Abstract
Strains of Extraintestinal Pathogenic 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 Trait Enrichment Analysis (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.
Highlights
Within the Escherichia coli lineage there are several distinct virulent subgroups that are principally classified by an ability to cause a common set of diseases
Delineation of the genetic elements utilized by Extraintestinal Pathogenic E. coli (ExPEC) to infect such a diverse spectrum of host niches and cause disease promises to advance our understanding of pathogen evolution and behavior while highlighting more effective strategies to combat these pervasive opportunistic pathogens
With mutant pools derived from the ExPEC reference isolate F11 and multiniche zebrafish infection models, we found that,18% of the F11 genome significantly affected bacterial fitness under the pathogenic conditions tested
Summary
Within the Escherichia coli lineage there are several distinct virulent subgroups that are principally classified by an ability to cause a common set of diseases. Extraintestinal Pathogenic E. coli (ExPEC), is typically thought to be a benign inhabitant of the lower intestinal tract of warmblooded vertebrates. Outside this niche, ExPEC strains have the ability to persist in an array of secondary host-associated habitats where they can cause urinary tract infections (UTIs), meningitis, bacteremia and sepsis in both humans and domesticated animals [1,2,3,4]. Further complicating the search for ExPEC-associated traits is the 30– 40% of genes within their genomes that still require functional annotation [7] This problem will likely persist as genome sequencing continues to outstrip rates of experimental characterization.
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