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Novel Algorithms Reveal Streptococcal Transcriptomes and Clues about Undefined Genes
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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) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.
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Introduction
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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 experimental variability, and often result in arbitrary thresholds for significance [1,2]. In addition, unknown bacterial genes can confound the interpretation of expression profiles, restricting many microarray studies to the differential expression of well-characterized genes. Several methods are available to organize gene expression profiles and to assist in extracting functional or regulatory gene information from microarray datasets. Clustering algorithms group genes by similarities in expression patterns, based on the assumption that co-expressed genes share common function or regulation [3,4]; however, clustering solely by co-expression patterns may not reveal a considerable amount of information contained in array data. These methods often: (1) produce unreliable data by missing known gene members of biological pathways; (2) fail to distinguish truly related gene clusters from coincidental groupings; and (3) identify clusters containing only unknown genes that may lack either common function or regulation, a considerable limitation for genomes containing a large percentage of undefined genes [1,2]. Because no tools exist to interpret unknown gene clusters or to assess their significance and completeness, a significant portion of bacterial expression profiles are not interpretable using current clustering methods. We introduce neighbor clustering as a new tool for analyzing bacterial microarray data that addresses some of these limitations by incorporating the physical position of genes on the bacterial chromosome into the analysis of expression data. Information about gene function and regulation is stored intrinsically in the bacterial genome structure, as genes with common function or regulation tend to be physically proximate on the chromosome and often linked as operons [5,6]. We incorporated these positional data into a series of neighbor clustering algorithms, named GenomeCrawler, that identifies groupings of potentially related genes from array data by combining two informative characteristics of bacterial genes that share common function or regulation [3-6]: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. The algorithms also recalculate the statistical significance of each gene as a member of a particular cluster, as well as the significance of each resulting grouping as a whole, to ensure accuracy of cluster assignments. This process ultimately identifies significant clusters of co-expressed gene neighbors that likely share common function or regulation. We used this approach to analyze microarray expression data from group A streptococci (Streptococcus pyogenes) during adherence to human pharyngeal cells, the first overt infection step [7]. The ability of all bacterial pathogens to infect the human host depends upon coordinated regulation of diverse gene sets that are required for survival in host environments. Although recent microarray studies have highlighted the molecular responses of streptococci in relevant host conditions [8-10], characterizing differentially expressed loci during pharyngeal cell adherence is critical for understanding the molecular basis for host colonization. Studies from our laboratory [11,12] and others [13] have demonstrated that in vitro association with pharyngeal cells results in streptococcal phage induction and the increased expression of phage-encoded virulence factors. Although the mechanisms mediating these responses are not known, the results of these studies indicate that streptococci sense and, on a transcriptional level, respond to various signals and cues in the pharyngeal cell environment. We undertook the present study to understand and to assess more accurately the genome-wide transcriptional responses of streptococci during one of the earliest recognized stages of infection, namely adherence to human pharyngeal cells. We compared data generated before and after neighbor clustering to show that this method provides a more comprehensive view of transcription by: (1) identifying more differentially expressed genes than even traditional, rigorous statistical analyses; (2) reconstructing intact biological pathways that statistical significance analysis could not reconstruct; and (3) providing preliminary insight and clues about the function or regulation of uncharacterized genes by associating their co-expression with physically proximate, functionally defined genes.
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Results/Discussion
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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 assays were performed as described [15] with modifications to minimize eukaryotic cell disruption. We replicated experiments independently and used dye-swaps to incorporate biological and technical variation [16,17]. Following filtering and normalization [18,19], we analyzed data from four biological replicates [16] with robust summary statistics [20], Bayesian statistics [21,22], and permutation algorithms [19] to identify genes differentially expressed with significance during pharyngeal cell adherence. This analysis identified 79 genes (4% of the genome) exhibiting statistically significant fold changes in expression (PF value < 0.05) during adherence from 1,769 open reading frames represented on the array (Table 1). We refer to such genes as "differentially expressed." We present the entire dataset from all experiments as Table S1. Genes demonstrating upregulation (n = 45) and downregulation (n = 34) included virulence factors, prophage-encoded transcripts, metabolic genes, and transcriptional regulators (Table 2). Undefined or hypothetical genes comprised 27% of differentially expressed genes (n = 21; 11 chromosomally encoded genes, ten phage-encoded genes).
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Verification by Quantitative Real-Time PCR
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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 < 0.05; two upregulated, three downregulated). The remaining six genes (four upregulated, two downregulated) did not have significant PF values, but were statistically significant as members of particular neighbor clusters in subsequent analyses (PE < 0.05) as detailed in later sections). We averaged the data to generate a value for each gene, creating a set of 11 paired values from quantitative real-time (qRT)-PCR and microarray analyses (Table S3). Results of standard linear regression analysis demonstrated a strong positive correlation (r = 0.9) between data obtained using the different techniques (see Figure S1).
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Virulence Factors
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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 SpeB protease (spy2039) were downregulated, while genes encoding pyrogenic exotoxin H (speH or spy1008) and a putative fibronectin-binding protein (spy0130) were upregulated. We verified the differential expression of spy2039 and spy0130 by qRT-PCR. The downregulation of virulence loci during presumably inappropriate stages of infection was not surprising. Streptolysin O is a cytotoxin that damages human tissue and increases host cell cytotoxicity [7,25]. The resulting cellular damage, particularly to polymorphonuclear leukocytes [26], decreases internalization and subsequent intracellular killing of streptococci [27]. Based on its downregulation during adherence, we infer that slo was transcribed during pre-adherence associations, perhaps, as previously reported, to protect streptococci from phagocytic killing in vivo [27]. However, once adhered, our data suggest that streptococci downregulate production of this cytotoxin, presumably to prevent further host tissue destruction that could interfere with adherence. SpeB (encoded by spy2039) is a multifunctional cysteine protease implicated in numerous infection strategies [28,29]. Although few studies have examined gene expression patterns during adherence, SpeB production (as detected by Western blot analysis) decreases during co-culture with human peripheral blood mononuclear cells [30] and in a mouse infection model [31]. When SpeB expression is limited, several streptococcal proteins necessary for adherence remain intact [24,32,33]; thus, decreased SpeB production (as indicated here) may promote pharyngeal cell attachment. Furthermore, SpeB abolishes internalization (following adherence) of certain streptococcal strains by epithelial cells (including Detroit 562 cells), a process mediated in part by the fibronectin-binding protein F [34,35]. We observed significant upregulation of the gene spy0130, encoding a protein recently found to be associated with the production of surface-exposed pili on strain SF370 [36]. The protein shares 60% sequence similarity to protein F, suggesting that it may coordinate a similar internalization mechanism or may be involved directly in adherence (discussed later in detail). SpeB downregulation also coincides with increased expression of pyrogenic exotoxins [33,37] that reportedly increase streptococcal survival in vivo. We observed that the exotoxin-encoding speH gene [38] was upregulated. Taken together, our results agree with previous reports on SpeB production during host cell interactions, suggesting that decreased expression may promote streptococcal adherence (by preventing proteolytic degradation of key virulence factors or adhesins), enhance internalization (perhaps through a fibronectin-mediated pathway), and increase survival (through increased pyrogenic exotoxin production, discussed below).
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Phage-Encoded Genes
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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, and the remaining genes, hypothetically involved in replication and regulation [39], were downregulated. The speH gene encodes a mitogenic exotoxin [38] reportedly induced during polymorphonuclear leukocyte phagocytosis [8] but not implicated previously in adherence.
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Allelic Replacement of speH
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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 analysis were downregulated. To determine if SpeH plays a direct role in the adherence process, we created a deletion mutant in strain SF370 (SF370DeltaspeH), which was confirmed by PCR (unpublished data) and RT-PCR (Figure 1A) and tested in vitro for adherence to human pharyngeal cells. We observed no significant difference in adherence between the wild-type (SF370) and mutant strains (Figure 1B), indicating that SpeH is not involved directly in attachment to the pharyngeal cell. The significant upregulation of the speH gene during adherence suggests that the gene product may function instead during a subsequent stage of infection.
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Differential Expression of Genes from Diverse Functional Categories
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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 biosynthesis [40] were upregulated, suggesting that certain cofactors that may be necessary during adherence were unavailable. Also upregulated were genes encoding subunits of the F0F1 ATPase [41] (discussed in more detail later), which may indicate an acid stress response to maintain cytoplasmic pH or a need to generate ATP in response to increased energy requirements. We also identified the adherence-mediated upregulation of four transcriptional regulators (Table 1), suggestive of an adaptive response to host cell contact that is dynamic and complex. For example, RopB (encoded by spy2042), a member of the Rgg family of response regulators, interacts with a number of regulatory networks throughout the streptococcal genome (e.g., mga, csrRS, sagA, and fasBCA), affecting the transcription of numerous proteins, virulence factors, and two-component regulatory systems [42,43]. Although the delineation of genes influenced by RopB (or any identified transcriptional regulator) is beyond the scope of this study, our initial analysis did identify the upregulation of a two-component regulatory system, encoded by spy1236-1237. The functions of these particular loci are not yet known, and their adherence-mediated upregulation represents new targets in the study of regulators that function during host cell contact.
