ROS-inhibitory activity of YopE is required for full virulence of Yersinia in mice

Abstract

YopE, a type III secreted effector of Yersinia, is a GTPase Activating Protein for Rac1 and RhoA whose catalytic activity is critical for virulence. We found that YopE also inhibited reactive oxygen species (ROS) production and inactivated Rac2. How YopE distinguishes among its targets and which specific targets are critical for Yersinia survival in different tissues are unknown. A screen identifying YopE mutants in Yersinia pseudotuberculosis that interact with different Rho GTPases showed that YopE residues at positions 102, 106, 109 and 156 discern among switch I and II regions of Rac1, Rac2 and RhoA. Two mutants, which expressed YopE alleles with different antiphagocytic, ROS-inhibitory and cell-rounding activities, YptbL109A and YptbESptP, were studied in animal infections. Inhibition of both phagocytosis and ROS production were required for splenic colonization, whereas fewer YopE activities were required for Peyer's patch colonization. This study shows that Y. pseudotuberculosis encounters multiple host defences in different tissues and uses distinct YopE activities to disable them.

Fig. 1. Inhibition of ROS, and Rac2 by YopE

(A-B) HL-60 cells infected with wild-type Yptb (WT) + pBAD (■), Δ5 + pBAD (▲), Δ5 + pYopE (▼) at an MOI of 20 or uninfected HL-60 cells (◆) were stimulated for ROS production with 1μg/ml of PMA. (A) One representative experiment in which the y-axis shows the relative light units (RLU) produced during 3 minutes. (B) Plot of average relative ROS level compared to Δ5+pBAD from three experiments. The data was analyzed using ANOVA with Tukey's multiple comparison. * indicates statistical significance with P<0.05. (C-D) HL-60 cells were infected with ΔyopE + pBAD or ΔyopE + pYopE at an MOI of 20. The level of Rac2-GTP was determined by effector pull-down assay. (C) The ratio of the GTP bound form of Rac2 to the total level in uninfected cells was set to 1.0. The numbers below the western blots reflect the ratio of GTP-bound/total GTPase in infected cells compared to that of uninfected cells. (D) The ratio of Rac2-GTP to total Rac2 in uninfected cells was set to 1 and then the ratio of GTP-Rac2 to total Rac2 in cells from the indicated condition (x-axis) relative to the ratio from uninfected cells was calculated. The plot shows the average ratio ± SEM from three independent experiments. The data was analyzed using ANOVA with Tukey's multiple comparison as the post test. * indicates statistical significance with P<0.05.

Fig. 2. Structure model of YopE interacting with Rac1

The YopE sequence was threaded onto the SptP-Rac1 crystal structure (Evdokimov et al., 2002; Stebbins and Galan, 2000) with YopE (grey), Rac1 (yellow), switch I (pink) and switch II (cyan) depicted. YopE point mutants listed in Tables 1 were tested in three cell-culture assays. When mutated residue results in no change in phenotypes, the residue is colored dark blue. When mutated residue results in a change in all phenotypes, the residue is colored red. When mutated residue affected some but not all YopE phenotypes, the residue is colored purple.

Fig. 3. GAP activities of purified L109A and ESptP and YptbL109A and YptbESptP

Purified GST-YopE mutants were incubated with purified His₆-RhoA (A), His₆-Rac1 (B), or GST-Rac2 (C) at a ratio of 10 Rho: 1 GAP in the presence of GTP for 20 minutes at 37°C. CytoPhos reagent was added, and the level of free phosphate released as a result of GTP hydrolysis was measured by OD₆₅₀. Experiments were repeated at least three times and shown as mean ± SEM. * indicates that the value was statistically different from the indicated protein using ANOVA with Tukey's multiple comparison as the post test with P<0.05. (D) HEp-2 cells were infected at MOI of 20 for 1 hour, lysed, run on SDS-PAGE and probed for YopM, YopE, and actin by Western blot. Translocation from each strain was normalized to the actin loading control and compared to the wild-type Yptb level, which was set to 1.0. The experiment was repeated twice and a representative blot is shown.

Fig. 4. Competition infection of YptbKan and Yptb, YptbL109A, YptbESptP, or ΔyopE in BALB/c mice

(A-D) BALB/c mice were infected orogastrically with a total of 2×10⁹ CFU/mouse of a 1:1 mixture of YptbKan and either Yptb, YptbL109A, YptbESptP, or YptbΔyopE. Day 5 post infection (A) PP, (B) MLN and (C) spleens, were harvested, homogenized, and plated on Yersinia selective plates. The CI from each mouse is represented as a closed circle. Open circle indicates that one strain of bacteria was not recovered and the value was calculated as if one colony was recovered from that strain. The geometric mean is shown as a horizontal bar in the graph and is listed below as the mean. Statistical analysis was determined by ANOVA followed by Tukey's as the post test with P value < 0.05. * under the strain name indicates that the strain is statistically different from the strain in the left column.

Fig 5. Competition infection with YptbKan and YptbL109A, YptbESptP, or YptbΔyopE in C57Bl/6 and Cybb−/− mice

C57BL6/J mice and Cybb^−/− mice were infected intravenously with a 1:1 mixture of YptbKan and each of the mutants at a total dose of 100 CFU/ mouse. Day 5 post infection, spleens were harvested, homogenized, and plated. Each closed circle represents the CI from one mouse. The fold difference between geometric mean of a strain's CI in Cybb^−/− mice compared to its CI in wild-type mice is indicated below each strain. * indicates the P value <0.05 as determined by Mann-Whitney test.

Fig. 6. YopE residues that confer differential phenotypes interact with conserved residues in Rac1 and RhoA which are oriented differently

Models of YopE residues interacting with Rac1 (A, D), RhoA (B, E), and merge of Rac1 and RhoA (C, F) are shown. YopE residues that confer differential phenotypes are colored purple. Rac1 residues are colored yellow. RhoA residues are colored white. Structures of SptP-Rac1 complex (Stebbins and Galan, 2000) and RhoA-RhoGAP (Rittinger et al., 1997) were used for Rac1 and RhoA models.