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 infection (CDI) is a leading cause of health care-associated diarrhea and has increased in incidence and severity over the last decade. Pathogenesis is mediated by two toxins, TcdA and TcdB, which cause fluid secretion, inflammation, and necrosis of the colonic mucosa. TcdB is a potent cytotoxin capable of inducing enzyme-independent necrosis in both cells and tissue. In this study, we show that TcdB-induced cell death depends on assembly of the host epithelial cell NADPH oxidase (NOX) complex and the production of reactive oxygen species (ROS). Treating cells with siRNAs directed against key components of the NOX complex, chemical inhibitors of NOX function, or molecules that scavenge superoxide or ROS confers protection against toxin challenge. To test the hypothesis that chemical inhibition of TcdB-induced cytotoxicity can protect against TcdB-induced tissue damage, we treated colonic explants with diphenyleneiodonium (DPI), a flavoenzyme inhibitor, or N-acetylcysteine (NAC), an antioxidant. TcdB-induced ROS production in colonic tissue was inhibited with DPI, and both DPI and NAC conferred protection against TcdB-induced tissue damage. The efficacy of DPI and NAC provides proof of concept that chemical attenuation of ROS could serve as a viable strategy for protecting the colonic mucosa of patients with CDI.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

Background: C. difficile TcdA and TcdB glucosylate small GTPases. Results: Structural and functional studies reveal comparable activities with Rho substrates, enhanced activities following autoprocessing, and TcdA-specific modification of Rap2A. Conclusion: TcdA is a potent enzyme and modifies a broader array of GTPase substrates than TcdB. Significance: These findings highlight the importance of autoprocessing for activity and reveal differences in target specificity between the toxins.

The bicomponent leukotoxins produced by Staphylococcus aureus kill host immune cells through osmotic lysis by forming ␤-barrel pores in the host plasma membrane. The current model for bicomponent pore formation proposes that octameric pores, comprised of two separate secreted polypeptides (S and F subunits), are assembled from water-soluble monomers in the extracellular milieu and multimerize on target cell membranes. However, it has yet to be determined if all staphylococcal bicomponent leukotoxin family members exhibit these properties. In this study, we report that leukocidin A/B (LukAB), the most divergent member of the leukotoxin family, exists as a heterodimer in solution rather than two separate monomeric subunits. Notably, this property was found to be associated with enhanced toxin activity. LukAB also differs from the other bicomponent leukotoxins in that the S subunit (LukA) contains 33-and 10-amino-acid extensions at the N and C termini, respectively. Truncation mutagenesis revealed that deletion of the N terminus resulted in a modest increase in LukAB cytotoxicity, whereas the deletion of the C terminus rendered the toxin inactive. Within the C terminus of LukA, we identified a glutamic acid at position 323 that is critical for LukAB cytotoxicity. Furthermore, we discovered that this residue is conserved and required for the interaction between LukAB and its cellular target CD11b. Altogether, these findings provide an in-depth analysis of how LukAB targets neutrophils and identify novel targets suitable for the rational design of anti-LukAB inhibitors.