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Neighbor Clustering
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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 differential expression of a large number of unknown genes (n = 21) and a number of incomplete biological pathways (e.g., F0F1 ATPase [41] and folate biosynthesis [40]) by failing to detect the differential expression of a number of known gene pathway members (Table 1). To overcome these limitations and to extract more functional information from the array dataset (including more complete biological pathways), we developed the neighbor clustering algorithms to combine the physical position of genes on the streptococcal chromosome with gene expression data. Neighbor clustering was designed to identify expanded groupings of potentially related genes from our array data by incorporating two reliable predictors of genes that share common function or regulation, namely physical proximity and similar expression profiles [5,6]. We implemented this approach by developing an algorithm with dynamic windowing (GenomeCrawler) that sequentially stepped through the microarray data and identified clusters of adjacent genes exhibiting similar fold changes in expression. Because the genome contains many possible clusters, we restricted the algorithm's search space to identify only spatially related clusters. GenomeCrawler applied a separate permutation algorithm, using the sum of each gene's t-statistics to calculate adjusted P values (PK) for each cluster, which corresponded to the probability of assembling a cluster by chance. Significance was assigned to clusters with PK < 0.05, and the resulting groupings are listed in Table 3. Because individual genes could be members of many different significant clusters, GenomeCrawler then applied a distinct permutation algorithm to calculate the probability (PC) that a gene was clustered coincidentally. Calculation of PC values relies on Bayes' Theorem, in which the probability of a gene's log2-fold change (PF value) is combined with the cluster probability itself (PK value). We stress that PC reflects the significance of a gene based on its cluster context rather than a recapitulation of PF. This ensures a strong dependency between PF and PC, preventing a gene with a relatively low log2-fold change from being scored as significant simply because it is clustered with a gene with a highly significant PF value. Finally, GenomeCrawler calculated the overall significance of differentially expressed genes (PE values) by integrating differential expression probabilities (PF) and cluster context probabilities (PC). We developed a plotting application (GenomeSpyer) that represents the chromosome as a linear molecule to visualize GenomeCrawler output, with genes displayed on the x-axis and their log2-fold change magnitudes on the y-axis. Applications and all datasets are available for download at http://www.rockefeller.edu/vaf/streparray.php. We visually inspected the resulting clusters and disqualified those that violated our neighbor cluster definition (see Methods for details). All output prior to cluster disqualifications is included for comparison (see Table S4). Of the 309 qualifying clusters (Table S5), 197 (63.8%) were composed entirely of known, functionally defined genes; however, 26 (13%) of these were incorrectly assembled, as they contained known genes that are functionally unrelated. Because we did not incorporate functional annotations of genes into the algorithms (i.e., to keep the analysis "blind"), we anticipated the possibility that some groupings could be assembled incorrectly despite the statistical framework for assigning clusters. Of the remaining 283 (91.6%) groupings, a number of differently sized clusters contained the same gene (Table S5). We report such clusters first by highest significance (lowest PK value), then by largest number of genes. Thus, if clusters containing a particular gene were of equal significance, we report the cluster with the most gene members. This method identified 47 significant clusters containing 173 differentially expressed genes (listed in Table 3 and visualized in Figures 2 and S2-S4), a considerably larger group than could have been compiled using only the initial 79 significant genes. A total of 56 of the original 79 significant genes became components of significant clusters, whereas 23 remained unclustered. We subdivided all clusters into three qualitative types based on the functional annotation of gene members. We present examples of Type I and II clusters: Type I clusters (n = 25) contained only functionally defined and functionally related genes (as reported in published studies), such as biological pathways components (Figures 2B and S2); Type II clusters (n = 20) included both known and unknown genes (Figures 2C and S3). Type III clusters (n = 2) were composed entirely of unknown genes (Figures 2D and S4), and are not discussed in detail.
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Type I Clusters: Intact Metabolic Pathways and Multimeric Proteins
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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 clustered genes exhibited significant differential expression (PF value < 0.05). A total of 11 (52.4%) of the remaining 21 clusters would not have been identified in their entirety without GenomeCrawler because we initially identified significant fold-changes in only a subset of genes necessary to encode particular pathways or loci; this is intuitively unreasonable if all genes are essential for functionality. GenomeCrawler expanded these clusters to contain more genes that encode intact loci (Table 3). For example, we initially identified (Table 1) the significant upregulation of three of the five known gene members of the folate biosynthetic pathway [40] (spy1096-1100), but GenomeCrawler identified a significant cluster containing all five genes (Table 3 and Figure 2B). We obtained a similar result for the eight-gene operon encoding the F0F1-type proton translocating ATPase [41] (spy0754-0761). The initial significance analysis identified only four atp genes (Table 1), but neighbor clustering identified a significant cluster containing all eight genes necessary to encode a functional ATPase (Table 3). Each of the 11 neighbor clusters that could have been only partially identified by our initial analysis alone gained gene members after application of the algorithms and became more complete sets of functionally related genes than initially identified (Table 3). These clusters encompass various metabolic processes, including purine biosynthesis (spy0025-0028), lactose metabolism (spy1916-1923), fatty acid biosynthesis (spy1743-1747), lipoteichoic acid synthesis (spy1308-1312), and sugar phosphotransferase transport (spy1058-1060) [14], suggesting that specific changes occur in the streptococcal metabolic program as the bacteria adhere to human pharyngeal cells in vitro. Notably, the remaining ten Type I clusters were composed entirely of genes that individually were not significant; however, after applying our algorithms, the combined contribution of each gene resulted in a significant cluster. For example, the nine-gene operon that spans genes spy0738-0746 encodes streptolysin S, a potent cytolytic toxin that promotes internalization and host tissue dissemination [25,44]. Though the differential expression of the individual genes was not significant following our initial statistical analysis, GenomeCrawler identified a significant downregulated cluster containing all nine genes (Table 3). Adherence-induced downregulation of streptolysin S is consistent with its previously determined role in host cell internalization [25]; however, without neighbor clustering, expression of this operon was not evident immediately. Although individual gene members of Type I clusters may not be statistically significant as a result of technical variability within experiments [17], the genetic structure of certain Type I operons may provide an alternative explanation. For example, the streptolysin operon encodes an internal terminator downstream of the sagA gene (the first gene in the operon), which modulates the abundance of particular mRNA species (e.g., sagA mRNA versus the polycistronic message for all nine genes) under different environmental conditions [45]. If transcription is internally disrupted by such a terminator, the abundance of the sagA transcript may be much greater than the polycistronic message; such disproportionate transcript levels would affect log2-fold change values and impact the statistical significance of individual genes within these types of clusters. Thus, in addition to helping resolve clusters that would not be easily recognized because of experimental technical variability, the neighbor clustering method may help to resolve operons with such internal terminators and regulators. These results demonstrate that neighbor clustering effectively reconstructed a number of complete pathways and loci from processed array data. Importantly, because functional gene data are not incorporated into its algorithms, GenomeCrawler is not biased toward identifying "expected" clusters. Curating the dataset following its application may make the algorithms less user-friendly; however, the elimination of such bias is essential for this type of analysis.
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Type II Clusters
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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 associating their expression with neighboring genes of known and defined function. Alternatively, co-expression of genes results from common regulation, and Type II associations may suggest shared regulatory mechanisms for clustered genes. We note, however, that despite the statistical framework with which groupings are assigned, experimental evidence is necessary to confirm functional or regulatory relatedness. We do not suggest simply assigning either based on cluster membership; rather, cluster associations may provide some preliminary functional or regulatory clues for gene members. A total of 18 (90%) of 20 Type II clusters (Table 3 and Figure S3) may not have been identified without neighbor clustering: eight (44.4%) of 18 gained additional gene members; the remaining ten comprised genes that demonstrated significant differential expression only after applying GenomeCrawler. Only two clusters (spy0127-0130 and spy1701-1704) could have been identified without neighbor clustering; however, a number of these genes were initially annotated as hypothetical proteins, so a potential relationship between the gene members may not have been readily apparent. The upregulated spy0127-0130 cluster is part of a larger genomic region known as FCT (for fibronectin- and collagen-binding proteins and T antigen-encoding loci), which spans spy0123-0136 in the SF370 genome and encodes surface proteins and transcriptional regulators [46]. A search of both the PFAM database [47] (http://pfam.wustl.edu) and sortase database (http://www.doe-mbi.ucla.edu/Services/Sortase) predicted that spy0129 encodes a sortase enzyme, which are transpeptidases that cleave protein substrates at conserved C-terminal motifs (often LPXTG) and then anchor these proteins to the bacterial cell wall [48,49]. Recently, it was reported that the four genes spanning spy0127-0130 encode, and are responsible for, the formation of surface-localized, trypsin-resistant pili that induce protective immunity against a lethal dose of group A streptococci in a mouse model of infection [36]. This same report provided the first experimental evidence supporting the sortase prediction, indicating that the gene product of spy0129 is responsible for the cell-wall sorting of the proteins encoded by both spy0128 (annotated as a Cpa homolog [50]) and spy0130 (annotated as a protein F homolog [14]). Furthermore, the spy0128-encoded protein is the structural backbone of the pili, and the gene product of spy0130 may be involved in stabilizing the structure [36]. Together with the identification of this cluster by GenomeCrawler, these results prompted us to study this cluster and the contributions of the gene products to pharyngeal cell adherence. We determined experimentally that cluster spy0127-0130 is an operon, verifying both related function and regulation of the gene members. Reverse transcription of SF370 RNA, with primer combinations that spanned all four genes, produced cDNA fragments of sizes that could only result from a polycistronic mRNA template (Figure 3). In silico sequence inspection identified a single putative promoter sequence upstream of spy0127 (see Table S6). Although GenomeCrawler is not an operon-identifying algorithm, these results show that it could (1) identify this commonly regulated gene cluster and (2) define the cluster boundaries, excluding other proximate genes, such as an additional sortase-encoding gene, spy0135.
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Allelic Replacement of spy0129
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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 of the gene products of this cluster, since both the spy0128 and spy0130 gene products do not localize to the cell-wall surface in the absence of the sortase enzyme [36]. Allelic replacement created two putative deletion mutants; however, RT-PCR analysis (Figure 4A) revealed that only one such clone (SF370Deltaspy0129.2) was a true knock-out for the spy0129 gene and useful for further study. Because the gene cluster is also an operon, expression of the downstream gene spy0130, encoding the protein F homolog/pilus protein, was also eliminated in this mutant (Figure 4A). In vitro pharyngeal cell adherence assays revealed that the SF370Deltaspy0129.2 mutant was approximately 66% less adherent than the parental control strain, SF370 (Figure 4B; p = 0.03 as determined by the Student's t-test). These results suggest that either the spy0130 gene product is involved directly in adherence, or that due to the elimination of the sortase, the pili, which may function in their entirety as adhesins, were not assembled on the surface of the mutant. Because the spy0129 gene product is not expected to be found on the streptococcal surface (i.e., it lacks a cell-wall anchoring motif), it is not likely to be involved directly in adherence. We are working to produce an in-frame deletion of spy0128 and a spy0130 single knock-out mutant to delineate the contribution of each individual clustered gene product to adherence. These results show that neighbor clustering is able to identify biologically relevant gene clusters. This attribute may be particularly important for datasets in which the relationship between clustered genes is not obvious, and may facilitate the organization of larger datasets into more manageable packages.
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Additional Type II Cluster Example
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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 Escherichia coli [51]. We examined the spy1725 and spy1723 gene products (annotated as hypothetical proteins [14]) for similarities with known proteins that might indicate a role for these gene products in protein biosynthesis. BlastP analysis aligned the spy1725 gene product, which has homologs in all sequenced streptococcal genomes, with the SP14.3 protein from S. pneumoniae [52] (80% sequence similarity; 67% identity). Based on structural characterization, SP14.3 is a predicted RNA-binding protein. The spy1723 gene product has similar domain structure to the YlxR protein of S. pneumoniae, an RNA-binding protein implicated in transcription termination [53]. These results indicate that both genes likely encode RNA-binding proteins, in agreement with their functionally defined cluster members. Although domain and homology searches yielded the functional predictions, their membership within a protein biosynthetic cluster provided the initial indication of common function or regulation.
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Neighbor Clustering and Operons
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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 methods exist [54,55], we inspected clusters in silico for upstream regulatory elements and identified 17 candidates, including clusters such as streptolysin S that have been previously confirmed as operons [56]; the spy0127-0130 grouping, which was confirmed as an operon in this study; and others that have yet to be verified (Table S6). Experimental confirmation of each candidate is beyond the scope of this study, but Northern blot and RT-PCR analyses could provide such information.
