INVESTIGATING THE MOLECULAR MECHANISM OF ACTION OF Clostridium perfringens ENTEROTOXIN USING STRUCTURE-FUNCTION AND OLIGOMERIC ANALYSES

Clostridium perfringens, a Gram-positive and spore-forming anaerobe, is a significant pathogen of both humans and domestic animals. Among the many toxins produced by this bacterium, the C. perfringens enterotoxin (CPE) is one of the principal contributors to C. perfringens human disease via its role in both foodborne and non-foodborne gastrointestinal illness. Produced in massive quantities during sporulation in the intestine, CPE begins its action by binding to host cells and forming an SDS-sensitive small complex. At physiologic conditions, CPE then associates with additional proteins to form large SDS-resistant complexes in the plasma membrane, the formation of which coincide with membrane permeability alterations of the cell. The two species of large complex have been reported to have molecular masses of ~155 and ~200 kDa, and recent compositional analysis of the complexes has shown that the former complex contains both CPE and claudin, while the latter contains CPE, claudin, and occludin. Prior structure-function analysis of CPE has defined regions at the N- and C-termini involved with cytotoxic and binding activities of the toxin, respectively. Despite the important findings contributed from previous studies of CPE, several significant questions remain regarding the molecular aspects of CPE's mechanism of action. In this thesis dissertation, research is presented aimed to answer three specific structure-function and mechanistic questions about CPE action. Site-directed mutagenesis enabled the identification of two residues in the N-terminal cytotoxicity region of CPE that were crucial for the formation of the CPE large complexes, and likely function in oligomerization of the toxin. In addition, a novel pre-pore step was defined by deletion mutagenesis of a 25 amino acid region of CPE proposed to be involved in membrane insertion. Lastly, CPE was determined to have hexameric stoichiometry in both of the large complexes, prompting a reevaluation of their molecular masses. Several new insights into CPE activity have been gained by the work presented here within, and models for the molecular mechanism of action and structure-function relationships of CPE are updated