Date of Award

3-5-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

School of Biological Sciences

First Advisor

Jan-Ulrik Dahl

Abstract

The objective of this dissertation was to investigate how uropathogenic Escherichia coli (UPEC) responds to and defends one of the most potent oxidants produced in neutrophils, hypochlorous acid (HOCl).While most bacteria have no negative effect on human health or are even beneficial, some pathogenic species can cause serious infections, resulting in increased morbidity and mortality. However, the immune system allows the host to efficiently fight back and eradicate pathogens. Innate immune cells (i.e., neutrophils) generate reactive oxygen and chlorine species (RO/CS) to kill invading pathogens, among which HOCl is considered the most potent and abundant one. HOCl exerts its bactericidal activity primarily through oxidizing amino acid side chains in proteins, leading to widespread protein aggregation and cell death. Surprisingly, little is known about how pathogenic bacteria respond to and defend HOCl stress. Given that bacteria have evolved specific and highly regulated mechanisms that protect them from changes in their environment, a better understanding of bacterial HOCl-stress defense strategies may lead to the development of alternate therapeutic targets in this post-antibiotic era. One bacterial species that experiences HOCl during their course of infection is UPEC, the common etiological agent of urinary tract infections (UTIs). UPEC strains usually reside as harmless commensals in the gut but turn into serious pathogens upon entering the urinary tract, where they ascend from the urethra to the bladder, the primary site of colonization. In the bladder, UPEC encounters increasing levels of HOCl, which is generated by infiltrating neutrophils and dual oxidases that are expressed by bladder epithelial cells. Intriguingly, before I started my graduate training, it was completely unexplored how UPEC responds to and defends HOCl. My work revealed that UPEC is much less sensitive to HOCl compared to other E. coli pathotypes. I therefore hypothesized that UPEC has additional defense systems in place that protect the pathogen from HOCl-stress by increasing cellular integrity against oxidative damage. Transcriptomic analysis of HOCl-stressed UPEC strain CFT073 showed the upregulation of a gene cluster which appears to be controlled by putative transcriptional regulator RcrR. I mechanistically characterized RcrR as a redox sensing transcriptional repressor that uses the oxidation state of conserved cysteine residues to control the expression of rcrARB genes. Importantly, I found that one target gene of RcrR, rcrB, appears to be crucial for HOCl-resistance, as UPEC strains lacking rcrB (ΔrcrB) are more sensitive to HOCl and show defects in survival during phagocytosis by neutrophils. Bioinformatic analyses revealed that RcrB is particularly prevalent among UPEC pathotypes. Using biochemical and molecular analyses, I present evidence that RcrB works as a “sponge” that quenches HOCl and protects cells from oxidative macromolecular damage. Collectively, this dissertation makes an important contribution to our incomplete understanding of how pathogens defend HOCl. My research outlines a novel HOCl-detoxification strategy, which to the best of my knowledge is the first report of this type. This work will allow us to better understand the way how UPEC thrive in HOCl-rich environments, which is relevant to human health, as it may help to explain UPEC’s unique extraintestinal fitness and add information on how the interface between the bacteria and their host is regulated.

Comments

Imported from Sultana_ilstu_0092E_12556.pdf

DOI

https://doi.org/10.30707/ETD2024.20240618063951332952.999920

Page Count

203

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