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 A 2 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 ⌬pla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or ⌬pla 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.

Proteolysis of plasminogen activator inhibitor‐1 by Yersinia pestis remodulates the host environment to promote virulence

“…A). Therefore, to assess the impact of Pla on PAI‐1 levels when bacterial loads were equivalent, we increased the inoculum (‘input CFU’) of Y. pestis ∆ pla to 10 8 , so that the output CFU at 48 h was matched to the output CFU of the wild‐type strain . We found no difference in the levels of total PAI‐1 in the lungs at 48 h when output CFUs were equivalent (Fig.…”

“…Mice were inoculated intranasally with Y. pestis , and at 48 h mice were killed and the lungs were inflated with 1 mL of 10% neutral buffered formalin via tracheal cannulation, removed, fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin, as previously described . Inflamed lesions were analyzed with imagej to calculate the area of inflammation.…”

“…BAL fluid (BALF) was collected by pooling five 1‐mL lavages with PBS per mouse; the first 1 mL of the BALF was reserved for analyses of cytokines or specific hemostatic proteins (see below). Samples were processed, stained, and analyzed by flow cytometry, as previously described .…”

“…Recent empirical evidence has shown that, in fact, multiple in vitro substrates of Pla have limited or no impact on the development of disease in vivo , including the group II substrate peroxiredoxin‐6 and the group III substrate A2AP . Indeed, to date only one in vitro ‐described Pla group I substrate, the apoptotic molecule Fas ligand (FasL), has been reported to contribute to disease during pneumonic plague .…”

Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence

“…Recently it was shown that Pla provides proteolytic processing of the Y . pestis autotransporters YapG and YapE [24, 25], cleaves and inactivates mammalian thrombin-activatable fibrinolysis inhibitor (TAFI) [26], plasminogen activator inhibitor 1 (PAI-1) [27, 28], endogenous anticoagulant tissue factor pathway inhibitor (TFPI) [29], host factor peroxiredoxin 6 [30], and degrades the apoptotic signaling molecule Fas ligand (FasL) [31]. Finally, murine C-type lectin receptor DEC-205 (CD205) expressed on both alveolar macrophages and pulmonary dendritic cells is a receptor for Pla of Y .…”