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J. Biol. Chem., Vol. 279, Issue 10, 9481-9489, March 5, 2004

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Glycosylphosphatidylinositol-anchored Proteins and Actin Cytoskeleton Modulate Chloride Transport by Channels Formed by the Helicobacter pylori Vacuolating Cytotoxin VacA in HeLa Cells^*

Nils C. Gauthier, Vittorio Ricci¶, Pierre Gounon||, Anne Doye, Michel Tauc**, Philippe Poujeol**, and Patrice Boquet

From the INSERM U 452, IFR 50, Faculté de Médecine 28 Avenue de Valombrose, 06107 Nice, France, the ^¶Department of Experimental Medicine, Human Physiology Section, University of Pavia, 27100 Pavia, Italy, the ^||Centre Commun de Microscopie Appliquée, Université de Nice, 06108 Nice, France, and ^**CNRS UMR 65-48, Faculté des Sciences, Parc Valrose, 06108 Nice, France

The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells (Ricci V., Galmiche, A., Doye, A., Necchi, V., Solcia, E., and Boquet, P. (2000) Mol. Biol. Cell 11, 3897-3909). Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level. Removal of GPI-Ps from HeLa cell surfaces did not impair VacA localization into lipid rafts but strongly reduced VacA channel-mediated cell influx and efflux of chloride. Disruption of the actin cytoskeleton of HeLa cells by cytochalasin D did not affect VacA localization in lipid rafts but blocked VacA cell internalization and inhibited cell vacuolation while increasing the overall chloride transport by the toxin channel at the cell surface. Specific enlargement of Rab7-positive compartments induced by VacA could be mimicked by the weak base chloroquine alone, and the vacuolating activities of either chloroquine alone or VacA were blocked with the same potency by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid shown to inhibit VacA channel activity (Tombola, F., Oregna, F., Brutsche, S., Szabò, I., Del Giudice, G., Rappuoli, R., Montecucco, C., Papini, E., and Zoratti, M. (1999) FEBS Lett. 460, 221-225). We suggest that formation of functional VacA channels at the cell surface required GPI-Ps and that endocytosis of these channels by an actin-dependent process increases the chloride content of late endosomes that accumulate weak bases, provoking their enlargement by osmotic swelling.

Received for publication, November 3, 2003 , and in revised form, December 11, 2003.

^* This work was supported by INSERM (Paris, France). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental videos.

Submitted in partial fulfillment of the requirements for a Ph.D. degree (Université de Nice Sophia-Antipolis, France). Supported by a Ph.D. grant from the Ministère de la Recherche et de la Technologie.

To whom correspondence should be addressed. Tel.: 33-4-9353-1755; Fax: 33-4-9353-3509; E-mail: boquet{at}unice.fr.

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