Figure 1.
Experimental strategy used to identify Y. pestis chromosomal genes involved in type III secretion of Yops.
In Step 1, transposon mutants were selected based on their ability to grow at 33°C in the absence of calcium. These mutants were subjected to a secondary screen (Step 2) evaluating their ability to secrete Yops. All mutants that displayed a defect in the synthesis or secretion of Yops were then sequenced to identify the location of the Mu insertion (Step 3). To eliminate mutants that carried unmarked mutations on the virulence plasmid, the tagged pCD1::KM was used to displace the original pCD1, and the transformants were then evaluated for their ability to secrete Yops (Step 4). Strains which retained the secretion defect after Step 4 were kept for further characterization.
Figure 2.
Identification of mutations in CHI strains.
The genetic locus carrying the Mu insertion, indicated by an arrowhead, is depicted for each CHI strain. The genome annotations of the disrupted genes (shaded) are shown. Arrows refer to primer locations used to amplify and clone each locus for complementation analysis. Expression of folD and pssA were driven by the nptII promoter.
Figure 3.
Secretion phenotypes of CHI strains.
Complementation plasmids were introduced into each mutant and the KIM5 parent, and the strains were evaluated by performing secretion assays. The TTSS mutant, yscU, was included as a negative control. Y. pestis 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.
Figure 4.
Translocation phenotypes of CHI strains.
CHO cells were infected with Y. pestis strains carrying either pMM83 (YopM-Bla, Panel A), pMM85 (YopE-Bla, Panel B) or pMM91 (Gst-Bla, not shown) at 37°C for 3 hours, followed by CCF2-AM staining and flow cytometry to quantify blue and green fluorescence. The level of translocated Bla reporter is indicated by white (no injection, green fluorescence), gray (intermediate level of injection, aqua fluorescence), or black (high level of injection, blue fluorescence) 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. *** P<0.001. To assess cytotoxicity due to Yop delivery, cell rounding was also evaluated (Panel C). HeLa cells were infected with the indicated Y. pestis strains for 3–4 hours. The percentage of round cells was determined for each infection. The infections were performed in triplicate and error bars represent standard deviation. The data shown is representative of two independent experiments. One-way ANOVA with Tukey post-hoc test was done to determine significance. *** Represents significant difference of mutants compared to WT (P<0.001); n.s. = no significance between indicated strains.
Figure 5.
Secretion phenotypes of strains carrying a heterologously expressed Yop reporter.
pAH83, which constitutively expresses YopM-Bla, was introduced into each mutant and the KIM5 parent, and the strains were evaluated by performing secretion assays as described in Figure 3. Proteins were visualized by immunoblotting with antibodies to β-lactamase (YopM-Bla), YopM, YopE (TTSS secreted proteins), YscD, YscF (TTSS apparatus components) Ail (outer membrane protein) and RpoA (cytoplasmic protein, loading and fractionation control). An asterisk indicates truncated Yops due to degradation by Pla protease.
Figure 6.
Translocation phenotypes of strains carrying a heterologously expressed Yop reporter.
CHO cells were infected with Y. pestis 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. *** P<0.001. Diamonds indicate statistically significant differences (P<0.001) compared to samples in Figure 4, Panel A.
Figure 7.
Trimethoprim inhibits the Yersinia TTSS.
A. HeLa cells were infected with KIM5 or CHI 30 (yscQ−) expressing YopE-Bla. Infections were carried out at the indicated MOI for three hours in the presence or absence of 500 µg/ml of trimethoprim. Following infection, cells were stained with CCF2-AM and visualized by microscopy to assess translocation of the YopE-Bla reporter. Blue fluorescence = YopE-Bla injection; green fluorescence = no injection. B. Y. pestis KIM8 and KIM8 Δ1234, carrying pMM207 (yopM expressed from native promoter), were subcultured into MM9 and grown at 26°C for 2 hours. Trimethoprim was then added at the indicated concentrations (0, 15, or 30 µg/ml) and the cultures were shifted to 37°C for 3 hours, followed by TCA precipitation of supernatant (S) and cell pellet (P) fractions and immunoblotting with antibodies against YopM, YopN, YopD, YopR (TTSS secreted proteins), YscD (TTSS machine), RpoA (cytoplasmic protein), Ail (outer membrane protein). Degradation products are not indicated since KIM8 and KIM8 Δ1234 lack Pla protease. C. Secretion assays and western blotting was performed as in Panel B using Y. pestis KIM8 carrying pAH83 (yopM expressed from nptII promoter).
Figure 8.
CHI 1800 cannot induce type III secretion in response to low calcium.
Y. pestis strains KIM5 and CHI 1800 (rfaL−) were subcultured into DMEM with or without EGTA and grown at 26°C for 2 hours and 37°C for 3 hours, followed by TCA precipitation of supernatant and cell pellet fractions and immunoblotting for early Yops (YopR, YopD, LcrV) and late Yops (YopH, YopM, YopE).
Table 1.
Mutant phenotypes.
Figure 9.
In vivo attenuation of CHI strains.
BALB/c mice were infected intravenously with serial dilutions Y. pestis KIM5 or transposon mutants. Moribund mice were euthanized and time to death was recorded. Survival curves are shown for KIM5 (WT) and two mutants (ctgA− and rfaL−).