Clostridium difficile Toxin B Causes Epithelial Cell Necrosis through an Autoprocessing-Independent Mechanism

Abstract

Clostridium difficile is the most common cause of antibiotic-associated nosocomial infection in the United States. C. difficile secretes two homologous toxins, TcdA and TcdB, which are responsible for the symptoms of C. difficile associated disease. The mechanism of toxin action includes an autoprocessing event where a cysteine protease domain (CPD) releases a glucosyltransferase domain (GTD) into the cytosol. The GTD acts to modify and inactivate Rho-family GTPases. The presumed importance of autoprocessing in toxicity, and the apparent specificity of the CPD active site make it, potentially, an attractive target for small molecule drug discovery. In the course of exploring this potential, we have discovered that both wild-type TcdB and TcdB mutants with impaired autoprocessing or glucosyltransferase activities are able to induce rapid, necrotic cell death in HeLa and Caco-2 epithelial cell lines. The concentrations required to induce this phenotype correlate with pathology in a porcine colonic explant model of epithelial damage. We conclude that autoprocessing and GTD release is not required for epithelial cell necrosis and that targeting the autoprocessing activity of TcdB for the development of novel therapeutics will not prevent the colonic tissue damage that occurs in C. difficile – associated disease.

Author Summary

Clostridium difficile is an anaerobic spore-forming bacterium that infects the human colon and causes diarrhea, pseudomembranous colitis, and toxic megacolon. Most people that develop disease symptoms have undergone antibiotic treatment, which alters the normal gut flora and allows C. difficile to flourish. C. difficile secretes two toxins, TcdA and TcdB, that are responsible for the fluid secretion, inflammation, and colonic tissue damage associated with disease. The emergence of hypervirulent strains of C. difficile that are linked to increased morbidity and mortality highlights the need for new therapeutic strategies. One strategy is to inhibit the function of the toxins, thereby decreasing damage to the colon while the patient clears the infection with antibiotics. Toxin function is thought to depend on an autoprocessing event that releases a catalytic ‘effector’ portion of the toxin into the host cell. In the course of trying to identify small molecules that would inhibit such a function, we found that TcdB induces a rapid necrosis in epithelial cells that is not dependent on autoprocessing. The physiological relevance of this observation is confirmed in colonic explants and suggests that inhibiting TcdB autoprocessing will not prevent the colonic tissue damage observed in C. difficile associated diseases.