Cytotoxicity of Clostridium septicum alpha-toxin: its oligomerization in detergent resistant membranes of mammalian cells

• Published in: Microbial pathogenesis 2004-12, Vol.37 (6), p.279-286
• Main Authors: Hang'ombe, Mudenda B., Mukamoto, Masafumi, Kohda, Tomoko, Sugimoto, Nakaba, Kozaki, Shunji
• Format: Article
• Language: English
• Subjects: Alpha-toxin; animal pathogenic bacteria; Animals; bacterial toxins; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Bacterial Toxins - toxicity; beta-Cyclodextrins - pharmacology; Biological and medical sciences; Cattle; Cell Line; Cell Membrane - drug effects; Cell Membrane - metabolism; Clostridium septicum; Cricetinae; Cytotoxicity; Detergent-resistant membranes; Detergents - pharmacology; Dimerization; Dogs; Fundamental and applied biological sciences. Psychology; Mice; Microbiology; Oligomerization; Rats; Recombinant Proteins - metabolism; Recombinant Proteins - toxicity
• ISSN: 0882-4010; 1096-1208
• DOI: 10.1016/j.micpath.2004.09.001

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.