Dominant-negative Inhibitors of the Clostridium perfringens ϵ-Toxin

Abstract

The Clostridium perfringens ⑀-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ⑀-toxin. Site-specific mutations were introduced into the gene encoding ⑀-toxin, and recombinant proteins were expressed in Escherichia 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 a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was 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 and mutant proteins were added to MDCK cells, 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/F118C mutant 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.The Clostridium perfringens ⑀-toxin is one of the most potent bacterial toxins (1, 2). The ⑀-toxin can lead to a fatal enterotoxemia characterized by widespread vascular permeability and edema in the heart, lungs, brain, and kidneys (3-6). The disease most frequently affects livestock animals, though the toxin may also affect humans (7-9). Because of its extreme potency and the possibility of intoxicating humans, the C. perfringens ⑀-toxin is considered a select agent by the United States Department of Health and Human Services. A vaccine currently is approved for veterinary use, though multiple immunizations are required to provide long-term immunity (10 -13). There also is an antitoxin approved for veterinary use. However, in the event that an animal exhibits symptoms of intoxication by ⑀-toxin, it is typically too late for the current antitoxin to be effective, and use of the antitoxin is typically limited to prophylactic treatment of unvaccinated animals within a herd (14). There is no treatment currently approved for use in humans. Thus, alternative countermeasures are needed that inhibit the activity of the toxin.One alternative method of countering the cytotoxic activity of bacterial toxins is through dominant-negative inhibitors. Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins a...

Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

“…The propidium iodide influx assay was used to characterize the cytotoxic activity of Etx fusion protein (with GFP) compared to wild type toxin, while the MTT assay was used to study the high dose-dependence of Etx mutants and the effect of reducing agent, as previously described [30]. Values were compared taking 0.5% of triton ×100 incubated cells as 100% mortality and cells incubated with GFP alone as 0% of cell mortality.…”

Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines

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Helicobacter pylori VacA Induces Programmed Necrosis in Gastric Epithelial Cells

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