Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity

Abstract

The vacuolating toxin VacA, a major determinant of Helicobacter pylori-associated gastric diseases, forms anion-selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low-conductance VacA pores in the cell plasma membrane and not to the opening of Ca(2+)- or volume-activated chloride channels. VacA-dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), while2-[(2-cyclopentenyl-6,7dichloro-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA-94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans-epithelial conductivity.

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

“…The presence of similar anionic channels formed by VacA was then detected at the level of the plasma membrane of HeLa cells and found only when the toxin was activated (11,12). There is evidence that links the formation of VacA anionic channel to the toxin-induced vacuolation (2).…”

Association of Helicobacter pylori Vacuolating Toxin (VacA) with Lipid Rafts

“…VacA is known to form anion-selective channels in the plasma membrane of HeLa cells, and the formation of these channels results in partial depolarization of the resting membrane potential (24). To determine whether methyl-␤-cyclodextrin pretreatment of cells inhibited the capacity of VacA to induce depolarization of the plasma membrane, we used bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol as a probe to monitor the membrane potential of HeLa cells.…”

“…Following binding of VacA to the surface of eukaryotic cells, the toxin can insert into the plasma membrane to form anionselective channels (23)(24)(25)(26) and can also be internalized (20,27,28). Intracellular expression of VacA by transient tranfection with VacA-encoding plasmids results in the formation of intracellular vacuoles that are indistinguishable from those that form when VacA is added to the outside of cells (29,30), suggesting that this toxin acts intracellularly.…”

“…It has been proposed that VacA toxicity requires localization of the toxin in either endosomes or mitochondria (28,32). One current model for understanding the cytotoxic effects of VacA proposes that its cell-vacuolating activity results from the formation of anion-selective channels in endosomal membranes (5,(23)(24)(25)(26). Alternatively, it is possible that VacA might have novel intracellular activities distinct from membrane channel formation (5,33,34).…”

Essential Domain of Receptor Tyrosine Phosphatase β (RPTPβ) for Interaction with Helicobacter pylori Vacuolating Cytotoxin

“…It is believed that VacA forms anion-selective channels in endosomal membranes through its interaction with lipid (26,27). Yeast two-hybrid analysis revealed an interaction between VacA and a 54-kDa protein of HeLa cells, consistent with an intracellular target for VacA action (28) and the hypothesis that VacA affects various cellular functions through association with intracellular molecules.…”