Biochemical and Immunological Characterizations of the Receptor Binding Domain of C. difficile Toxin B

Abstract

Clostridium difficile (Cd) is an emerging nosocomial pathogen responsible for antibiotic-associated pseudomembranous colitis and diarrhea in hospital acquired infections. Clostridial toxins A (TcdA) and B (TcdB) which specifically bind to unknown glycoprotein(s) on the surface of epithelial cells disrupt the intestinal barrier and ultimately lead to acute inflammation and diarrhea. There is still debate as to whether the receptor binding domains (RBD) of toxins can individually elicit protection in the hamster challenge model. In this study, a TcdB RBD which was derived from C. difficile strain VPI10463 with >95% amino acid sequence identity to hyper-virulent strain BI/ NAP1/027 was designed and expressed in Escherichia coli. Recombinant RBD (rRBD) was purified, characterized biologically and immunologically and found to have the following properties: (a) capable of binding to the cell surface of both Vero and Caco-2 cells and entering into the cytosol; (b) devoid of hemagglutinin activity (HA); (c) the ability to up-regulate cell surface markers expressions and cytokines secretions from dendritic cells; (d) eliciting anti-TcdB neutralizing antibody responses that could weakly cross-neutralize TcdA in the absence of adjuvant; (e) and inducing weak protection against a lethal dose of Cd spores in the hamster challenge model. Therefore, rRBD shows potential as an immunogen to be included in the development of vaccines against Clostridium difficileassociated diseases.

Recombinant lipoprotein-based vaccine candidates against C. difficile infections

“…The circular dichroism (CD) secondary structure analysis of rlipoA-RBD was also performed and found that rlipoA-RBD had correctly folded to form β-sheet structure similar to A-rRBD (>43 %) [ 30 ]. This result is consistent with other reports that RBD forms stable folded β-solenoid secondary structures independently of other functional domains in the TcdA [ 30 , 31 ]. Although a simple and rapid method for producing rlipoA-RBD with high purity was successfully developed, rlipoA-RBD was found to be unstable and showed a loss in biological function during the freeze-thaw process.…”

“…We have previously reported that both A-rRBD and B-rRBD at 0.8 −1 μM have strong abilities to up-regulate cell surface marker expression and cytokine secretion from BMDCs [ 30 , 31 ]. To enhance the effectiveness of A-rRBD as a recombinant subunit vaccine candidate against CDI, A-rRBD was rationally designed and lipidated (rlipoA-RBD) to contain a toll-like receptor 2 agonist (intrinsic adjuvant) [ 32 ].…”

“…The purification of A-rRBD and B-rRBD expressed in E. coli JM109 (DE3) strain have been previously described [ 30 , 31 ]. All purification steps were analyzed by 8 % SDS-PAGE.…”

“…The anti-TcdA neutralization assay was performed according to the protocol previously described by Huang et al [ 31 ]. Briefly, Vero cells (2 × 10 4 per well) were seeded into 96-well plates containing VP-SFM culture medium (Invitrogen, Carlsbad, CA) and 4 mM glutamine at 37 °C, and allowed to grow to confluent.…”

“…The results indicated that F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, but they do not express the same level of biological properties. In another study [ 31 ], a TcdB RBD derived from C. difficile strain VPI10463 which has >95 % amino acid sequence identity to BI/NAP1/027 hyper-virulent strains was designed and expressed in E. coli . Recombinant TcdB RBD (B-rRBD) was purified, characterized biologically and immunologically, and found to have the following properties: (a) capable of binding to the cell surface of both Vero and Caco-2 cells and entering into the cytosol; (b) showing no hemagglutinin activity (HA); (c) functioning as a toll-like receptor agonist activating dendritic cell maturation; (d) in the absence of adjuvant, eliciting anti-TcdB neutralizing antibody responses that could weakly cross-neutralize TcdA; and (e) inducing partial protection against a lethal dose of C. difficile spores in the hamsters challenge model.…”