Clostridium perfringens ε-Toxin Forms a Heptameric Pore within the Detergent-insoluble Microdomains of Madin-Darby Canine Kidney Cells and Rat Synaptosomes

Shigeru Miyata,Junzaburo Minami,Eiji Tamai,Osamu Matsushita,Seiko Shimamoto,Akinobu Okabe

doi:10.1074/jbc.m206731200

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.

Highlights

Most mammalian cells have at least two types of discrete lipid microdomains, lipid rafts and caveolae
Association of ET Heptamers with detergent-resistant membranes (DRMs)—To assess the possible association of ET heptamerization with the DRMs of Madin-Darby canine kidney (MDCK) cell and rat synaptosomal membranes, we first examined the distribution of ET heptamers in the detergent-soluble and detergent-insoluble fractions after TX-100 extraction at low temperature
The SDS-resistant ET heptamer formed in MDCK cell membranes was found almost exclusively in the precipitate after treatment with 1% TX-100 at 4 °C followed by centrifugation, there being very little in the supernatant (Fig. 1A, lanes 2 and 3)

Summary

Introduction

Most mammalian cells have at least two types of discrete lipid microdomains, lipid rafts and caveolae. When MDCK cells and synaptosomal membranes were incubated with the toxin and 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. cholesterol depletion by methyl-␤-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells.

Results

Conclusion