Date Published: July 14, 2016
Publisher: Public Library of Science
Author(s): Shelley A. Lyon, Melanie L. Hutton, Julian I. Rood, Jackie K. Cheung, Dena Lyras, Steven R. Blanke.
Clostridium difficile is a global health burden and the leading cause of antibiotic-associated diarrhoea worldwide, causing severe gastrointestinal disease and death. Three well characterised toxins are encoded by this bacterium in two genetic loci, specifically, TcdB (toxin B) and TcdA (toxin A) in the Pathogenicity Locus (PaLoc) and binary toxin (CDT) in the genomically distinct CDT locus (CdtLoc). Toxin production is controlled by regulators specific to each locus. The orphan response regulator, CdtR, encoded within the CdtLoc, up-regulates CDT production. Until now there has been no suggestion that CdtR influences TcdA and TcdB production since it is not carried by all PaLoc-containing strains and CdtLoc is not linked genetically to PaLoc. Here we show that, in addition to CDT, CdtR regulates TcdA and TcdB production but that this effect is strain dependent. Of clinical relevance, CdtR increased the production of TcdA, TcdB and CDT in two epidemic ribotype 027 human strains, modulating their virulence in a mouse infection model. Strains traditionally from animal lineages, notably ribotype 078 strains, are increasingly being isolated from humans and their genetic and phenotypic analysis is critical for future studies on this important pathogen. Here we show that CdtR-mediated toxin regulation did not occur in other strain backgrounds, including a ribotype 078 animal strain. The finding that toxin gene regulation is strain dependent highlights the regulatory diversity between C. difficile isolates and the importance of studying virulence regulation in diverse lineages and clinically relevant strains. Our work provides the first evidence that TcdA, TcdB and CDT production is linked by a common regulatory mechanism and that CdtR may act as a global regulator of virulence in epidemic 027 strains.
C. difficile antibiotic-associated diarrhoea is a toxin mediated disease [1,2]. During infection, TcdA, TcdB and CDT are secreted into the colonic epithelium by this bacterium, leading to diarrhoea that can progress to serious, life threatening inflammatory diseases, including pseudomembranous colitis and toxic megacolon . The production of these toxins varies between strains. TcdB is the most commonly encoded toxin and is most often co-located with the TcdA gene in the PaLoc region , both toxins act as monoglucosyltransferases that irreversibly modify Rho family members . PaLoc variants that produce TcdB and not TcdA are, however, becoming increasingly common, for example, they represented 23% of strains in one recent study of human strains in China . CDT is encoded in a specific locus, CdtLoc (Fig 1)  the carriage of which has also increased significantly over the last decade; in 2004 6% of clinical isolates encoded CDT whereas 33.5% now encode this toxin [7,8]. CDT is an ADP-ribosyltransferase that is not essential for disease, but may be important for colonisation during an infection . CdtLoc is not carried by all PaLoc-containing strains and it is not linked genetically to PaLoc.
The emergence of epidemic ribotype 027 strains over a decade ago prompted several investigations of the genetic and phenotypic characteristics that may have led to the global dominance of these strains. These features may include higher sporulation rates, resistance to key antibiotics and unique aspects of toxin regulation [11,12,29–31]. The presence of a full length CdtLoc was also initially considered to be important in this regard because it encodes CDT [21,23], however, despite numerous studies, the importance of this toxin in virulence remains undefined [2,32,33]. The results of the work presented here suggest that CdtLoc, and specifically CdtR, may play an indirect but significant role in disease pathogenesis of the ribotype 027 strains by regulating TcdA and TcdB production.