Characterizing anaerobic taurine metabolism by the human gut bacterium and opportunistic pathogen Bilophila wadsworthia

Abstract

The human gut microbiome performs diverse chemical transformations that impact host health, nutrition, and disease. While the field has discovered numerous correlations between gut microbiome composition, metabolite abundance, and health, very few of these links are understood at a mechanistic or correlative level. One such gut microbial metabolic transformation is the conversion of the abundant dietary- and host-derived sulfonate amino acid taurine into hydrogen sulfide, a disease-associated metabolite. This metabolic pathway is encoded in the human gut bacterium and opportunistic pathogen Bilophila wadsworthia. This thesis describes the further investigation of gut microbial taurine metabolism to gain insight into how this opportunistic pathogen can utilize taurine-related metabolites and how we can manipulate this metabolic pathway. Our studies encompass a mechanistic study of the enzyme responsible for the key C–S cleavage step in taurine metabolism, followed by an investigation of conjugated taurine as an upstream precursor to the previously characterized taurine metabolic pathway, and finally discuss our efforts to validate a small molecule inhibitor for utilization as a chemical tool to inhibit B. wadsworthia taurine metabolism. Chapter 2 describes our collaborative effort, together with the Drennan and Kulik labs at MIT, to understand the mechanism of isethionate sulfite-lyase (IslA), the glycyl radical enzyme responsible for C–S bond cleavage in taurine metabolism. We were able to obtain a substrate-bound crystal structure of IslA from B. wadsworthia. In comparison to other structurally characterized glycyl radical enzymes that perform similar chemical reactions, the IslA active site shows differences in substrate positioning and loop architecture. We then conducted biochemical assays on a panel of IslA variants to probe the role of active site residues in catalysis. Finally, utilizing the results of these assays with isotope-labeled substrate and computational approaches, we propose a mechanism for C–S bond cleavage by IslA. Chapter 3 discusses efforts to understand the extent to which taurine-conjugated bile acids are a source of taurine for B. wadsworthia. We first explore the ability of B. wadsworthia to utilize taurine-conjugated bile salts for growth. We determine the specificity of the enzymatic deconjugation reaction by testing glycine- and taurine-conjugated bile salts. Finally, we discuss several approaches used to identify the enzyme responsible for the release of taurine from bile salts. Chapter 4 describes our studies of the interaction of small molecules with B. wadsworthia using growth as a readout. Using previous literature, we rationally selected a small panel of potential compounds to screen as a starting place for the development of tool compounds to inhibit B. wadsworthia metabolism, and to understand the interaction between clinically-used drugs and B. wadsworthia. Both stimulatory and inhibitory molecules were identified. We further demonstrate that one small molecule inhibits a key enzyme involved in taurine metabolism in vitro and inhibits growth in a dose-dependent manner in B. wadsworthia cultures. Chapter 5 discusses our conclusions and ideas for future research directions that would transform our understanding of gut bacterial taurine metabolism and the biological roles of B. wadsworthia.