title: Clostridium perfringens epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes.
abstract: Clostridium perfringens epsilon-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled epsilon-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-beta-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When epsilon-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Miyata
authors.forename: Junzaburo
authors.initials: J
authors.lastname: Minami
authors.forename: Eiji
authors.initials: E
authors.lastname: Tamai
authors.forename: Osamu
authors.initials: O
authors.lastname: Matsushita
authors.forename: Seiko
authors.initials: S
authors.lastname: Shimamoto
authors.forename: Akinobu
authors.initials: A
authors.lastname: Okabe
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D002498:Centrifugation; D002784:Cholesterol / Q000378:metabolism; D003016:Clostridium perfringens / Q000378:metabolism*; D003505:Cyclodextrins / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology*; D017830:Octoxynol / Q000494:pharmacology; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism*; D013997:Time Factors; D047392:beta-Cyclodextrins
chemical_list: D001427:Bacterial Toxins; C039811:Clostridium perfringens epsilon-toxin; D003505:Cyclodextrins; D003902:Detergents; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C032676:aerolysin; D013395:Sucrose; D017830:Octoxynol; D002784:Cholesterol