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Role of Ca2+-binding motif in cytotoxicity induced by Clostridium perfringens iota-toxin
Authors:Kobayashi Keiko  Nagahama Masahiro  Ohkubo Noriko  Kojima Takashi  Shirai Hidenori  Iwamoto Shinobu  Oda Masataka  Sakurai Jun
Affiliation:Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima 7708514, Japan.
Abstract:
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 Ca(2+)-binding motif of Ib in the cytotoxicity of iota-toxin. The cytotoxicity of iota-toxin increased with an increase in the concentration of extracellular Ca(2+). A surface plasmon resonance analysis showed that the binding of Ia to the oligomer of Ib is dependent on the concentration of Ca(2+). However, the addition of Ca(2+) had no effect on the binding of (125)I-labeled Ib to the cells. We replaced Asp-8, -10, and -12 in the Ca(2+)-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 Ca(2+)-binding motif in the N-terminal region of Ib plays a role in the interaction of Ib with Ia in the presence of Ca(2+).
Keywords:Clostridium perfringens   Iota-toxin   Ca2+-binding site   Binding   Internalization
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