title: 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, United States of America.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, United States of America.
authors.forename: Kaustubh
authors.initials: K
authors.lastname: Parashar
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, United States of America.
authors.forename: Ananya
authors.initials: A
authors.lastname: Sitaram
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, United States of America.
authors.forename: James B
authors.initials: JB
authors.lastname: Bliska
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D058947:Bacterial Secretion Systems / Q000276:immunology*; D020690:GTPase-Activating Proteins / Q000276:immunology*; D007113:Immunity, Innate / Q000276:immunology*; D015151:Immunoblotting; D008264:Macrophages / Q000276:immunology* / Q000469:parasitology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015011:Yersinia pseudotuberculosis / Q000276:immunology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D058947:Bacterial Secretion Systems; D020690:GTPase-Activating Proteins; C063239:yopE protein, Yersinia