Date of Award
Spring 2020
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Crawford, Jason
Abstract
The human body is colonized by complex communities of microorganisms known as the human microbiota. These communities play an essential role in human health by modulating basic biological processes such as metabolism and immune, gastrointestinal, and cardiovascular development and homeostasis. Countless correlations have been made between specific microorganisms and host physiology/disease. However, the molecular mechanisms driving these phenotypes are largely undefined. This is in part due to our incomplete understanding of the microbiota metabolome. The members of the human gut microbiota express hundreds of diverse metabolic pathways that produce countless metabolites, many shared among diverse strains and the majority of which remain uncharacterized. Elucidating the structures of these metabolites is the first step towards defining the casual connections between specific organisms and host phenotypes at the molecular level. The work within this dissertation describes the discovery and structural elucidation of three families of microbial metabolites derived from Escherichia coli and their biological relevance. A commonality between these three families is their stimulation by the translation stress antibiotic, erythromycin. Like many of the small molecules produced by E. coli, the production of these metabolites can be observed across different bacterial species. Thus, our investigation of E. coli derived metabolites offers broad insight into the metabolic regulatory contributions at the host-microbe interface. Chapter 1 provides a review of E. coli-derived small-molecules, including details of their biosynthesis and their impact on human health. Those described are by no means the only metabolites produced by E. coli, but are selected to paint a picture of the diversity in structure and function of these small molecules and their significance in mediating microbe-microbe and host-microbe interactions. This chapter also describes several small molecules derived from non-enzymatic processes and highlights the importance of characterizing these often overlooked but biologically relevant products. Chapter 2 describes the use of translation stress to identify the structure of autoinducer-3 (AI-3), a pyrazinone metabolite involved in the pathogenesis of enterohemorrhagic E. coli (EHEC). AI-3 is a member of a larger family of pyrazinones derived from threonine dehydrogenase (Tdh) aminoketone products and “abortive” tRNA synthetase reactions. Along with regulating EHEC virulence, these novel AI-3 analogs exert diverse immunological effects on primary human tissues and are distributed across a variety of Gram-negative and Gram-positive bacterial pathogens, suggesting their involvement in broader interspecies signaling. The identification of the biochemical origins of these AI-3 metabolites provides a molecular foundation for future investigations into their diverse biological roles. Chapter 3 describes the stimulation of a family of lumazines, two previously characterized and two newly characterized, by translation stress. The biosynthesis of these lumazines is dependent on the conversion of tyrosine, phenylalanine, and tryptophan into their α-ketoacid analogs by aminotransferases AspC and TyrB, and the riboflavin precursor 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione (5-A-RU). The tryptophan-derived lumazine has been previously reported as an activating antigen for mucosal associated invariant T (MAIT) cells. The work within this chapter outlines new biological activities for the tryptophan analog, including antioxidant activity and the ability of this metabolite to modulate IL-10 production in T helper 17 (TH17) cells. Chapter 4 details the discovery of two naphthoquinone-derivatized metabolites termed MK-hCys and MK-Cys. Genetic and chemical studies reveal that these metabolites are derived from an intermediate of the menaquinone biosynthetic pathway, 4-dihydroxy-2-naphthoic acid (DHNA), and the amino acids homocysteine and cysteine, respectively. DHNA has been reported to display anti-inflammatory activity in the gut. However, DHNA is slightly toxic to mammalian cells, while it readily oxidized form, 1,4-naphthoquinone-2-carboxylic acid, is even more so. The trapping of cytotoxic 1,4-naphthoquinone-2-carboxylic acid with homocysteine to form the major product, MK-hCys, is a proposed detoxification strategy that allows DHNA to maintain its established beneficial roles.
Recommended Citation
Gatsios, Alexandra Marie, "The Elucidation of Novel Small Molecule Metabolites in Escherichia coli" (2020). Yale Graduate School of Arts and Sciences Dissertations. 781.
https://elischolar.library.yale.edu/gsas_dissertations/781
