Doctor of Philosophy

dissertationEscherichia coli is genetically and phenotypically diverse. Comparisons made between isolates reveal that they typically differ by up to 20-30% of their respective gene contents. A subgroup of E. coli, designated extraintestinal pathogenic E. coli (ExPEC), is responsible for eliciting a variety of diseases in a range of animal hosts including urinary tract infection, bacteremia, and meningitis. Understanding the biological significance of the intraspecific diversity that exists between ExPEC isolates and modeling the complex microenvironments they encounter is an important and challenging task. I present here, the development of an embryonic zebrafish infection model that recapitulates aspects of both tissue-localized and systemic infections. ExPEC require many of the same virulence determinants to grow and replicate within the zebrafish as they do when colonizing other vertebrate organisms. Using the high-throughput capacity of this surrogate host, I was able to conduct head-to-head genetic and phenotypic comparisons between multiple ExPEC strains. I found that ExPEC exhibit a gamut of distinct, virulence phenotypes and capacities, which proved to be both dependent and independent of bacterial genotype. Together with molecular and biochemical characterization of α-hemolysin and NeaT-two genes that underlie my observations-these findings provide evidence that ExPEC are utilizing, and possibly evolving, unique gene repertoires to occupy iv similar pathogenic niches. Finally, I carried out a transposon mutagenesis selection screen known as TnSeq to identify genes required for pathogen fitness during zebrafish infection. Using deep sequencing, TnSeq facilitates the tracking of specific insertion variants within bulk mutant pools. In this way, determining the fitness changes that result from chromosome-wide gene disruptions is possible. To parse broadly conserved candidate genes that may serve as bona fide virulence determinants, I developed a custom trait enrichment analysis (TEA). In total, this body of work fosters appreciation for the biological impact of strain-tostrain uniqueness and develops methods to elucidate the multigenic basis of ExPEC's pathogenic potential