Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis

Abstract

Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague.

FIG 1

Validation of Pla-dependent Prdx6 degradation within BALF. Immunoblot analysis of Prdx6 from C57BL/6 mouse BALF only or BALF following incubation with wild-type Y. pestis or Y. pestis Pla D206A for 6 h at 37°C. The density of each band relative to BALF only is indicated beneath. Numbers to the left of the blot indicate molecular masses in kilodaltons. The blot is representative of 3 independent experiments.

FIG 2

Cleavage of Prdx6 requires catalytically active Pla. (A) Immunoblot analysis of recombinant human Prdx6 following incubation with Y. pestis, Y. pestis Pla D206A, or Y. pestis Δpla at 37°C for the indicated times. (B) Immunoblot analysis of recombinant human Prdx6 following incubation with E. coli producing Pla or Pla D206A or E. coli carrying the corresponding empty expression vector at 37°C for the indicated times. (C) Amino acid sequence of human Prdx6. Black arrowheads indicate identified Pla cleavage sites. The lipase and peroxidase motifs are boxed, and residues for the active site for peroxidase activity (C47, H39, and R132) or the catalytic triad for lipase activity (S32, D140, and H26) are in bold. Lowercase letters in panels A and B represent Pla-cleaved Prxd6 products. Numbers to the left of the blots indicate molecular masses in kilodaltons. Data are representative of at least 3 independent experiments.

FIG 3

Cleavage of Prdx6 by Pla disrupts both phospholipase A₂ and peroxidase activities. (A) Peroxidase activity of Prdx6 following incubation with Y. pestis, Y. pestis Pla D206A, or trypsin or incubation alone for 2 h at 37°C. Prdx6 activity is calculated as the percentage of H₂O₂ removed, based on the ratio of the amount of H₂O₂ removed in the presence to that removed in the absence of Prdx6. (B) Phospholipase A₂ activity of Prdx6 following incubation with Y. pestis, Y. pestis Pla D206A, or the inhibitor MJ33 or incubation alone. One representative experiment of 2 biological replicates is shown, and error bars represent the SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Bonferroni's multiple-comparison test).

FIG 4

Pla-dependent changes in Prdx6 levels within the lung during Y. pestis respiratory infection. Abundance of extracellular Prdx6 recovered by BAL of C57BL/6 mouse lungs 48 h post-inoculation with PBS (mock), 10⁴ CFU of Y. pestis, 10⁴ CFU of Y. pestis Δpla, or 10⁸ CFU of Y. pestis Δpla as measured by ELISA (A) or immunoblotting (B). Data from mock- or wild-type Y. pestis-infected C57BL/6 prdx6^−/− mice are shown as controls in panel B. The input CFU and output CFU are included below the graph and blot to denote the number of bacteria inoculated and present at the time of assessment. The density of each band relative to BALF is indicated beneath the immunoblot. Data in panel A are combined from 2 independent experiments (n = 10), and error bars represent the SEM. Data in panel B are representative of 3 independent experiments (*, P < 0.05; ns, not significant by one-way ANOVA with Bonferroni's multiple-comparison test).

FIG 5

Absence of Prdx6 does not alter bacterial burden during Y. pestis respiratory infection. Bacterial burden within lungs (A) and spleens (B) of C57BL/6 or C57BL/6 prdx6^−/− mice infected via the i.n. route with 10⁴ CFU of Y. pestis or Y. pestis Δpla after 48 h. Each point represents the number of bacteria recovered from a single mouse. The median CFU is denoted by a solid line, and the dashed line indicates the limit of detection. Data are combined from two independent experiments (n = 10 for each group; ns, not significant by Mann-Whitney U test). WT, wild type.

FIG 6

Absence of Prdx6 does not alter the host immune response during Y. pestis respiratory infection. (A) Sections of formalin-fixed lungs stained with H&E from C57BL/6 or C57BL/6 prdx6^−/− mice inoculated with PBS (mock), 10⁴ CFU of Y. pestis, or 10⁴ CFU of Y. pestis Δpla after 48 h. Representative images of inflammatory lesions are shown (arrows; n = 3). Bars, 100 μm (inset images) and 50 μm (larger images). (B) Enumeration of total immune cells present in the BALF of C57BL/6 or C57BL/6 prdx6^−/− mice inoculated with PBS (mock), 10⁴ CFU of Y. pestis, or 10⁴ CFU of Y. pestis Δpla after 48 h. (C) Abundance of indicated inflammatory cytokines present in supernatants of the same BALF samples as those collected for panel B. Data are combined from 2 independent experiments (n = 10 for each group), and error bars represent the SEM (ns, not significant by one-way ANOVA with Bonferroni's multiple-comparison test).

FIG 7

Absence of Prdx6 does not alter lung damage during Y. pestis respiratory infection. Abundance of albumin present in supernatants of the same BALF samples as those collected for the experiment in Fig. 6 as measured by ELISA. Data are combined from 2 independent experiments (n = 10 for each group), and error bars represent the SEM (ns, not significant by one-way ANOVA with Bonferroni's multiple-comparison test).