Regulation of protein secretion in human gut commensals

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Project Summary Commensal bacteria in the human intestinal tract provide numerous benefits to their hosts, from digestion of foods inaccessible to the host to protection against pathogen invasion. Many host-microbiome interactions are mediated by bacterial proteins secreted into the gut (the secretome), but regulation and coordination of this process has not been studied. This proposal focuses on the dominant gut genus Bacteroides to test the hypothesis that these organisms use central mechanisms to control deployment of secreted proteins. One clue comes from studies of commensal-encoded toxins: two secreted toxins with no similarities in sequence or targets both use the same surface-associated cysteine protease to orchestrate their secretion. Homologs of this cysteine protease (but not its targets) are conserved across Bacteroides, suggesting a broader role for these enzymes in regulating protein secretion. This proposal will use an innovative N-terminome proteomic approach to determine the repertoire of protease substrates in Bacteroides and establish whether protease homologs share targets within and between species. Complementary studies in gnotobiotic mice will determine the contribution of protease processing to the in vivo secretome. This proposal will also use genetics, anaerobic microbiology, and gnotobiotics to elucidate environmental cues and signal transduction pathways that regulate protease expression. This will determine how these proteases might coordinate release of waves of secreted proteins in response to environmental signals in the gut. If successful, these studies will uncover fundamental mechanisms for secretome regulation in the microbiome, with implications for host-microbiome interactions and bioengineering therapeutic enzyme delivery in the gut. This proposal also presents a detailed training plan that will allow the candidate to develop new skills required for career advancement. This research will be conducted at the highly collaborative and multidisciplinary Microbial Sciences Institute (MSI) at Yale University. The sponsor has an established track record of mentorship, and the lab and the MSI provide state-of-the-art facilities. The diverse expertise of peers and faculty at MSI, ranging from chemists, biochemists, and geneticists, will support the candidate's research goals. A mentoring committee will provide technical guidance for the project and will support the candidate's career development. The candidate will also complete training in mentoring and teaching from the Poorvu Center for Teaching and Learning and the Yale Postdoctoral Association, and he will put this training into practice by mentoring students in their local science fair projects. By presenting his work to peers at Yale University and in conferences, the candidate will foster his communication skills and build a network of colleagues. Together, this training plan will build the skillset and connections required to succeed as an independent investigator.