Dominant-negative Inhibitors of the Clostridium perfringens -Toxin*

The Clostridium perfringens -toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the -toxin. Site-specificmutations were introduced into the gene encoding -toxin, and recombinant proteinswere expressed inEscherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in aMDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activitywas attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wildtype -toxin andmutant proteinswere added toMDCKcells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type -toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118Cmutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric poreforming toxins and provide the basis for developing new therapeutics for treating intoxication by -toxin.