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J. Biol. Chem., Vol. 277, Issue 37, 34642-34650, September 13, 2002
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From the A variety of extracellular ligands and pathogens
interact with raft domains in the plasma membrane of eukaryotic cells.
In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by
which Helicobacter pylori vacuolating toxin (VacA)
intoxicates cells. We first investigated whether GPI-anchored proteins
are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in
production of GPI-anchored proteins. Whereas wild-type and mutant cells
differed markedly in susceptibility to aerolysin (a bacterial toxin
that binds to GPI-anchored proteins), they were equally susceptible to
VacA. We next determined whether VacA physically associates with lipid
rafts. CHO or HeLa cells were incubated with VacA, and
Triton-insoluble membranes then were separated by
sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of
cell-associated toxin was associated with detergent-resistant membranes
(DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for
VacA cytotoxicity. Treatment of cells with methyl-
Association of Helicobacter pylori Vacuolating Toxin
(VacA) with Lipid Rafts*
§,§¶,,
, and¶**
Departments of Medicine and
¶ Microbiology and Immunology, Vanderbilt University School of
Medicine and ** Veterans Affairs Medical Center,
Nashville, Tennessee 37232 and the Department of Genetics and
Microbiology, University of Geneva, CH-1211 Geneva 4, Switzerland
-cyclodextrin (a
cholesterol-depleting agent) did not inhibit VacA-induced
depolarization of the plasma membrane, but interfered with the
internalization or intracellular localization of VacA and inhibited the
capacity of the toxin to induce cell vacuolation. Treatment of cells
with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the
absence of GPI-anchored proteins and suggest that association of the
toxin with lipid rafts is important for VacA cytotoxicity.
*
This work was supported in part by National Institutes of
Health Grants DK53623 and AI39657 and by the Medical Research Service of the Department of Veterans Affairs.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of
Infectious Diseases, A3310 MCN, Vanderbilt University School of
Medicine, Nashville, TN 37232. Tel.: 615-322-2035; Fax: 615-343-6160;
E-mail: COVERTL@ctrvax.vanderbilt.edu.
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