Cytotoxicity of Clostridium septicum alpha-toxin: its oligomerization in detergent resistant membranes of mammalian cells.
Alpha-toxin is an important agent of the virulence of Clostridium septicum. We examined cytotoxicity for alpha-toxin to various mammalian cells with recombinant toxin fused with a histidine-tag at the amino-terminal. The recombinant toxin retained the activity indistinguishable from the native form. Mammalian nucleated cells examined in this study are more sensitive to the protoxin than to the trypsinized toxin, except RAW 264.7 and P3U1 cells of myeloid lineage. Cellular proteins of various molecular sizes interacted with the toxin. The size and SDS-PAGE pattern of the proteins were different among cell lines but they were liberated from the cells by the treatment with phosphatidylinositol-specific phospholipase C. The toxin appeared to target and utilize detergent resistant membranes (DRMs) for binding and subsequent oligomerization. In discontinuous sucrose density gradient, we demonstrated by immunoblotting that the toxin bound to DRMs contained in L929 cells and caused the oligomer formation. Furthermore, cholesterol depletion with cholesterol-interacting agents reduced toxin oligomerization and lowered cytotoxicity of the toxin towards cells. These results suggest that alpha-toxin preferentially exploits DRMs for oligomerization.
Hang'ombe
Mudenda B
MB
Department of Veterinary Science, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.
Mukamoto
Masafumi
M
Kohda
Tomoko
T
Sugimoto
Nakaba
N
Kozaki
Shunji
S
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D003902:Detergents / Q000494:pharmacology*; D019281:Dimerization; D004285:Dogs; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism / Q000633:toxicity; D047392:beta-Cyclodextrins / Q000494:pharmacology
D001427:Bacterial Toxins; D003902:Detergents; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; C064270:hemolytic toxin, Clostridium septicum; C108732:methyl-beta-cyclodextrin