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Analysis of Previously Published Array Data
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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 mutant deficient for the Mga regulon were compared during exponential growth in culture broth. The Mga regulator is a growth-phase mediator of a number of surface-exposed molecules and secreted proteins involved in colonization and immune evasion during infection [58]. Although the authors of that study did not provide a statistical analysis of their data, we compared the published results for the magnitude and direction of fold-changes for each gene reported in this study with those obtained from our initial significance analysis of this dataset (presented as Table S7). A total of 256 genes reported in this study were also detected by our analysis, and the magnitude and log2-fold change were found to be in agreement for 81% of the genes. We suspect that this discrepancy results from different normalization methods used, or from different methods that were applied to analyze the ratio of signal intensities between sample and control (i.e., we analyzed the ratios of the median rather than the ratios of the mean [57]). Although the published report did not include statistical analysis of the data, we note that the statistical analysis that we performed identified four genes with significant log2-fold changes in expression (PF < 0.05; Table S8). We applied the GenomeCrawler algorithms to the statistically analyzed dataset, which identified an expanded group of genes (107 versus four) contained within 36 statistically significant clusters (PK < 0.05; Table S9). These groupings included clusters of genes that have been shown previously in streptococci to be functionally related, indicating that the algorithms were performing as expected. Two of the identified upregulated clusters (spy2009-2010 and spy2039-2040) encoding the well-studied virulence factors, C5a peptidase and SpeB, respectively, showed consistently large log2-fold changes of the genes across replicates [57]. GenomeCrawler confirmed these results by identifying both groupings as statistically significant neighbor clusters. GenomeCrawler also identified a number of clusters that contained genes known to share common function or regulation; however, they were not as apparent in the dataset without its application. For example, the algorithm identified a significant neighbor cluster spanning spy0711-0712. This grouping encodes two known virulence factors, pyrogenic exotoxin SpeC and the MF2 DNase, previously shown to be commonly regulated as an operon [11]. The algorithm also identified other neighbor clusters containing genes known to be functionally related, including spy0098-0100 (encoding the beta and beta' subunits of DNA-dependent RNA polymerase), spy2159-2160 (encoding the 50S ribosomal subunit proteins L32 and L33), and spy0741-0746 (six of the nine streptolysin S-encoding genes) [14]. Although the analysis of this previously published dataset did not reveal as many intact biological pathways as were identified from the pharyngeal cell adherence data, the inclusion of more replicates in the analysis to increase statistical power could resolve such loci. However, these results provided further supporting evidence that the GenomeCrawler algorithms can identify (1) a larger group of genes than a rigorous statistical analysis alone and (2) biologically relevant groupings in other microarray datasets, even if they contain fewer replicates than presented in our study.
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Concluding Remarks
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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 information (expression data and gene annotations). We recognize that incorporating intergenic distance and transcription direction into the algorithms would reduce processing time. Adding available clusters of orthologous groups (COG) information into a downstream processing step could decrease errors by minimizing clustering of unrelated genes. Nonetheless, neighbor clustering provided a more comprehensive view of the transcriptome of group A streptococci during adherence to human pharyngeal cells, a critical step in the infection program of this organism. We found that even a rigorous statistical analysis of well-replicated microarray data produced a dataset that was somewhat limited, although certainly more informative than assigning arbitrary thresholds for significance. As described in other microarray reports, we had initially identified a number of incomplete biological pathways in which we did not detect the differential expression of a number of known pathway members. Neighbor clustering was able to extend the results by identifying more differentially expressed genes and reconstructing more intact biological pathways. Neighbor clustering, despite the statistical framework with which it assigns groupings, would be valuable to microarray data analysis only if it produced biologically relevant data. Although biological testing of every identified gene or cluster is unrealistic, we provided evidence, through the creation and testing of isogenic deletion mutants and through the identification of clusters of known, functionally related genes from a published streptococcal array study, that the algorithms produce results that are pertinent to the biology of streptococci. This may be of particular importance for data in which the relationship between clustered genes is not obvious, and may facilitate the organization of larger datasets into more meaningful packages. It is also possible that GenomeCrawler (in its current form) could be used to interrogate intergenic portions of the genome (such as those encoding small noncoding RNAs or sRNAs), if probes representing such regions were included on the microarray, and experimental conditions were designed to promote their differential expression. Finally, because of the common architecture of bacterial chromosomes, the neighbor clustering algorithms may be applicable to microarray datasets from other prokaryotes.
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Porphyromonas gingivalis short fimbriae are regulated by a FimS/FimR two-component system
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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 that both fimbriae are required for formation of P. gingivalis biofilms. Here, the novel finding that FimR, a member of the two-component regulatory system, is a transcriptional activator of the mfa1 gene is promoted. Unlike the regulatory mechanism of FimA by FimR, this regulation of the mfa1 gene is accomplished by FimR directly binding to the promoter region of mfa1.
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Introduction
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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. gingivalis to initiate a periodontal infection is mainly dependent on the expression of fimbriae (Malek et al., 1994). Two distinct fimbriae are found on the surface of the organism (Dickinson et al., 1988; Hamada et al., 1996). The long (major) filamentous structure is comprised of a FimA subunit protein encoded by the fimA gene. The short (minor) fimbriae are made up of a 67 kDa protein (Mfa1). Both fimbriae appear to be involved in bacterial pathogenicity (Amano et al., 2004). The function of the FimA protein and regulation of fimA expression have been extensively studied. The FimA protein is required for P. gingivalis colonization on salivary coated surfaces, and the early colonization of dental plaque (Malek et al., 1994; Levesque et al., 2003; Maeda et al., 2004). A P. gingivalis fimA mutant shows impaired invasion capability of epithelial cells compared with wild-type strain, suggesting the involvement of FimA in the bacterial interaction with surface receptor(s) on gingival cells (Weinberg et al., 1997). Earlier studies by the authors showed that FimA expression was modulated by environmental cues, including temperature and hemin concentration, and by the presence of Streptococcus cristatus, an early colonizer of dental plaque (Xie et al., 1997, 2000b). FimR, a response regulator of the fimS/fimR two-component system was identified, and FimA expression was found to be dramatically reduced in fimR mutants (Hayashi et al., 2000). Investigation of the mechanism of regulation of fimA by FimR indicates that FimR does not bind directly to the fimA promoter, but rather binds to the promoter region of the first gene (pg2130) in the fimA cluster, suggesting that PG2130 is the FimR target gene, which in turn regulates expression of other genes in the fimA cluster, including the fimA gene (Nishikawa et al., 2004). The short fimbriae (Mfa1) also contribute to P. gingivalis colonization. Coadhesion and biofilm development between P. gingivalis and Streptococcus gordonii require the interaction of Mfa1 with streptococcal protein SspB (Park et al., 2005). The authors have recently reported that the short fimbriae are required for P. gingivalis cell-cell aggregation, an essential step in microcolony formation (Lin et al., 2006). A mutant with a deficiency in minor fimbriae binds to a saliva-coated surface but does not form microcolonies as the wild-type strain does. Mfa1 expression appears to fluctuate under various growth conditions (Masuda et al., 2006). In a nutrient-limited medium, expression of FimA and Mfa1 are inhibited in P. gingivalis, whereas such differences are not found in gingipain expression. A recent study has shown that expression of mfa1 is repressed in the presence of some common oral plaque bacteria such as S. gordonii, Streptococcus sanguinis and Streptococcus mitis (Park et al., 2006). However, very little is known about regulatory mechanisms of mfa1 expression. In this study, it is demonstrated that FimR is a positive regulator of Mfa1 expression. Evidence is provided that unlike FimR-dependent fimA expression, FimR regulates mfa1 expression by directly binding to the promoter region of mfa1.
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Results
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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 mutant was constructed by allelic replacement. Expression of fimA and mfa1 in the fimR- mutant was determined using real-time PCR analysis. Statistically significant differences of the level of gene expression in 33277 and the fimR- mutant were calculated by a Student's t-test. As shown in Fig. 1a, expression of the fimA gene was abolished in the fimR- mutant strain FRE. This result is consistent with previous observations (Hayashi et al., 2000; Nishikawa et al., 2004). The striking finding is that expression of the mfa1 gene was also repressed threefold in the fimR- mutant, although not to the degree observed with the fimA expression. However, the fimR- mutation had no effect on expression of rgpA, a gene encoding the arginine-specific protease, or the P. gingivalis 16S RNA gene. This analysis suggests the FimS/FimR system is required for expression of both major and minor fimbriae. To determine production of long (major) and short (minor) fimbriae in the fimR- mutant, western blotting was performed with a polyclonal anti-FimA or anti-Mfa1 antibody to compare fimbrial production in wild-type strain (33277), the fimR- mutant (FRE), the fimA- mutant (FAT) and the mfa1- mutant (MFAE). Density of protein bands was determined by UVP Bioimaging System (UVP, CA). This analysis revealed that the expression of the fimA and mfa1 genes was consistent at the mRNA level and protein level (Fig. 1b). FimA protein was not detectable in the fimR- mutant, while a 50% lower level of Mfa1 protein was found in the fimR- mutant compared with that in wild-type strain 33277. Similarly, there was no apparent change in RgpA production in the fimR- mutant, which was detected by anti-RgpA serum.
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Identification of the transcriptional start site of the mfa1 gene
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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 transcriptional start site (the A) of mfa1 was at 434 bp upstream from the potential start codon (Fig. 2b). To verify the result, the RACE experiment was repeated with mfa1-specific reverse primers MfaTSR3 and MfaTSR4 located at 237 bp upstream of the potential start codon. The same transcriptional start site was identified. To confirm the result of RACE, reverse-transcriptional PCR using three sets of primers was performed. As shown in Fig. 2c, the PCR product was generated only with primers MfaTSF1 (corresponding to +6 to +25) and MfaTSR5 (+805 to +824). There was no PCR product generated from RT-PCR using the primers (MfaTSF3, from -139 to -121 or MfaTSF2, from -66 to -47) which correspond to the DNA sequences upstream from the transcriptional start site. This transcriptional start site is 390 bp upstream of the site previously reported (Park et al., 2006). It is likely that mfa1 gene possesses two functional promoters, which are also detected in the fimA gene of P. gingivalis (Xie & Lamont, 1999; Nishikawa et al., 2004).
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Binding of FimR to the promoter region of mfa1
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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 observed for fimA expression. One possibility is that FimR modulates mfa1 expression by directly binding to the promoter region of mfa1. To test this hypothesis, electrophoretic mobility shift assays were performed. The mfa1 promoter (positioned from +18 to -138), fimA promoter (positioned from -22 to-190) (Xie & Lamont, 1999) and mfa1 coding DNA (positioned from +1253 to +1373) were generated by PCR with the 5' biotin-labeled primers (Table 1). The recombinant FimR (rFimR) was expressed in pET expression system and purified from E. coli. The rHGP44 protein, a binding domain of P. gingivalis gingipains (Xie et al., 2006), expressed in the same system and purified by the same procedures as rFimR was used as a control. Cold competitor chase experiments with a 100-fold excess of unlabeled DNA probe as a specific competitor were also used to demonstrate the specificity of rFimR binding. As shown in Fig. 3, the DNA fragment of the mfa1 promoter region was shifted in the presence of the rFimR. Retarded mfa1 promoter-rFimR complex was detected with as little as 10 pmol muL-1 rFimR (Fig. 3). As the concentration of rFimR increased, the retarded protein-DNA complex became evident, with complete loss of the mfa1 promoter DNA. The unlabeled mfa1 promoter fragments effectively competed with the labeled fragment, suggesting a specific interaction between rFimA and the mfa1 promoter. To investigate the role of phosphorylation of FimR in its binding to the mfa1 promoter region, EMSA experiments were also performed with the phosphorylated rFimR. No significant difference was detected in the level of DNA binding between the phosphorylated rFimR and unphosphorylated rFimR (data not shown). In agreement with a previous report (Nishikawa et al., 2004), rFimR did not bind to the fimA promoter region, suggesting that regulation of fimA expression by FimR is through a different mechanism. Moreover, incubation of rHGP44 with mfa1 promoter fragment did not retard the DNA movement in polyacrylamide gel. There was also no DNA shift detected when rFimR was incubated with the coding region of mfa1. These data clearly show that FimR protein can bind specifically to the mfa1 promoter region, acting as an activator of mfa1 transcription. EMSA experiments were also performed to examine whether the rFimR binds to the other promoter region identified by Park et al. (2006). The biotin-labeled DNA fragment corresponding to this promoter region did not shift in the presence of the rFimA protein (data not shown), suggesting that only the promoter identified here is involved for mfa1 expression mediated by FimR.
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Discussion
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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 target genes of P. gingivalis two-component systems are unknown, the role of the FimS/FimR in expression of the fimA gene is well defined. Expression of minor fimbriae (mfa1) in a fimR mutanthas been investigated. A comparison of the transcriptional level of the mfa1 in P. gingivalis wild-type strain and in the fimR mutant indicates that the FimS/FimR system is a positive regulator for the mfa1 gene, although the system controls two fimbrial genes at different levels. It is hypothesized that the FimS/FimR system regulates expression of each fimbrial gene through a unique mechanism. The previous study suggested that regulation of fimA expression by FimR is through a regulation cascade involving interaction of FimR and the promoter region of the first gene in the fimA cluster (Nishikawa et al., 2004). Here it is demonstrated that FimR binds directly to the promoter region of the mfa1 gene, suggesting a direct role of FimR in activation of mfa1 expression. It has also been reported previously that the transcriptional activity of fimA was reduced in the fimA mutant, indicating multiple levels of control of fimA expression in P. gingivalis (Xie et al., 2000a). This may explain the much tighter control of fimA expression by FimR. However, the possibility cannot be excluded that other regulatory elements are also involved in expression of the mfa1 gene. A two-component regulatory system typically contains a membrane-bound histidine kinase sensor and a cytosolic response regulator. Phosphorylation, mediated by histidine kinase at a specific aspartate residue, activates DNA-binding activity of the response regulator and initiates the corresponding cellular response. However, no apparent difference was found in DNA-binding affinity between rFimRs with or without acetyl phosphate treatment. Observation suggests that different mechanisms may be involved in P. gingivalis FimR activation. Activation of a regulatory protein not corresponding to phosphorylation was also observed in Streptococcus mutans (Biswas & Biswas, 2006). Phosphorylation of CovR, a global response regulator, had no effect on its DNA-binding affinity. The fact that FimR was not activated by phosphorylation may also be due to the short lifetime of the phosphorylated state, which has been observed in other bacteria (Stock et al., 2000). The transcriptional start site of the mfa1 gene located at 434 bp upstream of the putative start codon was detected, which is also 390 bp upstream of the site previously reported (Park et al., 2006). The transcriptional site revealed here is confirmed by RT-PCR analysis. Data of this study suggest that transcription of the mfa1 gene originated at a distal upstream transcriptional start site and read through the promoter region suggested by Park et al. (2006). Moreover, FimR appears to act on the promoter region identified here, suggesting that this promoter may make significant contributions toward mfa1 expression through the FimS/FimR system. Gene expression under the control of two promoters is not uncommon in bacteria. In E. coli, two promoters direct transcription of acs encoding, an acetate-scavenging enzyme required for fitness during periods of carbon starvation - the distal acsP1 and the proximal acsP2 (Beatty et al., 2003). It is suggested that each promoter may interact with different regulatory elements. Two promoter regions in the P. gingivalis fimA gene were also reported (Xie & Lamont, 1999; Nishikawa et al., 2004). A cascade regulation starting with FimR appears to act on the upstream promoter (Nishikawa et al., 2004). The observations that FimR binds only to the upstream promoter region of the mfa1 gene and that activity of the downstream promoter is inhibited by S. gordonii, S. sanguinis and S. mitis (Park et al., 2006) imply the complexity of regulation of mfa1 expression. It is possible that two promoters of mfa1 are regulated in response to different environmental signals. The hypothesis is currently under investigation. In conclusion, P. gingivalis fimbriae play a predominant role in the attachment of the organism to a variety of oral surfaces (Lamont & Jenkinson, 2000; Amano et al., 2004), although other surface proteins, such as gingipains, may also be involved in the bacterial colonization (Tokuda et al., 1996; Chen et al., 2001). It has been recently reported by the authors that both major fimbriae and minor fimbriae contribute to the formation of P. gingivalis biofilm (Lin et al., 2006). Major fimbriae are required for initial attachment and the minor fimbriae appear to play an important role in microcolony formation by facilitating cell-cell interactions. The data presented here provide evidence that these two distinct fimbriae are under the control of a two-component regulatory system: FimS/FimR. Expression of major fimbriae (FimA) is extremely low in the fimR mutant, and minor fimbriae production in this mutant is inhibited by least 50%. Therefore, it is proposed that FimR can be an attractive target for inhibition of P. gingivalis colonization.
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Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP
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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 (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra "knock-ins" that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis.
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Introduction
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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 threat to human life in most parts of the world. Indeed, M. tuberculosis has retained many secrets of its so successful pathogenic lifecycle. Among the different possibilities to obtain new insight into the mechanisms employed by M. tuberculosis to infect its host, the analysis of attenuated strains is one promising approach, and there are some well-documented examples of laboratory-attenuated strains. One of them is the "Bacille de Calmette et Guerin" (BCG), which was originally derived in 1921 from a virulent Mycobacterium bovis strain by 230 passages on potato-glycerol-ox bile medium [1]. The genetic lesions of BCG have recently been determined [2-4], revealing that the loss of region of difference 1 (RD1), which encodes part of the ESX-1 secretion system [5], was one of the key events in its attenuation. Another famous example of an attenuated strain is M. tuberculosis H37Ra ("a" for avirulent) (H37Ra). This strain was obtained in 1934 by serial passage of patient isolate M. tuberculosis H37 through media with different pHs [6] and since then has been widely used in many laboratories in the world. Despite its long use, the reasons for its stable attenuation have not yet been elucidated. As H37Ra is derived from the same parent strain as M. tuberculosis H37Rv ("v" for virulent) (H37Rv), the sequenced paradigm strain of tuberculosis research [7], genomic comparisons of the attenuated and virulent variants of M. tuberculosis H37 are particularly interesting and have the potential to identify subtle genetic changes that might be responsible for the phenotypic differences observed between the two strains. In a previous study we have tried to reveal these determinants [8], but the methods employed only identified large genetic polymorphisms, associated with IS6110, which were not found to be responsible for the attenuation of H37Ra [8]. In another study, Pascopella et al. [9] transformed a cosmid library of H37Rv into H37Ra and then selected for clones that were enriched on passage through the mouse. A number of overlapping cosmid clones that gave enhanced growth and survival in the spleens of infected mice relative to that of wild-type H37Ra were identified [9]. However, the effects of these complementation attempts on virulence remained limited, and no sequence information was described, which makes it difficult now to identify the genes implicated. H37Ra was also the subject of extensive micro-array based analyses, including whole genome comparative DNA/DNA analyses [10] and transcriptional studies [11,12], which have identified some candidate genes that were consistently downregulated. However, a definitive conclusion about the molecular determinants of the attenuation could not be drawn. As all these previous attempts have failed to identify the genetic basis for the attenuation, we subjected H37Ra to microarray-based DNA re-sequencing (NimbleGen Systems). This technique has previously permitted single nucleotide polymorphisms (SNPs) of a PA-824 drug resistant mutant strain of H37Rv to be detected [13]. This approach was combined with gene "knock-in" strategies, to complement selected lesions, that allowed recombinant H37Ra strains to be engineered, whose virulence and immunogenicity were then evaluated in in vitro, ex vivo and animal models. This strategy led us to identify and characterize a point mutation in the phoP/phoR two-component regulatory system of H37Ra that has uncovered novel regulatory links, which impact on the secretion and T cell recognition of the major T cell antigens ESAT-6 and CFP-10.
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Results
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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 of which the latter were verified by conventional dye terminator cycle sequencing. By this combined approach we identified 13 non-synonymous SNPs that differed between the H37Ra and H37Rv strains used (Table S1). We were particularly interested in a C to T mutation responsible for the serine to leucine replacement at position 219, S219L, of the two-component regulator PhoP as this protein is well known for its involvement in the virulent phenotype of M. tuberculosis [14]. Importantly, on inspection of the structure of the PhoP-ortholog from Bacillus subtilis, it was found that the equivalent residue Ser 207 is a main residue in the DNA-binding domain, helix alpha3 [15]. For the other 13 genes we found no indication that would identify them as potential virulence genes, as none of them belong to the 5% of genes that were previously determined by transposon site hybridization (TraSH) as being essential for in vivo growth of M. tuberculosis [16]. While writing this article, the whole genome sequence of H37Ra became publicly available (NC_009525), and comparison of the SNP data obtained from the H37Ra strain used in our laboratory (Table S1) with the NC_009525 data, showed that some differences existed between the H37Ra strains, a phenomenon which was also previously observed for BCG [4] and H37Rv [17]. Nevertheless, the S129L mutation in PhoP, which we consider an important SNP involved in the attenuation and reduced immunogenicity of H37Ra, is identical in all H37Ra strains.
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Rationale for Knock-Ins
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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 ordered H37Rv library of integrating shuttle cosmids [7,19] and individually electroporated into H37Ra, where they inserted stably into the attB site. By this approach we obtained appropriately complemented transformants for the SNPs in genes fadE5, rpsL, and phoP (Table S1), using cosmids I230, I563, and I36, respectively. Based on the known role of phoP in virulence [14], the H37Ra strain complemented with I36 (H37Ra::phoP) was accorded highest priority for further molecular characterization and functional analyses, whereas the two other recombinants (H37Ra::fadE5, H37Ra::rpsL) served as controls. Figure S1 shows part of the nucleotide sequence of a PCR-amplified fragment obtained from H37Ra::phoP. It is clearly visible that at nucleotide position 656 of phoP two peaks exist, one originating from the SNP present in H37Ra and one from the integrated cosmid I36 carrying the H37Rv wild-type copy of phoP. Similar results were also obtained for the SNPs in fadE5 and rpsL using cosmids I230 and I563, respectively (Figure S1). Correct integration of I36 was also confirmed by Southern blot. As depicted in Figure S2, hybridization of SpeI-digested genomic DNA with a 32P labeled phoP probe resulted in two bands of different sizes, one corresponding to the SpeI fragment harboring the H37Ra phoP gene (50 kb), and the other to the larger SpeI fragment created by integration of the I36 cosmid into the genome of H37Ra at the attB site. The successful integration of the cosmid was also reflected by a change in colony morphology that is shown in Figure S3. Indeed, Steenken and colleagues originally selected the H37Ra mutants mainly on the basis of the changes in colony morphology [6].
