Identification of Chromosomal Genes in Yersinia pestis that Influence Type III Secretion and Delivery of Yops into Target Cells

Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague

Terminal Restriction Fragment Patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities

Microbial populations in complex environmental samples are difficult to characterize; current techniques are incomplete and time consuming. We investigated a polymerase chain reaction (PCR)-based method for rapidly comparing bacterial communities independent of culture or cloning. Community 16S rRNA genes were amplified and fluorescently labeled by PCR. The labeled products were digested by a restriction enzyme and the labeled, terminal restriction fragments (TRFs) were separated by electrophoresis and detected by laser-induced fluorescence on an automated gene sequencer. PCR parameters were optimized using an in vitro model community of known organisms. Community comparisons were made between deer fecal pellets, petroleum hydrocarbon-contaminated sands and pristine sand. Principal components analysis (PCA) was used to compare the resulting TRF patterns (TRFPs). Patterns derived from a single enzyme digest did not result in accurate community characterizations. Accurate characterizations reflecting the expected bacterial community biology were only achieved by combining TRFP data derived from different enzyme digestions. Suggestions are offered for future use of this technique

The ttsA Gene Is Required for Low-Calcium-Induced Type III Secretion of Yop Proteins and Virulence of Yersinia enterocolitica W22703

Pathogenic Yersinia species use a virulence-plasmid encoded type III secretion pathway to escape the innate immune response and to establish infections in lymphoid tissues. At least 22 secretion machinery components are required for type III transport of 14 different Yop proteins, and 10 regulatory factors are responsible for activating this pathway in response to environmental signals. Although the genes for these products are located on the 70-kb virulence plasmid of Yersinia, this extrachromosomal element does not appear to harbor genes that provide for the sensing of environmental signals, such as calcium-, glutamate-, or serum-sensing proteins. To identify such genes, we screened transposon insertion mutants of Y. enterocolitica W22703 for defects in type III secretion and identified ttsA, a chromosomal gene encoding a polytopic membrane protein. ttsA mutant yersiniae synthesize reduced amounts of Yops and display a defect in low-calcium-induced type III secretion of Yop proteins. ttsA mutants are also severely impaired in bacterial motility, a phenotype which is likely due to the reduced expression of flagellar genes. All of these defects were restored by complementation with plasmid-encoded wild-type ttsA. LcrG is a repressor of the Yersinia type III pathway that is activated by an environmental calcium signal. Mutation of the lcrG gene in a ttsA mutant strain restored the type III secretion of Yop proteins, although the double mutant strain secreted Yops in the presence and absence of calcium, similar to the case for mutants that are defective in lcrG gene function alone. To examine the role of ttsA in the establishment of infection, we measured the bacterial dose required to produce an acute lethal disease following intraperitoneal infection of mice. The ttsA insertion caused a greater-than-3-log-unit reduction in virulence compared to that of the parental strain

Bacillus anthracis Sortase A (SrtA) Anchors LPXTG Motif-Containing Surface Proteins to the Cell Wall Envelope

Cell wall-anchored surface proteins of gram-positive pathogens play important roles during the establishment of many infectious diseases, but the contributions of surface proteins to the pathogenesis of anthrax have not yet been revealed. Cell wall anchoring in Staphylococcus aureus occurs by a transpeptidation mechanism requiring surface proteins with C-terminal sorting signals as well as sortase enzymes. The genome sequence of Bacillus anthracis encodes three sortase genes and eleven surface proteins with different types of cell wall sorting signals. Purified B. anthracis sortase A cleaved peptides encompassing LPXTG motif-type sorting signals between the threonine (T) and the glycine (G) residues in vitro. Sortase A activity could be inhibited by thiol-reactive reagents, similar to staphylococcal sortases. B. anthracis parent strain Sterne 34F(2), but not variants lacking the srtA gene, anchored the collagen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) BasC (BA5258/BAS4884) to the bacterial cell wall. These results suggest that B. anthracis SrtA anchors surface proteins bearing LPXTG motif sorting signals to the cell wall envelope of vegetative bacilli

Identification of Chromosomal Genes in <i>Yersinia pestis</i> that Influence Type III Secretion and Delivery of Yops into Target Cells

Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague.</p

In vivo attenuation of CHI strains.

<p>BALB/c mice were infected intravenously with serial dilutions <i>Y. pestis</i> KIM5 or transposon mutants. Moribund mice were euthanized and time to death was recorded. Survival curves are shown for KIM5 (WT) and two mutants (<i>ctgA</i>⁻ and <i>rfaL</i>⁻).</p

Secretion phenotypes of CHI strains.

<p>Complementation plasmids were introduced into each mutant and the KIM5 parent, and the strains were evaluated by performing secretion assays. The TTSS mutant, <i>yscU</i>, was included as a negative control. <i>Y. pestis</i> strains were grown in MM9 at 26°C for 2 hours and then shifted to 37°C for 3 hours to induce secretion. Proteins in the supernatant (S) and pellet (P) fractions were precipitated and visualized by immunoblotting with antibodies to YopM, YopE (TTSS secreted proteins), YscD (TTSS apparatus component) and RpoA (RNA Polymerase alpha subunit, loading and fractionation control). An asterisk indicates truncated Yops due to degradation by Pla protease.</p

Translocation phenotypes of strains carrying a heterologously expressed Yop reporter.

<p>CHO cells were infected with <i>Y. pestis</i> strains carrying pAH83 at 37°C for 3 hours, followed by CCF2-AM staining and flow cytometry. Cells were analyzed for blue and green fluorescence. The level of translocated Bla reporter is indicated by white (none), gray (intermediate level), or black (high level) bars. For each experiment, the assays were performed in triplicate, and error bars indicate the standard deviation. The data shown is representative of three independent experiments. One-way ANOVA with Tukey post-hoc test was done to demonstrate significant differences in injection (high-level injection, black bars) for mutants relative to the wild type parent. *** <i>P</i><0.001. Diamonds indicate statistically significant differences (<i>P</i><0.001) compared to samples in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034039#pone-0034039-g004" target="_blank">Figure 4</a>, Panel A.</p