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Date of Award

Summer 8-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Urinary tract infections (UTIs) are one of the most common bacterial infectious diseases worldwide, and most are caused by uropathogenic Escherichia coli (UPEC). UPEC and many bacterial pathogens synthesize and secrete chemically diverse secondary metabolites called siderophores to acquire iron, an essential nutrient, to survive and proliferate in the face of iron deprivation in human hosts. UPEC isolates may express multiple siderophore systems, almost always including the virulence-associated metallophore system yersiniabactin (Ybt). Disruption of the Ybt biosynthesis is thus a plausible therapeutic anti-virulence strategy. In this dissertation, I will describe results addressing substrate specificity during Ybt biosynthesis, an associated error-correcting function that ensures biosynthetic fidelity and activity, and the potential of therapeutic error incorporation as a means of inhibiting Ybt biosynthesis.Ybt biosynthesis is conducted by a hybrid nonribosomal peptide synthetase / polyketide synthase (NRPS/PKS) system, in which proteins and protein domains are arranged into an enzymatic assembly line that successively incorporates precursor metabolites into an increasingly complex synthetic intermediate. During biosynthesis, these substrates and intermediates are loaded onto the carrier protein (CP) domains of each module through formation of a thioester bond to the phosphopantetheine arm. Incorporation of an incorrect substrate at any step of this linear biosynthetic process can irreversibly inhibit the associated protein, stopping biosynthesis and potentially leading to a build-up of unprocessed intermediates. The genomic locus encoding these NRPS/PKS enzymes avoids this by encoding YbtT, a type II thioesterase (TEII) capable of restoring biosynthesis by removing covalently bound, “dead end” intermediates from CPs. Our structural, biochemical, and cellular studies suggest that YbtT helps maintain biosynthetic enzymes activity through a relatively nonspecific thioesterase activity that removes both nonproductive, inhibitory thioesters as well as canonical, “correct” substrates in growing, metabolically active E. coli. To better understand the specificity of the Ybt biosynthetic pathway for both cysteine and salicylate precursors in vivo, we monitored product generation by cultured UPEC following treatment with a series of substrate analogs. In this study, we evaluated the ability of fourteen L-cysteine analogs and over fifty salicylate analogs to interfere with the Ybt biosynthesis. In addition to product inhibition, we also monitored production of “mutasynthetic” products in which modified salicylates were used as alternative substrates to generate Ybt variants. These studies revealed a narrow structural tolerance for use of salicylate analogs as non-canonical substrates. Some cysteine and salicylate analogs exhibit stronger inhibitory effects on Ybt biosynthesis both with and without “mutasynthetic” incorporation. One such compound is 5-methyl-salicylate, which exhibits “broad-spectrum” Ybt biosynthetic inhibition in a genetically diverse panel of clinical E. coli isolates and also exhibits activity in a mouse model of E. coli urinary tract infection. These results suggest a novel strategy for preventing and treating antibiotic resistant UTIs.

Language

English (en)

Chair and Committee

Jeffrey P. Henderson

Committee Members

Timothy A. Wencewicz, Jeffrey P. Henderson, Thomas J. Brett, Michael L. Gross,

Available for download on Saturday, August 19, 2023

Included in

Biochemistry Commons

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