Chemoproteomic discovery of secreted microbial enzymes for engineered probiotics - PROJECT SUMMARY/ABSTRACT The gut microbiota produces a myriad of metabolites that have been shown to have wide effects on host biology. Specifically, peptidoglycan (PG) fragments are well established immunomodulators in many health issues including inflammatory diseases, microbial infections, and cancers and have been shown to be translationally relevant in immune cell differentiation, insulin processing, and the efficacy of checkpoint inhibitor immunotherapies. Although the microbiota’s ability to produce PG fragments may provide an underlying mechanism for host-microbial interactions, it remains difficult to selectively modulate the amount of host-available PG. Therefore, the development of new tools to upregulate PG fragments in circulation for gain of function studies are needed to understand certain fundamental host biologies. Previous efforts to decipher these signaling pathways have focused the phylogenetic relationships of correlated microbes rather than their shared metabolic outputs; however, these genomic data do not provide information on protein secretion levels, stability, or activity. Unlike previous work, this proposal will directly assay microbial enzymes through a chemoproteomic platform to uncover functional PG-degrading enzymes for the development of functionalized probiotics to control metabolite output in host systems. It is hypothesized that increased hydrolytic activity in the gut will selectively control PG fragment levels in circulation. The proposal’s main goals are to identify highly active, secreted, and stable PG-degrading enzymes (Aim 1) and to genetically engineer tools to upregulate PG metabolites in circulation (Aim 2). This research will allow for the discovery of high performing microbial enzymes to rationally control circulating glycan metabolites through gut colonization with engineered tool organisms. The potential discoveries in this proposal could be translationally applied to alter microbe-human dynamics through the utilization of mechanistically-defined probiotics. The Sponsor, Co-Sponsor and PI have developed a fellowship training plan that will enable the PI’s future goal of becoming an independent researcher through enhancing both her scientific expertise and professional skills. To accomplish the experimental aims as proposed, the PI will receive training in proteomics, probiotic genomic engineering, and animal handling. In addition, the training plan outlines key professional development events that will improve the PI’s scientific communication and writing abilities, which will enable a successful career trajectory for her. This research plan will ultimately develop the PI’s scientific and professional proficiencies so that she may obtain a post-doctoral research position, which will lead her toward becoming an independent scientist. The expertise that will be gained through her experimental work on this proposal as well as her participation in the didactic and career development activities planned will enable the PI to accrue the tools needed to have a successful career as a future independent researcher.
