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. 1999 Mar;76(3):1401–1409. doi: 10.1016/S0006-3495(99)77301-7

Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation.

F Tombola 1, C Carlesso 1, I Szabò 1, M de Bernard 1, J M Reyrat 1, J L Telford 1, R Rappuoli 1, C Montecucco 1, E Papini 1, M Zoratti 1
PMCID: PMC1300118  PMID: 10049322

Abstract

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.

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Selected References

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  1. Blaser M. J. Helicobacter pylori: microbiology of a 'slow' bacterial infection. Trends Microbiol. 1993 Oct;1(7):255–260. doi: 10.1016/0966-842x(93)90047-u. [DOI] [PubMed] [Google Scholar]
  2. Burns B. P., Hazell S. L., Mendz G. L. Acetyl-CoA carboxylase activity in Helicobacter pylori and the requirement of increased CO2 for growth. Microbiology. 1995 Dec;141(Pt 12):3113–3118. doi: 10.1099/13500872-141-12-3113. [DOI] [PubMed] [Google Scholar]
  3. Copass M., Grandi G., Rappuoli R. Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker. Infect Immun. 1997 May;65(5):1949–1952. doi: 10.1128/iai.65.5.1949-1952.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cover T. L., Blaser M. J. Purification and characterization of the vacuolating toxin from Helicobacter pylori. J Biol Chem. 1992 May 25;267(15):10570–10575. [PubMed] [Google Scholar]
  5. Cover T. L., Hanson P. I., Heuser J. E. Acid-induced dissociation of VacA, the Helicobacter pylori vacuolating cytotoxin, reveals its pattern of assembly. J Cell Biol. 1997 Aug 25;138(4):759–769. doi: 10.1083/jcb.138.4.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cover T. L., Reddy L. Y., Blaser M. J. Effects of ATPase inhibitors on the response of HeLa cells to Helicobacter pylori vacuolating toxin. Infect Immun. 1993 Apr;61(4):1427–1431. doi: 10.1128/iai.61.4.1427-1431.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cover T. L. The vacuolating cytotoxin of Helicobacter pylori. Mol Microbiol. 1996 Apr;20(2):241–246. doi: 10.1111/j.1365-2958.1996.tb02612.x. [DOI] [PubMed] [Google Scholar]
  8. Eisenman G., Horn R. Ionic selectivity revisited: the role of kinetic and equilibrium processes in ion permeation through channels. J Membr Biol. 1983;76(3):197–225. doi: 10.1007/BF01870364. [DOI] [PubMed] [Google Scholar]
  9. Galloway C. J., Dean G. E., Marsh M., Rudnick G., Mellman I. Acidification of macrophage and fibroblast endocytic vesicles in vitro. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3334–3338. doi: 10.1073/pnas.80.11.3334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garner J. A., Cover T. L. Binding and internalization of the Helicobacter pylori vacuolating cytotoxin by epithelial cells. Infect Immun. 1996 Oct;64(10):4197–4203. doi: 10.1128/iai.64.10.4197-4203.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HODGKIN A. L., KATZ B. The effect of sodium ions on the electrical activity of giant axon of the squid. J Physiol. 1949 Mar 1;108(1):37–77. doi: 10.1113/jphysiol.1949.sp004310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Leunk R. D., Johnson P. T., David B. C., Kraft W. G., Morgan D. R. Cytotoxic activity in broth-culture filtrates of Campylobacter pylori. J Med Microbiol. 1988 Jun;26(2):93–99. doi: 10.1099/00222615-26-2-93. [DOI] [PubMed] [Google Scholar]
  13. Lewis C. A. Ion-concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction. J Physiol. 1979 Jan;286:417–445. doi: 10.1113/jphysiol.1979.sp012629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Manetti R., Massari P., Burroni D., de Bernard M., Marchini A., Olivieri R., Papini E., Montecucco C., Rappuoli R., Telford J. L. Helicobacter pylori cytotoxin: importance of native conformation for induction of neutralizing antibodies. Infect Immun. 1995 Nov;63(11):4476–4480. doi: 10.1128/iai.63.11.4476-4480.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mendz G. L., Hazell S. L., van Gorkom L. Pyruvate metabolism in Helicobacter pylori. Arch Microbiol. 1994;162(3):187–192. doi: 10.1007/BF00314473. [DOI] [PubMed] [Google Scholar]
  16. Menestrina G., Schiavo G., Montecucco C. Molecular mechanisms of action of bacterial protein toxins. Mol Aspects Med. 1994;15(2):79–193. doi: 10.1016/0098-2997(94)90043-4. [DOI] [PubMed] [Google Scholar]
  17. Molinari M., Galli C., Norais N., Telford J. L., Rappuoli R., Luzio J. P., