Proteomic Characterization ofYersinia pestisVirulence

Abstract

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 ...

Identification of secreted bacterial proteins by noncanonical amino acid tagging

“…Outer membrane protein A (OmpA), also identified in our analysis, is known to be present in such vesicles and released by Gram-negative bacteria (11,30). OmpA has been detected in monocyte cell lysates after infection with Y. pestis (27), is known to bind scavenger receptors (31), and is considered a potent Yersinia virulence factor (30). Sif15, also known as systemic factor protein-a (Sfpa), is involved in systemic infection of Y. enterocolitica, is induced at 37°C, and is necessary for colonization of mesenteric lymph nodes in a mouse Peyer's patch infection model (32).…”

“…It seems likely that these highly abundant proteins are found in the HeLa cell lysate as a result of a low level of adventitious bacterial cell lysis, although we cannot rule out other mechanisms of transfer. Previous proteomic studies of factors secreted by Yersinia found subsets of these proteins, including HtpG, OmpA, GroL, and several elongation factors (27), as well as DnaK and Eno (28).…”

Nooks and Crannies in Type VI Secretion Regulation

“…Although no data for the P. aeruginosa HSI-2 locus within the host are available, the HSI-3 gene cluster is upregulated in the presence of epithelial cell extracts (32). In Yersinia pestis, the YPO0499 gene cluster is induced at low temperatures and repressed at 37°C, suggesting that the role of the T6SS gene cluster is probably more important for dissemination in the flea vector than in human cells (29,31,47,49,73,85,93).…”

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

“…To evaluate whether the levels of Pla remained unaltered in the ⌬ail mutants, Western blot analysis was performed. Essentially, similar levels of the Pla protein were noted for the various mutant strains tested (⌬ail single, ⌬lpp ⌬msbB double, ⌬lpp ⌬msbB ⌬ail triple, as well as ail-complemented mutant strains) compared to that of WT CO92 at 37°C, a temperature that increases Pla production and activity (36,68,69) (Fig. 2D).…”