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Date of Award

Summer 8-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Secondary metabolites, or natural products, are small molecules produced by bacteria, fungi, marine invertebrates, and plants that are not required for normal growth and development of the organism. Instead, these specialized metabolites possess activities that the organism uses to survive in specific, often harsh, environments. Siderophores are chemically diverse secondary metabolites produced by microorganisms that are capable of acquiring iron. Bacterial pathogens face significant iron deprivation within the human host, and consequently many have evolved the capacity to produce one or more siderophores. Uropathogenic E. coli (UPEC), the most common cause of urinary tract infections (UTIs), is a particularly prolific producer of siderophores, with strains possessing between one and four distinct siderophore systems. These siderophores are produced from common and structurally similar metabolites. Therefore, the biosynthesis of multiple siderophores poses a significant metabolic burden. It is curious why an organism would incur this metabolic burden to produce multiple siderophores, all with the seemingly redundant function of acquiring iron. We hypothesize that the siderophore systems of UPEC are not entirely redundant and that each system has characteristics that make it important in select pathogenic niches during UTI. We also hypothesize that mechanisms exist to maintain the integrity of these biosynthetic pathways in vivo, which would reduce the metabolic burden incurred during siderophore expression. In this dissertation, I will present work revealing how the biosynthetic pathway for the siderophore yersiniabactin (Ybt) has evolved to produce another secondary metabolite, named escherichelin, that plays a role in interspecies competition between UPEC and the uropathogen Pseudomonas aeruginosa. This is yet another example of a unique, non-canonical function of the Ybt siderophore system. I will also present work that elucidates mechanisms of maintaining the integrity of Ybt biosynthesis.

Ybt is produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. Both types of pathways are comprised of large, multidomain enzymes that act like an assembly line to condense precursor building blocks into complex, bioactive molecules. During biosynthesis, the building blocks and intermediates are covalently attached to carrier protein domains (CPs) via a phosphopantetheine cofactor. However, examples are being found of intermediates that are seemingly prematurely released from NRPS and PKS pathways. These molecules may be released because they serve a function for the producing organism. To determine if there are any additional products of the Ybt biosynthetic pathway, we used liquid chromatography-mass spectrometry (LC-MS)-based metabolomics to compare the secreted metabolomes of the model UPEC strain UTI89 and a Ybt-null mutant. This analysis identified a new bacterial product, which we named escherichelin. A previous publication reported that synthetic escherichelin inhibits transport of pyochelin, a virulence-associated siderophore produced by P. aeruginosa. Further investigations revealed that escherichelin is produced during human UTI and during experimental human colonization with a probiotic bacterial interference strain. The production of escherichelin, an anti-virulence compound targeting P. aeruginosa, provides a possible mechanism by which E. coli in the microbiome may help the host suppress UTI caused by pyochelin-producing organisms.

The covalent tethering of intermediates to the Ybt biosynthetic machinery is also significant to consider when incorrect precursor building blocks are mistakenly loaded onto CPs. This may create a stall on the assembly line and inactivate the large NRPS and/or PKS protein. Consequently, NRPS and PKS systems have evolved editing enzymes, called type II thioesterases (TEIIs), to remove these mistakes from CPs. YbtT is a TEII encoded within the Ybt biosynthetic gene cluster that has long been hypothesized to edit the pathway, but had never been characterized. Our biochemical characterization revealed that YbtT is a thioesterase with broad substrate specificity that can remove small molecules from CPs in the pathway. The likelihood of loading a CP with an incorrect precursor, which YbtT may need to remove, is increased when the other NRPS and PKS catalytic domains have relaxed substrate specificities. To understand better the specificity of the Ybt biosynthetic pathway for salicylate in vivo, we performed a mutasynthesis of Ybt. In this study, 26 salicylate analogs were tested for their ability to be incorporated into a variant Ybt by a strain lacking the salicylate synthase. The results revealed that the pathway can incorporate salicylate analogs with small modifications, such as halogens with small atomic radii and methyl groups, but excludes analogs with larger modifications. All together, this work builds on the published in vitro characterization of the Ybt biosynthetic pathway and advances our understanding of how Ybt biosynthesis operates inside a bacterial cell and even inside the human host. These advances may contribute to novel strategies for preventing and treating UTI. Such strategies are necessary as the rising prevalence of antibiotic resistance in Gram-negative uropathogens threatens our ability to treat these infections.

Language

English (en)

Chair and Committee

Jeffrey P. Henderson

Committee Members

Thomas J. Brett, Michael L. Gross, Joseph M. Jez, Audrey R. Odom John,

Comments

Permanent URL: https://doi.org/10.7936/K7JM292S

Available for download on Thursday, July 06, 2119

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