The GAP Activity of Type III Effector YopE Triggers Killing of <i>Yersinia</i> in Macrophages

<div><p>The mammalian immune system has the ability to discriminate between pathogens and innocuous microbes by detecting conserved molecular patterns. In addition to conserved microbial patterns, the mammalian immune system may recognize distinct pathogen-induced processes through a mechanism which is poorly understood. Previous studies have shown that a type III secretion system (T3SS) in <i>Yersinia pseudotuberculosis</i> leads to decreased survival of this bacterium in primary murine macrophages by unknown mechanisms. Here, we use colony forming unit assays and fluorescence microscopy to investigate how the T3SS triggers killing of <i>Yersinia</i> in macrophages. We present evidence that <i>Yersinia</i> outer protein E (YopE) delivered by the T3SS triggers intracellular killing response against <i>Yersinia</i>. YopE mimics eukaryotic GTPase activating proteins (GAPs) and inactivates Rho GTPases in host cells. Unlike wild-type YopE, catalytically dead YopER144A is impaired in restricting <i>Yersinia</i> intracellular survival, highlighting that the GAP activity of YopE is detected as a danger signal. Additionally, a second translocated effector, YopT, counteracts the YopE triggered killing effect by decreasing the translocation level of YopE and possibly by competing for the same pool of Rho GTPase targets. Moreover, inactivation of Rho GTPases by <i>Clostridium difficile</i> Toxin B mimics the effect of YopE and promotes increased killing of <i>Yersinia</i> in macrophages. Using a Rac inhibitor NSC23766 and a Rho inhibitor TAT-C3, we show that macrophages restrict <i>Yersinia</i> intracellular survival in response to Rac1 inhibition, but not Rho inhibition. In summary, our findings reveal that primary macrophages sense manipulation of Rho GTPases by <i>Yersinia</i> YopE and actively counteract pathogenic infection by restricting intracellular bacterial survival. Our results uncover a new mode of innate immune recognition in response to pathogenic infection.</p>