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Novel Algorithms Reveal Streptococcal Transcriptomes and Clues about Undefined Genes |
Bacteria-host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro... |
Introduction |
Microarray technology is now commonly used to reveal genome-wide transcriptional changes in bacterial pathogens during interactions with the host. Several factors, however, limit the power of such analyses, including inadequate statistical analysis and insufficient sample replication, both of which do not account for e... |
Results/Discussion |
Adherence-Mediated Differential Expression We developed spotted oligonucleotide arrays of the S. pyogenes SF370 (an M1 serotype) genome [14] and compared the transcriptomes of streptococci that adhere to Detroit 562 human pharyngeal cells to non-adherent ("associated") streptococci within the same experiment. Adherence... |
Verification by Quantitative Real-Time PCR |
We conducted TaqMan (qRT-PCR) analysis [23] of 11 differentially expressed genes to validate selected microarray hybridization results (see Table S2 for genes and primer-probe sequences). Five genes chosen for validation demonstrated statistically significant fold changes in expression by microarray analysis (PF value ... |
Virulence Factors |
Streptococci elaborate several factors implicated in infection, including surface-exposed adhesins and secreted toxigenic proteins (reviewed in [7,14,24]). The initial statistical analysis identified four differentially expressed virulence genes (Tables 1 and 2). Genes encoding streptolysin O (slo or spy0167) and the S... |
Phage-Encoded Genes |
SF370 contains one inducible prophage (370.1) and three defective prophages (370.2, 370.3, and 370.4) that produce no infectious phage [39]. We identified 11 differentially expressed phage 370.2 genes, suggesting that this defective phage is not transcriptionally silent (Table 1). The speH gene (spy1008) was induced, a... |
Allelic Replacement of speH |
Increased expression of speH during pharyngeal cell adherence suggests that the SpeH exotoxin is either necessary for adherence, or is a component of a downstream infection process. Adherence-mediated upregulation of speH is likely not the result of phage induction, as the remaining phage 370.2 genes identified in our ... |
Differential Expression of Genes from Diverse Functional Categories |
We identified a number of genes encoding proteins involved in housekeeping processes (such as carbohydrate and coenzyme metabolism) that were differentially expressed, indicating a shift in metabolic processes due to host cell adherence (Tables 1 and 2). For example, genes encoding proteins involved in folate biosynthe... |
Neighbor Clustering |
Our initial analysis revealed the differential expression of a wide range of functionally diverse genes and provided insight into the adaptive response of streptococci to host cell contact. However, despite a rigorous statistical approach, this analysis, like many previous microarray studies, identified the differentia... |
Type I Clusters: Intact Metabolic Pathways and Multimeric Proteins |
We measured the performance of our algorithm by examining whether it identified gene groupings known to be functionally related (Type I clusters). Only four (16%) of 25 Type I clusters (spy0080-0081, spy1236-1237, spy1707-1711, spy2041-2042) could have been identified in entirety by significance analysis because all cl... |
Type II Clusters |
Based on the Type I cluster results, we speculated that genes contained in Type II clusters might be related by function or regulation. Type II groupings contain a combination of both known and unknown gene members and could provide preliminary clues about the function of unknown genes within a particular cluster by as... |
Allelic Replacement of spy0129 |
We created a spy0129 deletion mutant in strain SF370 (SF370Deltaspy0129) to determine if genes contained within the spy0127-0130 cluster were directly involved in adherence to pharyngeal cells. We posited that a deletion in the spy0129 sortase gene may have the greatest overall effect on the production and processing o... |
Additional Type II Cluster Example |
Another cluster, spy1725-1719, contained six genes that together (though not individually) exhibited significant downregulation. The genes spy1724, spy1722, spy1721, and spy1719 share transcriptional order and predicted function with homologs in the nusA-infB protein biosynthesis operon of Bacillus subtilis and Escheri... |
Neighbor Clustering and Operons |
