Cellular uptake of Clostridium botulinum C2 toxin requires oligomerizationand acidification

The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists o f two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a rece ptor on the cell surface and mediates cell entry of C2I via receptor-mediat ed endocytosis. Here we report that binding of C2II to the surface of targe t cells requires cleavage of C2II by trypsin, Trypsin cleavage causes oligo merization of the activated C2II (C2IIa) to give SDS-stable heptameric stru ctures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme compo nent C2I is blocked by bafilomycin bat Plot by brefeldin A or nocodazole, i ndicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells invo lves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cl eaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbo hydrate receptor on the cell surface and assembly with C2I, (iv) receptor-m ediated endocytosis of both C2 components into endosomes, and finally (v) t ranslocation and release of C2I into the cytosol after acidification of the endosomal compartment.