Ulcerative colitis (UC) is a chronic disease characterized by inflammation of the mucosa of the colon. UC has a significant global burden, and is characterized by an aberrant immune response directed towards the gut microbiota. Current treatment options exclusively target host inflammatory pathways and are often ineffective in managing disease. Nearly 1 million individuals in North America are currently living with UC and its prevalence in humans continues to grow worldwide. The considerable number of growing cases, coupled with the lack of an effective therapy, stresses the need for investigation into new therapeutic interventions against UC. Our group recently characterized host-microbiome interactions governing UC through six fecal or serum based –omic datasets from 40 UC patients displaying a wide range of clinical, endoscopic, and histologic disease activity. After broad-scale analyses, the six datasets provided powerful evidence towards a central hypothesis: Bacteroides vulgatus proteases can drive UC disease severity. Metaproteomics pinpointed B. vulgatus proteases as a distinguishing feature of severity. Shotgun metagenomics guided taxonomic inferences and revealed that the B. vulgatus association was driven primarily by protein regulation as opposed to microbial abundances. An abundance of serine protease inhibitors found in the patient serum suggested the importance of proteases. The metapeptidome showed increased peptide fragments correlated with UC disease severity. An independent 210-person cohort validated the strong connection between B. vulgatus proteases and UC disease severity. Testing our hypothesis, we demonstrate B. vulgatus can disrupt intestinal epithelial permeability in vitro, and protease inhibition was sufficient to restore epithelial barrier. Monocolonization of B. vulgatus into germfree IL-10 deficient mice demonstrated colonization induces colitis in these animals and protease inhibition is sufficient to prevent colitis development. Furthermore, transplantation of feces from UC patients with over-abundant B. vulgatus proteases into germ-free mice induced colitis dependent on protease activity (Mills et al., Nature Microbiology 2021). Based on our published work, we have formulated two testable hypotheses to further build on these new findings: 1) proteolysis is a central mechanism utilized by B. vulgatus to induce colitis in vitro and in vivo relevant to UC and 2) B. vulgatus proteases degrade key components of the intestinal barrier. The specific aims of this application are to define the genomic context, roles, and develop specific inhibitors against B. vulgatus proteases in UC (Aim 1), characterize the targets and dynamics of B. vulgatus mediated proteolysis in UC (Aim 2). To complete this proposal, we will use a multifaceted approach of state-of-the-art biochemical, cell culture, bacterial genetics, and in vivo models. Ultimately, this work has the potential to break new ground in UC basic and translational resea...