Functional profiling of microbial proteases that modulate host-bacterial interactions in the gut

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Ulcerative colitis and Crohn’s disease 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 easing the typical IBD symptoms such as severe diarrhea, weight loss, fatigue and pain. Although the exact mechanisms are unclear, excessive proteolysis and dysregulated signaling of protease-activated receptors (PARs) have been linked to IBD and additional gastrointestinal (GI) diseases. PARs are a unique class of four eukaryotic G-protein coupled receptors (GPCRs) that are directly regulated by proteases. In contrast to classical GPCRs, PARs are not activated by binding of soluble ligands. Instead, proteolytic cleavage of a peptide sequence in the extracellular N-terminal domain (NTD) reveals a neo-N-terminus that acts as a tethered activating ligand. As many GI diseases are accompanied with a dysbiosis of the human microbiome, proteases derived from commensal bacteria are likely to be important regulators of gut homeostasis and pathogenesis, thus making them potential therapeutic targets. Nevertheless, no study so far has globally characterized which proteases are produced by commensal strains and how these enzymes affect health and disease by proteolysis of co-localized human PARs. Beneficial commensal bacteria might counteract excessive inflammation in the gut by basal activation of PARs or by proteolytic desensitization of the receptor, while pathobiont species could secrete proteases that promote IBD via increased PAR activation. Preliminary studies in the group of Prof. Matthew Bogyo have already revealed three commensal strains that activate PAR2 via secreted proteases, thus demonstrating the need to further study these interactions. In this work I aim to globally characterize proteases from commensal bacteria that modulate human PAR signaling in the gut. First, I will develop an in vitro high-throughput assay for monitoring proteolysis of the NTDs of all four human PARs and for direct analysis of the respective cleavage sites. After its establishment, I will screen individual cultures of 250 gut commensal strains for proteolytic PAR-processing. Next, I will identify the responsible proteases using a multi-step approach of biochemical fractionation and mass-spectrometry based chemoproteomic techniques. I will validate that proteolytic cleavage also occurs in the context of full length, membrane-bound receptor and investigate how these events impact downstream signaling in mammalian cell lines. Finally, I will apply multicellular and organoid models of intestinal barrier integrity to study the biological impact of microbial PAR-processing on intestinal permeability. Ultimately, success in these aims will not only lead to the characterization of novel microbial proteases and their role in host-microbe interaction, but also lay the foundation for future microbiome-based treatment options.