Research Article: Genome-Wide Detection of Fitness Genes in Uropathogenic Escherichia coli during Systemic Infection

Date Published: December 5, 2013

Publisher: Public Library of Science

Author(s): Sargurunathan Subashchandrabose, Sara N. Smith, Rachel R. Spurbeck, Monica M. Kole, Harry L. T. Mobley, Andrew Camilli.

http://doi.org/10.1371/journal.ppat.1003788

Abstract

Uropathogenic Escherichia coli (UPEC) is a leading etiological agent of bacteremia in humans. Virulence mechanisms of UPEC in the context of urinary tract infections have been subjected to extensive research. However, understanding of the fitness mechanisms used by UPEC during bacteremia and systemic infection is limited. A forward genetic screen was utilized to detect transposon insertion mutants with fitness defects during colonization of mouse spleens. An inoculum comprised of 360,000 transposon mutants in the UPEC strain CFT073, cultured from the blood of a patient with pyelonephritis, was used to inoculate mice intravenously. Transposon insertion sites in the inoculum (input) and bacteria colonizing the spleen (output) were identified using high-throughput sequencing of transposon-chromosome junctions. Using frequencies of representation of each insertion mutant in the input and output samples, 242 candidate fitness genes were identified. Co-infection experiments with each of 11 defined mutants and the wild-type strain demonstrated that 82% (9 of 11) of the tested candidate fitness genes were required for optimal fitness in a mouse model of systemic infection. Genes involved in biosynthesis of poly-N-acetyl glucosamine (pgaABCD), major and minor pilin of a type IV pilus (c2394 and c2395), oligopeptide uptake periplasmic-binding protein (oppA), sensitive to antimicrobial peptides (sapABCDF), putative outer membrane receptor (yddB), zinc metallopeptidase (pqqL), a shikimate pathway gene (c1220) and autotransporter serine proteases (pic and vat) were further characterized. Here, we report the first genome-wide identification of genes that contribute to fitness in UPEC during systemic infection in a mammalian host. These fitness factors may represent targets for developing novel therapeutics against UPEC.

Partial Text

Uropathogenic Escherichia coli (UPEC), one of the most common bacterial pathogens infecting humans, is the primary etiological agent of urinary tract infections (UTI) in otherwise healthy individuals [1]. UPEC is a subset of extraintestinal pathogenic E. coli (ExPEC), which causes a broad spectrum of conditions including colibacillosis in poultry, and UTIs, bacteremia, and neonatal meningitis in humans [2]. A subset of patients with UTI develops pyelonephritis and is at risk for developing bacteremia that may result in life threatening sepsis. UTI is the source of E. coli in >70% of both young and elderly patients with bloodstream infections [3], [4]. E. coli strains isolated from the bloodstream are becoming increasingly resistant to trimethoprim/sulfamethoxazole and ciprofloxacin, two first line antibiotics used to treat bacterial UTIs [5]. Despite the prevalence of these infections and potential difficulties in treatment, little is known about the fitness and virulence mechanisms employed by E. coli to establish a systemic infection.

Uropathogenic Escherichia coli (UPEC) is a major cause of bacteremia in humans, yet, there is limited understanding of the fitness mechanisms used by this important pathogen during bacteremia and systemic infection. Here, we describe screening transposon mutants of E. coli CFT073 in a murine model of systemic disseminated infection and identifying 242 candidate fitness genes. Specific mutations were introduced in 11 candidate fitness genes and the contribution of the following nine gene or gene clusters in fitness was confirmed: pgaABCD (biosynthesis and export of poly-N-acetyl glucosamine), c2394-95 (major and minor pilin of type IV pilus two), oppA (oligopeptide uptake periplasmic-binding protein), sapABCDF (sensitive to antimicrobial peptide), yddB (putative outer membrane receptor), pqqL (zinc metallopeptidase), c1220 (a shikimate pathway gene), and pic and vat (autotransporter serine proteases). 82% of the specific mutants in representative candidate fitness genes were significantly outcompeted by the wild-type strain, validating the TraDIS approach in our murine model of systemic infection.

 

Source:

http://doi.org/10.1371/journal.ppat.1003788

 

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