Molecular Methods to Study Transcriptional Regulation of Clostridium difficile Toxin Genes

Toxin A (TcdA) and Toxin B (TcdB) are the major virulence factors that contribute to the pathogenesis of Clostridium difficile -associated diarrhoea (CDAD). These enterotoxins act by glucosylation of members of the Rho protein family of small GTP-binding proteins. This leads to the disorganization of the host cell actin cytoskeleton (cytopathic effect) and apoptosis (cytotoxic effect). Due to their glucosyltransferase activity, they are referred as “clostridial glucosylating toxins”. The severe form of CDAD has been recently correlated to the levels of toxin production. This reinforces the idea that regulation of toxin production is an important part of the C. difficile infection. Genes encoding TcdA (tcdA ) and TcdB (tcdB ) are present in a pathogenicity locus (PaLoc) that also includes three accessory genes: tcdR , tcdE and tcdC . TcdR is an alternative RNA polymerase sigma factor that positively regulates toxin gene transcription as well as its own. TcdE has high homologies with bacteriophage holin proteins. TcdC negatively regulates toxin synthesis by interfering with the RNA polymerase formed with TcdR. Therefore, TcdR and TcdC constitute specific regulators of toxin gene transcription thereby tightly regulating toxin synthesis. In addition a variety of environmental signals, such as the presence of carbon sources or amino acids in the growth medium, and temperature also regulate toxin synthesis.