identical acquisition settings ( S2 Appendix ) . Images were reformatted using SoftWorx ( Enterprise Softworks ( Pty ) Ltd . ) and were cropped and standardized for contrast using Adobe Photoshop CS6 ( Adobe Systems , Inc . ) . To determine the effect of the heat treatments on adult body size , the left wing of six females from each treatment group was removed and dry mounted on a slide . The distance from the axial notch to the wing tip , excluding the fringe scales , was used as a proxy for body size [75 , 81] . All analyses were performed in R [82] and GraphPad Prism v . 6 ( GraphPad Software , San Diego , California , USA ) . Normality and homogeneity of variances within treatments were tested using Shapiro-Wilk and Bartlett’s tests , respectively . Log10-transformed Wolbachia densities were used for all analyses . A two-way blocked analysis of variance ( ANOVA ) was performed to determine the effects of treatment and collection time point and their interaction on Wolbachia density . Replicate was included as a blocking factor to account for any variation between the two experiments . An analogous two-way blocked ANOVA was performed to determine the effects of treatment group and collection time point and their interaction on body size . Pair-wise post-hoc comparisons between treatments and controls and between collection time points were made for both ANOVAs , and P values were adjusted for multiple comparisons using Tukey’s honest significant difference test . Differences were considered significant if adjusted P values were < 0 . 05 . A nonlinear regression was performed using ordinary least squares fit for each stage of onset at the two collection time points to determine relationships between the heat treatment duration and Wolbachia density . Sum of squares F-tests were used to determine significant differences in slopes and y-intercepts .
We found significantly lower Wolbachia densities relative to wMel controls in 0–2 d-old females emerging from eight of the nine treatments ( Fig 2 ) , with only the mature instar treatment lasting three days ( M3 ) showing no significant reduction . Wolbachia levels in the 0–2 d-old females that were exposed to 30–40°C for seven days starting at the egg stage ( E7 ) were less than 0 . 1% of wMel control densities ( Fig 2 ) . Both treatment group and collection time point were significant predictors of Wolbachia density ( F ( 10 , 362 ) = 197 . 34 , MSE = 55 . 24 , P < 0 . 001 and F ( 1 , 362 ) = 397 . 21 , MSE = 111 . 20 , P < 0 . 001 , respectively ) . Compared with the 0–2 d adult collection time point , 4–7 d-old adult females in all treatment groups except the wMel control group and the M3 group had higher Wolbachia levels , with adults from three-day treatments ( E3 , I3 , and M3 ) showing Wolbachia densities that were not significantly different from wMel-infected controls ( Fig 2 ) . The Wolbachia levels in 4–7 d-old adults from the six other treatments remained significantly lower than in wMel-infected controls . There were inverse relationships between the duration of heat treatment and Wolbachia density and for all stages of onset; however , the relationships differed significantly both in their slopes and y-intercepts ( F ( 5 , 305 ) = 3 . 68 , P = 0 . 003 and F ( 5 , 305 ) = 2 . 79 , P = 0 . 02 , respectively ) . Duration of heat exposure had the greatest impact on Wolbachia density in emerging females when high temperatures began in the 3rd/4th instar stages . At 4–7 days of age the impact of heat duration on density was most pronounced when high temperatures began at the egg stage . We also investigated whether we could visualize reductions in Wolbachia levels in the ovaries of adult mosquitoes after exposure to high temperatures during development . Using FISH we visualized very low levels of Wolbachia in the ovaries of 0–2 d-old E7 females ( Fig 3B ) . We also noticed that the E7 ovaries were much less developed than in controls , a possible consequence of the heat exposure . In 4–7 d-old E7 females ( Fig 3D ) , Wolbachia remained at very low levels compared with 4–7 d-old wMel-infected controls ( Fig 3C ) . We found a significant effect of treatment group on wing length ( F ( 10 , 71 ) = 13 . 70 , MSE = 0 . 32 , P < 0 . 001 ) and of the treatment group–collection time point interaction ( F ( 9 , 71 ) = 2 . 81 , MSE = 0 . 07 , P = 0 . 007 ) . Collection time point and replicate were not significant predictors ( F ( 1 , 71 ) = 0 . 22 , MSE = 0 . 005 , P = 0 . 64 and F ( 1 , 71 ) = 1 . 21 , MSE = 0 . 03 , P = 0 . 27 , respectively ) . Treatment groups E7 , I5 , I7 , M3 , M5 , and M7 were all significantly smaller than wMel controls ( S3 Fig ) . There was no significant difference in wing length between wMel controls and Cairns controls .
We found that when A . aegypti infected with the wMel strain of Wolbachia were exposed to daily fluctuating temperatures of 30–40°C during early development , the emerging females had reduced Wolbachia levels compared with controls . The most affected group consisted of mosquitoes exposed to high temperatures starting at the egg stage and lasting for seven days ( E7 ) . In E7 emerging females , mean Wolbachia levels were less than 0 . 1% of the levels of wMel controls . Loss of Wolbachia density from a subset of the mosquito population may be a concern for Wolbachia-based dengue and Zika control efforts in regions where the aquatic habitats of juvenile A . aegypti can reach extremely high temperatures . It has previously been shown that different Wolbachia strains attain different infection densities and that density is correlated with the level of virus inhibition [68 , 71 , 73 , 78] . The relationship between wMel density and DENV and ZIKV inhibition can be assumed from near complete blockage of these viruses in Ae . aegypti harboring dense wMel infections [28 , 30 , 35] , but the relationship has not been specifically defined . A recent study found that exposure of adult wMel-infected A . aegypti to 28°C ± 4°C beginning at 5–8 d of adult age was associated with reduced Wolbachia densities; however , there was no interaction between the reduced densities and DENV infection , dissemination , or transmission [40] . Eggs and larvae exposed to high temperatures in our study produced adult A . aegypti with very low Wolbachia densities; therefore , the level of pathogen inhibition in adult mosquitoes that were subject to impacts of heat exposure during early development deserves investigation . The partial recovery of Wolbachia density by 4–7 days of age suggests that any impacts of heat exposure during mosquito development on subsequent virus inhibition may be attenuated with age . This study is the first to investigate the duration and timing of heatwave conditions in relation to immature development of mosquitoes infected with Wolbachia . To achieve this we simulated normal and heatwave conditions based on temperature data from a city selected for Wolbachia biocontrol . We found an inverse relationship between the duration of heat exposure and Wolbachia density in adult females , raising the possibility that longer periods of heat might be capable of clearing Wolbachia . The slope of this relationship varied by the stage of heat onset and by the age of adult females collected . Duration of heat exposure had the greatest impact on Wolbachia density in emerging females when high temperatures began in the 3rd/4th instar stages; however , the impact of heat duration on density at 4–7 days of age was most pronounced when high temperatures began at the egg stage . In addition to reducing bacterial densities , high temperatures resulted in smaller adult body sizes , with more prominent effects in the later stages of heat onset and the longer durations . This is likely due to the known inverse relationship between larval rearing temperature and adult body size [83] . We controlled for the effect of body size by standardizing Wolbachia density measurements with the host gene Actin5c . Loss of Wolbachia density in response to heat has also been reported in T . urticae [49] O . scapulalis [84] , D . simulans [54] , D . bifasciata [53] , A . albopictus [58] , the predatory mite Metaseiulus occidentalis [85] , and the wasp Leptopilina heterotoma [86] . The mechanism behind the loss of Wolbachia in response to high temperatures is not fully understood , but deformation of the Wolbachia cellular membrane could be a contributing factor [87] . Our FISH visualization confirms the loss of Wolbachia from the ovaries of mosquitoes exposed to high temperatures . Partial recovery of Wolbachia in the ovaries after the mosquito returns to normal temperatures suggests that Wolbachia replication continues even after the ovaries are fully developed . It is uncertain whether replication continues throughout the female lifespan and at what age Wolbachia densities would be restored to control levels in heat-exposed females . Our results support the notion that wMel has a more restricted thermotolerance than its mosquito host A . aegypti . Loss of thermotolerance in insect symbionts can be due to point mutations that occur as the symbiont co-evolves with the host [88] . In the case of the obligate symbiont of aphids Buchnera aphidicola , a point mutation affecting heat-shock protein transcription leads to death of the symbiont following a heat treatment [89] . Compared with other symbionts of insects , wMel has experienced far less reductive evolution , as evidenced by its large genome with very high levels of repetitive DNA and mobile DNA elements [90] . Because of the low mutation rate of wMel [90] , loss of thermotolerance is less likely than for other symbionts [91] . If reductive evolution of wMel does occur , then rearing wMel-infected A . aegypti under constant temperatures in the lab might accelerate loss of wMel thermotolerance . More studies are needed to understand the co-evolution of wMel and A . aegypti . Wolbachia may hold the potential to reduce and even eliminate dengue and Zika transmission in endemic areas . The advent of a promising control tool for dengue fever and Zika could not have come at a better time , as currently many tropical countries have no options to control the massive arbovirus outbreaks they experience . The strategy of releasing wMel-infected A . aegypti is being tested in dengue-endemic regions around the globe , including Australia , Vietnam , Brazil , Indonesia , and Colombia [31] , although substantial epidemiological data is still needed to assess the impacts on dengue and Zika transmission . The importance of measuring Wolbachia density in field trials , as opposed to presence or absence of Wolbachia , is highlighted by our results and other investigations [61 , 64 , 78] . We found that the high temperatures that A . aegypti may experience during early development can attenuate wMel Wolbachia levels . Consequently , wMel Wolbachia might be less effective as a dengue or Zika control strategy in regions experiencing periods of extreme heat . If the effectiveness is compromised , increased surveillance and supplementary mosquito control may be required in these regions . Further estimates of Wolbachia recovery rates after heat exposure are needed to understand the impacts on DENV and ZIKV inhibition and the spread of wMel through naïve A . aegypti populations . In summary , we showed that fluctuating daily temperatures of 30–40°C experienced during wMel-infected A . aegypti egg and larval development significantly reduced Wolbachia levels in emerging adult females . However , Wolbachia recovered to differing degrees after adults returned to 20–30°C . These findings suggest that the effectiveness of Wolbachia-based arbovirus control might be compromised in ecosystems that experience periods of extreme heat , but given that Wolbachia levels partially recover after temperatures return to normal , any effects may be temporary . Greater understanding of environmental variables that affect Wolbachia can inform release site selection and help to better predict the impacts of Wolbachia on arbovirus transmission . | The proposed arbovirus biocontrol strategy of releasing mosquitoes infected with the wMel strain of Wolbachia bacteria promises to reduce the transmission of dengue and Zika viruses , but its utility in the field may be limited by the local environment . We show that when Aedes aegypti infected with wMel experience high temperatures during egg and larval development , they have lower Wolbachia levels as emerging adults . High temperatures starting at the egg stage and lasting for seven days reduce Wolbachia levels in emerging females to less than 0 . 1% of control levels . However , partial recovery of Wolbachia occurs by 4–7 days of age . The spread of Wolbachia in wild A . aegypti populations and any resulting impacts on dengue and Zika transmission could be limited by periods of extreme heat , but Wolbachia levels may subsequently recover . | [
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"vir... | 2016 | Heat Sensitivity of wMel Wolbachia during Aedes aegypti Development |
Changes in tissue homeostasis , acquisition of invasive cell characteristics , and tumor formation can often be linked to the loss of epithelial cell polarity . In carcinogenesis , the grade of neoplasia correlates with impaired cell polarity . In Drosophila , lethal giant larvae ( lgl ) , discs large ( dlg ) , and scribble , which are components of the epithelial apico-basal cell polarity machinery , act as tumor suppressors , and orthologs of this evolutionary conserved pathway are lost in human carcinoma with high frequency . However , a mechanistic link between neoplasia and vertebrate orthologs of these tumor-suppressor genes remains to be fully explored at the organismal level . Here , we show that the pen/lgl2 mutant phenotype shares two key cellular and molecular features of mammalian malignancy: cell autonomous epidermal neoplasia and epithelial-to-mesenchymal-transition ( EMT ) of basal epidermal cells including the differential expression of several regulators of EMT . Further , we found that epidermal neoplasia and EMT in pen/lgl2 mutant epidermal cells is promoted by ErbB signalling , a pathway of high significance in human carcinomas . Intriguingly , EMT in the pen/lgl2 mutant is facilitated specifically by ErbB2 mediated E-cadherin mislocalization and not via canonical snail–dependent down-regulation of E-cadherin expression . Our data reveal that pen/lgl2 functions as a tumor suppressor gene in vertebrates , establishing zebrafish pen/lgl2 mutants as a valuable cancer model .
Tumor suppression is a concept first formulated in Drosophila after emerging evidence that recessive mutations can lead to the formation of cellular overgrowth [1] , [2] . To date , more than 50 tumor suppressor genes have been identified in Drosophila [3] . Their deficiencies result in benign hyperplasias to malignant neoplasms . Amongst these tumor suppressors , mutations in lethal giant larvae ( lgl ) , cause malignant neoplasias in imaginal discs and the brain when transplanted into wild type adult host flies [4] , [5] . In Drosophila neuroblasts , lgl function is essential for localization of the cell fate determinant Numb , mislocalization of which in lgl mutant larvae prevents the neuroblasts from dividing asymmetrically and therefore causes neuroblastoma [6]–[9] . Furthermore , it has been proposed that lgl prevents tumor formation by antagonizing the activation of Dpp signaling by semaphorin 5c in the brain [10] . Although epithelial overgrowth phenotypes have been reported in lgl mutant larvae in Drosophila [1] , the mechanism by which lgl manifests its effects on epithelial growth remains to be understood . Nevertheless , it is known that along with lgl , two other tumor suppressor genes , discs large ( dlg ) and scribble ( scrib ) , primarily act in the maintenance of apico-basal cell polarity in epithelial cells [11] . The establishment as well as the maintenance of apico-basal cell polarity and eventually the depolarization of a cell is a complex process , involving several factors . In recent years , a conserved mechanism for the establishment and maintenance of apico-basal cell polarization has emerged , which mainly involves two pathways . Accordingly , the formation of the apical domain is controlled by the Par ( partitioning defective ) pathway , which consists of the PDZ domain containing proteins Par3 , Par6 , and atypical protein kinase C ( aPKC ) . In contrast , a pathway consisting of disc-large ( dlg ) , scribble ( scrib ) and lethal giant larvae ( lgl ) regulates the formation and maintenance of the baso-lateral domain [12] , [13] . Intriguingly , only mutations in genes that act in the baso-lateral pathway ( e . g . lgl , dlg , scrib ) lead to a neoplastic growth phenotype in Drosophila [2] , [14]–[16] . The two vertebrate orthologs of the Drosophila lethal giant larvae gene have conserved functions in the maintenance of cell polarity and tissue homeostasis . Disruption of lgl1 function results in the loss of apical junctional complex in neuroblasts and hyperplasia of the brain in mouse [17] . Furthermore , it has been shown for human melanoma cell lines , that a human homolog of lgl , hugl1 is significantly down-regulated . Artificial induction of hugl1 in these cell lines reduces their migratory potential with concomitant transcriptional up-regulation of the cell adhesion molecule E-cadherin ( E-cad ) and a down-regulation of matrix-metalloproteinases ( mmps ) , both of which are known to be involved in suppression of epithelial-to-mesenchymal transition ( EMT ) , a process which enables an epithelial cell to gain mesenchymal or migratory properties [18] . Recently , a significant correlation between the loss of hugl1 and a poor clinical prognosis for cancer patients has been shown [19] . The forward genetic approach in zebrafish has revealed a novel function for the second lgl ortholog , pen/lgl2 , in maintenance of the epidermal integrity , which is a stratified epithelium . The pen/lgl2 deficiency primarily results in the loss of hemidesmosomes , cellular junctions that mediate cell-matrix adhesion [20] . It has been shown that Lgl2 localizes to the lateral domain of the epidermal cells and regulates hemidesmosome formation by mediating the targeting of ITGa6 , a component of hemidesmosomes , to the membrane [21] . Furthermore , epithelial cells in the pen/lgl2 mutant exhibit altered epidermal cell morphology as well as cell polarity and enhanced epidermal growth [20] , [21] . Recently , it has been shown that in colorectal and breast carcinoma cell lines , a member of the ZFH family of repressors ZEB1 regulates the levels of Lgl2 . The loss of ZEB1 function restores Lgl2 levels and the epithelial phenotypes in tumor cells , which suggests that Lgl2 acts as an effector of ZEB1 in tumor suppression [22] . From the analysis of several cancer models it is evident that autocrine self-stimulation with growth factors is one of the hallmarks of tumorigenicity [23] , [24] . However , whether activation of growth factor signaling is a consequence of the loss of baso-lateral pathway components remains unclear to date . Moreover , whether lgl1 and lgl2 deficient clonal cell populations can promote tumor formation in vertebrate tissues , a typical characteristic of tumor suppressor genes , remains unresolved . Here , we show that lgl2 deficient clones indeed promote tumor formation in the zebrafish epidermis . Moreover , pen/lgl2 mutant basal epidermal cells undergo EMT . Using biochemical analysis , chemical inhibitors and genetic interaction studies , we demonstrate that these phenotypes are a consequence of an over-activation of erbB signaling involving at least one erbB family member , erbB2 . Our microarray and immuno-histological analysis reveal that activation of erbB signaling facilitates EMT by transcriptional up-regulation of key EMT regulators and a reduction in the membrane localization of E-cad , a known suppressor of EMT .
We have previously shown that zebrafish pen/lgl2 larvae show overgrowth of epidermal cells ( Figure 1A and 1B; and [20] ) . As pen/lgl2 mutant larvae die at 4–5 days post fertilization ( dpf ) , it was not clear whether these hyper-proliferating cells would be able to form tumor like structures . To test this , we transplanted cells from pen/lgl2 homozygous mutant donor individuals , into wild type hosts during blastula stage . In order to trace the donor cells in the wild type recipients , donor embryos were transgenic for ubiquitously expressed GFP . The recipient larvae with epidermal cells from mutants or wild type siblings were monitored for potential tumor development . On the 7th day after the transplantation , 17 of the 24 ( 71% ) larvae that had received pen/lgl2 deficient cells ( GFP marked ) in the epidermis developed epidermal tumors ( Figure 1C and 1D ) . In contrast , none of the larvae that had received cells from wild type siblings developed epidermal tumors ( n = 77 ) . Fluorescence microscopic analysis of these tumors revealed that they contain GFP positive cells , indicating that the lgl2−/− cells are inducing the formation of epidermal tumors ( Figure 1E ) . Interestingly , lgl2−/− cells in other tissues , including brain , did not show any visible hyperplasia at this stage . We conclude that pen/lgl2 mutant cells are capable of inducing epidermal tumor formation , even if surrounded by wild type tissue . The levels of GFP expression varied in the tumor cells ( Figure 1E ) . This may reflect a variegated expression of GFP . However , we cannot rule out the possibility that some wild type cells also contribute to the neoplastic tissue . Our results support the notion that lgl2 acts as a tumor suppressor gene specifically in the epidermis . Along with uncontrolled cell proliferation , migratory behavior mediated by EMT is another hallmark of cancer cells [25] . In pen/lgl2 mutant larvae , epidermal cells not only hyper proliferate but also exhibit different morphological shape as evident from the changes in the keratin organization from basal polygonal in wild type to peri-nuclear and spindle shaped in pen/lgl2 mutants ( Figure 2A and 2B , [20] ) . Such alterations in cell morphology are indicative of EMT . Since E-cad membrane levels are inversely correlated with acquisition of EMT [26]–[28] , we investigated the localization of E-cad in pen/lgl2 mutants . We found that the membrane localization of E-cad was severely reduced in the mutant epidermis , while the cytoplasmic fraction appeared to be increased compared to the wild type siblings ( Figure 2C and 2D ) . To observe the behavior of basal epidermal cells of pen/lgl2 mutants , we generated a transgenic line driving the expression of GFP in basal epidermal cells under the control of ΔN-p63 promoter [29] . Accordingly , tg ( ΔN-p63::Gal4 , UAS::GFP ) zebrafish larvae show GFP expression exclusively in this cell type ( Figure S1 ) . To test whether pen/lgl2 mutant cells acquire migratory properties , we performed time lapse studies of tg ( ΔN-p63::Gal4 , UAS::GFP ) ;lgl2−/− larvae . These revealed that , in contrast to their wild type siblings , basal epidermal cells in pen/lgl2 mutant larvae dramatically alter their shape , form numerous lamellipodia like structures and exhibit net displacement over time ( Figure 2 and Videos S1 , S2 , S3 , S4 ) . From an apical perspective , the observed changes in cell morphology also lead to an increase in average cell area ( Figure 2G ) . The mutant epidermal cells appear larger when compared to wild type cells as they exhibit a highly flattened morphology and develop lamellipodia like cell protrusions . We conclude that in absence of pen/lgl2 function the basal epidermal cells lose their epithelial morphology and acquire the morphology of migratory ( mesenchymal ) cells indicating that these cells undergo the morphological changes associated with EMT . To understand if pen/lgl2 mutants also show molecular signatures of EMT and if so , which of the known EMT related genes are active in pen/lgl2 mutant basal epidermal cells , we performed a genome wide expression profiling of pen/lgl2 mutant larvae showing neoplastic overgrowth at 108hpf using the Agilent microarray platform . Subsequent statistical analysis revealed 117 genes to be significantly differentially regulated in pen/lgl2 mutant larvae ( FDR; P≤10−6 ) ( Figure 3A and Table S1 ) . Amongst these differentially regulated genes , we found a very strong transcriptional induction of mmps such as mmp9 ( 11 . 1 fold ) and mmp13 ( 3 . 1 fold ) , which are known regulators of EMT , mainly in the context of malignancy [30]–[32] . Further , we found a set of cytokeratins , krt5 , ckrt1 , ckrt2 , and collagens to be down-regulated within a range of 3 . 5 to 4 . 4 fold , which is consistent with the previous analysis of the role of cytokeratins in EMT [28] . Moreover , genes involved in cell cycle regulation ( e . g . histone-b , jun-b , N-ras ) , cell survival ( e . g . sgk1 ) and tight-junction formation ( e . g . cldn-7 , cldn-e , cldn-c , cldn-i ) were also up-regulated from 3 . 6 to 5 . 6 fold ( Figure 3A ) . The canonical way to achieve EMT is to down-regulate E-cad at the transcriptional level . The transcriptional repressors , mainly those of the snail family , play an important role in this process [27] , [33]–[35] . Intriguingly , we observed robust up-regulation of E-cad expression at the mRNA level ( Figure 3A ) . By performing quantitative RT-PCR in pen/lgl2 mutant larvae , we detected 8 times higher E-cad RNA levels in mutants compared to the wild type sibling controls ( Figure 3B ) . Further , examination of E-cad by western blot analysis revealed increase in protein levels in pen/lgl2 mutant larvae compared to their wild type siblings ( data not shown ) . Thus , although the membrane localization is drastically perturbed ( Figure 2C and 2D ) , E-cad protein levels are higher in pen/lgl2 mutants . We further estimated the expression levels of snail family members . Consistent with up-regulation of E-cad levels , none of the snail family members shows increased expression in pen/lgl2 mutants . These data indicate that in pen/lgl2 mutant larvae EMT of basal epidermal cells is facilitated by mis-localizing E-cad rather than its snail mediated repression . We confirmed the tissue specificity of differential expression of the genes mmp9 , sgk1 , cldn7 , krt5 and E-cad by in-situ hybridization ( ISH ) ( Figure 3C–3L ) . We verified the up-regulation of mmp9 as well as sgk1 and down-regulation of krt5 specifically in the basal epidermal cells . Interestingly , the up-regulation of the tight-junction gene cldn7 was observed mainly in the cells that form epidermal cell aggregates in the ventral jaw region and in the fin-fold ( Figure 3H ) . Our analyses indicate that basal epidermal cells in pen/lgl2 larvae exhibit both the morphological and transcriptional characteristics of cells undergoing EMT . We conclude that pen/lgl2 function is essential to suppress epidermal neoplasia and EMT . Thus , pen/lgl2 acts as a recessive tumor suppressor gene in vertebrates . Furthermore , canonical snail mediated repression of E-cad is not involved in EMT in pen/lgl2 mutant . Instead , EMT is facilitated by removal of E-cad from the plasma membrane . As human carcinomas are often a consequence of massive over activation of growth factor signaling [36] , we assayed pen/lgl2 mutant larvae for the phosphorylation level of the mitogen activated protein kinase Erk , a common member of growth factor signaling cascades , by western blot [37] . We found elevated levels of phosphorylated Erk in the mutants compared to their wild type siblings ( Figure 4A ) . To identify which growth factor signaling is activated , we treated larvae from heterozygous pen/lgl2 carriers with inhibitors for three different receptor tyrosine kinases ( RTKs ) , FGFR , IGFR and EGFR , starting at 96 hpf just prior to the appearance of the pen/lgl2 mutant phenotype . These treatments revealed that an inhibitor of ErbB ( PD168393 ) reduced the levels of phosphorylated Erk ( Figure 4B ) and rescued the epidermal neoplasia phenotype as well ( Figure 4D and 4E ) . Similar rescue in the epidermal phenotype was observed with another ErbB inhibitor AG1478 ( data not shown ) . In contrast , Inhibition of FGFR ( SU5402 ) neither affected the levels of phosphorylated Erk ( Figure 4C ) nor did it rescue the epidermal overgrowth phenotype ( data not shown ) . Similarly , there was no rescue in the epidermal overgrowth phenotype when IGFR signaling was inhibited using AG1024 ( data not shown ) . Genotyping of an entire clutch treated with ErbB inhibitor ( PD168393 ) revealed the expected Mendelian proportion ( 27 out of 120 ) of pen/lgl2 homozygous larvae . We further asked whether the EMT and over-proliferation phenotypes were suppressed after treatment with PD168393 . Indeed , cytokeratin and E-cad antibody stainings of inhibitor treated pen/lgl2 mutant larvae were indistinguishable from wild type siblings ( Figure 4H and 4K ) . Consistently , epidermal cell proliferation and mmp9 transcript levels were strongly reduced ( Figure 4L–4N , Figure 4O–4Q ) . However , hemidesmosomes did not form in PD168393 treated pen/lgl2 mutant larvae as revealed by electron microscopic analysis ( Figure S4 ) . Our data indicate that loss of functional pen/lgl2 results in over activation of ErbB signaling which promotes over proliferation of basal epidermal cells as well as cellular EMT by transcriptional modulation of EMT regulators . We further conclude that over-activation of ErbB signaling is not the cause for the absence of hemidesmosomes in the pen/lgl2 mutant . This observation further indicates that disruption of pen/lgl2 primarily affects hemidesmosome formation , which is consistent with the previous analysis [20] , [21] . In mammals four ErbB receptors are known , ErbB1 to ErbB4 , which get activated upon the binding of ligands such as EGF , HB-EGF , neuregulins , betacellulin [38] . Ligand binding leads to the formation of homo- or heterodimers amongst these receptors , resulting in signal transduction [38] ) . Our bioinformatic and phylogenetic analysis coupled with the previous analysis of some of the family members [39] , [40] revealed that with the exception of erbB2 , all other members of this family exist in duplicates in the zebrafish ( Figure 5A ) . erbB2 as well as pcs/erbB3b zebrafish mutants exhibit defects in glia development and regeneration [39]–[42] . We found erbB2 to have an additional epidermal phenotype in the fin-fold ( Figure 5C ) and it is also expressed in the epidermis ( Figure 5E ) . To determine which of the ErbB receptors is activated in pen/lgl2 , we performed loss of function studies in a pen/lgl2 mutant background . We knocked down erbB1a with a splice site antisense morpholino . Injections of erbB1a morpholino did not interfere with the pen/lgl2 phenotype but reproduced the cardiovascular phenotype published earlier [43] ( data not shown ) . However , we cannot exclude the involvement of erbB1a in promoting the EMT and growth phenotype in pen/lgl2 mutant larvae as we found the effect of the morpholino to decrease beyond 48hpf , possibly due to dilution effects ( Figure S2 ) . Double mutants of pcs/erbB3b−/− , pen/lgl2−/− genotype did not show suppression of the pen/lgl2 neoplastic phenotype and immuno-histological analysis using the pan 1–8 Cytokeratin antibody did not reveal any reduction in strength and initiation of EMT phenotype ( data not shown , Figure 5I ) . Taken together , these results demonstrate that erbB3b function is not essential for neoplasia and EMT phenotype in pen/lgl2 mutants . In zebrafish , lgl2 and erbB2 are both located on chromosome 12 ( 23 . 6mb distance ) . To study double mutants , chromosomal recombinants were made ( see Materials and Methods ) . We investigated progeny from lgl2−/+ , erbB2−/+ double heterozygous fish ( 3 crosses; n = 296 ) , which were sorted for the morphological epidermal lgl2 phenotype at 108hpf as well as 132hpf and subsequently genotyped . We identified 40 larvae that were homozygous for both lgl and erbB2 . In 37 of these , no epidermal phenotype was detected up to 132hpf ( Figure 6C ) . In contrast , all lgl2 larvae with erbB2+/− or erbB2+/+ genotype showed strong phenotypes or lethality at the same stage ( Figure 6B , Table S2 ) . In a separate experiment , we analyzed Cytokeratin and E-cadherin localization , mmp9 expression and BrdU incorporation in lgl2−/− , erbB2−/− double mutants at 108hpf when the lgl2 phenotype becomes apparent . The morphology of the epidermal cells in the double mutant larvae , as revealed by Keratin staining , appeared completely normal at 108hpf Figure 6D–6F ) . Occasionally , epidermal cells in pen/lgl2; erbB2 double mutant larvae exhibited milder changes in Keratin organization , indicative of altered cellular morphology at 132 hpf ( data not shown ) . E-cadherin localization appeared normal in the epidermis of the double mutants ( Figure 6G–6I ) and mmp9 expression levels in the epidermis of double mutant larvae were comparable to those in wild type ( Figure 6M–6O ) . Interestingly , however , the BrdU incorporation analysis revealed that ( Figure 6J–6L ) there is no significant decrease ( t-test , p>0 . 05 ) in the epidermal cell proliferation in a predefined area in lgl2−/− , erbB2−/− double mutants ( 29±1 . 0 , n = 3 ) as compared to lgl2−/− , erbB2−/+ ( 32±9 , n = 3 ) larvae . Our data suggest that erbB2 but not erbB3b mediated signaling is responsible mainly for the EMT phenotype in pen/lgl2 mutant basal epidermis . We did not observe rescue in the epidermal cell-proliferation phenotype in pen/lgl2; erbB2 double mutants and epideramal cell morphology was altered as well at later stages indicating that erbB2 deficiency doesn't completely rescue the phenotype . Since the inhibitors completely rescue both the proliferation as well as EMT phenotypes , we propose that other ErbB receptors might be involved in promoting epidermal cell-proliferation in pen/lgl2 mutants .
Impaired cell polarity is one of the hallmarks of carcinoma but was considered to be a secondary effect for a long time . Recent findings however suggest it can also be a cause rather than a consequence of tumor progression [18] , [19] , [44] , [45] . In many epithelia , the apico-basal polarity is established by the activity of the apical aPKC-Par3-Par6 pathway and baso-lateral Lgl-Scrib-Dlg pathway . Surprisingly , only loss of function in baso-lateral pathway components leads to epithelial or brain neoplasia phenotypes in Drosophila [45] . In vertebrates , two components of this pathway , lgl and dlg , have multiple orthologues [45] . Loss of lgl1 function results in brain hyperplasia in the mouse [17] . In zebrafish loss of lgl2 function leads to over-proliferation of epidermal cells [20] . Here , we demonstrate that transplantation of pen/lgl2 homozygous mutant cells during blastula stage into wild type embryos results in the formation of tumor like structures in the epidermis after 7 days . This analysis clearly shows that lgl2 behaves as a tumor suppressor gene in vertebrates . The basal epidermal cells in pen/lgl2 mutants do not form hemidesmosomes and display an altered cell morphology instead of the polygonal cell shape found in wild type larvae [20] . E-cad localization at the plasma membrane is reduced in the mutant epidermal cells indicating that epidermal cells undergo EMT . Indeed , GFP labeling of basal epidermal cells revealed that in pen/lgl2 mutant larvae these cells show continuous changes in their morphology , project lamellipodia and move as a sheet . This sheet like movement has been described as a metastable cell state in the context of EMT [28] . Consistent with the cellular analysis , the expression profiling of lgl2 mutant basal epidermal cells revealed differential expression of the known EMT markers and regulators such as matrix-metalloproteinases and keratins . Intriguingly , the levels of E-cad RNA are high in the pen/lgl2 mutant . This is in contrast to known developmental scenarios of EMT such as the delamination of neural crest cells or gastrulation where E-cad expression is downregulated at the transcriptional level by a transcriptional repressor , snail [34] , [35] . In addition , structural components of tight junctions , such as several claudins , are also up-regulated in pen/lgl2 mutants . Thus , the expression profiles of cell adhesion molecules in pen/lgl2 larvae resemble several human breast carcinomas including an inflammatory breast cancer ( IBC ) , a highly aggressive subtype of human breast cancer , which has been characterized by E-cad and erbB2 over-expression [46]–[51] . The up-regulation of claudin genes is intriguing . However , it is not clear whether Claudins localize properly in the epidermal cells . Even if the Claudins do localize properly , the in-situ expression analysis suggests that claudins are up-regulated mostly in the cellular clumps that are formed in the median finfold or in the ventral jaw region . It is thus clear that claudins are not expressed during the process or EMT but rather when the mesenchymal cells re-acquire partial epithelial phenotypes while forming tumor like aggregation . However , further analysis is required to test this notion . How does deficiency in components of the baso-lateral pathway lead to epithelial cell proliferation and EMT ? It has been suggested that the protein Scrib stabilizes the coupling between E-cad and the Catenins and thus behaves as a regulator of epithelial cell adhesion and migration [52] . Our data show that for Lgl2 the mechanism to suppress EMT and tumor formation is fundamentally different . Lgl2 may not manifest its function by stabilizing the coupling between E-cad and Catenins . Previously , we have shown that neither the loss of maternal nor zygotic lgl2 function primarily affect E-cad localization [20] , [21] . Consistent with mammalian data , knockout of E-cad in the mouse or zebrafish epidermis does not result in EMT [21] , [53]–[55] . This indicates that although the loss of E-cad facilitates EMT [56] , it may not be sufficient to induce it in animal models . We show that inhibition of erbB2 signaling , either genetically or by small chemical inhibitors , leads to suppression of EMT and a neoplastic phenotype in pen/lgl2 mutant larvae . Intriguingly , E-cad membrane localization is restored in these larvae , indicating that the loss of E-cad is a consequence of activation of ErbB signaling rather than a cause of it . Thus , our analyses presented here suggest that Lgl2 acts as a tumor suppressor by regulating the amplitude of ErbB signaling in the epidermis . Since we did not observe snail-mediated down-regulation of E-cad , we propose that an erbB2 dependent pathway in pen/lgl2 mutants leads to the destabilization of E-cad at adherens junctions . Indeed in pen/lgl2 mutant epidermal cells , known modifiers of E-cad function , such as mmp9 and sgk1 are up-regulated ( Figure 3 ) . While Mmps are known to be involved in ecto-domain shedding of E-cad [57] , [58] , Sgk1 functions in the phosphorylation of Ndrg1 , a protein involved in vesicular recycling of E-cad [59] , [60] . Additionally , recently published data from cell culture suggests an involvement of RTK signaling in the destabilization of adherens junctions via Numb [61] . Here , Numb functions as an adapter protein coupling E-cadherin to the Par polarity-complex . This interaction was shown to be sensitive to elevated levels of RTK signaling , leading to mislocalization of E-cadherin [61] . Further analysis involving loss of function of mmps and sgk1 as well as studies of Numb localization in the pen/lgl2 mutant larvae would be necessary to clarify their contributions to the EMT phenotype . The primary function of Lgl2 in hemidesmosome formation [20] , [21] is not dependent on ErbB signaling as hemidesmosomes do not form in pen/lgl2 mutants even after the inhibition of ErbB signaling ( Figure S4 ) . This suggests that the loss of hemidesmosomes might be the cause but not a consequence of the activation of ErbB signaling . This hypothesis is supported by the fact that components of hemidesmosomes ( e . g . Itga6/Itgb4 ) , physically interact with the ErbB2 receptor tyrosine kinase [62]–[64] . Thus , it is possible that mislocalization of hemidesmosomal components in pen/lgl2 mutants lead to an activation of ErbB2 . ErbB signaling plays an important role in the development of human carcinomas , as it is able to induce proliferation and EMT and further is able to suppress apoptosis [36] , [65] . This is in particular true for ErbB2 which is over-expressed in more than 25% of all breast carcinoma [66] . A causal link between ErbB activation and loss of cell polarity , which is one of the hallmarks of carcinomas , has recently been established . It has been shown that activated ErbB2 associates with Par6-aPKC leading to disruption of the apico-basal polarity [67] . While ErbB2 regulates apico-basal cell polarity by interacting with apical polarity components like Par6-aPKC , our analyses presented here suggest that one of the basolateral pathway components , Lgl2 , regulates the activation of ErbB2 . Thus , there seems to be a reciprocal interaction between cell polarity regulators and ErbB2 signaling . Interestingly , although ErbB2 activation induces cell proliferation , the association of ErbB2 with Par6-aPKC was not essential for regulating cell proliferation indicating that ErbB2 affect the cell proliferation independent of disruption of apico-basal cell polarity [67] . Our data suggest that at the organismal level loss of ErbB2 does not affect the epidermal cell proliferation phenotype in lgl2 mutants but only prevents EMT . Since inhibitors , which do not discriminate between various ErbB family members rescue both , proliferation as well as EMT phenotypes , it appears that activation of more than one ErbB family member might be involved in promoting epidermal neoplasia in pen/lgl2 mutants . Thus , cell proliferation and EMT phenotypes are not coupled in pen/lgl2 mutant larvae . The ErbB signaling pathway includes multiple ligands and receptors in vertebrates . ErbB1 and ErbB4 represent discrete RTKs , as they contain ligand binding sites as well as a kinase activity , essential for auto-phosphorylation and signal transduction . In contrast , ErbB2 and ErbB3 do not independently transduce extracellular signals as homodimers since ErbB2 has no ligand-binding domain and ErbB3 has no kinase domain . Thus , ErbB2 and ErbB3 must interact with each other or with other ErbB receptors to transduce signals [38] . Analysis of the Danio rerio genome revealed that genome duplication events have led to the duplication of erbB1 , erbB3 and erbB4 making the situation even more complex . We found that homozygous mutations in erbB2 but not erbB3b dramatically rescued the EMT phenotype , establishing ErbB2 signaling to be responsible for the onset of EMT . However , the direct activator of ErbB2 , in promoting the EMT phenotype in lgl2 mutants remains unidentified at this point . To summarize , lgl2 function is essential for tumor suppression and EMT in the vertebrate epidermis . pen/lgl2 mutant cells are able to induce the formation of epidermal tumors when surrounded by wild type cells . In the absence of Lgl2 , hemidesmosomes do not form and ErbB signaling is activated , which results in induction of pathways involved in promoting EMT via mislocalization of E-cad as well as proliferation . Amongst several erbB paralogues , erbB2 is more directly involved in transducing the signal . Thus , the pen/lgl2 mutant would serve as a very good model to perform chemical screens for compounds that are able to suppress erbB signaling or important downstream effectors and thus have high potential for cancer therapeutic research .
The morphological and immuno-histological analysis of pen/lgl2 mutant was carried out in Tuebingen ( TUE ) and WIK background . For transplantations , embryos from pen/lgl2 mutants in the background of an β-actin::GFP transgenic line were used as donors and from albino fish as recipients . Live imaging was performed in transgenic ΔNp63::Gal4 , UAS::GFP line . The erbB2 mutant allele used in this work is st61 [39] . The erbB3b mutant allele is HJ036 , which contains a C to A point mutation at position 156 leading to a premature stop after 50 amino acids [41] . As lgl2 and erbB2 are located on the same linkage group , to generate double mutants , we crossed pen/lgl2+/− fish with erbB2+/− fish and out-crossed the obtained trans-heterozygote F1 fish to albino fish . The F2 generation were then screened by genotyping for both , the pen/lgl2 and erbB2 mutations . Protein sequences for ErbB zebrafish paralogues and human orthologues were obtained from Genebank ( http://www . ncbi . nlm . nih . gov ) and through Ensembl ( http://www . ensembl . org/Danio_rerio ) databases . Alignments were performed using ClustalW . Phylogenetic analysis was run using neighbor joining , maximum parsimony and minimum evolution algorithms , ( MEGA4 , http://www . megasoftware . net/ ) . All analysis methods showed similar tree morphology with comparable bootstrap values ( 1000 replicates ) . Human orthologues ( Genebank ID ) : ERBB1: 1956 , ERBB2: 2064 , ERBB3: 2065 , ERBB4: 2066 . Zebrafish paralogues ( Ensembl transcript ID ) : erbB1a: ENSDART00000027219 , erbB1b: ENSDART00000031151 , erbB2: ENSDART00000003932 , erbB3a: ENSDART00000014892 , erbB3b: ENSDART00000049893 , erbB4a: ENSDART00000092114 , erbB4b: ENSDART00000100398 The ΔNp63 promoter was cloned by enzymatic restriction of BAC dkey-13d19 with HhaI ( NEB ) and subsequent blunt-end cloning of a resulting 4 . 96 kb fragment into a plasmid containing a Gal4 , UAS::GFP expression cassette and mini-TOL2 sites ( based on [68] , [69] ) . Transgenesis was achieved by simultaneous injection of plasmid DNA and transposase RNA at 1-cell stage using WPI PV830 pneumatic injection system followed by F1 screen for GFP positive larvae . 5-day-old wild type and pen/lgl2 mutant larvae were incubated with 10 mM BrdU solution in 2% DMSO in embryonic medium ( E3 ) for 2 hours . After treatment , larvae were washed several times in E3 fixed overnight in 4% PFA in PBS at 4°C . Staining was performed as described below . For IHC procedures the following antibodies were used: Anti-BrdU antibody ab6326 ( abcam ) ; anti Cytokeratin antibody Ks pan 1–8 ( Progen Biotechnik ) and anti E-cad antibody ( BD Transduction Laboratory ) . Embryos were either fixed in 4% PFA ( E-cad , BrdU ) or in Dent's fixative ( cytokeratin ) . After downgrading the larvae to 0 . 1 M phosphate buffer ( PB ) , they were washed with PBT ( PB+0 . 8% Triton X-100 ) five times and blocked in 10% normal goat serum . For BrdU staining , larvae were treated with 4 N HCl for 20 min . , washed in PB and blocked in 1% BSA for 1–3 hours . Antibodies were diluted as: Ks pan 1–8 ( 1∶10 ) , anti-E-cad ( 1∶250 ) , anti-BrdU ( 1∶50 ) and samples were incubated at room temperature for 4 hours or overnight at 6–8°C . Afterwards , larvae were washed five times in PBT , incubated with Cy3 or Alexa 488 anti-mouse or anti-rat antibodies , post fixed in 4% PFA and upgraded in 70% glycerol for fluorescence/light microscopy . DIG-labeled RNA probes for cldn7 , krt5 , mmp9 and sgk1 were prepared from larval 5dpf total cDNA obtained from pen/lgl2−/− mutants . In situ hybridization was performed using Intavis in-situ robot ( model: insituPro VSi ) . Probe templates have been amplified with the following primer combinations by PCR reaction and subsequently cloned into pGEM-T Easy Vector ( Promega ) . DIG-labeled probes were synthesized using T7 and Sp6 RNA polymerases ( Roche ) . E-cad: 5′-TTACTTCTGCTATTGCTTGCT-3′; 5′-TCATAGTCTTGGTCGTTTCCT-3′ cldn7: 5′-TGGCACATAAAGGACTGCAA-3′; 5′-CGATGAAAATAGCTGCACCA-3′ erbB2: 5′-GTCATCCAGAACGAAGATCAG-3′; 5′-CATCAGTCTCCAGATCTCCA-3′ krt5: 5′-CAGGAGCTCAGTGTCCTTCC-3′; 5′-CGGTTGTTGAGGGTCTTGAT-3′ mmp9: 5′-GCTGCTCATGAGTTTGGAC-3′; 5′-CCGAGCTTCTCGATTTTACG-3′ sgk1: 5′-ATGGAACGACGTCAACCTTC-3′; 5′-GCGTAAGCTTCTTGGCATTC-3′ Transplantations were carried out at blastula stage . After transplantations , donor and corresponding recipients embryos were cultured together ( 3–5 in number ) in a 24 well plate . The pen/lgl2 mutant donors were identified at 4 . 5dpf by phenotype or by molecular genotyping at blastula stage ( RFLP ) . The host larvae that received mutant or wild type clones ( GFP-positive ) in the skin were sorted and further raised up to 7–8 dpf and analyzed by microscopy for tumor phenotypes . For screening , all inhibitors ( SU5402; AG1024; PD168393; AG1478 all Calbiochem ) were used at 10–50 µM concentration in 1% DMSO in E3 Medium starting at 96hpf . For BrdU , IHC and ISH experiments , PD168393 and AG1478 were used at a concentration of 10 µM starting at 96hpf . For western blots , mutants were identified by the development of the characteristic lgl2 mutant phenotype at 108hpf . Three times 40 mutants and equivalent number of wild type larvae were collected at 108hpf and than treated with either DMSO as a control , or inhibitors for ErbB signaling ( 10 µM ) or FGFR signaling ( 20 µM ) for 12 h . Subsequently , larval tails were collected by cutting posterior to the swimming bladder on ice . Proteins were extracted in DXB ( 25 mM Hepes , pH 6 . 8 , 50 mM KCl , 1 mM MgCl2 , 1 mM DTT , 250 mM sucrose ) containing Roche Complete protease inhibitors ( Cat No . 11836153001 ) and Pierce Halt phosphatease inhibitors ( Cat No . 78420 ) . Protein concentration was determined using OD at 280 nm ( NanoDrop ) . For PAGE , NuPage 4–12% gradient gels ( Invitrogen ) were used . After transfer , equal loading was re-checked by poinseau red staining . Antibodies used were: p-ERK ( M9692; 1∶600 ) , Erk ( M5670; 1∶500 ) , α-Tubulin ( T9026; 1∶10000 ) ( all from Sigma ) . Secondary antibodies used were HRP conjugated anti-mouse ( p-Erk , Tubulin ) and anti-rabbit ( Erk ) . Detection was performed using chemiluminiscence ( ECL+; GE Healthcare ) . The microarray experiment was conducted using tissues ( tail ) posterior of the anal opening of wild type ( Tuebingen ) and pen larvae at 5 dpf . RNA for microarray analysis was extracted from four biological replicates using TRIZol Reagent ( Gibco BRL , Eggenstein , Germany ) . Complementary RNA was prepared from 1 µg total RNA from each replicate as described in the Agilent Low RNA Input Linear Amplification kit manual ( Agilent Technologies , Palo Alto , CA , USA ) . Double-stranded cDNA was synthesized using the reagents from this kit , and Cy3- or Cy5-labeled cRNA was prepared by cDNA in vitro transcription in the presence of cyanine 5-CTP or cyanine 3-CTP dyes . Fluorescently labeled RNA was then purified with the Qiagen RNEasy spin columns , according to the manufacturer's protocol ( Qiagen , Hilden , Germany ) . After purification , cRNA was fragmented and used to hybridize to the zebrafish G2519F 4X44 microarray platform containing 4 duplicated arrays of the 22 , 000 probe-set design ( Agilent Technologies , Palo Alto , CA , USA ) . Four biological replicates were conducted , including two dye swap experiments to minimize the effect of any potential dye bias . Hybridization , washing , and scanning were performed according to the manufacturer's protocol . The microarrays were scanned on a Genepix Axon 4000B scanner ( Molecular Devices , Union City , CA , USA ) at five micron resolution with five-line averaging . Raw expression values from each probeset were extracted using the Genepix Pro 6 . 0 feature extraction software , and features were flagged manually for poor quality . The data were then analyzed in the R statistical programming environment using the Bioconductor module Limma [70] , [71] . Duplicate probesets on each array were considered as technical replicates for the analysis in addition to the 4 biological replicates on separate arrays . A standard linear model for differential expression , with the Limma module in Bioconductor , was used to identify genes up and down regulated in the mutant versus wild type experiment . The resulting p-values from the hypothesis tests were adjusted for multiple testing with the false discovery approach ( FDR ) to control for false positives [72] . In addition , the empirical Bayes approach automatically adjusts raw p-values for multiple testing and generates a B-statistic that may also be used for ranking differentially expressed genes [70] . All microarray data may be accessed through the ArrayExpress repository on the European Bioinformatics Institute database website ( http://www . ebi . ac . uk/microarray-as/ae/ ) . As for the microarray experiment , larval tails posterior to the anal opening were used as tissue sample ( 3 technical replicates of each biological replicate; 3 biological replicates ) . Total RNA was isolated using TRIZOL reagent ( Invitrogen ) and cDNA was synthesized using AMV cDNA Synthesis Kit ( Invitrogen ) with oligo-dT15 primers ( Promega ) . SYBR Green ( BioRad ) was used for quantitative real-time PCR . Time emission readings were recorded with DNA Engine Opticon 2 ( MJ Research ) , and analyzed as described [73] . gapdh was used for normalization ( ΔΔC ( t ) method ) as described [74] . Statistical significance was determined using student's t-test . Genotyping to indentify mutants was done by a PCR based restriction fragment length polymorphism ( RFLP ) method for lgl2- and erbB2- or by DNA sequencing for erbB3b mutants . The RFLP analysis was done as follows- the mutations in erbB2 cause a loss whereas in lgl2 a gain of a restriction site . PCR product from genomic DNA samples obtained by the primer pairs: 5′- ATGCATACCTTCCTGGAGTAG-3′; 5′-TGTGGTTCTAGTGGAGGAGGA-3′ ( for lgl2 ) or 5′-TGAAGAATGCTGGTAGCTGG-3′ and 5′-GGACTCAGCAAAGG ACTTAC-3′ ( for erbB2 ) was digested with either SfcI ( for lgl2 mutation ) or BsrGI ( for erbB2 mutation ) resulting in a genotype specific DNA band pattern ( Figure S3 ) . The erbB3b genotyping was done by scoring for the premature stop at position 156bp by DNA sequencing of a PCR product obtained from the genomic DNA using the primer pair 5′-CGCTCTCCTGTTCCTCTGTG-3′; 5′-ACCCTCTTCCTCCATTGTCC-3′ ( Figure S3 ) . In case of PFA fixation , individual genomic DNA samples were sampled before fixation ( head tissue ) . The genotype of larvae used in ISH experiments was scored by the development of the characteristic lgl2 phenotype . Time-lapse movies were acquired using AxioVision 4 . 6 ( Zeiss ) and compressed in the Codec H264 . For encoding in H264 , the OSS Virtual Dub was used . Cell area ( Figure 2G ) was determined using the “outline” tool included in Zeiss AxioVision 4 . 6 ( Zeiss ) on three biological replicates each from wild type and pen/lgl2 . Scale bars indicate equal sizes within one figure . | In metazoans , the body surface and linings of several organs are formed from membranous tissue called epithelia . The functions of epithelia include secretion , absorption , and protection . Epithelial cells exhibit polarized distribution of several proteins , which is essential for their function . In carcinomas , which are cancers of epithelial origin , this epithelial cell polarity is impaired . Intriguingly , defects in cell polarization can also lead to tumorigenesis in some animal model systems . It is thus important to understand how cell polarization and epithelial growth control are linked so as to treat the carcinomas better by identifying new drug targets . Here we show that in zebrafish a gene named lethal giant larvae 2 ( lgl2 ) , which is essential for the establishment of epithelial cell polarity , also acts as a suppressor of malignant growth properties in the epidermis , an epithelial component of the skin . We further show that in absence of lgl2 function increased epidermal growth factor receptor activity imparts malignant properties to the epidermal cells . Thus , we report here a mechanism by which epithelial cells acquire malignant characteristics when cell polarity is impaired in absence of lgl2 function . | [
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"genetics"... | 2009 | Lgl2 Executes Its Function as a Tumor Suppressor by Regulating ErbB Signaling in the Zebrafish Epidermis |
Rift Valley Fever ( RVF ) is a mosquito-borne zoonosis , which may cause significant losses for the livestock sector and have serious public health implications . Egypt has been repeatedly affected by RVF epidemics , mainly associated to the importation of animals from sub-Saharan countries , where the disease is endemic . The objective of our study was the improvement of the surveillance and control strategies implemented in Egypt . In order to do that , first we evaluated the legal trade of live animals into and within Egypt . Then , we assessed the risk of Rift Valley Fever virus ( RVFV ) transmission within the country using a multi-criteria evaluation approach . Finally , we combined the animal trade and the risk of RVFV transmission data to identify those areas and periods in which the introduction of RVFV is more likely . Our results indicate that the main risk of RVFV introduction is posed by the continuous flow of large number of camels coming from Sudan . The risk of RVFV transmission by vectors is restricted to the areas surrounding the Nile river , and does not vary significantly throughout the year . Imported camels are taken to quarantines , where the risk of RVFV transmission by vectors is generally low . Then , they are taken to animal markets or slaughterhouses , many located in populated areas , where the risk of RVFV transmission to animals or humans is much higher . The measures currently implemented ( quarantines , vaccination or testing ) seem to have a limited effect in reducing the risk of RVFV introduction , and therefore other ( risk-based ) surveillance strategies are proposed .
Rift Valley Fever virus ( RVFV ) is an arbovirus which belongs to the Phlebovirus genus , within the family Phenuiviridae ( order Bunyavirales ) . It is the causative agent of Rift Valley Fever ( RVF ) that may affect both humans and animals , mainly ruminants , but also camels . In livestock RVF causes large number of abortions ( abortion storms ) and high mortalities in young animals . In humans , RVFV infection generally causes a self-limiting , acute and febrile illness , although in some cases disease progresses to more severe forms , with neurological disorders , blindness , hemorrhagic fever or thrombosis [1] . RVFV infection of animals may occur by the bite of an infected mosquito ( mainly of the Culex or Aedes genera ) or through direct contact with infected animal tissues or fluids [2] . Infection via mosquito bites is considered the primary mode of transmission in the first stages of the epidemic , while contact with contaminated material is more relevant during the amplification stage . RVFV within infected tissues is quite resistant to inactivation and may remain infectious for a few days [2] . In fact , the majority of human cases are likely to occur in farmers , veterinarians or slaughterhouse employees due to contact with infected material [3] . RVF may have a dramatic consequence on producers and livestock industries , including the impact on international trade [4] . The public health impact of RVF may also be severe , as demonstrated by the 200 , 000 people infected and 600 fatal cases reported in the 1977 epidemic in Egypt [5] . Besides that epidemic , Egypt has been affected by RVF in 1993 , 1994 , 1997 , and finally in 2003 [6] . In fact , Mroz and collaborators [7] have recently reported the low level circulation of RVFV in some areas of Egypt . The continuous importation of viraemic ruminants or camels , mainly from Sudan , was considered the main source of introduction of RVF into Egypt [6] . Furthermore , those importations may also result in the introduction of other exotic diseases such as the Middle East Respiratory Syndrome coronavirus ( MERS-CoV ) [8] . RVF is considered to be endemic in sub-Saharan African countries ( including Sudan ) , with major outbreaks associated with periods of heavy rainfall and flooding [9] . Socio-economic changes , with human population growth and the associated increased demand for meat , or changes in the meat market prices could result in greater controlled and uncontrolled transboundary movements of livestock . A consequence of all those changes would be an increase of the risk of introduction of RVFV into areas of the Mediterranean basin and the Middle East [3] . While surveillance and diagnostic methods for RVFV are available , RVFV control is hindered by the difficulties of vector control , and the fact that vaccines are only available for ruminants , with either inactivated vaccines ( with a limited efficacy ) or live attenuated vaccines ( with a residual pathogenic effect ) [3 , 10] . Therefore , the best strategy to protect countries at risk of RVFV introduction is the implementation of regional monitoring and control programmes in endemic countries , as well as the establishment of early warning systems in the countries at risk . The Egyptian authorities implement both measures to prevent the introduction of RVFV ( vaccination of imported animals , quarantines and testing of a proportion of imported animals ) and measures to prevent the spread ( vaccination of the Egyptian susceptible population ) . If those measures were effective , there would be no risk of RVFV introduction and spread , however , their efficacy has not been evaluated , and some deficiencies have been identified in the past [6] . Given the limitation of resources , risk-based methods ( i . e . focusing on those areas and/or practices that pose the highest risk ) may be considered as a cost-effective alternative for the surveillance of RVF in Egypt . Many of those risk-based methods are data-driven , which rely on comprehensive information about disease-associated events . In a data-scarce context , knowledge-driven methods , which rely on the previous information on the factors associated with disease occurrence , offer an alternative approach to identify areas at risk [11] . The main objectives of this study were: That will allow the improvement of the surveillance and control measures currently being implemented by the Egyptian Authorities , and make a better use of the resources available . Improvement of RVFV surveillance should allow the early detection of the disease , while the improvement of control measures should help prevent cases of RVFV infection in both animals and humans .
The spatial risk of RVFV transmission within Egypt was estimated using a GIS-based multi-criteria evaluation ( MCE ) approach [12] , which allows to combine the ( spatial ) data on the different factors that may influence the risk of a given disease . In the case of RVF it is essential to account for the distribution of both the susceptible hosts and the vectors of RVFV [13] . All layers were transformed into a raster format , with 1 km × 1 km spatial resolution and the common UTM 35N projection . R software [14] was used for both the analyses and generation of maps . Besides the favorability for transmission in a given area , the actual risk of RVF occurring in that area is determined by the probability of the virus being introduced into that location . Given that in Egypt imported animals are quarantined on specific premises on arrival to the country , and that transmission within those quarantine stations and to animals in their surroundings may occur , those will be the risk locations of primary interest . No measures to protect animals from mosquitos are taken on those quarantines . After quarantine , imported camels may be taken to markets or to slaughterhouses , where transmission within the premises and to animals in their surroundings may also occur , and therefore those will be the risk locations of secondary interest . Studies on the dispersal of Cx . pipiens in urban habitats found maximum flight ranges of 1 . 98 km [29] and 2 . 48 km [30] . However , given the low probability of recapturing a marked mosquito at large distances from the point of release , we used a conservative radius of up to 5km for the buffer area . We also assessed the effect of changing the size of the radius on the risk of RVF transmission .
Animals imported into Egypt are not randomly distributed throughout the country , instead they are transported to specific locations where the risk of RVFV transmission by vectors will be quite variable . First , they are taken directly to specific quarantine premises depending on the species and origins ( Fig 10A ) . After quarantine , imported camels also follow an established movement pattern: they may be either be taken to slaughterhouses ( Fig 10B ) or to animal markets ( Fig 9B ) . In contrast , all cattle are slaughtered within the quarantine facilities . The summary estimates of the risks of RVFV transmission by vectors at those main locations after the importation of animals , i . e . markets , quarantines and camel slaughterhouses , considering buffers of either 5000 or 2000 meters , are shown in Table 3 . Of the quarantines ( Fig 3 and Fig 10A ) , Abu Simbel , where a significant proportion of the camels and all the cattle imported from Sudan arrived , seemed to have a low risk of RVFV transmission by vectors ( between 0 . 04 and 0 . 08 depending on the buffer considered ) . For Shelateen quarantine , where camels imported from eastern Sudan arrived , the risk was 0 . For Sahl Hasheesh , where cattle from Ethiopia arrived , the risk was extremely low ( between 0 and 0 . 01 depending on the buffer ) . For Adabiya , where camels from Ethiopia arrived , the risk was also relatively low ( between 0 . 07 and 0 . 14 ) . In contrast , for Al Qata , where also camels from Ethiopia arrived , the risk of RVFV transmission by vectors was quite high ( between 0 . 70 and 0 . 83 ) . Of the two markets with the highest risk-ranking score ( Fig 5 ) , Aswan had a risk of RVFV transmission by vectors relatively low ( between 0 . 15 and 0 . 16 ) , while Birqash market had risk of RVFV transmission by vectors quite high ( between 0 . 70 and 0 . 74 ) ( Fig 9B ) . Of the remaining high-risk markets there are 10 with risk of RVFV transmission by vectors estimates above 0 . 7 . Of them , 7 are located in risk-area 1 in Fig 8 , in the governorates of Gharbia ( 3 ) , Qalyubia ( 3 ) and Monofia ( 1 ) . The other 3 are located in Minya governorate ( risk-area 2 ) . In risk areas 3 and 4 , there is only one high-risk market in each with risk of RVFV transmission by vectors above 0 . 5 , in Qena and Aswan governorates , respectively . The camel slaughterhouses ( Fig 6A and Fig 10A ) are mostly located in the proximities of Cairo , where conditions are favourable for RVFV transmission and therefore their risk are relatively high ( Table 3 ) . Of the three slaughterhouses that killed the most animals , Elbasateen had a risk 0 . 49 regardless of the buffer distance , while Elwarak had a risk between 0 . 47 and 0 . 50 , and Kerdassa between 0 . 57 and 0 . 69 .
Camel meat is an important source of cheap protein for the Egyptian people . However , as the resident camel population in Egypt is really small , 66 , 233 animals in 2015 ( according to OIE data ) and located mainly in remote areas , large number of camels need to be regularly imported into Egypt , the majority of which come from Sudan ( 86% of the total in 2015 ) and the rest from Ethiopia . The flow of camels is continouos throughout the whole year and the number of animals imported per year seem to have an increasing trend . In 2015 , an average of 18 , 000 camels were imported per month . During the first RVF epidemic in Egypt in 1977 , mortality and abortions were reported in camels in Aswan governorate ( in the south of the country ) , and RVFV was isolated from this species [32 , 33] . Therefore , even though the source of the outbreak could not be determined , the possibility of introduction of the disease from Sudan by camels was considered the most likely explanation . In the following epidemics ( 1993 , 1994 , 1997 and 2003 ) , importation of camels , mainly from Sudan , was also considered as a possible source of introduction , although no proper epidemiological investigations were carried out , and alternative mechanisms were considered [6 , 26] . As a result , the role of camels in the epidemiology of RVFV remains unclear . In fact , it has been suggested that camels may be essential for RVFV transmission in some ecosystems where their density is high , which would allow viral amplification and transmission to alternative hosts , but not in others [34] . Furthermore , data on factors such as duration of viraemia , infectiousness to mosquitos , or the risk of infection of humans from contaminated products remain unknown , and further studies on camels would be required . Despite those uncertainties , camels are considered susceptible to RVFV , and the virus has been isolated from blood from healthy , naturally infected camels in Egypt and Sudan [32 , 35] . While RVFV infections in camels are usually mild or asymptomatic , abortion waves were observed in RVF outbreaks in Kenya and Egypt [34] . In the 2010 epidemic in Mauritania , clinical signs other than abortions were observed in camels , either a peracute form with sudden death within 24 hours; or an acute form with fever and systemic lesions , that resulted in death when haemorrhagic signs occurred [36] . RVFV infections in camels have been recorded in most sub-Saharan African countries , with prevalences ranging from 3 . 0 to 51 . 9 percent [34] . In fact , RVF is thought to be endemic in sub-Saharan African countries ( including Sudan ) , with large epidemics occurring after heavy rainfall and flooding [9] . Phylogenetic analyses carried out in Sudan with RVFV strains from the 2007 and 2010 epidemics , indicated that they were the result of multiple introductions from eastern Africa [37] . Since then , there were evidences of circulation of RVFV during the inter-epidemic period: in 2014–2015 , 9 . 6% of camels in Khartoum State ( Sudan ) had RVFV antibodies , including at least one positive born after the last reported epidemic [38] . Besides , camels may be involved in the introduction of other diseases such as MERS-CoV , which may cause severe disease in humans . MERS-CoV was detected in Egypt in camels imported from Sudan and Ethiopia [8] , and seems to still be present in Ethiopian camels [39] . Camels provide food ( milk and meat ) , fibre ( wool and leather ) and draft power ( for transportation and cultivation ) to human communities located in dry environments [34] . As a result , in some societies they are essential from a cultural and socio-economic point of view . The world camel population has steadily increased in recent years to reach almost 28 million heads in 2014 ( according to FAO ) . Besides , camel farming practices are changing , with a significant increase of intensive production in peri-urban areas , which results in a closer contact with the human population [34] . As camels are used for trade allowing the connection distant populations , in particular in arid and semi-arid ecosystems , they may play a role in the large-scale dissemination of pathogens . The number of cattle legally imported into Egypt , 4074 on average per month in 2015 , is small as compared to camels . The flow of cattle became more regular by the second half of the 4-years period , coinciding with the intensification of the trade with Sudan . In contrast to camels , the data revealed a more seasonal pattern , with an increase of importations in the months of July , August and September , just before the major Islamic feast of Eid al-Adha ( “Sacrifice Feast" ) in those years . Importation of cattle also poses a threat for Egypt , in fact , the epidemics of foot and mouth disease ( serotype A ) and of lumpy skin disease that affected Egypt in 2006 were both linked to the importation of animals from Ethiopia [40] . Increase of importations of cattle and camels into Egypt may be explained by the growing demand of meat . While between 2006 and 2015 the human population in Egypt grew a 19 . 9% [41] , the population of ruminants and camels in the country only grew a 3 . 2% [42] . According to the World Bank projections , the human population in Egypt is expected to continue growing at a rate of almost 2% per year in the following decade , and therefore legal ( and potentially illegal ) trade of animals into Egypt is expected to increase . After quarantine , camels may be transported to markets or slaughterhouses throughout Egypt . Those movements within Egypt would contribute the spread of RVFV in the event of introduction of the virus , as evidenced in other countries [43 , 44] . The EVS characterized the network of animal markets by collecting information on the location , but also on several characteristics of 273 animal markets distributed throughout Egypt . That combined with the slaughterhouse data allows having a basic picture of the animal movement patterns within the country , which offers an alternative approach in countries without the resources to have a comprehensive recording of animal movements . That information is very useful for planning risk-based surveillance strategies ( i . e . target those markets where detection of a particular disease is more likely ) , with the advantage the criteria may be adapted to each specific disease . The collection of more comprehensive information on for example the number of animals by species or the origins and destinations of the animals that went across those markets , would allow a much clearer picture of the animal movement network , and is therefore recommended The animal market network in Egypt is mainly composed of small markets , which basically operate once a week . Interestingly , almost 40% of the markets traded animals from different governorates , which means that infectious diseases are likely to spread efficiently once introduced into the country . In relation to the species , 2% of markets traded exclusively camels , and a 17% further traded camels plus other species susceptible to RVFV . Most of the markets are located in the areas beside the Nile river and its delta , but there are also animal markets alongside the Mediterranean coast , or even in the Sahara desert ( Farafra market ) . Of the 273 markets , only 17 were in the governorates bordering Sudan , where smuggling of small ruminants was considered more likely . The risk-ranking score for markets varied between 5 and 15 , with a mean value of 7 . 9 . The markets with the highest risk of RVFV transmission were in the proximities or within the main cities ( e . g . Cairo , Minya or Aswan ) , while most of the camel slaughterhouses were near Cairo . That proximity to urban areas results in an increased risk of RVFV transmission to humans ( zoonotic risk ) . In contrast to camels , all cattle imported into Egypt are slaughtered within the quarantines to which they arrive , which are located in remote areas , far from populated areas . In order to prevent the introduction of RVFV by camels , the EVS implement a series of measures that include vaccination of imported animals , quarantines and testing of a proportion of them . However , the vaccination strategy is unlikely be effective , as camels imported from Sudan receive a single dose and camels imported from Ethiopia receive two doses , but with only 7 days difference . The inactivated vaccines against RVFV require a booster 3–6 months after initial vaccination , followed by yearly boosters [45] . Besides , the efficacy of the inactivated vaccine has not been tested in camels [45] , and further research on that field would be needed . While it has been argued that the long distance that the camels from Sudan need to walk to reach the Egyptian border would be enough to prevent the importation of infected animals , there are still many uncertainties in relation to RVFV infection in camels , and in any case , that measure does not comply with OIE rules for importation of live animals [6] . On the other hand , means of transportation seem to be changing as at least some of the camels are currently transported on trucks rather than on foot ( MALRE personal communication ) and transmission ( either direct or indirect ) during the transport of camels cannot be ruled out . Seroconversions in Madagascar highlands evidenced the local circulation of RVFV in periods in which mosquitoes were rare or inactive , suggesting that other forms of transmission seem likely [46] . Even though camels are quarantined , for 3 days if coming from Sudan and 10–16 days if coming from Ethiopia , the efficiency of clinical detection of RVFV infection is limited by the fact that infections in camels are often unapparent . Besides , about 10% of the camels imported from Sudan and Ethiopia in the last 5 years were tested against RVF by RT-PCR , and were negative ( MALRE personal communication ) . However , the sensitivity of such non-statistically based testing protocol for detecting the infection , if present , is questionable . While the measures currently implemented in camels are likely to reduce some of the risk of RVFV introduction into Egypt , their effect is likely to be limited . Similar conclusions may be drawn from the strategies implemented in cattle with the particularities that all cattle are slaughtered within the quarantine premises , where the risk of RVFV transmission by vectors was estimated to be very low . Therefore , vaccination of imported cattle is unlikely to have a significant effect on the reduction of the risk of RVFV introduction into Egypt . On the other hand , to prevent the transmission of RVFV within Egypt , all the animals of the susceptible species ( cattle , sheep , goats , buffaloes and camels ) are meant to be vaccinated twice a year . However , the analysis of the vaccination data evidences a great heterogeneity in the level of vaccine coverage among species and governorates . Despite the increase in the number of vaccine doses administered in 2015 as compared to 2014 , the level of vaccine coverage ( 30% in sheep and goats , 35% in buffaloes and 60% in cattle ) would not be enough to prevent the transmission in the event of RVFV introduction into the country , in particular in those governorates where the level of vaccination is below average . Differences in the density of susceptible species throughout the country combined with the heterogenous application of vaccination programs among areas and species results in the identification of some areas of high density of susceptible hosts of RVFV , which is where efforts should be focused . Failures in the local application of vaccination programs were identified as one of the causes of previous epidemics of RVFV in Egypt [6] . There were also areas where high levels of vaccine coverage were reached resulting in a significant reduction of the risk of RVFV transmission . Conclusions in relation to vaccine coverages should be taken with caution , as the censuses used for their calculation may not be totally accurate/updated . Even though we used the distribution of Cx . pipiens to map vector distribution , we believe it is an accurate representation of the RVFV transmission by vectors . Entomological surveys indicate that Cx . pipiens is the most common mosquito species in Egypt [19 , 47] and was considered the primary vector of RVFV in previous epidemics [5 , 16] . Besides , the primary breeding areas of other RVFV vectors ( e . g . Cx . antennatus ) coincide with those of Cx . pipiens [16] . The map of RVFV transmission by vectors in Egypt evidences that the majority of the country surface has a risk of zero , as it corresponds to bare soil land cover ( i . e . desert ) , which is not suitable for the presence of neither hosts nor vectors . Therefore , RVFV transmission by vectors is basically restricted to the wet areas surrounding the Nile River and its delta . Within that area there is a great variability on the risk depending on the density of vectors and hosts . Transmission via mosquitos is considered the most important mode of transmission during the enzootic cycle [2] . The evaluation of the seasonality of Culex pipiens indicated that in general there was not much variation in Cx . pipiens favorability between the different months . Therefore the risk of RVFV transmission by vectors in the suitable areas of Egypt seem to be more or less constant throughout the year . However , results also indicate that in some specific locations there may be significant variations in Cx . pipiens density . Zayed and collaborators [17] demonstrated that in the areas where the seasonal flooding of the Nile Delta occurred , the population of vectors changed significantly , mainly in late summer-early fall . The validation of the risk map evidenced that the risk of RVFV transmission by vectors obtained at the location of human and animal cases and RVFV-positive vectors from the previous RVFV epidemics in Egypt , varied between 0 . 18 and 0 . 85 , with a mean value of 0 . 49 . That would indicate that RVFV transmission by vectors is not restricted only to the areas with the highest risks , but that transmission may occur also in areas where the risks estimated were moderate or even low . In other words , in Egypt RVFV transmission would be possible in areas where the density of vectors or susceptible hosts is far from their maximum values . However , the limitations of the model should also be taken into account . Model results may be influenced by the accuracy of some model inputs ( e . g . vaccine coverage ) , or the uncertainty about some model parameters ( e . g . susceptibility of the different species ) . Besides , knowledge-driven models are always subject to some sort of subjectivity , for example in relation to the factors ( layers ) chosen . On the other hand , the location of outbreaks used for model validation was not exact , and given that some of them were as old as 1979 , the landscape , and therefore the suitability for Cx . pipiens may have changed since then . As expected , the linear regression analysis indicated that the distribution of Cx . pipiens was the most determinant factor in the risk of RVFV transmission by vectors . The only other statistically significant parameter was the density of susceptible goats . Relevance of goats in the risk of RVFV transmission is probably linked to their higher susceptibility , and therefore higher weight , as compared to other species ( cattle , buffaloes and camels ) , as well as with the fact that the areas with the highest density of goats coincided with very low levels of vaccine coverage ( 7% and 14% , respectively ) . The risk of RVFV transmission by vectors in the surroundings of the five quarantines available in Egypt was estimated to be quite low except in the case of Al Qata ( where some of the camels imported from Ethiopia arrive ) . There is no data on the duration of viraemia in camels , but as camels imported from Sudan stay in the quarantine for only 3 days , it seems possible that they may remain infectious after being released from the quarantine , even if RVFV transmission by vectors within the quarantine was not possible . On the other hand , RVFV transmission within those quarantines may also occur by direct contact with tissues , body fluids or fomites of infected animals . While all cattle are slaughtered within quarantine facilities , imported camels ( potentially infected ) may be taken to animal markets or to slaughterhouses after the quarantine period , and those would be the places where RVFV transmission would be more likely . The risk of RVFV transmission by vectors at the 273 animal markets was rather variable ( between 0 and 0 . 84 ) , but the mean risk was quite high , 0 . 60 . In the case of the 18 camel slaughterhouses , the risk of RVFV transmission by vectors was quite high ( mean risk of 0 . 59–0 . 60 ) , and there was in fact less variability than in the case of animal markets . Establishment of an effective surveillance system in the areas surrounding key markets and camel slaughterhouses may allow the early detection of RVF and the timely establishment of control measures . That may include improving passive surveillance by raising farmers’ awareness for the reporting of any symptom compatible with RVF and providing the mechanisms that allowed a prompt laboratory confirmation of any suspected case . Also , the establishment of some active ( sentinel ) surveillance in the locations identified as having the highest risk would be essential . Besides the risk of RVFV transmission by vectors , the slaughtering of animals , which in the case of cattle is carried out within quarantine premises , poses an important risk for humans by exposure to body fluids or tissues of infected animals . Infectivity of blood during the acute phase of infection in mammals is high [2] . Moreover , given that RVFV is quite resistant to inactivation , infected tissues may remain infectious for a period of a few days [2] . In fact , Nicholas and collaborators [48] concluded that practices such as skinning or slaughtering animals were significantly associated with the risk of RVFV infection . A cross-sectional survey on 1181 abattoir workers in 15 governorates of Egypt after the 1993 RVFV epidemic evidenced a 2% prevalence of anti-RVF IgM antibodies [49] . Antibodies were found in 9 of 31 slaughterhouses and 8 of 15 governorates , including several areas where no clinical disease had been reported . Therefore , Egyptian abattoirs in which imported cattle or camels are slaughtered should implement measures ( e . g . use of personal protective equipment ) to prevent the infection of the personnel . Even though we focused on the risk posed by the legal trade of animals , other mechanisms for RVFV introduction into Egypt are also possible . Illegal trade of animals through the borders is likely to occur , but its relevance in relation to the risk of RVFV introduction is difficult to assess , and would require further studies . On the other hand , the possibility of RVFV spread ( potentially to distant areas ) through the movement of infected ( viraemic ) humans and subsequent infection of competent vectors remains controversial . While for some authors humans infected by RVFV may develop a viremia sufficient to infect naïve mosquitoes [50] , others consider humans as dead-end hosts [3] . Also , wind-borne transportation of infected mosquitos has been proposed [5] , but not demonstrated scientifically . | Rift Valley Fever ( RVF ) is a mosquito-transmitted disease , which may affect animals ( ruminants and camels ) and cause severe disease . Egypt has been affected by RVF in the past , and it is considered at risk because of the importation of animals from endemic countries . We developed a map of the risk of RVFV transmission by vectors using current knowledge on the disease , and then combined it with animal trade data to identify the areas at the highest risk for RVFV introduction . Our results indicate that the main risk is posed by camels imported from Sudan , when they are transported to animal markets or slaughterhouses , which are generally located in areas where the risk of RVFV transmission is much higher . In fact many of them are located in the proximities or within the main cities , resulting in an increased risk of RVFV transmission to humans . | [
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... | 2018 | Understanding the legal trade of cattle and camels and the derived risk of Rift Valley Fever introduction into and transmission within Egypt |
In multicellular organisms , cell type diversity and fate depend on specific sets of transcript isoforms generated by post-transcriptional RNA processing . Here , we used Schmidtea mediterranea , a flatworm with extraordinary regenerative abilities and a large pool of adult stem cells , as an in vivo model to study the role of Uridyl-rich small nuclear RNAs ( UsnRNAs ) , which participate in multiple RNA processing reactions including splicing , in stem cell regulation . We characterized the planarian UsnRNA repertoire , identified stem cell-enriched variants and obtained strong evidence for an increased rate of UsnRNA 3’-processing in stem cells compared to their differentiated counterparts . Consistently , components of the Integrator complex showed stem cell-enriched expression and their depletion by RNAi disrupted UsnRNA processing resulting in global changes of splicing patterns and reduced processing of histone mRNAs . Interestingly , loss of Integrator complex function disrupted both stem cell maintenance and regeneration of tissues . Our data show that the function of the Integrator complex in UsnRNA 3’-processing is conserved in planarians and essential for maintaining their stem cell pool . We propose that cell type-specific modulation of UsnRNA composition and maturation contributes to in vivo cell fate choices , such as stem cell self-renewal in planarians .
The cell types composing a multicellular organism differ significantly in morphology and function , despite their identical genetic information . These features are determined in a differentiation process starting from a common progenitor , a stem cell , through a number of cell fate decisions . While cell fate can be influenced by regulation at every step of gene expression as well as of function and turnover of the gene products , the role of transcriptional changes has been investigated in greatest detail . The regulation of pluripotent stem cell fate , in particular , has been attributed to networks of transcription factors and chromatin modifiers [1–3] . However , recent studies identified key functions of post-transcriptional RNA processing in stem cell self-renewal and fate regulation including alternative splicing ( AS ) [4–7] and -polyadenylation [8] as well as chemical nucleotide modifications [9] . Intriguingly , core components of the spliceosome , including Uridyl-rich small nuclear RNAs ( UsnRNAs ) , were recently implicated in the regulation of pluripotency [10 , 11] . The studies uncovering these mechanisms employed mammalian embryonic stem cells ( ESCs ) or induced pluripotent stem cells ( iPSCs ) in vitro . However , comparable cell types are present in vivo only during a short period of development or are exceedingly rare . The role of these factors in vivo remains , thus , largely obscure . In contrast to mammals , some invertebrates maintain highly potent stem cells lifelong . The stem cells of the planarian S . mediterranea , collectively termed neoblasts , constitute around 25% of all adult body cells and a substantial fraction of them are pluripotent [12] . This abundance of adult stem cells is the basis of the outstanding regenerative abilities and tissue plasticity of adult planarians ( reviewed in [13] ) . The ability to modify neoblast dynamics by tissue amputation or feeding and to transplant them between individual animals , allowing full repopulation of a neoblast-depleted animal from a single donor neoblast , together with their amenability to RNA interference ( RNAi ) make planarians a powerful model organism for studying stem cell regulation in vivo . In a recent study , we identified distinct AS patterns of planarian neoblasts and of their differentiated progeny and showed that they are shaped by the RNA binding protein bruli , a CELF family member , and its antagonists of the MBNL family , respectively [14] . Depletion of these factors disturbs not only splicing patterns but also the balance of neoblast self-renewal and differentiation confirming their role in fate regulation . Similarly , a member of the Sm family of proteins , which associate with UsnRNAs forming small nuclear ribonucleoproteins ( snRNPs ) of the spliceosome , has been implicated in neoblast proliferation [15] . UsnRNAs are short , highly-structured , non-coding RNAs mediating splice site recognition and catalyzing the splicing reaction . In addition to these essential functions as constituents of the splicing machinery , some UsnRNAs regulate splice site choice and alternative polyadenylation [16] . UsnRNA sequence variants are differentially expressed during development [17] and U1 snRNA variants in particular , were shown to control the expression of cell fate regulators [10 , 18] . Except for U6 and U6atac , UsnRNAs are transcribed by RNA polymerase 2 ( RNApol2 ) but , in contrast to mRNAs , they are not polyadenylated . Instead , the 3’-ends of their primary transcripts are formed by a co-transcriptional cleavage reaction catalyzed by the Integrator complex [19] . This 3’-processing step is required for their nuclear export , their association with Sm proteins and , consequently , for their function in splicing . The Integrator complex consists of at least 14 subunits ( ints ) of which ints9 and ints11 form an endonuclease that cleaves nascent pre-UsnRNAs close to a sequence element termed 3’ box [19 , 20] . By mediating U7 snRNA maturation , the Integrator complex also indirectly facilitates the 3’-processing of replication-dependent histone mRNAs [21 , 22] . In addition to this canonical function , the complex or a subset thereof was implicated in enhancer RNA ( eRNA ) processing , pause-release of transcription and the response to DNA damage ( reviewed in [23] ) . Given these important functions , it is not surprising that inactivation of ints severely affects development in a range of animal models [24–28] . Here , we analyzed the differential expression and 3’-processing of UsnRNAs , as central components of the mRNA processing machinery , in neoblasts in vivo . We characterized the UsnRNA repertoire of S . mediterranea and identified neoblast-enriched sequence variants . In line with higher expression levels of ints , we found evidence for enhanced UsnRNA 3’-processing in neoblasts compared to differentiated cells . Using RNAi-mediated ints depletion , as a means of disrupting UsnRNA maturation , we analyzed their role in neoblast fate regulation and identified an essential function in the maintenance of the neoblast pool .
UsnRNA variants are differentially expressed during development and have been implicated in pluripotency maintenance in vitro [10 , 17 , 18] . To analyze their function in adult stem cells in vivo , we first characterized the S . mediterranea UsnRNA repertoire employing a combination of in silico identification of putative UsnRNA genes and validation of their expression by small RNA sequencing . Since UsnRNA secondary structures are evolutionary conserved , we performed a structure-guided similarity search for candidate genes in the S . mediterranea genome [29–31] . This search identified a total of 217 spliceosomal UsnRNA candidates , of which 55 met the statistical threshold ( S1 Table ) . Small RNA sequencing validated the expression of 22 of them . Among these were multiple variants of U1 ( 11 ) , U2 ( 2 ) , U4 ( 3 ) and U6 ( 2 ) snRNA , many of which were present as multiple copies in the genome . Additionally , we identified a putative U4atac candidate , which did not meet the statistical threshold but was validated by RT-PCR ( S1 Table , S1A Fig ) . The predicted secondary structures of the identified UsnRNA types and sequence alignment to their orthologs of other Platyhelminthes species and of Homo sapiens showed substantial conservation of both structural and functional sequence elements , such as the splice site recognition motifs of U1 , U5 , U6 and U11 snRNA , the branch point recognition motif of U2 and U12 snRNA as well as the sequences mediating the dimerization of U4- and U6-type snRNAs ( Fig 1 , S1C and S1D Fig , S2 Fig ) . As expected , the Sm protein binding site ( PuA ( U ) 5-6Pu ) required for incorporation into snRNP particles is present in all Sm-class UsnRNAs and is highly similar to its vertebrate counterpart [32 , 33] . Altogether , our analysis identified S . mediterranea orthologs for all spliceosomal UsnRNA types ( S1B Fig ) . To test whether UsnRNA variants are differentially regulated between neoblasts and differentiated cells , we compared their expression levels in intact planarians with those in neoblast-depleted ones , by qRT-PCR . We employed h2b RNAi [34] and γ-irradiation [35] as two independent and efficient means of neoblast eradication . Interestingly , while the majority of the identified UsnRNAs were expressed uniformly ( S3A Fig ) , three U1- and one U2 snRNA variants were significantly reduced in both stem cell-depletion conditions ( Fig 2A ) . U1C1 , U1C2 and U2A1 showed the most prominent reduction ( 23–63% ) , which suggests a substantial enrichment in neoblasts . We corroborated these findings by comparing UsnRNA variant expression between cell populations isolated by fluorescence-activated cell sorting ( FACS ) . Dissociated cells were sorted according to their size and DNA content into a fraction of small cells with high DNA content ( >2n ) consisting mainly of neoblasts in G2/M phase of the cell cycle ( X1 population ) and a fraction of larger cells with 2n DNA content , comprised mainly of differentiated cells ( Xins population ) [36] . Whereas both variants of U1B and U1C snRNA were significantly enriched in the X1 compared to the Xins cell fraction ( 2–4 fold , Fig 2B ) , the mild enrichment of U2A1 snRNA in X1 cells ( 13% higher than in Xins cells , S3B Fig ) was not statistically significant . While the predicted secondary structures of the neoblast-enriched variants are overall very similar to those of the uniformly expressed U1A snRNAs ( Fig 2C ) , sequence alignment revealed base changes in both 5’ splice site recognition motif and in loop sequences mediating the interactions with RNPs ( Fig 2D ) . Notably , U1C1 and U1C2 , the U1 snRNA variants with the strongest neoblast enrichment , possess an alternative base within the 5’ splice site recognition motif ( Fig 2D , S1 Table , S3C Fig ) raising the possibility that these UsnRNAs may play a specialized role , perhaps in the recognition of specific splice sites . To evaluate the role of the identified UsnRNA variants in planarians , we employed dsRNA-mediated RNA interference ( RNAi ) . However , in contrast to planarian mRNAs [37] , UsnRNAs could not be efficiently depleted using this approach ( S4A Fig ) . As functional maturation of most UsnRNAs requires a 3’ cleavage reaction catalyzed by the Integrator complex [19 , 38] , depletion of Integrator complex subunits ( ints ) by RNAi should constitute an efficient means for inducing a UsnRNA loss-of-function condition in planarians [19 , 22] . We performed BLAST searches for ints in the S . mediterranea transcriptome [39] and identified potential homologs for 12 out of 14 known subunits ( S2 Table ) . For Smed-ints5 and -ints10 , homologous candidates with low sequence similarity were identified using the Planmine database BLAST annotation . Based on their BLAST scores and their previous identification in an S . mediterranea stem cell proteome [40] , we focused on Smed-ints3 and -ints9 ( ints3 and ints9 ) for further analysis . To test for a requirement of the Integrator complex for UsnRNA 3’-processing in planarians , we inhibited ints3 or ints9 expression by RNAi , followed by qRT-PCR quantification of 3’-unprocessed UsnRNAs . To correct for potential effects of ints RNAi on UsnRNA transcription , we normalized the level of the 3’-unprocessed UsnRNA to that of the respective total pool . Importantly , depletion of ints3 or ints9 resulted in a significant accumulation of the unprocessed forms of all tested UsnRNA variants , except for U11 and U12 ( Fig 3A ) . We observed the highest accumulation of the unprocessed form for U2 snRNAs ( 20- and 35-fold in ints3- and ints9 RNAi animals , respectively ) . These results show that ints3 and ints9 are functional planarian homologs of Integrator complex subunits and that their depletion disrupts UsnRNA maturation . Next , we employed the UsnRNA processing assays to RNA of X1 cells isolated by FACS from ints RNAi animals . For all UsnRNAs analyzed , we observed even higher average accumulation of the unprocessed forms in ints RNAi X1 cells than for RNA extracted from entire ints RNAi animals . Except for U4A2 , all unprocessed forms were significantly increased in either ints9 or ints3 RNAi X1 cells , or both , compared to X1 cells of control RNAi animals ( Fig 3B ) . The requirement of the Integrator complex for UsnRNA 3’-processing in X1 cells tempted us to analyze the expression of ints in different cell populations . Thus , we compared the mRNA levels of the putative ints homologs in previously published RNA expression data of FACS-isolated cell fractions [41] . Interestingly , all of the putative ints homologs showed higher expression in the X1 FACS fraction than in the Xins fraction , with an average enrichment of 4 . 8-fold , and five of the subunits had been previously classified as neoblast-enriched [41] ( S3 Table ) . We confirmed the significantly higher expression of ints3 and ints9 in the X1 compared with the Xins population by qRT-PCR ( Fig 3C ) . Consistently , γ-irradiated animals showed strongly reduced levels of both transcripts ( Fig 3D ) . Increased mRNA expression of ints may translate into a higher activity of the snRNA 3’-processing machinery . We therefore quantified the levels of 3’-unprocessed UsnRNA in X1 and Xins cell fractions isolated by FACS . To be able to correct for differential snRNA transcription- and degradation rates in the different cell fractions , we developed a qRT-PCR assay that quantifies the total nascent transcript level by detecting a short 3’ extension present in 3’-unprocessed UsnRNA and in an Integrator-processed , short-lived intermediate but not in the mature UsnRNA . We focused on U2A2 snRNA since the Integrator-processed intermediate of its counterpart in other species ( U2+10 nt ) was reported to have a relatively long 3’-extension [42 , 43] , allowing to distinguish it from the mature form by PCR ( Scheme in Fig 3E ) . Sanger sequencing identified position 187 as the mature end of U2A2 snRNA ( S4B Fig ) and we employed a reverse primer annealing to positions 165–192 to quantify the total nascent transcript level . After normalizing to this level , we found that the 3’-unprocessed U2A2 snRNA was , on average , 14% less abundant in RNA of the X1 compared to the Xins fraction ( p = 0 . 02 , two-sided heteroscedastic t-test ) , indicating that neoblasts possess not only a distinct composition of spliceosomal UsnRNAs but likely also a higher UsnRNA processing activity than differentiated cells . Importantly , both ints3 and ints9 RNAi resulted in an approximately 3-fold increase in the level of unprocessed U2A2 snRNA in the X1 population ( Fig 3E ) , demonstrating the importance of the Integrator complex for UsnRNA processing in neoblasts . Neoblasts are strictly required for tissue turnover and regeneration in planarians [13] . Hence , an essential role of Integrator complex-mediated UsnRNA processing in neoblast function would be reflected by defects in these processes upon ints depletion . Monitoring intact ints RNAi animals for several weeks , we found that they developed head regression and ventral curling , phenotypes indicative of a neoblast defect [44] , and died after a median survival time of 27 days ( ints9 RNAi ) or 33 days ( ints3 RNAi ) after the first dsRNA injection ( Fig 4A , S5A Fig ) . Interestingly , amputated ints RNAi animals initiated regeneration but developed only a very small regeneration blastema and completely failed to regenerate photoreceptors or pharynges ( Fig 4B ) . 7 days following amputation , when 1 or both eyes were already visible in the majority of control trunk and tail fragments , none of the ints RNAi fragments had regenerated eyes ( Fig 4C ) . Like homeostatic RNAi animals , they eventually underwent ventral curling and died approximately 3 weeks after the first RNAi treatment . To test whether ints depletion indeed affects the neoblast population , we performed flow cytometry of cell suspensions prepared from regenerating ints RNAi vs . control RNAi animals and observed a strong reduction of the X1 fraction at 7 days post amputation ( dpa; 17 days of RNAi; Fig 4D ) . Interestingly , while we observed a strong effect of ints RNAi on UsnRNA processing in neoblasts already at 13 days of RNAi ( Fig 3B ) , the proportion of X1 cells at that time-point was still comparable to that of control RNAi animals . This demonstrates that neoblasts are progressively lost during regeneration of ints RNAi animals and suggests that the disruption of UsnRNA processing is an indirect cause of this loss . To exclude that the neoblast loss is merely a consequence of the observed regeneration defect , we performed the same analysis on homeostatic ints RNAi animals , which showed a similar , progressive reduction of the neoblast fraction ( Fig 4D ) . In line with a slower turnover of neoblasts during homeostasis , their proportion decreased at a slower rate in homeostatic compared to regenerating ints RNAi animals . We next confirmed the observed neoblast loss using Fluorescent In Situ Hybridization ( FISH ) of smedwi-1 mRNA , the prototypical stem cell marker in planarians [44] , which was strongly diminished in regenerating ints3 and ints9 RNAi animals at 20 days of RNAi ( Fig 4E ) . Immunofluorescence co-staining of the SMEDWI-1 protein , which is maintained in the immediate progeny of neoblasts ( smedwi-1-/SMEDWI-1+ cells ) , revealed that in both ints RNAi conditions these cells were strongly reduced , albeit to a lesser extent than neoblasts , likely as a consequence of defective neoblast maintenance [45] ( Fig 4E ) . Depletion of a third putative Integrator complex subunit , Smed-ints11 , pheno-copied the regeneration defects and neoblast loss observed upon ints3 and ints9 RNAi ( S5B Fig ) . Recent studies identified neoblast subpopulations with different lineage potential [46] . To determine whether ints depletion affects sigma neoblasts , which are collectively pluripotent and therefore the most potent subtype known , we analyzed the expression of soxP-1 , a marker of these cells , by Whole-mount In Situ Hybridization ( WISH ) and qRT-PCR at different times following amputation . Strikingly , the sigma neoblast population was already substantially reduced at 2 dpa ( 15 days of RNAi ) in both ints RNAi conditions and decreased further during the following days ( Fig 4F and 4G; S6 Fig ) , suggestive of a progressive loss of sigma neoblasts in ints RNAi animals . Together with increased ints expression and lower levels of unprocessed U2A2 snRNA in neoblasts , these results may suggest a function of UsnRNA processing in the maintenance of the neoblast pool . Disruption of UsnRNA maturation may affect neoblast maintenance by altering different UsnRNA-mediated molecular processes . U7 snRNA , for instance , is required for histone mRNA 3’-end processing [21 , 22] and histone expression was implicated in neoblast maintenance [34 , 45] . Hence , we tested the effect of ints depletion on planarian histone processing . We designed qRT-PCR assays to quantify the 3’-unprocessed and the total pool of 5 canonical histone transcripts in RNA of ints- and control RNAi animals at 3 dpa ( 16 days of RNAi ) . Normalizing to the total transcript levels , we observed a significant accumulation of 3’-unprocessed mRNAs of histones H2A and H4 for both ints RNAi conditions as well as of H3 for ints3 RNAi animals ( Fig 5A ) , indicating that the known function of the Integrator complex in U7 snRNA- and , thus , indirectly in histone mRNA-processing is conserved in planarians . Given the key function of UsnRNAs in splicing , we hypothesized that the ints RNAi-induced loss of neoblasts is , at least in part , due to the disruption of their splicing patterns . To globally characterize the effects of ints depletion on the splicing patterns of neoblasts , we sequenced the transcriptomes of X1 cells isolated from ints3- and ints9 RNAi animals at 3 dpa ( 16 days of RNAi ) and compared their splicing patterns to that of control RNAi X1 cells . As described recently , we quantified the inclusion frequency ( PSI; Percent Spliced In ) of alternative exons and introns as well as the differential usage of alternative donor and acceptor splice sites [14 , 47 , 48] . This analysis identified 295 and 152 splicing events that showed a significant change in inclusion levels ( │PSI ints RNAi-PSI ctrl RNAi│>15% , see Methods ) in X1 cells of ints3 and ints9 RNAi animals , respectively , compared to their control RNAi counterparts ( Fig 5B , S4 Table ) . Although the total number of splicing changes differed , a strong bias towards exon skipping ( exclusion of exons ) was observed for both RNAi conditions ( 68 . 8% and 51 . 9% of all events , respectively ) and only a very small number of intron retention events were detected ( 2 . 7% and 1 . 3% , respectively ) . Moreover , a core set of 63 splicing events were affected in the same direction in both RNAi conditions ( S4 Table ) . 14/15 ( 93% ) tested exon skipping events shared by ints3 and ints9 RNAi X1 cells were validated by RT-PCR ( Fig 5C , S7A Fig ) . These results suggest that the defects in UsnRNA maturation induced by ints RNAi affect primarily the exon definition step of splicing in neoblasts . Remarkably , a comprehensive analysis of sequence and structural features of exons skipped upon either RNAi treatment revealed that these are very AT-rich ( S8D Fig ) . We analyzed the sequencing data of isolated X1 cells further for ints RNAi-induced changes of gene expression , which identified a total of 1351 or 477 transcripts differentially expressed between ints3 or ints9 RNAi and the control , respectively ( padj<0 . 05 , │log2 fold-change│>1 , S5 Table ) . Importantly , the overlap between the mis-spliced and the differentially expressed transcripts was minimal ( 15 and 0 for ints3 and ints9 RNAi , respectively; S7B Fig ) , suggesting that the majority of the splicing defects did not directly affect mRNA stability . The overlap between ints3 and ints9 RNAi-induced changes in transcript levels , however , was substantial ( S7C Fig ) indicating that these changes are indeed caused by a loss of Integrator complex function . We next compared these changes to expression data of Labbé et al . [41] , who analyzed the transcriptomes of FACS sorted cell fractions . Interestingly , among the transcripts down-regulated in ints3 or ints9 RNAi X1 cells , we observed a highly significant over-representation of those classified as stem cell-specific ( X1 ) by Labbé et al . ( Fig 5D , S5 Table ) . While these expression changes affected only a subset of all stem cell-specific transcripts , they may indicate a disruption of the normal neoblast gene expression pattern . Together , these data suggest that defects in histone mRNA processing and splicing collectively contribute to the neoblast loss observed after ints RNAi . We next analyzed the effects of ints depletion on the self-renewal of neoblasts . Since the high density of these cells and of early neoblast progeny complicates their quantification directly within the RNAi animals , we transplanted FACS-isolated X1 cells of ints or control RNAi animals into neoblast-depleted wild-type hosts and analyzed the resulting colonies 5 and 7 days after transplantation ( Fig 6A ) . We identified neoblasts and their differentiating progeny by FISH staining of smedwi-1 and NB . 32 . 1g [35] , respectively , and quantified their proportions . As shown in Fig 6A and 6B , the colonies derived from ints3 and ints9-depleted neoblasts were smaller than those derived from control RNAi neoblasts and contained a significantly higher proportion of differentiating NB . 32 . 1g+ cells ( p<0 . 01 and p<0 . 001 , respectively ) , demonstrating that ints depletion disrupts the ability of neoblasts to self-renew and changes their fate towards differentiation . To test the capacity of ints-depleted neoblasts to differentiate , we pulse-labelled proliferating cells with the thymidine-analog Ethynyl-deoxyuridine ( EdU ) and analyzed their progeny after different chase periods for the expression of cell type markers representing all germ layers by FISH staining . We normalized the number of marker+EdU+ cells to the total number of EdU+ cells , to account for the substantially lower number of EdU-incorporating neoblasts of ints RNAi animals ( Fig 7A ) . The median numbers of EdU-positive cells expressing early ( NB . 21 . 11e+ ) , intermediate ( AGAT-1+ ) [35] and late ( zpuf-6+ ) [49] markers associated with the epidermal lineage were substantially lower in ints9-depleted animals compared with controls . A similar reduction was observed for myoD+EdU+ muscle cell progenitors [50] and collagen+EdU+ muscle cells [51] . The numbers of gata4/5/6+EdU+ gut precursors [12] and of terminally-differentiated intestinal cells expressing porcupine [52] were reduced in ints3 RNAi animals . Furthermore , we observed a mild reduction in the number of EdU+ neurons expressing prohormone convertase 2 ( pc2 ) [53] in both ints RNAi conditions . Importantly , EdU-positive cells expressing markers of late progeny and of terminally differentiated cell types were still observed in ints RNAi animals ( Fig 7B–7D ) . These findings indicate that neoblast progeny is able to differentiate even at very low Integrator complex activity , yet gives rise to a lower number of differentiated cells , possibly due to a reduction in general cell fitness .
Here , we describe the UsnRNA repertoire of the planarian S . mediterranea and investigate the impact of disturbed Integrator complex-mediated UsnRNA processing on the fate of its stem cells . We first identified spliceosomal UsnRNAs using a bioinformatics approach that has been successfully employed in other non-mammalian model organisms [30 , 54 , 55] . The validation of their expression by small RNA sequencing effectively removed pseudogenes , as shown independently by RT-PCR . While this approach might have excluded some UsnRNA candidates that are expressed at low levels and/or have high sequence similarity to other candidates , the numbers of validated genes encoding the different UsnRNA types are in the range of those predicted for their counterparts in the closely-related Schistosomes [56 , 57] suggesting that they represent the majority of the S . mediterranea UsnRNA repertoire . As was recently reported for U1 snRNA in cultured mammalian stem cells [10 , 18] , our analysis showed that neoblasts express higher levels of several U1 snRNA variants when compared to differentiated cell types . The similarity of their predicted structure and the conservation of functional sequence motifs suggests that these variants could function in splicing . Given that neoblasts also express several spliceosome components at higher levels [14] and that increased spliceosome activity has been linked to the maintenance of pluripotency [11] , we speculate that increased UsnRNA levels support a higher spliceosome activity . Moreover , the modified 5’ splice site recognition motif present in the neoblast-enriched U1C1 and U1C2 snRNA variants indicates that they might facilitate the use of specific 5’ splice sites or , alternatively , act as dominant negative regulators suppressing differentiation-related AS events . A role for these variants in alternative polyadenylation is also plausible [16] . Of note , even minor U1 snRNA variants may exert substantial effects on gene expression [18] . In FACS-sorted cell fractions enriched for neoblasts , we found lower levels of 3’-unprocessed U2A2 snRNA , relative to the total nascent transcript , than in cell fractions consisting of differentiated cells . This raises the possibility that the 3’-processing of UsnRNAs constitutes a cell type-dependent control mechanism for UsnRNA abundance and hence for UsnRNA-dependent processes , such as splicing . While lower levels of unprocessed UsnRNAs could alternatively be explained by higher degradation rates , the observed increase of unprocessed U2A2 in neoblasts upon inhibition of the UsnRNA 3’-processing machinery supports our hypothesis . Together with our qRT-PCR results and previously published RNAseq data [41] , which show that ints-encoding transcripts are enriched in neoblasts , it is likely that this increased processing activity is , at least in part , due to higher ints expression in these cells . Testing the function of UsnRNAs directly requires efficient and highly sequence-specific methods for UsnRNA depletion or over-expression , both of which are currently not available in S . mediterranea . In contrast to mRNAs , UsnRNAs could not depleted by dsRNA-mediated RNAi , which is likely due to differences in expression levels , subcellular localization or structure of these RNA types . We therefore used RNAi against Integrator complex subunits as efficient means to disrupt UsnRNA maturation and hence function . This caused a plethora of splicing defects and the accumulation of unprocessed histone mRNAs . The latter is likely a consequence of defective U7 snRNA processing by the Integrator complex , since U7 snRNA is known to mediate the maturation of replication-dependent histone mRNAs [21 , 22 , 58] . Given the importance of histone expression for neoblast maintenance [34] , the reduced processing of H2A , H4 and H3 mRNAs is likely to contribute to the neoblast maintenance defect of ints RNAi animals . While splicing defects are an expected consequence of the loss of UsnRNA processing , the very high proportion of exon skipping , but not intron retention , events among the splicing changes observed in both ints RNAi conditions was surprising and suggests that ints depletion impairs primarily exon definition . Interestingly , very similar splicing changes were observed in ints8-mutant mammalian cells [59] , substantiating our results and supporting an evolutionary conserved function of the Integrator complex in facilitating splicing fidelity . As many of the exon skipping events are likely to disrupt the reading frame of the corresponding transcripts , reducing the production of functional proteins and causing the accumulation of potentially cytotoxic proteins , they may interfere with neoblast maintenance . Indeed , depletion of either ints3 or ints9 disrupted tissue homeostasis and regeneration , likely as a consequence of the gradual neoblast loss . While the Integrator complex has not been implicated in stem cell maintenance to date , knock-out of ints genes causes early developmental lethality in diverse animal models that could well be explained by defects of stem cell maintenance [24–27] . Investigating a putative effect of ints RNAi on the transcriptional profile of neoblasts we revealed a considerable de-regulation of gene expression . Whether this was due to defective histone processing or the disruption of a putative transcriptional regulator function of the Integrator complex [60–62] is unclear but the minimal overlap between mis-spliced and differentially expressed transcripts suggest that defective splicing is not a major contributor . Despite a stronger effect of ints9 RNAi on the survival of planarians , we observed a higher number of both mis-spliced and differentially expressed transcripts in ints3- compared to ints9 RNAi neoblasts , which may be due to differences in knock-down kinetics or efficiency . Neoblast-specific transcripts were strongly over-represented among those down-regulated in both ints3 and ints9-depleted neoblasts . It is important to point out that these transcriptomic changes occur in neoblasts and , thus , do not represent changes in cell type composition . However , they might be early indicators of a neoblast fate change . Transplantation of ints RNAi neoblasts into irradiated host animals confirmed the fate change of these cells towards differentiation . Importantly , since the recipient animals in this experiment were not injected with dsRNA , this cell fate change is likely not due to a loss of Integrator complex function in the host tissue , but rather represents a cell-autonomous phenotype of the transplanted cells . In line with the considerably smaller colonies derived from transplanted ints RNAi neoblasts , EdU pulse-chase experiments revealed that the production of a number of progenitor and differentiated cell types is reduced upon ints depletion . Since the number of neoblasts initially labelled with EdU cannot be determined in these experiments , it is unclear whether the reduced proliferation of ints-depleted neoblasts contributes to this lower cell output . Nonetheless , the presence of EdU-labelled pc2+ , porcupine+ and zpuf-6+ cells in the brain , the gut and underneath the epidermis of ints-depleted animals , respectively , suggests that the low levels of ints expression remaining after the incomplete knock-down achieved by RNAi allows for differentiation , at least of a portion of the neoblast progeny . Neoblasts , similar to germ line stem cells in other organisms , are highly dependent on post-transcriptional regulators of mRNA localization , splicing and/or translation [14 , 15 , 58 , 63–66] as well as on factors generating regulatory RNAs [44 , 67] . Our study now extends this list by the Integrator complex , which appears to have a dual function in the post-transcriptional regulation of transcripts important for neoblasts: By processing UsnRNA 3’-ends , it mediates splicing and histone mRNA maturation , both of which were previously linked to neoblast maintenance [14 , 15 , 34 , 45] . We propose a model ( Fig 8 ) in which the increased expression of U1 snRNA variants together with enhanced , Integrator-mediated UsnRNA maturation facilitate a higher activity and/or modified specificity of neoblast spliceosomes as well as the efficient U7-dependent processing of histone mRNAs . This may contribute to neoblast maintenance by generating a specific set of protein isoforms as well as by enabling the high turnover of mRNAs and proteins in these mitotically active cells .
UsnRNA multiple sequence alignments annotated with consensus secondary structures were obtained from the Rfam database v12 . 0 [31] . For each spliceosomal UsnRNA type , a covariance model was built , calibrated and then searched against the genome of the asexual Schmidtea mediterranea biotype ( Asxl v1 . 1 ) [29] using the INFERNAL 1 . 1 . 1 programs cmbuild , cmcalibrate and cmsearch [30] , respectively , with default settings . The identified candidate sequences were aligned to the respective covariance model using cmalign of INFERNAL and the resulting , predicted secondary structures were visualized using VARNA 3 . 93 [68] . For U6atac structure prediction , the candidate sequence was extended at the 3’-end by adding 32 nucleotides from the genomic sequence to fit the model length . Multiple sequence alignments generated by cmalign were visualized with Jalview 2 . 10 . 4b1 [69] . Phylograms were generated using Clustal Omega [70] . Variants of the same UsnRNA type were clustered according to sequence similarity and the clusters were named in alphabetical order , starting from the one with the highest number of uniquely-mapping RNAseq reads . Within clusters , variants were given consecutive numbers according to best-mapping read count . Sequence logos were generated using WebLogo generator [71] . Animals of the asexual , clonal Schmidtea mediterranea line BCN-10 ( originally provided by E . Salo ) , were maintained as described [72] and treated as specified in S7 Table . Double-stranded RNA ( dsRNA ) was transcribed from PCR amplicons using T7 RNA polymerase as described previously [73] . Control RNAi animals were injected with dsRNA targeting a region of the green fluorescent protein ( GFP ) coding sequence . For regeneration experiments , animals were amputated pre- and postpharyngeally into head , trunk and tail fragments . Neoblast depletion by h2b RNAi was performed as described previously [34] . For lethal γ-irradiation , animals were exposed to a dose of 60 Gy using a Gammacell 40 Exactor ( Nordion ) . For EdU pulsing , animals were incubated in ( 2′S ) -2′-Deoxy-2′-fluoro-5-ethynyluridine ( F-ara-EdU , Sigma Aldrich ) [74] dissolved in planarian artificial medium ( PAM ) containing 3% ( v/v ) DMSO for 16 hours . F-ara-EdU concentrations of 0 . 4 , 0 . 2 and 0 . 1 mg/ml were used for 3 , 7 and 9 day chase , respectively . Animals were maintained in the dark in PAM containing 50 μg/ml gentamycin throughout the chase period and then fixed with formaldehyde [75] , subjected to FISH and then EdU was stained using the EdU DetectPro Imaging Kit 647 ( baseclick GmbH ) according to the manufacturer’s protocol . RNA was isolated using the TRIzol or TRIzol LS reagent . RNA for qRT-PCR analysis of UsnRNAs or for mRNA sequencing was treated with TURBO DNase followed by either phenol-chloroform extraction and ethanol precipitation or by purification on NucleoSpin RNA XS columns ( Macherey-Nagel ) . For the latter , RNA was adjusted to a final ethanol concentration of 53% before loading onto the column to improve recovery of small RNA species . First strand cDNA was transcribed using the RevertAid H Minus First Strand cDNA Synthesis Kit with random hexamers ( for UsnRNA and histone mRNA assays ) or oligo dT primers ( for all other mRNA assays ) , respectively . For the analysis of unprocessed UsnRNA in wild-type X1 and Xins fractions , RNA prepared as described above was subjected to whole transcriptome amplification using the Ovation qPCR system ( NuGEN ) , which was performed with 45 ng of RNA in technical duplicates according to the manufacturers protocol and then pooled . qRT-PCR analysis was performed on the Real-Time PCR System 7500 or 9100 ( Applied Biosystems ) using either the Universal Probe Library ( Roche ) together with TaqMan Universal PCR Master Mix or the iTaq universal SYBR Green supermix ( Bio-Rad ) . Primers and probes are summarized in S6 Table . Relative expression levels were calculated as previously described [76] . 3’-unprocessed U2A2 levels were normalized to those of the nascent transcript . End-point RT-PCR was performed in a Mastercycler pro ( Eppendorf ) using Quick-Load Taq 2X Master Mix ( NEB ) and analyzed by agarose gel electrophoresis . For S6A Fig , the intensity of the gel bands was quantified using Fiji [77] . RNA species of 50–500 nucleotides were isolated from total RNA of intact , wild-type animals by electrophoresis on a 7% polyacrylamide gel containing 7M Urea , extracted from the homogenized gel piece in gel extraction buffer ( 300mM sodium acetate , 1mM EDTA , 0 . 01 U/μl SUPERase• In RNase Inhibitor ) at 4°C overnight , filtered through a Spin-X centrifuge tube filter ( Corning ) and concentrated by ethanol precipitation . 500 ng of size-selected RNA was de-capped using RNA 5’ pyrophosphohydrolase ( NEB ) according to the manufacturers recommendations and then concentrated by ethanol precipitation . The de-capped RNA was then subjected to sequencing library preparation using the NEBNext Small RNA Library Prep Set for Illumina ( NEB ) according to the manufacturers protocol and analyzed on an Illumina HiScan SQ . Using Bowtie v1 . 1 . 2 [78] , the RNAseq reads were mapped onto a compilation of all UsnRNA candidate sequences identified by Infernal cmsearch , each extended by 25 nucleotides on the 3’ end to facilitate the mapping of pre-snRNA-derived reads . To account for the high sequence similarity between variants of the same UsnRNA type , a dual mapping strategy was employed by counting either only uniquely-mapping reads ( for validating candidate gene expression ) or all best mapping reads ( for estimating expression levels ) . The Bowtie parameters for reporting best- or unique alignments were -a—best -v2 or -a -m1 -v2 , respectively . To quantify uniquely-mapping reads , identical candidate sequences in the compilation ( colour-coded in S1 Table ) were represented by a single sequence . The number of mapped reads were quantified using HTseq-count [79] . For Sanger sequencing ( GATC Biotech ) , individual UsnRNAs were PCR-amplified from the sequencing library described above using a forward primer specific for the respective UsnRNA and the SR RT Primer of the NEBNext Small RNA Library Prep Set for Illumina as the reverse primer ( S6 Table ) . Flow cytometry was performed as described previously [80] , except that Hoechst 33342 was used at 10 μg/ml and cells were stained at 24°C . X1-cell isolation was performed on a FACSAria Cell Sorter with its respective software ( BD Biosciences ) using the protocol described in [12] . Cell transplantations were performed 10 days after the first dsRNA injection as described in [40] , except that approximately 3750 X1 cells in 100 nl were injected per host animal . 5 and 7 days after cell injection , animals were formaldehyde-fixed and processed as described below ( S7 Table ) . For subsequent RNA isolation from cell fractions , cells were stained as described above for flow cytometry and sorted directly into TRIzol LS reagent . cDNA libraries were prepared from total RNA of the X1 FACS-fraction in biological triplicates using the TruSeq Stranded mRNA Library Prep Kit ( Illumina ) according to the manufacturers recommendations . Paired-end sequencing ( 2x75 cycles ) was performed on an Illumina NextSeq500 . Analysis of alternative splicing was performed using vast-tools v2 . 0 . 2 . ( https://github . com/vastgroup/vast-tools ) , as recently described [14 , 47] . Differentially spliced AS events were identified using vast-tools compare , with the following parameters:—min_dPSI 15—min_range 5—noVLOW—p_IR—paired ( i . e . │average ΔPSI│>15 and a minimum pairwise difference between paired replicates of | ΔPSI | > 5 in the same direction , excluding events with read coverage score VLOW or lower , and filtering introns that did not pass the binomial test for read balance at the exon-intron junctions ) . S4 Table contains a summary of the splicing analysis results . For differential expression ( DE ) analysis , RNAseq reads were mapped to the reference transcriptome ( http://vastdb . crg . eu/libs/vastdb . sme . 16 . 02 . 18 . tar . gz ) using vast-tools [47] to obtain raw read counts . DEseq2 [81] was then used for read count normalization using size factors and pairwise-comparison using contrasts . Genes with padj<0 . 05 and log2 fold-change ≤ -1 or ≥ 1 were considered significantly differentially expressed . For comparison to previously published RNA expression data of FACS-isolated cell fractions [41] , the corresponding transcripts in the to_Smed_v2 transcriptome were identified by BLAST search and the classification of the highest scoring hit was used for enrichment analysis . The enrichment ratio of class X among DE transcripts was calculated according to: ( No . DE transcripts with class X / Total No . DE transcripts ) / ( No . transcripts with class X in background / Total No . transcripts in background ) , where background are all transcripts evaluated for DE . The significance of the enrichment was determined using a one-sided hypergeometric test . Venn-diagrams representing overlaps were generated using the VennDiagram package in R [82] . Immunostaining was performed as described previously [73] with an anti-SMEDWI-1 antibody ( 1:1000 ) , generated as described in [83] , and Alexa Fluor-conjugated goat-anti-rabbit IgG secondary antibodies ( 1:400 ) . Chromogenic whole-mount ( WISH ) - and fluorescent in situ hybridizations ( FISH ) were performed using the protocols described in [75 , 84–86] and [87] , respectively . RNA probes were in vitro-transcribed , using T7 or SP6 RNA polymerase , from PCR amplicons generated with the primers listed in S6 Table and labelled with digoxigenin ( DIG RNA Labeling Mix , Roche ) or dinitrophenol ( Label IT Nucleic Acid Labeling Kit , DNP , Mirus Bio ) . Hoechst 33342 was used at 5 μg/ml as a nuclear counterstain and animals were mounted using Aqua Poly/Mount ( Polysciences ) . Bands on agarose gels were visualized using a GEL IX 20 ( intas ) . Live animals and WISH stainings were imaged using a Leica M80 and M165 FC microscope , respectively . Confocal microscopy was performed on a Zeiss LSM700 using the ZEN software . CellProfiler [88] was used for the automated identification and quantification of EdU+ and marker+EdU+ cells . The Classifier functionality of CellProfiler Analyst [89] was applied to the CellProfiler output to improve identification accuracy for NB . 21 . 11 . e , MyoD , gata4/5/6 , AGAT-1 , zpuf-6 and collagen-positive cells . Images were processed using Fiji [77] or Adobe Photoshop . Box-Whisker plots were generated using the BoxPlotR web-tool ( http://shiny . chemgrid . org/boxplotr/ ) . Figures were prepared in Adobe Illustrator . Next generation sequencing data is available under Bioproject PRJNA397855: SAMN07490271 ( X1_gfp_RNAi ) , SAMN07490272 ( X1_ints3_RNAi ) , SAMN07490273 ( X1_ints9_RNAi ) , SAMN07490274 ( small RNAs_wt_intact ) . Partial coding sequences of ints were deposited in Genbank: MF671814 ( ints3 ) and MF671815 ( ints9 ) . | Stem cells not only give rise to all specialized cell types of the body through differentiation but also to new stem cells through the process of self-renewal . Modifications of messenger RNAs ( mRNAs ) following their transcription play an important role in a stem cell’s decision between self-renewal and differentiation . However , since these cells are rare and difficult to access in mammals , the underlying mechanisms have mostly been studied in cultured cells . Here , we employ the planarian flatworm Schmidtea mediterranea that maintains a large pool of highly potent stem cells throughout life as an in vivo model to analyze the function of UsnRNAs , important regulators of multiple mRNA processing steps , in stem cell regulation . We show that stem cells express more UsnRNA sequence variants compared to differentiated cells . Moreover , the activity of the Integrator complex , which mediates UsnRNA maturation , is increased in stem cells . Depletion of Integrator complex components disrupts their global splicing pattern and affects the processing of histone mRNAs resulting in the loss of stem cell self-renewal and , consequently , in defects of tissue homeostasis and regeneration . Thus , we propose that planarian stem cell self-renewal in vivo depends on differential variant expression and efficient processing of UsnRNAs . | [
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"epigenetics",... | 2018 | The Integrator complex regulates differential snRNA processing and fate of adult stem cells in the highly regenerative planarian Schmidtea mediterranea |
The London Declaration on neglected tropical diseases was based in part on a new World Health Organization roadmap to “sustain , expand and extend drug access programmes to ensure the necessary supply of drugs and other interventions to help control by 2020” . Large drug donations from the pharmaceutical industry form the backbone to this aim , especially for soil-transmitted helminths ( STHs ) raising the question of how best to use these resources . Deworming for STHs is often targeted at school children because they are at greatest risk of morbidity and because it is remarkably cost-effective . However , the impact of school-based deworming on transmission in the wider community remains unclear . We first estimate the proportion of parasites targeted by school-based deworming using demography , school enrolment , and data from a small number of example settings where age-specific intensity of infection ( either worms or eggs ) has been measured for all ages . We also use transmission models to investigate the potential impact of this coverage on transmission for different mixing scenarios . In the example settings <30% of the population are 5 to <15 years old . Combining this demography with the infection age-intensity profile we estimate that in one setting school children output as little as 15% of hookworm eggs , whereas in another setting they harbour up to 50% of Ascaris lumbricoides worms ( the highest proportion of parasites for our examples ) . In addition , it is estimated that from 40–70% of these children are enrolled at school . These estimates suggest that , whilst school-based programmes have many important benefits , the proportion of infective stages targeted by school-based deworming may be limited , particularly where hookworm predominates . We discuss the consequences for transmission for a range of scenarios , including when infective stages deposited by children are more likely to contribute to transmission than those from adults .
In January 2012 , a high-level meeting brought together 13 pharmaceutical companies and the global health community in London , UK to announce a new public-private partnership to eliminate or control the seven preventable neglected tropical diseases ( NTDs ) by 2020 , based largely on a new NTD roadmap from the World Health Organization ( WHO ) [1] . The pledge by pharmaceutical companies to sustain and extend donation programmes facilitates a large portion of the necessary supply of drugs and other interventions to help achieve this goal [2] . Such commitments raise the question of how best to use these resources to induce maximum impact , given that many treatments for NTDs must be administered repeatedly to individuals living in endemic areas due to re-exposure to infection and the absence of fully protective acquired immunity . The most common NTDs worldwide are the soil-transmitted helminths ( STH: Ascaris lumbricoides , Trichuris trichiura and the hookworms , Necator americanus and Ancylostoma duodenale ) , with an estimated 5 . 3 billion people worldwide , including 1 . 0 billion school-aged children , living in areas of stable transmission for at least one STH species [3] . STHs are easily treated with one of four drugs: albendazole and mebendazole , and to a lesser extent , levamisole , and pyrantel pamoate [4]–[5] . However , reinfection commonly occurs [6] due to the inability of the human host to mount protective immunity to reinfection by intestinal helminths [7]–[8] , combined with inadequate hygiene and sanitation to restrict or eliminate re-exposure in environments continuously contaminated with the egg or larval free-living transmission stages of these parasitic worms [9] . Following treatment , average worm loads in the population return to their pre-treatment equilibiria in a monotonic manner . The exact dynamics will depend on a number of density-dependent processes that influence parasite reproduction , infection and mortality ( in part related to the build-up of a degree of acquired immunity ) , plus the relatively long life expectancies of established worms in the human host ( measured in years ) [10] . It will also depend on the proportion of worms in the entire human community in a defined location which are exposed to treatment in a particular control programme . Deworming programmes for the STHs are often centred on school delivery because of the large burden of morbidity and concomitant developmental consequences for these children [11]–[13] , as well as relative ease of access to children in poor rural areas through schools and the cost-effectiveness of school-based deworming [14]–[15] . A number of countries have programmes which additionally include adults , as part of lymphatic filariasis treatment campaigns providing mass treatment with albendazole and ivermectin ( or diethylcarbamazine ) , with associated large impacts on transmission [16] . However , there are large areas where this STHs are not co-endemic for lymphatic filariasis and these areas the WHO-recommended treatment strategies prioritise school-aged children , but also recommend preventive chemotherapy of preschool children , women of childbearing age and adults at high risk [4] . A number of countries are currently implementing only school-based deworming [11] . The large donation of 600 million doses per year announced in the London Declaration almost completely covers the estimated 610 million school-aged children in need of preventive chemotherapy [1]–[2] , [11] , [17] , but it does not cover pre-school children , women of child-bearing age or treatments more than once a year . This paper therefore examines the effectiveness of school-targeted programmes in restricting transmission within the larger community of pre-school children , school-aged children and adults . We use analytical methods deriving from the description of the transmission dynamics of these parasitic worms [18] and demographic plus school attendance information to calculate what fraction of the total population is treated . We also use mathematical models to discuss the impact of school-based programmes on transmission , including scenarios in which infective stages deposited by children are more likely to contribute to transmission than those from adults . We discuss how the impact of a treatment programme could affect the infection dynamics in the population as a whole depending on the , as yet unknown , details of between age-group mixing .
