title: Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral sphingomyelinase.
abstract: BACKGROUND
  Clostridium perfringens epsilon-toxin is responsible for fatal enterotoxemia in ungulates. The toxin forms a heptamer in the lipid rafts of Madin-Darby Canine Kidney (MDCK) cells, leading to cell death. Here, we showed that epsilon-toxin requires neutral sphingomyelinase (nSMase) activity during oligomerization.
  
  
  METHODS
  We tested the role of nSMase in the oligomerization of epsilon-toxin using specific inhibitors, knockdown of nSMase, formation of ceramide, and localization of epsilon-toxin and ceramide by immunofluorescence staining.
  
  
  RESULTS
  Epsilon-toxin induced the production of ceramide is a dose- and time-dependent manner in ACHN cells. GW4869, an inhibitor of nSMase, inhibited ceramide production induced by the toxin. GW4869 and knockdown of nSMase blocked toxin-induced cell death and oligomer formation of epsilon-toxin. Confocal microscopy images showed that the toxin induced ceramide clustering and colocalized with ceramide.
  
  
  CONCLUSIONS
  These results demonstrated that oligomer formation of epsilon-toxin is facilitated by the production of ceramide through activation of nSMase caused by the toxin.
  
  
  CONCLUSIONS
  Inhibitors of nSMase may confer protection against infection.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima 770-8514, Japan.
authors.forename: Teruhisa
authors.initials: T
authors.lastname: Takagishi
authors.affiliation: Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Oda
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima 770-8514, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Takehara
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima 770-8514, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima 770-8514, Japan. Electronic address: nagahama@ph.bunri-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000633:toxicity; D001597:Benzylidene Compounds / Q000494:pharmacology; D002460:Cell Line; D002518:Ceramides / Q000819:agonists* / Q000096:biosynthesis; D003016:Clostridium perfringens / Q000737:chemistry; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects*; D055503:Protein Multimerization; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D001427:Bacterial Toxins; D001597:Benzylidene Compounds; D002518:Ceramides; C039811:Clostridium perfringens epsilon-toxin; D004791:Enzyme Inhibitors; C468773:GW 4869; D034741:RNA, Small Interfering; D013108:Sphingomyelin Phosphodiesterase
keywords: C. perfringens epsilon-toxin; Ceramide; Neutral sphingomyelinase; Oligomer