Student Theses and Dissertations

Date of Award

2015

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hang Laboratory

Abstract

The intestinal microbiome can mediate host resistance to enteric pathogens by modulating host immunity or by directly inhibiting pathogen growth and virulence. Although the abundance and diversity of bacterial metabolites in the intestine is appreciated, the majority of intestinal bacteria and bacterial products have not been characterized in the context of microbiota-mediated pathogen resistance. In this thesis, we describe two projects aimed at developing new methodologies to study how intestinal bacteria, and the metabolites they generate, inhibit bacterial pathogenesis. In Chapters 2 and 3, we use a chemical reporter strategy to analyze fatty acid protein modifications in bacteria. Dietary and bacterially-produced fatty acids can modulate the virulence of various pathogens, such as Salmonella typhimurium, possibly through covalent protein modification. We demonstrate that alkynyl-functionalized fatty acids can be metabolized and covalently attached to known fatty-acylated proteins in E. coli. Proteomic analysis of modified proteins revealed an unconventional fatty-acid modification on the metabolic enzyme YjgF, highlighting the utility of this method for the discovery of novel sites of fatty-acylation. Using these reporters, we also explored the effects of fatty acids on S. typhimurium virulence. Our results in S. typhimurium underscored the extensive fatty acid metabolism of this pathogen as compared to E. coli, and revealed a potential fatty acid modification on the virulence factor HilA. In addition, we demonstrated that alkynyl-functionalized short chain fatty acids, like natural fatty acids, can inhibit the secretion of Salmonella virulence factors in vitro. Overall, our results suggest that alkynyl-functionalized fatty acids may be useful for analyzing Salmonella metabolism and virulence. In Chapter 4, we describe a C. elegans model system that we used to investigate the effects of Enterococcus faecium on bacterial pathogenesis. Probiotic strains of E. faecium can inhibit the virulence of several intestinal pathogens, including Salmonella typhimurium, but it is unknown if E. faecium directly targets pathogens or modulates host immunity. Through a combination of genetic, biochemical, and proteomic approaches, we identified a secreted peptidoglycan hydrolase (SagA) from E. faecium that confers protection against Salmonella pathogenesis. Our results indicate that SagA does not directly inhibit pathogen growth, but instead remodels peptidoglycan in vivo to enhance host resistance to pathogenesis. We define specific peptidoglycan fragments that are sufficient for mediating host protection, and show that this protection requires the host gene tol-1. Notably, introduction of SagA into a non-protective intestinal bacteria, E. faecalis, is sufficient to mediate host protection in C. elegans as well as germ-free mice, suggesting that SagA acts through evolutionarily conserved host pathways. We hypothesize that SagA-generated peptidoglycan fragments may strengthen intestinal barrier integrity through local innate immune activation to confine pathogens to the intestinal lumen. Our results suggest that commensal bacteria can restrict intestinal pathogens by directly modifying microbial-associated molecular patterns in the intestine. Overall, the projects described in this thesis underscore the role of bacterial metabolites, such as fatty acids and peptidoglycan fragments, in restricting intestinal pathogens.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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