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Date of Award
Doctor of Philosophy (PhD)
ABSTRACT OF THE DISSERTATIONVirulence Genes and Iron Acquisition Pathways in Escherichia coli in Catheter-Associated Urinary Tract Infections by Zongsen Zou Doctor of Philosophy in Energy, Environmental and Chemical Engineering McKelvey School of Engineering Washington University in St. Louis, 2021 Professor Jeffrey P. Henderson, Chair
The field of urinary tract infections (UTI), as well as many other infectious diseases, is moving into a post-antibiotic era with increasing antimicrobial resistance and fewer antibiotic options, which leads to more treatment failures, and complicates the medical decision-making and health care. With the motivation, also an unequivocal public health imperative, to develop truly new drugs and therapies to prevent persistent and recurrent UTI, this dissertation investigates the virulence genes and biofilm formation that contribute to catheter-associated UTI (CAUTI), as well as the biochemical network including multiple enterobactin-homologous siderophores and derived products for mediating bacterial iron uptake, in the context of uropathogenic Escherichia coli (UPEC)
This dissertation spans the fields of engineering, microbiology, biochemistry and clinical medicine and employs diverse tools during the investigation, e.g., engineered continuous flow model for biofilm characterization, genomic approaches such as genetic modification, whole genome sequencing, and comparative genomic analysis, metabolomic approaches such as untargeted profiling and targeted quantitative analysis using mass spectrometry, plus the comparative metabolomic analysis, mathematical computations in R and Python programming, and a wide spectrum of molecular biology and analytical chemistry assays.
Urinary tract infections (UTI) are among the most common bacterial infections worldwide, and catheter-associated UTI (CAUTI) with patients carrying implanted catheters in the urinary tract are the most common UTI in hospitalized patients. Escherichia coli is the most common urinary isolate in CAUTI patients, among which the increasing antibiotic resistance and virulence may lead to severe clinical outcomes. To identify E. coli virulence genes contributing to the occurrence of symptomatic CAUTI, we collected 41 clinical isolates from three diagnostic cohorts, including catheter-associated urinary tract infection (CAUTI), catheter-associated asymptomatic bacteriuria (CAASB), and rectal colonization (RC). We next assembled and analyzed their whole genome sequences in computational comparison analysis for targeted virulence genes identification. A group of genes and gene clusters, including the aerobactin siderophore iron acquisition system (iucD), secreted autotransporter toxin (sat) , and iron-regulated adhesin (iha), as well as two flagella related islands (flg and fli), were associated with CAUTI pathogenesis , serving as the new candidate targets for antimicrobial drug development.
Curiously, biofilm formation on catheter surface has been identified with both cons and pros in chronically catheterized patients, by acting as a source of persistent infections but also preventing colonization by more virulent or more antibiotic resistant strains, an interaction termed “bacterial interference”. To refine and advance our knowledge about the CAUTI-biofilm’s role in CAUTI pathogenesis, we examined the catheter biofilm formation in a broader spectrum of clinical UPEC strains using an ex vivo continuous flow system, with the results demonstrating widespread capacity for catheter biofilm colonization among clinical E. coli strains. Comparative genomic analysis revealed that prominent among the biofilm-associated genes was the ferric citrate uptake (fec) system, which was expressed during biofilm gowth and independently associated with biofilm formation. Moreover, to explore the possible further application of bacterial interference therapy in future clinical trials, we next examined the efficacy of employing low-virulence CAASB biofilm pre-colonizing the catheter surface to prevent the infection by high-pathogenicity CAUTI strains in the continuous flow bacterial competition model. We found that biofilm formation by some CAASB isolates could inhibit catheter colonization by CAUTI strains, suggesting that elimination of competing CAASB strains with lower pathogenic potential in catheterized patients may paradoxically increase the CAUTI risks. This reminded us again of the utmost importance of antimicrobial stewardship and the need to develop alternative treatment approaches to UTIs.
Prominent among various UPEC virulence factors are multiple iron-responsive pathways, such as the siderophore iron acquisition systems, which facilitate bacterial iron uptake during bladder infections and involved in diverse pathogenesis-associated functions. Catecholate siderophores, enterobactin and salmochelin, have been identified to play important roles in many bacterial functions related to UPEC pathogenesis. In addition, inspired by the development of one new antibiotic drug, cefiderocol, which possesses an iron-binding catechol moiety to facilitate its sneaking through the siderophore-iron import channel in a “Trojan horse” for fast and efficient inhibition, we sought to investigate the catecholate siderophores iron acquisition pathway in UPEC model strain UTI89 to advance our understanding and gain more novel insights on this key topic. Using comparative metabolomics, we first detected multiple iron-regulated products related to siderophore enterobactin, that dominated the iron-responsive extracellular metabolome in low iron cultures of UTI89. In addition to observing the canonical enterobactin and salmochelin siderophores, we also detected a group of structurally associated, lower molecular weight catechol metabolites, but with unknown biological origins and functions. We further investigated the enterobactin-associated (ent) sub-metabolome using siderophore import-deficient UTI89∆tonB mutant. In this mutant the short-length ent catechols were substantially attenuated, which favored a catabolic source of these products resulting from the import and incomplete hydrolysis of large enterobactin and salmochelin siderophores. In addition, we next purified small ent catechols and characterized their iron-sequestration functions, with the results revealing that they can mediate iron uptake to support bacterial growth under iron deprivation, similar to enterobactin and salmochelin. Moreover, we also discovered that full glucosylation of catechol moieties in the ent compounds might diminish its iron-binding ability, resulting in the dysfunction of iron sequestration. Finally, isotope-labelling experiments revealed that complete enterobactin hydrolysis product, DHB, might be recycled by the E. coli cells for subsequent rounds of enterobactin biosynthesis. Consequently, our findings reveal a biochemical network of catecholate compounds representing both anabolic and catabolic products. Some of these products may mediate iron uptake in UPEC bacteria, an interaction that may suggest applications for developing new drugs and therapies to treat or prevent UTI.
In summary, the studies in this dissertation identify a group of CAUTI-associated virulence genes as new antimicrobial targets, demonstrates the efficacy of employing catheter-biofilm in bacterial interference therapy to prevent recurrent CAUTI, and resolves the catecholate iron uptake pathway managed by a biochemical network including the biosynthesis, sequestration, hydrolysis and recycling of enterobactin-homologous molecules. It is worth noting that four iron acquisition pathways, including aerobactin iron-sequestration (iucD), ferric citrate transport (fec), enterobactin iron-sequestration (ent), and salmochelin iron-sequestration (sal) were identified for their involvement in diverse UPEC pathogenesis-associated functions. For example, aerobactin iron-sequestration (iucD) was identified with contribution to E. coli uropathogenesity in CAUTI, also operated cooperatively with ferric citrate transport (fec) system to enhance catheter-biofilm formation. To determine the key iron-regulated functions in UPEC bacteria, we identified another two siderophore systems, enterobactin and salmochelin, as the dominant bacterial response to assist its survival under low iron stress. Together, these results suggested the essential importance of scavenging critical iron nutrient in many colonization and infection mechanisms in UPEC pathogens. Consequently, these findings contribute novel insights and directions for developing new management strategies and pharmacotherapies to prevent and treat UTIs.
Jeffrey P. Henderson
Henderson P. Henderson, Pratim Biswas, Margaret A. Olsen, Yinjie Tang,
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