DNA microarrays encompassing the entire genome of Yersinia pestis were used to characterize global regulatory changes during steady-state vegetative growth occurring after shift from 26 to 37°C in the presence and absence of Ca 2؉ . Transcriptional profiles revealed that 51, 4, and 13 respective genes and open reading frames (ORFs) on pCD, pPCP, and pMT were thermoinduced and that the majority of these genes carried by pCD were downregulated by Ca 2؉ . In contrast, Ca 2؉ had little effect on chromosomal genes and ORFs, of which 235 were thermally upregulated and 274 were thermally downregulated. The primary consequence of these regulatory events is profligate catabolism of numerous metabolites available in the mammalian host.Bubonic plague caused by Yersinia pestis is generally recognized as the most devastating acute infectious disease experienced by mankind. It is therefore of interest that this organism has evolved within the last 10,000 years from Yersinia pseudotuberculosis (1), known to cause chronic enteropathogenic disease. Despite their very close resemblance, plague bacilli have both lost central genes of intermediary metabolism retained in its predecessor and acquired unique genes by lateral transfer (7). For example, even though early studies showed that Y. pestis possesses functional Embden-Meyerhof (28) and Entner-Doudoroff (20) pathways plus a complete tricarboxylic acid (TCA) cycle (13, 27), the species-specific absence of detectable glucose 6-phosphate dehydrogenase (Zwf) prevents use of hexose via the pentose-phosphate pathway (21). Similarly, loss of aspartase (AspA) activity in Y. pestis but not Y. pseudotuberculosis prevents complete catabolism of L-glutamic acid, which undergoes conversion and excretion as L-aspartate (12). In addition, Y. pestis possesses additional species-specific mutations that cause nutritional requirements at 26°C, prevent utilization of potential metabolites, and eliminate host cell invasins and adhesins (7); these events are now characterized by genomic sequencing (11, 23). The nature of nutritional requirements at 37°C is more complex and, as noted below, dependent upon plasmid profile, the presence or absence of Ca 2ϩ , Na ϩ , dicarboxylic amino acids, and regulatory functions addressed in this report.Established functions unique to Y. pestis are encoded by species-specific ϳ10-kb pPCP and ϳ100-kb pMT. The former encodes plasminogen activator (Pla) required for tissue invasion from dermal sites infected by fleabite whereas the structural genes for anti-phagocytic capsular fraction 1 (Caf1) and murine toxin (MT), required for survival in the flea, reside on pMT (7,25). Plague bacilli and the enteropathogenic yersiniae share a ϳ70-kb plasmid (pCD in Y. pestis) encoding a type III protein secretion system (TTSS) that delivers cytotoxins termed Yops to the cytosol of professional and nonprofessional phagocytes (8) and excretes soluble LcrV (V antigen), which inhibits generation of proinflammatory cytokines by upregulating interleukin-10 (6). These functions provide th...
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The Yersinia pestis proteome was studied as a function of temperature and calcium by two-dimensional differential gel electrophoresis. Over 4,100 individual protein spots were detected, of which hundreds were differentially expressed. A total of 43 differentially expressed protein spots, representing 24 unique proteins, were identified by mass spectrometry. Differences in expression were observed for several virulence-associated factors, including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as several putative virulence factors and membrane-bound and metabolic proteins. Differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants.Yersinia pestis, the etiological agent of plague, is a gram-negative bacterium that is both a natural environmental pathogen and a biothreat agent (4,8,32). Early studies of Yersinia physiology uncovered the low calcium response (LCR), whereby bacterial cultures grown in rich medium at an elevated temperature (37°C) exhibit a growth defect upon chelation of calcium ions. The growth arrest was shown to be a result of one of the two type III secretion systems (TTSSs) in Y. pestis, the Ysc TTSS, and is responsible for the secretion of virulence factors known as Yersinia outer proteins, or Yops (21, 29; for a review, see reference 61). This TTSS can be activated in vitro and virulence factors can be released into the medium when Y. pestis is grown at 37°C with submillimolar calcium (for a review, see reference 16). Upon interaction with the host, the TTSS enables virulence factors to enter the host cell through a specialized apparatus, the injectisome (15). Once inside the host cell, Yops affect a variety of host pathways, with detectable expression changes in the pathogen as well as the host (14,52,82).The Y. pestis proteome was previously examined using twodimensional electrophoresis (57,60,71,72). These studies demonstrated that virulence factors were not induced at 26°C or 37°C in the presence of calcium concentrations similar to that found in mammalian plasma (2.5 mM) (71). More recently, the introduction of two-dimensional differential gel electrophoresis (2-D DIGE) has significantly improved the quality of gel-based proteomics through fluorescence-based multiplex analyses providing relative quantitation of expression differences and improved gel-to-gel comparisons (75). Several examples of 2-D DIGE bacterial proteomics have been reported (23), including characterizations of the gram-negative bacterium Escherichia coli (1, 76, 81). Here we report the characterization of the soluble cell-associated proteome of Y. pestis as a function of temperature and calcium, which were used to effect virulence induction. Differentially expressed proteins include virulence-associated factors, membrane-bound proteins, metabolic and housekeeping proteins, and potential new virulence determinants.Bacterial ...
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