Project Summary and Abstract Salmonella enterica is the leading cause of food-borne hospitalizations in the United States. The Salmonella enterica species includes over 2500 circulating sub-species, or serovars, which cause disease ranging from self- limiting gastroenteritis to severe systemic disease and death. Studies of Salmonella pathogenicity and host immune responses to Salmonella rely on a limited number of commonly used reference S. enterica serovar Typhimurium (STm) or Typhi strains. While these studies have defined fundamental mechanisms of host- Salmonella interactions, they do not reflect the broad genotypic or phenotypic diversity among Salmonella enterica species, suggesting a crucial gap in our understanding of host responses to Salmonella. How the innate immune system recognizes and responds to other Salmonella serovars is of particular interest, as a significant fraction of disease is caused by serovars other than Typhi or Typhimurium. The innate immune system plays a crucial role in host defense against infection, and therefore represents a particularly attractive target for study. Salmonella invades and replicates within macrophages and utilizes a conserved type III secretion system encoded by a genomic region termed the Salmonella Pathogenicity Island 2 (SPI-2), to inject virulence factors, known as ‘effectors’, into the host cytosol. These effectors manipulate the host environment and permit Salmonella maintenance of an intracellular niche. SPI-2 effectors display intra- and inter-serovar genetic diversity, which contributes to important differences in host responses to infection. While effectors are important for bacterial virulence, they also enable the host to detect intracellular bacteria. One mechanism of innate immune recognition involves cytosolic innate immune sensors that detect bacterial ligands or the activity of effectors within the cytosol which activate the inflammatory caspases -1 and/or -11 (Casp1/11). Casp1/11 cleave and activate IL-1 family cytokines and the pore-forming protein Gasdermin-D (GSDMD), leading to an inflammatory cell death termed pyroptosis. Casp1/11 are necessary for pyroptosis in response to commonly studied strains of S. Typhimurium. Intriguingly, in contrast to prior studies, my preliminary studies show for the first time that multiple S. enterica clinical isolates of different serovars, including S. Enteritidis, trigger Casp1/11- independent GSDMD cleavage, IL-1 release, and cell death. Moreover, my preliminary studies further demonstrate that S. Enteritidis, and other clinical isolates, induce this Casp1/11-independent cell death in a SPI- 2-dependent manner. These data and as well as additional preliminary findings discussed in this proposal, provoke the hypothesis that Salmonella Enteritidis possesses a unique SPI-2 effector which triggers Casp8-mediated pyroptosis. In this proposal, I aim to define the host pathways mediating Casp1/11- independent cell death in response to clinical Sal...