Clostridium perfringens iota-toxin: Mapping of receptor binding and ia docking domains on Ib

Clostridium perfringens iota-toxin is a binary toxin consisting of iota a ( Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fus ion proteins of recombinant Ib and truncated variants were tested for bindi ng to Vero cells and docking with Ia via fluorescence-activated cytometry a nd cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were cr itical for cell surface binding, and truncated Ib variants containing great er than or equal to 200 amino acids of the C terminus were effective Ib com petitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an eff ective competitor of iota cytotoxicity. Further studies showed that Ib lack ing just the N-terminal 27 residues did not facilitate Ia entry into a targ et cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent ass ay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal eff ects of iota-toxin in mice.