For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

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.

No abstract