Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile

Abstract

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdBHV) was more cytotoxic in vitro than TcdB from historical strains (TcdBHIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdBHV and TcdBHIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdBHV were found to be substantially broader and more pronounced than those caused by TcdBHIST. The increased toxicity of TcdBHV was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdBHIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdBHV undergoes acid-induced conformational changes at a pH much higher than that of TcdBHIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile.

A chimeric protein comprising the glucosyltransferase and cysteine proteinase domains of toxin B and the receptor binding domain of toxin A induces protective immunity againstClostridium difficileinfection in mice and hamsters

“…It was reported that TcdA is relatively well conserved, while TcdB has much variability, 38,39 especially the RBD region. 38 The N terminus encompassing the GTD and CPD domains is more conserved between historical and epidemic strains.…”

“…38 The N terminus encompassing the GTD and CPD domains is more conserved between historical and epidemic strains. In our previous study 37 and consistent with others, 35,36 we indicated that the N-terminus of TcdB was able to elicit a protective antibody response.…”

“…32 Moreover, the N terminus of TcdB is more conserved than its RBD. 38,39 In contrast, the RBD of TcdA has potent adjuvant activity, e.g., it has been reported that a TcdA receptor peptide fragment enhanced mucosal antibody responses in mice when coadministered with heterologous protective antigens 40 . The RBD of TcdA has been used as an immunogen in several studies.…”

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

“…pH-induced conformational changes of TcdB were assessed as described previously (34). Assay plates were read in Spectramax M5 plate reader (Molecular Devices).…”

Clostridium difficile Infection: Clinical Challenges and Management Strategies

“…These include genetic divergence from other strains that enhances cell tropism, mutation in the tcdC gene that may result in a removal of logphase repression of toxin expression, and increased sporulation. [14][15][16] Finally, NAP-1/027 and other hypervirulent strains have high-level resistance to fluoroquinolones. 10 Currently, this strain accounts for more than 35% of all strains recovered from patients with CDIs.…”