Monitoring and evaluation of the impact of community-based preventive chemotherapy programmes is based on two epidemiological measures , the prevalence and the intensity of infection . Prevalence represents the fraction or percentage of the population infected and is typically stratified by factors such as age and gender . The intensity of infection or worm burden for STHs is typical measured indirectly by counts of eggs expelled in faeces ( eggs per gram of faeces or EPG ) and is similarly typically stratified by age and sex . Less commonly , worm burden may be measured by the worms expelled in total faecal output over a defined period post curative chemotherapy [24]–[26] . The two epidemiological measures are statistics of the probability distribution of worm numbers per person . These distributions are typically highly aggregated in form , with the variance exceeding the mean in value . They are well described by the negative binomial probability model [27] . For this distribution the relationship between prevalence ( as a proportion ) and mean intensity is given by: ( 1 ) Here , the negative binomial parameter varies inversely with the degree of parasite aggregation within the human population and for values in excess of five the distribution is approximately random in form with the variance approaching the mean in value . Some typical estimates of the magnitude of are recorded in Table 1 . A plot of the relationship between and for the negative binomial is presented in Figure 1 . It is clear from this figure that the prevalence is a very poor measure of the impact of community-based chemotherapy . Large changes in average worm load as a consequence of treatment will only have a small effect on prevalence unless the mean worm burden is low ( i . e . when transmission is very low ) . For example , in a study in Myanmar two villages with mean EPGs of about 4000 and about 400 , an order of magnitude difference in intensity , had almost no difference in prevalence ( Figure 1B [28] ) and so highly effective treatment of the high intensity village , reducing the burden by a factor of 10 , might be viewed as a failed programme if only prevalence were monitored . The cost and difficulty of monitoring intensity , as opposed to prevalence , is of course greater . However , if monitoring is to have any value , intensity must be measured in some fraction of the treated population . The issue of how best to sample to gain an accurate picture of the impact of treatment , while attempting to keep sample size and the concomitant costs low , requires careful thought . The underlying distribution of parasite numbers per host is central and given its heterogeneity , small sample sizes will not provide robust measures of trends [29] . One compromise is to monitor impact in a subset of age classes – one of children and one in the adult age groups , to see how the treatment of the school-aged children impacts on transmission to adults [30] . The greatly expanded deworming programmes seen in many regions of the world in recent years have not been accompanied by systematic recording of treatments delivered and the associated impact on prevalence and intensity . Recently , however , some progress has been made on the generation of open access databases recording global and national spatial distributions of helminths based on estimates of the infection prevalence , as illustrated , by the Global Atlas of Helminth Infection ( http://www . thiswormyworld . org [12] ) and the Global Neglected Tropical Diseases database ( http://www . gntd . org [31] ) . The Global Atlas of Helminth Infection will be expanded in the near future to include measures of the intensity of infection and treatments delivered . The website will also be extended to encourage the electronic deposition of data on STHs collected in association with the current expanded efforts on community-based control using mass or school-aged targeted anthelminthic treatment . Such data are collected by a number of excellent NTD programmes but is rarely subject to detailed analysis on trends in transmission [32] . The present absence of international databases on treatment of STH and impact of such treatment does make analysis of questions concerning the optimal delivery strategy for community-based programme somewhat challenging . We therefore base our analyses on a small number of available well-designed studies that record prevalence and intensity of infection , stratified by age and sex , before and after various treatment programmes . The age-profiles used are from studies of A . lumbricoides in Myanmar [33] , India [24] and Iran [34]; T . trichiura in St Lucia [35]; hookworm in Uganda [36] and Vietman [37] and , for comparison , studies of Schistosoma mansoni in Uganda [38] and Brazil [39] . Epidemiological studies that record the mean intensity of infection stratified by age , when combined with demographic plus school enrolment data , provide information on the fraction of the total worm population exposed to treatment . Along with measures of drug efficacy , this in turn gives the fraction of the total worm population removed by school-aged targeted chemotherapy . The importance of this fraction to the overall transmission dynamics of the target parasites cannot be overstated – and in current control programmes it is an unmeasured parameter . If is the proportion of the total population in age class a , the proportion of the human population in the school age classes 5 to 14 years of age , is given by: ( 2 ) The proportion of the total worm population harboured at time by people between the ages of 5 to 14 years , , is then given by: ( 3 ) where is the mean worm burden in age-group at time . If the measure of intensity of infection is eggs per gram of faeces ( EPG ) , then the proportion of egg output produced by school-aged children is: ( 4 ) Here is the density-dependent egg output function , which gives the expected egg output for an individual with mean worm burden for age and negative binomial aggregation parameter . In practice egg output is usually measured , rather than mean worm burden , so we can approximate this by ( 5 ) where the mean egg output in age-group at time . We calculate these fractions for some example datasets on parasite distributions , together with demographic and school enrollment data . Demographic data from various countries where STHs are endemic provides an initial template to assess these issues [40] . Table 2 records the fraction of various populations in the school ages of 5 up to 14 years . In general , within countries where helminth infections are endemic , the fraction of the total population in the school going groups is between 11% and 30% . UNESCO and the World Bank provide data on school enrolment by sex , location ( urban or rural ) and country . Recent data are recorded in Figure 2 for rural and urban areas for a selection of countries [41] and in Table 6 for enrollement of female students . Over the past decade there has been a steady increase in school enrolment in most countries throughout the world . Progress has been less good in poor rural areas by comparison with urban districts in developing countries . Generally , the most recent data ( 2005 and beyond ) suggest figures in the 80% to 90% range for most urban areas , but with a range of 20% to 60% in some sub-Saharan African countries in rural areas . There is often a gender bias in many poor countries , with attendance figures for females lower than those for males in the primary and secondary school enrolment data . Poor attendance could severely reduce the population-level impact of school-based deworming . Conversely however , there is anecdotal evidence that there may be higher attendance to schools for deworming days due to awareness of the health benefits . These effects have not yet been quantified , to our knowledge . For a given coverage of a school-based programme , as defined by proportion of estimated treatment of a proportion of worms , , or egg output , , the impact on transmission will depend on the particular dynamics of the parasite . Here we outline general insights on the non-linear effects of limited coverage on transmission . We then use heterogeneous mixing models to investigate how different mixing patterns between adults and children will affect the impact of targeted programmes . Simple theory provides some important general insights into the factors controlling helminth transmission and the impact of community-based chemotherapy [18] . For directly transmitted helminths with a free living larval or egg stage outside the human host , the basic dynamics of the system can be described by the following differential equation , ( 6 ) where 1/μ is human life expectancy , 1/μ1 is adult parasite life expectancy , M is the mean number of worms in the population , f ( M ) is the mean egg output per gram of stool , given a mean worm burden of M , the dispersal parameter , k , and fecundity coefficient , z: ( 7 ) The basic reproductive number defined as the average number of female worm offspring that survive to reproduce in the absence of density-dependent constraints ( ignoring the complexities of mating probabilities and age structure in the human population ) is given by equation 8 ( 8 ) Here , denotes the fraction of female worms , the per capita egg production rate , the proportion of female worms that survive in the human host to reproductive maturity , the fraction of eggs or larvae that survive to the infective state , is human life expectancy , is the adult worm life expectancy in the human host , is infective stage life expectancy and is the per capita transmission coefficient for the infective stage . Table 3 records published estimates of for various helminth species . A rough approximation of the growth rate of the parasite population post extensive treatment , again ignoring the effects of density-dependence and worm mating probabilities , is given by: ( 9 ) Where ( ) is the parasite life expectancy in the human host . Equation 9 is based on the assumption that is much shorter than human life expectancy , which is true for all STHs ( see Table 4 ) . Ignoring age structure in the human population , the effect of treatment on the effective reproduction number , , is described by ( 10 ) where is the interval between successive rounds of treatment and is the efficacy of the drug , defined as the proportion of worms killed by the drug . The critical fraction of the human population that must be treated , , to reduce the effective reproductive number to less than unity in value ( assuming that the breakpoint in transmission is close to zero due to the aggregated distribution of worms per person [27] ) is given by: ( 11 ) And thus , the fraction is simply determined by , , and adult worm life expectancy . We simulate these approximations for a range of parameter values to illustrate the non-linearities in these relationships . These approximations for homogenous mixing models are very useful to illustrate the impact of limited coverage on transmission . To gain a more accurate picture of how the treatment of just school-aged children influences the overall transmission dynamics of the parasite , the impact of not only age structure but also exposure to infective stages produced by all age groups in the population must be taken into account . The homogeneous model described above can be extended to an age-structured model to describe the characteristic age-profiles of intensity [18] . Such models usually assume that exposure to infective stages across all age classes is random and independent of which age class produces the eggs or infective larvae . However , the spatial structure of egg deposition and infective stage development arising from one age group , plus their concomitant contact with other age groups , is in reality unlikely to be random in a defined community . Instead , it may be more likely that infective stages produced by school-aged children are deposited in areas closer to habitation , and hence acquired by all age groups of the population , while those arising from adults are less likely to come into contact with children . Therefore the model should include heterogeneous mixing [42] . A simple way to mimic non-random contact is to stratify the population into two age groups , namely; school-aged children ( 5–14 years ) , and the rest ( 0–4 and ≥15 years combined for simplicity , although patterns may also be different between these two groups ) , and assume different contact patterns with the infective stages within and between these larger age groupings . Such a stratification of hosts has the further advantage of facilitating the modelling of school-based treatment programmes . We assume that the child and adult age groups have negative-binomially distributed worm distributions with the same shape parameter , k , but different means , Mc and Ma , respectively . The means evolve independently according to the degree of contact of each group with a common infectious ‘reservoir’ . The model equations are ( extending the approach by Chan et al . [42] ) ( 12 ) The quantity , , is the per capita infectiousness of the shared reservoir . The parameters and determine the strength of contact with the reservoir for children and adults respectively . The dynamics of the infectious reservoir are described by the following equation ( 13 ) The parameters in equation 13 are as defined earlier . The parameters and represent the proportion of the population in the two age classes and and the fraction of egg output that enters the reservoir from children aged 5–14 years and other age groups , respectively . We investigate the effect of regular school-based treatment on the evolution of worm burdens in the community for three scenarios: It should be noted that under scenario C there will be higher intensity of parasites in children at equilibrium , or baseline , as a consequence of their higher exposure to infection . We use the mathematical model described above ( equations 12 and 13 ) to simulate how the mean intensity of infection would vary for a setting with a given mean intensity and treatment coverage under each of these different scenarios . We run the mathematical model for A . lumbricoides and hookworm , which have contrasting transmission dynamics , to illustrate the importance of age-dependent mixing in the success of an age-targeted treatment programme .
The proportion of worms or egg-output in school-aged children , as calculated using equations 3 and 5 above , is best illustrated by reference to a set of published studies covering the main parasites in different country settings . The first example is that of A . lumbricoides in Myanmar from a study by Thein-Hliang et al . [33] . The demography and age intensity data are presented in Figure 3 . Demographic surveys for the year of the study reveal that treatment of all children in the 5–14 years age range would expose roughly 49 . 4% of the worm population to anthelminthics . In terms of the fraction of the parasites killed by a school-based programme assuming a drug efficacy of 95% [5] and school attendance of 95% on the days of treatment gives an overall estimate of 44 . 5% of the total worm population removed by one round of treatment . Calculations for a study by Elkins et al . [24] of A . lumbricoides in Tamil Nadu in India yield a very similar figure for the percentage of worms potentially removed by the treatment of children attending school . Calculations for hookworm ( largely N . americanus ) in Uganda from a study by Pullan et al . [36] are significantly different , as shown in Figure 4 . For this population only 15% of the egg-output is generated by school-aged children . A drug efficacy of 95% and school attendance figure of 85% in a rural region ( see Figure 2 ) , produces an estimate of 12% of egg output treated by one round of treatment . These contrasting figures for A . lumbricoides and hookworm illustrate the importance of the shape of the age intensity profile to both the efficiency of school-aged targeting and the degree to which such treatment programmes will impact on overall transmission in a population . Analyses of epidemiological studies of Schistosoma mansoni produce figures similar to A . lumbricoides , given marked convexity in the intensity by age profiles ( age intensity data from Kabatereine et al . [38] ) . T . trichiura is somewhat intermediary between A . lumbricoides and hookworm with a degree of convexity but significant worm loads in adults [35] , [43]–[44] . The more convex the age intensity profile is , the greater the impact of school-based deworming on overall transmission , provided the peak intensity lies within the age ranges 5–14 years . The continued rise of hookworm intensity in adult age classes yields a low fraction of the total worms in the treated classes . The calculations presented in Figures 3 and 4 and Table 5 are approximations to the values defined in equations 3 and 5 , based on the assumptions that human population size is constant but with an age distribution as documented by the US Bureau of Population and [40] , and that the effects of density-dependent fecundity ( ) are negligible . Polynomial fits are employed to calculate integrals of the functions and respectively from cross sectional studies of the intensity of infection , and age pyramids ( both sexes combined ) from national demographic data . The calculations are conservative estimates since they are based on the assumption that all who are eligible attend school . In practice , the percentages recorded in Table 5 must be multiplied by the school attendance data from recent UNESCO studies [45] . The impact of the parasite coverage levels presented in Table 5 on transmission under the assumption of homogeneous mixing can be seen using the approximations to the critical treatment coverage , ( derived above in relation to the parasite life expectancy , , and the basic reproduction number , , as in Figure 5 ( equation 11 ) ) and the impact of increasing on the mean worm load , , and the prevalence of infection , ( Figure 6 , equation 6 ) . Figure 6 reveals that the mean worm load decays approximately linearly as the fraction treated rises , while prevalence only begins to fall steeply as the fraction treated approaches the critical value and effective transmission ceases . Note that from Figure 5 the critical fraction treated reaches 1 for high /low parasite life expectancy . This indicates the parasite may be very difficult to eradicate by treatment with the given efficacy ( = 0 . 9 ) when is large ( ∼>4 , estimated values for particular parasites in Table 3 ) and is short ( ∼<1 . 2 years , values in Table 4 ) e . g . for A . lumbricoides and T . trichiura in high transmission settings . Further insight on the effect of targeting school-aged children can be gained by considering differential mixing patterns between children and the rest of the population , as outlined above . The results of example simulations are presented in Figures 7 and 8 , where the worm burdens in school-aged children and other age groups ( where applicable ) and averaged across the community are presented for different modelled scenarios , helminths and treatment intervals . The columns of the figures correspond to the scenarios A ( homogeneous population ) , B ( homogeneous mixing ) and C ( heterogeneous mixing ) . The heterogeneous mixing ( scenario C ) results in a higher worm burden in the children than in the adults , as is seen in several settings ( note this model does not include any immunity ) . All models have the same mid-range value of 3 . Treatment in the homogeneous model is made comparable with the heterogeneous model by setting coverage to . We have simulated these scenarios for A . lumbricoides , with a life expectancy of 1 year ( Figure 7 ) and hookworm with a life expectancy of 2 . 5 years ( Figure 8 ) . The most striking feature of Figure 7 and Figure 8 is the very modest impact of treatment of children on transmission , even at high levels of efficacy ( 95% ) and coverage ( 85% ) . The treated children do have large benefits in terms of periods free of worms or with low worm burdens . However , the effect of treating children on worm burdens in the larger community is small . This reflects the proportion of the worm population actually reached by treatment , even though the chosen value ( 30% ) is at the high end of school-attending fraction of the population ( Table 5 ) . Decreasing the treatment interval ( bottom row in each figure ) has only a moderate effect . The two group equal mixing model ( scenario B ) shows the direct effect of school-based treatment on school-aged children and also the indirect effect on adults through the reduction in infectious material in the community . The rate of bounce-back after treatment is slightly reduced in the heterogeneous model as compared to the homogeneous one ( scenario B versus scenario A ) . This means that homogeneous descriptions ( e . g . the homogeneous model , A ) of non-uniform treatment regimens ( targeted at some portion of the population ) will always underestimate the time to recover to pre-treatment levels . Scenario C mimics what we believe to be a more realistic epidemiological scenario , with school-aged children contributing twice as much infectious material as other age groups and also being twice as exposed , resulting in higher worm burdens in school-aged children . This is more likely to be observed for A . lumbricoides ( e . g . Figure 3 ) than for hookworm ( e . g . Figure 4 ) . As such , the effect of treatment on the school-aged group is quite pronounced , but the impact at the community level , sometimes termed the ‘herd impact’ is only marginally improved , due again to the small proportion of worms treated . These simulations highlight the importance of mixing patterns in determining the effectiveness of school-based treatment programmes .
School-based approaches to deworming children have many advantages in terms of ease of access in urban and rural regions and the ability to link with other nutritional , health and education initiatives in order to try and minimize delivery and logistic costs . Advocacy for this approach to the control of STHs and the morbidity they induce has been made by many over the past decade [1] , [30] , [46] . However , with increased drug donations to support such programmes , it is now crucial to evaluate the benefits and disadvantages of such an approach . The limitations of this approach has already been implicitly acknowledged in the WHO recommendations to additionally target pre-school children , women of child-bearing age and high risk adults where possible [4] and previous identification of adults as a possible reservoir of infection [47] . However , there are many countries where only school-based deworming is currently under consideration [11] . Of particular importance in this context is the impact of school-based treatment on transmission of the parasites in the entire community , including the pre-school and adult age groups . In particular , sustained transmission ( and thus production of infective stages ) in other age groups will influence the frequency of treatment in school-aged children required to sustain infection at very low levels . A limited number of field-based studies of mass chemotherapy have suggested that adult age groups who do not have access to treatment still benefit from school-based deworming as a result of its impact on the overall intensity of transmission within the population [30] , [33] , [48] . The treatment of a few ( heavily infected individuals ) can impact on the effective reproductive number and therefore reduced exposure to infective stages in those untreated . This effect is analogous to the concept of herd immunity in community-based vaccination programmes where vaccination reduces transmission to those still susceptible to infection . In the context of worms and chemotherapy , the herd impact arises from the reduction in the output of infective stages in faeces that result in the contamination of the environment in which the community of all age groups lives . A similar indirect protection among untreated individuals is the impact upon older children and adults seen after age-targeted ( 1–10 year olds ) large-scale treatment of azithromycin against trachoma [49] . We employed two approaches to examine the impact of school deworming programmes on infection within the entire community , the ‘herd impact’ of the treatment programme . The first was empirical and based on the calculation of the proportions of the population in the school attendance age groups , the fraction of these age groups who attend school and the fraction of the total worm population harboured by in these school-aged children . The results of a set of calculations are summarised in Table 5 . It should be noted that the age-profile and population pyramids used here were not exactly matched , and therefore the calculations will not be precise for a particular point in time . It highlights the need for more recent data for these pathogens , as highlighted in recent articles [50]–[51] . In many of the low-income settings in which STHs are highly prevalent , the population pyramid is approximately exponential in shape , meaning that from 20–30% ( Table 2 , Figure 3A and Figure 4A ) are of school age . However , for some STHs , such as A . lumbricoides , the highest intensity infections are in this age-group ( Figure 3B ) and therefore a large proportion of worms are targeted by school-aged treatment ( up to 50% in our examples , Table 5 ) . These predictions are similar to empirical findings by Bundy et al . [30] who evaluated the impact of age-target chemotherapy on community transmission on the island of Montserrat , West Indies , where T . trichiura , which has similar transmission dynamics to A . lumbricoides , was the predominant species and had a prevalence of 12% ( A . lumbricoides and hookworm occurred at <2% ) . The authors found that 4-monthy treatment of 2–15 year olds had a subsidiary effect on intensity of untreated 16–25 year olds . In contrast to A . lumbricoides and T . trichiura which exhibit age-convexity in intensity , hookworm intensity tends to monotonically increase with age ( e . g . Figure 4B ) , as has been seen in several studies [52]–[54] , and therefore a smaller proportion of worms or egg output are targeted by school-aged treatment ( <10% in one of our examples , Table 5 ) . The different age profiles for these helminth species are a result of differing behaviour patterns , force of infection , heterogeneous exposure and , arguably , immunity and genetic pre-disposition . The details of the biology which generate these patterns do not need to be understood for the calculation of the proportion of worms treated . However , to estimate the impact of treating children on transmission in the larger community we need a combination of additional studies and novel modelling analyses . Our simulations ( Figures 7 and 8 ) show the importance of understanding the nature of the interaction between school-aged children and the rest of the community in order to optimise treatment programmes in the longer term . It is possible that school-aged deworming will give little benefit outside the children being treated and that intensifying either the treatment coverage within this age-group and/or increasing the frequency of treatment ( bottom rows in Figures 7 and 8 ) may not lead to the desired benefits in the larger community . In this case , other strategies will be needed to increase the potential for long-term control and , if the breakpoint can be crossed by combinations of interventions , eventual elimination of these helminths . As the schematic , Figure 9 , shows , the impact of school-based treatment programmes on transmission in the larger community is diluted by a number of effects . The benefits of deworming for the affected children are many , but if we are to plan for long-term control and , in the longer term , elimination of these pathogens we need to consider strategies that will reduce transmission from year to year , as is already being discussed in some settings [55] , particularly for schistosomiasis [56] . We also require an understanding of the species mix in each setting so as to tailor the design of interventions according to the underlying transmission dynamics . As this paper shows , there are many outstanding data gaps and needs for new modelling studies both to understand the dynamics of transmission under such programmes , and to design optimal treatment strategies for the future . | Large donations of drugs to treat soil-transmitted helminths ( STHs , intestinal worms ) means that many more school-aged children will be treated , improving their well-being and development . These children will have to be repeatedly treated since reinfection will occur due to contaminated environments in the absence of improvements in hygiene and sanitation . Repeated treatment of school-aged children may have the added benefit of reductions in levels of infection for the whole community . This will in part be determined by the proportion of the total worms harboured or eggs output by school-aged children , a product of how heavily infected school-aged children are and how many school-aged children there are in the community . In one setting school-aged children output as little as 15% of hookworm eggs whereas in another setting they harbour up to 50% of roundworms . Thus , whilst school-based programmes may have important health benefits , the community-level impact on transmission could be limited unless school-aged children over-contribute to infection . We use mathematical models to show that if children contribute more infective stages to the environment which adults are exposed to than adults do , the reductions in transmission resulting from treating children will be larger , but may still be limited . | [
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"com... | 2013 | How Effective Is School-Based Deworming for the Community-Wide Control of Soil-Transmitted Helminths? |
Molting , or the replacement of the old exoskeleton with a new cuticle , is a complex developmental process that all insects must undergo to allow unhindered growth and development . Prior to each molt , the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle . We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf ( Knk ) protects the new cuticle from degradation . Here we demonstrate that , in Tribolium castaneum , the protein Retroactive ( TcRtv ) is an essential mediator of this protective effect of Knk . TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle . Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth , consistent with the loss of TcKnk function . Given the ubiquity of Rtv and Knk orthologs in arthropods , we propose that this mechanism of new cuticle protection is conserved throughout the phylum .
A critical feature of the insect molting process is the simultaneous synthesis and degradation of chitin at different sites associated with the cuticle [1] , [2] . Chitinolytic enzymes ( molting fluid chitinases and N-acetylglucosaminidases ) dissolve parts of the old chitinous exoskeleton into oligomeric and monomeric N-acetylglucosamine ( GlcNAc ) units , which are subsequently recycled into the collective pool of biosynthetically derived activated precursors of GlcNAc monomers ( UDP-GlcNAc ) used for the synthesis of new cuticular chitin [3] , [4] . The enzyme that catalyzes the addition of these monomers onto the growing chitin oligosaccharide is the membrane-bound chitin synthase [3] , [5] , [6] . Upon synthesis and secretion into the extracellular ( subcuticular ) space by epidermal cells , nascent chitin aggregates into microfibrils and accumulates within the assembly zone ( the layer immediately above the apical surface of the epidermal cells ) to eventually organize into new cuticular laminae [7] , [8] . At the ultrastructural level , the new cuticle extends from its inner boundary on the apical side of the epidermal cell membrane to its outer “envelope” layer that appears to partition it from the overlying old procuticle [9] . However the envelope layer functions only as a structural boundary at the early stages of molting , and does not function as a barrier against molting fluid chitinases [10] . Indeed , the molting fluid chitinase of T . castaneum ( TcCht-5 ) was observed throughout the new procuticle co-localized with chitin . The mysterious stability of the new cuticle in intimate contact with enzymatically active chitinases was shown to be due to its physical association with the cuticle assembly protein , Knickkopf ( TcKnk; homologue of D . melanogaster knickkopf , DmKnk ) [10] . Like DmKnk , the protein Retroactive from Drosophila melanogaster ( DmRtv ) was also previously demonstrated to affect cuticle differentiation and tracheal tube morphogenesis [11] , [12] , [13] . DmRtv was predicted to form a membrane-anchored , three-finger loop structure that interacts with chitin via two aromatic residues situated in each loop [11] . Although a direct role for this protein in cuticular chitin filament organization in embryonic tracheal tubules of D . melanogaster was envisaged , the mechanism of this ordering process is unclear . In the current study we show that a direct effect of the T . castaneum Rtv homologue ( TcRtv ) on cuticular chitin organization is unlikely , given its predominant intracellular distribution within epidermal cells . Instead , its activity is shown to be crucial for the proper trafficking of TcKnk to the procuticle where the latter protein associates with and protects chitin . Hence , TcRtv promotes cuticle synthesis and organization by facilitating TcKnk's localization in the integument .
TBLASTN and BLASTP searches of the T . castaneum genome using the DmRtv protein sequence as query identified only a single gene coding for an orthologous protein . T . castaneum retroactive ( TcRtv ) ( JX470185 ) maps on LG4 , position 24 . 3 cM . TcRtv is composed of three exons and encodes a 150- amino acid residue long protein with a predicted C-terminal GPI anchor and an ω-asparagine at position 124 ( Figure S1A and S1B ) ( GPIPred ) . A predicted cleavable signal peptide at its N-terminus suggests that this protein enters the ER secretory pathway prior to GPI-anchoring and is transported to the cell surface via intracellular secretory vesicles . Expression analysis revealed the presence of TcRtv transcripts throughout development including embryonic , larval , pupal and adult stages ( Figure 1A ) . Lower levels of TcRtv expression were detected at the embryonic and young larval stages relative to later stages of development . Tissue-specific expression of TcRtv revealed that TcRtv transcripts could only be detected in the hindgut and carcass but not in midgut tissues indicating that this gene is expressed in tissues of ectodermal origin ( Figure 1B ) . TcRtv transcripts were also detected in pharate adult elytra and hindwings ( Figure 1B ) . When other fully sequenced arthropod genomes were queried with DmRtv or TcRtv sequences , only a single homolog was identified in all the insect species . The presence of an ortholog of TcRtv gene in all sequenced insect genomes suggests an essential function for this protein in insect survival and development ( Figure 1C ) . Interestingly , a single Rtv ortholog is also present even in non-insect arthropod species including the water flea , Daphnia pulex , and the deer tick , Ixodes scapularis , suggesting that Rtv may have a conserved biological function in all species that produce a chitinous exoskeleton . On the other hand , we could not identify Rtv homologs in the genome of the nematode , Caenorhabditis elegans , which does synthesize chitin in its eggshells and the pharyngeal lumenal walls . Thus Rtv may have appeared early during arthropod evolution . Given the ubiquitous presence of TcRtv transcripts during insect growth , we hypothesized a critical role for this protein in post-embryonic development . To test this hypothesis , we depleted TcRtv transcripts via RNA interference ( RNAi ) by administration of TcRtv-specific dsRNA ( dsRtv ) during multiple developmental stages of T . castaneum . RNAi of TcRtv led to molting arrest at larval-larval , larval-pupal or pupal-adult stages , depending upon the developmental stage at the time of injection , with significant reductions in transcript levels at each stage tested ( Figure 1D and 1E ) . Apolysis and partial slippage of the old larval cuticle proceeded at the subsequent molt after injection of TcRtv dsRNA into penultimate-instar larvae , but the larvae failed to complete the larval-larval molt and remained entrapped within their old larval cuticle . Similarly , insects injected with TcRtv dsRNA at later stages were arrested at the larval-pupal or pupal-adult molts and failed to shed their old cuticle . These phenotypes were reminiscent of those that we have reported previously following RNAi for TcChs-A [5] . Control insects injected with the same dose of dsRNA for the eye pigmentation gene , TcVer ( T . castaneum Vermilion ) , developed normally except for the loss of eye color . These results demonstrate that Rtv is essential for molting in T . castaneum . Because of the terminal phenotypic resemblance of insects treated with dsRNAs against TcRtv or TcChs-A [5] , we suspected a reduction of procuticular chitin content resulting in loss of mechanical strength of the new cuticle may account for the failure to complete ecdysis . To test this hypothesis , insects at the pharate pupal stage were treated with TcRtv-specific dsRNA and their chitin contents were probed qualitatively and quantitatively just prior to the adult molt ( pharate adult ) . Confocal microscopic analysis of the pharate adult elytra stained with a chitin-binding domain ( CBD ) probe ( rhodamine-conjugated CBD ) showed a near complete loss of chitin in the new cuticle of TcRtv-depleted insects when compared to control insects ( Figure 2A ) . A similar reduction in new cuticular chitin was also detected in the pharate adult insect body wall after RNAi for TcRtv compared to TcVer-depleted insects ( Figure 3B , column 1- row 2 ) . Independently , quantitative analysis of total body chitin content by a modified Morgan-Elson assay confirmed chitin depletion in these insects during both larval-pupal and pupal-adult molts ( Figure 2B ) . The reduction of chitin content following RNAi of TcRtv was comparable to that observed in TcChs-A-depleted insects , and varied depending on the stage of insect development from ∼2- to 10-fold relative to control insects ( Figure 2B ) . Collectively , these data reveal that TcRtv affects molting by modulating the level of chitin , predominantly in the newly forming cuticle . The total amount of chitin in an insect is likely to change dynamically during periods of growth as a result of repeated cycles of cuticle deposition and turnover . The dynamics are more complex during molting when there is an overlap of the period of chitin synthesis in the new cuticle with that of chitin degradation in the old cuticle . As a result , several possible mechanisms by which TcRtv may regulate chitin levels in the procuticle can be envisioned . The loss of chitin following RNAi for TcRtv suggests that this protein might be an activator of chitin synthesis or an inhibitor of chitin degradation . In the first scenario , TcRtv might affect chitin synthesis via its effect on TcChs-A at the transcriptional , post-transcriptional , translational or post-translational levels . However , our observation that steady-state levels and cellular distribution of TcChs-A protein were similar in control and TcRtv-depleted insects ( Figure S2 ) argues against a direct effect of TcRtv on TcChs-A expression and localization . An alternative hypothesis that would explain the observed reduction in chitin in the newly forming procuticle following TcRtv RNAi is that TcRtv protects newly synthesized chitin from molting fluid chitinases . Indeed , we have shown recently that chitin is co-localized with chitinases , which are not excluded from the newly forming procuticle as had been assumed in the past . We have further shown that the chitin-binding protein , TcKnk , is needed to protect the nascent chitin in the procuticle from chitinase-mediated degradation [10] . To test the hypothesis that TcRtv also protects chitin , we down-regulated the expression of the major molting fluid chitinase ( TcCht-5 ) at the pharate adult stage either alone or in combination with TcRtv and subjected these samples to quantitative chitin content analysis . Remarkably , the analysis revealed that insects subjected to RNAi for TcRtv alone exhibited significant chitin reduction , but co-depletion of both TcRtv and TcCht-5 transcripts resulted in nearly a complete rescue of cuticular chitin content relative to control levels ( Figure 3A ) . These results also rule out a requirement for TcRtv for the synthesis and transport of chitin to the procuticle by Chs-A and suggest that Rtv-mediated stabilization of chitin involves a step after the synthesis of chitin . This conclusion was further supported by confocal microscopy , which showed a near complete loss of chitin in the procuticle following depletion of TcRtv transcripts compared to the dsRNA TcVer-treated insects . But RNAi for both TcCht-5 and TcRtv , rhodamine-CBD staining indicated that the level of chitin within the new cuticle of body wall was restored to the same level as dsTcVer controls ( Figure 3B , compare row 3 with row 1 ) . To gain a better understanding of the nature of the restored chitin in the procuticle following depletion of transcripts for both TcCht-5 and TcRtv , we performed Transmission Electron Microscopic ( TEM ) analysis of the elytra , body wall , tracheae and denticles of insects treated with the indicated combination of dsRNAs . Ultrathin sections of elytral and body wall cuticle from control insects were characterized by a well-organized laminar architecture . Along with the drastic reduction in procuticular chitin , the laminar architecture was completely disrupted following RNAi for TcRtv . Although simultaneous down-regulation of TcRtv and TcCht-5 transcripts resulted in the restoration of chitin within the procuticle , the laminar organization was not reestablished ( Figure 4 ) . Interestingly , this phenotype was indistinguishable from the phenotype that we have recently observed in elytra of pharate adults depleted of transcripts for both TcKnk and TcCht-5 . Additional analyses also revealed structural deformities of denticles protruding at the lateral body wall that is consistent with loss of laminar architecture in denticular procuticle following down-regulation of TcRtv in these insects ( Figure 4 ) . Similarly the taenidial cuticle that lines the tracheal lumen and stabilizes the tracheal tube in these insects also appeared to exhibit structural defects possibly due to the disorganized procuticle ( Figure 4 ) and electron-dense inclusions they harbored . Simultaneous down-regulation of TcRtv and TcCht-5 transcripts , which restores chitin back to normal levels , did not rescue these phenotypes in either the denticles or taenidia , indicating the importance of TcRtv in maintaining the morphology of both taenidia and denticles ( Figure 4 ) . Since the phenotypes of RNAi for TcRtv and TcKnk are indistinguishable , we considered the possibility that these two proteins may be involved in the same linear pathway . Immunohistochemistry carried out on sections of control insects ( dsTcVer-treated ) and those treated with dsRNAs specific for TcRtv , using a polyclonal antibody against Knk indicated that depletion of TcRtv transcripts alone resulted in the mislocalization of TcKnk from its normal distribution predominantly in the procuticle to an exclusive presence within the epidermal cells , perhaps with some TcKnk in the plasma membrane ( Figure 5 , compare row 3 with row 1 ) . However it is likely that the altered localization of TcKnk may have been brought about indirectly by a reduction of chitin which acts as a signal for TcKnk's release into the procuticle and not by a direct effect of TcRtv on TcKnk trafficking . To distinguish between these two possibilities , TcKnk localization was examined in insects depleted of both TcRtv and TcCht-5 transcripts by co-injection of both dsRNAs . This enabled us to disable TcRtv function without concomitant depletion of procuticular chitin , and thus observe the direct effect of TcRtv on localization of TcKnk independently of any possible contributing effects of procuticular chitin itself . Although chitin levels remained at near normal levels in the procuticle after such treatment , TcKnk was mislocalized , remaining entirely inside the cell rather than being secreted into the procuticle ( Figure 5 , compare row 4 with row 1 ) . The distribution of TcKnk in these double-knockout insects was indistinguishable from that observed in those treated with dsRNA for TcRtv alone . These observations support the hypothesis that TcRtv facilitates the trafficking of TcKnk into the procuticle .
The assembly of chitin into the laminated procuticle of insects is a complex process that involves the coordinated action of multiple proteins involved in the synthesis and modification of chitin as well as other chitin-binding and chitin-organizing proteins . While the functions of chitin synthases , deacetylases and chitinases have been studied in some detail [5] , [14] , [15] , [16] , the role of non-enzymatic chitin-binding and organizing proteins are less well understood [10] , [17] . Mutational analysis in D . melanogaster has implicated two genes , Knk and Rtv , in the organization of tracheal tube chitin and of the embryonic cuticular matrix [11] , [12] . More recently , we have shown that the TcKnk protein is localized primarily in the procuticle , possibly bound to chitin [10] . Furthermore , we have shown that procuticular TcKnk is crucial to the protection of chitin from the action of chitinases , which are not excluded from chitin in the newly forming procuticle as previously believed . In addition , TcKnk is needed for the organization of procuticular chitin into an ordered laminar array . However , the role of the protein TcRtv has not been studied extensively even though the phenotypes of Rtv and Knk mutants of D . melanogaster are virtually indistinguishable [11] , [12] . Both TcKnk and TcRtv are predicted to encode proteins with GPI anchors by bioinformatic prediction tools ( big-PI Predictor , PredGPI and GPI-SOM ) . However , experimental evidence for the GPI anchor is available only in the case of Knk . TcKnk and DmKnk are released from the plasma membrane of cells expressing this protein by treatment with phosphatidylinositol-specific phospholipase C ( PI-PLC ) , an enzyme known to cleave the GPI anchor of membrane-bound proteins [10] , [12] . TcRtv expressed in insect cell lines infected with a recombinant virus containing the Rtv coding sequences is not released by PI-PLC treatment ( Figure S3 ) . We suspect that TcRtv does not have a true GPI anchor and may merely have a membrane-spanning segment at the C-terminus , suggesting that this protein remains embedded in the ER or attached within vesicles and plasma membranes . Therefore , we propose that TcRtv may not have a direct role in protecting chitin from chitinases and that the loss of procuticular chitin observed after RNAi of TcRtv expression must be via an indirect effect . The finding that there are no significant differences in the amount or localization of TcChs-A in control and dsRNA TcRtv-treated insects also suggests that TcRtv does not have a direct effect on TcChs-A expression or localization . Finally , the full recovery of chitin levels in the procuticle in the absence of TcRtv upon down-regulation of chitinases rules out any role for TcRtv in the synthesis and transport of chitin to the procuticle . Then how does TcRtv affect cuticle integrity ? We have demonstrated that the absence of chitin in the procuticle alone does not account for the mislocalization of the TcKnk protein after RNAi-mediated transcript depletion of TcRtv , because simultaneous RNAi of TcRtv and chitinase genes , while restoring procuticular chitin , fails to rescue the intracellular mislocalization of TcKnk . Rather , it appears that the targeting of TcKnk protein to the procuticle requires the presence of TcRtv . Whether this is due to a direct protein-protein interaction between TcRtv and TcKnk or through a more complex targeting pathway remains to be established . A search of the SCOP database revealed that the insect Retroactive proteins share broad structural similarity with the snake toxin-like superfamily of proteins with the three-finger domain ( TFD ) or Ly-6/uPAR domain implicated in protein-protein interactions ( Figure S4 ) [11] , [18] . The Ly-6 protein family is composed of several membrane-bound receptors , GPI-anchored proteins , and soluble ligands . These proteins are characterized by the presence of an approximately 100-residue long extracellular motif called the three-finger domain ( TFD ) [18] . In the prototypical structure described for proteins of this family , the sea snake venom neurotoxin or erabutoxin b , 10 cysteines form a series of disulfide bridges that stabilize the protein core and allow three finger-like loops to protrude [18] , [19] . These finger-like extensions with variable sequences are believed to enable interaction of these proteins with a broad range of substrates or ligands , each with a high degree of specificity . Insect Rtv proteins contain 10 cysteines that are predicted to adopt the TFD fold ( Figure S4 ) [11] , suggesting that they may be members of the snake toxin-like superfamily of proteins . Our work has uncovered a novel mechanism involved in cuticular chitin maintenance , wherein presence of TcRtv inside the cells triggers the accretion of TcKnk to the growing chitinous matrix of the procuticle whereupon the latter protein facilitates chitin organization and confers protection from chitinolytic enzymes during the process of molting [10] . The mechanism described herein is probably conserved in all chitinous invertebrates .
The GA-1 strain of T . castaneum was used for all experiments . Insects were reared at 30°C in wheat flour containing 5% brewer's yeast under standard conditions as described previously [20] . An extensive , genome-wide search for homologs of DmRtv in the T . castaneum genome database was performed using NCBI programs TBLASTN and BLASTP and using the amino acid sequence of DmRTV as query . A DNA fragment containing the complete coding sequence of TcRtv ( 453 bp ) was amplified by reverse transcriptase-PCR ( RT-PCR ) using the gene-specific primers ( forward primer 5′- ATGGGTCTGTTTAGATCAATTT-3′ and the reverse primer 5′- TTACAAAAATCGTAAAATCAGTCT-3′ ) using cDNA prepared from the RNA extracted from different stages of beetle development as template . Additional 5′ and 3′ UTR sequences were obtained by 5′- and 3′- RACE . The amplified fragment was cloned into the pGEMT vector . Sequencing of the cDNA clone was carried out at the DNA sequencing facility at Kansas State University . Rtv-like proteins were identified by TBLASTN searches of the fully sequenced genomes of T . castaneum , Anopheles gambiae , Aedes aegypti , Culex quinquefasciatus , Bombyx mori , Acyrthosiphon pisum , Nasonia vitripennis and Ixodes scapularis using the amino acid sequence of DmRtv as query . Multiple sequence alignments of TcRtv and the related Rtv-like proteins from other insects and arthropods were carried out using the ClustalW software prior to phylogenetic analysis . A consensus phylogenetic tree was constructed using MEGA 4 . 0 neighbor joining method [21] . The isolation of RNA and preparation of cDNA from different stages of T . castaneum development were carried out as described previously [10] . These cDNA templates and the pair of TcRtv-specific primers , forward primer 5′-ATGGGTCTGTTTAGATCAATTT-3′ and reverse primer 5′- TTACAAAAATCGTAAAATCAGTCT -3′ , were used for RT-PCR to determine the TcRtv expression profile . The T . castaneum ribosomal protein-6 ( TcRpS6 ) gene was used as an internal control for equal loading of cDNA templates [22] . Two different regions of the TcRtv gene were selected for making dsRNAs . Pairs of forward and reverse primers for the chosen regions ( Table S1 ) were used to amplify the dsRNA sequences by using the cloned TcRtv cDNA as template . The Ampliscribe T7-Flash Transcription Kit ( Epicentre Technologies ) was used to synthesize dsRNA as described previously [5] . dsRNA for the tryptophan oxygenase , dsVermilion ( dsVer ) gene , which is required for eye pigmentation , was used as a control . dsRNAs for TcRtv or TcVer were injected into animals in the young larval , last instar larval , or pharate pupal stages of T . castaneum development ( 200 ng per insect , n = 30 ) . Five insects were collected at the young larval , pharate pupal and pharate adult stages of development 3–5 d after dsRNA treatment . Total RNA was extracted from the collected insects for measuring transcript levels by RT-PCR using gene-specific primer-pairs . The full-length TcRtv cDNA clone was used as template to amplify the complete coding region of the TcRtv gene . A primer pair containing appropriate restriction enzyme sites [forward primer 5′-TATCCCGGGATGGGTCTGTTTAGATC-3′ ( Sma I ) and a reverse primer 5′-CAGACTGATTTTACGATTTTTGTAATCTAGATAT-3′ ( Xba I ) ] was used to facilitate directional cloning of the TcRtv open reading frame ( ORF ) DNA in the pVL1393 baculovirus expression vector ( BD Pharmingen ) . PCR-amplified , full length TcRtv DNA and the pVL1393 vector DNA were digested with the same pair of restriction enzymes and ligated as described previously [15] . Hi-5 cells ( Trichoplusia ni cell line ) were used to express the TcRtv protein as described earlier [15] . TcRtv dsRNA-treated pharate adult insects were collected and fixed as described previously [10] . Cryosections of 20 µm thickness were made and stained for specific proteins using chicken antiserum to T . castaneum Rtv ( 1∶100 ) , rabbit antiserum ( 1∶100 ) to D . melanogaster Knk and rabbit antiserum to T . castaneum Chs-A ( 1∶50 ) as primary antibodies . Alexa 488 goat-anti-chicken IgG ( 1∶1000 ) or Alexa 488 goat-anti-rabbit IgG ( 1∶1000 ) were used as secondary antibodies for the fluorescence detection of TcRtv , TcKnk and TcChs-A proteins . Rhodamine conjugated chitin-binding probe ( 1∶100 ) and DAPI ( 1∶15 ) were used for staining of chitin and nuclei , respectively . Confocal microscopy was performed with an LSM META 510 laser scanning confocal microscope using laser lines of 405 nm , 488 nm and 543 nm for excitation . Images were taken using an oil objective ( 40×/1 . 3 NA ) with 8× zoom and processed in Adobe Photoshop 7 . 0 . Hi-5 cells were infected with a recombinant baculovirus containing the TcRtv and TcKnk open reading frame and incubated at 30°C for 3 d for expression of proteins . Three days post-infection , Hi-5 cells expressing respective proteins were treated with PI-PLC as described previously [10] . Following dsRNA treatment , pharate pupae and pharate adults of T . castaneum were collected for chitin content analysis by a modified Morgan-Elson method as described previously [5] . TcVer dsRNA and TcChs-A dsRNA-treated insects were used as negative and positive controls for the experiment , respectively . Pharate pupae were injected with dsRNA and pharate adult samples were collected on pupal day 5 , fixed and embedded in EMBED 812/Araldite resin as described previously [10] . Resin embedded samples were then thin-sectioned ( silver to gold section ) and imaged on a CM-100 TEM . cDNA sequence for T . castaneum Retroactive ( TcRtv ) is deposited at NCBI with accession number JX470185 . | The outer shell of an insect serves both as protective skin and rigid exoskeleton that must be periodically replaced with a new , larger one during development . During this molting process , the inner layers of the old exoskeleton are digested and recycled , while the outer layers are discarded . Secretion of the new skin necessarily commences before the partial recycling and shedding of the old shell . This creates a problem for the insect , namely how to protect the new skin from digestive enzymes intended for the old shell that closely enwraps it . Previously we showed that such protection is afforded by the Knickkopf ( Knk ) protein , which is secreted from the epidermis and infiltrates the new skin , rendering it resistant to enzymatic degradation . In this work , we show that another protein , called Retroactive ( Rtv ) , ensures the proper trafficking of Knk into the newly secreted skin . Rtv remains inside the epidermal cells , while directing the transport of Knk to the cell surface and ensuring its export into the new skin . Digestive enzymes are then secreted and target the old exoskeleton while leaving the new one intact . This dependence of Knk on Rtv function is probably true for all insects and other arthropods . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"developmental",
"biology",
"biology"
] | 2013 | Retroactive Maintains Cuticle Integrity by Promoting the Trafficking of Knickkopf into the Procuticle of Tribolium castaneum |
CD8+ T cells mediate immunity against Plasmodium liver stages . However , the paucity of parasite-specific epitopes of CD8+ T cells has limited our current understanding of the mechanisms influencing the generation , maintenance and efficiency of these responses . To identify antigenic epitopes in a stringent murine malaria immunisation model , we performed a systematic profiling of H2b-restricted peptides predicted from genome-wide analysis . We describe the identification of Plasmodium berghei ( Pb ) sporozoite-specific gene 20 ( S20 ) - and thrombospondin-related adhesive protein ( TRAP ) -derived peptides , termed PbS20318 and PbTRAP130 respectively , as targets of CD8+ T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge . While both PbS20318 and PbTRAP130 elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice , only PbTRAP130-specific CD8+ T cells exhibit in vivo cytotoxicity . Moreover , PbTRAP130-specific , but not PbS20318-specific , CD8+ T cells significantly contribute to inhibition of parasite development . Prime/boost vaccination with PbTRAP demonstrates CD8+ T cell-dependent efficacy against sporozoite challenge . We conclude that PbTRAP is an immunodominant antigen during liver-stage infection . Together , our results underscore the presence of CD8+ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes . Our identification of antigen-specific CD8+ T cells will allow interrogation of the development of immune responses against malaria liver stages .
Malaria is responsible for an estimated 250 million episodes of clinical disease and 600 , 00 to 1 . 2 million deaths each year [1] , [2] . Notwithstanding recent reductions in the burden of malaria in some endemic areas , sustained control , elimination or eradication of the disease will require a highly efficacious vaccine that prevents malaria transmission as well as reducing the burden of disease . As a benchmark in malaria vaccination , multiple immunisations of γ-radiation-attenuated Plasmodium sporozoites ( γ-Spz ) can protect both mice and humans against sporozoite challenge [3] , [4] . The elicited protection targets the development of liver stages and completely prevents blood stage infection , resulting in sterile immunity . This experimental vaccine approach has now been replicated using other whole sporozoite immunisation strategies that include infection under drug cover and genetically arrested parasites [5]–[8] . Naturally acquired pre-erythrocytic immunity is likely multifactorial [9] , involving both antibodies and T cells . However , CD8+ T cells are the prime mediators of protection after γ-Spz vaccination in mice [10] , [11] , and interferon ( IFN ) -γ is a signature of effector function [12] . How CD8+ T cells are primed , modulated , and maintained following immunisation , and how these cells execute protective functions , are key considerations for vaccine design and can only be addressed with antigen-specific tools . The circumsporozoite protein ( CSP ) , the major surface protein of the sporozoite , has been at the forefront of vaccination studies for more 20 years – being the basis of RTS , S , the most advanced malaria vaccine to date [13] . Furthermore , CSP-specific responses have been the standard in measuring cellular responses to malaria liver stages in fundamental immunological studies in mice [14] , [15] . Murine models of sporozoite immunisation have largely focused on two strains , BALB/c and C57BL/6 ( B6 ) . Immunisation with Plasmodium berghei ( Pb ) or P . yoelii ( Py ) γ-Spz induces highly protective , H2d-restricted CD8+ T cell responses to defined CSP epitopes in BALB/c mice [16] , [17] . However , protection can also be obtained in the absence of PyCSP-specific T cells: ( a ) PyCSP-transgenic BALB/c mice - that are tolerant to CSP - can be completely protected by Py γ-Spz immunisation [18] and ( b ) there is cross-species immunity to sporozoites despite lack of cross-reactivity of the CSP-derived CD8+ T cell epitopes [19] . These data highlight the importance of non-CSP antigens in generation of protective immunity to liver stages . However , the paucity of liver-stage specific antigens for CD8+ T cells , and the limited availability of gene-targeted mice on the BALB/c background , has limited both the evaluation of subunit vaccine candidates in murine malaria models and the characterisation of the mechanisms underlying CD8+ T cell mediated protection . In contrast to the ease of inducing protective immunity in BALB/c mice , B6 mice expressing H-2b can only be protected against Pb ( or Py ) infection by multiple rounds of γ-Spz immunisation . Most importantly , protection is entirely independent of CSP-specific CD8+ T cells [18] . Indeed , the CSP seems to contain no naturally processed and presented H-2b-restricted epitopes . We propose , therefore , that the Pb-B6 model is a more relevant model of liver stage immunity than the BALB/c model . It more closely resembles the situation in humans , where CD8+ T cell responses to the CSP are infrequent [9] . These immune-epidemiological findings in malaria-endemic areas are reflected by the fact that multiple immunisations are needed to elicit sterilising immunity [20] Moreover , B6 mice are particularly attractive for immunological studies due to the availability of a large collection of sub-strains with targeted gene deletions . In order to develop a Pb-B6 model of antigen-specific CD8+ T cell-mediated anti-liver stage immunity , we employed an unbiased genome-wide approach for screening H-2b ( Kb and Db ) restricted Pb-derived peptides that are recognised by CD8+ T cells from B6 mice immunised with whole sporozoite immunisation strategies known to induce protection . Our results identify two novel liver stage immunogenic targets of effector CD8+ T cells in immunised B6 mice . Of these two , CD8+ T cell responses to PbTRAP confers partial efficacy against sporozoite challenge in vivo . Considering that P . falciparum TRAP ( PfTRAP ) is a major target of human malaria vaccine development , our results emphasize the translational relevance of the Pb-B6 model .
The data and tools available at the Immune Epitope Database and Analysis Resource ( www . iedb . org ) were used for the identification of putative CD8+ T cell epitopes [21] . Systematic epitope profiling has previously identified previously unrecognized CD8+ T cell responses to a number of viral infections including vaccinia , dengue and herpes viruses [22]–[24] . To assemble a genome-wide peptide library , Pb open-reading frames , based on published sporozoite and liver stage transcriptomic and proteomic data [25]–[29] , were scanned in silico using artificial neural network methods [30] for major histocompatibility complex ( MHC ) Class I H2-Kb and Db restricted peptides . In addition , predictions were performed using stabilised matrix methods [31] on the entire Pb draft genome [32] . Finally , Pb orthologs of Pf proteins that were reported to be antigenic for either human antibodies or T cells from individuals immunised by irradiated Pf sporozoites ( Pf γ-Spz ) [33] , [34] were also analysed . From this in silico analysis , 600 unique peptide sequences ( 288 8-mers , 311 9-mers and one 10-mer ) , which correspond to >350 Pb antigens , were identified and subsequently produced by solid phase synthesis ( Table S1: Summary of datasets and Table S2: Complete list of peptides ) . Individual peptides were tested and CD8+ T cell-derived IFN-γ was quantified by two complementary read-outs: ( 1 ) an enzyme-linked immunospot ( ELiSpot ) assay [35] ( Figure 1A , C ) , and ( 2 ) direct peptide-stimulation followed by intracellular cytokine staining ( ICS ) ( Figure 1B , C ) . Animals received two immunisation doses of one of four whole sporozoite vaccination strategies: ( i ) Pb γ-Spz [3] and live sporozoites ( PbSpz ) given concomitantly with anti-malaria drugs ( ii ) azithromycin ( AZ ) [8] , ( iii ) primaquine ( PQ ) [7] or ( iv ) chloroquine ( CQ ) [6] , [36] . As negative controls , CD8+ T cells were isolated from mice immunised with heat-killed sporozoites ( PbHKSpz ) , known to elicit sub-optimal T cell responses [37] , [38] , and naïve mice . Two peptides consistently elicited robust IFN-γ responses in a proportion of CD8+ T cells isolated across all four whole sporozoite vaccine strategies ( Figure 1A , B ) but not from PbHKSpz ( Figure 1B ) and naïve mice ( data not shown ) . Several other peptides were weakly reactive during initial screens but were not confirmed upon re-screening . The first peptide , VNYSFLYLF , contains motifs for Kb and is derived from amino acids 318–325 of the PbS20 protein [PbS20318 ( or PbS20318–325 ) ] ( PBANKA_142920; gi: 40950503 ) , an uncharacterised protein that is conserved in Pf ( Figure S1 ) . S20 was first identified as a sporozoite-specific gene in Py [26] . PbS20318 is located within a galactose oxidase ( central domain ) superfamily motif of the protein ( Figure S1 ) . The second peptide , SALLNVDNL , is restricted for Db and is derived from amino acids 130–138 of the PbTRAP [PbTRAP130 ( or PbTRAP130–138 ) ] ( PBANKA_134980; gi: 1813523 ) [39] , also known as sporozoite surface protein 2 [40] or sporozoite gene 8 ( S8 ) [26] ( Figure S1 ) . Conserved in Pf ( Figure S1 ) , TRAP is a secreted transmembrane protein of sporozoites that plays a vital role in parasite motility and invasion of hepatocytes [41] . PbTRAP130 is located within the von Willebrand factor type A domain ( Figure S1A ) , the key motif for parasite locomotion and target cell entry [42] . Reactive CD8+ T cells to PbTRAP130 are considerably more abundant than those reactive to PbS20318 ( Figure 1A–C ) . The identification of a PbTRAP-derived peptide as a target of CD8+ T cells in B6 mice is of interest since PfTRAP has been a major target for malaria vaccine development in humans [43]–[46] . Thus far , no CD8+ T cell epitope in TRAP has been identified in a murine model , meaning that fundamental studies on the induction , differentiation and long-term persistence of protective TRAP-specific cells following parasite immunisation could not be carried out yet . PbS20318 and PbTRAP130 represent the first reported endogenously processed CD8+ T cell epitopes of malaria liver stages in the B6 model . To determine expansion and contraction of PbS20318- and PbTRAP130-specific CD8+ T cells over time , we quantified the responses in the spleen and the liver after one or two immunisations with Pb γ-Spz ( Figure 2A ) . After a single immunisation , CD8+ T cell responses in both the spleen and the liver reach the highest magnitude on day 7 ( Figure 2A , B ) . The responses were slightly decreased on day 14 as contraction of the response occurs but they remained quantifiable for up to 180 days after immunisation . The percentages of antigen-specific CD8+ T cells were generally higher in the liver that in the spleen . More robust responses were observed after two immunisations with Pb γ-Spz ( Figure 2A , B ) Polyfunctional analysis of PbS20318- and PbTRAP130-specific CD8+ T cells revealed the induction of IFN-γ positive cells and IFN-γ/tumour necrosis factor ( TNF ) double positive CD8+ T cells ( Figure S2 ) . Consistent with the generation of effector and effector memory responses , PbS20318 and PbTRAP130-specific IFN-γ-producing CD8+ T cells were immunophenotyped as CD62Llo , CD44hi , CD11ahi , and CD49dhi ( Figure 3 , S3 ) . Cells stimulated with no peptide or cells from naïve mice stimulated with either peptide did not respond to either PbS20318 or PbTRAP130 ( data not shown ) . Together , these results indicate that immunisation with Pb γ-Spz recruits antigen-specific CD8+ T cells to undergo differentiation , proliferation , and long-term persistence . To determine the in vivo cytotoxic potential of PbS20318- and PbTRAP130-specific CD8+ T cells , we utilised an assay that allows the quantification of rapid killing of adoptively transferred target cells by activated CD8+ T cells in vivo [47] . CFSE-labelled and peptide-pulsed syngeneic targets were transferred to Pb γ-Spz-immunised mice 14 days after the last immunisation ( Figure 4A ) . We observed considerable ( ∼90% ) disappearance of PbTRAP130 , -pulsed ( Figure 4B , C ) , but not PbS20318-pulsed , target cells when transferred to mice that were immunised twice with Pb γ-Spz . To corroborate our finding that PbTRAP130-specific CD8+ T cells exhibit significant cytotoxic activity , we repeated the cell transfer to mice that were immunised only once and to naïve controls ( Figure S4 ) . Cytotoxicity against cells presenting PbTRAP130 , but not PbS20318 , was already apparent after a single immunisation . To test whether the newly identified targets of CD8+ T cells contribute to protection , we first performed peptide-tolerisation experiments . This method of depleting antigen-specific CD8+T cells , being performed in a malaria model for the first time , was adapted from previous studies aimed at inducing and maintaining antigen-specific tolerance [48]–[51] . Mice were subjected to repeated high dose administrations of adjuvant-free PbS20318 and PbTRAP130 peptides prior to and during the immunisation protocol ( two immunisations with Pb γ-Spz ) ( Figure 5A ) . These mice , along with sham-tolerised mice , were challenged with sporozoites and liver parasite loads were measured 42 hours later . As shown in Figure 5B , the levels of protection , i . e . very low parasite load , in PbS20318-tolerised mice were similar to sham-tolerised controls . In contrast , a significantly increased liver parasite load was observed in PbTRAP130-tolerised mice . These data indicate that a substantial degree of protection in whole sporozoite-immunised animals , measured by a reduction of parasite liver load over four orders of magnitude , can be attributed to PbTRAP130-specific responses . These results are in agreement with the in vivo cytotoxicity experiments ( Figures 4 , S4 ) , where we observed the potent in vivo cytotoxic function of PbTRAP130-specific , but not PbS20318-specific , CD8+ T cells . However , it remains to be determined whether parasite killing per se requires the lytic capacity of antigen-specific CD8+ T cells . To corroborate our finding that PbTRAP130-specific CD8+ T cells contribute to protection against malaria liver stages , the tolerisation experiments were also performed in mice that were immunised only once ( Figure S5A ) . As shown in Figure S5B , the contribution of PbTRAP130 , -specific CD8+ T cells to protection was already apparent after a single immunisation . Together , our results show a critical and immunodominant contribution of PbTRAP130-specific CD8+ T cells in parasite killing . To determine the efficiency of tolerisation , we measured PbS20318- and PbTRAP130-specific CD8+ T cell responses in the tolerised mice ( Figure 5C and S5C ) . Indeed , in spleens of mice injected with the respective peptides , IFN-γ secretion was greatly reduced and was comparable to naïve mice . Importantly , PbS20318-tolerised mice mounted PbTRAP130-specific CD8+ T cell responses comparable to non-tolerised immune mice . Similarly , PbTRAP130-tolerised mice mounted PbS20318-specific CD8+ T cell responses indistinguishable from controls . These results demonstrate that induction of tolerance was peptide-specific and did not interfere with induction of CD8+ T cell responses against other antigens . Of note , the absence of a CD8+ T cell response to one antigen did not increase the response to another , suggesting the lack of compensation in the immunodominance hierarchy in our infection model , in contrast to other infections [51]–[54] . At least three immunisations with Pb γ-Spz are needed to induce sterile protection in the B6 model [8] , [55] . To ascertain whether the development of sterile immunity is dependent on responses to the identified antigens , peptide tolerisation experiments were repeated to mice that were immunised three times with Pb γ-Spz ( Table S3 ) ; 14 days after the last immunisation , mice were challenged with sporozoites . Similar to control sham-tolerised mice , multiply immunised PbS20318- and PbTRAP130-tolerised mice were completely protected and did not develop patent parasitaemia . The results are reminiscent of observations in transgenic Balb/c mice tolerant to T cell responses to PyCSP where complete protection was achieved following three immunisations with Py γ-Spz despite an immunodominant and protective role for PyCSP after one and two immunisations [18] . Taken together , it is likely that additional antigens contribute to protection in both the B6 and Balbc models . Since PbTRAP130-specific , but not PbS20318-specific , CD8+ T cell responses are cytotoxic in vivo and contribute to protection after one or two immunisations with Pb γ-Spz , we evaluated the immunogenicity and protective efficacy of PbTRAP in a heterologous prime-boost vaccine regimen with viral vectors . Priming with adenovirus ( Ad ) carrying a foreign antigen and boosting with orthopoxvirus modified vaccinia Ankara ( M ) expressing the same antigen has consistently been shown to induce strong T cell responses capable of inducing high levels of efficacy against intracellular pathogens [56]–[58] . Adenovirus chimpanzee serotype 63 ( Ad ) and Modified Vaccinia Ankara ( M ) vaccines expressing a mammalian codon-optimised fragment of PbTRAP were generated . Referred to as Ad-M PbTRAP combination vaccine , they were used to vaccinate B6 mice with an 8-week resting period between priming and boosting ( Figure 6A ) . Since the recombinant PbTRAP vaccines contained sequences in addition to PbTRAP130 , we used both PbTRAP130 and a pool of overlapping peptides to PbTRAP ( PbTRAPpool ) in stimulation assays to verify if other PbTRAP-derived sequences were able to induce T cell responses . As shown in Figure 6B–D , the frequencies of IFN-γ secreting CD8+ T cells were indistinguishable between the two stimulations; approximately ∼17% ( range: 14%–50% ) of the total CD8+ T cells produce IFN-γ specific for PbTRAP130 or PbTRAPpool . Consistent with the induction of effector responses , these activated IFN-γ-producing cells coincided with the modulation of the corresponding expression markers CD62Llo and CD11ahi ( Figure 6B ) . Polyfunctional analysis revealed that the responses were predominantly IFN-γ positive cells and IFN-γ/TNF double positive cells ( Figures 6C , D ) . This intracellular cytokine pattern of PbTRAP130-specific CD8+ T cells were similar to that measured by immunizations with irradiated sporozoites ( Figure S2 ) . Cells stimulated with no peptide did not respond to either PbTRAP130 or PbTRAPpool . No cytokine-producing CD4+ T cells were detected following PbTRAP130 or PbTRAPpool stimulation . These results demonstrated the induction PbTRAP130-specific CD8+ T cells by vaccination and confirmed that PbTRAP130 is the only T cell epitope in PbTRAP in this infection model . To determine protective efficacy , mice vaccinated with Ad-M PbTRAP were challenged with PbGFP-Luccon . This permitted us to perform in vivo imaging in order to quantify hepatic parasite development after challenge and to subsequently follow the development of patent parasitaemia in the same animal . Mice vaccinated with Ad-M PbTRAP show significant efficacy against sporozoite challenge as shown by a considerable decrease ( ∼95% reduction in liver parasite load; range: 91%–99% ) in liver parasite load as compared to mice given Ad-M vector controls ( Figures 7 ) . However , Ad-M PbTRAP-vaccinated mice did not develop sterile immunity; rather , they developed patent parasitaemia on day 3 after challenge . To further assess vaccine efficacy , we performed survival analysis by measuring time to reach 1% parasitaemia . This measurement has been reported to reflect the number of merozoites that egress from the liver under the assumption that the vaccine has no efficacy against malaria blood stage parasites [44] . Consistent with lower liver parasite load , Ad-M PbTRAP-vaccinated mice showed significant delay in parasite growth as compared to controls ( Figure S6 ) . Finally , to establish whether the observed decrease in liver parasite load in mice vaccinated with Ad-M PbTRAP is solely mediated by CD8+ T cells , groups of vaccinated mice were administered CD8+ depleting antibodies or control rat IgG prior to sporozoite challenge ( Figure 8 ) . In addition , a group of mice vaccinated with Ad-M PbTRAP were given anti-CD4+ T cell antibodies . Efficacy was abrogated in the group that received anti-CD8+ T cell , but not anti-CD4 T cell , antibodies after vaccination . Taken together , these results provide a striking correlation between the high levels of PbTRAP130-specific CD8+ T cells and the CD8+ T cell-mediated efficacy elicited by Ad-M PbTRAP vaccination .
