PROJECT SUMMARY/ABSTRACT: Bacterial secretion system effectors are essential for the virulence of many bacterial pathogens, and a number of effector proteins block key immune signaling pathways. However, these effectors or the alterations they cause can also be sensed by the innate immune system. For example, effector mediated modifications of host proteins can be recognized as pathological and trigger immune responses, a process known as effector- triggered immunity (ETI). While ETI was first discovered in plants, it is now clear that ETI is a common defense mechanism that is a major driving factor in the constant arms race between host and pathogens. Knowledge about these processes is central to understanding bacterial virulence as well as host defense. Conventional inflammasomes are multi-molecular complexes that control caspase-1 and/or 11-mediated pyroptotic cell death and maturation of inflammatory cytokines IL-1b/IL-18. Gasdermin D (GSDMD) was recently shown to be a key mediator of inflammasome triggered pyroptosis by cleavage at residue D276 by caspase-1/11, and the released N-terminal fragments create a pore in cell membranes compromising membrane integrity. These pores are also linked to IL-1β and IL-18 release. Infection of macrophages with pathogenic Yersinia spp., causative agents of plague and gastroenteritis, trigger cell death as well as IL-1β/IL- 18 release. We have previously described a novel unconventional inflammasome pathway, requiring RIP1 kinase and caspase-8, which are typically associated with apoptotic death, that control cytokine release and macrophage cytotoxicity after Yersinia infection. Specifically, the Type III secretion system (T3SS) effector YopJ strongly activates caspase-8 and drives this unconventional pathway. Thus, Yersinia is an excellent physiologically relevant model system to investigate the role of caspase-8 in the context of bacterial infection. In particular, our supporting data indicate that YopJ inhibition of TAK1 and IKKβ kinases activates caspase-8 and induces a non-canonical inflammasome pathway, that is not dependent on caspase-1 or -11. Similar responses are observed when inhibiting TAK1 and IKK activity, mimicking YopJ action. We show that GSDMD is cleaved at D276 and activated by Yersinia in a caspase-8 dependent manner and that GSDMD strongly regulates cell death and IL-1β/IL-18 release. These findings lead us to hypothesize that Yersinia YopJ triggers a novel effector-mediated immune response, via RIPK1 and caspase-8, leading to GSDMD cleavage, cell death and IL-1β release. How RIPK1 and caspase-8 are activated and how these events activate GSDMD are critical questions addressed herein