Role of Ca2+-binding motif in cytotoxicity induced by Clostridium perfringens iota-toxin

Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). We investigated the role of the conserved Ca2+-binding motif of Ib in the cytotoxicity of iota-toxin. The cytotoxicity of iota-toxin increased with an increase in the concentration of extracellular Ca2+. A surface plasmon resonance analysis showed that the binding of Ia to the oligomer of Ib is dependent on the concentration of Ca2+. However, the addition of Ca2+ had no effect on the binding of 125I-labeled Ib to the cells. We replaced Asp-8, -10, and -12 in the Ca2+-binding motif of Ib with alanine. D8A, D10A, and D12A bound to the cell and formed an oligomer at about half of the wild-type Ib. The cytotoxicity of Ib variants in the presence of Ia was about 500-fold less than that of wild-type Ib. Immunofluorescence study showed that these variants were internalized in the early endosomes like wild-type Ib. However, wild-type Ib-induced internalization of Ia in the cells, but these variants did not. The result indicates that the conserved Ca2+-binding motif in the N-terminal region of Ib plays a role in the interaction of Ib with Ia in the presence of Ca2+.