Journal
PLOS PATHOGENS
Volume 18, Issue 2, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.ppat.1010323
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Funding
- National Institutes of Health (NIH)
- United States Department of Veterans Affairs (VA)
- NIH [T32 DK007673, AI957555]
- VA grant [VA BX002943]
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Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. This study investigates the role of two protein toxins, TcdA and TcdB, produced by the pathogen in causing disease. The study finds that both toxins have an additive role in disease pathogenesis and produce both glucosyltransferase-dependent and independent phenotypes. These findings will contribute to the development of toxin-based therapeutics for CDI.
Author summaryClostridioides difficile is an anaerobic spore-forming bacterium that is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in the USA. This pathogen produces two protein toxins, TcdA and TcdB, that enter host colon cells to cause inflammation, fluid secretion, and cell death. The enzymatic activity of TcdB is a target for novel C. difficile infection (CDI) therapeutics since it is considered the major factor in causing severe CDI. However, necrotic cell death due to non-enzymatic TcdB-host interactions has been reported in cell culture and colonic explant experiments. Here, we generated C. difficile mutant strains with enzyme-inactive toxins to evaluate each toxin's role in an animal model of CDI. We observe an additive role for TcdA and TcdB in disease, and both glucosyltransferase-dependent and independent phenotypes. These findings are expected to inform the development of toxin-based CDI therapeutics. Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.
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