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Ex Vivo Virulence Studies
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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 multiplicity of infection (MOI) of 1:1 and 10:1 bacteria per cell. As depicted in Figure 1, macrophages were able to control ex vivo growth of H37Ra, but failed to control growth of H37Rv, confirming results from Freeman and colleagues [20]. When H37Ra knock-ins were tested, important differences in their ex vivo growth characteristics were found. Whereas H37Ra::fadE5 (Figure 1) and H37Ra::rpsL (unpublished data) showed very little or no growth over the 7-d period, the H37Ra::phoP mutant grew more vigorously, with a 7.5-fold increase in colony-forming units (CFU) over the 7-d period. From these experiments we concluded that complementation of H37Ra with the phoP wild-type copy partially restored its virulence, but not to the extent of the fully virulent H37Rv strain.
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Virulence Studies of H37Ra Complemented Mutants in a Mouse Model
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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 and 0.5 log increase in CFU number in lungs and spleens, respectively. In contrast, no effects on the virulence were observed when H37Ra was complemented with fadE5 (Figure 2) or rpsL (unpublished data). However, as already observed in macrophages, integration of phoP did not restore levels of virulence to those of the reference strain H37Rv (Figure 2). This situation is also reflected in the sizes of spleens, which correlate with the CFU data in spleens (Figure S4). Together with the data from the macrophage infection assay, the results from the mouse infection show that the S219L mutation in the phoP gene definitely represents one genetic lesion that contributed to the attenuation of the H37Ra strain.
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Link between Mutation in phoP and Secretion of ESAT-6
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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 constitute a major interface between the bacterium and its host [23], we analyzed the different strains for their potential to induce T cell responses against ESAT-6 and its binding partner, the 10-kD culture filtrate protein CFP-10. Groups of C57BL/6 mice were subcutaneously inoculated with H37Rv, H37Ra, or one of three recombinant H37Ra strains complemented with phoP, fadE5, or rpsL, respectively. Two weeks after vaccination we assessed the interferon-gamma (IFN-gamma) production of splenocytes mounted against ESX-1 antigens or controls. As anticipated, all tested strains induced IFN-gamma production in response to purified protein derivative (PPD) but not to a control peptide (Mal-E), indicating successful vaccination (Figure 3). Most importantly, the various strains differed extensively in their potential to induce antigen specific T cell responses towards ESAT-6 and CFP-10. As shown in Figure 3, splenocytes from mice that were inoculated with H37Rv produced high amounts of IFN-gamma upon stimulation with ESAT-6 or CFP-10, whereas the responses of H37Ra, H37Ra::fadE5, and H37Ra::rpsL infected mice were extremely weak. In contrast, splenocytes from H37Ra::phoP inoculated mice showed very strong IFN-gamma production in response to incubation with ESAT-6 and CFP-10. Exactly the same trend was observed when a highly sensitive T cell hybridoma assay was used to investigate ESAT-6 secretion. This test is based on the presentation of the immunodominant epitope contained in the first 20 amino acids of ESAT-6 by H37Ra, recombinant H37Ra, or H37Rv infected bone marrow-derived dendritic cells (BM-DC) to an ESAT-6-specific T cell hybridoma (NB11), restricted by I-Ab. Figure 4A shows that infection of BM-DC with H37Ra or H37Ra::rpsL induced no stimulation of the hybridoma as judged by IL-2 production, while H37Ra::phoP or H37Rv triggered high amounts of IL-2 production in this very sensitive and specific test. All strains behaved comparably towards the Ag85A:241-260 control, emphasizing the specificity of the observed phenomenon for ESAT-6 (Figure 4B). To further evaluate the involvement of phoP and the ESX-1 system in immunogenicity, C57BL/6 mice were vaccinated with additional strains. Firstly, we constructed a partially diploid H37Ra knock-in strain carrying cosmid RD1-ppe68-ko that, in BCG yields the greatest amounts of ESAT-6 expression and secretion [24]. However, in H37Ra the presence of this cosmid could not increase the weak ESAT-6 and CFP-10 T cell responses (Figure 3B) in the splenocyte IFN-gamma assay. Secondly, we also tested the previously described M. tuberculosis MT103 phoP knock-out (ko) strain SO2 [14,25] in this assay and found that this strain induced a potent PPD response as previously reported [25]. However, in comparison to the corresponding MT103 wild-type strain, the SO2 strain showed a strongly reduced ESAT-6 and CFP-10 specific T cell response, corresponding to a similar low level as the H37Ra strain (Figure 3B). Altogether, our data obtained from multiple replicate experiments indicate that a direct link exists between a fully functional PhoP and the ability to generate T cell responses against ESAT-6 and CFP-10.
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Functional Characterization of Knock-In Mutants
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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 strains were tested for their potential to express and secrete ESAT-6 in vitro. The western blot analysis presented in Figure 5 shows that the cell lysates of H37Ra and H37Ra::rpsL contain large amounts of ESAT-6, indicating that the antigen is properly expressed. However, hardly any ESAT-6 was present in the culture filtrates of these cultures, indicating that these strains were unable to secrete ESAT-6 under the in vitro conditions employed in spite of proper expression. Similar results were obtained for H37Ra::fadE5 (unpublished data). In contrast, from examination of the western blots of M. tuberculosis H37Ra::phoP and H37Rv it is clear that a large portion of their ESAT-6 protein is secreted into the culture filtrates. Analysis of the M. tuberculosis MT103 phoP ko strain SO2 showed strong expression of ESAT-6, but only very little secreted ESAT-6 (Figure 5). In a previous report [25], the presence of ESAT-6 in cell free extracts was described for this strain, but no secretion assay was performed. Together, our findings correlate perfectly with the in vivo data described above and suggest that the lack of ESAT-6 specific T cell recognition for H37Ra, H37Ra::fadE5, H37Ra::rpsL, and SO2 is not caused by a loss of ESAT-6 expression, but rather due to a failure of (phoP dependent) ESAT-6 secretion. The observation that knocking-in the ESX-1 region of H37Rv into H37Ra (H37Ra::RD1-ppe68-ko), and the resultant diploidy, did not improve the ESAT-6-specific T cell responses (Figure 3B), further argues that PhoP-dependent ESAT-secretion might be regulated via a mechanism that lies outside the RD1 region. As closer inspection of the available literature on transcriptional analyses of a H37Rv-phoP ko strain [21] and H37Ra [12] suggested that the expression of gene cluster rv3612c-rv3616c is reduced in both strains, we evaluated the expression level of rv3614c in H37Rv, H37Ra, and H37Ra::phoP by quantitative real time PCR (qRT-PCR). In previous studies rv3614c, rv3615c, rv3616c (espA) were independently shown to be essential for proper ESAT-6 secretion [27,28]. We confirmed the trend that rv3614c was expressed much lower in H37Ra compared to H37Rv by qRT-PCR (Figure 6), whereas expression of rv3614c in H37Ra::phoP was restored to wild-type levels, suggesting that the rv3612c-rv3616c gene cluster is regulated by PhoP. Interestingly, expression of phoP was higher in H37Ra than in H37Rv (Figure 6). These findings are consistent with data from a previous transcriptional study [12] and suggest that the proposed ability of PhoP to downregulate its own expression [22] is affected by the S219L phoP mutation [29]. They also indicate that it is not a lack of phoP expression that is causing the phoP-associated effects in H37Ra.
