Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities

Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1(V)) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1(V) and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1(V) is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1(V) strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdB(NAP1) and TcdB(NAP1V) share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdB(NAP1) induced a stronger proinflammatory response than TcdB(NAP1V) as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.