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Infection and Immunity, November 2009, p. 5139-5148, Vol. 77, No. 11
0019-9567/09/$08.00+0     doi:10.1128/IAI.00638-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Binding and Internalization of Clostridium botulinum C2 Toxin{triangledown}

Masahiro Nagahama,1 Tohko Hagiyama,1 Takashi Kojima,1 Kouhei Aoyanagi,1 Chihiro Takahashi,1 Masataka Oda,1 Yoshihiko Sakaguchi,2 Keiji Oguma,2 and Jun Sakurai1*

Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima 770-8514, Japan,1 Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan2

Received 4 June 2009/ Returned for modification 16 July 2009/ Accepted 20 August 2009

Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic component (C2I) and a binding component (C2II). The activated binding component (C2IIa) forms heptamers, and the oligomer with C2I is taken up by receptor-mediated endocytosis. We investigated the binding and internalization of C2IIa in cells. The C2IIa monomer formed oligomers on lipid rafts in membranes of MDCK cells. Methyl-beta-cyclodextrin inhibited the binding of C2IIa and the rounding of the cells induced by C2I plus C2IIa. C2I was localized to the rafts in the presence, but not the absence, of C2IIa. Surface plasmon resonance analysis revealed that C2I bound to the oligomer of C2IIa, but not the monomer of C2IIa. C2I and C2IIa were rapidly internalized in the cells. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited the internalization of C2IIa in the cells and the rounding activity in the presence of C2I plus C2IIa. Incubation of the cells with C2I plus C2IIa resulted in the activation of PI3K and in phosphorylation of phosphoinositide-dependent kinase 1 and protein kinase B/Akt (Akt), but that with C2IIa alone did not. Akt inhibitor X, an Akt phosphorylation inhibitor, inhibited the rounding activity but not the internalization of C2IIa. The results suggest that the binding of C2I to the oligomer of C2IIa on rafts triggers the activation of the PI3K-Akt signaling pathway and, in turn, the initiation of endocytosis.

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan. Phone: 81-088-602-8483. Fax: 81-088-655-3051. E-mail: sakurai{at}ph.bunri-u.ac.jp

{triangledown} Published ahead of print on 31 August 2009.

Editor: J. B. Bliska

Infection and Immunity, November 2009, p. 5139-5148, Vol. 77, No. 11
0019-9567/09/$08.00+0     doi:10.1128/IAI.00638-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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