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Discussion
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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 propagate in an ordinarily susceptible host while the virulent variant H37Rv was able to proliferate [6]. Although these different H37 daughter strains have been available for many years now and belong to the standard strain collections of almost every tuberculosis diagnostic or research laboratory in the world [30,31], the genetic basis of the decreased virulence of H37Ra remained unknown. The identification of the amino acid change S219L in the predicted DNA binding region of the C-terminal effector domain of PhoP [15,22] in H37Ra was therefore a particularly exciting finding that has the potential to explain part of the attenuation of the H37Ra strain and to elucidate the PhoP/PhoR associated complex regulatory network in M. tuberculosis. In the bacterial world the PhoP/PhoR like two-component systems are widely distributed and fulfill a large variety of functions including sensing phosphate and magnesium [32,33], or regulating virulence genes [34]. For M. tuberculosis, several studies have described a link between the inactivation of phoP and loss of virulence, suggesting that the phoP gene is required for intracellular growth of M. tuberculosis [14,25]. It was also found that the PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids [35] and determines the amount of triacylated and monoacylated lipoarabinomannans in M. tuberculosis [36]. However, according to very recent information, these polyketide-derived (Pks2-4) lipids do not have a strong impact on the virulence of M. tuberculosis [29]. Finally, microarray-based trancriptome analysis using the wild-type versus a phoP ko mutant of H37Rv has provided an overview of the transcriptional regulation linked to PhoP [21]. That study identified 44 genes in the phoP-ko strain that were expressed at least 2.5-fold lower in the mutant than their counterparts in the M. tuberculosis wild-type strain. Most interestingly, about two thirds of these 44 genes were also found substantially lower expressed in H37Ra in comparison to H37Rv, when the transcriptional profiles of H37Ra and H37Rv cultures grown in various media were examined [12]. This finding argues that the PhoP S219L mutation likely has a broad impact on gene regulation in H37Ra with effects similar to those seen for complete inactivation of phoP. Among the genes expressed at low level in the phoP-ko strain and in H37Ra were several that are involved in lipid metabolism; for example, the polyketide synthases pks2 (rv3825c) or the Pks-associated protein papA1 (rv3824c). There were also several ppe genes that seem to be regulated by phoP but not ppe68 [21]. In addition, NirA (Rv2391), a putative nitrite reductase and MmpL10-FadD21 (Rv1183-85c), which were all identified by TraSH analysis as potentially implicated in virulence [16], seem to be regulated by PhoP [12,21]. However, the most striking link in relation to our results on phoP and ESAT-6 secretion that was revealed by these two transcriptional analyses, were the data on gene cluster rv3612c-rv3616c. Closer inspection of the data published in the corresponding supplemental materials showed that rv3612c-rv3616c were strongly downregulated in the H37Rv phoP ko mutant as well as in H37Ra [12,21]. These transcriptome data corroborate our results obtained by quantitative RT-PCR using probe rv3614c, which showed that gene rv3614c was definitely expressed much lower in H37Ra than in H37Rv and H37Ra::phoP strains (Figure 6). Since in previous, independent studies it has been demonstrated that functions of the rv3614c-rv3616c (espA) gene cluster were essential for the secretion of ESAT-6 and CFP-10 [27,28], it is quite probable that the lack of ESAT-6-specific T cell responses observed for strains H37Ra and SO2 (Figure 3) might be caused by the insufficient expression of rv3612c-rv3616c, which results from the lack of a fully functional PhoP in these strains. Moreover, the expression values of genes rv3612c-rv3616c in H37Rv described in the article by Gao et al. [12] show substantial differences between cultures grown in roller bottles relative to cultures grown in shake flasks, arguing that this gene cluster might be regulated by environmental conditions through the PhoP/PhoR system. It is not clear yet which factors might contribute to this effect. However, upregulation of phoP and rv3614c-rv3616c was observed after incubation of M. tuberculosis in 5mM H2O2 [37]. Furthermore, rv3614c-rv3616c were reported to be transiently upregulated in the phagosome and under acidic stress [38]. Altogether, from these reports it seems plausible that the pleiotropic regulator PhoP is involved in the regulation of rv3612c-rv3616c expression and thereby influences ESAT-6 secretion and virulence [16], but to unambiguously answer this question, virulence tests with recombinant H37Ra and/or phoP ko strains that express rv3616c-3612c under a constitutive promoter or a tetracycline inducable expression system [39] will be needed. Construction of such strains has recently been initiated as a first step of a future study to gain deeper insight into signaling events and regulation of the ESX-1 secretion system in different tubercle bacilli, including members of the putative progenitor species Mycobacterium prototuberculosis [40]. As in previous DNA/DNA microarray analyses [10], and from inspection of the very recently released genome sequence of H37Ra, no significant differences in the ESX-1 encoding region were found, the assumption that the observed lack of ESAT-6 secretion in H37Ra may be caused by factors outside of the RD1 region is very plausible. In contrast, in another transcriptome analysis, Mostowy and colleagues [11] found several genes of the RD1 region, including that encoding ESAT-6, among the genes that were at least 2.5-fold lower expressed in H37Ra than in H37Rv. Our results obtained for ESAT-6 expression (Figure 5) and the transcriptome data from Gao and colleagues [12] argue against direct downregulation of ESAT-6 in H37Ra. However, it is also possible that the observed differences may be caused by variations of in vitro culture conditions. In conclusion, in this work we report the identification and profound consequences of the PhoP S219L mutation on the widely used H37Ra strain. BLASTP comparisons of the PhoP sequence showed that the predicted DNA binding region, where the mutation occurred, is perfectly conserved among a wide range of actinobacteria, with H37Ra being the only strain showing such a mutation. By complementation with the wild-type copy of PhoP, we obtained a significant increase in virulence in ex vivo and animal models as well as restoration of ESAT-6 secretion and the accompanying antigen-specific immunological recognition by sensitized splenocytes or T cell hybridomas. It should be mentioned here that the SO2 phoP ko strain, which showed strong ESAT-6 expression but little ESAT-6 secretion (Figure 5), induced only very reduced ESAT-6- specific T cell responses (Figure 3). This strain was previously used to vaccinate mice and guinea pigs and conferred good protection against a challenge with virulent M. tuberculosis [25]. As ESAT-6-specific T cell responses induced by this strain are low, ESAT-6-based IFN-gamma production assays might be used to differentiate between vaccination with SO2 and infection with wild-type M. tuberculosis. With its well documented role in virulence and strong T cell antigenicity, ESAT-6 is one of the most important proteins of M. tuberculosis involved in host pathogen interaction [5,41-43]. However, as previously observed in complementation studies of BCG [18], the level of virulence displayed by the phoP complemented H37Ra strain was still substantially below that of wild-type H37Rv, arguing that the strong attenuation of the H37Ra strain is likely based on several different genetic lesions, such as the one responsible for the absence of phthiocerol dimycocerosates (PDIM) [29], that were probably introduced into this strain during years of continued culture following the original event of attenuation described by Steenken et al. in 1934 [6]. Indeed, preliminary comparison of the 4.4-Mb genome of H37Rv [7] (NC_000962) with the recently accessible H37Ra genome (NC_009525) revealed 194 differences, including 126 SNPs, 16 deletions, 27 insertions, and 25 substitutions. At present, we do not know how many of these differences actually contribute to the attenuation of H37Ra, and it also remains to be determined how many differences exist among the various H37Ra strains that are presently used in the worldwide tuberculosis research laboratories. The link described here between mutation of phoP and consequences on ESAT-6 secretion, however, appears to be one factor that significantly contributes to attenuation. Finally, it is intriguing that both BCG and H37Ra, the two most often used attenuated tubercle bacilli, are attenuated for reasons linked to loss of ESAT-6 functions. Thus, our results once more emphasize the great importance of the ESX-1 system for tubercle bacilli and provide important new information about the phoP-associated virulence regulation in M. tuberculosis.
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Comparative analysis of the Photorhabdus luminescens and the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity
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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 known to cause gastroenteritis in men, but has only recently been shown to be also toxic for insects. It is expected that both pathogens share an overlap of genetic determinants that play a role within the insect host. Results A selective genome comparison was applied. Proteins belonging to the class of two-component regulatory systems, quorum sensing, universal stress proteins, and c-di-GMP signalling have been analysed. The interorganismic synopsis of selected regulatory systems uncovered common and distinct signalling mechanisms of both pathogens used for perception of signals within the insect host. Particularly, a new class of LuxR-like regulators was identified, which might be involved in detecting insect-specific molecules. In addition, the genetic overlap unravelled a two-component system that is unique for the genera Photorhabdus and Yersinia and is therefore suggested to play a major role in the pathogen-insect relationship. Our analysis also highlights factors of both pathogens that are expressed at low temperatures as encountered in insects in contrast to higher (body) temperature, providing evidence that temperature is a yet under-investigated environmental signal for bacterial adaptation to various hosts. Common degradative metabolic pathways are described that might be used to explore nutrients within the insect gut or hemolymph, thus enabling the proliferation of P. luminescens and Y. enterocolitica in their invertebrate hosts. A strikingly higher number of genes encoding insecticidal toxins and other virulence factors in P. luminescens compared to Y. enterocolitica correlates with the higher virulence of P. luminescens towards insects, and suggests a putative broader insect host spectrum of this pathogen. Conclusion A set of factors shared by the two pathogens was identified including those that are involved in the host infection process, in persistence within the insect, or in host exploitation. Some of them might have been selected during the association with insects and then adapted to pathogenesis in mammalian hosts.
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Background
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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 comparative study of the genomes of Photorhabdus luminescens and Yersinia enterocolitica. The former bacterium is a representative of pathogens highly virulent towards insects, but apathogenic against men. Y. enterocolitica, an example of a primarily human pathogen, also confers toxicity to insects, but is less toxic towards these hosts than P. luminescens. Members of the genus Yersinia are primarily considered as mammalian pathogens. However, Y. pestis, a blood-borne pathogen and the etiological agent of human plague, has long been known to be transmitted by insects, specifically by rat fleas. Y. enterocolitica strains have been isolated from flies that are assumed to play an important role in food contamination by this pathogen [1-3], and Y. pseudotuberculosis strains were recovered from fly larvae isolated in the wild [4]. More recent data strongly support the idea that yersiniae are capable to interact with insects. Loci encoding the insecticidal toxin complexes (Tc) have been identified in the genomes of Y. pestis KIM [5], Y. pseudotuberculosis [6], and Y. enterocolitica [7]. Y. pseudotuberculosis, in contrast to Y. pestis, has been shown to be orally toxic to flea [8]. This toxicity revealed to be independent of tc genes, suggesting that loss of one or more insect gut toxins is a critical step in the change of the Y. pestis lifestyle compared with the Y. pseudotuberculosis and thus in evolution of flea-borne transmission [8]. While Y. enterocolitica and Y. pseudotuberculosis have diverged within the last 200 million years, Y. pestis has emerged from Y. pseudotuberculosis only 1,500-20,000 years ago [9]. Bacterial lysates both of Y. enterocolitica and Y. pseudotuberculosis are toxic for Manduca sexta neonates, and significant levels of natively or heterologously expressed toxins were observed in both species at 15degreesC, but not at mammalian body temperature [7,10]. Furthermore, Y. pseudotuberculosis and Y. enterocolitica have been demonstrated to adhere to and invade cultivated insect cells [10]. Thus, the interaction of Y. enterocolitica with insects is an important link in the ecological range of bacteria-host interactions extending from entomopathogenic to humanpathogenic bacteria. In contrast, Photorhabdus luminescens is predominantly an insect pathogenic enterobacterium which maintains a mutualistic interaction with heterorhabditid nematodes, and can infect a wide range of insects [11,12]. Interestingly, another Photorhabdus species, P. asymbiotica, has been described as a human pathogen. It was isolated from human clinical specimens where the cells caused locally invasive soft tissue infections [13,14]. It is assumed that these strains are associated with spiders, because spider bites where attended with Photorhabdus human infections [15]. However, bacteria of the species P. luminescens are exclusively known to be associated with nematodes and insects. Generally, the bacteria colonise the gut of the infective juvenile stage of the nematode Heterorhabditis bacteriophora. Upon entering an insect host, the nematodes release the bacteria by regurgiation directly into the insect hemocoel, the open circulatory system of the insect. Once inside the hemocoel, the bacteria replicate rapidly and establish a lethal septemica in the host by the production of virulence factors such as the insecticidal toxin complexes that kill the insect within 48 hours. Bioconversion of the insect's body by P. luminescens produces a rich food source for the bacteria as well as for the nematodes. Nematode reproduction is supported by the bacteria, probably by providing essential nutrients that are required for efficient nematode proliferation [16]. Further properties of P. luminescens are the production of many antimicrobial substances to defend the insect cadaver from bacterial competitors, and glowing due to bacterial luciferase production. When the insect cadaver is depleted, the nematodes and bacteria reassociate and emerge from the carcass in search of a new insect host (Fig. 1, right circle)[17,18]. Photorhabdus species exist in two forms, designated as primary and secondary phenotypic colony variants, which differ in morphological and physiological traits. Primary variants are found to produce extracellular protease, extracellular lipase, intracellular protein crystals CipA and CipB, antibiotics, and are bioluminescent. Secondary variants lack protease, lipase and antibiotic activity, and bioluminescence is strongly decreased. They also differ in colony morphology, pigmentation, dye adsorption, metabolism, and the ability to support growth and reproduction of the nematodes. It is assumed that primary variants correspond to the nematode-associated form, and secondary variants to the insect-associated form of the bacteria [19,20]. Therefore, P. luminescens serves as an ideal model to study the switch from a symbiotic state with nematodes to one in which the bacterium is pathogenic to insects [21,22]. In the following comparative genome analysis, we examined the extent to which P. luminescens and Y. enterocolitica share factors that are probably attributed to insect association. We identified genes and the corresponding proteins involved in signalling, regulation, pathogenicity, as well as in metabolism, and suggest their possible function during colonization and infection of non-mammals. The results obtained not only improve our understanding of the biology of both pathogens, but also reveal some implications on the evolution of invertebrate and vertebrate virulence factors.
