Health-Relevant Functions of the Gut Microbiome

Abstract

The gut microbiome serves vital functions in host nutrition, immunity, and metabolism.Perturbations in the gut microbiome, commonly referred to as dysbiosis, have been correlated to a wide range of diseases. However, the mechanistic basis of these relationships are poorly understood. Here, I elucidated the enzymatic mechanisms underlying three important, health-relevant functions of the gut microbiome using our lab’s integrated approach, combining experimental techniques with bioinformatic analysis. In Chapter 2, I identify the novel bilirubin reductase BilR using a fluorescence assay to measure bilirubin reduction to urobilinogen. We delineate BilR from similar reductases via my identification and mutation of key residues to find that BilR is primarily encoded by Bacillota species. Our analysis of human gut metagenomes revealed that BilR is ubiquitous in healthy adults, but shows decreased prevalence in neonates and individuals with Inflammatory Bowel Disease (IBD). Our discovery sheds light on the role of the gut microbiome in bilirubin metabolism and highlights the significance of the gut-liver axis in maintaining healthy serum bilirubin homeostasis. In Chapter 3, I demonstrate that disparate taxa can recapitulate the metabolism of oxidized sugars utilizing enzymatically divergent, yet functionally equivalent gud/gar pathways. We identify novel enzymes in the divergent commensal pathway, including putative 5-KDG aldolase GudL and an uncharacterized ABC transporter GarABC that can recapitulate the function of their nonhomologous pathogen counterparts. Our findings reveal a phenomenon parallel to the pathogen growth promotion from inflammation-derived nitric oxide: oxidized sugars, which are also produced during inflammation, serve as alternative carbon sources for commensal microbes and may contribute to their increased relative abundance during gut inflammation. In Chapter 4, I discover SpiR, a microbial steroid Δ5-4 isomerase/3-keto reductase responsible for cholesterol conversion to coprostanone in the gut. We verify that SpiR catalyzes the stereospecific oxidation of both cholesterol and pregnenolone, but that it preferentially binds to cholesterol over other related steroids. Phylogenetic analysis revealed SpiR is restricted to an uncultured Actualibacteraceae clade, and multi-omics analysis shows SpiR is not only enriched in cholesterol-converting individuals, but is a predictor of cholesterol conversion. Our findings refine the enzymatic model of gut cholesterol metabolism and establish spiR as a driver for microbial cholesterol reduction in the gut.