Mechanisms of Dysbiosis by Attaching and Effacing Pathogens

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DESCRIPTION (provided by applicant): Enteropathogenic E. coli (EPEC) are common causes of gastrointestinal illness in very young children in developing nations. These and the related murine pathogen, Citrobacter rodentium, are classified as attaching and effacing (A/E) bacteria because upon ingestion, they intimately attach to and cause effacement of host intestinal cells. A/E bacteria induce shifts in the bacteria groups typically present naturally in the gastrointestinl tract, resulting in dysbiosis and disease. While the molecular basis of A/E bacteria-induced pathology is well studied, the mechanisms that enable these pathogens to successfully compete for resources and drive dysbiosis are not well understood. Additionally, the fitness benefit of intimate adherence to host cells through formation of pedestal-like structures, a feature unique to A/E pathogens, is currently unknown. The long-term objectives of this study are to define the mechanisms that result in pathogen-induced intestinal dysbiosis. Specifically, we aim to 1) illustrate the role of respiration to outgrowth of A/E pathogens during infection and 2) define the mechanisms that govern the fitness benefit provided by C. rodentium intimate attachment to host cells. Our hypothesis states that close adherence to epithelial cells provides A/E pathogens with access to oxygen for respiration, therefore allowing for more efficient energy generation over fermenting commensal microbes. To address our hypothesis, C. rodentium strains will be constructed with disruptions in key respiratory enzymes (bd oxidase and formate dehydrogenase) and attachment genes (pedestal-inducing effector protein EspH) and characterized in vitro with a combination of growth assays, tissue culture assays, and confocal microscopy. Mice will then be orally gavaged with single or mixed inocula for competitive infections to test the benefit derived from respiration and attachment in vivo. Germ-free mouse infections will then allow us to isolate interactions between pathogen and specific members of the microbiota. Quantitative PCR on host and pathogen genes will provide information on the levels of cytokine expression as part of the host response as well as a gauge to judge the metabolic state of C. rodentium under different experimental conditions. Completion of this study will provide valuable information on the mechanisms that result in pathogen-induced gut dysbiosis and a strong foundation on which to develop future strategies to prevent A/E pathogen transmission and disease.