Strains of Ex traintestinal P athogenic Escherichia c oli (ExPEC) exhibit an array of virulence strategies and are a major cause of urinary tract infections, sepsis and meningitis. Efforts to understand ExPEC pathogenesis are challenged by the high degree of genetic and phenotypic variation that exists among isolates. Determining which virulence traits are widespread and which are strain-specific will greatly benefit the design of more effective therapies. Towards this goal, we utilized a quantitative genetic footprinting technique known as transposon insertion sequencing (Tn-seq) in conjunction with comparative pathogenomics to functionally dissect the genetic repertoire of a reference ExPEC isolate. Using Tn-seq and high-throughput zebrafish infection models, we tracked changes in the abundance of ExPEC variants within saturated transposon mutant libraries following selection within distinct host niches. Nine hundred and seventy bacterial genes (18% of the genome) were found to promote pathogen fitness in either a niche-dependent or independent manner. To identify genes with the highest therapeutic and diagnostic potential, a novel T rait E nrichment A nalysis (TEA) algorithm was developed to ascertain the phylogenetic distribution of candidate genes. TEA revealed that a significant portion of the 970 genes identified by Tn-seq have homologues more often contained within the genomes of ExPEC and other known pathogens, which, as suggested by the first axiom of molecular Koch’s postulates, is considered to be a key feature of true virulence determinants. Three of these Tn-seq-derived pathogen-associated genes—a transcriptional repressor, a putative metalloendopeptidase toxin and a hypothetical DNA binding protein—were deleted and shown to independently affect ExPEC fitness in zebrafish and mouse models of infection. Together, the approaches and observations reported herein provide a resource for future pathogenomics-based research and highlight the diversity of factors required by a single ExPEC isolate to survive within varying host environments. Author Summary Antibiotic resistance is an increasingly serious problem, especially among pathogenic strains of Escherichia coli that cause urinary tract infections, sepsis and meningitis. It is important to obtain a more comprehensive genome-wide understanding of bacterial virulence because it has the potential to uncover novel and alternative therapeutic targets. Therefore, we probed the genome of a pathogenic E. coli isolate using transposon mutagenesis, deep sequencing and comparative pathogenomics in an effort to define its virulence gene repertoire. Using this multilayered approach in combination with high-throughput zebrafish infection models, we identified hundreds of genes that affect pathogen fitness during localized and/or blood-borne infections. We also developed a bioinformatics-based method to systematically sift through our datasets for genes that are broadly conserved among an assortment of pathogenic species. Follow-up analysis of several pathogen-associated candidate genes using zebrafish and mouse infection models highlighted the capacity of our approach to identify novel fitness determinants. The results from this study are available via an interactive online data viewer ( http://pathogenomics.path.utah.edu/F11_TnSeq/ ) so that investigators can more effectively search and utilize these findings.
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