title: Detergent-resistant membrane microdomains facilitate Ib oligomer formation and biological activity of Clostridium perfringens iota-toxin.
abstract: Clostridium perfringens iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na(+)/K(+)-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol. Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown. In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100. Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types. The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions. Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation. While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-beta-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity. These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.
authors.affiliation: Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011, USA. martha.hale@amedd.army.mil
authors.forename: Martha L
authors.initials: ML
authors.lastname: Hale
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Marvaud
authors.forename: Michel R
authors.initials: MR
authors.lastname: Popoff
authors.forename: Bradley G
authors.initials: BG
authors.lastname: Stiles
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism* / Q000633:toxicity*; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003016:Clostridium perfringens / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D019281:Dimerization; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects* / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D014709:Vero Cells
chemical_list: D001427:Bacterial Toxins; D003902:Detergents; C050737:iota toxin, Clostridium perfringens; D017830:Octoxynol; D036002:ADP Ribose Transferases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase