title: Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells.
abstract: 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. 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. 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.
authors.affiliation: INSERM U 452, IFR 50, Faculté de Médecine 28 Avenue de Valombrose, 06107 Nice, France.
authors.forename: Nils C
authors.initials: NC
authors.lastname: Gauthier
authors.forename: Vittorio
authors.initials: V
authors.lastname: Ricci
authors.forename: Pierre
authors.initials: P
authors.lastname: Gounon
authors.forename: Anne
authors.initials: A
authors.lastname: Doye
authors.forename: Michel
authors.initials: M
authors.lastname: Tauc
authors.forename: Philippe
authors.initials: P
authors.lastname: Poujeol
authors.forename: Patrice
authors.initials: P
authors.lastname: Boquet
mesh_terms: D000199:Actins / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology*; D018408:Patch-Clamp Techniques; D014617:Vacuoles / Q000502:physiology
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D002712:Chlorides; D017261:Glycosylphosphatidylinositols; D007473:Ion Channels; D008565:Membrane Proteins; C074689:VacA protein, Helicobacter pylori