Over 40 years have passed since the observation that immunisation of mice with a whole organism vaccine , i . e . Pb γ-Spz , induces complete protection against normal sporozoite challenge [3] . Although a crucial role for CD8+ T cells in this protection was demonstrated more than 25 years ago [10] , [11] , information regarding the precise targets of these cells remains remarkably incomplete . This paucity of naturally processed parasite-specific epitopes of CD8+ T cell responses has thwarted efforts to characterise the induction of protective and possibly non-protective responses that can be rigorously studied in murine vaccination models for malaria liver stages [59] . CSP , the major protein that coats the sporozoite's surface , has been at the forefront of malaria vaccination studies for more than two decades , and CSP-specific responses have been the benchmark in measuring cellular responses to malaria liver stages [20] . However , PbCSP and PyCSP are targets of immunodominant and protective CD8+ T cell responses only in the BALB/c model [16] , [60] . The need to identify non-CSP targets of CD8+ T cell responses comes from accumulating evidence suggesting that protection against malaria infection can be obtained in the absence of T cell responses to CSP [18] , [19] . In this study , we employed an unbiased approach for screening Pb-derived peptides that are recognised by CD8+ T cells from B6 mice immunised with whole sporozoite strategies , which are known to induce protection . Out of 600 peptides predicted to contain H-2b motifs , we report for the first time the identification of two peptides as signature targets of CD8+ T cells from sporozoite-immunised B6 mice . Notably , we provide evidence that responses to PbTRAP confers partial efficacy in vivo . Thus , we anticipate that pre-clinical development of an anti-malarial vaccine based on whole sporozoites or sub-unit vaccines that incorporate TRAP will be facilitated by the identification of quantifiable signatures of effective immunisation . Plasmodium parasites are complex pathogens with a ∼23 Mb genome [32] , [61] . The identification of targets of CD8+ T cells has important implications for understanding the hierarchy and the scope of responses to complex pathogens . When we initiated this study , we expected the CD8+ T cell responses to be relatively widely dispersed over many sporozoite and liver stage antigens that together can account for the protection offered by sporozoite immunisation . However , we only identified two targets - PbS20318 and PbTRAP130 . Our inability to identify additional targets is indicative of immune evasion once sporozoites invade target cells , a hallmark that might generally apply to parasitic infections . Coincidentally , vaccines against these complex pathogens remain elusive . In marked contrast , a similar epitope prediction approach revealed broad and abundant H2b-restricted CD8+ T cell epitopes of vaccinia virus [22] . One potential limitation in our in silico analysis is the inclusion of only the top hits of potential liver stage peptides . In addition , by utilising IFN-γ as a read-out of our assays , we cannot formally exclude other cytokines or cellular markers that may serve as additional signatures of protection . The quantification of IFN-γ expression by CD8+ T cells is a reliable read-out in murine and human vaccine studies . Consistent with the observation that few antigens are recognised by CD8+ T cells from immunised mice , an assessment of 34 candidate non-CSP sporozoite antigens in the BALB/c model failed to reveal any additional epitopes [62] . In experimental studies in humans , antigenic analysis of genomic and proteomic data revealed 16 new antigens recognised by volunteers immunised with Pf γ-Spz [33] . However , there was no significant overall difference between protected and non-protected volunteers , indicating that T cell proliferation is associated with pathogen exposure rather than protection . By measuring the responses to PbS20318 and PbTRAP130 , we report the first characterisation of the development of CD8+ T cell responses following sporozoite immunisation in B6 mice . PbS20318- and PbTRAP130-specific CD8+ T cells persist to long-term memory while displaying effector and effector memory phenotypes in both the spleens and livers of immunised mice . The ability of PbS20318 and PbTRAP130-specific CD8+ T cells to produce IFN-γ after peptide stimulation , coinciding with activation phenotypes , suggested their ability to exert cytotoxic functions in vivo . Remarkably , only PbTRAP130-specific , but not PbS20318-specific , CD8+ T cells , are able to lyse target cells pulsed with the respective peptides . These results provide the first evidence that liver stage infection evokes antigen-specific CD8+ T cells that are both cytolytic and non-cytolytic . In support of this notion , tolerisation induction via high dose intravenous injection of peptides revealed that PbTRAP130-specific , but not PbS20318-specific , CD8+ T cells contribute to parasite killing . Owing to the complexity of immune responses to sporozoites , it was not surprising that PbTRAP130-tolerised mice that received multiple immunisations with Pb γ-Spz were completely protected against sporozoite challenge . The residual efficacy obtained in the challenged PbTRAP130-tolerised mice is likely due to responses to unidentified targets of both CD8+ and CD4+ T cells . It is noteworthy that in the Py-B6 and Py-C57Bl/10 ( both expressing H2b ) models , both CD8+ and CD4+ T cells equally participate in the inhibition of parasite development [63] . The results are in agreement with earlier findings on the immunodominant but imperfect CSP epitope in the Balb/c infection model [18] . In our prime-boost vaccination experiments with Ad-M PbTRAP in B6 mice , we were able to generate very high levels ( 14–50% ) of circulating PbTRAP130-specific CD8+ T cells , yet complete protection was not achieved . This level of antigen-specific CD8+ T cells is not achieved following multiple immunisations with Pb γ-Spz in B6 mice , yet complete protection is attained ( Figure 2 ) . It is likely that additional antigens help consolidate the protection afforded by whole sporozoite immunisation . Very high levels of PyCSP- or PbCSP-specific CD8+ T cells generated by various vaccination strategies in Balb/c mice have been shown to induce complete protection [64]–[69] . We anticipate that generating high levels of PbS20318-specific CD8+ T cells through a similar Ad-M vaccine protocol is unlikely to confer any quantifiable protection in B6 mice . Further studies are needed to characterise the complex mechanisms of protection . In a recent study , it was suggested that strain-specific background genes in nonhematopoietic cells can control the threshold of antigen-specific CD8 T cells necessary for protection [70] . Taken together , our results raise the intriguing and important question as to what factors govern the protective efficacy of responding antigen-specific CD8+ T cells . Numerous possibilities exist to explain these findings , including quantitative and functional differences in CD8+ T cells , distinct expression of cognate proteins and/or MHC class I presentation . Future work is warranted to identify the underlying mechanisms that distinguish cellular correlates of sporozoite exposure ( PbS20318-specific CD8+ T cells ) , from signatures of protection ( PbTRAP130-specific CD8+ T cells ) . Our work suggests that the mechanisms involved can now be studied in a tractable animal model . The outcome of our work has obvious relevance for vaccine development . We provide a clearly defined model system , in which to investigate fundamental aspects of the CD8+ T cell response and to manipulate this response to enhance protective immunity . Our identification of TRAP as major target of protective CD8+ T cells in the Pb-B6 model lends strong support for PfTRAP as leading vaccine candidate to elicit strong cellular immune responses . Initial and partial results from ongoing phase III clinical trials of the RTS , S/AS01 malaria vaccine candidate , which is based on PfCSP , demonstrated only modest efficacy [71] , [72] . RTS , S/AS01 immunisation elicits high concentrations of anti-CSP antibodies [73] and induces CD4+ , but not CD8+ , T cell responses [74] . Towards a second-generation , >80% effective malaria vaccine , rational vaccine design to elicit superior and lasting immune responses is critical . TRAP has been identified as a viable target for Pf vaccines . While PyTRAP was suggested as a target of CD8+ T cells in BALB/c mice [66] , [75] , the epitopes have been elusive so far . PfTRAP induces large numbers of polyfunctional CD8+ T cells in experimental vaccination studies [76] , and these T cells are associated with partial but significant efficacy in human vaccine trials [43] , [45] . However , when tested in larger Phase IIb trials in endemic areas , efficacy was lost despite moderate immunogenicity of the vaccine , suggesting that the T cell response that is induced is insufficient in some way [46] . Further work is needed to improve the immunogenicity of TRAP-based vaccines for malaria-exposed individuals . More recently , vaccination regimes with viral vectors have induced much stronger CD8+ T cell responses in phase I trials [76] and greater efficacy in controlled challenge studies ( Ewer et al . , submitted for publication ) . Based on our identification of a major protective CD8+ T cell epitopes in PbTRAP , we propose that the parasite-host combination Pb-B6 is an attractive and relevant model to further systematically explore subunit vaccination strategies , focusing on the TRAP antigen with the aim of increasing potent and durable sterilising immunity .
Animal procedures were performed in accordance with the German ‘Tierschutzgesetz in der Fassung vom 18 . Mai 2006 ( BGBl . I S . 1207 ) ’ , which implements the directive 86/609/EEC from the European Union and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes . Animal protocols were approved by the ethics committee and the Berlin state authorities ( LAGeSo Reg# G0469/09 ) . Experiments performed at the University of Oxford were performed under license from the United Kingdom Home Office under the Animals ( Scientific Procedures ) Act 1986 and approved by the Animal Care and Ethical Review Committee . Female B6 mice 6–8 weeks of age were purchased from either Charles River ( Sulzfeld , Germany ) or Harlan ( Derbyshire , UK ) . The complete life cycles of two parasite strains , Pb ( strain ANKA , clone cl15cy1 ) and PbGFP-Luccon ( strain ANKA , clone 676m1cl1 ) , were previously cloned and maintained by continuous cycling between rodent hosts and Anopheles stephensi mosquitoes [77] . Sporozoites were isolated from salivary glands . For Pb γ-Spz , the radiation dose was 1 . 2×104 cGy , and mice were immunised intravenously with 1 . 5×104 parasites . For Pb infection and treatment strategies , 1 . 5×104 sporozoites were injected intravenously while anti-malaria drugs were given intraperitoneally: 160 mg AZ/kg ( days 0 , 1 and 2 ) [8] , 60 mg PQ/kg ( days 0 , 1 and 2 ) [7] and 40 mg CQ/kg ( days 0–7 ) [6] . For PbHKSpz , parasites were subjected to 95°C for 15 minutes [38] . Immunised mice were challenged with 104 Pb sporozoites . In some experiments involving imaging , mice were challenged with 5×103 PbGFP-Luccon normal sporozoites . For mice receiving >1 immunisations , sporozoites were administered >7 days apart . Peptide libraries were generated by solid phase synthesis ( Peptides and Elephants , Potsdam ) . PbS20318 ( VNYSFLYLF ) and PbTRAP130 ( SALLNVDNL ) peptides were additionally synthesised at large scale . Peptides were synthesised as peptide amides and lyophilised peptides were resuspended in DMSO at a concentration of at least 1 mg/ml and stored at −80°C . Purity was confirmed by mass spectrometry . Peptides were diluted in PBS and tested individually at 10 µg/ml for the ELISpot assay and T cell stimulations with the exception of Figure 6 in which peptides were used at 5 µg/ml . A 20-mer peptide library ( pooled ) spanning the entire sequence of PbTRAP was generated , in which each peptide was overlapped 10 amino acids to another peptide ( Mimotopes , Victoria , Australia ) . The final concentration of the peptide pool used for stimulations was 5 µg/ml for each peptide . For the tolerisation experiments , mice received 300 µg peptide ( either PbS20318 or PbTRAP130 ) on day −7 and 100 mg on days −4 and −1 as described [51] . Mice were immunised on day 0 and received 100 µg peptide weekly until the end of the experiment . AdCh63 and MVA vaccines expressing a mammalian codon-optimised fragment of PbTRAP [39] ( GeneArt , Regensburg , Germany ) were constructed and propagated based on previously published viral vectors [76] , [78] . The TRAP signal peptide sequence was replaced with the human tissue plasminogen activator signal peptide sequence and the 3′ transmembrane region deleted . The viral vectors , referred to as Ad-M PbTRAP as a combination vaccine , were administered intramuscularly in endotoxin-free PBS at a concentration of 109 viral particles for Ad PbTRAP and 106 plaque-forming units for M PbTRAP . ELiSpot assay was performed as described [35] with minor modifications . CD8+ T cells were purified from spleen cells by positive selection using mouse CD8 microbeads ( MACS Miltenyi Biotec , Bergish Gladbach , Germany ) . Syngeneic spleen cells from naïve B6 mice were coated with peptides and were used as antigen-presenting cells . Anti-IFN-γ [AN18] and biotin-anti-IFN-γ [R4-6A2] were obtained from Mabtech ( Nacka Strand , Sweden ) . For T cell stimulations followed by ICS , splenic and liver-infiltrating lymphocytes were incubated with peptides for 5–6 hours in the presence of Brefeldin A ( eBioscience , California , USA ) , followed by standard surface and intracellular staining procedures . Data was acquired using either a LSRII or a LSRFortessa ( BD Bioscience , Heidelberg/Oxford ) . Antibodies for stainings were obtained from eBioscience: anti-mouse CD8 [53-6 . 7] , CD62L [MEL14] , CD44 [IM7] , CD11a [M17/4] , CD49d [R1-2] , IFN-γ [R4-6A2] , TNF [MP6-XT22] and interleukin ( IL ) -2 [JES6-5H4] . Data analysis was performed using FlowJo 7 . 6 . 3 ( Tree Star Inc . , Oregon , USA ) . SPICE 5 . 21 , a gift from Dr . Mario Roederer ( NIAID , NIH , Maryland , USA ) was used to analyse polyfunctional data . The cytotoxic potential of antigen-specific CD8+ T cells was assayed as described [47] . Livers were excised 42 hours after challenge and total RNA was isolated . cDNAs , generated by reverse transcription , were used as templates for quantitative real-time PCR [8] of Pb 18S rRNA ( gi: 160641 ) and GAPDH sequences ( gi: 281199965 ) using the Applied Biosystem Step One Plus Real Time PCR System ( Darmstadt , Germany ) . Relative parasite loads were calculated using the ΔΔCt method . For in vivo imaging , an IVIS200 imaging system ( Caliper Life Sciences , Chesire , United Kingdom ) was utilised to monitor parasite development in the liver 42 hours after challenge [78] . Mice were anaesthetised , injected subcutaneously with D-luciferin ( Synchem Laborgemeinschaft OHG , Felsberg/Altenberg ) ( 100 mg/kg in PBS ) , and 8 minutes later , imaged for 120 s at binning value of 8 and fields-of-view ( FOV ) of 12 . 8 cm . Bioluminescence in the liver was quantified using Living Imaging 4 . 2 software ( Caliper Life Science ) and expressed as total flux of photons per second of imaging time . The development of patent parasitaemia was determined based on Giemsa-stained blood smears . Relationships between log percentage parasitemia and time after challenge were plotted . Kaplan Meier analysis was performed to compare the parasite growth rate , and protection was measured as a delay in reaching 1% parasitaemia [44] . Statistical analysis ( see Figure Legends ) , unless otherwise specified , was performed using Prism 5 . 0c ( GraphPad Software Inc . , CA , USA ) . Mixture model calculations were performed using Stata 12 ( StataCorp LP , TX , USA ) . | Vaccination against malaria is feasible , as demonstrated with radiation-attenuated sporozoite vaccine , which protects experimental animals and humans by targeting the clinically silent liver stages . Potent protection largely depends on CD8+ T cells , a type of white blood cell that is tailor-made to kill obligate intracellular pathogens . Malaria-infected cells display fragments of parasite proteins , which are then recognised and targeted by CD8+ T cells . How CD8+ T cells are activated following immunisation and how they execute protective functions are key considerations for vaccination . However , characterisation of CD8+ T cells is hampered by the lack of identified malaria protein targets . Of concern , the circumsporozoite protein , which is the basis of the most advanced malaria vaccine candidate ( RTS , S ) , is not an essential target of CD8+ T cells induced by attenuated sporozoites in several mouse strains . In this study , we have made considerable advances by identifying for the first time , fragments of malaria proteins that are targeted by CD8+ T cells generated by vaccination in a relevant mouse strain , C57BL/6 . Notably , CD8+ T cells against one of the target proteins elicit partial protection against infection . Our study exemplifies how immunisation by complex pathogens can be dissected to identify distinct antigens for subunit vaccine development . | [
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... | 2013 | Identification of Targets of CD8+ T Cell Responses to Malaria Liver Stages by Genome-wide Epitope Profiling |
TIA-1 positive stress granules ( SG ) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response . In this report , we provide evidence that Kaposi’s sarcoma-associated herpesvirus ( KSHV ) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57 . By immunofluorescence analysis , we found that B lymphocytes with KSHV lytic infection are refractory to SG induction . KSHV ORF57 , an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny , inhibits SG formation induced experimentally by arsenite and poly I:C , but not by heat stress . KSHV ORF37 ( vSOX ) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation , but KSHV RTA , vIRF-2 , ORF45 , ORF59 and LANA exert no such function . ORF57 binds both PKR-activating protein ( PACT ) and protein kinase R ( PKR ) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production . Consistently , knocking down PKR expression significantly promotes KSHV virion production . ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation , leading to inhibition of eIF2α phosphorylation and SG formation . Homologous protein HSV-1 ICP27 , but not EBV EB2 , resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway . In addition , KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation . Altogether , our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection .
Mammalian somatic cells produce two types of RNA granules , processing bodies ( P-bodies , PB ) and stress granules ( SG ) [1 , 2] . Both granules are physically and mechanistically distinct compartments with many unique biomarkers . While GW182 is confined to PB , RNA-binding proteins TIA-1 , poly ( A ) binding protein ( PABP ) and G3BP are specific markers of SG . PB appear during normal cell growth and contain enzymes for RNA de-capping and degradation [1 , 3] , and have been shown to store and degrade siRNA- or miRNA-guided mRNA [4 , 5] . SG on the other hand , lack de-capping/de-adenylating machinery and appear during cell stress to play a role in global translational arrest by storing mRNA [1] . Therefore , SG represent a central and dynamic warehouse where stored mRNA is protected and exchanged with polysomes or PB for further translation or degradation , respectively [3 , 6] . SG contain 40S ribosomal subunits , mRNAs , dozens of RNA-binding proteins and many translation initiation factors including eIF4G , eIF4E , eIF3 , and PABP [3 , 7 , 8] . SG assembly is initiated by phosphorylation of the α subunit in eIF2 at a specific serine ( Ser 51 ) residue [9] . eIF2 is a translation initiation factor which forms a ternary complex with GTP and the initiator methionine-tRNA ( eIF2-GTP-tRNAi-Met ) [10] and in turn loads the initiator tRNAmet onto the small ribosomal subunit [11–13] ) . Different types of stress ( oxidative , heat , or nutrient deprivation ) can induce eIF2α phosphorylation by activation of four different eIF2α kinases ( GCN2 , PKR , PERK , and HRI ) [14 , 15] . Phosphorylation of heterotrimeric eIF2 on its regulatory α subunit increases its affinity with eIF2β ( the subunit responsible for GTP binding ) and thus reduces its availability for GTP exchange . This deficiency in GTP exchange inhibits the ability of eIF2 to reach its active GTP-bound state and therefore prevents ternary complex formation and arrests translation initiation [16] . Consequently , polysomes disassemble leaving translationally arrested mRNPs to be recognized by the RNA binding proteins , TIA-1 and TIAR , and sequestered through their prion-like aggregation property to nucleate SG [17 , 18] . Many RNA binding proteins including tristetrapolin ( TTP ) , and fragile X mental retardation proteins ( FMRP ) also join the assembly in SG [19 , 20] . Virus infection imposes stress on multiple biosynthetic pathways in host cells , including translation [21] , and captures the host translation machinery to ensure virus translation and production [22] . During infection , the presence of viral double stranded RNA ( dsRNA ) activates host cell PKR to induce eIF2α phosphorylation and SG formation and thereby , triggers the host cell antiviral response and shutting down host cell translation [23 , 24] . To bypass this response , however , many RNA viruses utilize alternative ways of translation [25] . Notably , several RNA viruses suppress the formation of SG by a viral factor [26–30] . Poliovirus C3 cleaves Ras-GAP ( Ras GTPase activating protein ) SH3 domain-binding protein ( G3BP ) , a component of SG that initiates the assembly of SG and interacts with inactive PKR [28 , 31 , 32] . Semliki Forest virus nsP3 targets G3BP [33] , and influenza virus NS1 inactivates dsRNA-activated PKR [26 , 27] . Hepatitis C Virus instead induces SG formation , but co-opts SG proteins for its replication and production [34 , 35] . The regulation of SG formation during infection with large DNA viruses is poorly understood although herpesviruses are proposed to produce viral proteins to regulate SG formation [33 , 36 , 37] . Kaposi’s sarcoma-associated herpesvirus ( KSHV ) is a γ-2 herpesvirus [38] and infects human B lymphocytes and endothelial cells . KSHV infection leads to development of Kaposi’s sarcoma , primary effusion lymphoma ( PEL ) , and multicentric Castleman’s disease ( MCD ) [39 , 40] . Like other herpesviruses , KSHV infection undergoes two alternative life-cycle programs . Viral lytic infection is characterized with the expression of all viral genes to produce infectious virions; whereas latent viral infection features highly restricted expression of only a few viral genes . Although the underlying mechanism responsible for the switch between lytic and latent infection , or vice versa , remains an unresolved topic , it is known that both viral and host factors are involved in the shift of KSHV infection [41 , 42] . A viral replication and transcription activator ( RTA or ORF50 ) is an immediately early protein and is essential for transactivation of almost all other viral genes in the lytic infection . RTA expression from the latent KSHV infection can be induced experimentally by chemicals such as sodium n-butyrate ( Bu ) [43] or valproic acid ( VA ) [44 , 45] . Another important KSHV protein is ORF57 ( mRNA transcript accumulation or MTA ) which is an early lytic RNA-binding protein responsible for posttranscriptional processing of viral transcripts and virus production [46 , 47] . ORF57 stabilizes viral RNAs [48–51] ) , promotes splicing of intron-containing viral mRNA [52 , 53] , and inhibits miRNA function to promote viral gene expression [48 , 54] through its interactions with targeted RNA and numerous host factors [47] . In this report , we discovered a novel function of ORF57 to inhibit SG formation during KSHV lytic infection . Mechanistically , ORF57 directly interacts with PACT and PKR and prevents phosphorylation of PKR and eIF2α and , thereby , prevents both SG formation and the stalling of RNA translation .
To determine whether KSHV is able to modulate SG formation in infected cells , we employed a defined strategy ( Fig 1A ) to study SG formation in KSHV-infected BCBL-1 cells [55] and HEK293-derived Bac36 cells [45] by arsenite , a common chemical inducer which causes oxidative stress and robust formation of SG [10] . Both BCBL-1 and Bac36 cells harbor an episomal KSHV genome at the latent stage and can be reactivated to lytic KSHV infection in the presence of 1 mM Valproic acid ( VA ) or 3 mM Butyrate ( Bu ) ( Fig 1A ) [45 , 55 , 56] . On cells with or without virus lytic induction , we performed IF staining for SG-specific TIA-1 , an RNA-binding protein that promotes the assembly of SG [18] . In the absence of arsenite treatment BCBL-1 cells either with latent or VA-induced lytic KSHV infection showed no visible SG ( S1A Fig ) . In contrast , after arsenite treatment the majority of cells displayed ~3–6 TIA-1-positive SG per cell ( Fig 1B ) . These arsenite-induced SG were also stained positive for PABPC1 and G3BP , two other SG-specific markers ( S1B and S1C Fig ) . However , BCBL-1 cells expressing viral lytic genes , indicated by the presence of ORF57 , exhibited a remarkable reduction of arsenite-induced SG ( Fig 1B , S1B and S1C Fig ) , both by numbers of SG per cell and by numbers of cells with SG ( Fig 1B and 1D bar graph ) . We also assessed the ability of Bac36 cells containing a wt KSHV genome ( Bac36-wt ) or ORF57-null KSHV genome ( Bac36-Δ57 ) to prevent arsenite-induced SG formation during virus lytic infection , although viral lytic induction in Bac36 cells is less efficient than BCBL-1 cells [45] . We detected RTA in Bac36-Δ57 cells and ORF57 in Bac36-wt cells for butyrate-induced expression of viral lytic genes . While the ORF57 expressing Bac36-wt cells showed complete abrogation of arsenite-induced SG formation , the Bac36-Δ57 cells with RTA expression in lytic infection did not ( Fig 1C and 1E bar graph ) , nor in LANA or ORF45-expressing cells ( S1D Fig ) . The TIA-positive granules induced by arsenite in Bac36-Δ57 cells are bona fide SG and were sensitive to cycloheximide [6] by which blocks the flux of molecules between fully formed SG and polysomes ( S1E Fig ) . These data indicate that , unlike latent infection , the presence of viral early protein ORF57 , but not viral RTA , ORF45 or LANA during KSHV lytic infection is capable of preventing the arsenite-induced SG formation . We also stained Bac36 cells ( wt or Δ57 ) for TIA-1-positive SG in the absence of arsenite treatment , but in the presence of ectopic RTA expression to induce viral lytic infection . We did not see any visible SG in Bac36 wt or Δ57 cells ( Fig 2A , control ) , unless the cells were treated with arsenite ( Fig 2A , control ) . However , when cells are induced to the lytic phase by ectopic RTA expression , SG were found in approximate 20% of the RTA-expressing Bac36-Δ57 cells , but only in ~3% of the RTA-expressing Bac36-wt cells ( Fig 2A for RTA activation Image and bar graph ) . Again , the RTA-induced SG formation in Bac36-Δ57 cells was sensitive to cyclohemixide treatment ( S1F Fig ) [6] . Importantly , the presence of ectopic RTA alone does not induce SG in KSHV-negative HEK293 cells , from where Bac36 cells were originated [45] . Collectively , these results indicate that KSHV lytic infection provides a stress to the infected cells and the expression of ORF57 but not RTA during virus lytic infection is required for suppression of SG formation . The complex nature of lytic replication in KSHV makes it difficult to confirm that the observed suppression of SG formation is a result of ORF57 function . To examine whether ORF57 alone is sufficient to block SG formation in the absence of other viral lytic proteins , we transfected HeLa cells with an ORF57-expressing or an empty vector . Transfection of these plasmids did not induce SG formation as confirmed by staining for TIA-1 and PABPC1 ( S2A Fig ) . We then treated the cells with arsenite for 30 min and performed similar immunostaining . As expected , all cells without ORF57 displayed SG positive for TIA-1 , PABPC1 and G3BP1 staining , but a dramatic reduction in SG formation was found in ORF57-expressing cells ( Fig 2B , S2B Fig ) , indicating that ORF57 is a viral lytic protein responsible for efficient suppression of SG formation . There are three nuclear localization signals ( NLS ) present in the N-terminal domain of ORF57 that are important for the various functions of the viral protein . Introduction of point mutations in any two of the NLS renders ORF57 dysfunctional and unable to bind other partner proteins [57] . We next compared the ability of wild-type ORF57 ( ORF57 wt ) and an NLS mutant ORF57 ( ORF57 mt ) protein to abrogate arsenite-induced SG formation in HeLa cells . KSHV ORF59 , a DNA polymerase processivity factor , served as another viral lytic protein control . While ORF57 wt showed nearly complete abrogation of SG formation , ORF57 mt and ORF59 did not ( Fig 2C , panels on the left ) . By quantification , we measured ~20 SG per cell in all cells transfected with an empty vector , ORF57 mt , or ORF59 , but ~80% of the ORF57-expressing cells did not exhibit any SG and the remaining ~20% of the cells showed a dramatically reduced number of SG ( 2–5 SG/cell ) ( Fig 2C , bar graphs on the right ) . Three-dimensional image reconstructions acquired by confocal microscopy verified the absence of SG in ORF57 expressing cells ( S1 & S2 Videos ) . As an initial investigation into how ORF57 may prevent SG formation , we checked whether ORF57 affects the expression of TIA-1 , PABPC1 , G3BP , eIF4E and its phosphorylated form ( p-eIF4E ) and found no difference in the protein levels for all of these SG components , compared to control cells ( S2C–S2E Fig ) . During stress , TIA-1 aggregates in a similar manner to prion-like proteins to form SG [18] . The concentrated TIA-1 in SG can be detected in the insoluble pellets after high speed centrifugation of cellular extracts ( S3A Fig ) . As expected , arsenite treatment of HeLa cells led to remarkable increase of TIA-1 in the pellet ( Fig 2D and S3B Fig ) in a time-dependent manner ( Fig 2E and S3C Fig ) . Of particular interest , ORF57 wt , but not ORF57 mt , could be found in the pellets ( S3B Fig ) and prevent TIA-1 recruitment into the pellets ( Fig 2D and S3C Fig ) . We calculated that ORF57 wt blocked ~75% of total TIA-1 from being aggregated in 30 min of arsenite treatment ( Fig 2E ) and this function of ORF57 begins even at 10 min of arsenite treatment of HeLa cells in this study ( Fig 2E and S3C Fig ) . All together , these results indicate that a novel function of ORF57 is to establish the conditions that maintain the solubility of TIA-1 to prevent SG formation during stress . The formation of SG is initiated as a downstream event after elF2α phosphorylation which leads to the prion-like aggregation of TIA-1 . Normally , eIF2α is required to initiate mRNA translation by promoting the binding of tRNAmet to the 40S ribosome in a GTP-dependent manner . Stress induces phosphorylation of eIF2α to attenuate eIF2α activity ( Fig 3A ) and thereby promote TIA-1 aggregation to form the SG where mRNA translation is stalled [14] . Accordingly , we examined whether ORF57 could affect eIF2α phosphorylation . HeLa cells transfected with an ORF57-expressing or empty vector and treated with arsenite were blotted for the phosphorylation status of eIF2α ( p-eIF2α ) . As expected , arsenite was found to induce eIF2α phosphorylation ~15 times greater than basal level ( Fig 3B , lane 3 vs lane 1 ) , whereas ORF57-expressing cells remarkably inhibited eIF2α phosphorylation upon arsenite induction ( Fig 3B , lane 4 vs lane 3 , and Fig 3C ) , with the total level of eIF2α protein remaining the same . To correlate kinetic production of KSHV ORF57 with both total eIF2α and phosphorylated eIF2α , we induced KSHV-infected BCBL-1 cells with 1 mM VA for the indicated time and then treated the cells with arsenite for 30 min before collecting cell lysates for Western blotting analysis . Although lytic KSHV infection did not increase eIF2α phosphorylation over that of latent KSHV infection in the cells without exogenous stress ( Fig 3D and 3E ) , we found that arsenite-induced phosphorylation of eIF2α was in reverse correlation with kinetic ORF57 expression ( Fig 3F and 3G ) . Higher ORF57 expression resulted in less eIF2α phosphorylation , while the total eIF2α remained unchanged ( Fig 3F and 3G ) . Altogether , these data indicate that ORF57 is inhibitory for eIF2α phosphorylation both when expressed alone and when present with other viral proteins during viral lytic infection . During cellular stress eIF2α can be phosphorylated by four different kinases , and which kinase is activated depends on the cause of stress ( Fig 4A ) [14] . Of particular interest , both viral infection [24 , 58] and arsenite [59] commonly activate PKR although GCN2 could be activated by Sindbis virus in a report [60] . PKR is well known for its antiviral activity by induction of interferon and is both a cytoplasmic and a nuclear dsRNA-binding protein [61 , 62] . We confirmed that arsenite did induce phosphorylation of PKR and SG formation in both HeLa and BCBL-1 cells , which could be specifically blocked by a PKR inhibitor ( S4A–S4D Fig ) . These observations exclude the possibilities of other three pathways being involved in the studied SG formation in this report , although arsenite was also reported to mediate eIF2α phosphoryaltion through HRI in erythroid cells [63] and mouse fibroblast cells [64] . Virus infection activates PKR through the binding of viral dsRNA to the dsRNA-binding domain ( RBD ) of PKR , whereas arsenite activates PKR by inducing PACT to bind with and activate PKR [65 , 66] . PKR contains at least 15 autophosphorylation sites , but phoshorylation at both Thr 446 and Thr 451 is critical for its activation , and subsequent phosphorylation of eIF2α [67 , 68] . To elucidate the mechanism of ORF57-mediated inhibition of eIF2α phosphorylation , we investigated whether ORF57 could inhibit PKR activation and phosphorylation in HeLa cells under three different stress conditions ( arsenite , double-stranded poly I:C , or heat stress ) ( Fig 4B ) . An optimized poly I:C dose was used to mimic dsRNA [69] to specifically activate PKR-mediated eIF2α phosphorylation ( S5A Fig ) . Heat treatment at 44°C for 40 min served as an alternative route of cell stress resulting in eIF2α phosphorylation . As expected , both arsenite and poly I:C induced PKR phosphorylation along with eIF2α phosphorylation ( Fig 4B , compare lanes 1 vs 3 and 5 vs 7 ) . However , heat shock induced eIF2α phosphorylation without PKR phosphorylation . Thus , the heat shock-induced phosphorylation of eIF2α is not related to PKR activity ( Fig 4B , compare lanes 9 vs 11 ) as has been reported in reticulocytes [63] . We did not see any induction in phosphorylation of PERK ( PKR-like endoplasmic reticulum kinase ) by arsenite ( S5B Fig ) . Interestingly , we observed a dramatic reduction ( ~75% ) of both phosphorylated PKR ( p-PKR ) and p-eIF2α when ORF57-expressing cells are exposed to either arsenite or poly I:C ( Fig 4B , compare lanes 3 vs 4 and 7 vs 8 , and Fig 4C ) , with only minimal or no change in overall PKR and elF2α protein levels . In contrast , ORF57-expressing cells exhibited no effect on the phosphorylation of eIF2α induced by the heat shock ( Fig 4B , compare lanes 11 vs 12 , and Fig 4C ) , but a dose-dependent , increased expression of ORF57 exhibited a steady decrease in PKR phosphorylation ( Fig 4D and 4E ) . The ORF57 prevention of arsenite- and poly I:C-induced PKR phosphorylation with no change in total PKR levels was also observed in HEK293 cells ( S5C Fig ) , indicating that the inhibitory effect of PKR phosphorylation by ORF57 is not cell-specific and could take place as early at 15 min of arsenite treatment , the earliest time point of the sample collection in this study ( S5D Fig ) . Moreover , ORF57 was found to block the phosphorylation of TLR3 ( toll-like receptor 3 ) induced by dsRNA ( poly I:C ) ( S5E Fig ) to activate Interferon regulatory factor 3 ( IRF3 ) and production of type 1 interferon [70–73] . By IF microscopy , we found that both poly I:C and heat stress induce SG formation in HeLa cells ( Fig 4F and S6 Fig ) , however , ORF57 was found to inhibit SG formation when induced either by arsenite or by poly I:C , but not when induced by the heat stress ( Fig 4F and 4G and S6 Fig ) . The functional relevance of how ORF57 modulation of SG-formation impacts KSHV gene expression and replication was further explored in BCBL-1 cells . Because ORF57 does not affect heat shock-induced SG formation in BCBL-1 cells with lytic KSHV infection ( Fig 4H ) , the effect of heat-induced SG formation on the expression of viral RTA , a viral replication and transcription activator and ORF45 , a viral tegument protein , was investigated in the VA-induced cells . We found that 40-min heat shock-induced SG formation showed no effect on their RNA levels by RT-qPCR , but led to reduction of RTA protein expression by 30–40% in VA-induced BCBL-1 cells . The 40-min heat-shock had little effect on the expression of ORF45 , a less-sensitive viral early gene to RTA transactivation [74] and a relative stable and abundant protein [75] ( Fig 4I ) . Together with our previous findings [47] , these results reveal that ORF57 inhibits PKR activation , disrupts the PKR-mediated phosphorylation of eIF2α and , therefore , blocks SG formation to promote viral gene expression . Given that both viral infection [24 , 58] and arsenite treatment [66] activate PKR and induce SG formation , and arsenite activates PKR by inducing PACT to bind PKR [65 , 66] , we examined the mechanism by which ORF57 inhibits arsenite/poly I:C activation of PKR . By co-immunoprecipitation ( co-IP ) in combination with Western blot analysis we found that ORF57 , but not the ORF57-mt , interacts with both PKR and PACT independent of RNA ( Fig 5A–5C ) . Interestingly , the interaction of ORF57 individually with either PKR or PACT disrupts the interaction between PACT and PKR . As shown in Fig 5B and 5C , various co-IP experiments using an anti-PKR antibody ( Fig 5B ) revealed a remarkable reduction in the amount of PACT associated with PKR when ORF57 is present . Similarly , using an anti-PACT antibody ( Fig 5C ) , we observed a significant reduction in the association of PKR with PACT in the presence of ORF57 . This mechanistic function of KSHV ORF57 in blocking SG formation resembles that of the TRBP-PACT interaction and inhibition of PKR activation [76] , but differs from other viruses that rely on cleavage or direct interaction with G3BP to block SG formation [28 , 33] . In this regard , ORF57 did not interact with G3BP ( Fig 5D ) , nor alter G3BP expression ( S2D Fig ) . In addition , ORF57 failed to interact with eIF2α ( Fig 5E ) , TIA-1 or eIF4G1 ( Fig 5F ) , although ORF57 did interact with PABPC1 [50] ( Fig 5D ) and eIF4E ( Fig 5F ) , two common components of SG . Importantly , we further confirmed by co-IP and Western blotting that ORF57 interacts with both PACT and PKR in BCBL-1 cells during viral lytic infection ( Fig 5G ) . Altogether , these studies indicate that PACT , PKR , PABPC1 and eIF4E are ORF57-interacting proteins and ORF57 binds to PACT and PKR and blocks PKR activation . PACT contains two RNA-binding motifs ( RBM ) in its N-terminal half and a PKR-activation domain ( PAD ) in its C-terminal half . To determine the specific domain of PACT interacting with ORF57 , the cell lysates containing individual Flag-PACT deletion mutants were mixed with the cell lysates containing ORF57 protein and followed by RNase A/T1 treatment to avoid any possible RNA-mediated protein-protein interaction in the subsequent anti-Flag antibody co-IP for PACT-associated ORF57 or anti-ORF57 antibody co-IP for ORF57-associated PACT . Western blot analysis of the proteins pulled down by the co-IP revealed that ORF57 interacts with PACT through its two RBM motifs and this interaction is independent of RNA . As shown in Fig 6 , deletion of either RBM1 ( PACT-Δ1 ) or RBM2 ( PACT-Δ2 ) from PACT significantly reduced the binding of PACT to ORF57 ( Fig 6B and 6C , compare lanes 7 and 12 to lanes 8–9 and 13–14 ) , but deletion of the PAD ( PACT-Δ3 ) did not ( Fig 6B and 6C , compare lanes 7 and 12 to lanes 10 and 15 ) . This study also demonstrated that further deletion of both RBM1 and RBM2 from PACT ( PACT-Δ1 , 2 ) completely prevented the binding of PACT to ORF57 ( Fig 6D , compare lane 9 to lanes 7–8 and 10 ) . Based on these data , we concluded that ORF57 interacts with PACT via its two RBM motifs . PKR has a N-terminal regulatory domain containing two dsRNA-binding motifs ( RBM1 & RBM2 ) and a C-terminal kinase domain ( Fig 7A ) . To determine the specific domain of PKR interacting with ORF57 , we generated two deletion mutants of PKR either by deletion of the kinase domain ( ΔPK ) or by deletion of the dsRNA-binding domain ( ΔRBM ) . Both deletion mutants and the full-length ( FL ) PKR have a chimeric Myc-Flag tag and were individually expressed in HEK293 cells separately from untagged ORF57 . By mixing the PKR cell extract with the ORF57 cell extract , followed by RNase A/T1 treatment to avoid any possible RNA-mediated protein-protein interaction in the subsequent anti-Flag or anti-Myc co-IP for PKR-associated ORF57 , we found that the FL PKR and the N-terminal RBM domain ( ΔPK ) , but not the kinase domain ( ΔRBM ) interact with ORF57 ( Fig 7B , compare lanes 6–7 to 8 for ORF57 ) . Interestingly , the ΔPK exhibited ~6 . 5-fold greater binding to ORF57 than did FL PKR ( Fig 7B , compare lanes 7 to 6 ) . Moreover , we found that the phosphorylated FL PKR increased its binding capacity toward ORF57 ~3 times more when activated in cells treated with arsenite ( Fig 7C , compare lanes 6 to 5 ) . Overall , we find that ORF57 interacts with the N-terminal RBM-containing domain of PKR , and the conformational change that occurs in PKR during activation allows ORF57 to bind to the N-terminus of PKR with greater affinity . PKR activation depends on its binding to dsRNA via its two RBMs . This interaction induces PKR dimerization at the C-terminal kinase domain , which in turn leads to autophosphorylation of PKR ( Fig 8A ) . Once phosphorylated , each subunit kinase domain in the dimerized PKR can independently phosphorylate the substrate eIF2α [14 , 77] ( Fig 8A ) . To investigate the mechanism of how the ORF57-PKR interaction prevents phosphorylation of PKR and eIF2α , we first performed an in vitro competitive binding assay by which Myc-Flag-tagged PKR and its ΔRBM mutant immobilized separately on the anti-Myc beads were compared for competitive binding with 32P-poly I:C and recombinant ORF57 . To do this , 32P-poly I:C was first mixed with recombinant ORF57 or BSA before allowing to interact with the immobilized PKR . As shown in Fig 8B , we observed that while BSA didn’t compete with 32P-poly I:C to interact with PKR , the recombinant ORF57 protein significantly reduced this interaction to ~50% . The PKR ΔRBM mutant and Flag control both served as negative controls , and ORF57 had no effect on their basal level of binding to 32P-poly I:C . These data suggest that ORF57 interacts with the N-terminal domain of PKR to prevent PKR binding to dsRNA . We next examined that ORF57 prevention of PKR binding to dsRNA might affect PKR autophosphorylation . Subsequently , an in vitro autophosphorylation assay was conducted by incubation of recombinant ORF57 or BSA ( a negative control ) with immobilized PKR beads first before adding poly I:C and [γ-32P]-ATP . By examining the 32P-labelled PKR , we demonstrated that poly I:C did stimulate PKR autophosphorylation in the absence or presence of BSA , but this poly I:C induction of PKR autophosphorylation could be reduced by ~50% in the presence of ORF57 ( Fig 8C ) . In a separate experiment , we also tested whether ORF57 prevents the phosphoryated-PKR ( p-PKR ) from in turn phosphorylating its substrate eIF2α . To do so , we performed an in vitro kinase assay using a GST-eIF2α . The Myc-Flag-tagged PKR expressed in HeLa cells treated with or without arsenite was immobilized on anti-Myc beads and used to phosphorylate GST-eIF2α in the presence of recombinant ORF57 or BSA in a kinase reaction containing [γ-32P]-ATP] . By examining the amount of 32P-labelled GST-eIF2α , we found that while the inactive PKR from the cells without arsenite treatment did not exert much kinase activity on GST-elF2α , the arsenite-activated p-PKR did actively phosphorylate GST-eIF2α equally well both in the presence of BSA or ORF57 ( Fig 8D ) . The inability of ORF57 to inhibit phosphorylation of GST-elF2α was not due to a lack of interaction between ORF57 and the active p-PKR . In fact , ORF57 was found to associate more efficiently with the activated p-PKR ( Fig 7C ) . From these results presented above , we conclude that ORF57 interacts with the RBM motifs of PKR and prevents PKR binding to dsRNA or PACT and PKR activation by autophosphorylation , consequently preventing eIF2α phosphorylation and SG formation . However , once PKR is phosphorylated , ORF57 is unable to prevent p-PKR from phosphorylating eIF2α by its C-terminal kinase domain . In the course of drafting our manuscript for publication , Finnen and colleagues reported that viral vhs protein encoded by UL41 in herpes simplex virus type 2 ( HSV-2 ) suppresses SG formation [78] . Although α-herpesvirus vhs and γ-herpesvirus vSOX are not homologs , both are RNA endonucleases and exert their host shutoff function by digesting host mRNAs [79–82] which are fundamental for SG formation [8] . In assumption of KSHV vSOX encoded by viral ORF37 in blocking SG formation via a mechanism similar to HSV-2 vhs [78] , we transfected both HEK293 and HeLa cells with a Flag-tagged KSHV vSOX expression vector for 24 h and stained the cells for TIA-1-specific SG formation in the presence or absence of vSOX after induction by arsenite for 30 min . We confirmed vSOX expression in HEK293 cells by anti-Flag antibody staining of HEK293 cells ( Fig 9A ) and by anti-Flag antibody Western blot ( Fig 9B ) , but had difficulty to express vSOX in HeLa cells . As expected , HEK293 cells expressing no vSOX displayed SG formation induced by arsenite , but all cells expressing vSOX did not ( Fig 9A ) . In contrast to ORF57 , vSOX in transfected HEK293 cells had no effect on arsenite-induced phosphorylation of eIF2α . These results clearly indicate that KSHV vSOX inhibits SG formation in HEK293 cells , similar to HSV-2 vhs [78] by degradation of RNA [80–82] . Considering that KSHV interferon regulatory factor 2 ( vIRF-2 ) might play an inhibitory role in PKR activation and PKR-mediated phosphorylation of eIF2α [83] , we compared ORF57 with vIRF-2 in regulation of PKR activation and phosphorylation of eIF2α in arsenite-treated HeLa cells . Because the actual vIRF-2 ORF splits into two separate exons , with exon 1 in K11 . 1 and exon 2 in K11 [84] and encodes a full-length vIRF-2 having 680 aa residues , the annotated vIRF-2 ORF encoding 163 aa residues in an early report [83] based on initial ORF annotation in the KSHV genome [85 , 86] was not an authentic vIRF-2 ORF . Thus , we cloned a full-length vIRF-2 encoding 680 aa residues and expressed as a Flag-vIRF-2 in HeLa cells with or without arsenite treatment . As shown in Fig 9C , vIRF-2 was expressed predominantly as a cytoplasmic protein , but exhibited no effect on arsenite-induced SG formation . When compared with ORF57 , we found that vIRF-2 in HeLa cells did not inhibit arsenite-induced PKR phosphorylation , nor eIF2α phosphorylation ( Fig 9D , compare lane 4 to lane 3 ) . In contrast , ORF57 at the same condition displayed the expected inhibition on phosphorylation of both PKR and eIF2α ( Fig 9D , compare lane 6 to lane 2 ) . According to these results , we conclude that the full-length vIRF-2 which modulates the host antiviral response [87–89] has no inhibitory function in activation of PKR pathway . Previously , we and others demonstrated that KSHV ORF57 is essential for KSHV replication and virus production [45 , 90] . Our observations in this study showed that KSHV ORF57 inhibits PKR activation and disrupts the PKR-mediated phosphorylation of eIF2α to block SG formation ( Fig 4B–4G ) . A well-recognized outcome of SG formation is to trigger the host cell antiviral response and inhibit virus production [91] . Therefore , we postulate that PKR might be a host inhibitory protein to block KSHV production and therefore , one of the ORF57 functions in blocking PKR activation and SG formation is to promote KSHV gene expression and virus production . To confirm this hypothesis , we examined KSHV virion production in a newly established iSLK-BAC16 cell line [92] with or without siRNA-mediated PKR knockdown . By using a PKR-specific siRNA , we found that efficient knockdown of PKR expression from iSLK-BAC16 cells ( Fig 10A ) resulted in significantly increased production of KSHV virions and led the iSLK-BAC16 culture supernatants being highly infectious for HEK293 cells ( Fig 10B ) . Quantitative analyses by flow cytometry indicate that siRNA knockdown of PKR expression in iSLK-BAC16 cells led to ~78-fold increase of KSHV virion production over the cells with the normal level of PKR expression ( Fig 10C ) . Given that HSV ICP27 and EBV EB2 ( SM ) proteins are homologues to ORF57 , we reasoned that the ability to block the PKR/eIF2α/SG axis by KSHV ORF57 might be a conserved function in other herpesviruses . To investigate this possibility , we expressed ICP27 from HSV-1 and EB2 from EBV in HeLa cells and examined their influence on SG formation and phosphorylation of both PKR and eIF2α . The expression of ORF57 , ICP27 or EB2 protein failed to induce SG in HeLa cells , but the three homologues exhibited a functional disparity in cells treated with arsenite . While both ORF57 and ICP27 abrogated the formation of SG in ~85% of arsenite-treated cells ( Fig 11A and 11B ) , EB2 did not exert such an inhibitory function on SG formation ( Fig 11A ) . By Western blotting of the lysates prepared from untreated or arsenite-treated cells transfected with an empty control vector or a vector expressing individual viral proteins , we found that both ICP27 and ORF57 , but not EB2 , inhibited the phosphorylation of ~70% PKR and ~75% eIF2α ( Fig 11C , lanes 6–7 vs lanes 5–8 and Fig 11D ) , along with only a minimal effect on total PKR or eIF2α protein levels ( Fig 11C and 11D ) . Altogether , these data indicate that the ability to inhibit SG formation in herpesviruses is conserved in HSV-1 through the ORF57 homologue ICP27 .
Virus infection inevitably induces host cell stress . Thus , SG formation in the infected host cells has been widely appreciated as an antiviral defense mechanism [91 , 93] . SG formation is a downstream event of eIF2α phosphorylation which stalls translation initiation [10 , 94] . Of the four cellular kinases which can phosphorylate eIF2α and induce SG formation [77] , PKR is a major player during viral infection . Viral dsRNA activates PKR and , therefore , induces SG formation . Although counteracted by many RNA viruses , regulation of the PKR/eIF2α/SG pathway by DNA viruses is poorly understood . Besides its activation by viral dsRNA , PKR is activated by PACT during arsenite stress [65 , 66] . Here , we utilized arsenite stress to explore the differential ability of KSHV infection to block SG in B cells and HEK293-derived Bac36 cells with a wt or ORF57-null ( Δ57 ) KSHV genome . We found that KSHV , a DNA virus expressing the viral early protein ORF57 , confers the infected B cells and Bac36-wt cells refractory to SG induction during lytic infection . Viral ORF57 alone blocks activation and phosphorylation of PKR and thereby SG formation . KSHV vSOX bearing intrinsic endoribonuclease activity also affects SG formation by degrading RNA . Other KSHV-encoded proteins examined in this study , such as RTA , ORF45 , ORF49 , LANA and vIRF-2 , have no such function . TIA-1 protein is a robust marker of SG and the N-terminal RRM domain TIA-1 binds to targeted RNA transcripts that have an AU-rich or C-rich element in the 5’ or 3’ UTRs [95–97] . TIA-1 nucleates SG formation via its C-terminal glutamine-rich prion-related domain ( PRD ) responsible for self-association [18] . Consistently , we found TIA-1 enrichment in the insoluble cell pellets during arsenite treatment , but this enrichment is prevented when ORF57 is present . The ORF57-mediated reduction of TIA-1 in the pellet is most likely a consequence of ORF57 inhibiting SG formation , but why a proportion of ORF57 resides in the insoluble pellets remains unknown , presumably being associated with ribosomes , microtubes , or other cellular debris . ORF57 does not interact directly with TIA-1 or G3BP1 , another SG nucleator in addition to TIA-1 [98] and therefore , must indirectly influence the biochemistry of TIA-1 and G3BP1 . Unlike TIA-1 and G3BP1 , ORF57 does bind to PABPC1 and eIF4E , two important components of SG . ORF57 interaction with PABPC1 reduces the cytoplasmic pool of PABPC1 by promoting the redistribution of PABPC1 to the nucleus [50] . Although an important component of SG , PABPC1 is viewed as a passenger and does not function directly in SG formation [10 , 99] , but may influence indirectly the ability of TIA-1 and G3BP1 to form SG . We also find that ORF57-mediated inhibition of SG formation is accompanied by a significant reduction in the amount of cytoplasmic PABPC1 . PABPC1 binds to the 3’ poly ( A ) tail of eukaryotic mRNAs , and its interaction with the N-terminus of eIF4G stabilizes RNA and promotes both ribosome recruitment and translation initiation [100–102] . Whether the reduction in cytoplasmic PABPC1 mediated by ORF57 affects recruitment of the polyadenylated RNA transcripts by TIA-1 into SG needs to be investigated . Cap-binding protein eIF4E is a translation initiation factor which binds mRNA’s 5’ cap and mediates the cap structure of mRNA directly binding to the 40S ribosomes . Similar to PABPC1 , eIF4E could be a passenger protein and does not function directly in SG formation , although ORF57 interaction with eIF4E , but not with eIF4G1 , might be involved in regulation of protein translation initiation . The inability of ORF57 to block heat stress-induced SG formation indicates that the ORF57 interaction with PABPC1 and eIF4E is insignificant with regards to SG formation , at least under conditions of heat shock-induced cell stress . KSHV ORF57 is an RNA binding protein and a posttranscriptional regulator of viral RNA transcripts [47] . Our finding that ORF57 functions as an inhibitor of PKR/eIF2α phosphorylation is intriguing and surprising . Further characterization of this inhibitory function of ORF57 led us to discover that KSHV ORF57 interacts with both PACT and PKR via their RBM motifs to prevent activation and phosphorylation of PKR and thereby , to inhibit eIF2α phosphorylation and SG formation ( Fig 12 ) . PKR is a dsRNA-binding protein important for the antiviral action of IFN and is a major cellular kinase that controls translation by phosphorylation of eIF2α [14] . We demonstrated that knocking down PKR expression in iSLK-BAC16 cells significantly promoted KSHV lytic infection and virion production . PKR is activated during viral infections [24 , 58] and experimentally by arsenite treatment [59] . The activation mechanisms are slightly different in that , viral infections activate PKR through the binding of viral dsRNA to the RNA-binding motif of PKR [103–105] , whereas arsenite activates PKR via PACT , a PKR activating protein which heterodimerizes with PKR and activates PKR in the absence of dsRNA [65 , 66] . When we experimentally induce PKR phosphorylation by either arsenite treatment or incubation with poly I:C , the presence of ORF57 blocks PKR phosphorylation by binding directly to both PACT and PKR leading to a reduction in PACT-PKR and dsRNA ( poly I:C ) -PKR interactions . Other viral proteins , including TRS1 of cytomegalovirus [106] and UL41 of HSV-2 [36 , 37] , exhibit a similar function in blocking PKR phosphorylation and activation to block SG formation . Even though ORF57 interacts with the N-terminal RBM domain of PKR and blocks it’s binding to dsRNA and its autophosphorylation , it has no direct effect on phosphorylation of eIF2α once PKR is activated and autophosphorylated and does not interact with eIF2α . The enhanced interaction of ORF57 with the p-PKR might be a result of conformational changes in p-PKR . This mechanistic function of ORF57 resembles that of TRBP which inhibits PKR activity through the interaction with PKR RBMs [76 , 107] , but differs from poliovirus 3C protease which cleaves G3BP1 [28] , SFV nsP3 and HSV-2 ICP8 which suppress SG formation by their FGDF motifs interacting with G3BP1 [33] , and HSV-2 vhs which requires its endoribonuclease activity in disruption of SG formation [78] . KSHV ORF57 does not have a FGDF motif or interact with G3BP1 and bears no endoribonuclease activity . Although α-herpesvirus vhs and γ-herpesvirus SOX are not homologs , both are RNA endonucleases that digest host mRNAs [79 , 81] . Thus , it is not to our surprise that KSHV vSOX is capable of blocking SG formation by a mechanism similar to HSV-2 vhs [78] . Moreover , poxviruses induce a SG-like antiviral granule formation which does not entirely depend on PKR or eIF2α [108 , 109] and can be induced in the cells lacking eIF2α [109] or TIA-1 [110] , although vaccinia virus E3L protein antagonizes PKR function and blocks the antiviral granule formation [110] . The discrepancy between EBV EB2 ( SM ) and both KSHV ORF57 and HSV-1 ICP27 in regulating the phosphorylation of PKR and eIF2α is another interesting result described in this report . KSHV ORF57 , HSV-1 ICP27 and EBV EB2 are three well-known homologous proteins in regulation of viral RNA biogenesis at various lytic stages of viral infections . Although ORF57 deviates from ICP27 in protein structure and the function of RNA splicing , EBV EB2 is a more closely matched homologue to ORF57 , with many functional similarities [47] . Here we show that both ICP27 and ORF57 , but not EBV EB2 , inhibits arsenite-induced SG formation by blocking phosphorylation of PKR and eIF2α ( Fig 11 ) . EBV EB2 was described as a dsRNA-binding protein and inhibits PKR activation via its RXP triplet repeats [111] , but this domain with the RXP triplet repeats doesn’t exist in EB2 homologs and the function of EB2 in promoting protein translation appears independent of the PKR pathway [112] . ICP27 has not been characterized as a protein being able to block SG formation , but does appear in spontaneous SG during virus infection [36 , 37] and inhibits IFN signaling [113] . Our data show an extreme similarity of HSV-1 ICP27 with KSHV ORF57 in preventing SG formation by blocking phosphorylation of PKR and eIF2α , providing evidence for ectopic ICP27 being directly involved in regulating the PKR pathway in the absence of HSV infection . KSHV encodes many proteins that function to evade the host immune system in multiple ways . Most of these viral proteins are homologous to cellular proteins and interfere with both innate and adaptive immune responses . In particular , immune evasion of the interferon pathway and the TLR pathway by KSHV vIFNs and other host mimics is one of the important strategies for KSHV to escape from the host innate immune response [114] . In this regard , KSHV vIRF-1 , -2 , or -3 blocks TLR3-mediated activation of IFN-responsive promoter activity [73] and vIRF1 decreases phosphorylation and nuclear translocation of IRF-3 in response to TLR3 activation [73] . Although a non-existing short-form vIRF-2 of KSHV was found to interact directly with PKR and inhibit PKR autophosphorylation and eIF2α phosphorylation [83] , the full-length vIRF-2 in our study does not exhibit such a function or inhibition of SG formation . In addition , we found ORF57 also blocks poly I:C-induced phosphorylation of TLR3 . Together with the finding that KSHV ORF57 regulates PKR pathway to suppress SG formation and PKR inhibits KSHV production , our data provide the first evidence that KSHV ORF57 plays a critical role in modulation of PKR/eIF2α/SG axis to enhance KSHV lytic infection .
