Regulation of toxin production in Clostridium difficile by CdtR

Clostridium difficile is an important nosocomial and animal pathogen. During the early 2000s, the emergence of ribotype 027 strains responsible for widespread epidemics throughout Europe and North America resulted in the increased incidence and severity of C. difficile infections (CDI). Three toxins are encoded by C. difficile in two genomically distinct loci that are variably present in different strains. Two closely related members of the large glucosylating toxins, Toxin A (TcdA) and Toxin B (TcdB), are encoded within the pathogenicity locus known as PaLoc. TcdB is essential for virulence and causes the majority of severe damage and disease observed during CDI, while TcdA plays a minor but non-essential role in pathogenesis. Some strains of C. difficile including the epidemic 027 isolates also produce binary toxin or CDT, which is encoded in the CDT locus (CdtLoc). Although CDT is not essential for virulence, studies suggest that it may contribute to colonisation during infection by modifying the host intestinal epithelia. Regulation of TcdA and TcdB production is co-ordinated and occurs primarily via TcdR and TcdC, a sigma and anti-sigma system encoded within PaLoc. Several global regulatory genes encoded outside of PaLoc also regulate production of TcdA and TcdB, linking toxin production to sporulation, motility and metabolism. An orphan response regulator, CdtR, encoded within the CdtLoc has previously been shown to regulate CDT production. However, the regulation of CDT production has not been extensively studied in different strain backgrounds, including the epidemic 027 strains. In addition, no regulatory link has been shown between the three toxins encoded in the two separate pathogenicity loci. This thesis describes the first construction of cdtR mutants in two epidemic 027 strains. Analysis of these mutants showed that they produced less CDT than wild type, confirming the importance of CdtR in regulating CDT production for the first time in an 027 strain background. Further phenotypic analysis of these cdtR mutants yielded unexpected results. It was found that the cdtR mutants produced less vii TcdA and TcdB than wild type and these results represent the first time that regulation of the two major toxins has been linked to the regulation of CDT. Importantly, the cdtR mutants were severely attenuated in a mouse infection model, implicating CdtR as an important regulator of toxin production and virulence in the 027 strains. To determine whether CdtR function is conserved across evolutionarily diverse backgrounds, two strains from different ribotypes, a ribotype 078 strain and the historical, ribotype 012 strain 630 were investigated. It was found that CdtR regulates the production of CDT in the ribotype 078 strain but is not important for the regulation of TcdA and TcdB production in either strain background. This data is in accordance with other studies showing that toxin regulation in C. difficile is strain specific. Collectively, this study establishes CdtR as an important virulence regulator in the 027 strains and further highlights the need to investigate regulatory mechanisms of virulence factors in diverse strain backgrounds.