Identification of Escherichia coli genes required for bacterial survival and morphological plasticity in urinary tract infections

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Urinary tract infections (UTIs) are the second most common bacterial infectious disease affecting humans, after pneumonia. A range of pathogens have been implicated in causing UTIs, however strains of uropathogenic Escherichia coli (UPEC) are the predominant etiological agents. UPEC originate within the intestine, but have adapted the ability to disseminate and colonise the human urinary tract via a multi-stage intracellular infection cycle within the cells of the bladder. This infection cycle is a complex pathway involving epithelial cell attachment, invasion and intracellular biofilm-like proliferation, leading to the formation of a sub-population of filamentous bacteria. Bacterial filamentation occurs when rod-shaped cells grow without dividing. This filamentation accompanies bacterial dispersal and the rupture of the host bladder cell. UPEC filaments have the potential to revert to bacillary, rod-shaped morphology and can thereafter divide as normal bacterial cells, thereby initiating a new infection cycle. There is also the potential for UPEC to ascend to the kidneys and enter the bloodstream causing urosepsis. The rapid emergence of antibiotic resistance has greatly influenced the severity of UTIs and financial burden on the health-care sector. This further complicates UTI therapies and highlights the urgent need to advance our understanding of the biological mechanisms and requirements underpinning UPEC survival and pathogenesis. The work carried out for this PhD thesis aimed to expand our existing knowledge surrounding UPEC survival and morphological plasticity (cell shape changes). Each Chapter within this thesis focused on distinct, but related aspects of this overarching aim. Firstly, we developed and applied a high-throughput sequencing-based method for the genome-wide identification of genes and genomic DNA fragments that induce filamentation in E. coli. This revealed genes from several prophages, carbon metabolic pathways, as well as endogenous bacterial genes or loci that have known and novel roles in cell division or bacterial filamentation. A large number of short predicted peptides that trigger filamentation were also identified for the first time, which are not expected to have evolved with the purpose of causing filamentation, but could be used as synthetic, artificial inhibitors of cell division. A transposon-insertion mutant library in the uropathogenic E. coli cystitis isolate UTI89 was constructed at a moderate scale. We combined this library with a modified transposon-directed insertion-site sequencing (TraDIS) technique to define the genes required for UPEC growth and survival in M9-glycerol minimal medium compared to a rich LB medium. We identified 60 mutants with a significant fitness defect and reduced capacity to survive in the M9-glycerol, the majority of which encode gluconeogenic and amino acid catabolism proteins. We also highlight novel differences and several apparent discrepancies in the metabolic requirements between uropathogenic and commensal E. coli. Several uncharacterised and UPEC-specific genes were identified that likely underlie metabolic capacities of UPEC strains during infection. Two of these genes, neuC and his were confirmed and verified as important during UPEC infection of human bladder epithelial cells in vitro. Finally, we employed TraDIS to an up-scaled in vitro UTI model to identify the UTI89 genes required for survival at distinct phases of the multi-stage bladder infection cycle, focusing particularly on the later infection events, when bacterial physiology appears to most substantially change. This revealed a total of 143, 333 and 322 statistically significant genes required for the IBC, dispersal and recovery phases of bladder cell infection. We additionally characterised the extent and distribution of UPEC filamentation in the dispersal phase of our up-scaled model and implicate, through TraDIS, known and novel cell division regulators to potentially be involved in the UTI-filamentation response pathway. Further, the catalog of genes identified through our TraDIS experiments provides a foundation for further characterisation of UPEC factors needed for survival, both in laboratory culture conditions and during human bladder infection. The extensive gene functional identification reported in this PhD thesis represents both a substantial resource and some of the first steps towards the understanding of UPEC gene function during infection. This is expected to facilitate the development of treatments for UTIs or other bacterial infections in a future faced with increasing resistance to current antibiotics.
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