Date Published: December 6, 2012
Publisher: Public Library of Science
Author(s): Nicole M. Chumbler, Melissa A. Farrow, Lynne A. Lapierre, Jeffrey L. Franklin, David Haslam, James R. Goldenring, D. Borden Lacy, Steven R. Blanke.
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.
Clostridium difficile is a gram-positive, spore-forming anaerobe that infects the colon and causes a range of gastrointestinal disorders including diarrhea, pseudomembranous colitis, and toxic megacolon , . This is a major healthcare concern as the number and severity of C. difficile-associated disease (CDAD) cases have increased dramatically in recent years . Two large toxins, TcdA and TcdB (308 kDa and 270 kDa, respectively), are recognized as the main virulence factors of C. difficile, . The C-terminal portion of these toxins is responsible for delivering an N-terminal glucosyltransferase domain (GTD) into the host cell , . The GTD inactivates Rho family GTPases including Rho, Rac1, and Cdc42 , .
TcdB is a multi-functional protein with a central role in CDAD pathogenesis. Our goal at the outset of this study was to conduct a screen for small molecule inhibitors that could aid in the dissection of the TcdB mechanism and the generation of new leads for therapeutic intervention. Our strategy was to combine a cell-based phenotypic screen with target-specific secondary assays. In the course of setting up our screening assays, we made two unexpected observations that warranted further investigation.