Project Summary The intestinal microbiome is made up of trillions of microorganisms that inhabit the gastrointestinal tract. Metabolites produced by the microbiome reach extraintestinal tissues, and can be found in the bloodstream, and are thereby able to influence almost all tissues of the body. Interestingly, the expression of a number of genes of the gastrointestinal tract is dependent on colonization by the microbiome. In this project, we will explore the mechanisms through which the gut microbiome controls gene regulation of intestinal epithelial cells (IECs), through the study of a microbiome-modulated gene involved in IEC cell death. Salmonella enterica serovar Typhimurium (STm) is a pathogen that is adept at overcoming host defenses in order to cause disease. STm triggers an inflammatory response that benefits growth of the pathogen by taking advantage of the host response to infection. Our preliminary findings suggest that STm is able to benefit from a homeostatic microbiome- mediated cell death pathway that has yet to be described. Under steady state conditions, metabolites produced by the microbiome lead to pyroptosis of IECs, which helps maintain normal epithelial turnover. However, upon STm infection, the pathogen harnesses this pathway to elicit increased IEC pyroptosis, leading to increased STm numbers in the gastrointestinal tract. Our early results indicate loss of components of the IEC pyroptosis pathway led to lower STm luminal outgrowth and dissemination to extraintestinal organs, yet, it is unclear how STm activates IEC pyroptosis, and whether this pathway indeed leads to an increase in pyroptosis of intestinal epithelial cells during infection. Thus, I hypothesize that during infection of the gastrointestinal tract, STm takes advantage of a microbiome-controlled homeostatic IEC-specific cell death pathway to bloom to high numbers. In order to elucidate how the microbiome and STm activate IEC pyroptosis, and how this activation leads to cell death and downstream pathogen expansion within the gastrointestinal tract and in extraintestinal sites, we propose two specific aims. In AIM 1 we will assess the contribution of the gut microbiome to induction of IEC pyroptosis, by using a combination of sequencing and in vivo mouse models using conventional and gnotobiotic mice. In AIM 2 we will use mouse infection models to determine how STm elicits IEC pyroptosis during infection, and define the upstream activation pathway. Our mechanistic approach will provide a causal link between the microbiome, a host cell death pathway, and pathogen expansion. Successful completion of this proposal will identify a previously undefined IEC cell death pathway that plays a crucial role under steady state and infectious conditions. This project will additionally expand my training to include key methods and concepts in IEC biology and in the study of the microbiome. Altogether, the research and training plan proposed will facilitate a better underst...