Receptor-binding domain 2 of Clostridioides difficile binary toxin as a promising vaccine component against C. difficile infection

Clostridioides difficile infection (CDI) is primarily caused by two major protein toxins - toxin A (TcdA) and toxin B (TcdB). A third toxin, C. difficile binary toxin (CDT), is found in about 5-30% of strains and is linked to heightened virulence and severe disease. CDT is composed of an enzymatic component CDTa and a binding and translocation component CDTb, which delivers CDTa into host cells.

CDTb has two receptor-binding domains, RBD1 and RBD2. Recent structural research suggests RBD2 is crucial for the formation and stability of the di-heptameric CDTb assembly necessary for effective cell intoxication.

This study examined the immunogenicity and protective potential of RBD1 and RBD2 utilizing in silico, in vitro, and in vivo methods. Analysis revealed that RBD2 is highly conserved among various CDT-producing C. difficile ribotypes and toxinotypes. Mice immunized with RBD2, but not RBD1, showed effective protection against direct CDT challenge.

Furthermore, RBD2 immunization shielded hamsters from infection by a CDT-only-producing C. difficile strain, DSM 101085 (TcdA-TcdB-CDT). Mechanistically, anti-RBD2 serum, unlike anti-RBD1 serum, effectively neutralized CDT-induced cytotoxicity, as evidenced by inhibition of cell rounding in Vero cells. Overall, these results identify RBD2 as a promising vaccine antigen targeting CDT and provide functional evidence confirming its essential role in CDT-mediated host-cell intoxication.

Including RBD2 in multivalent C. difficile vaccines could enhance protection against virulent, CDT-producing strains.