Analysis of the Transcriptomics, Physiology, and Invasiveness of Uropathogenic and Non-pathogenic Escherichia Coli


August 2023

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Escherichia coli is a disease-causing species that can be divided into several phylogenetic groups (PGs). The B2 group of the Clermont classification scheme causes up to 65% of E. coli caused urinary tract infections (UTIs), with the remaining 35% caused by the summation of groups A, B1, and D. A common set of virulence genes has not been identified. To determine the features of B2 strains that contribute to virulence, I analyzed 35 strains by comparative RNA sequencing. The strains were non-pathogenic and pathogenic, i.e., isolated from individuals with a UTI, from groups A, B2 and D. I established transcriptomic differences of core gene expression between groups B2 and A/D that involve genes for all aspects of macromolecular synthesis, pathways of energy generation, and environment-sensing transcription factors. I propose that these differences are responsible for the B2 group’s enhanced virulence potential. Virulence requires attachment to and the formation of intracellular bacterial colonies within epithelial cells. I found that five of five uropathogenic and one of three nonpathogenic B2 strains had 25-50 times more intracellular colonies than strains from either groups A or D. My analyses of bacterial appendages and the receptors for bacteria on epithelial cells show multiple entry mechanisms through which E. coli can utilize to invade human urothelium. A UTI involves multiple environments where bacteria must grow, including urine. Analyzing growth in urine can be difficult as urine is a highly unreproducible mixture, so pools of human urine are commonly used to minimize individual variation. I analyzed how the bacteria respond to growth in an individual’s urine and in pools of urine from multiple classes of patients. I identified pooled urine as a highly variable environment that does not necessarily mimic the average growth of urine from individual patients. These results suggest that better urine mimics are needed for future studies and that variations in urinary nutrient content could affect the host- pathogen interaction and the outcome of the infection. In the same study, I also observed that group B2 uropathogens are better at obtaining low levels of nutrients. Genetic analyses of uropathogens are difficult because many are resistant to a common form of genetic exchange mediated by a virus, known as transduction. We developed a method by which uropathogens will accept the transducing phage P1 and subsequently allow for alterations of the pathogenic chromosome. With the use of this method, metabolic mutants were constructed, and the use of the genes were studied in motility, another prominent virulence function in uropathogens. This led to the identification of a mechanism of motility, using the pili appendage, previously thought not to be used by E. coli. The regulation of this motility mechanism and alanine synthesis were also studied in part to help identify novel treatment avenues for UTIs. In summary, I provided evidence that suggests the basis for group B2’s virulence: Group B2 strains have an altered transcriptome, and increased invasiveness, metabolism, and nutrient acquisition capabilities. A new approach to the transduction protocol allows genetic alterations that allows easier genetic analysis of these prominent pathogens.



Biology, Microbiology, Biology, Genetics, Biology, Physiology, Biology, Molecular, Biology, General, Biology, Cell