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<OL> <LI> Determine how a DNA damage response defect due to targeted disruption of the mouse Atm gene affects the susceptibility of T and B lymphocyte subsets to the cytolethal distending toxin produced by H. hepaticus and C. jejuni. <LI> Compare the disease susceptibility of wild-type Atm+/+, null Atm-/- and heterozygous Atm+/- mice following inoculation with wild-type H. hepaticus and C. jejuni or their respective isogenic toxin-inactivated mutant and complemented strains.
NON-TECHNICAL SUMMARY: Campylobacter jejuni and Escherichia coli are major primary pathogens of livestock and poultry, and collectively responsible for the majority of food-borne and water-borne bacterial intestinal illnesses worldwide. In common with Helicobacter hepaticus, a cause of intestinal infection in mice, these pathogens produce a newly identified toxin known as cytolethal distending toxin. Identification of basic mechanisms of toxicity associated with this toxin in cultured cells in vitro and inbred strains of mice has the potential to provide tools necessary for implementation of improved control strategies for these bacterial diseases, and increase our basic understanding of disease mechanisms for clinically important human and animal bacterial pathogens.
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APPROACH: Cellular susceptibility to cytolethal distending toxin will be evaluated by incubation of single cell suspensions isolated from re-derived H. hepaticus-free Atm+/+, Atm+/-, and Atm-/- mice with reconstituted recombinant toxins obtained from either H. hepaticus or C. jejuni, followed by fluorescent flow cytometric analysis of cell cycle, apoptosis, and histone H2AX phosphorylation parameters. Protein lysates will also be prepared from intoxicated and control lymphocytes that vary in Atm status, and the kinetics and amplitude of DNA damage signaling induced by the toxins will be determined by immunoblotting to measure the phosphorylation and activation of Atm and Chk2, as well as the accumulation of p53, as indicators of signaling in response to DNA damage. The in vitro studies will be extended to assess how infection with toxin-producing bacteria is influenced by DNA damage repair mechanisms in vivo. For these studies, control wild-type Atm+/+ and Atm-defective Atm+/- heterozygous or Atm-/- null mice will be assessed for the presence and severity of lesions over time post challenge-inoculation with wild-type, toxin-mutants, and toxin-complemented toxin-mutants of H. hepaticus and C. jejuni strains.