title: Cellular Uptake of Clostridium botulinum C2 Toxin Requires Acid Sphingomyelinase Activity.
abstract: Clostridium botulinumC2 toxin consists of an enzyme component (C2I) and a binding component (C2II). Activated C2II (C2IIa) binds to a cell receptor, giving rise to lipid raft-dependent oligomerization, and it then assembles with C2I. The whole toxin complex is then endocytosed into the cytosol, resulting in the destruction of the actin cytoskeleton and cell rounding. Here, we showed that C2 toxin requires acid sphingomyelinase (ASMase) activity during internalization. In this study, inhibitors of ASMase and lysosomal exocytosis blocked C2 toxin-induced cell rounding. C2IIa induced Ca2+influx from the extracellular medium to cells. C2 toxin-induced cell rounding was enhanced in the presence of Ca2+ASMase was released extracellularly when cells were incubated with C2IIa in the presence of Ca2+Small interfering RNA (siRNA) knockdown of ASMase reduced C2 toxin-induced cell rounding. ASMase hydrolyzes sphingomyelin to ceramide on the outer leaflet of the membrane at acidic pH. Ceramide was detected in cytoplasmic vesicles containing C2IIa. These results indicated that ASMase activity is necessary for the efficient internalization of C2 toxin into cells. Inhibitors of ASMase may confer protection against infection.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan nagahama@ph.bunri-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Takehara
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan.
authors.forename: Teruhisa
authors.initials: T
authors.lastname: Takagishi
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan.
authors.forename: Soshi
authors.initials: S
authors.lastname: Seike
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Miyamoto
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism* / Q000633:toxicity; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D004285:Dogs; D004705:Endocytosis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism*
chemical_list: D002518:Ceramides; D034741:RNA, Small Interfering; D013108:Sphingomyelin Phosphodiesterase; D001905:Botulinum Toxins; C096324:botulinum toxin type C; D002118:Calcium
keywords: C. botulinum C2 toxin; acid sphingomyelinase; endocytosis