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The TGF-beta type II receptor in chronic myeloid leukemia: analysis of microsatellite regions and gene expression.
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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 (replication error phenotype, RER+). Furthermore, TGF-beta RII mutations in RER+ tumors have been associated with decreased TGF-beta RII mRNA levels. As TGF-beta is a potent negative growth regulator of hematopoietic cells, investigations were undertaken to determine whether inactivation of the receptor by microsatellite alteration might be involved in the progression of CML. Analysis of TGF-beta RII mRNA expression by RNase protection, with comparison of cells from the chronic, accelerated and blast phases of CML, showed no change in TGF-beta RII transcript levels during disease progression. However, during each phase of the disease, low levels of TGF-beta RII were detected when compared with the hematopoietic cells of normal donors. Furthermore, this decreased expression was also observed in the other myeloproliferative disorders, polycythemia rubra vera (PRV) and essential thrombocythemia (ET). The leukemia cell lines K562 and HL-60 had no detectable TGF-beta RII mRNA. Two microsatellite regions found altered in RER+ colon cancers were analyzed to establish if these sequences were aberrant in CML. No alteration was detected in either of these regions in any phase of the disease. These results suggest that alterations of the microsatellite regions in the TGF-beta RII gene are not involved in the progression of CML. Decreased expression of TGF-beta RII in CML cells and leukemia cell lines raises the possibility that altered expression of the receptor may play a role in the initiation and/or maintenance of the disease state.
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N-glycosylation of glucose transporter-1 (Glut-1) is associated with increased transporter affinity for glucose in human leukemic cells.
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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-deoxyglucose uptake and this was associated with a 2-2.5-fold decrease in transporter affinity for glucose (Km) without a change in Vmax. Leukemic K562, U937 and HL60 cells expressed Glut-1 transporter protein. With K562 cells Glut-1 appeared as a broad band of 50-60 kDa, whereas with U937 and HL60 cells a diffuse band was observed at approximately 55 kDa. Treatment of K562 cells with tunicamycin for 18 h, resulted in extensive loss of the 50-60 kDa glycoprotein, appearance of a 30-40 kDa band and increased staining of a 45 kDa band. With U937 cells, tunicamycin treatment resulted in the appearance of a 30-40 kDa band and increased staining of a 45 kDa band. With HL60 cells loss of the 55 kDa Glut-1 band was observed and a band of 45 kDa appeared. Tunicamycin-treatment resulted in 75-90% inhibition in [3H]mannose incorporation but only 20-25% inhibition in [3H]thymidine and [3H]leucine incorporation. In contrast, tunicamycin had little effect on the viability and MTT responses of the cells used. These results suggest that in leukemic cells N-glycosylation of Glut-1 plays an important role in maintaining its structure and functional integration.
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Immunohistochemical analysis of nm23-H1 gene product in node-positive lung cancer and lymph nodes.
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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 expression of nm23-H1 gene product in surgically resected 46 pairs of primary lung cancers and metastatic lymph nodes by immunohistochemistry. The positive staining of nm23-H1 gene product in primary cancers and metastatic lymph nodes were observed in 56.5 and 67.4%, respectively. The heterogeneity of nm23-H1 gene product expression between primary cancers and metastatic lymph nodes was observed in 41.3%. No correlations were found between the nm23-H1 gene product expression in lung cancers and the patients survival. No significant association was also observed between nm23-H1 gene product expression in lymph nodes and the patients survival. There was, furthermore, no correlation between the heterogeneity of nm23-H1 gene product expression and the patients survival. In conclusion, the level of nm23-H1 gene product expression does not significantly reveal prognostic value in node-positive lung cancers. Expression of nm23-H1 gene product in metastatic lymph nodes was also unrelated to patients survival.
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Bradycardia-induced coronary angiogenesis is dependent on vascular endothelial growth factor.
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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 induced in rats by administering the bradycardic drug alinidine (3 mg/kg body weight) twice daily. Heart rate decreased by 32% for 20 to 40 minutes after injection and was chronically reduced by 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment, respectively. Arterial pressure and cardiac output were unchanged. Left ventricular capillary length density (mm/mm(3)) increased gradually with alinidine administration; a 15% increase after 2 weeks and a 40% increase after 3 weeks of alinidine treatment were documented. Left ventricular weight, body weight, and their ratio were not significantly altered by alinidine treatment. After 1 week of treatment, before an increase in capillary length density, VEGF mRNA increased greater than 2-fold and then declined to control levels after 3 weeks of treatment. VEGF protein was higher in alinidine-treated rats than in controls after 2 weeks and increased further after 3 weeks of treatment. Injection of VEGF-neutralizing antibodies over a 2-week period completely blocked alinidine-stimulated angiogenesis. In contrast, bFGF mRNA was not altered by alinidine treatment. These data suggest that VEGF plays a key role in the angiogenic response that occurs with chronic bradycardia. The mechanism underlying this VEGF-associated angiogenesis may be an increase in stretch due to enhanced diastolic filling.
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External beam radiotherapy for subretinal neovascularization in age-related macular degeneration: is this treatment efficient?
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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 AND MATERIALS: The prospective trial included 287 patients with subfoveal neovascularization due to AMD which was verified by fluorescein angiography. Patients have been treated between January 1996 and October 1997. All patients received a total dose of 16 Gy in 2-Gy daily fractions with 5-6 MeV photons based on computerized treatment planning in individual head mask fixation. This first analysis is based on 73 patients (50 women, 23 men, median age 74.3 years), with a median follow-up of 13.3 months and a minimum follow-up of 11 months. RESULTS: All patients completed therapy and tolerability was good. First clinical control with second angiography was performed 6 weeks after irradiation, then in 3-month intervals. Eighteen patients with SRNV refusing radiotherapy served as a control group and were matched with 18 irradiated patients. After 7 months median visual acuity (VA) was 20/160 for the irradiated and 20/400 for the untreated patients. One year after radiotherapy final median VA was 20/400 in both groups. CONCLUSION: These results suggest that 16 Gy of conventionally fractionated external beam irradiation slows down the visual loss in exudative AMD for only a few months. Patients' reading vision could not be saved for a long-term run.
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Increased serum levels of vascular endothelial growth factor in patients with renal cell carcinoma.
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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 investigated the serum VEGF level (s-VEGF) in patients with renal cell carcinoma (RCC). s-VEGF in peripheral blood samples was analyzed in 40 RCC patients and 40 patients without cancer (controls) using a sandwich enzyme-linked immunoassay. In 20 RCC patients, serum samples were obtained separately from the bilateral renal veins. s-VEGF was also measured before, 4 and 8 weeks after nephrectomy in 11 patients. There were significant differences in s-VEGF between the RCC patients and the controls (207.3+/-32.9 vs. 71.5+/-9.1 pg/ml, mean+/-SE) (P less than 0.005), between the tumor-bearing renal veins and the contralateral ones (P less than 0.01), between the pre- and post-nephrectomy situations (P less than 0.01) and among the various parameters of tumor status such as tumor extent (P less than 0.001) and existence of metastasis (P less than 0.001). s-VEGF significantly correlated with the tumor volume obtained by a three-dimensional measurement (r=0.802, P less than 0.0001). The sensitivity and specificity of s-VEGF at the cut-off level of 100 pg/ml, as determined by the receiver-operating-characteristics curve, were 80.0% and 72.5%, respectively. The results indicate that tumor tissue of RCC liberates VEGF into the systemic blood flow and that s-VEGF is a possible marker for RCC.
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Insulin-induced vascular endothelial growth factor expression in retina.
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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 the pathogenesis of diabetic retinopathy. The current study was conducted to characterize the effect of insulin on retinal VEGF gene expression in vitro and in vivo. METHODS: The effect of insulin on VEGF expression in vivo was examined by in situ hybridization studies of rat retinal VEGF transcripts. To examine the mechanisms by which insulin regulates VEGF expression, human retinal pigment epithelial (RPE) cells were exposed to insulin, and VEGF mRNA levels were quantified with RNase protection assays (RPAs). Conditioned media from insulin-treated RPE cells were assayed for VEGF protein and capillary endothelial cell proliferation. The capacity of insulin to stimulate the VEGF promoter linked to a luciferase reporter gene was characterized in transient transfection assays. RESULTS: Insulin increased VEGF mRNA levels in the ganglion, inner nuclear, and RPE cell layers. In vitro, insulin increased VEGF mRNA levels in human RPE cells and enhanced VEGF promoter activity without affecting transcript stability. Insulin treatment also increased VEGF protein levels in conditioned RPE cell media in a dose-dependent manner with a median effective concentration of 5 nM. The insulin-conditioned RPE cell media stimulated capillary endothelial cell proliferation, an effect that was completely blocked by anti-VEGF neutralizing antibody. CONCLUSIONS: Insulin increases VEGF mRNA and secreted protein levels in RPE cells through enhanced transcription of the VEGF gene. Intensive insulin therapy may cause a transient worsening of retinopathy in patients with diabetes through increased retinal VEGF gene expression.
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The antiangiogenic agent linomide inhibits the growth rate of von Hippel-Lindau paraganglioma xenografts to mice.
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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 "prevention" experiments; and (b) 2-3-mm fragments allowed to grow to 1 cm for "intervention" studies. Both groups received either 0.5 mg/ml linomide in drinking water or acidified water and were followed until tumor diameter reached 3 cm or for 4 weeks. In both the prevention and intervention experiments, a significant diminution of tumor size and weight was observed in the drug-treated animals. In vivo nuclear magnetic resonance analysis of tumor blood flow in linomide-treated animals showed localization of blood vessels almost exclusively to the periphery of the poorly vascularized tumors with a significant reduction of both vascular functionality and vasodilation. Histological examination of tumors from linomide-treated animals revealed marked avascularity. Treated animals also displayed a 2.4-fold reduction of tumor vascular endothelial growth factor mRNA levels. Taken together, our data indicate that in VHL disease, therapy directed at inhibition of constitutively expressed VEGF induction of angiogenesis by VHL tumors may constitute an effective medical treatment.
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LMP1 of Epstein-Barr virus induces proliferation of primary mouse embryonic fibroblasts and cooperatively transforms the cells with a p16-insensitive CDK4 oncogene.
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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 tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recombinant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the proliferation of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4(R24C) oncogene in transforming MEF cells. Our results provide the first evidence of the abilities of the LMP1 gene, acting alone, to effectively induce the proliferation of primary MEF cells and of its cooperativity with another cellular oncogene in transforming primary cells.
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Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells.
