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RpoS contributes to phagocyte oxidase-mediated stress resistance during urinary tract infection by Escherichia coli CFT073.

ABSTRACT: UNLABELLED:Uropathogenic Escherichia coli (UPEC) is the most common causative agent of community-acquired urinary tract infection (UTI). In order to cause UTI, UPEC must endure stresses ranging from nutrient limitation to host immune components. RpoS (?(S)), the general stress response sigma factor, directs gene expression under a variety of inhibitory conditions. Our study of rpoS in UPEC strain CFT073 began after we discovered an rpoS-frameshift mutation in one of our laboratory stocks of "wild-type" CFT073. We demonstrate that an rpoS-deletion mutation in CFT073 leads to a colonization defect during UTI of CBA/J mice at 48 hours postinfection (hpi). There is no difference between the growth rates of CFT073 and CFT073 rpoS in urine. This indicates that rpoS is needed for replication and survival in the host rather than being needed to address limitations imposed by urine nutrients. Consistent with previous observations in E. coli K-12, CFT073 rpoS is more sensitive to oxidative stress than the wild type. We demonstrate that peroxide levels are elevated in voided urine from CFT073-infected mice compared to urine from mock-infected mice, which supports the notion that oxidative stress is generated by the host in response to UPEC. In mice that lack phagocyte oxidase, the enzyme complex expressed by phagocytes that produces superoxide, the competitive defect of CFT073 rpoS in bladder colonization is lost. These results demonstrate that ?(S) is important for UPEC survival under conditions of phagocyte oxidase-generated stress during UTI. Though ?(S) affects the pathogenesis of other bacterial species, this is the first work that directly implicates ?(S) as important for UPEC pathogenesis. IMPORTANCE:UPEC must cope with a variety of stressful conditions in the urinary tract during infection. RpoS (?(S)), the general stress response sigma factor, is known to direct the expression of many genes under a variety of stressful conditions in laboratory-adapted E. coli K-12. Here, we show that ?(S) is needed by the model UPEC strain CFT073 to cope with oxidative stress provided by phagocytes during infection. These findings represent the first report that implicates ?(S) in the fitness of UPEC during infection and support the idea of the need for a better understanding of the effects of this global regulator of gene expression during UTI.

SUBMITTER: Hryckowian AJ

PROVIDER: S-EPMC3573659 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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RpoS contributes to phagocyte oxidase-mediated stress resistance during urinary tract infection by Escherichia coli CFT073.

Hryckowian Andrew J AJ Welch Rodney A RA

mBio 20130212 1

<h4>Unlabelled</h4>Uropathogenic Escherichia coli (UPEC) is the most common causative agent of community-acquired urinary tract infection (UTI). In order to cause UTI, UPEC must endure stresses ranging from nutrient limitation to host immune components. RpoS (σ(S)), the general stress response sigma factor, directs gene expression under a variety of inhibitory conditions. Our study of rpoS in UPEC strain CFT073 began after we discovered an rpoS-frameshift mutation in one of our laboratory stocks ...[more]

PMID: 23404396

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Project description:Urinary tract infections (UTI), the second most diagnosed infectious disease worldwide, are caused primarily by uropathogenic Escherichia coli (UPEC), placing a significant financial burden on the health care system. High-throughput transposon mutagenesis combined with genome-targeted sequencing is a powerful technique to interrogate genomes for fitness genes. Genome-wide analysis of E. coli requires random libraries of at least 50,000 mutants to achieve 99.99% saturation; however, the traditional murine model of ascending UTI does not permit testing of large mutant pools due to a bottleneck during infection. To address this, an E. coli CFT073 transposon mutant ordered library of 9,216 mutants was created and insertion sites were identified. A single transposon mutant was selected for each gene to assemble a condensed library consisting of 2,913 unique nonessential mutants. Using a modified UTI model in BALB/c mice, we identified 36 genes important for colonizing the bladder, including purB, yihE, and carB Screening of the condensed library in vitro identified yigP and ubiG to be essential for growth in human urine. Additionally, we developed a novel quantitative PCR (qPCR) technique to identify genes with fitness defects within defined subgroups of related genes (e.g., genes encoding fimbriae, toxins, etc.) following UTI. The number of mutants within these subgroups circumvents bottleneck restriction and facilitates validation of multiple mutants to generate individual competitive indices. Collectively, this study investigates the bottleneck effects during UTI, provides two techniques for evading those effects that can be applied to other disease models, and contributes a genetic tool in prototype strain CFT073 to the field.IMPORTANCE Uropathogenic Escherichia coli strains cause most uncomplicated urinary tract infections (UTI), one of the most common infectious diseases worldwide. Random transposon mutagenesis techniques have been utilized to identify essential bacterial genes during infection; however, this has been met with limitations when applied to the murine UTI model. Conventional high-throughput transposon mutagenesis screens are not feasible because of inoculum size restrictions due to a bottleneck during infection. Our study utilizes a condensed ordered transposon library, limiting the number of mutants while maintaining the largest possible genome coverage. Screening of this library in vivo, and in human urine in vitro, identified numerous candidate fitness factors. Additionally, we have developed a novel technique using qPCR to quantify bacterial outputs following infection with small subgroups of transposon mutants. Molecular approaches developed in this study will serve as useful tools to probe in vivo models that are restricted by anatomical, physiological, or genetic bottleneck limitations.

Dataset Information

RpoS contributes to phagocyte oxidase-mediated stress resistance during urinary tract infection by Escherichia coli CFT073.

Publications

RpoS contributes to phagocyte oxidase-mediated stress resistance during urinary tract infection by Escherichia coli CFT073.

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