Understanding and Preventing Disease-Associated Anaerobic Choline Metabolism by the Human Gut Microbiota

Abstract

The consortium of microorganisms inhabiting the human gastrointestinal tract, our gut microbiota, has an extensive set of metabolic capabilities that directly influence human health. Over the past decade, DNA sequencing has significantly improved our knowledge of this microbial community’s composition. However, the molecular details of how gut bacterial metabolism impacts human health and disease are largely unknown. We propose that by combining chemical knowledge with bioinformatics, we can uncover microbial metabolic pathways that contribute to human disease. By developing small molecule inhibitors specifically targeting these pathways, we can further elucidate the roles of bacterial metabolism in disease and create a new paradigm for therapeutics. To illustrate this strategy’s potential, we are investigating the catabolism of the essential nutrient choline by anaerobic gut microbes. Within the human gastrointestinal tract, bacteria can process choline in an entirely different manner than human cells, cleaving its C–N bond to produce trimethylamine (TMA), which is subsequently oxidized to trimethylamine N-oxide by hepatic enzymes. This microbial-human co-metabolic pathway has been linked to several diseases, including non-alcoholic fatty liver disease and atherosclerosis. Even though anaerobic microbial choline conversion into TMA has been known for over a century, its genetic and biochemical bases had not been identified prior to our work. This thesis presents the discovery and validation of a gene cluster responsible for anaerobic choline utilization (cut gene cluster), as well as the characterization and inhibition of the key TMA-forming enzyme, choline TMA-lyase (CutC). Chapter 2 details the bioinformatic approach used to discover the cut gene cluster, and the genetic, spectroscopic and cultivation-based strategies to connect these genes to anaerobic choline metabolism. We also reveal the involvement of a C–N bond cleaving glycyl radical enzyme (CutC) in TMA production, an unprecedented reactivity for this enzyme family. Overall, our experimental and computational findings suggest that the cut pathway may be a major mechanism for the direct conversion of choline to TMA by gut bacteria. Chapter 3 describes the in vitro reconstitution and study of choline-TMA lyase CutC and its activating protein, the radical S-adenosylmethionine dependent enzyme CutD. We demonstrate that CutC can be activated to the glycyl radical form by CutD under anaerobic conditions, and can process choline to TMA and acetaldehyde with high specificity. Homology modeling and mutagenesis experiments further allow us to conclude that CutC is a glycyl radical enzyme of unique function and a molecular marker for anaerobic choline metabolism. Chapter 4 presents the results of a close collaboration with the Drennan group at Massachusetts Institute of Technology. They elucidated four high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline, while I characterized the impact of mutations on choline binding and catalysis. Our data uncover unexpected interactions between the trimethylammonium group of choline and polar amino acids side chains and provide new insight into the mechanism of C–N cleavage by CutC. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production. Chapter 5 depicts initial efforts towards discovering small molecule inhibitors of CutC-mediated choline cleavage. A first round of structure-guided rational design revealed betaine aldehyde as a promising lead. This molecule inhibited choline conversion to TMA by a panel of cut gene cluster-containing bacteria and by a human fecal sample. I also describe our high throughput screening approach to identify new inhibitors of choline metabolism by gut microbes, as an orthogonal strategy to rational design. Towards this end, we optimized a media formulation containing choline as sole carbon source, such that survival of gut bacteria grown in this media would be dependent on the cut pathway. Overall, our preliminary results show that small molecules can interfere with choline metabolism by anaerobic gut microbes and set the stage for more extensive efforts to discover potent inhibitors of this pathway.