Chemoproteomic strategies for probing outer membrane vesicles and their role in gut microbial interactions

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The human gut microbiota is a complex and dynamic ecosystem of microorganisms that profoundly affects health and disease. An imbalance of the gut microbiota, known as dysbiosis, has been linked to a range of gastrointestinal diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Excessive proteolysis in the gut by host or microbial proteases has been suggested as a major contributor to IBD, but the underlying mechanisms remain largely unclear. Initial reports have suggested that bacteria from the order Bacteroidales excrete significant amounts of outer membrane vesicles (OMVs), small spherical structures that are derived from outer membranes of Gram-negative bacteria. OMVs act as long-range vehicles for membrane-bound and encapsulated cargo and thus might play crucial roles in microbe-microbe as well as microbe-host communication. Intriguingly, the studied Bacteroidales OMVs featured high numbers of membrane-anchored lipoproteins, particularly proteases, that are located at the surface, thus allowing them to directly interact with their environment. This makes Bacteroidales proteases potentially powerful mediators for microbial interactions. However, we still have limited knowledge on which proteins are trafficked via OMVs from individual Bacteroidales strains and how external factors modulate the composition and the protein localization within OMVs. Moreover, it is unknown how bacterial proteases affect the composition of the gut microbiota and thereby modulate pathogenesis. In this project, we will develop and apply chemical tools to comprehensively characterize Bacteroidales OMVs and their function in microbial interactions. First, we will use activity-based protein profiling to identify bacterial strains and conditions that result in strongest OMV secretion. Next, we will apply chemoproteomic methods to characterize the proteomes of OMVs with a particular focus on serine hydrolases and the lipidation status of predicted lipoproteins. Select proteases found in OMVs will be recombinantly produced and biochemically characterized. Subsequently, we will synthesize fluorogenic substrates, covalent inhibitors and chemical probes tailored towards individual proteases. Having these powerful chemical tools in hand, we will elucidate how OMVs and their associated proteases affect individual strains and bacterial co-cultures. Ultimately, we will apply fluorescent chemical probes to study the dynamic biogenesis and trafficking of OMVs via fluorescence microscopy. Overall, success in these aims will result in a comprehensive understanding of extracellular microbial proteins, the characterization of novel enzymes, and the development of tailored tools to probe the role of OMVs in microbial interactions.