Although neighbor clustering is not an operon-predicting method, we wanted to identify additional putative operons among the groupings since neighbor clusters by definition share certain operon characteristics (tandemly arranged genes, separated by <300 bp, with similar expression patterns). Although operon-modeling me... |
Analysis of Previously Published Array Data |
We applied the statistical analysis and the GenomeCrawler algorithms to data from a recently published streptococcal microarray study that is relevant for comparison to our own data (same streptococcal strain, similar array platform) [57]. In this study, the transciptomes of S. pyogenes strain SF370 and an isogenic mut... |
Concluding Remarks |
Although GenomeCrawler improves bacterial array analyses, it has limitations: it cannot identify regulons comprising genes dispersed throughout the genome by virtue of its design, it does not specifically interrogate single-gene operons, and it only applies to genomes with available and accurate experimental informatio... |
Porphyromonas gingivalis short fimbriae are regulated by a FimS/FimR two-component system |
Porphyromonas gingivalis possesses two distinct fimbriae. The long (FimA) fimbriae have been extensively studied. Expression of the fimA gene is tightly controlled by a two-component system (FimS/FimR) through a cascade regulation. The short (Mfa1) fimbriae are less understood. The authors have recently demonstrated th... |
Introduction |
Porphyromonas gingivalis is a gram-negative bacterium, which is considered to be a major periodontal pathogen (Socransky & Haffajee, 2005). It is also a pathogen that may be involved in coronary heart disease and preterm births (Boggess et al., 2005; Brodala et al., 2005; Chou et al., 2005). The ability of P. gingivali... |
Results |
Role of FimR in mfa1 expression The fimA gene is the only gene known to be tightly controlled by the FimS/FimR system. It was postulated that the expression of other genes may also be controlled by this two component regulatory system. To investigate effects of FimR on expression of the mfa1 gene, an insertional fimR m... |
Identification of the transcriptional start site of the mfa1 gene |
To identify the promoter region of mfa1, the transcriptional start site was first determined. The RACE experiment was first conducted with mfa1-specific reverse primers MfaTSR1 located at 135 bp up-stream of the potential start codon and MfaTSR2 located at 37 bp downstream of the potential start codon (Fig. 2a). The tr... |
Binding of FimR to the promoter region of mfa1 |
The previous study has shown that the mechanism of FimR activation of the fimA gene involves a regulatory cascade (Nishikawa et al., 2004). It was postulated that different mechanisms might be involved in FimR-mediated mfa1 expression, since expression regulation of mfa1 by FimR was not controlled as tightly as observe... |
Discussion |
The two-component regulatory system is a major mechanism of signal transduction and is widespread in bacteria. Six putative two-component regulatory systems were detected by surveying the P. gingivalis W83 genome database for homologues of the two-component sensor histidine kinase (Hasegawa et al., 2003). Although most... |
Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP |
Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BC... |
Introduction |
125 years of intense research on the major human pathogen Mycobacterium tuberculosis have passed since its discovery by Robert Koch, resulting in a huge body of knowledge. In spite of the great progress that has been made in the understanding of some basic features of its pathogenesis, tuberculosis remains a major thre... |
Results |
Microarray-Based Comparative Genome Sequencing of M. tuberculosis H37Ra The genome-wide comparative mutational analysis of H37Ra and H37Rv was carried out by NimbleGen Systems following a previously published method [13]. Putative SNPs with high probability scores were separated into synonymous and non-synonymous SNPs ... |
Rationale for Knock-Ins |
To evaluate the phenotypic effect of the different SNPs and to assess their potential contribution to the attenuation process, we undertook functional genomic analyses using knock-ins of H37Ra, as described previously [18]. Clones spanning the different genomic regions of non-synonymous SNPs were selected from an order... |
Ex Vivo Virulence Studies |
Changes in the regulatory potential of a pathogen are often accompanied by altered virulence. In a first attempt to determine the virulence of the complemented H37Ra knock-in strains relative to wild-type H37Ra and H37Rv, bone marrow-derived murine macrophages (BMM) were infected with the different strains at a multipl... |
Virulence Studies of H37Ra Complemented Mutants in a Mouse Model |
