PROJECT SUMMARY/ABSTRACT Salmonella species (especially the S. enterica serotypes typhi and typhimurium) cause disease in ~90 million people every year worldwide. Salmonella infects both phagocytic immune cells (such as macrophages), as well as non-phagocytic cells. When Salmonella infects macrophages, it triggers a rapid, pro-inflammatory form of death called pyroptosis. Unlike the case with macrophages, less is known about Salmonella-triggered cell death responses in non-phagocytic cells, such as intestinal epithelial cells and fibroblasts. As these are the some of the first cell types infected by Salmonella, understanding how they respond to this bacterium, and whether pyroptotic clearance mechanisms are at play in these cells, is an important objective. Our preliminary data demonstrate that infecting epithelial and fibroblastic cells with Salmonella and later exposing them to IFN- γ, mimicking the environment of infection in the intestine, induces a novel form of cell death that is neither pyroptosis nor any of the other reported mechanisms of programmed cell death, including apoptosis, necroptosis, or autophagy. Mechanistically, we find that IFN-γ ruptures the Salmonella-containing vacuole in non-phagocytic cells, releasing Salmonella into the cytosol, from where the bacterium induces cell death. Results from a CRISPR screen suggest that such cell death is the consequence of toxified mitochondria. Based on these and other observations, we hypothesize that IFN-γ-induced cell death in non-phagocytic cells is a host defense mechanism that destroys infected intestinal epithelial cells to limit Samonella spread in vivo. To test this hypothesis, we have generated mice in which IFN-γ signaling can be selectively ablated in either intestinal epithelial cells or phagocytes. The Aims of this proposal are to (1) elucidate the mechanism by which Salmonella kills non- phagocytic cells upon IFN-γ exposure; and (2) test the in vivo role of IFN-γ signaling and cell death in Salmonella- infected intestinal epithelial cells during acute Salmonella infection. The successful completion of these Aims will reveal the mechanism and in vivo role of a IFN-γ-driven new death pathway engaged by the host during anti- Salmonella immune responses.