The GAP Activity of Type III Effector YopE Triggers Killing of Yersinia in Macrophages

Abstract

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 Yersinia pseudotuberculosis 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 Yersinia in macrophages. We present evidence that Yersinia outer protein E (YopE) delivered by the T3SS triggers intracellular killing response against Yersinia. 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 Yersinia 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 Clostridium difficile Toxin B mimics the effect of YopE and promotes increased killing of Yersinia in macrophages. Using a Rac inhibitor NSC23766 and a Rho inhibitor TAT-C3, we show that macrophages restrict Yersinia 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 Yersinia 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.

Author Summary

The type III secretion system (T3SS) is a macromolecular protein export pathway found in gram-negative bacteria. It delivers bacterial toxins into eukaryotic cells to promote pathogenic infection. T3SSs and the bacterial toxins delivered are critical arsenals for many bacterial pathogens of clinical significance, such as Yersinia, Salmonella and Shigella. On the other hand, the mammalian immune system may recognize the T3SS as a danger signal to signify pathogenic infection, and to stimulate appropriate defense against pathogens. Here, we show that the innate immune system recognizes the activity of YopE delivered by the Yersinia T3SS. Modulation of host Rho GTPases by YopE elicits a defensive response, which results in killing of bacteria in host cells. Inhibition of host Rho GTPases by Clostridium difficile Toxin B, another bacterial toxin, mimics the YopE-triggered killing effect. Our study demonstrates that host cells sense manipulation of Rho GTPases by bacterial toxins as a surveillance mechanism, revealing new insights into innate immune recognition of pathogenic infections.