Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Hatzios, Stavroula
Abstract
All host-associated microorganisms require mechanisms to adapt to stress encountered in the host. In particular, host immunity generates reactive oxygen species (ROS) as a measure to regulate microbial expansion in the gut and to kill invading pathogens. ROS are broadly toxic to cellular biomolecules, and consequently both microorganisms and the host employ molecular mechanisms to contend with their harmful properties. Accordingly, all life forms rely on low- molecular-weight (LMW) thiols, a family of ubiquitous small molecule antioxidants, to maintain intracellular redox homeostasis and resist oxidative damage caused by ROS. Chapter 1 reviews the molecular and biological functions of LMW thiols, including their roles as cellular antioxidants and enzyme cofactors, which facilitate microbial adaptation and resistance to stress in the host. Recently emerging roles of LMW thiols in regulating microbial virulence are also discussed, as well as how LMW thiol metabolism can contribute to the production of bioactive metabolites that influence human physiology. Chapter 2 details investigations into the presence of LMW thiol antioxidants in thegastric pathogen Helicobacter pylori, which unexpectedly lacks LMW-thiol biosynthetic pathways. Using reactivity-guided metabolomics, we identify the unusual LMW thiol ergothioneine (EGT) in H. pylori, a dietary compound that accumulates to millimolar levels in human tissues and is broadly implicated in mitigating disease risk. Whereas certain microorganisms synthesize EGT, we show that H. pylori acquires this LMW thiol from the host environment using a highly selective ATP-binding cassette transporter, EgtUV. EgtUV confers a competitive colonization advantage in vivo and is widely conserved in gastrointestinal microbes. We also demonstrate that human fecal bacteria metabolize EGT, which may contribute to the production of the disease-associated metabolite trimethylamine N-oxide. Altogether, the findings in this chapter illustrate a previously unappreciated mechanism of microbial redox regulation in the gut and suggest that inter-kingdom competition for dietary EGT may broadly impact human health. Finally, Chapter 3 describes the biochemical characterization of a serine protease secreted by Vibrio cholerae that may facilitate its adaptation to the host. Enzymes exported by V. cholerae can facilitate adaptation to intestinal and aquatic niches by mediating nutrient acquisition, biofilm assembly, and V. cholerae interactions with the host. The V. cholerae-secreted serine protease IvaP is produced in V. cholerae-infected rabbits and human choleric stool, where it alters the activity of several host and pathogen enzymes in the gut, including that of the host immune carbohydrate-binding protein, intelectin. Here we detail the mechanism of extracellular IvaP maturation, which involves a series of sequential N- and C- terminal cleavage events. We determined that both inter- and intra-molecular proteolysis contributes to the formation the mature enzyme, and which involves the removal of an N- terminal propeptide domain that can temporarily inhibit and be cleaved by the purified enzyme. We also show that IvaP directly cleaves purified intelectin, inhibiting intelectin binding to V. cholerae cells. Altogether, these data suggest that IvaP plays a role in modulating host intelectin-V. cholerae interactions.
Recommended Citation
Dumitrescu, Daniel G., "Molecular Mechanisms of Microbial Adaptation to the Host" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1071.
https://elischolar.library.yale.edu/gsas_dissertations/1071
