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

Abstract

The Gram-positive obligate anaerobe Clostridium 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 of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. 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 genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; 28 ) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.

CodY-Dependent Regulation of Sporulation in Clostridium difficile

“…Toxin gene expression is also regulated by the SigD flagellumspecific sigma factor (53). Toxin production is greatly decreased in the related nonmotile 012 JIR8094 strain due to a significant decrease in sigD expression (53).…”

“…S6 in the supplemental material, the 630 codY mutant consistently displayed lower motility than the corresponding parent strain, but no significant differences in motility between the UK1 and UK1 codY strains were observed (mean final diameters: strain 630⌬erm, 51 Ϯ 1.7 mm; strain 630codY, 42.3 Ϯ 0.7 mm; strain UK1, 48.0 Ϯ 1.2 mm; strain UK1codY, 52.0 Ϯ 2.1 mm). CodY is not known to directly regulate the SigD motility sigma factor or any other known factors that affect motility in C. difficile (37,53). Considering that the growth rate defect of the 630 codY mutant was more pronounced than that of the UK1 codY mutant (Fig.…”

“…Toxin production is greatly decreased in the related nonmotile 012 JIR8094 strain due to a significant decrease in sigD expression (53). Because the toxin and motility phenotypes share some regulatory components and because CodY influences motility in the related organism C. perfringens (36), we asked whether CodY controls motility in the motile 630⌬erm strain (012 ribotype) and UK1 strain (027 ribotype).…”

“…Western blot analyses were performed as previously described (53), with minor modifications. Strains were grown in BHIS medium supplemented with 0.1% taurocholate and 0.5% fructose until the cultures reached mid-log phase.…”

Clostridium difficile Infection

“…The sigma factor SigD, which is associated with the expression of motility genes, promotes toxin gene expression by binding to a SigD-dependent promoter sequence upstream of tcdR 93,94 . The master regulator of sporulation in both Bacillus and Clostridium species, Spo0A, can also regulate toxin production in C. difficile, but only in some strains 95 .…”