Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation.
The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.
Centro CNR per lo Studio delle Biomembrane and Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy.
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F
Tombola
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C
Carlesso
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I
Szabò
M
M
de Bernard
J M
JM
Reyrat
J L
JL
Telford
R
R
Rappuoli
C
C
Montecucco
E
E
Papini
M
M
Zoratti
D001426:Bacterial Proteins / Q000737:chemistry / Q000633:toxicity*; D001427:Bacterial Toxins / Q000737:chemistry / Q000633:toxicity*; D055592:Biophysical Phenomena; D001703:Biophysics; D004047:Diethyl Pyrocarbonate; D004553:Electric Conductivity; D005759:Gastroenteritis / Q000209:etiology; D006367:HeLa Cells; D016481:Helicobacter Infections / Q000209:etiology; D016480:Helicobacter pylori / Q000472:pathogenicity*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry / Q000187:drug effects*; D008051:Lipid Bilayers / Q000737:chemistry; D008564:Membrane Potentials; D008954:Models, Biological; D014617:Vacuoles / Q000187:drug effects*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007473:Ion Channels; D008051:Lipid Bilayers; C074689:VacA protein, Helicobacter pylori; D004047:Diethyl Pyrocarbonate