Human HEK293 and HeLa cells ( ATCC , Manassas , VA ) were cultivated in DMEM ( Thermo Fisher Scientific , Waltham , MA ) supplemented with 10% fetal bovine serum ( FBS , GE Healthcare , Logan , UT ) . Primary effusion lymphoma BCBL-1 cells ( KSHV+ ) [55] obtained from the AIDS Research and Reference Reagent Program , Division of AIDS , NIAID , NIH , were grown in RPMI 1640 ( Thermo Fisher Scientific ) containing 10% FBS . KSHV lytic infection in BCBL-1 cells was reactivated by 1 mM sodium valproate ( cat . no . P4543 , Sigma-Aldrich , St . Louis , MO ) for 24 h . HEK293-derived Bac36 cell lines stably harboring a wt KSHV genome ( Bac36-wt ) or an ORF57-null KSHV genome ( Bac36-Δ57 ) were established in our lab as described [45] . KSHV lytic infection in Bac36 cells was reactivated with 3 mM sodium butyrate ( cat . no . B5887 , Sigma-Aldrich ) for 24 h . All plasmid transfections were performed using LipoD293 transfection reagent ( SignaGen Laboratories , Gaithersburg , MD ) according to the manufacturer’s instruction . Unless indicated , for IFA and Western analysis , HeLa ( 2 . 5 ×105 ) and HEK293 ( 5 × 105 ) cells were plated a day prior to 1 μg ( 2 μg for vSOX ) of plasmid DNA transfection in a 6-well plate . For IP , HeLa ( 2 × 106 ) and HEK293 ( 5 × 106 ) cells were plated a day prior to 5 μg plasmid DNA transfection in a 10-cm Petri dish . The custom made rabbit polyclonal and mouse monoclonal anti-ORF57 antibodies were described earlier [45] . Mouse monoclonal and rabbit anti-RTA and mouse monoclonal anti-ORF45 antibodies were kindly provided by Drs . Yoshi Izumya and Fanxiu Zhu , respectively . Mouse monoclonal anti-β-tubulin ( cat . no . T5201 ) , anti-Flag M2 ( cat . no . F1804 ) , anti-c-Myc ( 9E10 , cat . no . M4439 ) and rabbit polyclonal anti-Flag antibody ( cat . no . F7425 ) were obtained from Sigma-Aldrich . Rabbit polyclonal antibodies anti-eIF2α ( cat . no . 9722S ) and anti-phospho-eIF2α ( ser51 ) ( cat . no . 9721S ) and rabbit monoclonal anti-GAPDH ( cat . no . 2118 ) , anti-phospho-PERK ( Thr 980 ) ( 16F8 , cat . no . 3179 ) and anti-PERK ( C33E10 , cat . no . 3192 ) were obtained from Cell Signaling Technology . Other antibodies used were: rabbit polyclonal anti-TLR3 antibody ( phospho-Tyr759 ) ( cat . no . LS-C19344 , LifeSpan BioScience Inc . Seattle , WA ) , anti-PABPC1 ( cat . no ab21060 , Abcam , Cambridge , MA ) , anti-PACT/PRKRA ( cat . no 10771-AP , ProteinTech group , Rosemont , IL ) , anti-phospho ( Ser 209 ) -eIF4E ( cat . no . ab47605 , Abcam ) , and anti-phospho PKR ( pThr451 ) ( cat . no . 527460 , EMD Millipore , Billerica , MA ) ; mouse monoclonal anti-eIF2AK2 ( PKR ) ( cat . no . H000005610-M01 , Abnova ) , anti-eIF4E ( cat . no . 610269 , BD Biosciences , San Jose , CA ) , anti-PACT/PRKRA ( cat no . H00008575-M01 , Abnova , Taipei , Taiwan ) , and anti-eIF4G1 ( cat . no . ab54970 , Abcam ) ; goat polyclonal anti-TIA-1 ( cat . no . sc-1751 , Santa Cruz Biotechnology , Dallas , Texas ) , and rat monoclonal anti-HHV-8 LANA ( cat . no . MABE1109 , EMD Millipore ) . The peroxidase-conjugated secondary antibodies used in Western blotting were obtained from Sigma-Aldrich and all Alexafluor-conjugated secondary antibodies used in IFA were purchased from Thermo Fisher Scientific ( Waltham , MA ) . Sodium arsenite ( cat . No 38150 ) , valproic acid sodium salt ( cat . no . P4543 ) , sodium butyrate ( cat . no . B5887 ) and cycloheximide ( cat . No . C-7698 ) were obtained from Sigma-Aldrich . PKR inhibitor ( PKRi , cat . no . 527451 ) and its negative control ( PKRc , cat . no . 527455 ) were obtained from EMD Millipore . The expression vectors were used to express recombinant proteins: KSHV ORF57 ( pcDNA-ORF57 ) , ORF57-FLAG ( wt , pVM7; mt NLS 2+3 , pVM89 ) , ORF57-GFP ( wt , pVM8; mt NLS 2+3 , pVM36 ) , KSHV ORF59-FLAG ( pVM18 ) , pKY15 ( HSV1-ICP27-FLAG ) and pGS113 ( myc-EBV-EB2 ) [48 , 52 , 57] . Empty pcDNA 3 . 0 and pCMV-FLAG 5 . 1 ( Sigma-Aldrich ) were used as a negative control . KSHV vSOX ( ORF37 ) ORF was amplified by PCR using a primer pair of oVM400 ( 5’-CCGGAATTCACC/ATGGAGGCCACCCCCACAC-3’ , nt 57273–57291 ) and oVM401 ( 5’-ACTGTCTAGA/CGGGCTGTGAGGGACGTTTG-3’ , nt 57291–57273 ) on total DNA from BCBL-1 cells . The resulting PCR product was cloned into pFLAG-CMV-5 . 1 vector ( Sigma-Aldrich ) via EcoRI and XbaI sites to create plasmid pVM116 . The identity of inserted ORF was verified by Sanger sequencing . The expression of vSOX-Flag fusion protein ( 486 aa + Flag tag , ~52 kDa ) was confirmed by Western blot using anti-Flag antibody upon transfection in HEK293 . Full-length Protein Kinase R encoding plasmids ( p-CMV-Entry , PKR-Myc-Flag or pPKR-FL ) ( cat . no . , RC210792 ) was obtained from Origene ( Rockville , MD ) . PKR deletion mutants ( ΔRBM , aa 1-12/153-551 ) was generated by overlapping PCR using following primers: oVM78 5’-CCGTTGACGCAAATGGGC-3’ and oNS1 ( 5’-TGCTTCCTGTTT/CTCCATGAAGAAACCTGC-3’; PKR nt 1487-1476/1064-1047 ) for first PCR and oNS2 ( 5’-TTCTTCATGGAG/AAACAGGAAGCAAAACAATT-3’; PKR nt 1053-1064/1476-1495 ) and oNS3 ( 5’-CATCACTGGTCTCAGGATC-3’; PKR nt 2043–2025 ) for second PCR . Obtained two PCR products were combined and re-amplified with oNS3 and oVM78 . The final PCR product was used to replace full length PKR using Asp718 and BclI sites in pPKR-FL plasmid . The PKR ΔPK mutant ( aa 1–270 ) was generated by PCR using oVM78 and oNS4 ( 5’-AGTATTACGCGTATCCATGCCAAACCTCTTG-3’ , PKR nt 1826–1808 ) as forward and reverse primers , respectively . The resulting PCR fragment was cloned to replace FL-PKR at BamHI and MluI sites in PKR-FL plasmid . Full-length PACT-Myc-Flag encoding plasmid ( pPACT-FL ) was obtained from Origene . Deletion of PACT-RBM1 ( Δ1 , aa 1-34/100-313 ) , RBM2 ( Δ2 , aa 1-126/193-313 ) or both RBM1 and RBM2 ( Δ1 , 2 ) was generated by overlapping PCR on pPACT-FL using following primers: oVM78 5’-CCGTTGACGCAAATGGGC-3’ in the CMV IE promoter region and oNS16 ( 5’- TGCATTGGCTTT/TGTTTTCCCTGGCTTAGCT-3’; PACT nt 1337-1326/1130-1112 ) for first PCR and oNS17 ( 5’-AGCCAGGGAAAACA/AAAGCCAATGCAAGTATTT-3’ , PACT nt 1117-1130/1326-1344 ) and oNS18 ( 5’-CACTGGAGTGGCAACTTC3-3’; PACT nt 2166–2149 ) for second PCR were used for deletion of RBM1; oVM78 and oNS19 ( 5’-AGAAATATTACT/ATTAAGCTGGTTCTTTGGT3’-3’; PACT nt 1616-1605/1406-1388 ) for the first PCR and oNS20 ( 5’-AGAACCAGCTTAAT/AGTAATATTTCTCCAGAGA-3’; PACT nt 1393-1406/1605-1623 ) and oNS18 for the second PCR were used for deletion of RBM2 . Obtained two PCR products in oligo-mediated deletion of RBM1 or RBM2 were combined and re-amplified with oVM78 and oNS18 . The final PCR product ( PACT-Δ1 or– Δ2 ) was used to replace full length PACT using BamHI and MluI sites in the plasmid pPACT-FL . To make the PACT-Δ1 , 2 ( aa 1-34/100-126/193-313 ) , oVM78 and oNS16 were used for the first PCR and oNS17 and oNS18 were used for the second PCR on plasmid PACT-Δ2 . The overlapped PCR products from oVM78 and oNS18 were cloned as above . The PACT- ΔPAD mutant ( Δ3 , aa 1–239 ) was generated from the parent pPACT-FL plasmid by PCR using oVM78 and oNS21 ( 5’-AGTATT/ACGCGT/TGTATTTGGAATACTAAGGA-3’ , PACT nt 1745–1726 ) as forward and reverse primers , respectively . The resulting PCR fragment was cloned to replace the PACT-FL into the parent plasmid pPACT-FL using BamHI and MluI sites . Because vIRF2 is a split gene in the KSHV genome [84] , the overlapping PCR was also performed by using oVM386 ( 5’-TACTCAGAATTCACC/ATGCCTCGCTACACGGAGT-3’ , vIRF2 nt , 94127–94109 ) and oVM389 ( 5’- TCGCTCTGTGACCGTGATGAA-3’ , vIRF2 nt 93435–93453 ) for the first PCR and oVM388 ( 5’-AGTCGCCACGCCCACAACAT-3’ , vIRF2 nt 93496–93472 ) and oVM387 ( 5’-ATCGTGGATCC/GTCTCTGTGGTAAAATGGG-3’ , vIRF2 nt 93435–93453 ) for the second PCR on cDNA derived from VA-activated BCBL-1 cell total RNA . Following by annealling the two PCR products and re-amplification using oVM386 and oVM387 to generate a spliced isoform of full-length vIRF2 in size of 2161 bps , the final PCR product was digested by EcoRI and BamHI and cloned into p-Flag-CMV-5 . 1 . The resulting plasmid was subsequently named as pVM105 . All plasmids were verified by restriction digestion and sequencing . Unless indicated otherwise , protein samples for Western blot were prepared by direct lysis of the cells in 2 × SDS sample buffer ( Quality Biological ) containing 5% 2-mercaptoethanol ( Sigma-Aldrich ) . Samples were resolved on a 4%-12% SDS-PAGE gel in 1 × MOPS buffer ( Thermo Fisher Scientific ) . The signal was detected with SuperSignal West Pico or Femto Chemiluminescent Substrate ( Thermo Fisher Scientific ) . Sodium arsenite ( cat . No . 38150 , Sigma-Aldrich ) solution ( 0 . 83 M ) was prepared in water to serve as a 1660× stock solution . To induce oxidative stress the cells were cultivated in fresh culture medium containing 0 . 5 mM of sodium arsenite for 30 min . To mimic the cellular stress induced by dsRNA , double-stranded poly I:C ( lyophilized polyinosinic–polycytidylic acid sodium salt ) obtained from Sigma-Aldrich ( cat . no . P0913 ) was dissolved in sterile DEPC-treated H2O containing 0 . 98% NaCl to make 5 mg/ml stock solution . Before use , poly I:C was denatured by incubation at 50°C water bath for 20 min followed by slowly cooling to room temperature for at least 45 minutes for proper annealing . The size of poly I:C was determined on a 1% agarose gel and was in size of ~250–300 bp . The amount of poly I:C required for induction of eIF2α phosphorylation was experimentally determined by transfection of HeLa cells with 0 . 1–5 μg of annealed poly I:C using Lipojet transfection reagent ( SignaGen ) . To study inhibitory effect of ORF57 on PKR activation by poly I:C , HeLa or HEK293 cells with or without ORF57 expression were transfected with 1 μg poly I:C and incubated additional 8 h before harvesting . The heat stress was induced by incubation of cells at 44°C for 40 min . Before harvesting , the cells were washed with 1 × phosphate-buffered saline ( PBS , Thermo Fisher Scientific ) . The activation of PKR was inhibited by pre-incubation of cells for 1 h with increasing dose ( 1 , 10 and 100 μM ) of PKR inhibitor ( PKRI ) or PKR inhibitor control ( PKRC ) dissolved in DMSO . DMSO alone was used as a negative control . Cells were subsequently washed and incubated with fresh medium containing 0 . 5 mM of sodium arsenite for 30 min before harvesting . BCBL-1 cells ( 2 × 106 cells ) were induced with 1mM VA for 8 h followed by 40 min incubation at 44°C . The cells cultivated at 37°C were used as negative controls . Subsequently , the cells were harvested for preparation of total proteins in SDS sample buffer and total RNA extracted by TRIzol . Quantitative real-time RT-PCR ( RT-qPCR ) was performed using customized TaqMan probes and TaqMan Gene Expression Master Mix ( Thermo Fisher Scientific ) with standard 2-Δ ( ΔCT ) protocol . HeLa cells with or without ORF57 expression were treated with 0 . 5 mM arsenite for 30 min to induce SG . After washing with ice-cold 1 × PBS , cells were lifted from plates using a cell scrapper and in RSB-200 buffer ( 10 mM Tris-HCl [pH 7 . 5] , 200 mM NaCl , 2 . 5 mM MgCl2 , 0 . 1% NP-40 ) supplemented with 1 × protease and phosphatase inhibitor cocktails ( Roche ) followed by sonication using 10 strokes at level 4 on a sonic dismembrator ( Model 100 , Fisher Scientific ) . The cell lysates were incubated on ice for 15 min and centrifuged at 15871 × g for 15 min at 4°C to separate soluble ( supernatants ) and insoluble ( pellets ) fractions . The cells without arsenite treatment were used as a negative control . IP was performed as described earlier [50] . Briefly , ectopic expression of proteins was obtained by individual plasmid transfection in HeLa or HEK293 cells grown in a 10-cm plate . Cells with or without plasmid transfection were washed with 1× PBS , lysed in 500 μl of 1× RSB-200 lysis buffer ( 10 mM Tris-HCL [pH 7 . 5] , 200 mM NaCl , 2 . 5 mM MgCl2 , 0 . 1% NP-40 and protease inhibitor cocktail [Roche] ) , sonicated ( 10 strokes at level 4 ) and cleared by centrifugation at 11500 × g for 10 min at 4°C . In the assays designed to see the interaction of multiple overexpressed proteins , 100 μl total cell extract containing one overexpressed protein was mixed with 100 μl total cell extract with another overexpressed protein . The mixed cell lysates ( 200 μl ) were then incubated with 5 μl of RNase A/T1 mixture containing 1 . 25 U RNase A and 50 U RNase T1 ( Thermo Fisher Scientific ) for 10 min at room temperature followed by pre-cleaning with pre-washed sepharose CL-4B beads ( Sigma-Aldrich ) . The pre-cleaned cell lysates were mixed with 80 μl of antibody-coated protein A/G beads ( 50% slurry , Sigma-Aldrich ) in 1 ml of IP buffer ( 50 mM HEPES [pH 7 . 5] , 200 mM NaCl , 1 mM EDTA , 2 . 5 mM EGTA , 10% glycerol , and 0 . 1% NP-40 , 1 × Roche’s protease inhibitor cocktail ) and incubated overnight at 4°C followed by extensive wash with IP buffer . Immunoprecipitated complexes on the beads were dissolved in 70 μl of 2 × SDS protein sample buffer containing 50 mM DTT . Alternatively , the immunoprecipitated protein complexes on the beads were eluted by incubation with 50 μl of 100 μg/ml 3×Flag ( Sigma-Aldrich ) in IP buffer for 2 h at 4°C . The eluant in the supernatant after spinning was collected and mixed with 20 μl of 5 x SDS protein buffer containing 50 mM DTT . All samples were heat-denatured at 95°C for 5–10 min before SDS-PAGE . Western blotting for individual proteins was carried out using the 3–5% input lysates and 30%-50% immunoprecipitated proteins . Densitometric quantification of the individual protein band intensity was performed using Image J software ( NIH ) . Adherent HeLa and HEK293 cells were grown directly on glass coverslips . The non-adherent BCBL1 cells were immobilized by spotting of cells suspension on poly-D-lysine-treated glass coverslips . Immunofluorescence staining was performed as described previously [50 , 57 , 115] . Briefly , the cells were washed with PBS , fixed with 4% paraformaldehyde , permeabilized with 0 . 5% Triton X-100 and blocked with 2% BSA ( bovine serum albumin , Promega , Madison , WI ) dissolved in Tris-buffered saline containing 0 . 05% of Tween-20 ( TTBS ) . Primary antibodies diluted in blocking buffer were incubated with slides overnight at 4°C . AlexaFluor-conjugated secondary antibodies ( 1: 500 , ThermoFisher Scientific ) were diluted in blocking solution and incubated with slides at 37°C in humidified chamber . The slides were washed with TTBS and before mounting the cells nuclei were visualized by 5 min counterstaining with wash buffer containing Hoechst dye 33342 ( Sigma-Aldrich ) . Confocal fluorescence images were collected with a Zeiss LSM780 laser-scanning microscope ( Carl Zeiss , Inc . , Thornwood , NY ) equipped with 20x Plan-Apochromat ( numerical aperture , 0 . 8 ) and 63x Plan-Apochromat ( numerical aperture , 1 . 4 ) objective lenses . The x-y pixel sizes of 0 . 4 and 0 . 07 μm and optical slice thicknesses of 1 . 5 and 0 . 9 μm were used to acquire confocal images with the 20× and 63× objectives lenses , respectively . Volume reconstructions were generated using the Imaris ( version 8 . 0 . 2 ) image processing software ( Bitplane , Inc . , Concord , MA ) . One microgram of reconstituted poly I:C was labelled using 10 units of T4 polynucleotide kinase ( T4 PNK , Thermo Fisher Scientific ) for 10 min at 37°C in a 25-μl reaction containing 25 μCi [γ-32P]-ATP ( Perkin Elmer ) . The reaction was terminated by adding 5 mM EDTA to the reaction mixture and the labeled poly I:C was purified using an illustraMicrospin G-25 column ( GE Healthcare , Marlborough , MA ) . In a pull-down assay , HeLa cells in a 10-cm culture dish were transfected with a Myc-Flag-tagged PKR expression vector or an empty vector . The Myc-Flag-tagged PKR protein in the HeLa cell lysate was immunoprecipitated using 80 μl of anti-mycEZview beads ( Sigma-Aldrich ) overnight at 4°C . The beads were then washed four times with IP buffer and subsequently resuspended in 100 μl of IP buffer . Approximately , 40 μl of the resuspended beads coated with PKR were incubated with a mixture of 100 ng [γ-32P]-labelled poly I:C and 500 ng of purified recombinant ORF57-Flag protein or BSA ( negative control ) in 750 μl of IP buffer for 2 h at room temperature . The beads were extensively washed with IP buffer , resuspended in 5 ml of liquid scintillation cocktail ( CytoScint , MP Biochemicals , Santa Ana , CA ) and radioactivity was counted by a liquid scintillation counter . Recombinant full-length PKR was purified from HeLa cells in a 10-cm petri dish by by immunoprecipitation using 80 μl of anti-mycEZview beads as described above and subsequently dissolved in 100 μl of 1x kinase buffer ( 10 mM Tris-HCl [pH7 . 6] , 50 mM KCl , 2 mM magnesium acetate , 20% glycerol ) . In a 32-μl kinase reaction , 12 μl of beads-attached PKR protein were first incubated with 200 ng of recombinant ORF57-Flag or BSA proteins for 10 min at room temperature . The resulting mixture was sequentially supplemented with 50 ng poly I:C , 1x kinase buffer supplemented with 0 . 83 mM MgCl2 and 20 μCi [γ-32P]-ATP . The reaction was incubated at 30°C for 10 min and terminated by addition of equal volume of 2 × SDS protein sample buffer . After SDS-PAGE the gel was mounted in an exposure cassette and analyzed by a PhosphorImager ( GE Healthcare ) . HeLa cells with expression of Myc-Flag-tagged PKR in a 10-cm Petri dish were treated with arsenite to phosphorylate PKR . Myc-Flag-tagged PKR purified by immunoprecipitation as described above from the cells with ( for activated PKR ) or without ( for inactive PKR ) arsenite treatment was finally resuspended in 100 μl of 1x kinase buffer . Ten ul of the resuspended beads coated with PKR were mixed with 500 ng of recombinant ORF57 protein or BSA and incubated for 10 min at room temperature . The mixture was sequentially supplemented with 1x kinase buffer , 0 . 83 mM MgCl2 , 50 ng GST-eIF2α ( cat# H00001965-P01 , Abnova ) , and 20 μCi [γ-32P]-ATP to a final volume of 32 μl . The kinase reaction involving eIF2α phosphorylation by phosphorylated ( activated ) PKR was allowed to proceed for 40 min at 30°C . The reaction was terminated by addition of 2x SDS sample buffer and the samples were resolved by SDS-PAGE . The gel was exposed to a PhosphorImager and an X-ray film for signal quantification . KSHV infected iSLK-BAC16 cells [92] growing in a six-well plate ( 2 . 5 x 105 cells/ well ) were transfected twice , respectively , at an interval of 24 h with 40 nM of ON-TARGETplus SMART-pool PKR siRNAs targeting human PKR/EIF2AK2 ( L-003527-00-0005 , Dharmacon , GE Healthcare , Lafayette , CO ) or ON-TARGETplus Non-targeting siRNA #1 negative control ( D-001810-01 , Dharmacon , GE Healthcare ) using LipoJet transfection reagent ( SL100468 , SignaGen Laboratories , Gaithersburg , MD ) . Total cell extract was collected 24 h after the second siRNA transfection to measure the knockdown efficiency by immunoblotting . For KSHV virus production and titration assays , iSLK-BAC16 cells in a six-well plate without or with PKR siRNA transfection at 24 h of the second round of siRNA transfections described above were induced for KSHV lytic infection with 1 mM sodium Butyrate and 1 ug/ml doxycycline in 2 ml of DMEM medium . On the third day , fresh 2 ml of DMEM medium containing the same amount of sodium butyrate and doxycycline were added to make the culture medium in total of 4 ml per well for another two more days . For virus production and titration , the iSLK-BAC16 culture supernatants were harvested on the 5th day after induction , cleared by centrifugation at 2000 rpm for 10 min , and filtered through Sterile Millex 0 . 45 μM filter units ( cat# SLHA033SS , Millipore , Billerica , MA ) . 400 μl of the supernatants were used to infect HEK293 ( 5 x 105 cells/ well ) in a six-well plate . KSHV-infected GFP+ HEK293 cells at 48 h after infection were observed by a fluorescent microscopy and analyzed by flow cytometry . | Mammalian RNA granules , including stress granules ( SG ) , are important components of the host cell antiviral responses and their assembly is widely counteracted by RNA viruses . In Kaposi’s sarcoma-associated herpesvirus ( KSHV ) lytically infected B cells , we found that KSHV infection inhibits the assembly of SG by expression of viral lytic protein ORF57 and ORF37 ( vSOX ) . KSHV ORF57 blocks arsenite-induced SG formation by binding to and preventing PACT from activating PKR . ORF57 also interacts with two double-stranded RNA binding motifs of PKR and prevents its binding with poly I:C and autophosphorylation , subsequent inhibition of eIF2α phosphorylation and SG formation . Consistently , knockdown of PKR increases production of KSHV virions . This function of KSHV ORF57 is conserved in homolog HSV-1 ICP27 , but not in EBV EB2 . We conclude that KSHV ORF57 antagonizes host antiviral defenses for virus lytic infection and production . | [
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"mol... | 2017 | KSHV inhibits stress granule formation by viral ORF57 blocking PKR activation |
A multi-phased study was conducted in Cambodia from 2005–2011 to measure the impact of larviciding with the bacterial larvicide , Bacillus thuringiensis israelensis ( Bti ) , a water dispersible granule ( WG ) formulation on the vector , Aedes aegypti ( L . ) and the epidemiology . In our studies , all in-use containers were treated at 8 g/1000 L , including smaller containers and animal feeders which were found to contribute 23% of Ae aegypti pupae . The treated waters were subjected to routine water exchange activities . Pupal production was suppressed by an average 91% for 8 weeks . Pupal numbers continued to remain significantly lower than the untreated commune ( UTC ) for 13 weeks post treatment in the peak dengue vector season ( p<0 . 05 ) . Suppression of pupal production was supported by very low adult numbers in the treated commune . An average 70% of the household harbored 0–5 Ae aegypti mosquitoes per home for 8 weeks post treatment , but in the same period of time >50% of the household in the UTC harbored ≥11 mosquitoes per home . The adult population continued to remain at significantly much lower numbers in the Bti treated commune than in the UTC for 10–12 weeks post treatment ( p<0 . 05 ) . In 2011 , a pilot operational program was evaluated in Kandal Province , a temephos resistant site . It was concluded that 2 cycles of Bti treatment in the 6 months monsoon season with complete coverage of the target districts achieved an overall dengue case reduction of 48% in the 6 treated districts compared to the previous year , 2010 . Five untreated districts in the same province had an overwhelming increase of 352% of dengue cases during the same period of time . The larvicide efficacy , treatment of all in-use containers at the start of the monsoon season , together with treatment coverage of entire districts interrupted disease transmission in the temephos resistant province .
Cambodia located in Southeast Asia has a tropical monsoon climate with distinct dry ( November–April ) and wet ( May–September ) seasons . Dengue fever ( DF ) is widely prevalent in Cambodia with year-round transmission , but it peaks during the rainy season which is accompanied with high humidity . Since the 1990’s , there have been nationwide cyclical dengue outbreaks , once every 3 to 4 years [1] . Transmission is caused by all 4 serotypes of dengue virus [2] . The principal dengue vector is Aedes aegypti ( L ) [3] . The vector population is supported throughout the year by the presence of various types of domestic water containers that are used for daily household water consumption . In 1995 the National Dengue Control Program ( NDCP ) was established and has since used temephos as the primary means of vector control , targeting areas with high population density or dengue high-risk areas [1] . Temephos is applied twice per year during the wet season . An estimate of 66 , 000 kg of temephos is used to cover 1 . 7 million containers per treatment in 22 provinces [1] . Development of temephos resistance in field populations was first reported by Polson in 2001 [4] . In 2013 , larvae collected from sentinel sites in 7 provinces exhibited different levels of mortality ( 11–90% ) at WHO diagnostic dosage of 0 . 02 mg/L temephos [5] . The susceptibility status of the F2 –F4 larvae from the same provinces was further confirmed by the World Health Organization ( WHO ) Collaborating Centre for Vectors , Institute for Medical Research , Malaysia ( IMR ) with 4–36% mortality at the diagnostic dosage [6] . In search of new larvicide tools , Cambodia’s National Malaria Center ( CNM ) evaluated VectoBac WG , a bacterial larvicide based on Bacillus thuringiensis israelensis strain AM65-52 ( Bti ) , for its efficacy against Ae aegypti . VectoBac WG is a water dispersible granule formulation , which has been reviewed by World Health Organization Pesticide Evaluation Scheme ( WHOPES ) and is approved for introduction into finished drinking waters [7 – 8] . Since 2001 , municipalities in Brazil used this Bti formulation as a substitute to temephos [9] . In 2004 , a semi field study conducted in Phum Thmei village within Phnom Penh province showed that at the dosage of 0 . 4 g/50 L , effective control of Ae aegypti in cement containers containing river , rain and well waters was achieved for 3 months , without any water exchange activity [10] . This paper reports the results of a multi phased study that was conducted from 2005–2011 . In 2005 and 2006 , CNM expanded their study to an operational scale to determine the impact of Bti strain AM65-52 applications on field populations of Ae aegypti pupae and adults . In 2007 , a large scale pilot program was carried out in Kandal Province . A post treatment survey was conducted to determine the community acceptance for this new larvicide introduced in potable waters and also the capability of the applicators to handle this new larvicide . Following these evaluations from 2005–2007 , in 2010 the Ministry of Health ( MOH ) purchased 10 Mt VectoBac WG with the support of World Bank for the treatment of water containers in Kandal Province to control temephos resistant vector population . The larvicide was used to conduct 2 treatments per year in 2010 and in 2011 . The impact of larviciding on the number of dengue cases was determined in 2011 .
Cambodia is administratively divided into 24 provinces . The provinces are subdivided into districts , which in turn are further divided into communes . Communes consist of several villages . In 2005 ( June-November ) and 2006 ( March-September ) studies were conducted to measure the impact of VectoBac WG applications on field populations of Ae aegypti pupae and adults . The studies were conducted with the approval from the National Ethics Committee for Health Research of Cambodia . The study was conducted in Peani and Ou Ruessei communes during both years . The communes were located in Kampong Tralach district of Kampong Chhnang Province at N 11° 55' 41" , E 104° 42' 38" . The communes were separated by a dual carriage road known as National Road 5 . Peani commune measured within 5 . 7 km x 4 . 5 km ( 2565 ha ) and had 9 villages , 1343 household and a population of 5439 people . Containers in this commune were treated with Bti . Ou Ruessei commune measuring 3 . 6 km x 5 . 6 km ( 2016 ha ) with 9 villages , 1595 household and 7371 people was used as the untreated control ( UTC ) . The environment and houses in both communes were similar . They were farming communities living in rural traditional houses built on stilts . The walls were made from wood or woven dried palm leaves or rice straws . The houses were clustered in each village , with paddy fields surrounding the villages . Typically each house contained one room which was used as living/sleeping quarters in the cooler hours of the day . All other activities including cooking were conducted in the space under the house , which provides a cool place in the heat of the day . Animals were also kept in the space outside the enclosed walls of the house or below the house . Surveillance was conducted once every 2 weeks , with 3 surveillance rounds in the pretreatment phase and 9 surveillance rounds in the post treatment phase . Each surveillance was conducted in 50 households per commune . The 50 houses were from 3 different villages in the commune . Each surveillance covered 50 different households . No one house was repeated in the surveillance rounds during the year , as this methodology allowed broader surveillance coverage . In 2006 , the 2nd year of study , a GPS unit ( Garmin GPSMAP 60Cx ) was used to map the houses surveyed in both communes for each of the 12 surveillance rounds . During the 2 years of study , a total of 24 surveillance rounds were conducted in both communes using the same two entomology teams to conduct the pupal and adult surveillance . Pupal and adult populations were measured in the same household . All pupae were collected from in-use and discarded containers and were allowed to emerge in the CNM insectarium . The emerged adults were then identified . Adult mosquitoes were collected in the field with an aspirator . A technician with a battery operated aspirator ( 100 cm long ) swept the entire indoor for 10 minutes per house . The adults collected in the insect bag were transferred to CNM for identification and Ae aegypti mosquitoes were counted . In 2006 , the percent ( % ) houses in both communes were categorized according to the numbers of collected indoor adult Ae aegypti mosquitoes . The categories were set at 0 , 1–5 , 6–10 , 11–20 , 21–30 , 31–40 , and ≥ 41 mosquitoes per household . There are no previous reports or publications to correlate the threshold adult mosquito numbers to dengue outbreaks in Cambodia or elsewhere . The authors in this study made an arbitrary decision to categorize as such to reflect the impact of Bti treatment on the infestation of indoor Ae aegypti adult mosquitoes . During the 2005 and 2006 surveys , the productivity of water receptacles for Ae aegypti pupae was determined in the untreated Ou Ruessei commune in order to characterize production by container type . The containers were categorized into 3 types , following the guidelines used by the Cambodian National Dengue Control Program ( NDCP ) in their annual larviciding program with temephos . The first category included key containers normally recommended by WHO for treatment , i . e . the cement jars with volume capacity 100 L to 500 L . The second category included key containers that were recommended and included in the treatment program by NDCP: cement tanks with volume capacity ≥ 200 L , earthen jars with volume capacity ≥ 80 L , metal drums ( 200 L volume capacity ) , and cooking utensils of 30–50 L . The third category were containers that were not under the annual treatment program , such as earthen jars with volume capacity ≤ 80 L , cooking utensils of ≤ 30 L volume capacity which included bamboo containers and also any other containers used for animal feed which included tires and coconut shells . VectoBac WG , Bti strain AM65-52 , ( manufactured by Valent BioSciences , IL , USA ) was used to treat the larval habitats in Peani commune in both years of the study . Lot 125–516 PG was used in 2005 and Lot 139-345-3L was used in 2006 . Direct application of the product was made at 8 gm/1000 L , based on the container volume . The same team of 8 applicators worked in 4 groups , with 2 applicators per group to conduct the operation in both years . In 2005 , treatment was conducted from July 25–29 and in 2006 it was conducted from May 1–5 . At the start of the treatment operation , the applicators were given a review of the target larval habitats . The applicators carried about 2 . 5 kg material in plastic containers with lids . They dispersed the Bti material over the entire surface of each container using a pre-calibrated teaspoon . One full teaspoon ( scooped flat ) held 1 . 6 g of the WG formulation , which was sufficient to treat a 200 L container . All target larval habitats with or without water were treated , with a dose based on their volume capacity . The treated containers were marked with permanent paint as soon as the container was treated with Bti . All treated containers were subject to their routine water exchange activity . Prior to the operational programs in each year , training sessions were held for local supervisors and applicators . The training sessions were held about 2–3 days before the treatment was initiated . Leaflets describing the product , its efficacy and safety were distributed to each household . All target containers were treated and marked as described in the section above under “Larviciding with Bacillus thuringiensis israelensis ( Bti ) ” . In 2007 , the Bti treatment in Kandal Province was completed in selected communes in May . The communes were selected based on high population density and dengue incidence rate . The NDCP team from CNM conducted their routine survey for 14 days to evaluate the treatment coverage of the larviciding program . During this survey they questioned the householders on the acceptance or rejection of the new larvicide and the reason ( s ) for the acceptance or rejection . In 2010 and 2011 , the impact of larviciding with Bti on dengue transmission in Kandal Province was determined . This province has 11 districts . In each year two Bti treatments were made during the season . No other larviciding or adulticiding measures were conducted . In 2010 , the treatment covered only selected communes in the 11 districts . Communes were selected based on high population density and dengue incidence rate . While in 2011 , six districts with the highest dengue incidence rate in the previous year 2010 were chosen for the treatment program . All communes in the six districts received full coverage of Bti treatment . The remaining 5 districts were not under any mosquito intervention program during this study period . Dengue case numbers are routinely collated by NDCP in CNM from all 24 provinces in Cambodia . The health department in each province tabulates the weekly data from all government hospitals in the province and forwards the data to NDCP . NDCP also collects case numbers from 7 sentinel hospitals: Kantha Bopha Hospital and National Pediatrics Hospital from Phnom Penh city; Takeo , Kompot and Kg Cham hospitals from the 3 respective provinces; and Angkor and Javavarman VII hospitals from Siem Reap province . All reported cases are diagnosed by clinical symptoms , and some are serologically identified by Institute Pasteur , Cambodia . The impact of larviciding with Bti on dengue transmission was not determined by using the population based epi indicator , such as Incidence Rate ( IR ) , because the annual population census data was lacking in Cambodia . So we determined the impact of larviciding by comparing the dengue case numbers between 2 consecutive years for 3 different phases: pre Bti treatment; window; and post Bti treatment . After the Bti treatment a 4 week lapse was given to allow the existent adult mosquito population and the circulating dengue virus in human and adult mosquitoes to diminish . This 4 week period is denoted here as the ‘window’ period . For studies conducted in the year 2005 and 2006 , comparisons were made for each surveillance for mean number of containers , Ae aegypti pupae and adults between the untreated commune , Ou Ruessei and Bti treated commune , Peani . SPSS ver . 17 was used for this purpose . Normality tests indicated that the data was not normally distributed for both communes ( p<0 . 05 ) . Therefore a non-parametric test , Mann-Whitney U test was performed to determine the differences in the 3 parameters mentioned above between the Bti treated and untreated commune . The impact of Bti treatment on dengue cases in the year 2011 was also statistically analyzed . The comparisons were made for each district for dengue cases between year 2010 and 2011 at 3 different phases: pre Bti treatment phase ( weeks 1–20 ) ; window phase ( weeks 21–24 ) ; and post Bti treatment phase ( weeks 25–36 ) . Test of normality indicated that the dengue cases were not normally distributed for both years ( p<0 . 05 ) . Therefore , the significant difference in the dengue cases between the year 2010 and 2011 was determined using a non—parametric test , the Mann-Whitney U test .
The single direct application exercise to treat all in-use larval habitats in Peani commune required 19 kg of VectoBac WG in 2005 and 23 kg in 2006 . More than ten thousand containers were treated in each year . Efficacy of the larviciding exercise was determined by measuring the Ae aegypti pupae and adult mosquito numbers . Twelve surveillance rounds were conducted in each year covering a total of 600 houses per commune . These houses comprised 38% and 45% of the total household in Ou Ruessei and Peani communes , respectively . In 2006 , the total 1200 houses surveyed in both communes were mapped as shown in Fig 1 . The mean number of containers surveyed in Peani and Ou Ruessei communes for each surveillance for the years 2005 and 2006 are as shown in Table 1 . In 2005 , it was an average of 3–4 containers per household , and in 2006 most homes had an average of 4 containers per household . In both years , for several surveillances the number of containers significantly differed between the 2 communes , and generally the difference is by one container . A significant difference of one container did not give any significant difference in the pupal numbers between the 2 communes , as observed during the pretreatment phase in 2006 ( Fig 3 ) . Mean numbers of Ae aegypti pupae and Ae aegypti adults collected per house from Peani and Ou Ruessei communes are as shown in Fig 2 for year 2005 and in Fig 3 for year 2006 . In 2005 , surveillance was conducted from June-November . Pre treatment surveillance was conducted from June-July and post treatment surveillance from August-November . Both communes had similar numbers of Ae aegypti pupae and adults during the pre treatment phase ( p>0 . 05 ) . Two weeks into post treatment phase no Ae aegypti pupae were collected from the Bti treated commune , while 50 . 42 ± 12 . 04 pupae were collected from the UTC commune . Pupae numbers remained lower in treated commune for 18 weeks post treatment in 2005 . Significant reduction of adult mosquitoes was observed in the treated commune for continuous 12 weeks following the Bti post treatment phase ( p<0 . 05 ) . Seasonal mosquito population reductions were observed in both the study communes beginning in August . But , the Ae aegypti adult numbers continued to trend lower over time in the Bti treated commune for continuous 12 weeks post treatment with 1–2 adult mosquitoes collected per house ( p<0 . 05 ) . Entomological surveillance data from 2006 is presented in Fig 3 . Surveillance was conducted from March-September . Pre treatment surveillance was conducted from March-April and post treatment surveillance from May-September . Both communes had similar numbers of Ae aegypti pupae and adults during the pre treatment phase ( p > 0 . 05 ) , except in the 2nd pre treatment surveillance where Peani commune had significantly higher adult population at 9 . 58 ± 1 . 19 ( p<0 . 05 ) . Two weeks into the post treatment phase , containers in Bti treated commune had zero pupal production , and production continued to be suppressed to very low numbers for 10 weeks post treatment . After 10 weeks the numbers increased , but remained significantly lower than in untreated commune for 13 weeks post treatment , that is during the peak dengue vector season ( p<0 . 05 ) . The Ae aegypti indoor adult population was significantly suppressed from the period of Bti treatment until the end of surveillance at 19 weeks post treatment ( p<0 . 05 ) . The population was lowest at 1 . 82 ± 0 . 42 per house at 4 weeks post treatment . In the untreated commune the adult numbers during the peak rainfall season from 6–13 weeks post treatment ( mid-June to end July ) was in the average of 15–26 mosquitoes per house . In the same period there were only 3–12 mosquitoes per house in the Bti treated commune , peaking at 12 . 52 ± 2 . 45 at 13 weeks post treatment . The effect of larviciding with Bti on suppression of indoor adult Ae aegypti populations was supported by data as shown in Table 2 . The percent ( % ) houses in both communes were categorized according to the numbers of indoor adult Ae aegypti mosquitoes collected during the study period in 2006 . During the pre treatment sampling period , Peani commune had more houses ( 90 . 67% ) harboring adult mosquitoes than the Ou Ruessei commune and it also had more houses ( 27 . 34% ) with higher density of adult mosquito population ( ≥11 mosquitoes per house ) in comparison to Ou Ruessei commune . On the first month after Bti treatment , the percentage of houses with zero mosquitoes increased to 27% in the Bti treated commune and continued to remain at a far higher percentage in comparison to the untreated commune through the rainy season until September . Most of the houses ( ≥ 40% ) in the Bti treated commune had 1–5 mosquitoes through the 4 months ( May 16th -September 16th ) of post treatment . In the same months , in the untreated commune only 3–6% of the houses did not harbor any dengue vector and the remaining homes had ≥ 6 adult mosquitoes per home . During the peak rainfall season ( May-July ) most homes in Ou Ruessei had 11–20 Ae aegypti adults per home and the percent homes with ≥ 21 mosquitoes per household increased by more than 2 folds in June and July . Total number of containers surveyed in Ou Ruessei commune and the total number of pupae collected which successfully emerged into Ae aegypti adults are shown in Table 3 . A total of 2413 and 3059 containers were surveyed in 2005 and 2006 , respectively . For the 2 years , 60+ ( % ) of the Ae aegypti pupae were produced by key containers of the type recommended for larviciding by WHO . The next most productive category was containers that were not recognized as key containers by WHO or NDCP and were not under the routine larviciding program . These containers produced 23% of the pupae , with an average of 3732 pupae per study period . The Bti treatment was done over 14 days in 58 communes in 11 districts from May 5–19 . A 2 . 9 metric tons Bti material was applied in 68 , 241 households with 461 , 693 containers . A post treatment survey was conducted by the NDCP team from CNM in 1299 households and they observed through 7864 containers . A 95 . 55 ± 1 . 38% of the households accepted the Bti treatment because the larvae died within 2 hours and the treated waters did not undergo any physical change in color and odor . A 92 . 45 ± 1 . 16% of the containers were treated , showing the ability of the applicators to recognize and treat the many and varied larval habitats . In 2010 , two Bti treatment cycles were conducted , in June and in September . Both treatments took 14 days each and covered only selected communes in the 11 districts in Kandal province . The first treatment covered 47 communes in 11 districts with 2 . 09 metric tons of Bti material to treat 532 , 745 containers in 96 , 528 households . The second treatment covered 20 communes in 4 districts with 0 . 75 metric tons to treat 303 , 017 containers in 24 , 220 households . We were not able to determine the impact of Bti treatment on dengue transmission in 2010 because several of the recorded dengue cases had incomplete patient address i . e . the records stated the district name , but not the name of the commune/village ( within the district ) where the patient resides . This prevented us from knowing the origin of the transmission , whether from a Bti treated or untreated commune . In 2011 , six districts with the highest dengue incidence rate in 2010 were larvicided with Bti . The entire six districts with 59 communes were treated with the first cycle in May , followed by a second cycle in August . The first cycle used 2 . 63 metric tons of Bti material for 754 , 098 containers in 85 , 239 household . The second cycle used 2 . 77 metric tons to treat 754 , 111 containers in 85 , 257 households . So in 2011 , the pre Bti treatment phase was from weeks 1–20 . The next 4 weeks was the window period , weeks 21–24 . The impact of larviciding in 2011 was measured by comparing dengue case numbers between 2010 and 2011 for weeks 25–36 , during the peak dengue season . In 2011 , the 5 untreated districts had an increase in dengue cases ( Fig 4 ) , with a significant peak of 351 . 52% for weeks 25–36 ( p < 0 . 05 ) ( Table 4 ) . In 2010 from weeks 1–36 , these same 5 districts had lower number of dengue cases than the 6 districts which were used for Bti treatment in 2011 ( Table 4 ) . As for the 6 Bti treated districts during the weeks 25–36 , a significant overall reduction of 47 . 48% cases was achieved ( p<0 . 05 ) ( Fig 5 and Table 4 ) . Three of the Bti treated districts ( Ponhea Lueu , Leuk Daek , Angk Snoul ) had a 66–85% reduction . But , one of the district , Kien Svay had an 83% increase and this was due to untreated communes in the district . These communes were not treated as they administratively belonged to an untreated district .
Dengue is endemic in the tropics and subtropics , with as many as 400 million infections globally per year . The dengue virus is transmitted by Ae aegypti , Ae albopictus or any other related Aedes species . For the moment there is no specific treatment for the disease , nor any suitable vaccine to prevent infection by any one of the four dengue viruses . Thus , the protective initiative to prevent dengue or subdue the transmission relies completely on mosquito vector control . To date , there is only one published paper describing reduction in vector populations with an impact on dengue transmission following larviciding with Bti [11] . The study was in a dengue endemic site in Malaysia and it achieved dengue suppression by larviciding with VectoBac WG ( Bti strain AM65-52 ) [12] . Bti was applied by spray application into all target larval habitats . The adult vector population was suppressed to below 10% ovitrap index ( OI ) and no transmission was reported during the peak dengue season . It was contrary in the untreated site , with more than 40% OI and 15 serologically confirmed dengue cases . This paper reports a multi phased study conducted in Cambodia from 2005–2011 and it would be the second report confirming that larviciding with Bti strain AM65-52 suppressed the adult dengue vector population in the peak season in the treated areas and also impacted the dengue transmission in the treated districts . Cambodian economy is heavily dependent on agriculture , keeping the masses in suburban and rural areas . Water supply to these areas remains a challenge . Homeowners are dependent on waters collected from rain , groundwater and river . The waters are commonly stored in >200 L cement containers . Smaller in volume earthen containers are also found in the household vicinity together with animal feeders . A preceding semi-field study conducted in a village in Phnom Penh , Bti strain AM65-52 at 8 g/1000 L was found to significantly suppress the formation of Ae aegypti pupae in all 3 water types for 10–12 weeks ( p<0 . 05 ) [10] . This same dosage was used for the 2 years study in 2005 and 2006 . The Bti treated waters in 10 , 000+ containers per year was exposed to frequent flooding from rainfall , UV light , ambient temperature , and routine domestic activities . The long residual efficacy at 8 g/1000 L could be due to the nature of the formulation , enhanced by the container material . Almost all containers in the test commune were made from cement and clay . As soon as the applicator dispersed the Bti evenly across the water surface , the granules sediment to the base of the containers and some could have been embedded into the porous cement and clay matrix . Significantly longer persistence of Bti toxins has been demonstrated in earthen containers than in plastic or HDPE containers , with or without regular water exchange activity [13] . When containers were emptied completely of the Bti treated water , a near complete larval mortality was still achieved on the introduction of fresh untreated water into earthen jars , and this could be due to the release of embedded Bti toxins from the container surface . The long residual efficacy could also be due to sedimentation of the dispersible granules , leaving very minimal amounts of Bti toxins in the water column to be removed during any water exchange activity . In Australia pupae suppression was achieved for 23 weeks with the same larvicide , but at a much higher dose of 400 g/1000 L in plastic containers that were exposed to rainfall [14] . In Brazil , a 11 weeks pupae suppression was achieved with 2 g/1000 L in covered containers with no water exchange activity [15] . This same dose was used by the NDCP of Brazil to treat all containers in temephos resistant areas at 60 day intervals . Generally , in Cambodia during the monsoon season there is an increase of Ae aegypti larval habitats ( storage containers ) . This is accompanied by an exponential increase in dengue cases with a peak in August and September . In our study in 2006 , we did observe in the rainy season the adult Ae aegypti population increased by 4 to 5 folds in the untreated commune ( Ou Ruessei ) from mid-June to end July 2006 . The seasonal abundance of the adult population could also be due to increased larval survival rate and pupation rate , as observed in a survey conducted in a neighboring country , Myanmar [16] . In 2006 , the rise of the adult vector population in the peak rainfall season was well suppressed in the Bti treated commune which received the treatment in early May . The Bti treatment made at the onset of the rainy season significantly suppressed the adult mosquito population for 4 months . Most homes in the treated commune had 0–5 mosquitoes per household . On the contrary most homes in the untreated commune had more than ≥ 6 mosquitoes per household . It is necessary that the larvicide distribution is conducted at the onset of the rainy season to curb the rise of the adult vector population during the rainy season , thus preventing outbreaks . In Cambodia 2003 , a delay in temephos distribution , intended for May-June , but conducted in July–August , accounted for outbreak of dengue cases in the village that year [17] . The success of this study was also due to treating all in-use containers , irrespective of container size and its water content . The 2 year ( 2005 and 2006 ) data confirmed that small in-use containers , e . g . jars of size ≤ 80 L and cooking utensils of ≤ 30 L including animal feed containers , contributed to 23% of Ae . aegypti pupae production . Thus , it is important that these containers to be included for larvicide treatment under the routine NDCP . Treating the smaller in-use containers did not require much effort and time , because these containers are usually found placed around the bigger containers or just behind the home among the animal dwelling . NDCP cannot expect an impact on dengue transmission if efforts and resources are invested to treat only the key containers which contribute to 80% of Ae . aegypti pupae . There will always be a population of adult mosquitoes emerging from the smaller untreated containers , which will actively bite and transmit dengue . Dry containers were also treated with Bti , because on flooding they serve as larval habitats or the dried Ae aegypti eggs from the previous season in the containers will hatch into fresh larvae . Dry containers can be treated at least 8 weeks before flooding with no significant decrease in larvicide efficacy [14] . In Brazil , temephos resistance was first detected in 1999 and the resistant vector populations are widespread throughout the country . Since 2001 , the resistance was managed by replacing temephos with Bti in some localities [18] . The temephos resistance ratio was reduced by 30% where Bti was used , but municipalities which continued to use temephos in spite of resistance detection had increased resistance ratios in the same period of time [19] . Temephos resistant populations are susceptible to Bti and there is no cross-resistance between temephos and Bti [20] . In Cambodia , since 2009 temephos resistance has been detected in several provinces . Beginning the year 2010 , the Cambodian Ministry of Health ( MOH ) under the Second Health Sector Support Program ( HSSP2 ) with financial support from World Bank purchased VectoBac WG ( Bti strain AM65-52 ) for treatment in provinces with confirmed temephos resistance . The decision to purchase was based on the local data collected in 2005–2006 on the suppression of adult Ae aegypti population by Bti strain AM65-52 , the high acceptance of the local community in Kandal Province for this new larvicide as shown by the post Bti treatment survey in 2007 and the success of the temephos replacement program in Brazil [9] . The first procurement was used to evaluate Bti larviciding on the interruption of dengue transmission in a pilot program in Kandal Province . The 2011 pilot operational program confirmed that 2 cycles of Bti treatment in the 6 months monsoon season with a complete coverage of the target district achieved an overall reduction of 47 . 5% in 6 treated districts compared to the previous year , 2010 . Five untreated districts in the same province had an overwhelming increase of 351 . 5% during the same period of time . Subsequent purchases by the Cambodian MOH were based on the results achieved for interruption of dengue transmission in Kandal Province . In the following years the NDCP used Bti strain AM65-52 in Kandal Province , and 2 other provinces with temephos resistance , Kampong Thom and Siem Reap . The introduction of the new larvicide in each province required intense training of the larvicide distribution team . The team consisted of hundreds of applicators and supervisors under the NDCP , who have also worked to disburse temephos . Training focused on the identification of larval habitats and the importance of treating all in-use dry and wet containers . Each team member was provided with a pocket size application guide manual . The post treatment Bti survey in 2007 showed that 93% of the larval habitats were treated , concluding that the applicators were able to work efficiently with the new larvicide . Leaflets were also distributed to each household describing the safety and specificity of this new larvicide . Over the years , households in Cambodia have increased and so has the number of storage water containers . Thus , Cambodia requires the annual larviciding campaign against dengue . The annual campaign is effective and cost-effective intervention to reduce epidemiologic and economic burden of dengue . The reduced number of dengue cases in 2001–2005 in comparison to 1995–2000 was attributed to intervention by larviciding with temephos . It averted dengue hospitalizations , ambulatory cases and dengue deaths , which resulted in a saving of 997 disability adjusted life years ( DALYs ) [21] . Widespread resistant to temephos in Cambodia requires for an alternative larvicide . This multi phased study in Cambodia from 2005–2011 concludes with good supporting evidence , that larviciding with Bti strain AM65-52 by a single dose of 8g per 1000 L in all in-use containers significantly suppresses Ae aegypti pupae production and adult mosquitoes for a continuous 13 weeks in the peak rainfall and vector season . During the 6 months rainfall season , two Bti treatment cycles with coverage of all larval habitats within the community will interrupt dengue transmission . | Aedes aegypti is responsible for global dengue outbreaks in the recent years . It is also the vector of Zika virus , which may be associated with the thousands of microcephaly cases in Latin America . At the moment , dengue prevention and control solely depends on vector control measures . Ae aegypti thrives in all types of artificial water storage containers . Countries lacking piped water systems and depending on water storage containers are required to implement preventive measures regularly . Temephos has been the mainstay of Ae aegypti larval control for 40 years in the Asian region . Temephos resistance is established in the vector population . Here in Cambodia , we assessed a bacterial larvicide , Bacillus thuringiensis israelensis ( Bti ) strain AM65-52 in communes and districts with temephos resistant mosquito population . A single dose of Bti treatment was sufficient to suppress the pupal and adult vector population for three months . Moreover , two cycles of Bti treatment over six months suppressed disease transmission in the treated districts . As this study was conducted over multiple years in multiple sites , we know that these data are reproducible . Our data confirm that Bti strain AM65-52 is an efficient ammunition , and when used with complete coverage of the target habitats it can prevent dengue outbreaks . | [
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"... | 2016 | Bacterial Larvicide, Bacillus thuringiensis israelensis Strain AM 65-52 Water Dispersible Granule Formulation Impacts Both Dengue Vector, Aedes aegypti (L.) Population Density and Disease Transmission in Cambodia |
Brassinosteroids ( BRs ) regulate rice plant architecture , including leaf bending , which affects grain yield . Although BR signaling has been investigated in Arabidopsis thaliana , the components negatively regulating this pathway are less well understood . Here , we demonstrate that Oryza sativa LEAF and TILLER ANGLE INCREASED CONTROLLER ( LIC ) acts as an antagonistic transcription factor of BRASSINAZOLE-RESISTANT 1 ( BZR1 ) to attenuate the BR signaling pathway . The gain-of-function mutant lic-1 and LIC–overexpressing lines showed erect leaves , similar to BZR1–depleted lines , which indicates the opposite roles of LIC and BZR1 in regulating leaf bending . Quantitative PCR revealed LIC transcription rapidly induced by BR treatment . Image analysis and immunoblotting showed that upon BR treatment LIC proteins translocate from the cytoplasm to the nucleus in a phosphorylation-dependent fashion . Phosphorylation assay in vitro revealed LIC phosphorylated by GSK3–like kinases . For negative feedback , LIC bound to the core element CTCGC in the BZR1 promoter on gel-shift and chromatin immunoprecipitation assay and repressed its transcription on transient transformation assay . LIC directly regulated target genes such as INCREASED LEAF INCLINATION 1 ( ILI1 ) to oppose the action of BZR1 . Repression of LIC in ILI1 transcription in protoplasts was partially rescued by BZR1 . Phenotypic analysis of the crossed lines depleted in both LIC and BZR1 suggested that BZR1 functionally depends on LIC . Molecular and physiology assays revealed that LIC plays a dominant role at high BR levels , whereas BZR1 is dominant at low levels . Thus , LIC regulates rice leaf bending as an antagonistic transcription factor of BZR1 . The phenotypes of lic-1 and LIC–overexpressing lines in erect leaves contribute to ideal plant architecture . Improving this phenotype may be a potential approach to molecular breeding for high yield in rice .
Brassinosteroids ( BRs ) are plant steroid hormones that have been used to increase the yield of crops [1] , [2] . BRs function in multiple developmental and physiological processes , including vascular differentiation , reproductive development , photomorphogenesis , and stress responses [3]–[5] . BR-deficient and -insensitive mutants show dwarfism , dark-green leaves , reduced fertility , and altered photomorphogenesis in the dark [6]–[9] . In rice ( Oryza sativa ) , leaf-angle response to BRs is a specific physiological process . For example , the erect leaves of BR-deficient rice allow for greater growth density and higher grain yield [10] . Thus , analysis of genes involved in rice BR signaling could shed light on the molecular mechanisms of BR-regulated growth in monocots and help identify feasible approaches to increase rice yield by genetic engineering . The BR signaling pathway has been well studied in Arabidopsis . Most of the signaling components of this pathway , from the BR receptor BRI1 and co-receptor BAK1 to nuclear transcription factors BZR1 and BES1/BZR2 , have been identified [11] , [12] . During the early events of BR signaling , BRI1 perceives BRs , thus inducing dissociation of the inhibitory protein BKI1 , which results in association with and transphosphorylation of the co-receptor BAK1 [13]–[17] . BR signal kinases ( BSKs ) mediate signal transduction from BRI1 to BSU1 phosphatase through association with and phosphorylation of BSU1 [18] . BSU1 positively regulates BR signaling by dephosphorylating the negative regulator BR-insensitive 2 ( BIN2 ) . This process facilitates accumulation of unphosphorylated BZR1 and BES1/BZR2 in the nucleus [19]–[23] , which directly or indirectly activate the expression of BR-responsive genes and regulate plant growth [21] , [24] , [25] . BZR1 is also responsible for the negative feedback of BR biosynthetic genes such as CPD by directly repressing transcription [26] . BZR1 and BES1 are major transcription factors in the BR signaling pathway [27] . BZR1 binds to the BR-responsive element ( BRRE , CGTGT/CG ) and mainly represses gene expression . BES1 binds to E-box by interacting with BIM1 or MYB30 to promote target gene expression [28]–[30] . BZR1 could also bind to E-box and BES1 to BRRE , so the functions of the family members may overlap [31] , [32] . These are key transcription factors activating the BR signaling pathway in plants . Phosphatase 2A ( PP2A ) dephosphorylates BZR1 and also BRI1 in mediating BR signaling . BRI1 degradation depends on PP2A–mediated dephosphorylation that is specified by methylation of the phosphatase , thus leading to the termination of BR signaling [33]–[35] . However , how BR signals are repressed at the transcriptional level to elicit a “turn-off” pathway is less well known . Rice , as a model monocot plant and one of the major crops , has been used to study the BR action mechanism . Both rice and Arabidopsis share primary BR biosynthesis and signal transduction pathways . A series of Arabidopsis orthologs of biosynthetic genes identified in rice include D2 , D11 , BRD1 , BRD2 , and CPD [36]–[38] . However , only a few members in the BR primary signaling pathway have been reported [39]–[41] . OsBRI1 and OsBAK1 are cell-surface receptor kinases that perceive BR signals . Os GLYCOGEN SYNTHASE KINASE 1 ( OsGSK1 ) , an ortholog of AtBIN2 , is a negative regulator of rice BR signaling . Although the direct targets of BZR1 and BES1 have been identified in Arabidopsis , only a few targets have been identified in rice [31] , [32] . The transcription factor OsBZR1 , the closest ortholog of both BZR1 and BES1 , has similar functions as its Arabidopsis orthologs [42] . OsBZR1 translocated from the cytoplasm to the nucleus in response to BR treatment in a process mediated by 14-3-3 proteins [42]–[44] . A pair of antagonizing HLH/bHLH factors , INCREASED LEAF INCLINATION ( ILI1 ) and ILI1 BINDING bHLH ( IBH1 ) , function downstream of OsBZR1 to regulate cell elongation and lamina joint bending [45] . These studies suggest a conserved BR signaling mechanism in rice and Arabidopsis . BZR1 is a key component of the transcription pathway that activates BR signaling in both species . However , how to halt BR signaling at the node of transcription factors including BZR1 remains unclear . A CCCH-type zinc finger protein , LEAF AND TILLER ANGLE INCREASED CONTROLLER ( LIC ) is involved in sterol homeostasis in rice [10] . Here , we studied the phenotypes of a rice LIC gain-of-function mutant lic-1 and LIC-overexpressing rice lines to explore a novel mechanism of BR signaling . Both groups showed erect leaves and reduced BR sensitivity as compared with antisense lines . LIC was further characterized as an antagonistic transcription factor of BZR1 in regulating rice architecture . Furthermore , LIC is phosphorylated by GSK1/BIN2 ( GSK3-like kinases ) , which affect translocation from the nucleus to cytoplasm . LIC may mediate a novel mechanism that represses the BR signaling pathway .
