Chemical profiling of secreted proteases from the gut microbiota and their impact on microbial communications

The human gut microbiota, the diverse community of microbes inhabiting the intestinal tract, has emerged as a key mediator of human health and disease. Long-term studies have observed associations between various diseases and changes in the microbiota, but the underlying molecular mechanisms that cause these shifts in the microbiota remain unknown. At the molecular level, both human and bacterial enzymes are essential for the breakdown of food but can also function as regulatory elements that modulate immune function. Elevated bacterial serine hydrolase abundance is implicated in inflammation and gut barrier dysfunction and has been correlated with gut-related diseases, including inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer. Recent work has highlighted serine proteases as particularly important mediators in microbe-microbe and host-microbe interactions. This project therefore investigates secreted serine hydrolases, with a particular focus on serine proteases, and their role in shaping bacterial interrelationships within the gut. Cultivating gut bacteria in vitro often requires complex media which can affect downstream processes, and secreted proteins are present in very low concentrations. This project will first focus on establishing optimised proteomics sample preparation protocols that will allow for robust removal of interfering media components, and high-throughput analysis even with low sample inputs. Having established this method, we will combine it with activity-based protein profiling to evaluate the presence and activity states of individual enzymes in the secretomes of a variety of commensal bacteria. The most active bacterial secretomes will be further characterised using enzymatic assays and co-cultivation experiments. Lastly, candidate proteins will be subjected to recombinant overexpression and purification for biochemical characterisation. Overall, this work will contribute to a detailed understanding of how secreted bacterial hydrolases impact microbial communication and potentially reshape the gut microbiota with important implications for host health and disease.

PhD start: 10/01/2024