Human bile has been studied for over 170 years and yet we are routinely reminded of how little we know. Early medicine considered bile an essential component of the four ‘humors’ governing health. This gradually transitioned to determining the structures of cholic acid and chenodeoxycholic acid, the two primary bile acids (BAs) present in humans, followed by investigating the nuances behind further modifications to these BA by bacteria in our intestines. Yet, prior to 2019, scientific dogma was that BA conjugation with glycine and taurine was solely performed by the host in the liver. Then, Quinn and colleagues described how bacteria in our gut are capable of ligating amino acids leucine, phenylalanine, and tyrosine to cholic acid. This was the first description of microbially conjugated bile acids (MCBAs). Given the recency of their discovery, the mechanisms behind MCBA production and their physiological relevance remained unknown prior to the work presented here.In Chapter 2, I describe the in vitro acyl transfer of amino acids to BA by the enzyme bile salt hydrolase/transferase (BSH/T). I show that purified BSH/T from Clostridium perfringens is capable of transferring amino acids to taurocholic acid, glycocholic acid, and free cholic acid. I identify the pH optimum for this transfer and show that all 20 proteinaceous amino acids are not used. Finally, I examine the reaction kinetics of phenylalanine transfer to taurocholic acid. In Chapter 3, I begin teasing apart the taxonomic diversity of bacteria capable of BA conjugation within the gastrointestinal tract. Because Enterocloster bolteae was the first bacterium implicated in MCBA production, culture-based screening for other producers focused on members of the Lachnospiraceae family. 19 of 29 species screened demonstrated the ability to produce MCBAs and clustered based on amino acid use and total abundance of MCBAs produced. However, these groups did not correlate with taxonomy. Further analysis revealed instead that MCBA profiles correlated based on BSH/T amino acid sequence, leading to three distinct classes based on MCBA profile. I then compared MCBA production between wild type and variants containing active site substitutions to further understand how active site structure impacts MCBA production. Chapter 4 begins to describe the physiological relevance of MCBAs from the level of individual bacteria to microbial communities, to human health and development. Given the antimicrobial effects of free BAs, I show how the hydrophobicity of the ligated amino acid impacts overall MCBA antimicrobial efficacy. I then show how high oral MCBA dosing correlates with shifts in the gut microbiome and that, at a lower dose, MCBAs are capable of entering enterohepatic circulation and infiltrating several tissues. Transitioning to direct human relevance, I show that MCBAs are enriched in a patient cohort undergoing sleeve gastrectomy surgery and shift dramatically following the operation. Finally, I shift from analyzing the BA pool in the context of gut dysbiosis to gastrointestinal development and describe fecal microbiome and metabolome changes through the first 12 months of life in an infant cohort. Certain classes, such as those resulting from host BA detoxification, show marked changes with time. These include MCBAs, where prevalence decreases as the infant matures.
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