The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

The Gram-positive obligate anaerobeClostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility ofC. difficile . Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the twoC. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genestcdA andtcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. InC. difficile , increasing c-di-GMP levels reduced the expression levels oftcdA andtcdB , as well as that oftcdR , which encodes an alternative sigma factor that activatestcdA andtcdB expression. We hypothesized that theC. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ28 ) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression ofsigD inC. difficile resulted in increased expression levels oftcdR ,tcdA , andtcdB . Furthermore,sigD expression enhanced toxin production and increased the cytopathic effect ofC. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activatestcdR expression and that SigD cannot activatetcdA ortcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes inC. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator inC. difficile .