A T-DNA insertion line of lic-1 was obtained from the Rice Mutant Database ( http://rmd . ncpgr . cn ) [46] . Molecular analysis revealed that the T-DNA was inserted in the eighth exon near the 3′ terminus of LIC and was predicted to cause the deletion of 110 amino acids ( Figure 1A and Figure S1A ) . The inserted gene encodes a truncated LIC protein containing the CCCH DNA binding domain , EELR activation domain and the putative phosphorylation sites ( Figure S1B and S1C ) . Segregation analysis of the heterozygous lic-1 with molecular evidence revealed an approximate 3∶1 ( 76/24 ) ratio of lic-1 mutants to the wild-type , which indicates that lic-1 is a dominant mutant . The crossed progenies of lic-1 and the LIC antisense lines showed increased leaf angles that were similar to those of the antisense lines ( Table S1 ) . As compared with wild type , the lic-1 line showed reduced leaf angles from tillering stage ( Figure S2A and S2B ) . All LIC-overexpressing lines also showed erect leaves at tillering ( Figure S2A , Figure S3 , and Table S2 ) . During the seedling stage , the wild type and lic-1 , as well as overexpression lines , did not differ in leaf angle . Therefore , the phenotype of the lic-1 mutant was consistent with the LIC-overexpression lines in terms of leaf angle . BR biosynthetic genes D2 and D11 had repressed expression in antisense lines . In contrast , the expression of the biosynthetic gene BRD1 , as well as the receptor gene BRI1 , was enhanced in lic-1 ( Figure S4 ) . In rice , the physiologic processes of leaf bending and root growth are sensitive to BR [47] , [48] . In the wild type ( WT ) , increased leaf angle depended on the concentration of BR ( Figure 1B and 1C ) . The overexpressing lines and lic-1 showed reduced dependence on BR concentration in leaf bending . In contrast , the antisense line was more sensitive to BR dosage than the WT . In the antisense lines , the root growth patterns in response to 24-eBL ( an active form of BR ) were similar to leaf angle patterns ( Figure S5A and S5B ) . Thus , LIC overexpression reduced the BR response , and LIC depletion caused hypersensitivity to BR in terms of leaf bending and root growth . Therefore , LIC may negatively regulate BR signaling in rice . Bioinformatics analysis revealed the BZR1 binding site BRRE ( CGTGT/CG ) [26] present in the promoter of LIC ( Figure 2A ) . EMSA was used to examine BZR1 binding to the cis-elements in the LIC promoter in vitro . When the purified BZR1 protein was incubated with the reaction mixture , a shifted band appeared in the upper part of the gel but not in the control MBP . The greater the amount of BZR1 in the incubation , the greater the amount of shifted band on the gel . When the competitive unlabeled probe ( Co ) was added to the system , the shifted band was suppressed . In contrast , neither mutated P1 ( MP1 , CGAAAA ) nor P2 ( CGTGTG ) shifted under the same conditions ( Figure 2B ) . We performed chromatin immunoprecipitation ( ChIP ) assay with WT rice ( Figure S9 ) . Real-time PCR revealed a fragment of the LIC promoter containing the P1 binding element significantly enriched as compared with the reference gene promoter ( UBQ5 ) and control fragments ( P2 , P3 and P4; Figure 2C ) . In the RNAi lines of BZR1 , LIC transcription was increased ( Figure 2D ) . Thus , BZR1 binds to the cis-element in the LIC promoter , and knockdown of BZR1 leads to upregulation of LIC . We crossed the BZR1 RNAi lines with erect leaves to the LIC antisense lines with increased leaf bending to explore the genetic relationship of the lines . By molecular identification ( Figure S6A and S6B ) , phenotypic analysis revealed an increased leaf bending phenotype in the progenies , which was similar to that of the LIC antisense lines ( Figure 2E and 2F ) . Therefore , BZR1 may functionally depend on LIC in terms of genetics . Transformed LIC-GFP fusion protein was used to investigate subcellular localization . With BR treatment , GFP-tagged LIC was rapidly weakened in the cytoplasm within 30 min but was enhanced in the nucleus ( Figure 3A a and b ) . The ratio of GFP-tagged LIC in the nucleus to that in the cytoplasm ( N/C ratio ) was significantly increased with BR treatment . Although LICm , mimicking the C-terminus-truncated protein , was distributed in the nucleus and cytoplasm , the cytoplasmic signal of LICm was clearly weaker than that of intact LIC ( Figure 3A c and d; 3C ) . Digital signal assay demonstrated a lower ratio of LIC than truncated LICm in the nucleus . The LICm pattern showed a similar increased N/C ratio in response to BR treatment . In contrast , the truncated protein LICp , lacking the putative phosphorylation sites ( designated P site in Figure 1A and Figure 3C ) was localized only in the nucleus ( Figure 3B ) . Western blot analysis revealed a greater LIC band in the nucleus of lic-1 as compared with the WT . Intensity of the nuclear band was enhanced by treatment with 24-eBL ( 1 µM ) for both lic-1 and the WT . At the total protein level , the signal intensity of LIC in the WT and lic-1 was not significantly different after treatment ( Figure 3D ) . Thus , the translocation of LIC from the cytoplasm to the nucleus may be regulated by BR treatment and depend on the phosphorylation status of LIC . The GSK3-like kinase BIN2 phosphorylates BZR1 through the conserved GSK3 kinase phosphorylation sites ( S/TxxxS/T ) and promotes its cytoplasmic retention in Arabidopsis [49] . In rice , whole-genome screening analysis revealed two putative orthologs of BIN2 , OsGSK1 and OsSKETHA [43] . Yeast two-hybrid assay revealed that LIC but not forms LICm and LICp interact with GSK1 and SKETHA , as well as AtBIN2 . The mutated form did not interact with them ( Figure S7 ) . Western blot analysis revealed that LIC was two bands and the larger one was enhanced by incubation with BIN2 . Furthermore , the intensity of the larger band was reduced by the addition of λ-phosphatase 1 ( Figure 4A ) , which agreed with the prediction that 5 typical phosphorylation sites of GSK3-like kinases ( S/TxxxS/T ) were deposited in the P site domain of LIC protein . When plants were treated with 24-eBL ( 1 µM ) , the phosphorylated form of LIC was suppressed ( Figure 4B ) , whereas the dephosphorylated form was increased . The P-LICm was weaker than that for LICm ( Figure 4C ) . Western blot revealed dephosphorylated LIC accumulated in the nuclear fraction , with the phosphorylated form mainly in the cytoplasm ( Figure 4D ) . Transformed cells with GFP-tagged LIC showed the N/C ratio of LIC with digital fluorescence signals was 3 . 0 ( Figure 5A and 5B ) . The nuclear distribution of LIC was enhanced with 24-eBL treatment . In contrast , in cells co-transformed with both GSK1 and LIC , less of LIC localized in the nucleus . Western blot analysis demonstrated that phosphorylated LIC was upregulated after incubation with GSK1 but downregulated with λ-phosphatase 1 ( Figure 5C ) . This result suggested that GSK1 phosphorylated LIC , which might repress its localization in the nucleus . Expression pattern assay demonstrated BZR1 and LIC with overlapping and distinct expression patterns in different organs ( Figure S10 ) . The expression of LIC was distributed from the abaxial to adaxial sides in leaves . In contrast , the expression of BZR1 was dominant in the abaxial sides of leaves ( Figure 6A ) . The effect of BR on LIC transcription showed repression at low ( 1 nM ) and activation at high ( >100 nM; Figure 6B ) 24-eBL concentrations . This matches the phenotype of root growth ( Figure S5C ) . The peak of LIC transcription occurred with 1 µM 24-eBL . In contrast , the mRNA level of BZR1 was increased with low levels of 24-eBL ( 1 nM ) and decreased with high levels ( >100 nM; Figure 6B ) . Western blot analysis demonstrated that the pattern of LIC protein level was similar to the mRNA pattern with low levels of 24-eBL . However , the reduced negative-peak occurred with a higher 24-eBL concentration ( 100 nM ) than for the RNA ( 1 nM ) . Additionally , the increased protein expression was sustained with up to 10 µM 24-eBL ( Figure 6C ) . Thus , LIC expression may be downregulated by a low level of BR but upregulated by higher concentrations . In the mutant d2 , BR deficiency caused LIC expression reduced to only 20% the WT level . BZR1 depletion resulted in increased LIC expression ( Figure 6D ) . Time-course assay revealed LIC expression gradually increased from 15 min up to 3 h during BR treatment ( 1 µM 24-eBL ) ( Figure 6E ) . In the LIC antisense lines , BZR1 expression was enhanced with the treatment , which was opposite to that in the WT . Transcription expression of the BZR1 target gene CPD was greatly repressed by BR treatment in the antisense lines ( Figure 6F ) . Thus , LIC may be involved in the negative regulation of BZR1 . To screen LIC potential target motifs , genes with altered expression of the LIC antisense lines in microarray data were re-sorted . In previous microarray analysis [10] , the expression of 1 , 175 genes was altered by at least 2-fold in the LIC antisense lines . We extracted 1 kb of upstream sequences of the genes with altered expression patterns as the predicted promoters and then used MEME ( http://meme . sdsc . edu/meme/cgi-bin/meme . cgi ) to locate the recurrent motifs . We extracted 14 motifs representing the potential regulatory cis-elements from the altered genes ( Figure S8A ) . EMSA results suggested that 3 elements ( S1–3 ) containing a core sequence TCGC bound to LIC ( Figure S8B ) . Therefore , the core sequence TCGC is one of the LIC-binding elements . The core sequence was deposited in BZR1 gene . ChIP data revealed that LIC bound to the BZR1 promoter in various regions , such as , a , c , e , f , g and j , which were upregulated by BR treatment in the WT ( Figure 7A and Figure S9 ) . In contrast , the remaining fragments , which lacked TCGC , such as , b , d , h , I and k , had lower binding affinities . In the lic-1 mutant , the bound patterns were similar to that of the BR-treated WT . We used MEME to locate the recurrent motifs among the multiple region sequences identified on ChIP . The motif CTCGC ( denoted as S , containing the TCGC core sequence ) was consistently found with high values ( Figure 7B and Figure S8C ) . EMSA demonstrated that LIC bound specifically to CTCGC ( Figure S8D ) . Mutated probes M1 ( ATCGCG ) and M2 ( CTCGCT ) led to decreased intensity of the shifted band . In contrast , mutated M3 ( CAAAAG ) caused the band to disappear . The fragments with multiple copies of the element on the BZR1 promoter were used to further confirm the binding activity . EMSA results suggested that binding affinities of LIC were related to copy numbers of the elements in the BZR1 promoter ( Figure 7C ) . Transient transfection assay revealed that LIC protein repressed the expression of BZR1pro:LUC in Arabidopsis protoplasts as compared with the control ( vector; Figure 7D ) [26] . Therefore , LIC may be a primary transcription factor targeting OsBZR1 to regulate the BR signaling pathway . To determine the potential antagonistic functions of both genes , we analyzed the expression patterns of their potential downstream genes . BZR1 mainly binds IBH1 to affect the balance of a pair of antagonistic HLH/bHLH transcription factors ILI1 and IBH1 in rice [45] . In an LIC-depleted line ( AS2 ) , ILI1 expression was higher than in the WT , whereas IBH1 transcription was not significantly altered ( Figure 8A ) . The core motif sequence CTCGC of LIC target was present as a glomerate pattern in ILI1 but as a sparse pattern in IBH1 . EMSA data indicated that the fragment containing the sequence B2 in ILI1 strongly bound to LIC . In contrast , the signal of C3 in IBH1 with a single core element was weaker ( Figure 8B ) . ChIP analysis of the potential target ILI1 after BR treatment in the WT demonstrated significant changes ( >2 . 5-fold ) in binding in diverse regions such as a , d , e , f and n , but not in regions such as b , c , h , i , j and l ( Figure 8C ) . IBH1 exhibited a similar pattern as ILI1 on ChIP analysis , but the copy number of the core element on the IBH1 fragments , such as c and k , was much lower than that for ILI1 ( Figure 8D ) . Unexpectedly , the change appeared in the region without the core motif such as j , so other unknown motifs may be involved . To further explore the potential activity of the transcription factor with its targets ILI1 and IBH1 , we used a protoplast transfection assay . LIC repressed the expression of ILI1pro:LUC but activated that of IBH1pro:LUC ( Figure 8E ) . Competitive binding assay showed that the repression activity of LIC on ILI1 was weakened by co-expression of BZR1 ( Figure 8F ) . Thus , LIC dominantly repressed ILI1 expression and weakly bound to IBH1 to enhance expression to balance the regulation activity of BZR1 .
Leaf bending is a specific phenotypic response to BR in rice [47] . LIC-depleted rice lines show increased leaf bending , which mimic the phenotypes of enhanced responses to BR , such as the OsBAK1-overexpressing lines [40] . Consistently , the gain-of-function mutant lic-1 and overexpressing lines show erect leaves similar to OsBZR1- and OsBAK1-depleted lines . Reverse expression patterns of downstream genes such as ILI1 and IBHI1 were found in the silenced lines of BZR1 and LIC . Similarly like BZR1 , LIC acts in an early BR response , because its expression was induced by BR treatment within 15 min . BR-induced LIC accumulation in the nucleus was a rapid response to BR and acts as an upstream component in BR signaling . Therefore , LIC functions negatively in the BR-mediated regulation of leaf bending . LIC , with phosphorylation sites of GSK1/BIN2 kinases , interacts with BIN2 and its rice orthologs GSK1 and SKETHA and could be phosphorylated . The C-terminus-truncated LICm , as well as P-site-truncated LICp , show decreased interaction with GSK1/BIN2 and consequently display lower phosphorylation levels and greater accumulation in the nucleus to constitutively regulate downstream genes . The shuttle of LIC between the nucleus and cytoplasm was regulated by BR treatment and might depend on the phosphorylation status of LIC . This shuttle localization pattern depended on BIN2/GSK1 , as seen with BZR1/BES1 [23] , [42] , [50] . Our results suggest that LIC directly mediates BR signaling from GSK3 kinases . BZR1 is a key component positively regulating BR signaling , whereas LIC plays a negative role in the signaling pathway . With BR treatment , LIC and BZR1 accumulate in the nucleus and regulate downstream genes . BZR1 represses BR-downregulated genes through the downregulation of BIN2 phosphorylation and decreases cytoplasmic retention mediated by 14-3-3 proteins during BR-mediated induction [42] , [50] . BZR1 and BES1 gain-of-function mutants in Arabidopsis are hypersensitive to BR . Depleted AtBES1 leads to reduced BR sensitivity [51] . The RNAi lines of OsBZR1 are insensitive to BR and show erect leaves [42] . Our genetic analysis and molecular data suggested that LIC and BZR1 work on rice leaf bending in a genetic pathway , but their roles are opposite to each other . EMSA and ChIP data , as well as transcription assay data , indicated that the BZR1 protein directly represses LIC expression via the specific BRRE motif ( CGTGCG ) . We found that LIC protein recognizes the BZR1 gene through the core element ( CTCGC ) to repress its transcription . The expression of both genes may be induced by BR treatment at various concentrations . BR treatment at low concentrations ( 10−9 M ) induced the expression of BZR1 and promoted the dephosphorylation of BZR1 protein as an activation mechanism . However , BR treatment at high concentrations ( up to 10−7 M ) induced LIC expression . The repression of BZR1 transcriptional expression by LIC is enhanced by high BR levels . Therefore , LIC and BZR1 antagonize each other in controlling BR-mediated leaf bending . LIC , with only one CCCH domain , binds DNA or RNA in vitro ( Figure S11 ) [10] . It prefers to recognize the core sequence CTCGC , which is present in genes such as BZR1 and ILI1 , to regulate BR signaling in rice . Our finding of the motif binding to LIC with specificity will provide new insights into this family . BZR1 is a transcription factor that represses the expression of downstream genes such as OsIBH1 , which is responsible for leaf bending . ILI1/PRE1 and IBH1 promote or repress cell elongation downstream of BZR1 in rice and Arabidopsis [45] , [52] . Overexpression of ILI1 causes increased leaf bending , whereas overexpression of IBH1 results in erect leaves in rice . EMSA and ChIP results suggested that LIC greatly represses ILI1 , the positive partner of OsIBH1 . As well , LIC weakly binds to OsIBH1 promoter to enhance its transcriptional expression . This pattern is similar to BZR1 weakly binding to the promoter of ILI1 , which is induced by BR [45] . In regulating downstream genes , LIC may play a major role in repressing positive regulators such as ILI1 , and BZR1 may function to repress negative regulators such as IBH1 . Therefore , a novel negative regulation module of BR signaling is parallel to and antagonizes the BZR1 signaling pathway to regulate leaf bending . In plant development , LIC and BZR1 show various spatial and temporal expression patterns . BZR1 acts in the presence of low levels of BR , whereas LIC is predominantly activated by high levels of BR and antagonizes BZR1 to prevent intense activation of the BR cascade . The novel negative regulation module of LIC and the positive one of BZR1 in mediating leaf bending may help in designing ideal plant architecture for improving photosynthesis efficiency during rice development . The approach may have potential in rice molecular breeding for high yield .
Rice ( Oryza sativa ssp . japonica var . Zhonghua 10 ) plants were grown in the field or in the greenhouse at 30°C/25°C ( day/night ) cycles . For the analysis of BR induction in leaf bending and root growth , rice seeds were sterilized with 1% NaClO and grown in half-strength Murashige and Skoog ( MS ) medium with the indicated concentrations of 24-eBL ( Sigma-Aldrich , St . Louis , MO , USA ) at 30°C under continuous light . Seedlings were examined 7 days after germination . For every transgenic rice plant , 3 lines were used . Sterilized seeds were grown for 8 days in a dark chamber . Uniform seedlings were then sampled by excising segments of approximately 2 cm that contained the second-leaf lamina joint under dim light conditions . These were floated on distilled water containing various concentrations of 24-eBL . After incubation in a dark chamber at 30°C for 72 h , the angle between the lamina and the sheath was measured [47] . Total RNA was extracted from 2-week-old seedlings by using the Trizol RNA extraction kit ( Invitrogen , Carlsbad , CA , USA ) . The first-strand cDNAs were synthesized by use of M_MLV reverse transcriptase ( Promega ) and used as RT-PCR templates . Quantitative real-time PCR analysis involved an Mx3000P ( Stratagene ) with a SYBR green detection protocol . RT-PCR was repeated at least 3 times for each harvested samples with gene-specific primers and ACTIN1 as the reference gene ( see Table S1 ) . The data were analyzed by the CT formula considering amplification efficiencies for every PCR [53] . The cDNA of LIC from a rice cDNA library was amplified by PCR and ligated into pUN1301 binary vectors for overexpression . Full-length cDNAs of LIC , GSK1 , SKETHA , and AtBIN2 without the stop codon were amplified by PCR from rice or Arabidopsis and cloned into pGADT7 or pGBDT7 vectors . All binary vector constructs were transformed into Agrobacterium tumefaciens strain GV3101 or EHA105 , then transformed into rice calli by A . tumefaciens-mediated transfection [54] , [55] . Primers are in Table S4 . For tobacco transformation , full-length cDNAs of LIC and GSK1 were ligated into pBI121 and pRT105-3×flag vectors [56] , respectively . The binary vector constructs were transformed into A . tumefaciens strain GV3101 and then transformed into tobacco by A . tumefaciens-mediated transfection . Full-length LIC sequence was inserted into the pBI221 vector to generate pBI221-LIC . To generate the BZR1pro:LUC reporter gene , the BZR1 promoter was amplified with the rice genomic DNA used as a template and then inserted into the pGEM-T Easy vector to produce pGEM-BZR1p . The BZR1 promoter was released from pGEM-BZR1p by digestion with HindIII and BamHI and inserted into the corresponding sites of the YY96 vector [57] to produce BZR1pro:LUC . The ILI1pro:LUC and IBH1pro:LUC reporter genes were constructed as for BZR1pro:LUC . Isolation of Arabidopsis protoplasts and PEG-mediated transfection were as described [58] . The reporter constructs BZR1pro:LUC , ILI1pro:LUC and IBH1pro:LUC; effector plasmid; and 35S:GUS construct ( internal control ) were co-transformed into protoplasts . After transformation , the protoplasts were incubated at 23°C for 12–15 h , then pelleted and resuspended in 100 µL of 1× CCLR buffer ( Promega ) . For the ß-glucuronidase enzymatic assay , 5 µL extract was incubated with 50 µL 4-methylumbelliferyl ß-d-glucuronide assay buffer ( 50 mM sodium phosphate , pH 7 . 0 , 1 mM ß-d-glucuronide , 10 mM EDTA , 10 mM ß-mercaptoethanol , 0 . 1% sarkosyl , 0 . 1% Triton X-100 ) at 37°C for 15 min , and the reaction was stopped by adding 945 µL of 0 . 2 M Na2CO3 . For luciferase activity assay , 5 µL extract was mixed with 50 µL luciferase assay substrate ( Promega ) , and activity was detected with use of a Modulus Luminometer/Fluometer with a luminescence kit ( Promega ) . The reporter gene expression was expressed as relative ratio of LUC to ß-glucuronidase . The cDNA of LIC was cloned into the pGADT7 vector . The cDNAs of GSK1 , SKETHA , and AtBIN2 were cloned into pGBDT7 ( Stratagene ) and then transformed into yeast strain AH109 . Transformants were screened for growth on medium lacking Leu , Trp , and His . Recovered clones were then assayed for LacZ activity by a filter lift assay . For the transactivation activity assay , LIC , AtBIN2 , GSK1 , and SKETHA were cloned into the pGBDT7 vector and co-transformed with pGADT7 into yeast cells . Yeast that could grow on SD/-Leu/-Trp/-His medium with ß-galactosidase activity exhibited transactivation activity . Western blot analysis involved extracts prepared from yeast cells as described [14] . The yeast cells were collected , ground to a fine powder in liquid nitrogen , and further ground in cold grinding buffer ( 50 mM HEPES ( pH 7 . 4 ) , 10 mM EDTA , 0 . 1% Triton X-100 , 1 mM PMSF ) . After the addition of an equal volume of 2× sample buffer , the samples were boiled for 10 min , separated by 15% SDS–PAGE , and transferred to a polyvinylidene fluoride membrane . The blots were incubated with the antibodies mouse anti-Myc ( Neo-Marker , UK ) or mouse anti-HA ( Santa Cruz , Germany ) , then goat anti-mouse IgG HRP-conjugated secondary antibody ( Santa Cruz , Germany ) . Chromatin immunprecitipation ( ChIP ) was performed as described [26] with 3-week-old seedlings . The antibody polyclonal anti-BZR1 or anti-LIC was used for immunoprecipitation . Untagged purified LIC protein was used to inject rabbit , and polyclonal serum was affinity-purified with its target antigen . ChIP products were analyzed by quantitative real-time PCR , and enrichment was calculated as the ratio of transgenic to wild-type sample or BR-treated and control seedlings . Data are mean±SD from 3 biological replicates . The primers for UBQ5 ( LOC_Os04g57220 ) promoter were 5′-TATCCAACATGAATGCCACA-3′ and 5′-CAGCACGAGATGAGTAAAACAA-3′ . Sequences used in bioinformatics analysis are in Table S3 . EMSA was performed essentially as described [59] . Briefly , the OsBZR1 coding region was cloned into a maltose-binding protein ( MBP ) fusion vector ( pETMALc-H vector , Pryor and Leiting , 1997 ) with the primers for OsBES1NAsp718 , 5′-CTCGGTACCGGAGCTGGTGGGTATGACGTC-3′ , and OsBES1CHind3 , 5′-CGCAAGCTTTCATTTCGCGCCGACGCCGAGC-3′ . The recombinant MBP–OsBZR1 was purified from Escherichia coli with amylose resin ( NEB , http://www . neb . com ) according to the manufacturer's instructions [60] . The coding sequence of LIC was cloned into the expression vector pGEX-4T-1 [10] . The construct was transformed into E . coli BL21 ( DE3 ) . Cells were grown at 30°C and induced by the addition of isopropyl β-d-thiogalactopyranoside at a final concentration of 1 mM when the optical density ( OD ) 600 of the cultured cells was 0 . 5–0 . 9 . The fusion protein was purified with Glutathione Sepharose 4B ( GE Healthcare ) . The nucleotide sequences of the double-stranded oligonucleotides for EMSA were for LIC P1 ( 5′-CGA CGT CGT GCG GCC GCG-3′ and 5′-CGC GGC CGC ACG ACG TCG-3′ ) and LIC P2 ( 5′-CGG GCG CGT GTG TGG CGG-3′ and 5′-CCG CCA CAC ACG CGC CCG-3′ ) . The oligonucleotides were annealed and then labeled with the Biotin 3′ End DNA Labeling Kit ( Pierce ) . Standard reaction mixtures ( 20 µL ) for EMSA contained 2 µg purified proteins , 2 µL biotin-labeled annealed oligonucleotides , 2 µL 10×binding buffer ( 100 mM Tris , 500 mM KCl , 10 mM DTT , pH 7 . 5 ) , 1 µL 50% glycerol , 1 µL 1% NP-40 , 1 µL 1 M KCl , 1 µL 100 mM MgCl2 , 1 µL 200 mM EDTA , 1 µL 1 µg/µL poly ( dI-dC ) and 8 µL ultrapure water . The reactions were incubated at room temperature ( 25°C ) for 20 min and loaded onto a 10% native polyacrylamide gel containing 45 mM Tris , 45 mM boric acid , 1 mM EDTA , pH 8 . 3 . The gel was sandwiched and transferred to an N+ nylon membrane ( Millipore ) in 0 . 5× TBE buffer at 380 mA in a 4°C refrigerator for 60 min . The detection of biotin-labeled DNA by chemiluminescence followed the manual of the LightShift Chemiluminescent EMSA Kit ( PIERCE ) . GFP fluorescence was visualized under a confocal microscope ( Zeiss LSM510 META , Germany ) equipped with an argon laser ( 488 nm ) . GFP was excited by an Argon laser at 488 nm , and images were acquired using a 512b Roper Cascade EMCCD camera and MetaMorph software ( Molecular Devices , Sunnyvale , CA ) . Images of LIC- , LICm- , and LICp-GFP were obtained with identical image acquisition settings . A series of images at different points along the z-axis were collected from the top to the bottom . Projection of the z-series of images results in a 3D view of the cell . To quantify the effect of 24-eBL on OsLIC-GFP localization in the time-course experiment , images were obtained with a 500-ms exposure time . Quantification of the fluorescent protein signal involved use of ImageJ ( http://rsb . info . nih . gov/ij ) . To measure the ratio of nuclear to cytoplasmic signals ( N/C ratio ) for LIC-GFP for each cell , small areas were drawn , and measurements of integrated densities were taken from representative areas within the nucleus , cytoplasm , and background ( central vacuole ) of each cell . Each sample of at least 20 cells was measured 3 times; the average N/C ratio were then calculated [42] . Total protein samples were extracted from 2-week-old rice seedlings with 2× SDS loading buffer; cytoplasmic and nuclear fractions were extracted as described [20] , [61] . Tissues were lysed with use of a buffer ( 20 mM Tris-HCl , pH 7 . 0 , 250 mM sucrose , 25% glycerol , 20 mM KCl , 2 mM EDTA , 2 . 5 mM MgCl2 , 30 mM ß-mercaptoethanol , 13-protease inhibitor cocktail , and 0 . 7% Triton X-100 ) and fractionated by centrifugation at 3000×g . The supernatant was taken as the cytosolic fraction . The pellet was further washed with a resuspension buffer ( 20 mM Tris-HCl , pH 7 . 0 , 25% glycerol , 2 . 5 mM MgCl2 , and 30 mM ß-mercaptoethanol ) and reconstituted as the nuclear fraction . All proteins were separated on SDS-PAGE gels , transferred to a nitrocellulose membrane , and probed with anti-LIC antibody . For the in vitro kinase assay , purified LIC-GST protein was incubated with BIN2/GSK1 protein or λ-phosphorylase 1 at 30°C for 30 min and loaded onto SDS-PAGE gels . SDS-PAGE gels of 8% or 15% were used to analyze the total LIC level or its phosphorylation , respectively . The proteins were transferred to a nitrocellulose membrane until the 35-kDa protein marker ran out of the gel during electrophoresis . Tissues were fixed in 4% ( w/v ) paraformaldehyde and 0 . 25% glutaraldehyde in 0 . 1 M sodium phosphate buffer; samples were vacuum-infiltrated for 30 min and then stored overnight at 4°C . The dehydrated samples after a graded ethanol series were embedded in Paraplast Plus ( Oxford Labware , St . Louis , MO ) . A fragment of 232 bp was amplified from the second exon of LIC with the primers 5′-GGATCCGCAAGTACGGAGCGCAGTG-3′ and 5′-AAGCTTTTCG CAGGACCAGGAGCA-3′ , subcloned into the pGEM-T-easy vector ( Promega ) , and used as a template for RNA probe synthesis . A fragment of BZR1 was amplified with the primers 5′-ATCAGGAAGCCGGACTGGG-3′ and 5′-GGTTGACGAGGTTGTAGGTGGG-3′ . Hybridization in situ with digoxigenin-labeled sense or antisense RNA of LIC and BZR1 was conducted as described [62] . MEME software ( http://meme . sdsc . edu/meme/cgi-bin/meme . cgi ) was used to find recurrent motifs among multiple sequences in Affymetrix microarray data for the LIC antisense lines that were up- or downregulated by at least 2-fold [10] . We extracted 1-kb genomic sequences upstream of 1 , 175 genes to screen the potential motifs . Differentially expressed genes were divided into those up- or downregulated . Randomly generated sequences of the same length were used as controls to remove false-positive results . Sequence data from this article can be found in the GenBank or EMBL database under the following accession numbers: LIC , Os06g49080; GSK1 , Os01g10840; SKETHA , Os06g35530; ILI1 , Os04g54900; Os IBH1 , Os04g0660100 . | Brassinosteroids ( BRs ) are phytohormones mediating multiple biological processes , such as development and stress response . They have been used in crops to produce high yield . In rice , the ideal plant architecture for high yield includes effective tillers , as well as height and leaf angle , which is modulated by BRs . Activation of BRI1–mediated BR signaling is well understood , but much less is known about its inactivating mechanism . Here , we found a gain-of-function mutant lic-1 with the phenotype of the ideal rice plant architecture . The C3H-type transcription factor LIC antagonizes BZR1 to repress BR signaling in rice . We used BR to induce the negative regulator LIC and found that it functioned at high BR level , which may restrain plant development . LIC was phosphorylated by GSK3–like kinases . Phosphorylated LIC mainly localized in cytoplasm , whereas dephosphorylated LIC was in nucleus , which was regulated by BR treatment . LIC regulated transcription patterns of the downstream genes in an opposite direction to BZR1 . BZR1 activated BR signaling , but the brake module of LIC repressed BR cascade amplification . LIC and BZR1 may balance BR signaling to control growth and development in rice . | [
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... | 2012 | Dynamics of Brassinosteroid Response Modulated by Negative Regulator LIC in Rice |
The highly conserved bone morphogenetic protein ( BMP ) signaling pathway regulates many developmental and homeostatic processes . While the core components of the BMP pathway have been well studied , much research is needed for understanding the mechanisms involved in the precise spatiotemporal control of BMP signaling in vivo . Here , we provide evidence that two paralogous and evolutionarily conserved tetraspanins , TSP-12 and TSP-14 , function redundantly to promote BMP signaling in C . elegans . We further show that the ADAM10 ( a disintegrin and metalloprotease 10 ) ortholog SUP-17 also functions to promote BMP signaling , and that TSP-12 can bind to and promote the cell surface localization of SUP-17 . SUP-17/ADAM10 is known to be involved in the ligand-induced proteolytic processing of the Notch receptor . We have evidence that the function of SUP-17 , and of TSP-12/TSP-14 in BMP signaling is independent of their roles in Notch signaling . Furthermore , presenilins , core components of the γ-secretase complex involved in processing Notch , do not appear to play a role in BMP signaling . These studies established a new role of the TSP-12/TSP-14/SUP-17 axis in regulating BMP signaling , in addition to their known function in the Notch signaling pathway . We also provide genetic evidence showing that a known BMP signaling modulator , UNC-40/neogenin/DCC , is one of the substrates of SUP-17/ADAM10 in the BMP signaling pathway .
The highly conserved bone morphogenetic protein ( BMP ) pathway is repeatedly used in metazoan development to regulate multiple distinct processes in different cellular contexts . In canonical BMP signaling , secreted BMP ligands bind to the heteromeric type I/type II receptor complexes and induce the phosphorylation of type I receptors by type II receptors , which in turn phosphorylate the R-Smads . The phosphorylated R-Smads then complex with Co-Smad and enter the nucleus to regulate gene transcription . Increasing evidence has shown that BMP signaling is tightly regulated spatiotemporally and that misregulation of this pathway can cause many different disorders in humans , such as cardiovascular diseases and cancers [1–5] . Recent studies have identified a number of factors that modulate BMP signaling at the level of the ligand-receptor complex [6–10] . In particular , we have previously shown that the small transmembrane tetraspanin proteins are important in promoting BMP signaling in C . elegans [7] . Tetraspanins are a large group of integral membrane proteins with a characteristic protein topology . They contain four transmembrane domains and a number of conserved residues , such as the featured CCG motif in the second extracellular loop [11] . Recent studies have shown that a subgroup of tetraspanins , the TspanC8 family of tetraspanins , functions to promote another highly conserved signaling pathway , the Notch pathway [12–15] . In particular , the TspanC8 tetraspanins physically associate with ADAM10 and promotes its cell surface localization . ADAM10 , in turn , cleaves the Notch receptor in a process called “ectodomain shedding” , releasing the intracellular domain of Notch for subsequent activation of downstream gene expression [12–14] . Previously , we have shown that in C . elegans , the TspanC8 tetraspanins TSP-12 and TSP-14 function in modulating BMP signaling [7] . However , whether they exert their functions in BMP regulation by acting through the C . elegans ADAM10 protein is not known . Similarly , because of their roles in both BMP signaling and Notch signaling , it is not clear whether TSP-12 and TSP-14 regulate these two pathways independently . In this study , we investigated how TSP-12 and TSP-14 regulate BMP signaling in C . elegans . The C . elegans BMP-like pathway is called the Sma/Mab pathway . This pathway is known to regulate body size , male tail patterning and mesoderm development [16] . Core members of this pathway include the ligand DBL-1/BMP , the type I and type II receptors , SMA-6/RI and DAF-4/RII , the R-Smads SMA-2 and SMA-3 , as well as the Co-Smad SMA-4 [16] . Loss-of-function mutations in any of these core members will result in a small body size , male tail sensory ray formation defects [17] , reduced expression of the RAD-SMAD reporter [18] , and suppression of the sma-9 ( cc604 ) M lineage defect [19] . In particular , mutations in the C . elegans schnurri homolog sma-9 cause a dorsal-to-ventral cell fate transformation in the postembryonic M lineage , thus the loss of two M-derived coelomocytes ( CCs ) . Loss-of-function mutations in any core members of the Sma/Mab pathway can suppress this M lineage defect ( Fig 1A and 1B ) , resulting in the reappearance of the two M-derived CCs . Thus , Sma/Mab pathway mutants exhibit a Susm ( suppression of the sma-9 M lineage ) phenotype ( [19]; Fig 1A and 1E ) . We have previously shown that the Susm phenotype can be used as a specific and sensitive assay to screen efficiently for mutants with defects in Sma/Mab signaling [7] . Using the Susm assay , we have identified several highly conserved factors that function to modulate BMP signaling at the level of the ligand-receptor complex . These include the RGM protein DRAG-1 [20] , the neogenin/DCC homolog UNC-40 [18] , the tetraspanin TSP-21 [7] , as well as two redundant tetraspanins , TSP-12 and TSP-14 . Here , we show that TSP-12 and TSP-14 function redundantly in BMP signaling by regulating the cell surface localization of the ADAM10 ( a disintegrin and metalloprotease 10 ) ortholog SUP-17 . We demonstrate that the function of SUP-17 in BMP signaling is independent of its well-established role in Notch signaling . Finally , we provide genetic evidence indicating that a known BMP modulator , UNC-40/neogenin/DCC , is one of the substrates of SUP-17/ADAM10 in the BMP signaling pathway .
The C . elegans genome encodes 21 tetraspanins . We have previously reported a weak Susm ( suppression of the sma-9 M lineage ) phenotype in the tsp-12 ( ok239 ) null mutants , which is enhanced by tsp-14 ( RNAi ) , but not by RNAi of any of the remaining 19 tsp genes in C . elegans [7] . To further determine mechanistically how TSP-12 and TSP-14 function in Sma/Mab signaling , we generated three deletion alleles of tsp-14 ( jj95 , jj96 and jj97 ) using the CRISPR/Cas9 system ( Fig 1C; see Materials and Methods ) . We used both jj95 and jj96 in all our analyses described below , and found them to behave identically . We therefore refer to both alleles as tsp-14 ( 0 ) . Similarly , we refer to the deletion allele tsp-12 ( ok239 ) as tsp-12 ( 0 ) . tsp-12 ( 0 ) and tsp-14 ( 0 ) single mutants are each fully viable and fertile . tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants produced by tsp-12 ( 0 ) /+; tsp-14 ( 0 ) mothers are also viable , but exhibit vulva morphogenesis defect ( Fig 1N’ ) and are egg-laying defective ( Egl ) . They also exhibit multiple Sma/Mab signaling defects: unlike the tsp-12 ( 0 ) or tsp-14 ( 0 ) single mutants , these tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants have a smaller body size ( Fig 1F and 1J ) , exhibit reduced RAD-SMAD reporter activity ( Fig 1K ) and double mutant males have severe tail defects ( Fig 1L’ and 1M’ ) , including crumpled spicules , fused and shortened sensory rays , and smaller fans . tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants also suppress the sma-9 ( cc604 ) M lineage defect at high penetrance ( Fig 1A , 1B , 1D , 1E and Table 1 ) . These observations indicate that TSP-12 and TSP-14 share redundant functions in promoting BMP signaling . In addition to these phenotypes , the tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants also exhibit 100% maternal-effect embryonic lethality: all their eggs die in late embryogenesis , with defects in ventral enclosure ( Fig 1O’ and 1R’ and SI Appendix , S1 and S2 Movies ) . The ability of tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants produced by tsp-12 ( 0 ) /+; tsp-14 ( 0 ) mothers to survive through embryogenesis is likely due to the perdurance of maternally loaded tsp-12 mRNA or TSP-12 protein by the tsp-12 ( 0 ) /+; tsp-14 ( 0 ) mothers . Thus , TSP-12 and TSP-14 share redundant functions that are essential for both embryonic and postembryonic development . To determine how TSP-12 functions to promote BMP signaling , we determined the expression and subcellular localization patterns of TSP-12 . We tagged the endogenous TSP-12 with GFP at either the N-terminus or at the C-terminus using CRISPR/Cas9-triggered homologous recombination ( [21 , 22]; see Materials and Methods ) . Both tagged proteins are fully functional because when introduced into the tsp-14 ( 0 ) background , neither GFP::TSP-12 nor TSP-12::GFP caused any defects observed for tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants as described above . Additionally , both GFP::TSP-12 and TSP-12::GFP exhibited the same expression and localization pattern . As shown in Fig 2 , GFP::TSP-12 is widely expressed from early embryos through larval development to adults in multiple cell types , which include the vulva precursor cells , the hypodermis , and the germline ( Fig 2A to 2H ) . Within a cell , GFP::TSP-12 is both localized to the cell surface and in the cytoplasm in what appears to be small vesicles ( Fig 2A , 2E and 2G ) . Because the fluorescence signal for the endogenously tagged GFP::TSP-12 in somatic cells is faint , we also generated integrated transgenic lines over-expressing a functional , C-terminally tagged TSP-12::GFP transgene ( see Materials and Methods ) . In these animals , we detected TSP-12::GFP in the pharynx , intestine , neurons and M lineage cells ( Fig 2I to 2R ) . The cell surface TSP-12::GFP in the intestinal cells appears to be restricted to the basolateral membrane but absent in the apical membrane ( Fig 2K and 2M ) . It has been well established that the Sma/Mab pathway receptors and downstream Smad proteins function in the hypodermal and M lineage cells to regulate body size and mesoderm development , respectively [19 , 23 , 24] . Since tsp-12 is expressed in the hypodermal and M lineage cells , we asked whether it functions in these cells to promote Sma/Mab signaling . We forced the expression of tsp-12 cDNA in specific tissues of the tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants using tissue specific promoters . Forced expression of the tsp-12 cDNA in the hypodermis ( elt-3p::tsp-12 cDNA and rol-6p::tsp-12 cDNA ) rescued the small body size of tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants , but forced expression in the pharynx ( myo-2p::tsp-12 cDNA ) , intestine ( elt-2p::tsp-12 cDNA ) or neurons ( rab-3p::tsp-12 cDNA ) had no effect ( Fig 2S ) . Forced expression of tsp-12 in the M lineage ( hlh-8p::tsp-12 cDNA ) partially rescued the Susm phenotype of tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants: transgenic tsp-12 ( 0 ) ; tsp-14 ( 0 ) mutants carrying the hlh-8p::tsp-12 cDNA transgene exhibited 15% ( n = 108 ) of the Susm phenotype , while non-transgenic tsp-12 ( 0 ) ; tsp-14 ( 0 ) mutants exhibited 59% ( n = 230 ) of the Susm phenotype . Thus , TSP-12 functions in the signal-receiving cells to promote Sma/Mab signaling in regulating body size and M lineage development . TSP-12 and TSP-14 orthologs are known to regulate the maturation and trafficking of ADAM10 ( a disintegrin and metalloprotease 10 ) to promote Notch signaling in Drosophila and mammalian cells [13] . We therefore tested whether the C . elegans ADAM10 ortholog , SUP-17 [25] , and its close homolog ADM-4 [26] , play any role in Sma/Mab signaling using the sma-9 M lineage suppression assay . A deletion allele of adm-4 , ok265 , failed to suppress the sma-9 M lineage defect; instead , two strong loss-of-function alleles of sup-17 , n1258 and n316 , both partially suppressed the sma-9 M lineage defect ( Fig 3A and Table 2A; sup-17 null mutants are embryonic lethal [25] and cannot be used in this assay ) . Both sup-17 ( n1258 ) and sup-17 ( n316 ) mutants also exhibit smaller body sizes ( Fig 3B , 3C and 3F ) . sup-17 ( n1258 ) males exhibit severe tail patterning defects , including shortened and fused rays , smaller fans ( Fig 3D and 3E ) and crumpled spicules . Thus , sup-17 strong loss-of-function mutants exhibit Sma/Mab signaling defects . We then performed double mutant analysis between sup-17 ( n1258 ) and null mutations in the Sma/Mab pathway by assaying for their body sizes . We found that sup-17 ( n1258 ) ; dbl-1 ( wk70 ) , sup-17 ( n1258 ) ; sma-6 ( jj1 ) and sup-17 ( n1258 ) ; sma-3 ( jj3 ) double mutants were as small as the corresponding dbl-1 ( wk70 ) , sma-6 ( jj1 ) or sma-3 ( jj3 ) single mutants , respectively ( Fig 3F ) . This lack of enhancement of the small body size phenotype of Sma/Mab pathway mutants by sup-17 ( n1258 ) is not because Sma/Mab pathway mutants have reached the minimal body size of C . elegans , because previous studies have shown that mutations in genes regulating body size but not functioning in the Sma/Mab pathway , such as sma-5 ( encoding a MAPK , [27] or wts-1 ( encoding a serine/threonine kinase , [28] ) , can make Sma/Mab pathway mutants much smaller . We therefore concluded that SUP-17 functions in the Sma/Mab pathway to regulate body size . To determine how SUP-17 functions in modulating Sma/Mab signaling , we tagged the endogenous SUP-17 by generating SUP-17::GFP knock-in strains using CRISPR/Cas9-triggered homologous recombination ( see Materials and Methods ) . The SUP-17::GFP fusion is functional , because a transgene with the same SUP-17::GFP fusion rescued the small body size and Susm phenotypes of sup-17 ( n1258 ) mutants ( Fig 4L ) . Like TSP-12 , SUP-17::GFP is also widely expressed from the early embryo through larval development to adults . Cells expressing SUP-17::GFP include the germline , developing vulva , hypodermis , M lineage cells and intestinal cells ( Fig 4B to 4M ) . Also like TSP-12 , SUP-17::GFP is localized both at the cell surface and in the cytoplasm in small puncta ( Fig 4B , 4D , 4F and 4H ) . In the early embryo , cell surface localized SUP-17::GFP is enriched in the basolateral , but not the apical , surfaces ( Fig 4B ) . Forced expression of sup-17 cDNA in the hypodermis ( rol-6p::sup-17 cDNA ) , but not in the intestine ( elt-2p::sup-17 cDNA ) , rescued the small body size of sup-17 ( n1258 ) mutants ( Fig 4N ) . Similarly , forced expression of sup-17 cDNA in the M lineage ( hlh-8p::sup-17 cDNA ) partially rescued the Susm phenotype of sup-17 ( n1258 ) mutants ( Table 2A ) . These findings indicate that SUP-17 also functions in the signal-receiving cells to modulate Sma/Mab signaling in regulating body size and mesoderm development . The shared expression/localization pattern and function of TSP-12 , TSP-14 and SUP-17 in Sma/Mab signaling suggest that TSP-12 and TSP-14 may be functionally linked with SUP-17 in their roles in the Sma/Mab pathway . Using the mating-based split ubiquitin yeast-two-hybrid assay for membrane protein interactions ( see Materials and Methods ) , we found that SUP-17 can bind to TSP-12 and TSP-14 ( Fig 5A ) . We then examined the localization of SUP-17::GFP in tsp-12 ( 0 ) and tsp-14 ( 0 ) single mutants , and in tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutants . We focused on the early embryo for these localization studies because of the large cell sizes and the brighter GFP signals in these embryos . Wild-type embryos expressing the endogenously-tagged SUP-17::GFP showed GFP localization both at the cell surface and in the cytoplasm ( Fig 5B and 5F ) . This localization pattern does not change in tsp-14 ( 0 ) mutant embryos ( Fig 5C and 5F ) . However , in tsp-12 ( 0 ) or tsp-12 ( 0 ) ; tsp-14 ( 0 ) mutant embryos , there is a significant reduction of cell surface SUP-17::GFP accompanied by an increase of intracellular SUP-17::GFP ( Fig 5D , 5E and 5F ) and an increase in the number of bright puncta in the cytoplasm ( Fig 5G ) . These findings indicate that TSP-12 and TSP-14 can bind to SUP-17 and that TSP-12 , but not TSP-14 , promotes the cell surface localization of SUP-17 in embryos . The involvement of SUP-17/ADAM10 in Sma/Mab signaling prompted us to test whether the γ-secretase functions in Sma/Mab signaling , because Notch undergoes sequential cleavage by ADAM and γ-secretase [29] . C . elegans has three paralogous genes encoding presenilins , sel-12 , hop-1 and spe-4 [30–32] , and presenilins are major components of the γ-secretase complex . Because spe-4 appears to be specifically expressed and functions in the germline during spermatogenesis , and because sel-12 and hop-1 share redundant functions , in particular in Notch signaling [30–32] , we tested whether mutations in sel-12 and hop-1 exhibited any Susm phenotypes . Using null alleles for each gene , we found that sel-12 ( ar171 ) , sel-12 ( ok2078 ) , hop-1 ( ar179 ) single mutants or hop-1 ( ar179 ) ; sel-12 ( ar171 ) and hop-1 ( ar179 ) ; sel-12 ( ok2087 ) double mutants did not exhibit any Susm phenotype ( Table 2B ) , suggesting that the γ-secretase is unlikely to be involved in Sma/Mab signaling in C . elegans . ADAM10 is known to cleave transmembrane or membrane-associated proteins in a process called ectodomain shedding [33 , 34] . We have previously shown that the extracellular domain of UNC-40 is sufficient to function in promoting Sma/Mab signaling , suggesting that UNC-40 may be cleaved for its function in Sma/Mab signaling [18] . We therefore tested genetically whether UNC-40 is a SUP-17/ADAM10 substrate . We reasoned that if UNC-40 is a SUP-17 substrate in BMP signaling , then the extracellular domain of UNC-40 , which is sufficient to function in promoting Sma/Mab signaling , should be able to , either completely or partially , rescue the Sma/Mab signaling defects of sup-17 mutants . We , therefore , tested whether two unc-40 alleles that lead to the production of truncated UNC-40 proteins lacking the intracellular domain ( ICD ) , ev495 and tr115 [18 , 35] , could rescue the Susm phenotype of sup-17 ( n1258 ) mutants . As shown in Table 2C , a null unc-40 allele , e1430 , did not rescue the Susm phenotype of sup-17 ( n1258 ) mutants , while both unc-40 ( ev495 ) and unc-40 ( tr115 ) partially suppressed the Susm phenotype of sup-17 ( n1258 ) mutants . The similar penetrance of the Susm phenotype shared by unc-40 ( e1430 ) and unc-40 ( e1430 ) sup-17 ( n1258 ) mutants , and the partial suppression of the sup-17 ( n1258 ) Susm phenotype by unc-40 ( ev495 ) and unc-40 ( tr115 ) suggest that: 1 ) UNC-40 and SUP-17 function in the same pathway to regulate Sma/Mab signaling , and 2 ) UNC-40 is a substrate of SUP-17 in the Sma/Mab pathway . The incomplete suppression of the Susm phenotype of sup-17 ( n1258 ) mutants by unc-40 ( ev495 or tr115 ) suggests that additional SUP-17 substrates must exist in the Sma/Mab pathway .
In this study , we extended our previous work [7] and provided additional evidence that TSP-12 and TSP-14 , two paralogous TspanC8 tetraspanins , function redundantly to promote BMP signaling in C . elegans . We further showed the ADAM10 ortholog SUP-17 also functions in the BMP pathway . Both tsp-12; tsp-14 double mutants and sup-17 single mutants exhibited BMP signaling defects , including a smaller body size , male tail abnormalities , reduced RAD-SMAD reporter activity , and suppression of the sma-9 mesoderm phenotype ( Susm ) . Both TSP-12 and SUP-17 are expressed and function in the signaling-receiving cells to promote BMP signaling in regulating body size and M lineage development . TSP-12 and TSP-14 can bind to SUP-17 . TSP-12 and SUP-17 share similar expression and subcellular localization patterns . Additionally , TSP-12 promotes the cell surface localization of SUP-17 in embryos . TspanC8 tetraspanins and ADAM10 are known to promote Notch signaling in C . elegans , Drosophila , and mammalian tissue culture cells [12 , 15 , 25 , 36 , 37] . Three lines of evidence suggest that the function of these proteins in promoting BMP signaling is independent of their role in Notch signaling . First , unlike lin-12 ( 0 ) mutants , which exhibit a ventral-to-dorsal fate transformation in the M lineage [38] , tsp-12 ( 0 ) , tsp-14 ( 0 ) , tsp-12 ( 0 ) ; tsp-14 ( 0 ) or sup-17 ( n1258 ) mutants do not exhibit any M lineage defects . Second , two lin-12 null mutants , lin-12 ( n941 ) and lin-12 ( ok2215 ) , do not exhibit any body size defects ( S1 Table ) . Third , we have previously reported that lin-12 ( 0 ) mutants do not suppress the M lineage defects of sma-9 ( 0 ) mutants [39] . Thus our findings uncover an additional function for TspanC8 tetraspanins and ADAM10 in promoting BMP signaling . Notably , TSP-12 and TSP-14 may have other functions in addition to modulating Notch and BMP signaling . In particular , tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutant embryos exhibited ventral enclosure defects , but Notch and BMP signaling pathways have not been previously implicated in the regulation of ventral enclosure in C . elegans [40] . Over-expression and knockdown experiments in Drosophila and mammalian tissue culture cells have shown that TspanC8 tetraspanins promote the cell surface localization of ADAM10 [12–14] . Our work demonstrates that this function of the TspanC8 tetraspanins is evolutionarily conserved . How TspanC8 tetraspanins regulate the cell surface localization of ADAM10 requires further investigation . Tetraspanins are known to regulate intracellular trafficking of various cargo proteins via multiple distinct mechanisms [11 , 41] . Previous work showed that TspanC8 tetraspanins function to promote the exit of ADAM10 from the endoplasmic reticulum ( ER ) [12–14] . We noted in our work that SUP-17::GFP from transgenic lines overexpressing this transgene exhibited a strong perinuclear , ER-like localization pattern with very little cell surface localization ( Fig 4J and 4K ) . However , endogenously-tagged SUP-17::GFP exhibited much more prominent cell surface localization ( Figs 4B , 4D , 4F , 4H and 5B ) . We showed that this cell surface localization is significantly reduced in tsp-12 ( 0 ) and tsp-12 ( 0 ) ; tsp-14 ( 0 ) mutants; while there was a concomitant increase of bright SUP-17::GFP-positive puncta in these mutant embryos ( Fig 5D to 5G ) . Whether these signals represent SUP-17::GFP trapped in the ER or in other intracellular organelles will need further investigation . The basolateral plasma membrane localization of TSP-12 in intestinal cells ( Fig 2K and 2N ) resembles the localization patterns of both SMA-6/RI and DAF-4/RII of the BMP pathway [6] . We have previously shown that both TSP-12 and TSP-14 can bind to SMA-6 in yeast [7] . Whether TSP-12 , TSP-14 or both of them are required for the proper localization of SMA-6 and DAF-4 will require further investigation . Our phenotypic analysis clearly showed that TSP-12 and TSP-14 function redundantly to promote BMP signaling ( Fig 1F and 1K ) . Both proteins can also bind to SUP-17 in yeast ( Fig 5A ) . However , only tsp-12 ( 0 ) mutant embryos showed severe defect in SUP-17::GFP localization , and this defect is not enhanced in tsp-12 ( 0 ) ; tsp-14 ( 0 ) double mutant embryos ( Fig 5D to 5G ) . Due to technical challenges , our SUP-17::GFP localization study was carried out in embryos but not in tissues relevant to Sma/Mab signaling . It is possible that TSP-14 may function similarly as TSP-12 to promote the cell surface localization of SUP-17 in other cell types , but not in the early embryo . Alternatively , it is possible that while TSP-12 and TSP-14 exhibit functional redundancy in regulating BMP signaling and other developmental processes at the organismal level , they may differ in their functions at the sub-cellular and/or molecular level . In worms , flies and mammals , ADAM proteases are well known for their role in processing the Notch receptor upon ligand binding [42–44] . Notch undergoes sequential cleavage , first by the ADAM protease , followed by subsequent cleavage by the γ-secretase . Although ADAM10 has not been previously implicated in BMP signaling in any organisms , the ADAM10 paralog ADAM17 or TACE ( tumor necrosis factor-alpha-converting enzyme ) has been shown to play a role in TGFβ signaling in mammals [45] . Earlier work by Liu and colleagues indicated that ADAM17/TACE-mediated cleavage of the type I TGFβ receptor ( TβR1 ) causes downregulation of TGFβ signaling in CHO cells [45] . Cleavage of TβR1 by ADAM17/TACE and Presenilin 1 , a γ-secretase catalytic core component , has also been found in multiple cancer cell lines , which results in the translocation of the intracellular domain ( ICD ) of TβR1 into the nucleus and activation of genes involved in tumor invasion [46 , 47] . More recently , polymorphic variants of the Adam17 gene have been found to differentially regulate TGFβ signaling and influence the severity of Tgfb1-dependent vascular pathology in mice and humans [48] . Because ADAM10 and ADAM17 are known to share a number of common substrates [34] , and in C . elegans SUP-17/ADAM10 and ADM-4/ADAM17 share redundant functions [26] , our findings raise the possibility that ADAM10 or ADAM17 may also play a role in regulating BMP signaling in mammals . Consistent with this notion , Jackson and colleagues reported that ADAM17/TACE-null mice exhibit defects in valvulogenesis that are associated with aberrant BMP signaling [49] . How SUP-17/ADAM10 functions to modulate BMP signaling is an open question . Our genetic studies suggest that the neogenin/DCC ortholog UNC-40 is one of the substrates of SUP-17 . UNC-40 , like its vertebrate ortholog neogenin , functions through the RGM ( repulsive guidance molecule ) proteins to promote BMP signaling [10 , 18] . We have previously shown that the extracellular domains of UNC-40 and neogenin are sufficient to mediate BMP signaling in C . elegans and in mammalian tissue culture cells , respectively [18] . There are other reports showing that neogenin or DCC can be cleaved by ADAM proteases . Earlier work using ADAM inhibitors and rat dorsal spinal cord explants showed that DCC undergoes ADAM-dependent proteolytic processing , which may affect its role in regulating axon migration [50] . Okamura and colleagues showed that ADAM17/TACE can bind to and cleave neogenin , regulating the behavior of rat cortical neurons in response to RGM [51] . Recently , van Erp and colleagues showed that ADAM17/TACE-mediated cleavage of neogenin is regulated by the leucine-rich repeats and immunoglobulin-like domains ( Lrig ) protein Lrig2 , and that this regulation is required for RGM-mediated neurite growth inhibition in vitro and cortical neuron migration in vivo [52] . How SUP-17 cleaves UNC-40 and whether similar types of regulatory interactions also occur in the BMP signaling pathway will require further experimentation . Nevertheless , cleavage of UNC-40 by SUP-17 may not be followed by subsequent cleavage by the γ-secretase , because hop-1 ( 0 ) ; sel-12 ( 0 ) double mutants , which lack two of the redundant C . elegans presenilins , do not exhibit any BMP signaling defects ( Table 2 ) . We are aware that we assayed the Susm phenotype in hop-1 ( 0 ) ; sel-12 ( 0 ) animals produced by hop-1 ( 0 ) /+; sel-12 ( 0 ) mothers due to the sterility of the hop-1 ( 0 ) ; sel-12 ( 0 ) double mutants . Thus , although unlikely , we cannot rule out the possibility that residual γ-secretase activity is still present in hop-1 ( 0 ) ; sel-12 ( 0 ) double mutants due to maternally loaded HOP-1 and that this residual activity is sufficient for proper Sma/Mab signaling . Our genetic studies suggested that UNC-40/neogenin is not the only substrate of SUP-17/ADAM10 in the BMP pathway . Identifying the additional substrate ( s ) and determining the functional consequences of ADAM processing of these substrates in vivo will provide insight into the intricate levels of modulation of BMP signaling . Notably , ADAM10 and ADAM17 mutations are associated with many diseases in humans , including a variety of cancers [34 , 53 , 54] . We speculate that some of these incidences could be due to abnormal processing of factors involved in BMP signaling caused by altered activities of ADAM10 and/or ADAM17 .
All strains were maintained at 20°C using standard culture conditions [55] unless otherwise noted . The following mutations , transgenes and balancers were used: Linkage group I ( LG I ) : drag-1 ( jj4 ) , hop-1 ( ar179 ) , unc-40 ( n1430 ) , unc-40 ( ev495 ) , unc-40 ( tr115 ) , sup-17 ( n316 ) , sup-17 ( n1258 ) , sup-17::gfp ( jj98 ) , arIs37 ( secreted CC::gfp ) ; LG II: sma-6 ( jj1 ) ; jjIs2437 ( RAD-SMAD ) ; LG III: lon-1 ( jj67 ) , sma-3 ( jj3 ) , lin-12 ( n941 ) , lin-12 ( ok2215 ) , ccIs4438 ( intrinsic CC::gfp ) , hT2[qIs48]; LG IV: tsp-12 ( ok239 ) , gfp::3×flag::tsp-12 ( jj181 ) , gfp::3×flag::tsp-12 ( jj182 ) ; nT1[qIs51]; LG V: dbl-1 ( wk70 ) , him-5 ( e1467 ) ; LG X: sel-12 ( ar171 ) , sel-12 ( ok2078 ) , lon-2 ( e678 ) , tsp-14 ( jj95 ) , tsp-14 ( jj96 ) , tsp-14 ( jj97 ) , adm-4 ( ok265 ) , sma-9 ( cc604 ) . Body size measurement and RAD-SMAD reporter assays were performed following the protocols described in Tian et al . [18] . Hermaphrodite worms at the L4 stage ( identified based on vulva development ) were used for body size measurement . Body sizes were measured using segmented lines in Fiji [56] after images were taken . Synchronized L3 hermaphrodites carrying the RAD-SMAD reporter jjis2437 II [18] were used for imaging . The reporter fluorescence intensity was measured using Fiji . Oligonucleotides used in this study are listed in SI Appendix , S2 Table . Plasmids generated are listed in SI Appendix , S3 Table . The tsp-12 cDNA was amplified from the Vidal RNAi library [57] , while yk23h2 contains the full-length sup-17 cDNA . Each clone contains a point mutation in the respective coding regions of tsp-12 and sup-17 , which were respectively fixed via site-directed mutagenesis , resulting in the generation of pJKL1079 ( containing the tsp-12 ORF ) , and pJKL1140 ( containing the sup-17 cDNA ) . pK3 , a rescuing plasmid containing 7kb of the sup-17 genomic region , is a kind gift from Iva Greenwald ( Columbia University ) . One of the following plasmids were used as co-injection markers to generate transgenic lines: pRF4 ( rol-6 ( su1006 ) ) , LiuFD61 ( mec-7p::mRFP ) , pJKL449 ( myo-2p::gfp::unc-54 3’UTR ) , or pCFJ90 ( myo-2p::mCherry::unc-54 3’UTR ) [58] . Stable integrants carrying pZL51 were generated using γ-radiation , followed by outcrossing with N2 animals five times . To generate deletion alleles of tsp-14 , we designed two sgRNAs targeting both the N-terminus ( ACCACCGCGCGAGCTCGCGT ) and the C-terminus ( AAGCGGCTGAAGGCATCCG ) of the tsp-14 coding region ( SI Appendix , S1 Fig ) . The sgRNA plasmids pZL8 and pZL9 ( S3 Table ) were generated by replacing the unc-119 gRNA sequence in the pU6::unc-119 sgRNA vector [59] with the tsp-14 sgRNA sequence . The following plasmid mix was injected into young hermaphrodite N2 adults: ( 1 ) the Cas9 expression plasmid pDD162 ( peft-3-cas9+empty sgRNA ) [60] , ( 2 ) two tsp-14-specific sgRNA plasmids pZL8 and pZL9 , and ( 3 ) the co-injection marker pRF4 ( rol-6d ) . Positive Rol F1 worms were picked and placed onto fresh NGM plates ( 3 worms/plate ) . After they have laid eggs , the F1 worms were pooled for PCR screening using primers ZL4 and ZL5 ( S2 Table ) . After screening 282 F1s , we obtained three tsp-14 deletion alleles , jj95 , jj96 and jj97 , which were confirmed by Sanger sequencing ( SI Appendix , S1 Fig ) . We tagged the endogenous TSP-12 at either the N-terminus or the C-terminus using CRISPR/Cas9 . To generate these TSP-12 knock-ins , we followed the strategy described in Arribere et al . [21] to generate two sgRNA expression plasmids for each tagging strategy ( see S2 and S3 Tables for oligo and plasmid information ) . We then used the method described in Dickinson et al . [22] to generate the homologous repair templates , and to subsequently obtain stable knock-in strains that carry either GFP::3×FLAG::TSP-12 ( jj181 and jj182 ) or TSP-12::GFP::3×FLAG ( jj194 and jj196 ) . We first confirmed the success of knock-ins by genotyping , and then tested jj181 and jj194 for functionality by crossing each of them into the tsp-14 ( 0 ) background and examined the strains for viability and fertility . The sup-17::gfp knock-in strain was generated by injecting the following mix of plasmid DNAs into N2 worms: ( 1 ) a Cas9 expression plasmid pDD162 [60] , ( 2 ) two sgRNA plasmids ( pLW4 and pLW5 , generated using the pU6::unc-119 sgRNA plasmid described in Friedland et al . [59] ) , ( 3 ) the homologous repair template pJKL1034 ( SUP-17::GFP translational fusion , with GFP inserted between amino acid 876 and 877 , see S3 Table ) , and ( 4 ) a co-injection marker pRF4 ( rol-6d ) . GFP knock-in events were screened via PCR using primers LW27 , LW30 and JKL269 ( see S2 Table ) . Single worm PCR of 33 F1 rollers failed to detect any germline integration event . However , we screened the F3 worms from six high transmission efficiency ( >30% ) rol transgenic lines and found that five of the six lines examined had a gfp knock-in event . After genotyping and confirming homozygous sup-17::gfp knock-ins , we kept three of the lines , jj98 , jj99 and jj100 , for further analysis . To examine the male tail , the developing vulva or developing embryos , animals were visualized under a Leica DMRA2 compound microscope equipped with a Hamamatsu Orca-ER camera using the iVision software ( Biovision technology , Inc . ) . An inverted Zeiss LSM880 confocal microscope with Airyscan was used to examine the expression and subcellular localization patterns of GFP::3×FLAG::TSP-12 or SUP-17::GFP . Fluorescence was detected with an Airyscan detector using the 40× objective ( NA 1 . 4 , WD 0 . 13 mm ) and in the super-resolution mode ( pinhole is around 2 . 25 AU ) . The laser line for GFP excitation is at 488 nm , and for mCherry is at 561 nm . Collected images were subsequently processed by ZEN ( Carl Zeiss ) , and further quantification of the fluorescence signals was performed using Fiji . To quantify the fluorescence intensity and bright intracellular puncta of SUP-17::GFP , live embryos from worms of different genotypes that were grown under the same condition were collected on the same day and imaged using the same settings as described above . Images of stage-matched embryos were further processed in the same way using Fiji . Average SUP-17::GFP fluorescence intensity was calculated by drawing a straight line of fixed length ( for cell surface signal ) or by drawing an oval of fixed width and height ( for intracellular signal ) . Only cells with focal plane going through the nucleus were used for this purpose . Bright puncta were defined as those signals that are above a threshold of brightness ( 3 , 500 pixels ) and size ( the area is above 0 . 07 μm2 ) . Signals on top of the cell membrane were excluded from the counting . The split-ubiquitin yeast two-hybrid experiments were carried out by following the protocol described in Grefen et al . [61] . The bait CubPLV and prey NubG fusion constructs used are listed in SI Appendix , S2 Table . After transforming into yeast , interactions among each pair of bait and prey constructs were visualized by streaking diploid cells on SC-Trp , -Leu , -Ade , -His , -Ura , -Met plates that were supplemented with four different concentrations of methionine: 0mM , 0 . 075mM , 0 . 150mM , 0 . 300mM , respectively . Growth was monitored for 2–9 days at 30°C . The plasmids KAT-1-Cub-PLV , KAT-1-NubG ( in XN21 vector ) , empty NubG vector , and the vector expressing soluble wild-type Nub ( NubWT ) were used as controls [7] . Confirmation of expression of each fusion protein was by western blot analysis using rabbit polyclonal anti-VP16 antibodies ( ab4808 , Abcam , for CubPLV fusions ) and monoclonal anti-HA antibodies ( Clone 12CA5 , Sigma , for NubG fusions ) . Statistical analyses were conducted with Prism 5 ( GraphPad ) , and the test for significance for groups of two were analyzed using the unpaired Student’s t-test . Two-tailed p values were calculated . Data was shown as mean ± 95% confidence interval ( CI ) . | Bone morphogenetic protein ( BMP ) signaling regulates multiple developmental and homeostatic processes . Misregulation of this pathway can cause various diseases , including cancers . Thus , it is essential to understand how BMP signaling is tightly regulated spatiotemporally in vivo . We have identified a highly conserved ADAM ( a disintegrin and metalloprotease ) protein , SUP-17/ADAM10 , as an important factor in modulating BMP signaling in C . elegans . We showed that the proper localization and function of this ADAM protease require two conserved tetraspanin proteins , TSP-12 and TSP-14 . We provided genetic evidence showing that one of the substrates of SUP-17/ADAM10 in the BMP signaling pathway is a known BMP signaling modulator , UNC-40/neogenin/DCC . Our studies established a new role of the TSP-12-TSP-14-SUP-17 axis in regulating BMP signaling , in addition to and independent of their known function in the Notch signaling pathway . | [
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"paramete... | 2017 | Two Paralogous Tetraspanins TSP-12 and TSP-14 Function with the ADAM10 Metalloprotease SUP-17 to Promote BMP Signaling in Caenorhabditis elegans |
It is often claimed that pair bonds preferentially form between individuals that resemble one another . Such assortative mating appears to be widespread throughout the animal kingdom . Yet it is unclear whether the apparent ubiquity of assortative mating arises primarily from mate choice ( “like attracts like” ) , which can be constrained by same-sex competition for mates; from spatial or temporal separation; or from observer , reporting , publication , or search bias . Here , based on a conventional literature search , we find compelling meta-analytical evidence for size-assortative mating in birds ( r = 0 . 178 , 95% CI 0 . 142–0 . 215 , 83 species , 35 , 591 pairs ) . However , our analyses reveal that this effect vanishes gradually with increased control of confounding factors . Specifically , the effect size decreased by 42% when we used previously unpublished data from nine long-term field studies , i . e . , data free of reporting and publication bias ( r = 0 . 103 , 95% CI 0 . 074–0 . 132 , eight species , 16 , 611 pairs ) . Moreover , in those data , assortative mating effectively disappeared when both partners were measured by independent observers or separately in space and time ( mean r = 0 . 018 , 95% CI −0 . 016–0 . 057 ) . Likewise , we also found no evidence for assortative mating in a direct experimental test for mutual mate choice in captive populations of Zebra finches ( r = −0 . 020 , 95% CI −0 . 148–0 . 107 , 1 , 414 pairs ) . These results highlight the importance of unpublished data in generating unbiased meta-analytical conclusions and suggest that the apparent ubiquity of assortative mating reported in the literature is overestimated and may not be driven by mate choice or mating competition for preferred mates .