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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-expressing cell lines, parental and ras-transformed NIH3T3 (clone7) cells. The effect of ATX secretion on in vitro cell motility was variable. The ras-transformed, ATX-secreting subclones had enhanced motility to ATX as chemoattractant, but there was little difference in the motility responses of NIH3T3 cells transfected with atx, an inactive mutant gene, or empty vector. In MatrigelTM invasion assays, all subclones, which secreted enzymatically active ATX, demonstrated greater spontaneous and ATX-stimulated invasion than appropriate controls. This difference in invasiveness was not caused by differences in gelatinase production, which was constant within each group of transfectants. In vivo studies with athymic nude mice demonstrated that injection of atx-transfected NIH3T3 cells resulted in a weak tumorigenic capacity with few experimental metastases. Combination of ATX expression with ras transformation produced cells with greatly amplified tumorigenesis and metastatic potential compared to ras-transformed controls. Thus, ATX appears to augment cellular characteristics necessary for tumor aggressiveness.
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Oncogenic epidermal growth factor receptor mutants with tandem duplication: gene structure and effects on receptor function.
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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 tyrosine kinase (TK) and calcium-mediated internalization (CAIN) domains. Here we describe a 185-kDa tandem duplication mutant (TDM) that is expressed in KE and A-1235 glioma cells, along with certain functional characteristics of the mutants. The corresponding transcripts in KE and A-1235 cells contain 1053 additional nucleotides representing an in-frame duplication of exons 18 through 25 which encode the entire TK region and a portion of the CAIN domain. As with duplication of the entire TK/CAIN region (exons 18-26) in A-172 cells, duplication of exons 18-25 is associated with a specific genomic rearrangement between flanking introns. Involved introns contain homology to recombination signal sequence (RSS) heptamers present in the V(D)J region of the T lymphocyte receptor gene. In defined medium, both oncogenic TDM are constitutively autophosphorylated and inefficiently downregulated. High-affinity binding is reduced in EGFR.TDM/18-26, although the t1/2 of receptor internalization is not prolonged.
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Ciprofloxacin mediated cell growth inhibition, S/G2-M cell cycle arrest, and apoptosis in a human transitional cell carcinoma of the bladder cell line.
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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 with recurrence rates reduced to 17-44%. In addition, most of these agents require instrumentation of the urinary tract and are delivered at a significant cost and potential morbidity to the patient. Fluroquinolone antibiotics such as ciprofloxacin, which can be administered p.o., may have a profound effect in bladder cancer management. This is primarily based on limited in vitro studies on tumor cells derived from transitional cell carcinoma of the bladder that revealed a dose- and time-dependent inhibition of cell growth by ciprofloxacin at concentrations that are easily attainable in the urine of patients. However, the mechanism(s) by which ciprofloxacin elicits its biological effects on bladder cancer cells is not well documented. Our experimental data confirm previous studies showing the in vitro cell growth inhibition of the transitional cell carcinoma of the bladder cell line HTB9 and further showed the induction of cell cycle arrest at the S/G2-M checkpoints. In addition, we found down-regulation of cyclin B, cyclin E, and dephosphorylation of cdk2 in ciprofloxacin-treated bladder tumor cells. There was also an up-regulation of Bax, which altered the Bax:Bcl-2 ratio, which may be responsible for mitochondrial depolarization reported to be involved prior to the induction of apoptosis. The cyclin-dependent kinase inhibitor p21WAF1 level was found to be decreased within 12 h of ciprofloxacin treatment and disappeared completely when HTB9 cells were treated with 200 microg/ml ciprofloxacin for 24 h. The down-regulation of p21WAF1 closely correlated with poly(ADP-ribose) polymerase cleavage and CPP32 activation. Recent studies revealed that p21WAF1 protects cells from apoptosis by arresting them in G1 and further binds to pro-caspase-3, preventing its activation and thus, inhibiting the apoptotic cascade. Hence, the down-regulation of p21WAF1, together with the alterations in Bax and cdk2 as observed in our studies, may define a novel mechanism by which ciprofloxacin inhibits tumor cell growth and induces apoptotic cell death. The results of our current studies provide strong experimental evidence for the use of ciprofloxacin as a potential preventive and/or therapeutic agent for the management of transitional cell carcinoma of the bladder.
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Vascular proliferation and enhanced expression of endothelial nitric oxide synthase in human peritoneum exposed to long-term peritoneal dialysis.
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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 of nitric oxide synthase (NOS) isozymes might play a role in these modifications, via enhanced local production of nitric oxide, was tested in this study. NOS activities were measured by the L-citrulline assay in peritoneal biopsies from seven control subjects, eight uremic patients immediately before the onset of PD, and 13 uremic patients on short-term ( less than 18 mo, n = 6) or long-term( greater than 18 mo, n = 7) PD. Peritoneal NOS activity is increased fivefold in long-term PD patients compared with control subjects. In uremic patients, NOS activity is positively correlated with the duration of PD. Increased NOS activity is mediated solely by Ca(2+)-dependent NOS and, as shown by immunoblotting, an upregulation of endothelial NOS. The biologic relevance of increased NOS in long-term PD was demonstrated by enhanced nitrotyrosine immunoreactivity and a significant increase in vascular density and endothelial area in the peritoneum. Immunoblotting and immunostaining studies demonstrated an upregulation of vascular endothelial growth factor (VEGF) mostly along the endothelium lining peritoneal blood vessels in long-term PD patients. In the latter, VEGF colocalized with the advanced glycation end product pentosidine deposits. These data provide a morphologic (angiogenesis and increased endothelial area) and molecular (enhanced NOS activity and endothelial NOS upregulation) basis for explaining the permeability changes observed in long-term PD. They also support the implication of local advanced glycation end product deposits and liberation of VEGF in that process.
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Active hair growth (anagen) is associated with angiogenesis.
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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 epithelial hair bulbs of anagen follicles display angiogenic properties, and that the follicular dermal papilla can produce angiogenic factors. Despite these suggestive observations, no formal proof is as yet available for the concept that angiogenesis is a physiologic event that occurs all over the mature mammalian integument whenever hair follicles switch from resting (telogen) to active growth (anagen). This study uses quantitative histomorphometry and double-immunohistologic detection techniques for the demarcation of proliferating endothelial cells, to show that synchronized hair follicle cycling in adolescent C57BL/6 mice is associated with substantial angiogenesis, and that inhibiting angiogenesis in vivo by the intraperitoneal application of a fumagillin derivative retards experimentally induced anagen development in these mice. Thus, angiogenesis is a physiologic event in normal postnatal murine skin, apparently is dictated by the hair follicle, and appears to be required for normal anagen development. Anagen-associated angiogenesis offers an attractive model for identifying the physiologic controls of cutaneous angiogenesis, and an interesting system for screening the effects of potential antiangiogenic drugs in vivo.
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Neuroblastoma and hepatocyte coculture conditioned media alter apoptosis.
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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 cell death, is thought to play a role in the dichotomous behavior of neuroblastoma. We hypothesize that neuroblastoma cells will interact with host tissues to release mediators that affect apoptosis. MATERIALS AND METHODS: Human neuroblastoma cells and human Chang hepatocytes are grown in a noncontact, coculture system. After incubation for 4 days, the medium from the coculture system is collected. Neuroblastoma cells and Chang hepatocytes are then plated separately with the conditioned medium and their own standard growth medium as controls. After 4 days, these cells are harvested and cytospins made for immunostaining. Tumor necrosis factor alpha (TNF-alpha), Fas ligand, and Bcl-2, are measured with immunohistochemistry. Apoptosis is detected with the TUNEL method. Immunostaining data are interpreted with computer image analysis and reported as stain index. TUNEL data are reported as percentage apoptotic cells. All data are reported as means +/- SEM. Statistical analysis is performed and P < 0.05 considered significant. RESULTS: Chang hepatocytes grown in the coculture conditioned media have an increase in TNF-alpha and Fas ligand. The neuroblastoma cells have a significant decrease in Fas ligand. There is a significant increase in the number of apoptotic hepatocytes when they are cultured in the conditioned media. In contrast, the neuroblastoma cells grown in the coculture conditioned media show no increase in apoptosis. Finally, Bcl-2 is significantly increased in the neuroblastoma cells cultured in the conditioned media. CONCLUSIONS: Neuroblastoma cells grown in coculture conditioned media show increased expression of Bcl-2 and decreased Fas ligand levels. These changes should diminish apoptosis activity in the tumor cells. In contrast, the conditioned media induce elevated levels of proapoptotic mediators in the Chang hepatocytes. A tumor's ability to successfully metastasize may be dependent on mediators generated in the tumor-host interaction, and may not be just an independent characteristic of the tumor itself.
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Neoplastic transformation by Notch requires nuclear localization.
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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 between neighboring cells. The prevailing model for Notch signaling suggests that ligand, presented on a neighboring cell, triggers proteolytic processing of Notch. Following proteolysis, it is thought that the intracellular portion of Notch (N(ic)) translocates to the nucleus, where it is involved in regulating gene expression. There is considerable debate concerning where in the cell Notch functions and what proteins serve as effectors of the Notch signal. Several Notch genes have clearly been shown to be proto-oncogenes in mammalian cells. Activation of Notch proto-oncogenes has been associated with tumorigenesis in several human and other mammalian cancers. Transforming alleles of Notch direct the expression of truncated proteins that primarily consist of N(ic) and are not tethered to the plasma membrane. However, the mechanism by which Notch oncoproteins (generically termed here as N(ic)) induce neoplastic transformation is not known. Previously we demonstrated that N1(ic) and N2(ic) could transform E1A immortalized baby rat kidney cells (RKE) in vitro. We now report direct evidence that N1(ic) must accumulate in the nucleus to induce transformation of RKE cells. In addition, we define the minimal domain of N1(ic) required to induce transformation and present evidence that transformation of RKE cells by N1(ic) is likely to be through a CBF1-independent pathway.
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Alpha-melanocyte-stimulating hormone modulates activation of NF-kappa B and AP-1 and secretion of interleukin-8 in human dermal fibroblasts.
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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 fibroblasts, in extracellular matrix composition, and in cytokine expression. By use of electric mobility shift assays, we demonstrate that alpha-MSH (10(-6) to 10(-14) M) activates AP-1 in human dermal fibroblasts, whereas coincubation with interleukin-1 beta (IL-1 beta) results in suppression of its activation. alpha-MSH also induces activation of NF-kappa B but does not modulate DNA binding on costimulation with IL-1 beta. Since AP-1 and NF-kappa B are key elements in controlling interleukin-8 (IL-8) transcription, human fibroblasts were treated with alpha-MSH and IL-1 beta for 24 hours, and cytokine levels in the supernatants were measured by ELISA. alpha-MSH alone had little effect, whereas coincubation with IL-1 beta led to marked downregulation of IL-8 secretion (at most 288 +/- 152 ng/mL) when compared to treatment with IL-1 beta alone (919 +/- 157 ng/mL). Our results indicate that alpha-MSH exerts modulatory effects on the activation of NF-kappa B and AP-1, and that it can regulate chemokine secretion in human dermal fibroblasts. These effects of alpha-MSH may have important regulatory functions in extracellular matrix composition, wound healing, or angiogenesis.
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