Clostridium perfringens enterotoxin (CPE) is a single polypeptide chain of 35,000 Mr. CPE has been shown to produce all symptoms associated with C. perfringens food poisoning and recent studies strongly suggest that CPE may also be involved in other forms of serious gastrointestinal disease. CPE has a unique action distinguishable from all other enterotoxins. CPE produces changes in membrane permeability which lead to secondary effects, such as inhibition of macromolecular synthesis. The principal goal of the proposed research is to extend understanding of the action of CPE to the biochemical/molecular level. To accomplish this goal the following research is proposed; 1) Identification and purification of the CPE receptor by use of receptor purification, antibody reactivity studies and CPE: CPE receptor crosslinking. 2) Insertion of CPE into intestinal brush border membranes (PFMs) will be further investigated using photoreactive membrane probes and protease- release studies. The importance of insertion in CPE action will be determined. 3) The action of CPE will be investigated to determine if CPE has any action beyond insertion. This will be determined by low- temperature studies, proteoliposome experiments and experiments with colonic cells (these cells bind but do not respond to CPE). 4) The intact CPE gene will be cloned and DNA sequenced. The cloned gene and gene fragments will be used as a probe in Southern or Northern blotting experiments to study CPE synthesis and regulation. 5) Defined mutations will be engineered into the CPE gene or gene fragments and, after expression, the effects of these mutations on CPE activities, particularly on binding, will be determined to help dissect steps in CPE action. These studies should significantly increase our understanding of how CPE acts at the molecular level. Increased knowledge of CPE action should allow valuable comparisons of CPE action with pathways used by other agents producing intestinal disease. As the membrane action of CPE is clarified, CPE should also become a useful probe to examine structure/function relationships in intestinal BBMs. These findings would have long-term significance to numerous medical disciplines seeking to understand intestinal physiology under normal and disease conditions. Additionally, these studies may contribute to improved diagnostic assays for CPE and possibly lead to development of a CPE vaccine.
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