Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells.
Clostridium perfringens beta toxin is an important agent of necrotic enteritis. Of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin. The toxin induced swelling and lysis of the cell. Treatment of the cells with the toxin resulted in K+ efflux from the cells and Ca2+, Na+, and Cl- influxes. These events reached a maximum just before the cells were lysed by the toxin. Incubation of the cells with the toxin showed the formation of toxin complexes of about 191 and 228 kDa, which were localized in the domains that fulfilled the criteria of lipid rafts. The complex of 228 kDa was observed until 30 min after incubation, and only the complex of 191 kDa was remained after 60 min. Treatment of the cells with methyl-beta-cyclodextrin or cholesterol oxidase blocked binding of the toxin to the rafts and the toxin-induced K+ efflux and swelling. The toxin-induced Ca2+ influx and morphological changes were inhibited by an increase in the hydrodynamic diameter of polyethylene glycols from 200 to 400 and markedly or completely inhibited by polyethylene glycol 600 and 1000. However, these polyethylene glycols had no effect on the toxin-induced K+ efflux. The toxin induced carboxyfluorescein release from phosphatidyl-choline-cholesterol liposomes containing carboxyfluorescein and formed an oligomer with 228 kDa in a dose-dependent manner but did not form an oligomer with the 191-kDa complex. We conclude that the toxin acts on HL 60 cells by binding to lipid rafts and forming a functional oligomer with 228 kDa.
Nagahama
Masahiro
M
Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan.
Hayashi
Shinya
S
Morimitsu
Shinsuke
S
Sakurai
Jun
J
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000494:pharmacology*; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D002713:Chlorine / Q000378:metabolism; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007477:Ions; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010713:Phosphatidylcholines / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014709:Vero Cells
D001427:Bacterial Toxins; C031884:CPB protein, Clostridium perfringens; D007477:Ions; D008081:Liposomes; D010713:Phosphatidylcholines; D011092:Polyethylene Glycols; D002713:Chlorine; D013395:Sucrose; D002784:Cholesterol; D007770:L-Lactate Dehydrogenase; D011188:Potassium; D002118:Calcium