Student Theses and Dissertations

Date of Award

2018

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hang Lab

Abstract

The intestinal microbiota plays critical roles in human physiology and diseases. While recent research has revealed many mechanisms by which gut microbiota influences host immunity to defend against invading pathogens, how microbiota directly antagonizes pathogen virulence is less studied. In particular, gut microbiota produces large amounts and varieties of small molecules that may impact both host immunity and pathogen virulence. In this thesis, I describe how fatty acids, derived from both gut microbiota and diet, contribute to attenuation of virulence of enteric pathogen Salmonella. In Chapter 1, I review how dietary and microbiota metabolites affect different aspects of host-microbe interactions. These metabolites are categorized into microbial-associated molecular patterns and microbiota-derived secondary metabolites. Small molecules reviewed in this chapter not only enhances host innate and adaptive immunity, but also directly inhibit virulence of invading pathogens, providing colonization resistance to the host. In some cases, pathogens could exploit these metabolites as environmental signals to enhance its survival and expansion. These findings highlight the importance of understanding the intricate interactions between host and microbiota, and should provide insights in developing microbiota-targeting therapeutics for host physiology, immunity, and pathogen resistance. In Chapter 2, I describe a mechanism by which microbiota-derived short-chain fatty acids inhibit virulence of Salmonella Typhimurium. Short-chain fatty acids can inhibit Salmonella virulence, but the molecular mechanism(s) remain poorly characterized. We use a chemical reporter strategy to identify molecular targets of short-chain fatty acids in Salmonella. I demonstrate that alkynyl-functionalized short-chain fatty acids can be metabolized and covalently attached to proteins in Salmonella. Proteomic analysis reveal that HilA, a key virulence transcription regulator, is short-chain fatty acylated. I employ Amber Suppression Technology and CRISPR-Cas9 genome editing to faithfully mimic butyrylation on endogenous HilA. Biochemical and functional characterization show that acylation of HilA has site-specific effect, and K90 butyrylation affect HilA DNA-binding activity and Salmonella invasion in mice. Overall, our results discover a mechanism by which gut microbiota provides resistance against Salmonella through short-chain fatty acids. In Chapter 3, I describe long-chain fatty acylation of HilA and biochemical characterization of HilA. I find that dietary long-chain fatty acids potently inhibit Salmonella virulence. Chemical proteomics with alkynyl-functionalized long-chain fatty acids reveal proteins that are long-chain fatty acylated in Salmonella, including HilA. Modification by long-chain fatty acids on HilA is post-translationally N-linked. Moreover, with photo-crosslinking unnatural amino acid, we discover that HilA forms homo-oligomers in Salmonella. Our data suggest that dietary long-chain fatty acids may interfere pathogenesis of Salmonella through post-translational modification, and further structural characterization of HilA may reveal novel target for treatment of Salmonella infection. The projects described in this thesis underscore the important roles microbiota and dietary metabolites have played in host immunity and enteric pathogen restriction.

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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