Project Summary Crohn’s disease and ulcerative colitis are forms of inflammatory bowel disease (IBD) that affect more than 6.8 million patients worldwide. Because no cure is available to date, treatment is limited to reducing IBD symptoms such as severe diarrhea, weight loss, fatigue and pain. Although the exact mechanisms that mediate the pathogenesis of the disease are unclear, excessive proteolysis in the gut combined with dysregulated signaling of protease-activated receptors (PARs) have been identified as important drivers of IBD and additional gastrointestinal (GI) diseases. PARs are a unique class of four eukaryotic G-protein coupled receptors (GPCRs) that are directly regulated by proteolytic cleavage of a peptide sequence in the extracellular N- terminal domain (NTD). Cleavage reveals a tethered activating ligand or alters the receptor conformation to induce activation. PAR-signaling can promote inflammation by disrupting the integrity of the intestinal epithelial barrier, which under physiological conditions allows permeability of nutrients but restricts the entry of bacterial pathogens and toxins. Many GI diseases are accompanied by loss of barrier function and dysbiosis of the gut microbiome. Proteases derived from commensal bacteria are likely to be important regulators of gut homeostasis and pathogenesis, thus making them potential therapeutic targets. However, it is unclear which extracellular proteases are produced by commensal strains in the gut and how these enzymes affect health and disease by proteolysis of co-localized PARs. We hypothesize that beneficial commensal bacteria secrete proteases that keep excessive inflammation in check by basal activation or proteolytic desensitization of PARs. Conversely, pathobiont bacteria species secrete proteases that promote inflammation via increased PAR activation. Therefore, proteases produced by the gut microbiota as well as pathogenic bacteria have the potential to be valuable new therapeutic targets for the treatment of various forms of IBD. To test our hypothesis, we will develop a robust in vitro assay to broadly screen for proteases with PAR-processing activity in both commensals and pathobiont bacterial species. We will then identify specific PAR processing proteases and assess their specific roles in regulating epithelial barrier integrity and inflammation using cell culture systems and gut organoids. Finally, we will establish the therapeutic relevance of the identified proteases by confirming their presence and elevated activity in clinical samples isolated from patients with active IBD. Ultimately, this work will identify specific PAR processing proteases produced by bacterial strains and define the mechanism by which they impact the pathogenesis of IBD.