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.

Bacteria have a tendency to attach to surfaces and grow as structured communities called biofilms. Chronic biofilm infections are a problem because they tend to resist antibiotic treatment and are difficult to eradicate. Bacterial biofilms have an extracellular matrix that is usually composed of a mixture of polysaccharides, proteins, and nucleic acids. This matrix has long been assumed to play a passive structural and protective role for resident biofilm cells. Here we show that this view is an oversimplification and that the biofilm matrix can play an active role in stimulating its own synthesis. Working with the model biofilm bacterium Pseudomonas aeruginosa , we found that Psl, a major biofilm matrix polysaccharide for this species, acts as a signal to stimulate two diguanylate cyclases, SiaD and SadC, to produce the intracellular secondary messenger molecule c-di-GMP. Elevated intracellular concentrations of c-di-GMP then lead to the increased production of Psl and other components of the biofilm. This mechanism represents a unique positive feedback regulatory circuit, where the expression of an extracellular polysaccharide promotes biofilm growth in a manner analogous to autocrine signaling in eukaryotes.

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