Members of a pair often resemble each other . For instance , in humans , partners have similar political attitudes [1 , 2] , level of education [3 , 4] , and body height [4–6] . In fact , assortative mating appears to be common across all animal taxa and across all phenotypic traits that have been investigated [7] . However , in most cases , the underlying processes that lead to mate similarity remain unclear . Similarity of pair members , quantified as the strength of the correlation between their trait values , may arise via at least three biological mechanisms . ( 1 ) Mate choice and mating competition; one or both sexes may prefer phenotypes similar to their own ( “like attracts like” ) . This may lead to the more frequent formation and enhanced stability of assortative pair bonds . ( 2 ) Spatial and temporal autocorrelation; individuals with different phenotypes may be separated in space and time , such that at the population level , even random mating would lead to partner similarity ( “like meets like” ) . For instance , in high-quality habitats , individuals may grow larger than in poor habitats . If individuals from different habitats are less likely to meet ( “nonpanmixis” ) , e . g . , because of low mobility [8] , a population-wide pattern of assortative mating may arise in the absence of choice for an assortative partner . Similarly , in migratory species , individuals that resemble each other in particular traits may have a higher probability to form a pair simply because they arrive at the breeding grounds closer in time ( e . g . , older individuals might arrive earlier , leading to assortative mating for age [9] ) . ( 3 ) Phenotypic changes over time; females and males may mate randomly for a certain phenotype but become similar to their partner over time ( “become alike” ) [10] . For instance , in humans , a positive correlation in body mass between couples may arise because they share the same food [11 , 12] . Because the three biological mechanisms can act together and can also interact with other processes , their relative importance may be difficult to tease apart [13 , 14] . Furthermore , if testing assortative mating that comes about via mate choice and mating competition is the goal of the study , then the other “biological” mechanisms could be considered as confounds [15] . Also , it should be noted that not all mechanisms may be relevant for all phenotypes ( e . g . , “becoming alike” does not apply to assortative mating with regard to age ) . Besides the influence of biological processes [16] , estimates of the strength of assortative mating can be confounded by three main methodological issues . ( 1 ) Observer bias; data sets often consist of measurements from multiple observers and may be taken over long periods . This introduces heterogeneity and temporal autocorrelation in the data because observers can differ in their measurements [17] , and they may unconsciously change their measuring technique over time . Hence , spurious trait correlations between pair members may arise when pair members are typically measured by the same observer and on the same day ( see our simulation in S1 Fig ) . Such methodological heterogeneity may be difficult to distinguish from biological heterogeneity in space and time . ( 2 ) Reporting bias; estimates found in the literature will be inflated when statistically significant estimates are more likely reported than nonsignificant ones [18 , 19] . ( 3 ) Search bias; if a meta-analysis is based on a literature search with keywords like “assortative , ” the strength of assortative mating may be overestimated because it is less likely null results are mentioned in the abstract of a publication [20]; hence , such searches may preferentially yield a subset of studies that have detected significant assortative mating . Similarly , when screening relevant publications , taking estimates from related studies that are being cited may also discriminate against null findings because studies with null findings tend to get cited less often than studies with significant and , hence , typically larger effects [21] . Assortative mating is often investigated with a focus on mate choice and mating competition [22 , 23] . However , a systematic review of assortative mating in arthropods [13] suggests that mechanisms other than “mating preferences for phenotypic similarity” are predominantly responsible for the mating patterns observed in these invertebrates ( such as mate availability and physical constraints ) . Whether mate choice and mating competition or other processes underlie assortative mating in vertebrates such as birds is unclear , particularly because birds often form strong social pair bonds for which the compatibility of partners may determine reproductive success [24] . Here , we quantify the strength of assortative mating in birds and assess how assortative mating estimates change with increasing control of the confounding factors discussed above . We then discuss biological implications of our findings and propose ways to minimize confounding effects if the aim is to investigate assortative mating due to mate choice and mating competition . For practical reasons ( data availability ) , we focus primarily on assortative mating for size in birds , yet our approach is relevant for most phenotypic traits and taxa . In birds , body size is measured and reported in a variety of ways ( body mass , wing length , tarsus length , bill length , body length , and first principal component of different measures ) . Thus , our measure of body size includes all size-related morphological measures but not measures of ornament size ( e . g . , plumage patch area or ornamental feather length ) and of body condition ( e . g . , mass residuals over tarsus length ) , which were categorized separately . First , we compare published estimates of the strength of assortative mating with estimates from our previously unpublished data from nine long-term field studies that are a sample of the species studied in the literature . This allows assessment of the effect of search and reporting bias , which should only affect the published data set . Second , we use the previously unpublished data set to explore causes of assortative mating patterns that are not due to mate choice and mating competition , such as effects of observer bias and spatial and temporal nonindependence . Finally , we present an analysis of experimental data from five studies of assortative mating in captive Zebra finches , Taeniopygia guttata [24 , 25] . In these experiments , we took standardized measurements of all birds before randomly allocating them to experimental aviaries . So far , this is the only avian study for which we can estimate the strength of assortative mating among individuals that encountered each other , excluding all known confounding factors .
Overall , the published literature showed striking evidence for positive assortative mating across all trait categories ( ranging from r = 0 . 156 to 0 . 434; none of the 95% CI overlap zero ) ( Fig 1 and S1 Table ) . The two random effects , “Study” and “Species , ” explained only 9% and 6% of the variance , respectively . Compared to other traits , assortative mating for body size was the second weakest ( r = 0 . 178 , 95% CI 0 . 142–0 . 215 , 83 species , 35 , 591 pairs ) , but it was the most frequently studied trait ( 54% of all estimates ) . The previously unpublished data from nine long-term field studies also showed a clear yet weaker tendency for positive assortative mating by size , whereby the magnitude depended on how the data were analyzed ( ranging from r = 0 . 067 to 0 . 103 ) ( S2 Fig and S2 , S3 and S4 Tables ) . When repeatedly measured individuals were represented by a randomly selected single measure ( i . e . , model of “random alignment” of male to female measure ) , the strength of assortment was weak ( mean r = 0 . 067 , 95% CI 0 . 042–0 . 093 , n = 16 , 545 pair–trait combinations ) ( S2 Fig and S2 Table ) . When average measures per individual were used ( “average model” ) , estimates of assortment were only slightly higher ( mean r = 0 . 080 , 95% CI 0 . 054–0 . 105 , n = 16 , 545 ) ( S2 Fig and S3 Table ) . Finally , when using the male and female measures taken closest to the presumed time of pair formation ( “nearest model” ) , the estimate of assortative mating was highest ( mean r = 0 . 103 , 95% CI 0 . 074–0 . 132 , n = 16 , 611 ) ( S2 Fig and S4 Table ) . The estimates from the “nearest” model were significantly higher than those from the “random alignment” model ( paired t test for 32 species–traits , t31 = 4 . 50 , P = 0 . 0001 ) and higher than estimates from the “average” model ( t31 = 3 . 32 , P = 0 . 002 ) . In the previously unpublished data set , levels of apparent assortative mating were significantly higher when measurements on the two members of a pair had been taken by the same observer ( r = 0 . 075 , 95% CI 0 . 033–0 . 118 , t = 3 . 45 , P = 0 . 0006 , n = 22 estimates , 34 , 672 pair–trait combinations ) than when measurements came from different observers ( r = 0 . 023 , 95% CI −0 . 022–0 . 067 , t = 1 . 01 , P = 0 . 3 , n = 22 estimates , 24 , 771 pair–trait combinations; tsame versus different observer = 2 . 48 , P = 0 . 01 ) ( Fig 1 and S5 Table: model 4 ) . Similarly , estimates for assortative mating for size were significantly higher when measurements on the two members of a pair had been taken within the same month ( within 30 days: r = 0 . 110 , 95% CI 0 . 079–0 . 142 , t = 6 . 92 , P < 0 . 0001 , n = 32 estimates , 51 , 995 pair–trait combinations ) than when the partners had been measured more than 30 days apart ( r = 0 . 014 , 95% CI −0 . 013–0 . 041 , t = 0 . 98 , P = 0 . 3 , n = 31 estimates , 20 , 729 pair–trait combinations; tsame versus different time = 5 . 7 , P < 0 . 0001 ) ( Fig 1 and S5 Table: model 5 ) . Finally , estimates of size-assortative mating were significantly higher when partners had been measured at the same site ( 0 . 073 , 95% CI 0 . 046–0 . 100 , t = 5 . 30 , P < 0 . 0001 , n = 32 estimates , 26 , 542 pair–trait combinations ) than when they were measured at different sites ( 0 . 017 , 95% CI −0 . 009–0 . 044 , t = 1 . 28 , P = 0 . 2 , n = 32 estimates , 44 , 112 pair–trait combinations; tsame versus different location = 3 . 90 , P < 0 . 0001 ) ( Fig 1 and S5 Table: model 6 ) . Data from the five experiments on Zebra finches showed an overall size-assortative mating close to zero ( r = −0 . 020 , 95% CI −0 . 148–0 . 107; weighted mean of 13 estimates based on 1 , 414 pair–trait combinations ) ( S6 Table and S8 Data ) . Note that the statistical power for detecting an effect of r = 0 . 20 was >0 . 99 for each of the three size phenotypes ( mass , tarsus , and wing length ) . The strength of assortative mating decreased with increasing control for confounding factors from published ( Web of Science search: r = 0 . 184; Cited studies: r = 0 . 140 ) to previously unpublished ( r = 0 . 107 ) to experimental data ( r = −0 . 004 ) . However , in such a joint model , CIs around the estimates were relatively wide despite high sample sizes ( Fig 1 and S7 Table ) . The random effect “Study” explained 7% of the variance , while the other two random effects ( “Species” and “Trait” ) explained zero variance ( S7 Table ) . Estimates of assortative mating from the literature data declined significantly with increasing sample size ( test for asymmetry in the funnel plot t = −2 . 76 , P = 0 . 006 , n = 449 ) ( Fig 2 ) . According to the estimated regression line ( Fig 2 ) , correlation coefficients from the “Published data” reached the same magnitude as those from the previously “Unpublished data” at a sample size of n = 822 pairs .
A recent analysis of assortative mating across the animal kingdom [7] did not find clear evidence for publication bias , as indicated by significant asymmetry in the funnel plot , while in our data set of published studies , the funnel plot asymmetry was statistically significant ( Fig 2 ) . This could indicate either that publication bias is limited or that tests for asymmetry in the funnel plot are sometimes inefficient in detecting it [26] . Publication bias is expected because ( a ) most studies emphasize positive findings rather than null results [27] despite statistical power being low ( selective reporting [28] ) , ( b ) incomplete reporting of nonsignificant outcomes is widespread [29] , and ( c ) the few meta-analyses that compared published and unpublished effect sizes ( including this one ) found that published effects were larger than unpublished ones ( Fig 1 ) ( e . g . , [30–32] ) . In this context , the increased availability of open access data [33] might allow an unbiased quantification of effect sizes ( particularly when the published data were part of a study addressing a different question ) , which might help to put the existing knowledge into perspective . In field studies , it is practically impossible to implement the optimal measuring design of experimental studies ( all individuals measured in random order by the same observer in the same environment on the same day before mate choice starts ) . This means that multiple observers , multiple environments , and multiple time points introduce heterogeneity into the data , which is partly of methodological and partly of biological origin . Such heterogeneity may ( 1 ) partly blur existing patterns of assortment due to sexual selection , thereby leading to underestimation , and ( 2 ) induce spurious correlations between pair members when these are often measured by the same observer and close in time and space , leading to overestimation of assortment due to sexual selection . As our simulation shows , the effect of an overestimation of assortative mating clearly exceeds the effect of underestimation when ( 1 ) levels of true assortment are relatively low and ( 2 ) a large fraction of the pair members are measured by the same observer ( see S1 Fig ) . In our empirical data , we found the expected difference between the “same” versus “different” observer conditions , as the simulation showed ( Fig 1 ) , and conclude that our “nearest” model tended to overestimate true levels of assortment because for 80% of the pairs , the partners had been measured by the same observer ( S1 Fig ) . When it is not practically feasible that all measurements are taken by a single observer , observer bias can be controlled for by calculating correlations between pair members after statistically removing observer effects ( see [34] for a solution ) . Measurement error may also fluctuate in time and thereby induce temporal autocorrelation and , hence , an apparent similarity of pair members ( see next paragraph ) . Finally , observers may have preconceptions about assortative mating , such that measurements suffer from confirmation bias [35] . This is perhaps less likely for data sets that were collected without hypotheses about assortative mating in mind ( such as ours ) . Preconception bias can be solved by blinding observers to the hypothesis of interest [36]; this is already ensured when the members of a pair are measured by independent observers or at different points in time . Our results show that the estimated strength of size-assortative mating is higher when individuals are measured within the same month or at the same site ( Fig 1 ) . Such results may arise for both biological and technical reasons . For example , measures of plumage coloration may show temporal autocorrelation because plumage color gradually changes after moult due to wear or because of changes in the white balance used for calibration of hand-held photo spectrometers [37] . Also , depending on the research question , effects that induce temporal and spatial autocorrelation can be of biological interest ( e . g . , for ecologists ) , or it can be a confounding factor ( e . g . , if the interest lies in studying mate choice and mating competition ) . In the latter case , one can use bivariate mixed models to disentangle the confounding effects of space or time , such that the correlation at the pair level will more closely reflect assortative mating due to mate choice and mating competition [34 , 38] . We found that mate choice is an unlikely driver of size-assortative mating in birds ( Fig 1 ) . The observed effect sizes were much lower than , for instance , the level of assortative mating for body height in humans ( r = 0 . 23 , [6] ) , for which body height is only of secondary importance for mate choice [39] . To robustly examine whether assortative mating arises from ( mutual ) mate choice and mating competition rather than from other processes ( e . g . , “like meets like” or “become alike” ) , an experimental approach is needed in which all individuals can be measured before they mate ( see “experimental study” in Methods ) . Further , such an experimental approach is inevitably needed when groups of individuals ( potential mates ) are spatially and temporally segregated ( nonpanmixis ) . This is because evidence for the role of mate choice requires knowledge about the potential partners available during pair formation ( who encountered whom ) . Also , an experimental approach allows disentangling scenarios in which separation in space or time according to certain phenotypic traits is already a consequence of choosiness ( i . e . , individuals separate in space because they have already been rejected , e . g . , during courtship displays ) . Often , however , an experimental approach is not feasible . In that case , a targeted analysis of binary mating decisions observed in the field ( e . g . , evidence for rejection versus acceptance of an individual ) may provide some clues . In the end , knowledge of a study system and the consideration of possible confounds ( “like meets like” and “become alike” ) may help separating mate choice and mating competition from other causal factors . Assortative mating for certain phenotypic traits may arise from various biological processes and methodological issues . We thus argue for careful consideration of mechanisms and confounding effects . Our results show ( a ) that assortative mating can arise through many processes other than mate choice and mating competition [8 , 13 , 40] and ( b ) that it might be less strong than suggested by meta-analyses of the published literature [7] . In this respect , the value of unpublished data sets deserves greater appreciation .
This study was carried out under the license of Max Planck Institute for Ornithology Animal Care; the Animal Experimentation Committee of the University of Groningen; Mexican authorities , including the administration of Isla Isabel National Park , the National Commission for Protected Natural Areas ( CONANP ) , and the Secretary of Environment and Natural Resources ( SEMARNAT , permit number SGPA/DGVS/08333/10 ) . Field protocols of Barn swallow were noninvasive and adhered to the laws and guidelines of the Czech Republic ( Czech Research Permit number 6628/2008-10001 ) . All protocols were approved by the Animal Care and Use Committees at the Czech Academy of Sciences ( 041/2011 and 09/2015 ) and Charles University in Prague ( 4789/2008-30 ) . The procedures required to collect the data of Tawny owl , which all fall under the ringing licenses of the authors , as provided by the relevant local authorities . Data collection on Western bluebird was carried out in accordance with the recommendations and guidelines approved by University Institutional and Animal Care and Use Committees and under state and federal permitting guidelines . We also assessed assortative mating for size experimentally , using captive populations of Zebra finches ( Taeniopygia guttata ) . As a rule , in each experiment , all birds were measured by a single observer prior to their release into breeding aviaries . Measurements were taken in an order that was independent of allocation to aviaries . This excludes systematic observer error as well as spatial and temporal heterogeneity . To minimize the “scale-of-choice-effect” ( when mate choice exists at a smaller scale than that of the investigator’s sampling , while the trait is heterogeneously distributed at the true scale-of-choice ) [8 , 15 , 59] , we analyzed the degree of assortative mating within aviaries , hence comprising only the birds that were available for pairing at the time of release . To avoid selective reporting , we summarize all available information from our laboratory ( partly published in [25] ) , comprising five experiments that largely fulfill the above criteria ( minor violations of criteria are described in the S1 Text; decisions about inclusion were made blind to the outcome of the analyses ) . The five experiments involved both a domesticated population ( experiments 1–3; [25] ) and a recently wild-derived population ( experiments 4–5 [24]; details are given in the S1 Text ) . In the domesticated population , we released six males and six females in each aviary ( n = 6 , 6 , and 72 aviaries in experiments 1–3 , respectively ) , while in the wild-derived population we used 20 males and 20 females per aviary ( n = 4 and 2 aviaries in experiments 4 and 5 , respectively ) . The formation of pair bonds was deduced from observations of pair-bonding behavior ( allopreening , sitting in body contact , or visiting a nest box together ) . We only considered monogamous pair bonds ( cases of polygyny and polyandry were excluded ) but allowed sequential bonds ( initial pairing followed by divorce and repairing; for details see [25] ) . In total , we recorded 510 pair bonds ( n = 44 , 35 , 342 , 58 , and 31 in experiments 1–5 , respectively ) . For the domesticated population , we measured body mass , tarsus length , and wing length; for the recently wild-derived population , we measured body mass and tarsus length . Thus , we had data for 13 experiment–trait combinations ( 3 x 3 plus 2 x 2 ) . For each of these combinations , we calculated the strength of assortative mating ( Pearson’s r ) within each experimental aviary . To summarize the multiple estimates for the same trait from multiple aviaries within an experiment , we Fisher’s z-transformed the r-values and calculated a weighted average ( weighted by [n − 3]0 . 5 , in which n is the number of pairs , requiring a minimum of four pairs within an aviary ) . The 13 average estimates ( based on 1 , 414 pair–trait combinations; morphological data missing for 27 pair–trait combinations ) were back-transformed to correlation coefficients to allow comparison with all other results . To compare the results across the different methods , we modeled the Pearson’s r estimates as a function of “data source” ( fixed effect with four levels: “Web of Science search , ” “Cited studies , ” previously “Unpublished data , ” and “Experimental data” ) . “Species” and “Study” were added as random effects to account for nonindependence of data . Moreover , different measures of size might yield different levels of assortment , so we fitted “Trait” as a third random effect—seven levels , reflecting the following categories: ( 1 ) body mass , ( 2 ) principal components of size , ( 3 ) bill measurements , ( 4 ) head measurements , ( 5 ) leg measurements , ( 6 ) tail measurements , and ( 7 ) wing measurements . Each Pearson’s r estimate was weighted by ( n − 3 ) 0 . 5 , in which n is the total number of unique pairs contributing to the estimate . The analysis included 392 r-values from “Web of Science search” ( based on 26 , 076 pairs ) , 57 r-values from “Cited studies” ( based on 9 , 515 pairs ) , 32 r-values from the previously “Unpublished data” ( based on 16 , 611 pairs ) , and 13 r-values from “Experimental data” on Zebra finches ( based on 1 , 414 pairs ) . For the previously “Unpublished data , ” we used estimates from the “nearest model 3” because this method is most frequently used in the published literature . Hence , differences between “Web of Science search , ” “Cited studies , ” and previously “Unpublished data” estimates should reflect search and reporting bias rather than methodological differences . To examine the estimates from the literature ( “Web of Science search” and “Cited studies” ) for signs of publication bias , we plotted all 449 correlation coefficients over their sample size ( number of pairs ) and tested for asymmetry in “funnel plots” using the package “meta” in R 3 . 1 . 1 [60] . For comparisons and contrasts between published and previously unpublished data sets , the same plotting was done for previously unpublished data as well . | Research on mate choice in birds has attracted much attention , partly because many birds form monogamous pair bonds like humans do . Human mate choice is characterized by the phenomenon of “like attracts like , ” meaning that partners resemble each other in multiple ways ( “assortative mating” ) . Assortative mating is also frequently reported for birds , but it is unclear whether this in turn implies that birds also have preferences for a similar partner . Here , we show that a range of methodological issues may provide a simpler and more accurate explanation for the frequent observation of assortative mating in birds . First , studies that report assortative mating may achieve greater visibility than studies that yield no such finding . Hence , the scientific literature may be biased toward positive results . Second , in field studies , it is logistically impossible to measure all birds accurately and under standardized conditions . Hence , fluctuations in , for instance , environmental conditions may induce a spurious similarity between partners when these are measured together in space or time . After accounting for such methodological issues , we conclude that mate preferences for a similar partner may be less common than previously thought . | [
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"zebra",
"... | 2019 | Scrutinizing assortative mating in birds |
Oxidative stress is unavoidable for aerobic organisms . When abiotic and biotic stresses are encountered , oxidative damage could occur in cells . To avoid this damage , defense mechanisms must be timely and efficiently modulated . While the response to oxidative stress has been extensively studied in plants , little is known about how the activated response is switched off when oxidative stress is diminished . By studying Arabidopsis mutant paraquat tolerance3 , we identified the genetic locus PARAQUAT TOLERANCE3 ( PQT3 ) as a major negative regulator of oxidative stress tolerance . PQT3 , encoding an E3 ubiquitin ligase , is rapidly down-regulated by oxidative stress . PQT3 has E3 ubiquitin ligase activity in ubiquitination assay . Subsequently , we identified PRMT4b as a PQT3-interacting protein . By histone methylation , PRMT4b upregulates the expression of APX1 and GPX1 , encoding two key enzymes against oxidative stress . On the other hand , PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome . Therefore , we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance .
Sessile plants cannot avoid harsh living conditions such as drought , salinity , cold and hot temperature . These stresses alter the normal cell homeostasis and increase the generation of reactive oxygen species ( ROS ) [1] . ROS could also be generated by paraquat , one widely used herbicide [2] . Accumulation of ROS directly destroys biological membranes and macromolecules , accelerates cell senescence , induces irreversible damages to cells and even leads to cell death [3] . The level of ROS is increased sharply under stress conditions [1 , 4–9] . Two protection systems , enzymatic and non-enzymatic , have evolved to scavenge ROS and protect plant cells from oxidative damage . Enzymatic system mainly includes the ascorbate peroxidase ( APX ) , glutathione peroxidase ( GPX ) , catalase ( CAT ) , superoxide dismutase ( SOD ) , and peroxiredoxin Q ( PRXQ ) [8 , 10 , 11] . The transcript level and activity of antioxidant enzymes correlate with paraquat tolerance [12] . The activation of oxidative response involves many layers of regulations [8 , 13] . Little is known about the regulation by histone methylation of the genes involved in oxidative stress response . Histone methylation plays important roles in the plant development and growth as well as in some stress responses [14–18] . The methylation marks are written on lysines or arginines respectively by protein arginine methyltransferases ( PRMTs ) and histone lysine methyltransferases ( HKMTs ) . In Arabidopsis , nine PRMTs are found in the genome [14] . It has been reported that PRMTs are involved in salt stress responses , flowering time as well as circadian cycle [19 , 20] . Two different types of PRMTs catalyze asymmetric di-methylation ( ADMA ) and symmetric di-methylation ( SDMA ) on the Arg residues , respectively [17 , 21] . The prmt5 mutant has decreased level of histone H4 Arg3-SDMA , leading to enhanced drought tolerance [22] . A pair of PROTEIN ARGININE METHYLTRANSFERASE4 ( PRMT4 ) homologs , AtPRMT4a and AtPRMT4b , is required for the asymmetrical di-methylation of Arg-2 , Arg-17 , and Arg-26 in histone H3 [14] . The prmt4aprmt4b double mutants are sensitive to salt stress [20] . Protein arginine methylation plays essential roles in diverse biological processes , such as RNA processing and transcriptional regulation [21 , 23] . Oxidative stress could be perceived by multiple mechanisms , including sensor or cellular receptor . The perception by receptors results in the activation of Ca2+–calmodulin and mitogen-activated protein kinase ( MAPK ) cascade signaling transduction pathway . The activation or suppression of different transcription factors regulates a variety of defense pathway subsequently , such as ROS-scavenging , heat-shock proteins ( HSPs ) , and photosynthesis [8 , 10 , 13] . Several paraquat tolerance mutants have been analyzed in Arabidopsis [24] . While much attention has been paid to how plants respond to oxidative stress , we know little about how plants switch off the activated responses when stress is diminished . A common regulatory mechanism is to control the protein level of the stress responsive factors . The most studied mechanism of protein degradation is the ubiquitin/26S proteasome system [25] . The ubiquitin/26S proteasome pathway is involved in different regulatory processes of eukaryotic cells , as it can rapidly eliminate the specific proteins in the cell [25–27] . Ubiquitin , containing 76 amino acids , could be attached to the target protein under the action of three different enzymes [27] . The ubiquitin system can identify and modify many intracellular proteins , such as proteins involved in signal transduction , transcription factors , and receptors on cell surface , to participate in the regulation of physiological processes [28] . The target proteins with ubiquitin have different fates . For monoubiquitination , one lysine residue of substrate is modified by a single ubiquitin . If several individual lysine residues of target protein are attached with single ubiquitin respectively , the protein modification is named multiubiquitination . Both mono- and multi-ubiquitination could affect protein activity and intracellular localization [29–31] . For polyubiquitination , one ubiquitin is attached to lysine residue of substrate firstly . The C-terminal glycine of next ubiquitin is linked with lysine residue of the preceding ubiquitin to form polyubiquitin chain subsequently [32] . As ubiquitin contains seven lysines , polyubiquitin chains are divided into different types according to different linkages between two adjacent ubiquitins [33] . Proteins with Lys48-Linkage polyubiquitination could be recognized and degraded by the 26S proteasome [34] . It has also been reported that Lys29-Linkage polyubiquitination involved in proteasome-dependent degradation [35] . In addition , Lys63-Linkage polyubiquitination plays roles in endocytosis , repair of DNA damage , protein synthesis and signal transduction [36 , 37] . In the ubiquitin degradation process , E3 played a crucial role . E3 is responsible for specific recognition of substrate protein and accurate positioning of the binding site between substrate protein and ubiquitin [38] . In Arabidopsis thaliana , the genes encoded the subunits of E3 ubiquitin ligases make up approximately 90 percent of about 1400 genes encoded the components of ubiquitin/26S proteasome pathway . [26 , 39] . The large and diverse family of plant E3 ubiquitin ligases can be divided into HECT domain- and RING/U-box domain-containing E3 ubiquitin ligases [25] . The HECT family is relatively small compared with the RING domain-containing family that contains several hundreds of proteins and can be further divided into single subunit RING/U-box E3 ligases and multi subunit RING E3 ligases [26 , 40] . The large number of E3 ubiquitin ligases in higher plants indicates their important regulatory roles in diverse biological processes [41] . Several RING E3 ligases play positive roles in ABA-mediated drought tolerance [42–45] . In addition , overexpression of RING E3 ligases Rma1 inhibited the trafficking of aquaporin PIP2;1 and promote protein degradation of PIP2;1 to enhance drought tolerance [46] . A few E3 ligases have also been identified to turn off the activated stress responses [47] . HOS1 , as a RING E3 ligase , acts as a negative regulator in cold tolerance . HOS1 inhibits the expression of CBFs and downstream cold-responsive genes through the degradation of ICE1 in ubiquitin-proteasome pathway [48–50] . Both PUB22 and PUB23 , as E3 ligases contained U-box , play roles as negative regulators in drought tolerance . They could recognize and degrade RPN12a , a subunit of 19S regulatory particle belonged to 26S proteasome , to affect Arabidopsis drought tolerance [51] . The negative function of PUB18 and PUB19 in ABA-mediated drought tolerance has also been reported [52] . The drought-induced AtERF53 could be degraded by RING E3 Ligase RGLG1 and RGLG2 that acts as negative regulators in drought tolerance [53] . Overexpression of salt-induced RING E3 ligase OsSIRP1 reduced salt tolerance in Arabidopsis [54] . In addition , several substrate receptors of CUL4 E3 ligases , DWAs , regulate ABA responses negatively [55 , 56] . The function of an enormous number of E3 ligases still remains to be identified . Here we report a novel negative regulator of oxidative stress response by PARAQUAT TOLERANCE3 ( PQT3 ) in Arabidopsis . We identified a paraquat tolerant mutant , paraquat tolerance3 ( pqt3 ) , and cloned the gene PQT3 that encodes an E3 ligase containing RING/U-box domain . The function of PQT3 was revealed , although PQT3 ( At4g17410 ) had been identified in previous microarray data on salt stress response and cold acclimation [57 , 58] . The expression of APX1 and GPX1 was up-regulated in pqt3 , while PQT3 was down regulated by oxidative stress . PQT3 was able to interact with PRMT4b . PRMT4b may catalyze histone methylation on APX1 and GPX1 chromatin and up-regulate their expressions , therefore protect plants from oxidative stress . When oxidative stress is diminished , PQT3 level increases and acts as E3 ubiquitin ligase to specifically target PRMT4b for degradation . Based on our results , PQT3 is a negative regulator that turns off the activated response of oxidative stress .
The mutant pqt3 was obtained after screening an activation-tagging library [59] . This library consisting of approximately 55 , 000 independent lines was screened for mutants with enhanced tolerance to different stresses [60 , 61] . To isolate tolerant mutant to oxidative stress , we germinated seeds on MS medium with 2 μM paraquat . Growing green seedlings were selected as putative mutants and were named paraquat tolerance ( pqt ) because of their enhanced tolerance to paraquat . The pqt3 , one of such mutants , was further characterized and marked as pqt3-1 . The enhanced oxidative tolerance of pqt3-1 mutant was confirmed by germinating seeds on MS medium containing 0 or 2 μM paraquat . Based on the observation of green cotyledons , survival ratio were counted . In presence of paraquat , more than 60% pqt3-1 seeds germinated with green cotyledons but only 2% wild type seeds did , while all seeds of both wild type and pqt3-1 survived on MS medium without paraquat ( Fig 1A and 1B ) . Genetic analysis showed that the mutation was recessive . All F1 backcross offsprings ( pqt3-1 x wild type ) were paraquat sensitive and F2 selfing population showed typical 3:1 segregation ratio ( sensitive: resistant; 85:27 , χ2 = 0 . 0476 ) . The result suggested that pqt3-1 mutant may have a more efficient mechanism of ROS scavenge , which was caused by loss-of-function mutation in a single nuclear gene PQT3 ( At4g17410 ) . In pqt3-1 mutant , a single T-DNA insertion was located in the fourth intron of At4g17410 ( S1A Fig ) . The exact integration site of the T-DNA right border was 803bp downstream of the ATG initiation codon of At4g17410 . As a result , the expression of At4g17410 was completely disrupted as confirmed by RT-PCR analysis ( S1B and S1D Fig ) . The expressions of its neighboring genes , At4g17390 and At4g17420 , were not affected ( S1B Fig ) . The At4g17410 locus includes 13 exons and 12 introns . By prediction , the open reading frame encodes a 91 kD polypeptide composed of 827 amino acids . Based on the conserved RING/U-box domain , this protein is predicted as an E3 ubiquitin ligase . To further determine whether the loss of At4g17410 resulted in the enhanced oxidative tolerance of pqt3-1 mutant , we used another allele of pqt3 , the T-DNA insertion mutant Salk_065409 , which was ordered from Arabidopsis Biological Resource Center ( ABRC ) and its T-DNA insertion was confirmed by RT-PCR ( S1A , S1C and S1D Fig ) . As the first identified pqt3 mutant was named as pqt3-1 , the Salk_065409 was marked as pqt3-2 . The pqt3-2 mutant showed similar enhanced oxidative tolerance to paraquat and had high survival ratio under different concentrations of paraquat treatment as pqt3-1 did ( Fig 1C ) . The survival ratio of pqt3-1 and pqt3-2 were 50% and 20% , respectively , under 2 μM paraquat treatment , while none of the wild type seedlings survived under the same condition . In addition , both pqt3-1 and pqt3-2 showed a late-flowering phenotype ( Fig 1D ) . To confirm further , we generated functional complementation ( FC ) lines and 35Spro:PQT3 overexpression lines ( S1E and S1F Fig ) . FC lines and 35Spro:PQT3 lines showed similar if not higher paraquat sensitivity to wild type under 2 μM paraquat treatment while the pqt3-1 and the pqt3-2 mutants displayed enhanced paraquat tolerance ( Fig 1E and 1F ) . These results indicate that PQT3 is a negative regulator of oxidative stress tolerance and is responsible for the phenotype of pqt3 mutants . In addition , we assayed H2O2 level in the leaf with DAB staining , in which the chemical reaction between hydrogen peroxide and DAB lead to the formation of brown precipitate that indicates hydrogen peroxide distribution and oxidative damage . After 6 μM paraquat treatment for 12 or 24 hours , the result of 3 , 3′- diaminobenzidine ( DAB ) staining showed that the brown precipitate in the leaves of the wild type was more than that of pqt3 mutants ( Fig 1G–1R ) . As several stresses could cause oxidative damage to plants , the sensitivity of pqt3 mutants to other environmental stresses was analyzed subsequently . The result indicated that pqt3 mutants have enhanced tolerance to CdCl2 , mannitol , NaCl , and drought stress ( S2 Fig ) . To investigate the spatiotemporal pattern of PQT3 expression , we generated PQT3pro:GUS reporter lines . GUS staining results showed that PQT3 was expressed in both shoot and root tissues under normal condition ( Fig 2A–2G ) . GUS expression was detected in the root tissues at all developmental stages we analyzed ( Fig 2A–2C ) . For the 1-week-old seedlings , strong GUS staining was observed in the cotyledons , hypocotyls and root tissues ( Fig 2A ) . For the 3-week-old seedlings , strong GUS staining was also detected in cotyledons , young leaves , and root tissues , but weakly stained in older leaves ( Fig 2B ) . In 7-week-old adult plants , GUS expression was detected in rosette leaves , cauline leaves , the tip and basal junction of siliques , and was significantly higher in the flower petals , stamens and stigma of pistil ( Fig 2D–2G ) . PQT3 has two predicted nuclear localization signals ( NLSs ) in the carboxyl terminus ( S3 Fig ) , implicating its nuclear localization . To confirm this , the 35Spro:PQT3-GFP construct was made and transiently expressed in onion epidermal cells . The 35Spro:GFP construct was used as control ( Fig 2H–2J ) . PQT3-GFP signal was indeed detected in the nucleus ( Fig 2K–2M ) . For further confirmation , we transformed the PQT3pro: PQT3-GFP fusion construct into the Arabidopsis and obtained transgenic plants . Fluorescent microscopy results showed that GFP signal was accumulated in the nucleus of root cells ( Fig 2N–2P ) , which is in agreement with the presence of two NLSs of the PQT3 . The expression of PQT3 was rapidly down-regulated by paraquat treatment and maintained at a low level as long as the paraquat treatment was applied ( Fig 3A ) . This result is supportive for our previous opinion that PQT3 is a negative regulator of plant oxidative tolerance . The suppressed transcript level of PQT3 was restored to the previous level when PQ stress diminished ( S4 Fig ) . By extrapolation , the expression of PQT3 could be down-regulated by other stress conditions . Indeed , our results showed that the expression of PQT3 was down-regulated by H2O2 ( Fig 3B ) , mannitol ( Fig 3C ) , drought ( Fig 3D ) and CdCl2 ( Fig 3E ) at the indicated time points . Among these stresses , CdCl2 treatment led to the most significant reduction of the expression of PQT3 ( Fig 3E ) . The expression of PQT3 could also be down-regulated by NaCl stress at 3h . However , unlike the above results , the expression of PQT3 was activated by salt treatment at other time points ( Fig 3F ) . The PQT3pro:GUS pattern was observed under different stresses subsequently ( Fig 3G–3DD ) . Compared with transgenic seedlings under normal conditions , GUS staining was weaker in the seedlings under paraquat , H2O2 , mannitol , CdCl2 , and NaCl treatment . The changed PQT3pro:GUS pattern was consistent with the altered expression of PQT3 detected by quantitative RT-PCR under different stresses . Enzymatic protection systems are very important for ROS elimination . The transcript levels of ascorbate peroxidase ( APX ) , glutathione peroxidase ( GPX ) , catalase ( CAT ) , cytosolic Cu/Zn SOD ( CSD1 ) , plastidic Cu/Zn SOD ( CSD2 ) , FeSOD ( FSD ) , atypical Cys-His rich thioredoxin ( ACHT ) , glutaredoxin C ( GRXC ) , 2-Cys peroxiredoxin B ( 2CPB ) , peroxiredoxin Q ( PRXQ ) and mitochondrial MnSOD ( MSD ) were analyzed by quantitative RT-PCR in pqt3 and wild type . The results show that transcript levels of APX1 and GPX1 were up-regulated in pqt3 under normal conditions compared with that in the wild type ( Fig 4A–4J ) . The elevated transcript levels of APX1 and GPX1 may contribute to the improved oxidative tolerance . The enzyme activity of APX and GPX in wild type and pqt3 mutant was also detected . The pqt3 mutant had higher enzyme activity of APX and GPX compared with wild type ( Fig 4K and 4L ) . To study the molecular mechanisms that underlie the enhanced stress tolerance of pqt3 , we screened cDNA library for potential candidate target proteins of PQT3 using yeast-two-hybrid ( Y2H ) . Several proteins were isolated from the screen . Among these candidate interactors , PRMT4b , a member of arginine methyl transferase family , was frequently presented . To reveal the domain of PQT3 responsible for the interaction with PRMT4b , the PQT3 protein was divided into four parts: N-terminal DWNN , zfCCHC , U-box ( RING finger ) , and C-terminal section containing the NLS1 and NLS2 domains , based on the predicted domains of PQT3 protein ( S3A Fig ) . Full-length PQT3 and four protein sections were used for Y2H assay as baits . Full-length protein and the C-terminus of PQT3 were able to interact with PRMT4b in Y2H assays ( Fig 5A ) . The interaction between PQT3 and PRMT4b was confirmed by colonies that grew on the SD-Leu-Trp-His plate with 50 mM 3-amino-1 , 2 , 4-triazole ( 3-AT ) and displayed the blue color in X-gal assay ( Fig 5A ) . However , PRMT4a was never isolated in the screening . The Y2H assay was also performed to further study the potential interaction between PQT3 and PRMT4a , since PRMT4a is a close related gene of PRMT4b . The result showed that the PQT3 did not interact with PRMT4a ( S5 Fig ) . The interaction between PQT3 and PRMT4b was further confirmed by protein pull-down assay in vitro . MBP-PQT3-C66 protein containing the NLS domain ( S3A Fig ) and His-PRMT4b protein were expressed in E . coli and purified subsequently . His-PRMT4b was incubated with amylose resin bound with recombinant MBP-PQT3-C66 protein . Pulled-down protein complex was detected by SDS-PAGE ( Fig 5B ) and western blotting using anti-His antibody ( Fig 5C ) . The pull-down result clearly shows that PQT3 interacts with PRMT4b in vitro . To determine whether the interaction also occur in vivo , we used the bimolecular fluorescence complementation ( BiFC ) system . The N-terminus of yellow fluorescent protein ( YFP ) was fused to full-length PQT3 cDNA , while full-length PRMT4b cDNA was fused to the C-terminal region of YFP . The empty plasmids were used as negative controls . Different plasmid combinations were co-infiltrated into epidermal cell of N . benthamiana leaves . The yellow fluorescence was observed in epidermal cell contained both NE-PQT3 ( the N-terminus of YFP fused with PQT3 ) and CE-PRMT4b ( the C-terminus of YFP fused with PRMT4b ) ( Fig 5D ) . No fluorescence was observed from the negative controls ( NE-PQT3/CE , NE/CE-PRMT4b and NE/CE ) ( Fig 5D ) . The nuclei were stained by Hoechst and detected by confocal . These results indicate that PQT3 can interact with PRMT4b in the nucleus of plant cell . Not all the proteins with the predicted RING domain function as an ubiquitin ligase [62] . The E3 activity of PQT3 was determined via self-ubiquitination system . Both full-length ( GST-PQT3 ) and C-terminal deletion ( GST-PQT3-N40 ) proteins showed the E3 ubiquitin ligase activity ( Fig 6A and S3B Fig ) . The ubiquitinated bands of PQT3 were detected by western blotting in the presence of E1 ( from wheat ) , E2 ( UBCh5b , from human ) , and 6×His-tagged ubiquitin ( UBQ14 , from Arabidopsis ) . When any of essential reaction components was missing , self-ubiquitination of PQT3 was not detected ( Fig 6A ) . To determine whether PRMT4b is a substrate recognized by PQT3 , we resorted to the in planta ubiquitination assay [63] . Leaf infiltration was conducted via Agrobacterium tumefaciens strains containing different combination of constructs . The infiltrated parts of N . benthamiana leaves were harvested . Total protein was extracted and detected via western blotting with anti-HA antibody . A smear of bands , which were larger than the size of HA-PRMT4b and showed the features of ubiquitinated form of the PRMT4b proteins , could be detected by anti-HA antibody in the samples co-infiltrated with PQT3 and HA-PRMT4b ( Fig 6B ) . The cell lysates were immunoprecipitated with anti-HA antibody subsequently . Immunoprecipitated samples were detected via western blotting with anti-ubiquitin antibody . In the PQT3-PRMT4b co-infiltration sample , these high molecular size bands could also be detected by anti-ubiquitin antibody ( Fig 6B ) . These results indicated that these high molecular size bands were ubiquitinated forms of PRMT4b . PQT3 protein could ubiquitinate the PRMT4b protein in tobacco . The decline of PRMT4b protein was also found in the samples co-infiltrated with PQT3 and HA-PRMT4b ( Fig 6B ) . In addition , PRMT4b protein levels in different PQT3 genetic background under MG132 treatments were also supportive of PRMT4b as a substrate of PQT3 . As shown in the Fig 6C and 6D , the protein level of PRMT4b in pqt3-1 was higher than that of the wild type under normal conditions . Under paraquat treatment , it remained lower in wild type than that of pqt3-1 at the same time point , although protein level of PRMT4b increased gradually in wild type and pqt3-1 with the prolonged paraquat treatment ( Fig 6C and 6D ) . The transcript level of PRMT4b was unlikely to cause the observed difference of protein levels between the mutant and wild type ( S6 Fig ) . Under MG132 treatment , the protein level of PRMT4b was increased in both wild type and pqt3-1 mutant . When the seedlings were co-treated with paraquat and MG132 for 12 h , PRMT4b was accumulated in both wild type and pqt3-1 mutant , and no significant difference of PRMT4b protein level was found between the wild type and pqt3-1 mutant ( Fig 6C and 6D ) because the degradation of the PRMT4b protein through ubiquitination-26S proteasome pathway was inhibited by MG132 . In order to confirm further the PQT3-dependent ubiquitination of PRMT4b , the phenotype of wild type and pqt3-1 mutant treated without or with paraquat in presence or absence of proteasome inhibitor MG132 was studied . Survival ratio of pqt3-1 seedlings was higher than that of wild type under paraquat treatment . No significant difference of survival ratio could be observed when the wild type and pqt3-1 seedlings were co-treated with paraquat and MG132 ( Fig 6E ) . The wild type gained enhanced paraquat tolerance under MG132 treatment to the level of pqt3-1 mutant . To reveal whether PRMT4b could play any roles in oxidative tolerance of plants , we obtained the prmt4b mutant and 35Spro:PRMT4b lines ( S1G , S1H and S1L Fig ) and observed the phenotypes of prmt4b and 35Spro:PRMT4b under different concentrations of paraquat treatment . Survival ratio of wild type , pqt3-1 , pqt3-2 , prmt4b and 35Spro:PRMT4b were counted ( Fig 7A ) . 35Spro:PRMT4b had similar phenotype as pqt3-1 and pqt3-2 , while the prmt4b mutant was more sensitive to paraquat treatment than wild type ( Fig 7A ) . Under CdCl2 treatment , primary root elongation of prmt4b mutant was also slower than that of wild type ( Fig 7B ) . Furthermore , the overexpression lines of PRMT4b were analyzed under other stress conditions . 35Spro:PRMT4b increased tolerance to CdCl2 and NaCl stresses compared with wild type . The 35Spro:PRMT4b showed the opposite phenotype of the 35Spro:PQT3 which was more sensitive to CdCl2 and NaCl stresses as compared with wild type ( S7 Fig ) . These results show that PRMT4b is a positive regulator for plant oxidative tolerance , which is also consistent with the function of PQT3 . The prmt4a mutant and prmt4aprmt4b double mutants were subsequently examined ( S1I–S1K Fig ) . The prmt4aprmt4b double mutants had the similar phenotype to prmt4b under paraquat treatment , and the knockout of PRMT4a did not affect the oxidative tolerance ( Fig 7C–7E ) . In addition , the transcript levels of APX1 , GPX1 and other antioxidant enzyme genes were down-regulated in prmt4b mutant and up-regulated in 35Spro:PRMT4b as compared with that in the wild type ( Fig 7F and S8 Fig ) . The transcript level of APX1 and GPX1 was higher in pqt3 than that in wild type under normal conditions ( Fig 4A and 4B ) , which could be regulated by PRMT4b . The modification status of H3R17me2a in the chromatin of APX1 and GPX1 was compared between wild type and pqt3 mutant via chromatin immunoprecipitation ( ChIP ) assays . ChIP assays were performed with wild type and pqt3 plants using antibody against H3R17me2a . As shown in the Fig 8A and 8B , the APX1 and GPX1 chromatin was divided into different regions and the enriched chromosome fragments were detected by quantitative RT-PCR . The results showed that histone H3R17me2a modification of APX1 and GPX1 chromatin was increased in pqt3 mutant ( Fig 8C and 8D ) . The enriched chromosome fragments of APX1 ( C , D and E fragments ) and GPX1 ( C , D and I fragments ) were further analyzed in prmt4b mutant and pqt3prmt4b double mutants without or with paraquat treatment ( Fig 8E–8J ) . Histone H3R17me2a modification of APX1 ( C , D and E fragments ) and GPX1 ( C , D and I fragments ) chromatin was decreased in prmt4b mutant and pqt3prmt4b double mutants without paraquat treatment as compared with wild type . It remained lower in wild type than that of pqt3 mutant , although H3R17me2a modification of APX1 ( C , D and E fragments ) and GPX1 chromatin ( C , D and I fragments ) in both wild type and pqt3 mutant were enhanced under paraquat treatment . H3R17me2a modification of APX1 ( C , D and E fragments ) and GPX1 chromatin ( C , D and I fragments ) was kept at a low level , although the modification in prmt4b mutant and pqt3prmt4b double mutants could also be affected by paraquat treatment . These results suggest that PRMT4b may target APX1 and GPX1 to enhance the oxidative tolerance by increasing asymmetric dimethylation of H3 at R-17 in APX1 and GPX1 chromatin . To confirm further that PRMT4b is the target of PQT3 , the pqt3prmt4b double mutants were obtained ( S1M and S1N Fig ) . The survival ratio of pqt3prmt4b under paraquat treatment was intermediate between pqt3 and prmt4b , which demonstrates that the PRMT4b protein is one of the targets of PQT3 and suggests that PQT3 may also target other proteins that contribute to the tolerance to oxidative stress ( Fig 9A–9C ) . We also obtained the pqt3prmt4a double mutants ( S1O and S1P Fig ) and found that the survival ratio of pqt3prmt4a under paraquat treatment had no significant difference from that of pqt3 mutant ( Fig 9D–9F ) , again indicating that PRMT4a is not involved in oxidative stress response .
PQT3 is a member of Arabidopsis RING-finger/U-box E3 ligase family . Secondary structure prediction using InterProScan protein sequence analysis software revealed four conserved domains including DWNN ( domain with no name ) , zinc finger domain , RING-finger domain , and U-box domain in N-terminus of PQT3 protein ( S9A and S9B Fig ) . DWNN is a novel ubiquitin-like domain , which is a highly-conserved domain in eukaryotic plants and animals [64] . The DWNN domain of PQT3 contains 76 amine acids . DWNN domain is only found in the N-terminus of the members in the splicing-associated RBBP6 ( Retinoblastoma Binding Protein 6 ) protein family [64] . The RING-finger domain was also found in RBBP6 protein family . The existence of the RING-finger domain suggests that DWNN domain may play its role as ubiquitin-like regulatory factors [64] . CCHC-type Zinc finger domain is also known as zinc knuckle , which can be found in a large number of RNA binding proteins [65 , 66] . As mentioned above , RING-finger domain can combine with the E2 in the cascade reaction of ubiquitination system , while U-box is a modified RING-finger [67] . In addition , two predicted NLS sequences ( 471–477 and 696–711 amino acids ) were also found in the C-terminus of PQT3 , which is consistent with nuclear localization of the protein ( Fig 2H–2P and S9A and S9B Fig ) . Phylogenetic tree analysis further revealed the high homology proteins of PQT3 in other species ( S9C Fig ) . DWNN domain and RING-finger/U-box domain in N-terminus of PQT3 were highly conserved in homologous proteins in different plant species ( S10 Fig ) . The PQT3 may play its role as an ubiquitin ligase in different species and its function may be conserved throughout the plant kingdom . In vitro ubiquitination assay shows that PQT3 has the E3 ligase activity ( Fig 6A ) . We noticed that the in vitro activity was not as high as expected , which could be contributed by suboptimal reaction conditions such as E2 source . This E3 activity was confirmed by in planta ubiquitination assay , in which PRMT4b protein could be ubiquitinated by expressed PQT3 protein in N . benthamiana ( Fig 6B ) . Consistent with this result , pqt3-1 mutant had higher protein level of PRMT4b than the wild type , as the PRMT4b protein was degraded by PQT3 in wild type under normal conditions ( Fig 6C and 6D ) . The transcript level of PRMT4b could be induced in wild type under paraquat treatment ( S6 Fig ) , while the transcript level of PQT3 was decreased by paraquat treatment ( Fig 3A ) . In this case , PQT3-mediated PRMT4b degradation could be weakened in wild type . Consequently , the level of PRMT4b protein in wild type was elevated , but lower than that of pqt3 ( Fig 6C and 6D ) . The proteasome inhibitor MG132 could block the degradation of the PRMT4b protein in wild type and enhance paraquat tolerance of wild type ( Fig 6C–6E ) . Taken together , PQT3 , as an E3 ubiquitin ligase , play its role in oxidative tolerance through the ubiquitination-degradation of PRMT4b . A series of environmental stress could lead to oxidative damage to plants [10 , 13 , 68] . Both biotic stress and abiotic stress result in the production of ROS which in excess cause oxidative stress . Besides paraquat tolerance , pqt3 mutants have enhanced tolerance to various environment stresses ( Fig 1 and S2 Fig ) . Further studies may reveal new mechanisms of PQT3 in multiple stress tolerance . Analysis of PQT3-interacting proteins may be a good start point to further understand the function of PQT3 . In the screen for PQT3-interacting proteins with Y2H , we isolated 66 positive colonies based on the expression of reporter genes included His3 and LacZ . Several proteins were identified from these positive colonies repeatedly . The most frequent interacting partner is 20S core protease subunit of 26S proteasome . The result suggested that PQT3 act as an E3 ubiquitin ligase . Among these proteins , PRMT4b , as previously mentioned , may be responsible for the increased degree of Arg-17 methylation which may further regulate APX1 and GPX1 genes to enhance oxidative tolerance of plants ( Figs 4A , 4B and 8 ) . It has been reported that arginine methylation was involved in signal transduction , transcriptional control , DNA repair , RNA processing , and nuclear transport [17 , 19 , 23 , 69–72] . The functions of PRMTs have been extensively analyzed [15 , 19 , 20] . Here , the prmt4b mutant was found to be more sensitive to paraquat and CdCl2 treatment than wild type ( Fig 7A–7E ) . Thus , PRMT4b plays a role in oxidative stress response of plants . It is known that PRMT4-mediated methylation at Arg-17 of histone H3 is linked to transcription activation [73] . The Arg-17 of histone H3 is the major site of PRMT4-mediated methylation , although it was reported that other protein site could also be methylated by PRMT4 [74–76] . We suggest that the increased Arg methylation degree of specific regions in APX1 and GPX1 chromatin was caused by PRMT4b ( Fig 8 ) . The increased transcript level of APX1 and GPX1 may be caused by PRMT4b-mediated histone Arg methylation ( Figs 4A , 4B and 8 ) . In presence of ascorbate as electron donor , the cytosolic enzyme , APX1 , catalyze the degradation of H2O2 [77] . The response of APX1 to oxidative stress has been studies in Arabidopsis . The crucial role of APX1 in multiple stress response was reported [78] . It could be activated by multiple stresses to protect plants against oxidative stress [79–81] . The tobacco overexpressing APX1 could be more tolerant against UV-C-caused oxidative damage [82] . GPXs also have important functions in oxidative signaling , which can protect plants from harmful effects of excessive oxidation [83] . It has also been reported that enhanced peroxide scavenging and decreased oxidative damage was found in transgenic tobacco seedlings overexpressing tobacco glutathione S-transferase that showed glutathione peroxidase ( GPX ) activity [84] . The pqt3 mutants also have late-flowering phenotype ( Fig 1D ) . It has been reported that prmt4aprmt4b double mutants display late-flowering phenotype [15] . The Y2H result showed that the PQT3 can not interact with PRMT4a ( S5 Fig ) . As compared with wild type , the prmt4a mutant also has no significant difference in the oxidative tolerance ( Fig 7C–7E ) . The phenotype of pqt3prmt4a double mutants demonstrate that PRMT4a protein was not involved in the response regulation of oxidative stress by PQT3 ( Fig 9D–9F ) . The late-flowering phenotype of pqt3 may be regulated by other mechanisms , rather than PRMT4a and PRMT4b . The flowering-related transcription factor AGAMOUS ( AG ) was found to be a potential target interacted with PQT3 in Y2H library screening . AG is involved in carpel development , leaf development , identification of floral organs , and stamen development [85] . Targeted removal of AG by PQT3 may be related to the late-flowering phenotype of pqt3 . In addition , one member of a large G protein family and the proteins with unknown function were also found to be potential targets interacted with PQT3 in Y2H library screening . By interacting with different partners , PQT3 may mediate multiple functions in diverse biological processes . The pqt3 has enhanced tolerance to multiple stresses . PQT3 is also down-regulated by various oxidative stresses . PQT3 act as negative regulator in multiple stress responses . The interacting partner will be further analyzed to reveal the molecular function of PQT3 . As research continues , other combinations , also targeted by PQT3 , may be revealed , where PQT3 act as a positive regulator . We could improve crop tolerance to multiple stresses through PQT3 mutation . More candidate genes could be further determined from the potential targets interacted with PQT3 . These genes may also have high application value after the revelation of molecular mechanisms . In conclusion , paraquat may enter the cell through plasma membrane-localized paraquat transporters PDR11 and RMV1 [59 , 86] . The intracellular paraquat may further activate different downstream signaling pathways to regulate the expression of PQT3 and PRMT4b . In addition , paraquat stress increases the generation of ROS . ROS is also an important signal molecule that mediate the responses to environmental stress [87] . ROS-activated signaling pathways may also be responsible for the regulation of PQT3 and PRMT4b . The oxidative stress activates the expression of PRMT4b , represses the expression of PQT3 , and weakens PQT3-mediated the ubiquitinated degradation of PRMT4b , synergistically resulting in increased accumulation of PRMT4b . Consequently , the increased level of PRMT4b protein may lead to higher degree of histone methylation on the APX1 and GPX1 chromatin . As a result , the transcription of APX1 and GPX1 is activated , leading to more APX1 and GPX1 enhancing oxidative tolerance of plants . When the stress disappears , transcription repression of PQT3 by oxidative stress is removed . The function of PQT3 , as a negative regulator of oxidative stress response , is restored . The PRMT4b is then degraded by PQT3 in ubiquitination pathway . The activated response to oxidative stress is switched off . We propose a working model for PQT3 as a negative regulator of oxidative stress response ( Fig 10 ) .
