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Inactivation of Clostridium difficile cytotoxin by the neutrophil myeloperoxidase system
Abstract:
The cytotoxin of Clostridium difficile was examined for sensitivity to oxidant secretory products of neutrophils. Exposure to myeloperoxidase, H2O2, and a halide resulted in loss of toxin activity measured by tissue-culture cytotoxicity. The peroxide requirement was provided by reagent H2O2, a peroxide-generating enzyme (glucose oxidase), or a peroxide-producing intestinal microorganism, Lactobacillus acidophilus. Human neutrophils stimulated by phorbol myristate acetate caused similar toxin inactivation. In both the cell-free and the neutrophil systems, inactivation of toxin required halides and was abrogated by azide, cyanide, or catalase. Neutrophils from patients with lack of myeloperoxidase or failure to produce H2O2 were impaired in toxin inactivation unless myeloperoxidase or H2O2, respectively, was added. The reducing agent 2-mercaptoethanol enhanced toxin activity. These data suggest a similarity between C difficile cytotoxin and the classic thiol-activated cytolysins. Moreover, they raise the possibility that neutrophils are involved in oxidative detoxification of microbial products.
Insights
Neutrophils inactivate Clostridium difficile cytotoxin using oxidative mechanisms involving myeloperoxidase and hydrogen peroxide (H2O2). This finding suggests neutrophils play a role in detoxifying microbial toxins.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Clostridium difficile is a significant cause of infectious diarrhea.
- Neutrophils produce potent antimicrobial oxidants.
- The susceptibility of C. difficile cytotoxin to neutrophil oxidants is unknown.
Purpose of the Study:
- To investigate the sensitivity of C. difficile cytotoxin to neutrophil-derived oxidants.
- To elucidate the mechanisms of toxin inactivation by neutrophils.
Main Methods:
- Assessing cytotoxin activity using tissue-culture cytotoxicity assays.
- Exposing the toxin to cell-free myeloperoxidase, hydrogen peroxide (H2O2), and halide systems.
- Utilizing stimulated human neutrophils to inactivate the toxin.
- Evaluating the role of specific inhibitors and enzymes (azide, cyanide, catalase, glucose oxidase).
Main Results:
- Myeloperoxidase, H2O2, and halide exposure inactivated C. difficile cytotoxin.
- Stimulated human neutrophils also inactivated the toxin via a similar oxidative mechanism.
- Toxin inactivation was dependent on halides and inhibited by azide, cyanide, and catalase.
- Neutrophils deficient in myeloperoxidase or H2O2 production showed impaired inactivation, which was restored by adding these components.
- 2-mercaptoethanol enhanced toxin activity, suggesting a thiol-dependent mechanism.
Conclusions:
- C. difficile cytotoxin is susceptible to oxidative inactivation by neutrophil secretory products.
- The inactivation mechanism involves myeloperoxidase, H2O2, and halides, characteristic of neutrophil antimicrobial activity.
- These findings suggest a potential role for neutrophils in the innate immune defense against C. difficile infection through oxidative detoxification of its cytotoxin.