title: Clostridium botulinum C2 toxin is internalized by clathrin- and Rho-dependent mechanisms.
abstract: Clostridium botulinum C2 toxin is an ADP-ribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft-dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant-negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA-mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin-independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho-mediated uptake of C2 toxin. In conclusion, C2 toxin is endocytosed by dynamin-dependent mechanisms and we provide evidence for involvement of clathrin and Rho.
authors.affiliation: Centre for Cancer Biomedicine and Institute for Cancer Research, Department of Biochemistry, Oslo University Hospital and University of Oslo, 0316 Oslo, Norway.
authors.forename: Sascha
authors.initials: S
authors.lastname: Pust
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
authors.forename: Kirsten
authors.initials: K
authors.lastname: Sandvig
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D001905:Botulinum Toxins / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D003014:Clostridium botulinum / Q000472:pathogenicity*; D034281:Dynamins / Q000172:deficiency / Q000378:metabolism*; D004705:Endocytosis; D017353:Gene Deletion; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D008565:Membrane Proteins; C106354:flotillins; D001905:Botulinum Toxins; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020741:rho GTP-Binding Proteins; D034281:Dynamins; C096324:botulinum toxin type C