Date of Award

Spring 5-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Urinary tract infections (UTI) are one of the most common bacterial infections, of which, the majority are caused by uropathogenic Escherichia coli (UPEC). Many bacterial pathogens including UPEC synthesize and secrete chemically diverse metabolites called siderophores, which are classically defined by their ability of bind and deliver iron(III), an essential nutrient, to pathogens during infections. UPEC isolates can express multiple siderophore systems, of which, the virulence-associated siderophore yersiniabactin (Ybt) binds to both iron(III) and copper(II) during urinary tract infections. In this thesis we show that Ybt interacts with multiple physiologic transition metals and acts as a metallophore system to deliver select metal ions to UPEC. We show Ybt transporters promote bacterial fitness during urinary tract infections, consistent with a pathogenic gain-of-function conferred by the Ybt system to UPEC.

Direct detection of both iron(III) and copper(II) complexes with Ybt during UTI led to the hypotheses that Ybt may interact with additional physiologic transition metals and that these complexes are transport substrates for the Ybt import system. Using a mass spectrometry based screen, we identified stable trivalent and divalent metal-Ybt complexes that were predicted to use similar coordination sites as iron(III)-Ybt. We used bacterial genetic and quantitative mass spectrometric approaches to show that these complexes are imported into UPEC by the TonB-dependent outer membrane transporter FyuA. Of the metal-Ybt complexes examined, copper(II)-Ybt was predicted to be structurally unique and was the only complex to not competitively inhibit iron(III)-Ybt transport. These results suggest that Ybt can preferentially transport iron in copper-rich environments where copper(II)-Ybt concentrations may exceed those of iron(III)-Ybt.

While the Ybt transport system has been shown to be critical for several Gram-negative pathogens, its role in UPEC during UTI has been unclear. We used bacterial genetics and quantitative mass spectrometric approaches to show that ATP-binding cassette transporters YbtP and YbtQ are necessary for iron(III)-Ybt dependent growth but not Ybt synthesis in UPEC. Using an experimental murine cystitis model and standard liquid culture conditions, we compared the outcome of isogenic wild type and ybtPQ mutant strains and found UPEC lacking ybtPQ to exhibit significant competitive fitness defect only during high titer infections. These results support a model in which YbtP and Q play an important role in the bladder microenvironment during UPEC pathogenesis.

Combining our findings with metal-Ybt import through FyuA and the importance of YbtP and Q during UPEC infections, we sought to investigate metal-Ybt import through YbtPQ and the fate of intracellular metal-Ybt complexes. Using a combination of bacterial genetics, quantitative mass spectrometry and radiolabeling approaches, we observed that in fyuA and ybtPQ-expressing E. coli, both iron and copper are released from their respective intracellular metal-Ybt complex through reduction, with metal-free apo-Ybt recycled to the extracellular medium. This was in contrast to intracellular gallium(III)-Ybt, which remained stable with no detectable apo-Ybt recycling. These findings are consistent with our model of Ybt as a metallophore system that delivers physiologic transition metals to UPEC. The requirements for iron and copper by UPEC metalloenzymes may therefore be targets for future investigations and therapeutic development.

Together, the work presented in this thesis describes the Ybt system as a multi-level virulence strategy used by UPEC to overcome varying host environments and challenges to metal homeostasis.

Language

English (en)

Chair and Committee

Jeffrey P. Henderson

Committee Members

Peter Burgers, Michael G. Caparon, James Fleckenstein, Jennifer K. Lodge,

Comments

Permanent URL: https://doi.org/10.7936/K7W0947H

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