: Clostridium perfringens enterotoxin (CPE) is responsible for the gastrointestinal (Gl) symptoms of C. perfringens type A food poisoning, the 3rd most common foodborne illness in the USA, as well as certain nonfoodborne human Gl diseases. The long-term objective of this project is to identify strategies to prevent or control CPE-associated Gl disease by understanding the pathogenesis of these illness via a focus on CPE's unique mechanism of action. To progress towards this goal, the next grant period will pursue the following aims: 1) further evaluating CPE interactions with claudin receptors by Western blotting electroeluted proteins from CPE complexes, using claudin-1 or -4 transfectants to distinguish whether CPE interacts with claudins from the same or apposing claudin fibrils, using site-directed mutagenesis to identify specific claudin regions/amino acids that bind CPE, and evaluating by immunohistochemistry/SDS-PAGE whether CPE interacts with claudins in vivo, 2) continued analysis of post-binding steps in CPE action by mass spectroscopy analyses of proteins in CPE complexes, determining via SDS-PAGE how many CPE proteins are present in each complex, testing for CPE internalization into host cells by immunofluorescent microscopy, and conducting Western blot/ELISA evaluations of the contribution of lipid rafts, activated calpain, or inflammation to CPE action in vivo and in vitro, 3) additional study of the CPE structure function relationship using site-directed mutagenesis/fluorescent spectroscopy techniques to identify specific CPE amino acids involved in receptor binding and CPE insertion into membranes, and x-ray diffraction to determine the 3-D structure of the CPE protein, and 4) further analyzing the molecular pathogenesis of CPE-positive C. perfringens type A isolates by using PCR, microarrays and Southern blots to study the diversity of the cpe plasmid and by evaluating the contribution of beta2 toxin to the pathogenesis of CPE-associated nonfoodborne Gl disease by constructing isogenic beta2 toxin mutants.
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