Toxin B is essential for virulence of Clostridium difficile

Clostridium difficile is the leading cause of infectious diarrhea in hospitals worldwide, because of its virulence, spore-forming ability and persistence1,2. C. difficile-associated diseases (CDAD) are induced by antibiotic treatment or disruption of the normal gastrointestinal flora3,4. Recently, morbidity and mortality resulting from CDAD have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns1,2,5,6. Since 2002, epidemic toxinotype III NAP1/027 strains1,2, which produce high levels of the major virulence factors, toxin A and toxin B, have emerged. These toxins have 63% amino acid sequence similarity7 and are members of the large clostridial glucosylating toxin family, which are monoglucosyltransferases that are proinflammatory, cytotoxic and enterotoxic in the human colon8–10. Inside host cells, both toxins catalyze the transfer of glucose onto the Rho family of GTPases, leading to cell death8, 11. However, the role of these toxins in the context of a C. difficile infection is unknown. Here we describe the construction of isogenic tcdA and tcdB mutants of a virulent C. difficile strain and their use in the hamster disease model to show that toxin B is a key virulence determinant. Previous studies showed that purified toxin A alone can induce most of the pathology observed following infection of hamsters with C. difficile8,9, 12 and that toxin B is not toxic in animals unless it is co-administered with toxin A, suggesting that the toxins act synergistically12. Our work provides evidence that toxin B, not toxin A, is essential for virulence, which represents a major paradigm shift. Furthermore, it is clear that the importance of these toxins in the context of infection cannot be predicted exclusively from studies using purified toxins, reinforcing the importance of using the natural infection process to dissect the role of toxins in disease.