Further assessment of the in vivo growth of different H37Ra knock-in strains was carried out by intravenous infection of severe combined immuno-deficient (SCID) mice. Complementation of H37Ra with the PhoP-expressing cosmid increased the virulence of the H37Ra::phoP recombinant relative to H37Ra, resulting in a 1.0 log... |
Link between Mutation in phoP and Secretion of ESAT-6 |
As PhoP fulfills important regulatory functions in M. tuberculosis [21,22], it was of primary interest to identify and study potential effector molecules whose involvement in host pathogen interaction were influenced by the point mutation in phoP of H37Ra. Since secreted proteins of the ESX-1 system of M. tuberculosis ... |
Functional Characterization of Knock-In Mutants |
We have previously shown that antigen-specific IFN-gamma production of splenocytes is a reliable readout system to evaluate whether or not ESAT-6 was secreted by recombinant strains [23,24,26]. Thus, in order to determine the reason for the observed failure of H37Ra to induce ESAT-6-specific responses, H37Rv and H37Ra ... |
Discussion |
The attenuated H37Ra strain was obtained at the Trudeau Institute in the 1930s in an attempt to dissociate virulent and avirulent forms of the tubercle bacillus H37. Steenken et al. have shown that the virulence of the H37 strain was associated with colony morphology and that the avirulent variant H37Ra failed to propa... |
Comparative analysis of the Photorhabdus luminescens and the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity |
Background Photorhabdus luminescens and Yersinia enterocolitica are both enteric bacteria which are associated with insects. P. luminescens lives in symbiosis with soil nematodes and is highly pathogenic towards insects but not to humans. In contrast, Y. enterocolitica is widely found in the environment and mainly know... |
Background |
Pathogenicity as well as symbiosis plays a key role in the interaction of bacteria with their hosts including invertebrates. Despite the relevance of this relationship for the evolution of bacterial pathogenicity, few studies have addressed this subject at the genomic level. We therefore decided to perform a comparativ... |
The TGF-beta type II receptor in chronic myeloid leukemia: analysis of microsatellite regions and gene expression. |
Genomic instability is one mechanism proposed to play a role in the disease progression of chronic myeloid leukemia (CML). Microsatellite regions in the type II transforming growth factor-beta receptor (TGF-beta RII) gene appear to be targets for mutation in some cancers displaying microsatellite instability (replicati... |
N-glycosylation of glucose transporter-1 (Glut-1) is associated with increased transporter affinity for glucose in human leukemic cells. |
To elucidate the role of N-glycosylation in the functional activity of the universal glucose transporter, Glut-1, we investigated effects of the N-glycosylation inhibitor, tunicamycin, on glucose transport by human leukemic cell lines K562, U937 and HL60. Treatment with tunicamycin produced a 40-50% inhibition of 2-deo... |
Immunohistochemical analysis of nm23-H1 gene product in node-positive lung cancer and lymph nodes. |
The nm23-H1 gene product has been considered as an anti-metastatic protein and the level of its expression has been reported to correlate inversely with metastatic potential in some cancers. However, the expression of nm23-H1 gene product in the metastatic sites have not been studied in detail. We examined the expressi... |
Bradycardia-induced coronary angiogenesis is dependent on vascular endothelial growth factor. |
A marked coronary angiogenesis is known to occur with chronic bradycardia. We tested the hypothesis that vascular endothelial growth factor (VEGF), an endothelial cell mitogen and a major regulator of angiogenesis, is upregulated in response to low heart rate and consequential increased stroke volume. Bradycardia was i... |
External beam radiotherapy for subretinal neovascularization in age-related macular degeneration: is this treatment efficient? |
PURPOSE: Control of the natural course of subretinal neovascularization (SRNV) in age-related macular degeneration (AMD) is difficult. Only a subset of patients is suitable for laser coagulation. This prospective study aimed to determine the efficacy and individual benefit of external beam radiotherapy (EBRT). METHODS ... |
Increased serum levels of vascular endothelial growth factor in patients with renal cell carcinoma. |
Neovascularization, an essential event for the growth of solid tumors, is regulated by a number of angiogenic factors. One such factor, vascular endothelial growth factor (VEGF), is considered to exert a potent angiogenic activity, as indicated by immunohistochemical and molecular evidence. In this study we investigate... |
