179 million people in the US suffer from infectious diarrhea every year, and deaths have increased four times since 1980. A major cause of infectious diarrhea in the US is enterotoxigenic E. coli, which produces heatstable enterotoxins (ST) to overactivate host guanylyl-cyclase C (GC-C), a major regulator of fluid balance in the gut. STs cause diarrhea in the host, benefiting the bacteria by facilitating spread to new hosts. Members of this team previously identified the ST-GC-C interface as an ongoing evolutionary conflict between hosts and pathogens, where GC-C was undergoing positive selection in primates and especially bats. The latter taxa also present compensatory evolution in the host ligand for GC-C, uroguanylin, maintaining its ability to activate GCC despite the evolution of GC-C in response to the bacterial evolutionary pressure. In this proposal, this unexpected compensatory evolution in a critical physiologic pathway will be leveraged to identify potential inhibitors of STs as a treatment strategy for infectious diarrhea in humans. The central hypothesis is that rapid evolution of uroguanylin and ST resulted in functional differences in binding and activation of GC-C and high affinity-low activity peptides that will competitively inhibit human-pathogenic enterotoxins from causing diarrhea can be found and leveraged to treat infectious diarrhea in the future. Aim (1) will characterize the effect of uroguanylin and enterotoxin variation on binding and activation of GC-C. Evolutionary analyses of GC-C ligands will be conducted to identify sites under positive selection, and ligands will be tested for binding and activation to identify high affinity and low activity candidates. Aim (2) will identify peptides that modulate GC-C activation in cellular and organoid models of diarrheal infection. High affinity-low activity peptides will be tested in competition assays to determine if enterotoxins can be blocked from causing diarrhea as a direct method of treating infectious diarrhea. The results will inform the design of future peptide treatments for infectious diarrhea that will greatly improve clinical care by using targeted therapy instead of mainly treating symptoms. This project will provide excellent multidisciplinary training in evolutionary analysis, biochemical assays, and organoid models, complemented by clinical mentorship and shadowing in infectious disease. The training plan was developed to complete the central goals of conducting independent, collaborative research; advancing communication and grant-writing skills; learning how to effectively mentor and teach trainees in a supportive environment; and honing clinical skills in preparation for the return to medical school. This training is ideal for a future physician-scientist with the goal of improving patient care at the intersection of basic research and clinical treatment of pediatric infectious diseases.