Approximately 55 , 000 individual lines were screened for paraquat tolerant mutant , which came from the activation-tagging library constructed with Arabidopsis Columbia ecotype and pSKI015 vector [59] . All Arabidopsis transgenic lines and mutants were based on the genetic background of Columbia ecotype used as wild type in the study . Salk_065409 ( pqt3-2 ) , Salk_097442C ( prmt4b ) and Salk_033423 ( prmt4a ) were obtained from ABRC ( Arabidopsis Biological Resource Center ) . The seeds were sterilized in 10% bleach for 10 min . Then the seeds were washed for 5 times at least with sterile water . For vernalization , the seeds were kept in the dark with water at 4°Cfor 3 days to ensure the synchronous germination . Sterile seeds were germinated on MS medium . The seedlings were grown at 22°C under 16-h-light /8-h-dark cycle with light intensity 100 μE m-2 s-1 . The constructs were electroporated into competent cell of Agrobacterium tumefaciens C58C1 . The floral-dip method was used to transfer these constructs into Arabidopsis as described [88 , 89] . The different tissues of plants were used for RNA extraction with Trizol method . Then RNA reverse reaction was carried out by TransScript Kit ( TransGen Biotech ) . Specific primers were designed for RT-PCR analysis and the PCR products were detected by agarose gel electrophoresis . Applied Biosystem Step One real-time PCR system was used for the quantitative RT-PCR detection with specific primers listed in the S1 Table and Premix Ex Taq II SYBR ( TaKaRa ) . UBQ5 was used as the internal control . The identification of homozygous mutants was performed by genomic PCR for T-DNA insertion lines: Salk_065409 , Salk_097442C and Salk_033423 [90] . RT-PCR was carried out as previously described to confirm the results of genomic PCR . The 35Spro:PQT3 and 35Spro:PRMT4b plasmids were transformed into Col-0 to obtain the overexpression lines of PQT3 and PRMT4b . For the FC line , the 35Spro: PQT3 was transformed into pqt3 mutant , and the line with the same expression level of PQT3 as wild type was chosen and used as FC line . 35Spro: PQT3 , 35Spro: PRMT4b and FC line were identified by glufosinate screening and quantified by RT-PCR or quantitative RT-PCR . The seeds were germinated on MS medium containing different concentrations of paraquat , mannitol , CdCl2 and NaCl , respectively . The phenotype was observed and survival ratio was scored at the indicated time points . For drought tolerance assay , the wild type , pqt3-1 mutant and pqt3-2 mutant seeds were germinated in one pot at same density . The 15-day-old seedlings were used for drought tolerance assay . Watering was withheld for another 15 days before re-watering . The photos were taken before re-watering and after re-watering for 1 day and 7 days . The survival ratio was scored after re-watering for 1 day and 7 days . DAB staining was performed as described [91] . DAB staining solution ( pH 6 . 0 ) was prepared by adding 0 . 05% ( v/v ) Tween-20 and 10 mM Na2HPO4 to the DAB solution ( 1 mg/ml DAB , pH3 . 0 ) . For each treatment condition , at least 3 leaves per plant were obtained from 3 independent plants for each line ( Col-0 , pqt3-1 and pqt3-2 ) . Arabidopsis leaves from different lines were treated using MS liquid medium without or with 6 μM paraquat for 12 hr or 24 hr . These leaves were stained in 6-well culture plates with DAB staining solution subsequently and 10 mM Na2HPO4 ( pH 6 . 0 ) was used as the negative control . The 6-well plates were covered with aluminum foil and placed on a shaker for 4–5 h . Follow the incubation , the bleaching solution ( glycerol: acetic acid: ethanol = 1:1:3 ) was used in the discoloration after DAB staining solution was removed . The 6-well plates were placed into boiling water bath at 95°C for 15–20 min . Discoloration process was repeated using fresh bleaching solution . The brown precipitate caused by the reaction between DAB and H2O2 could be observed on the leaves . Photos were taken using a camera . Special attention should be paid to light avoidance through the whole operation . The experiment was repeated for three times . The promoter of PQT3 was cloned into pCB308R [92 , 93] . The transgenic lines containing PQT3pro: GUS were isolated by glufosinate screening . The T2 population was used for GUS staining . Histochemical staining for GUS activity in Arabidopsis was carried out as described previously [94] and GUS staining solution was prepared as described before [59] . The experimental materials were incubated in staining solution at 37°C . Then Arabidopsis tissues were destained and stored in 70% ethanol . The GUS activities of individual parts were observed via a light microscope Axio skop2 plus ( ZEISS , Germany ) with a video camera . The full length CDS of PQT3 was cloned into the binary vector pCB2008E to construct the PQT3-GFP fusion vector [93] . After the detection of gene sequencing for inserted sequence , the recombinant plasmid was transducted into the epidermal cells of onion along with particle gun bombardment for transient expression assay . PQT3-GFP was also transferred into Col-0 via floral-dip method to create transgenic plant for the analysis of PQT3-GFP fusion protein localization . ZEISS fluorescence microscope ( Axio skop2 plus ) with video camera was used to observe the green fluorescence in both the epidermal cells of onion and the root cells of Arabidopsis transgenic lines . For APX and GPX enzyme activity assay , Arabidopsis seedlings were ground in liquid nitrogen and resuspended in precooling Enzyme extraction buffer ( 50 mM phosphate buffer ( Na2HPO4-NaH2PO4 ) , pH = 7 . 0; 2% ( w/v ) polyvinylpolypyrrolidone ( PVPP ) ; 0 . 05% ( v/v ) TritonX-100; 1 mM EDTA and 1 mM ascorbic acid ) on ice . Extraction solution was centrifuged for 20 min ( 16 , 000 g/min , 4°C ) . The protein concentration of supernatant was detected by One-drop micro-ultraviolet spectrophotometer and SDS-PAGE before the supernatant was used for enzyme activity analysis . The detection of APX activity was performed as described with modifications [95] . For APX activity , 50 μl enzyme and 2950 μl reaction mixture ( 50 mM Tris-HCl , pH7 . 0; 0 . 1 mM EDTA; 0 . 1 mM H2O2 and 0 . 5 mM ascorbic acid ) was mixed . The decreased OD290 was recorded per 10 s . The enzyme amount oxidized 1 mM AsA in one minute set as one activity unit ( U ) of APX . The APX activity was defined as U • g-1 protein . GPX activity was measured indirectly through the detection of glutathione reductase ( GR ) activity using GPX Activity Measurement Kit ( Beyotime Biotech , China ) . GR activity was detected as described with modifications [96] . The OD340 was recorded per 30 s . The enzyme amount consumed 1 mM NADPH in one minute set as one activity unit ( U ) of GPX . The GPX activity was also defined as U • g-1 protein . For western blot analysis , proteins were electroblotted from 12% acrylamide gel to nitrocellulose membrane ( Immobilon-P , MILLIPORE Corporation , USA ) after the separation of SDS-PAGE . Antibodies used in western blot were as follows: anti-HA antibody ( M20003 , Mouse mAb , Abmart , Shanghai , China ) , 1:1 , 000 for western blot; anti-PRMT4b antibody , 1:500 for western blot; anti-Ubiquitin antibody ( ab7254 , abcam , USA ) , 1:1 , 000 for western blot; anti-His antibody ( M30111 , Mouse mAb , Abmart , Shanghai , China ) , 1:1 , 000 for western blot and goat anti-mouse lgG-HRP ( Santa Cruz Biotechnology , USA ) , 1:5 , 000 for western blot . Image Quant LAS 4000 ( GE , USA ) , as the CCD camera system , was used for the result examination with Super Signal West Femto Trial Kit ( Thermo , USA ) . Band densities were quantified via Quantity One software ( Bio-Rad , USA ) . The in vitro E3 ubiquitin ligase activity assay was carried out as described previously [43] . GST-PQT3 fusion protein was obtained from E . coil and purified subsequently . His-tagged ubiquitin of Arabidopsis ( UBQ14 ) was also expressed using bacterial expression system and purified . In addition , the wheat ( Triticum aestivum ) E1 ( GI: 136632 ) and human E2 ( UBCh5b ) were also used in the reaction . Reactions were performed for 1 . 5 h at 30°C . For the immunoblot , His-tagged ubiquitin of Arabidopsis was detected using Nickel-HRP ( Kirkegaard & Perry Laboratories , nickel–nitrilotriacetic acid agarose conjugated to horseradish peroxidase ) . The bait plasmid was transformed into Mav203 strain of yeast , which was constructed with pDEST32 vector and the full-length PQT3 cDNA . Yeast two-hybrid screening was performed using two-hybrid cDNA library of Arabidopsis . The cDNA library containing fragments of fusion proteins composed of prey proteins and GAL4—AD was used to transform Mav203 cells harboring bait plasmid in Y2H assay . Positive clones were screened using SD/-Leu-Trp-His and X-gal assay . Then it was identified by nucleotide sequencing with corresponding primers . Two-hybrid screening was carried out based on the protocol described in Two-Hybrid System Manual ( Invitrogen , USA ) . The result of screening was further confirmed by the two-hybrid assay . The full-length CDS of PQT3 and the four segments of PQT3 were inserted into pDEST32 vector to construct the bait plasmid , while the prey plasmid was constructed with pDEST22 vector and the full-length CDS of PRMT4b . For two-hybrid assay , the primers could be found in S1 Table . MBP-PQT3-C66 and His-AtPRMT4b fusion protein were expressed using prokaryotic expression system and purified . MBP-PQT3-C66 fusion protein was incubated with MBP beads ( amylose resin ) at 4°C for 2 h , and the MBP tag was used as a negative control . The beads were cleaned with washing buffer for 4 times . Then the beads were incubated with His-AtPRMT4b at 4°C for 2 h respectively . The beads were cleaned with washing buffer for 4 times . Western blot was used to detect the SDS-PAGE separation results of pulled-down mixtures in nitrocellulose membrane with anti-His antibody . Agrobacterium tumefaciens strain C58C1 was used in the experiments . Agroinfiltration procedure was carried out as described previously [63] . At first , these strains were grown on LB medium with Gentamicin and Kanamycin . Single colony was transferred into 5 ml LB liquid medium containing same resistance and grown for 48 h in a 28°C shaker . The bacteria solution was inoculated into new LB liquid medium containing 40 μM acetosyringone ( 1:100 ratio , v/v ) and 10 mM 2- ( N-morpholine ) -ethanesulfonic acid ( MES; pH 5 . 6 ) . Bacteria were developed in a 28°C shaker until OD600 reached 3 . 0 approximately . The bacteria were collected gently by means of 10 min centrifugation ( 3 , 200 g/min ) , and the resuspension of pellets was performed with 10 mM MgCl2 until OD600 reached 1 . 5 approximately . The bacteria solution was kept at room temperature with a final concentration of 200 μM acetosyringone for at least 3 h without shaking . The different plastid combinations were transformed into epidermal cells of N . benthamiana leaves by disposable syringe . NE-PQT3 ( the N-terminus of YFP fused with PQT3 ) and CE-PRMT4b ( the C-terminus of YFP fused with PRMT4b ) were constructed . These constructs were transferred to Agrobacterium strains C58C1 respectively . As mentioned above , the different plastid combinations were transformed into epidermal cells of N . benthamiana leaves by agroinfiltration . YFP was observed 1–2 days after leaf infiltration using confocal . The nuclei were stained by Hoechst subsequently and fluorescence detection by confocal was performed . For BiFC assay , the primers could be found in S1 Table . Native extraction buffer 1 [10 mM EDTA; 50 mM TRIS-MES , pH 8 . 0; 1 mM MgCl2; 5 mM DTT; 0 . 5 M sucrose; protease inhibitor cocktail for plant cell and tissue extracts ( Sigma , USA ) ] was chosen for protein extraction buffer . Other steps of protein extraction were carried out as described previously [63] . N . benthamiana leaves was infiltrated using the mixture of Agrobacterium strains containing different constructs , from which total proteins were extracted 1 day after Agroinfiltration . The p19 was used as gene-silencing suppressor . The anti-HA antibody was used for western blot detection of the total proteins after the separation of SDS-PAGE . HA-PRMT4b is a fusion protein composed of HA-tag and PRMT4b . The protein level of PRMT4b could be analyzed via the HA-tag detection using anti-HA antibody . Ponceau S staining of the Rubisco protein was used as loading control . In the presence of MG132 , total proteins were immunoprecipitated with protein A agarose beads ( Millipore , USA ) and anti-HA antibody ( Abmart ) subsequently . Detailed steps of immunoprecipitation were carried out as described previously [63] . Immunoprecipitated samples were analyzed using western blot with anti-ubiquitin antibody ( Abcam ) . Image Quant LAS 4000 ( GE , USA ) , as the CCD camera system , was used for the result examination . The wild type , pqt3 mutant , prmt4b mutant and pqt3prmt4b double mutants were used for ChIP assay without or with 6 μM paraquat treatment for 24h . UBQ5 was chose for internal control . ChIP was performed as previously described [97 , 98] . The regions with Arg-17 methylation were precipitated via anti-H3R17me2a antibodies ( anti-Histone H3 asymmetric dimethyl R17 antibody-ChIP grade , ab8284 , Abcam , USA ) from input DNA . The corresponding primers were designed for quantitative RT-PCR to detect the enrichments of different DNA fragments in APX1 and GPX1 chromatins [99 , 100] . The primers used in ChIP assay were showed in S1 Table . | Oxidative stress is a major stress in plant cells when biotic and abiotic stresses are imposed . While the response to oxidative stress has been extensively studied , little is known about how the activated response is switched off when oxidative stress is diminished . By studying Arabidopsis mutant paraquat tolerance3 , we identified the genetic locus PARAQUAT TOLERANCE3 ( PQT3 ) as a major negative regulator of oxidative tolerance . PQT3 encodes an E3 ubiquitin ligase and is rapidly down-regulated by oxidative stress . Subsequently , we identified PRMT4b as a PQT3-interacting protein . PQT3 was demonstrated to recognize PRMT4b for targeted degradation via 26S proteasome . By histone methylation , PRMT4b may regulate the expression of APX1 and GPX1 , encoding two key enzymes against oxidative stress . Therefore , we have identified PQT3 as an E3 ubiquitin ligase that turns off the activated response to oxidative stress . Our study provides new insights into the post-translational regulation of plant oxidative stress response and ROS signaling . | [
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"pla... | 2016 | PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b |
Dengue virus ( DENV ) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide . DENV is divided into four major serotypes , and infection with one serotype leads to immunity against the same , but not the other serotypes . The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses , such as West Nile virus ( WNV ) , Yellow Fever virus ( YFV ) or Tick-borne encephalitis virus ( TBEV ) . Especially in areas where several flaviviruses co-circulate or in the context of vaccination e . g . against YFV or TBEV , this severely complicates diagnosis and surveillance . Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop ( FL ) domain in the viral envelope ( E ) protein . We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain . By using specific mixtures of these mutant antigens , cross-reactivity against heterologous flaviviruses was strongly reduced , enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements . These results have indications for the development of serological DENV-tests with improved specificity .
Dengue virus ( DENV ) is a mosquito-transmitted pathogen of the family Flaviviridae , a group of small , enveloped and positive stranded RNA-viruses . Besides DENV there are several other human pathogenic vector-borne flaviviruses , such as yellow fever virus ( YFV ) , West Nile virus ( WNV ) , tick-borne encephalitis virus ( TBEV ) or Japanese encephalitis virus ( JEV ) [1] . DENV is endemic to over a hundred tropical and subtropical countries worldwide , and the numbers of annual infections are strongly increasing with a current estimate of 400 million [2] . High fever is the most common clinical symptom of a DENV-infection , but also severe complications are observed , such as dengue shock syndrome ( DHS ) or dengue haemorrhagic fever ( DHF ) . Approx . half a million hospitalizations and several thousands of fatalities are caused by DENV every year [3 , 4] . Dengue viruses include four major distinct serotypes , named DENV-1 to DENV-4 , and survival of an infection with one of these serotypes leads to a lifelong immunity to this serotype , but not to the others [5] . In parallel to the increasing distribution of its vector , mosquitos from the genus Aedes , DENV is emerging or re-emerging in several areas including Europe and North America . In Europe local transmission of the virus has been demonstrated in France and Croatia in 2010 [6 , 7] . In addition , a DENV-outbreak occurred in Madeira in 2012 and resulted in over 2000 cases and transportation of the virus into several other European countries [8] . During the acute phase of infection , Dengue can be diagnosed by directly detecting viral RNA or the non-structural protein 1 ( NS1 ) , which is secreted by infected cells . About five days after onset of symptoms , these direct infection markers start to decrease in blood and DENV-IgM antibodies appear , followed by IgG a couple of days later . Therefore , except during the acute phase , DENV-infections are usually diagnosed by antibody-measurements , and several serological test systems are available [reviewed in 9 , 10] . Whereas IgM-detection can indicate recent primary infections , IgG-measurements are useful for detection of secondary infections , where IgM responses usually remain lower , and for serological surveillance activities . Acute dengue infections can be confirmed via a rise in IgM or IgG levels in paired samples [10] . One of the major problems in antibody-based diagnosis of dengue is the similarity of structural proteins of different flaviviruses which leads to cross-reactive antibodies and false positive test results [11–13] . Especially in areas where several flaviviruses co-circulate or in the context of vaccination against e . g . YFV or TBEV , this is of concern [1 , 14] . As the currently available tests cannot exclude flavivirus cross-reactivity , positive test results have to be confirmed by virus neutralization tests , which are time consuming and require BSL-3 laboratories . The E ( envelope ) protein is a major target of the human antibody response during DENV infections and is used in most available tests [15] . Cross-reactive antibodies target mainly the highly conserved fusion loop ( FL ) domain of the E protein which is involved in fusion of the viral and cellular membranes [16–19] . As a consequence , the insertion of mutations into the FL leads to a decrease in binding of cross-reactive antibodies , which has been employed to develop diagnostic methods on the basis of WNV- or JEV- virus-like particles ( VLPs ) [20–22] . Alternatively , other recombinant antigens than the E protein , such as NS1 , have been used to differentiate flavivirus antibodies via titer-determinations [23] . It was shown previously that a bacterially expressed E protein from WNV bearing four point mutations in the FL and an adjacent loop domain can be used to serologically distinguish WNV- from TBEV and DENV-infections [22 , 24] by reducing the binding of antibodies from heterologous flavivirus infections . Here , we expand this technology to DENV . Quadruple mutant forms of the E proteins from the different DENV-serotypes , containing the same mutations as the WNV-protein [24] , were generated in insect cells and analyzed as a diagnostic tool . The results show the high potential of this method for the development of a specific serological DENV-assay .
Drosophila S2 cells ( Invitrogen ) were propagated at 28°C in T-75 cm2 flasks in Schneider´s medium supplemented with 10% FCS and 1% Pen/Strep ( complete Schneider´s Medium ) . The sequences of the DENV-2 E wild type ectodomain ( E-protein amino acid residues 1–399; strain 16681 ) and the quadruple mutants ( Equad: T76R , Q77E , W101R; L107R ) of DENV serotypes 1–4 ( DENV-1: Nauru/West Pac/1974 , E-protein amino acid residues 1–399; DENV-3: Sri Lanka/1266/2000 , E-protein amino acid residues 1–397; DENV-4: Dominica/814669/1981 , E-protein amino acid residues 1–399; ) were synthesized ( Centic Biotec ) and cloned with BglII and EcoRI into the pMT/BiP/V5-His vector ( Invitrogen ) . Plasmids were transfected with a Ca-Phosphate transfection kit ( Invitrogen ) according to manufacturer´s instructions into Drosophila S2 cells . To generate stable cell lines 1 μg of pCoHygro ( Invitrogen ) , containing a hygromycin resistance gene , was co-transfected with each expression vector . Stably transfected polyclonal S2 cell populations were generated after 3 weeks of selection with hygromycin B ( 300 μg/ml ) in complete Schneider´s Medium . These cells were then propagated at 28°C in tissue culture flasks with complete Schneider´s medium containing 300 μg/ml hygromycin B and adapted to Sf900II medium containing 600 μg/ml hygromycin B . For an expression culture , cells were seeded at a cell density of 2–3 x106 cells/ml in 600 ml Sf900II medium in 2 l baffled Erlenmeyer shaker flasks at 28°C and 90 rpm and were induced with 700 μM CuSo4 at a cell density of 6 x 106 cells/ml . After 7 days the suspension culture was centrifuged for 15 min and 4000 g at 4°C and culture supernatant was concentrated and diafiltrated against His-binding buffer ( 20 mM sodium phosphate , 500 mM NaCl , 10 mM Imidazole , pH7 , 4 ) using Vivaflow 50R TFF cassettes ( Sartorius ) according to manufacturers´ instructions . The DENV E proteins were purified by immobilized metal affinity chromatography ( IMAC ) with 5 ml HisTrap FF crude columns ( GeHealthcare ) and size exclusion chromatography with a 16/600 HiLoad Superdex 200 pg column ( GeHealthcare ) using the ÄKTA pure 25 l chromatography system ( GeHealthcare ) . Purified proteins were quantified using a BCA protein assay ( Pierce ) . Conformation of the E-proteins and loss of the FL-domain structure in the Equad mutants were verified by binding studies of monoclonal antibodies recognizing DENV-2 epitopes in the domains DI-DII ( antibody DV2-44 ) , DIII ( DV2-76 , DV2-96 , DV2-106 ) and FL ( DV2-29 ) and WNV-specific FL-antibodies ( E18 and E60 ) [25 , 26] ( S1 Table ) . 22 DENV-positive serum samples were obtained from Padova University Hospital ( Italy ) . The confirmed DENV cases were international travelers returning from endemic countries with diagnosis of recent infection . 15 DENV-positive serum samples and one negative control were obtained from Seracare Life Sciences ( USA ) , and 8 DENV-infected serum samples and two negative controls were obtained from ZeptoMetrix Corporation ( USA ) . Three DENV-infected and one WNV-infected samples were from the Robert Koch Institute ( Berlin ) as part of an external quality assessment study [12] . Serum samples positive for DENV antibodies all fulfilled one of the following criteria: ( a ) positive in DENV-specific RT-PCR , ( b ) positive for both DENV IgM and IgG , ( c ) positive in a DENV-specific virus neutralization test ( VNT ) . Serum samples from confirmed WNV-infections as well as sera from TBEV-infected individuals and negative controls were derived from Italy ( University of Padova ) . The WNV-positive samples ( IgG and IgM ) were from seroprevalence studies , blood donors or patients with WNV- neuroinvasive disease . WNV-infections were confirmed by VNT . TBEV IgG-positive serum samples were selected from a seroprevalence study in forest rangers with a history of confirmed TBEV infection and no previous exposure to DENV . Serum samples from YFV-vaccinated individuals ( IgG positive ) participating in a randomized controlled vaccination study were obtained from the Robert Koch Institute ( Berlin , Germany ) . These were confirmed by VNT . Ethical approval for all serum samples from Padova University was obtained from the Padova University Hospital ethics committee . The randomized controlled vaccination study for YFV from the Robert Koch Institute was approved by the Charité medical ethics committee . All persons participating in this research provided informed consent and all samples were analyzed anonymously . Indicated protein amounts of DENV-2 Ewt and Equad or DENV1-4 Equad mixtures were coated overnight on Nunc polysorb plates ( Thermo Scientific ) in 100 μl coating buffer ( 15 mM Na2CO3 , 35 mMNaHCO3 pH 9 . 6 ) at 4°C . The plates were washed three times with 350 μl per well of PBS-0 , 05% Tween and blocked with 200 μl of 5% non-fat milk powder ( blocking solution ) for 2 h at room temperature . After a second washing step , human sera were diluted 1:100 in 100 μl blocking solution per well and incubated for 1 . 5 h at room temperature . Following a third washing step , the HRP-conjugated secondary goat anti human IgG ( Fisher Scientific , 1:10000 in 100 μl blocking solution per well ) or rabbit anti human μ-chain IgM ( Dianova , 1:7500 in 100 μl blocking solution per well ) antibody was added for 1 h at room temperature . After a fourth washing step , 100 μl TMB substrate ( Biozol ) per well were incubated for 30 min at room temperature . The reaction was stopped with 50 μl 1 M H2SO4 and signals were read out at 450 nm with background reduction at 520 nm in a micro plate reader ( Infinite M200 , Tecan ) . All tests were performed in duplicates and in two independent experiments . The Panbio Dengue IgG Indirect ELISA and was used according to the manufacturer´s instructions . All antibody measurements were performed in duplicates in at least two independent experiments . Graphical and statistical analysis of the data in boxplots was carried out using SigmaPlot .
To enhance specificity of serological DENV diagnosis we inserted 4 amino acid point mutations into the conserved fusion loop ( FL ) domain and an adjacent loop domain of DENV wildtype ( wt ) E proteins , yielding quadruple mutants for DENV serotypes 1 to 4 ( DENV 1–4 Equad-proteins , Fig 1 ) . The mutant proteins as well as the DENV-2 wt E-protein were overexpressed in Drosophila S2 cells and secreted into the culture´s supernatant . After purification by immobilized metal affinity chromatography several unspecific proteins were co-purified . Therefore a second purification step using size exclusion chromatography was performed to eliminate the visible unspecific bands ( Fig 2A ) . To determine the optimal antigen concentration per well for an IgG-ELISA increasing amounts of the proteins DENV-2 Ewt and DENV 1–4 Equad were incubated with three sera of DENV- and WNV- infected or flavivirus-uninfected individuals , respectively ( Fig 3 ) . Signal saturation of DENV-positive sera was observed with an antigen amount of approx . 200 ng per well for DENV -1 , 3 and -4 Equad and 300 ng per well for DENV-2 Ewt and Equad proteins . Two of the three DENV-positive sera showed a substantial decrease in binding to the DENV-1 and DENV-2 mutant proteins ( Fig 3A and 3B ) as compared to the DENV-2 wild type version , but the values were still detectable when 50 ng or more was used . Two WNV-positive samples bound to the wild type antigen as strongly as the DENV-positive sera , and the third started to show such cross-reactivity with more than 200 ng of antigen per well ( Fig 3E ) . However , all WNV sera lost binding almost completely when the mutant antigens were used except for DENV-4 Equad , where two WNV-positive sera still showed substantial signals for 200 ng of antigen per well or more ( Fig 3D ) . Negative sera showed negligible signal intensities for all proteins in all amounts tested . Based on these results an antigen amount of 300 ng per well was chosen for the analysis of a larger serum panel with DENV-2 wt and DENV-2 Equad in an IgG ELISA . First , sera from 38 DENV- , 13 WNV- , 19 TBEV- infected , 8 YFV vaccinated and 7 uninfected individuals were incubated with DENV-2 Ewt protein ( Fig 4A ) . Strong binding for DENV-positive samples was observed ( mean absorbance value 2 . 36 ) , however , several WNV- and TBEV- positive sera ( mean values 1 . 26 and 0 . 82 , respectively ) showed cross-reactive signals which were in the range of DENV-infected sera . Samples from YFV-vaccinated individuals showed a reduced cross-reactivity in comparison to WNV- and TBEV-infected serum samples . When using the DENV-2 Equad protein , the cross-reactivities of WNV- and TBEV-infected samples were significantly reduced ( mean values 0 . 106 and 0 . 160 , respectively ) ( Fig 4B ) . In addition , the signal intensities of the DENV-positive samples changed . The mean value was reduced to 1 . 9 and the overall signal range increased from 2 . 1 to 2 . 4 in comparison to DENV-2 Ewt . Next , a mixture of the Equad proteins of all four serotypes was prepared ( DENV 1–4 Equad Mix ) . Based on the titration curves ( Fig 3 ) a total amount of 160 ng was found to be optimal in specificity and sensitivity , consisting of 50 ng of DENV 1–3 Equad respectively and 10 ng of DENV-4 Equad . The proportional amount of DENV-4 Equad was reduced compared to the other serotypes because it elicited a higher cross-reactivity with heterologous flaviviral sera ( Fig 3D ) . The mixture was tested with the same serum panel . This resulted in an increase of the 25th percentile from 1 . 36 to 1 . 6 , demonstrating a higher sensitivity compared to using only DENV-2 Equad . At the same time , cross-reactivity of WNV- and TBEV-positive sera was even further reduced ( mean values 0 . 063 and 0 . 084 , respectively ) showing a higher specificity of the test in comparison to using DENV-2 Equad only . The stasticial analysis of the data is shown in S2 Table . The five DENV-positive sera detected as outliers on the lower end of the DENV-panel using the mutant antigens in Fig 4 were numbered . Whereas sera 1 , 2 ( both unknown DENV serotype infections ) and 3 ( DENV-3 infection ) showed decreased binding to the DENV-2 Equad-protein as compared to the wild type , their signals increased when using the DENV 1–4 Equad mix . Also the signals of samples 4 and 5 ( DENV-1 and -2 infections , respectively ) decreased with the DENV-2 Equad compared to the wild type but were even lower with the mutant mixture ( Fig 4C ) . To compare these results with a state-of-the-art diagnostic method , a number of the serum samples were analyzed with a commercially available DENV-IgG ELISA . The test detected 3/3 DENV-positive sera as positive and 3/3 negative sera as negative . However , 10/10 WNV-infected sera were detected as positive with several OD-values similar to DENV-infected sera . For TBEV-infected sera , 4/10 samples were detected as negative , 3/10 as equivocal and 3/10 as positive ( Fig 5 ) . Comparison with the values obtained using DENV Ewt showed a similar cross-reactivity . In contrast , when using the DENV 1–4 Equad mixture high signals were obtained only with the three DENV-infected sera ( mean values of 1 . 5 , 1 . 6 and 1 . 7 , respectively ) and cross-reactivity was strongly reduced in all samples from WNV- and TBEV-infections ( mean values all >0 , 3 , Fig 5 ) . Subsequently , the proteins were used to measure IgM antibodies in human sera . 300 ng of DENV 1–4 Equad mix were found to be optimal for IgM detection ( S3 Table ) , which was then compared to 300 ng of DENV-2 Ewt in binding of IgM-positive DENV and WNV sera . Generally , the cross-reactivity of heterologous flavivirus IgM antibodies was lower than for IgG , as demonstrated by less binding to the Ewt protein ( Fig 6A ) . By using the DENV 1–4 Equad mixture the mean value of signals for DENV sera was enhanced as compared for DENV-2 Ewt . On the other hand , WNV cross-reactivity was significantly reduced in comparison to DENV-2 Ewt ( Fig 6B and S2 Table ) .
A challenge for current serological dengue diagnosis is the high degree of cross-reactivity between antibodies produced during infections with related flaviviruses [27–29] , leading to false positive results in currently available serological test systems [11 , 12] . Especially in areas with different co-circulating flaviviruses , this is of concern [1 , 14] . Here , we present insect-cell derived recombinant DENV E proteins bearing mutations in the conserved FL domain to enhance the specificity of serological DENV diagnosis . When using the wild type E protein from serotype 2 ( which is the basis for several available test systems ) we found all the DENV-positive sera displayed high signals ( Fig 4A ) . However , the cross-reactivity problem was also confirmed , as the signals obtained with many WNV- and TBEV positive samples were in the range of DENV-signals , although the mean intensities were lower ( Figs 3E and 4A ) . As a consequence , several sera would be misdiagnosed as false-positive , which was indeed observed when a commercial assay was used ( Fig 5 ) . It has been shown before that a large proportion of antibodies to DENV-infections are produced against the FL domain [17 , 18 , 22] . Accordingly , two out of three dengue sera showed a clearly decreased signal when the FL-mutant version of the E-protein from DENV-2 was used ( Fig 3B and 3E ) . However , in the larger serum panel , the signals obtained with the DENV-sera remained detectable , but most of the binding of the heterologous flavivirus-antibodies was eliminated ( Figs 3B and 4B ) . This result confirms previous studies performed with WNV and JEV , using VLP-based systems with single- or double mutations in the FL domain [20 , 21] and underlines the role of the conserved FL domain in cross-reactivity between flavivirus-antibodies . It also demonstrates that the four mutations of the Equad proteins from WNV [24] can be used to eliminate cross-reactive epitopes in DENV E proteins . When the mixture of all four serotype-specific mutant proteins was used , the values of many DENV-positive serum samples increased as compared to using DENV-2 Equad only , although only half the amount of total protein was used ( Fig 4C ) . This increase in sensitivity might result from serum samples that were from non-serotype 2 infections and now bound to their corresponding E-protein , such as the three outliers in Fig 4B . However , in the absence of knowledge of the infecting serotype , this interpretation remains speculative . Data on the infecting serotypes were available only for a limited subset of the DENV-positive sera in this study . In addition to an increase in sensitivity , the cross-reactivity of TBEV and WNV sera was further reduced with the serotype mixture and lower amount of antigen . Under the conditions used , all DENV-infected sera of the panel led to high absorbance values with the DENV 1–4 mix . Nevertheless , to avoid single outliers , the exact relative contribution and amount of each serotype mutant protein in the mixture might still be optimized . The titration curves ( Fig 3 ) suggest that a doubling of the amount of the individual mutant antigens does still not lead to an increase in cross-reactivity . Using the DENV 1–4 mix for IgM detection resulted in a higher mean value of positive samples as compared to the wildtype antigen ( Fig 6 ) . At the same time , the cross-reactivity with WNV-positive sera was decreased . Due to the generally observed higher specificity of IgM-antibodies [12] , this improvement in specificity was less pronounced as for IgG-detection ( Fig 6 ) . Nevertheless , our data support the idea that recombinant E proteins bearing four point mutations are suitable antigens for the specific serological diagnosis of dengue . This is also supported by testing samples from a recent external quality assessment on serological dengue diagnosis [12] , where the vast majority of participating laboratories ( using various ELISA systems ) detected a cross-reactive WNV serum as false positive . The serotype mixture used here was able to detect the samples with 100% accuracy ( results are included in Fig 4 ) , including diluted samples for sensitivity determination . In addition , the direct comparison of the DENV-1-4 mixture antigen with a commercially available test clearly showed the increased specificity of the mutant antigens ( Fig 5 ) . Although no cut-offs were yet defined for the DENV1-4 Equad , it is clear from Figs 4 and 5 that cross-reactive sera cause absorbance values more similar to flavivirus-negative than to DENV-positive samples . We recently have performed a study using a bacterially expressed Equad mutant for WNV [24] . These data also showed a reduction in cross-reactivity . However , especially some DENV-positive samples with a high antibody titer still bound the mutant protein to some extent . Therefore , the ratio between signals obtained with the wildtype and mutant protein was calculated for each serum enabling correct diagnosis . In contrast , in the present study the mutant proteins alone were sufficient to exclude cross-reactivity , which might reflect higher sensitivity of the insect-cell derived antigen or specific differences between the antibody responses to WNV and DENV . In summary , a system for the sensitive and specific serological diagnosis of DENV-infections is presented , which consists of recombinant E-proteins from the four major serotypes , with mutations in the FL and an adjacent loop domain . The binding of cross-reactive antibodies from heterologous flavivirus-infections is strongly decreased . These antigens might form a valuable basis for improved antibody tests , especially in areas where co-circulation of different flaviviruses is observed . | The serological diagnosis of dengue is severely complicated by cross-reactivity between antibodies against different flaviviruses . Currently available tests cannot rule out false positive results due to infections or vaccinations with related pathogens such as West Nile virus or Yellow Fever virus . Most cross-reactive antibodies target the conserved fusion loop ( FL ) domain in the E protein ( the major component of the viral envelope ) . Therefore , we generated insect-cell derived E proteins of DENV from the four different serotypes with mutations in the FL and set up an ELISA-based platform . Using these antigens we were able to detect DENV-infections with high sensitivity . In addition , cross-reactivity with a variety of heterologous flavivirus-infections was eliminated . The results have strong indications for the development of simple and sensitive serological DENV-tests with greatly improved specificity . | [
"Abstract",
"Introduction",
"Materials",
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"Results",
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] | [] | 2015 | Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections |
Tumors are defined by their intense proliferation , but sometimes cancer cells turn senescent and stop replicating . In the stochastic cancer model in which all cells are tumorigenic , senescence is seen as the result of random mutations , suggesting that it could represent a barrier to tumor growth . In the hierarchical cancer model a subset of the cells , the cancer stem cells , divide indefinitely while other cells eventually turn senescent . Here we formulate cancer growth in mathematical terms and obtain predictions for the evolution of senescence . We perform experiments in human melanoma cells which are compatible with the hierarchical model and show that senescence is a reversible process controlled by survivin . We conclude that enhancing senescence is unlikely to provide a useful therapeutic strategy to fight cancer , unless the cancer stem cells are specifically targeted .
Cancer cells are characterized by their persistent proliferation , but just as for normal cells [1] tumor cells can go senescent , halting their growth [2] , [3] . The molecular basis for the induction of senescence appears to be a combination of several mechanisms such as telomerase shortening , DNA-damage and oxidative stress [3] . It has been suggested that senescence should be present only in pre-neoplastic cells [3] but there is evidence that senescence markers increase during tumor progression [4] . This is puzzling since it is usually assumed that tumors can only grow if senescence is avoided . Recent experiments are challenging this conventional view of cancer , showing that only a small fraction of cancer cells , the cancer stem cells ( CSCs ) [5] , actively drive tumor growth . The implications of this finding for tumor cell senescence have hitherto not been explored . According to the CSC hypothesis , tumors behave in analogy with normal tissues , whose growth is controlled by a small population of slowly replicating stem cells with the dual capacity of either self-renewal or differentiation into the more mature cells required by the tissue . The crucial difference between tissue stem cells and CSCs lies in their proliferation properties . Stem cells in tissues tend to keep their number constant either deterministically by enforcing asymmetric division , or stochastically by balancing proliferation and depletion probabilities [6] , [7] . This restriction does not hold for CSCs . The role of CSCs has important implications for therapeutic approaches . According to the conventional view , the success of a treatment is measured by the number of tumor cells killed; in contrast , according to the CSC hypothesis , only the CSC subpopulation matters in the end for complete eradication . First evidences for the existence of CSCs came from hematological tumors [5] and later from solid tumors such as breast cancer [8] and melanoma [9]–[16] . The presence of CSCs in melanoma is currently debated . It was argued that to obtain reliable estimates of the number of tumor initiating cells one should use highly immunocompromised mice for tumor xenografts [17] . Different groups , however , reported conflicting results with slight differences in assay conditions and mouse models [14] , [17] . Furthermore , several putative CSC markers appears to be reversibly expressed in melanoma [18] and in breast cancer [19] . In this paper , we analyze cell senescence during the growth of melanoma cells both in vitro and in tumor xenografts . We find that the fraction of senescent cells increases considerably after a few months of cultivation , slowing down the growth of the cell population . This process is , however , only transient: after some time senescence almost disappears and growth resumes at the initial rate . We also show that senescence is a reversible process controlled by survivin: by overexpressing survivin in senescent cells , we are able to decrease senescent markers and increase cell proliferation . These results can be interpeted in terms of the hierarchical cancer model where only CSCs replicate indefinitely and senescence reflects the loss of proliferative capacity of other cancer cells . To prove this point we sort cancer cells according to a putative CSC marker , ABCG2 , and show that senescence is more prevalent for negative cells , suggesting that CSC are able to rejuvenate an otherwise senescent cell population . To understand quantitatively the experimental results , we propose a mathematical model for cancer growth that is compatible with the existence of CSCs in melanoma , provides an unambiguous interpretation of experimental data , and explains quantitatively the occurrence of senescence in tumors . While in the stochastic cancer model , senescence is due to spontaneous mutations of tumor cells , in the hierarchical model only CSCs replicate indefinitely and senescence reflects the loss of proliferative capacity of other CCs . These observations can be recast in a mathematical framework using the theory of branching processes , a generic model for a growing population [20] , [21] . Branching processes were first proposed at the end of the XIX century and found wide application in physics and biology with examples ranging from nuclear reactions [20] to evolution theory [21] , tissue growth [6] , [22]–[24] and cancer progression [25] , [26] . The main limitation of branching processes is that they do not account for interactions between cells which could be relevant for the growth of a tumor , especially in vivo . Despite this shortcoming , our model allows for a good description of the experiments , providing an indirect confirmation of the CSC hypotesis for melanoma with important implications for therapeutic strategies based on the induction of senescence in cancer .
Our theory has biologically motivated ingredients characterizing the probabilities for cells to duplicate , become senescent or die: According to the CSC hypothesis , cells are organized hierarchically , with CSCs at the top of the structure . CSCs can divide symmetrically with rate giving rise to two new CSCs with probability , two CCs with probability , or asymmetrically with probability giving rise to a CSC and a CC . While CSCs can duplicate for an indefinite amount of time , CCs become senescent after a finite number of generations and then eventually die rate ( Fig . 1 ) . The model kinetics depends on the combination , the average relative increase in the number of CSCs after one duplication , rather than on , and separately . In normal tissues , the stem cell population should remain constant which implies that [6] , [27] , while in tumors we expect . The kinetics of the cell populations for this model can be solved exactly . To this end , we first derive recursion relations linking the average cell populations at each generation . Denoting by the average number of CSCs after generations , by the average number of CCs , where indicates the “age” of the CCs ( i . e . the number of generations separating it from the CSC from which it originated ) , and by the average number of senescent cells , we obtain ( 1 ) These relations are derived considering that CSCs can only originate from other CSCs either by a symmetric division – two CSCs are generated with probability – or by an asymmetric one , in which case a single CSC is generated with probability . Hence each CSC generates an average of new CSCs . CSCs also generate CCs ( ) by asymmetric CSC divisions , with probability or by symmetric CSC division with probability , yielding an average of CCs . Two CCs are generated by duplication of other normal cells with unit probability ( ) . Senescent cells accumulate at each generation when normal cells ( with ) lose the ability to duplicate and die with probability . Eqs . 2 can be solved explicitly . We consider first an initial condition with CSC only ( i . e . ) and obtain ( 2 ) Eqs . 2 implies that the average CSC population grows exponentially fast , and that after a large number of generations ( i . e . for ) , all cells populations , and hence the size of the tumor , are proportional to the CSC population . This result confirms that the population of CSCs is the driving force behind tumor growth . Note that the population of senescent cells is also driven by the growth of CSCs and therefore cancer growth and senescence are inextricably linked . To interpret experimental data , it is important to consider the case in which the initial condition is composed of a mixed population of CSCs and CCs . The solution in this case can be obtained as a linear superposition of the solution of Eqs . 2 and the solution with initial conditions , and . For simplicity , we consider the case of a uniform distribution for the ages of CSCs: , and obtain ( 3 ) The complete solution for the total number of cells , corresponding to an initial condition and , is given by ( 4 ) and is plotted in Fig . 2A , while in Fig . 2B we report the fraction of senescent cells . Eqs . 2 represent only a transient contribution to the cell population and does not influence the asymptotic fractions ( i . e . for ) of senescent cells and CSCs which are readily obtained dividing the results in Eqs . 2 by the total number of cells at each generation: ( 5 ) ( 6 ) The asymptotic solutions are plotted in Fig . S1 as a function of for different values of and . It is instructive to use the solution to compare how cells become senescent in normal tissues and in cancers . In the long time limit , the asymptotic fraction of senescent cells is equal to and is independent of the number of duplications needed to induce senescence ( see Fig . S1 ) . For normal stem cells , and the percentage of senescent cells is expected to be large , reaching 100% in the limit of , when senescent cells never die . For cancer cells , and the fraction of senescent cells is smaller but still non-vanishing . Similarly , the CSC fraction is given by which is smallest for normal stem cells and increases as a function of in tumors ( see Fig . S1 ) . Aggressive tumors such as melanoma should be characterized by high values of and therefore by relatively high values of the CSC fraction . Beside asymptotic fractions , the model allows one also to characterize the evolution of the populations , i . e . the time-dependent evolution of the senescent cell fractions ( see Fig . 2 ) . The results show that the level of senescence in tumors strongly depends on the fraction of CSCs and on time . To quantitatively confirm that the model is able to predict the growth properties of stem cells , we first compare its results with experimental data for normal stem cells , and then turn to cancers . It has been shown that long-term in vitro culture of mesenchymal stem cells ( MSC ) induces replicative senescence with important implication for the therapeutic application of MSC preparations [28] , [29] . In particular , the authors show a rapidly increasing fraction of cells that are positive to , a senescence marker; after roughly 50 days , the fraction of senescent cells reaches 80% of the population . By looking at the growth curves for cells extracted from donors of different ages , the authors could not establish a correlation between senescence and aging [29] . To validate our model , we have used it to fit the growth curves reported in Ref . [28] . In order to compare the model with the experiments , we should first express the evolution equations as a function of time rather than generation number . This is easily done introducing the rate of cell division per day and replacing in Eqs . 2–3 the generation index by , where is time expressed in days . In this way , Eq . 4 can directly be compared with the growth curves reported in Refs . [28] , [29] . In particular , we use Eq . 4 for , and . Under this conditions the expression reduces to a much simpler form ( 7 ) We then compute the CPD based on Eq . 14 and perform a two parameters fit in terms of and ( see Figs . 3 and S2 ) . We have also analyzed similar data reported in Ref . [29] for the growth of MSC isolated from human bone marrow for the iliac crest ( BM ) and from the femoral head ( HIP ) ( see Fig . 2 ) . Although significant fluctuations between donors are revealed , the results show in general a decrease of the parameter with the age of the donor ( see Fig . 4 ) , older people have fewer generations of reproduction before senescence . The plot also shows for donors of similar age that is smaller for BM-MSC than for HIP-MSC , suggesting that the former are more prone to become senescent . On the other hand , the cell division rate fluctuates around the value of divisions/day , as reported in Ref . [27] , without any apparent correlation with age . In recent publications , one of us identified aggressive subpopulations expressing two stem cell markers , ABCG2 [9] and CXCR6 [16] , in human melanoma cell lines , making this tumor a suitable candidate to test our theory . Here , we analyze the level of expression of the senescence marker in three human melanoma cell lines , WM115 , IGR39 and IGR37 [9] , [16] . IGR39 and IGR37 cells have been sorted into two subpopulations according to the expression of the ABCG2 marker [16] . In Fig . 5A and Table S1 , we report the level of the senescent marker as a function of time for ABCG2 positive ( ABCG2+ ) and negative cells ( ABCG2− ) . In both cases , we observe an increase in the percentage of positive cells after 80–90 days of growth followed by a decrease to the initial level after 120 days ( see Table S1 ) . While the data follow a similar trend for the two subpopulations , the peak for IGR39 ABCG2− cells is much higher , reaching 90% of the cells . In order to confirm that the difference in the expression of the senescence marker is related to a different proliferative behavior for the two subpopulations , in Fig . 5B we report the long-term growth curves corresponding to IGR39 . The results show that ABCG2+ cells grow substantially more than ABCG2− cells ( i . e . 23 times more ) . Furthermore , the peak percentage of coincides in time with a marked slow down in the growth ( Fig . 5 ) . When the level of expression falls back to the initial value , growth is also restored ( Fig . 5B ) . These data can be described by our theoretical model , if we assume that ABCG2+ and ABCG2− data are only distinguished by a different initial fractions of CSCs . Fitting simultaneously the growth curves and the gal concentrations with the model ( see Fig . 5 and Materials and Methods for details ) , we obtain for ABCG2− and for ABCG2+ cells . Hence , our model suggests that expression of ABCG2 is strongly correlated with CSCs ( almost a factor of thirty ) , but only a relatively small fraction of ABCG2 expressing cells are CSCs . Notice that the final CSC fraction is different from the initial one: using the fit parameters we estimate . As shown in Fig . 5A the fraction of senescent cells predicted by the model is in good agreement with the experimental data , predicting a large senescence peak for ABCG2− cells . Notice that we could easily find parameters producing a clear peak in the concentration also for ABCG2+ cells . Our multicurve fit , however , is dominated by the ( higher precision ) growth curves and the resulting best fit parameters yield no peak in the plot . For ABCG2− cells , the peak in the theory curves arises after generations . The experimental peak in senescence and the associated dip in doubling rate after generations directly implies a long-term memory of the initial preparation , embodied by our senescence generation , a parameter that we expect may be smaller for other growth conditions and tumor types . The peak in cellular senescence is quantitatively described by the hierarchical model and it would be hard to reconcile this result with the stochastic cancer model , where cancer cells are not organized hierarchically . To illustrate this point , we reformulate the stochastic model in mathematical terms assuming that senescence can only occur as a result of random mutation . In this model , we distinguish between two cell populations , cancer cells ( CC ) and senescent cells . CC duplicate with probability , die with probability and become senescent with probability , while senescent cells do not duplicate but can die with probability . The average numbers of CC and senescent cells after generations follow recursion relations ( 8 ) ( 9 ) We can solve the recursion relations explicitly obtaining: ( 10 ) ( 11 ) where we have assumed for simplicity that . The solution implies that the number of CC grows as long as and shrinks otherwise , yielding a condition for tumor progression . Dividing and by the total population size , we obtain the relative fractions and of CC and senescent cells , respectively . In the asymptotic limit ( i . e . large ) , we obtain steady-state solutions ( 12 ) ( 13 ) The approach to the steady-state is reported in Fig . 6A and the steady-state solutions are plotted in Fig . 6B in the case . These results show that the fraction of senescent cells should evolve rapidly towards a steady state in way that is incompatible with the experiments , providing additional evidence supporting the existence of CSCs in melanoma . The present stochastic model is clearly oversimplified and one could think of more elaborate models involving heterongeneous cell populations without a hierarchy . We do not see , however , how we can explain a peak in senescence after 90 days without assuming the presence of at least two distinct subpopulations one of which undergoes senescence after this long interval . In Fig . 7 , we report the evolution of the fraction of senescent cells in human melanoma WM115 cells according to the marker . Fig . 7 shows an increase in the fraction of cells expressing this marker , reaching more than 40% after 80 days of cultivation . We find a similar level of also in tumor xenografts after 60 days ( Fig . 7D ) . This confirms that our finding of senescent melanoma cells is not restricted to growth in vitro . In order to confirm that the observed cell senescence in xenografts ( Fig . 7 ) is not due to hypoxia , we grow WM115 cells under normal or hypoxic conditions ( see Materials and methods ) . Only a slight inhibition on cell growth was observed 48 hrs after incubation under hypoxic condition . This behavior is explained by the induction of the hypoxia inducible factor ( ) after 18 hours of hypoxic conditions ( see Fig . 3 ) . is a pleiotropic transcription factor typically activated in response to low oxygen tension as well as other stress factors in normoxic conditions . Upon activation mediates the transcriptional activation of target genes involved in a variety of processes comprising stress adaptation , metabolism , growth and invasion , but also apoptotic cell death . While we cannot exclude that some form of environmental stress factor affect our results , we conclude that hypoxia is not a likely cause for the senescence we observe in xenografts; our in vitro cells do not experience hypoxia . Since it has been recently shown that survivin allows cells to escape senescence [30] , we transfect IGR37 ABCG2− cells at the peak of ( corresponding to the 98th day for IGR37 cells , see Table S1 ) with a survivin-GFP or GFP alone [31] . We then quantify the resulting senescence with activity and visualize cell proliferation with crystal violet assays ( see supplement for more details ) . In Fig . 8A , we show that the overexpression of survivin induces a dramatic decrease of activity . Importantly , under the same conditions survivin enhances cell proliferation as shown by the colony size distributions ( Fig . 8B ) , the number of colonies and the fraction of area covered by colonies ( see Table S2 ) . The effect of survivin is absent for unsorted IGR37 cells when the level of is low ( see Fig . 8B ) . Moreover , when the percentege of positive cells is below the peak the effect is very small ( see Fig . 5 ) . These data provide a further confirmation of the presence of senescent cells in growing tumors . It is illuminating to use the CSC model to simulate the effect of a treatment on the progression of a tumor . We consider two possible strategies: ( i ) try to stop tumor growth by stimulating cell senescence , and ( ii ) eradicate the tumor by inducing cell death . Case ( i ) can be described by decreasing the parameter , representing the number of generation needed for a CC to turn senescent . The responses to treatment is summarized in Fig . 9A showing that the tumor size stops growing for a short while , but eventually the growth resumes at the previous rate . This is due to the action of CSC , whose fractional population increases dramatically in response to the treatment and sustains cancer growth . Our model quantifies the common-sense statement that if the cancer stem cells are the only parties that double forever , then a treatment that does not remove them will be fruitless in the long term . Case ( ii ) can be described by introducing a parameter , as the probability that a CC cell dies ( before senescence ) . If we assume that CSCs are drug resistant and therefore do not die , we predict that the tumor size would initially shrink , but the growth would starts again after some time ( Fig . 9C ) , due to the persistent growth of the CSC subpopulation ( Fig . 9D ) Again , the model predicts that the only possible strategy to stop cancer growth is to target CSCs .
The present results have powerful implications for our picture of cellular senescence in tumors . The stochastic cancer model suggests that since cancer cells proliferate indefinitely , they must have evaded senescence by some yet unknown biological mechanism . Our results show instead that cancer cells in general become senescent , but this fact is ultimately irrelevant for tumor progression since long-term proliferation eventually resumes . This finding can be explained by the hierarchical cancer model where a subpopulation of CSCs drives tumor growth and the other cancer cells turn senescent after a fixed number of duplications . The peak in cell senescence should correspond precisely to the time at which the initial population of cancer cells turn senescent slowing tumor growth . At this point , CSCs are able to restart the growth process by symmetric duplication leading eventually to the decrease of the fraction of senescent cells as observed in the experiments . It would be hard to reconcile our experimental data with the conventional cancer model hypothizing that a random mutation would lead to massive senescence roughly at the same time for all the different cancer cell populations considered . The fact that CSCs do not become senescent is probably a signature that they originate from normal stem cells and are therefore already immortal . This observation may have implications for therapeutic strategies trying to stop tumor growth by inducing senescence: such an approach is bound to fail unless senescence is induced in CSCs . The data on the ABCG2 sorted population can be explained by the hierarchical cancer model if we assume that ABCG2+ and ABCG2− data are distinguished by a different initial fractions of CSCs . A larger fraction of CSCs in ABCG2+ cells should reduce the occurrence of senescence and increases proliferation with respect to ABCG2− cells . Furthermore , the observation that both cell populations eventually resume their growth at the same rate suggests that some CSCs are also present in ABCG2− cells , whose growth would otherwise stop . This implies that while ABCG2 is able to select a CSC rich population , by itself it is not a clean sorting criterion . This is in agreement with the results of Ref . [16] showing that ABCG2− cells yield a tumor xenograft in immunodeficient mice that is smaller than the one produced by ABCG2+ cells . The fact that ABCG2 is not an absolute CSC marker implies that its expression is reversible after sorting . We have checked this by sorting ABCG2+ and ABCG2− cells after 139 days finding that both express ABCG2 ( with percentage 0 . 92% and 0 . 45% , respectively ) . The same problems are likely to affect putative CSC markers shown in the literature to be reversibly expressed and hitherto considered incompatible with the CSC model [18] . Similar observations in breast cancer cells have been recently interpreted as a signature of stochastic phenotypic switching , assuming that CC have a small probability to revert to the CSC state [19] . The same data could possibly be interpreted in our framework by assuming that the marker was not perfect . It would be interesting to develop quantitative tools to distinguish between phenotypic switching and imperfect markers . The presence of CSC in melanoma is debated because conflicting data are reported in the literature [14] , [17] , [18] showing that slightly different assay conditions lead to different CSC fractions , that can sometimes be relatively large [17] . There is in fact no reason to believe that the CSC population must be small . This idea comes from the analogy with tissue stem cells that replicate homeostatically , keeping their population constant either by asymmetric division or stochastically [6] , [7] , leading to a vanishing concentration of stem cells in the total cell population . CSCs do not replicate homeostatically and therefore their population grows exponentially . Changes in assay conditions can change the duplication rate of CSC , leading for extreme conditions ( i . e . for example the use of matrigel , mice permissive conditions etc . ) to a relatively large concentration of CSCs , perhaps resolving previous controversies [14] , [17] , [18] .
Human IGR37 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and cultured as previously described [16] . IGR39 was derived from a primary amelanotic cutaneous tumor and IGR37 was derived from an inguinal lymph node metastasis in the same patient . Human primary melanoma WM115 ( ATCC CRL 1675 ) was cultured as in previous work [9] . Cells were analyzed for fluoroscein isothiocyanate ( FITC ) mouse anti-human ABCG2 ( R&D Systems , Minneapolis , MN ) expression . All samples were analyzed using one- or two-color flow cytometry with non-specific mouse IgG used ( Invitrogen , Carlsbad , CA ) as isotype controls . For each flow cytometry evaluation , a minimum of cells were stained and at least 50000 events were collected and analyzed ( cells were stained for sorting ) . Flow cytometry sorting and analysis was performed using a FACSAria flow cytometer ( Becton , Dickinson and Company , BD , Mountain View , CA ) . Data were analyzed using FlowJo software ( Tree Star , Inc . , San Carlos , CA ) . Expression of pH-dependent senescence associated activity was analysed simultaneously in different passage of cells using the Senescence galactosidase Staining Kit ( Sigma ) according to the manufacturers protocol . cells were injected subcutaneously into five-week-old NOD-SCID mice ( Charles River Laboratories , Boston , MA ) . Animals were maintained on standard laboratory food ad libitum fed and free access to water . Tumor mass was excised after two months and digested with 0 . 2% collagenase type I ( Gibco ) , 0 . 2% bovine serum albumin ( BSA; SIGMA , St . Louis , MO ) for 30 minutes at on an orbital shaker . Cells are maintained in culture for no more than two passages . WM115 cells were grown in standard medium under normal or hypoxic conditions ( 99 . 8% /0 . 2% ) for up to 48 hours . Total RNA was extracted from WM115 cells or prostate cancer cell lines ( PC-3 used as control ) [32] under normal and hypoxic conditions . Reverse transcription ( RT ) -PCR reactions were done as previously described [16] . The oligonucleotide primer sequences and gene-specific PCR amplification programs used are following: sense -CCTATGTAGTTGTGGAAGTTTATGC; antisense -ACTAGGCAATTTTGCTAAGAATG for 40 cycles at for 30 s , for 15 s , and for 30 s . The integrity of each RNA and oligodeoxythymidylic acid-synthesized cDNA sample was confirmed by the amplification of the -actin housekeeping gene . Ten microliter of each RT-PCR sample was run on a 2% agarose gel and visualized by ethidium bromide staining . 200000 cells were plated on 6 multiwell and the day after transfected by lipofectamin with cytoplasmic survivin-GFP plasmid or GFP ( both plasmids were a gift of Dr Wheatley , [31] ) . 24 hours later the GFP-positive cells were counted by flow cytometry . More than 95% of cells were GFP-positive . These cells were immediately used for crystal violet or actosidase assays . Cells were plated on 6 multiwell and stained after 8 days . Briefly , they were fixed with 3 . 7% paraformaldeide ( PFA ) for 5 minutes and then stained for 30 min with 0 . 05% crystal violet solution . After two washings with tap water , the plates were drained by inversion for a couple of minutes and then photographed . To quantify cell proliferation , we threshold the images and apply the Hoshen-Kopelman algorithm to identify individual clusters ( see Fig . 4 for a visual explanation of the method ) . We then compute the number of colonies , the area fraction covered by colonies ( see Table S2 ) and the colony size distribution ( see Figs . 8B and S5 ) . The last method is the most accurate and complete , avoiding possible experimental artifacts due to local cell detachments . Long-term growth curves are obtained by splitting the cells at each passage and rescaling the result of the counting by an appropriate factor . Cells are counted using a burken chamber in the presence of trypan blue . It is convenient to express the growth curves in terms of cumulative population doublings ( CPD ) , expressing the number of times the populations has doubled . CPD is directly related to the number of cells at time by the expression: ( 14 ) where is initial number of cells . In order to fit the data with model , the total number of cells can be expressed as the sums of two terms derived from Eq . 2 and Eq . 3 , respectively . In particular , we have ( 15 ) and ( 16 ) Furthermore , we can also compare the model with the concentration , assuming that it is equal to the fraction of senescent cells . The experimental comparison involves the fit of four curves in terms of the parameters , , , and . Since ABCG2+ and ABCG2− cells only differ by their relative fraction of CSC , we use the same values of , , , for the two subpopulations and vary only the parameter . We thus perform a multiple curve fitting in which the two growth curves and the two curves are fitted simultaneously in terms of six parameters ( , , , , and ) , where is the value of for ABCG2 cells . To this end , we use the pyFitting software ( https://github . com/gdurin/pyFitting ) developed by G . Durin . The result is ( 17 ) ( 18 ) ( 19 ) ( 20 ) ( 21 ) ( 22 ) with a reduced of . In Table S3 , we report the covariance matrix and the t-statistic . We can express the parameters and in terms of the initial fractions of CSC as for positive and negative cells , where the arbitrary scale is set equal to one in the fits . We obtain ( 23 ) ( 24 ) Hence the ABCG2+ cells have more CSCs than ABCG2− cells , as expected . We have also checked how much the choice of the initial condition affect the results . For simplicity , we have imposed a uniform distribution of ( for ) to obtain a closed form solution ( Eqs . 2–3 ) . To assess the robustness of our results , we have compared the growth curves obtained under this assumption with those obtained using the same fit parameters and imposed as the steady-state solution of the model with the same parameters but smaller . The resulting curves are very close to each other indicating that at least for this case there is a weak dependence on the initial conditions . | It is commonly believed that cell senescence – the loss of replicative capacity of cells – acts as a barrier for tumor growth . Here we follow the evolution of senescence markers in melanoma cells and find that while most cancer cells eventually turn senescent , this is at root irrelevant for the long-term growth rate of a tumor . To demonstrate this , we construct a mathematical population dynamics model incorporating cancer stem cells which is able to reproduce quantitatively the experimental data . Our results support the existence of cancer stem cells in melanoma and explain why it is difficult to fight cancer by inducing senescence in cancer cells . Only a fraction of the cells are susceptible to senescence , but those cells are irrelevant for tumor growth . A successful therapeutic strategy should instead target cancer stem cells , which are , however , likely to be strongly resistant to drug induced senescence . | [
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] | [
"oncology",
"medicine",
"mathematics",
"cell",
"biology",
"cellular",
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"biology",
"molecular",
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] | 2012 | Senescent Cells in Growing Tumors: Population Dynamics and Cancer Stem Cells |
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