Insulin-induced vascular endothelial growth factor expression in retina. |
PURPOSE: Clinical studies have demonstrated that intensive insulin therapy causes a transient worsening of retinopathy. The mechanisms underlying the initial insulin-induced deterioration of retinal status in patients with diabetes remain unknown. Vascular endothelial growth factor (VEGF) is known to be operative in th... |
The antiangiogenic agent linomide inhibits the growth rate of von Hippel-Lindau paraganglioma xenografts to mice. |
The aim of this study was to ascertain the potential usefulness of the antiangiogenic compound linomide for treatment of von Hippel-Lindau (VHL)-related tumors. Paraganglioma tissue fragments obtained at surgery from a VHL type 2a patient were transplanted s.c. to male BALB/c nu/nu (nude) mice: (a) 2-3-mm fragments for... |
LMP1 of Epstein-Barr virus induces proliferation of primary mouse embryonic fibroblasts and cooperatively transforms the cells with a p16-insensitive CDK4 oncogene. |
The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkin's lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumor... |
Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells. |
Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, was originally isolated as a potent stimulator of tumor cell motility. In order to study whether ATX expression affects motility-dependent processes such as invasion and metastasis, we stably transfected full-length ATX cDNA into two non-expressi... |
Oncogenic epidermal growth factor receptor mutants with tandem duplication: gene structure and effects on receptor function. |
A number of epidermal growth factor receptor (EGFR) deletion mutants have been identified in gliomas, in which the EGFR gene is frequently amplified and rearranged. We have previously characterized the structure of a gene in A-172 human glioma cells that encodes a 190-kDa EGFR mutant with tandem duplication of the tyro... |
Ciprofloxacin mediated cell growth inhibition, S/G2-M cell cycle arrest, and apoptosis in a human transitional cell carcinoma of the bladder cell line. |
The second most prevalent urological malignancy in middle aged and elderly men is bladder cancer, with 90% of the cases being transitional cell carcinomas. The success of current systemic and intravesical therapeutic agents, such as cisplatin, thiotepa, Adriamycin, mitomycin C, and bacillus Calmette-Guerin, is limited ... |
Vascular proliferation and enhanced expression of endothelial nitric oxide synthase in human peritoneum exposed to long-term peritoneal dialysis. |
Long-term peritoneal dialysis (PD) is associated with alterations in peritoneal permeability and loss of ultrafiltration. These changes originate from increased peritoneal surface area, but the morphologic and molecular mechanisms involved remain unknown. The hypothesis that modifications of activity and/or expression ... |
Active hair growth (anagen) is associated with angiogenesis. |
After the completion of skin development, angiogenesis, i.e., the growth of new capillaries from pre-existing blood vessels, is held to occur in the skin only under pathologic conditions. It has long been noted, however, that hair follicle cycling is associated with prominent changes in skin perfusion, that the epithel... |
Neuroblastoma and hepatocyte coculture conditioned media alter apoptosis. |
BACKGROUND: Neuroblastoma is a childhood tumor that often displays unusual biological behavior. The tumor may present with widespread metastases that are unresponsive to aggressive treatment. At other times, both the metastases and the primary tumor may spontaneously regress without treatment. Apoptosis, or programmed ... |
Neoplastic transformation by Notch requires nuclear localization. |
Notch proteins are plasma membrane-spanning receptors that mediate important cell fate decisions such as differentiation, proliferation, and apoptosis. The mechanism of Notch signaling remains poorly understood. However, it is clear that the Notch signaling pathway mediates its effects through intercellular contact bet... |
Alpha-melanocyte-stimulating hormone modulates activation of NF-kappa B and AP-1 and secretion of interleukin-8 in human dermal fibroblasts. |
Alpha-melanocyte-stimulating hormone (alpha-MSH) has evolved as a mediator of diverse biological activities in an ever-growing number of non-melanocytic cell types. One mechanism by which alpha-MSH exerts its effects is modulation of AP-1 and NF-kappa B. These two transcription factors also play an important role in fi... |
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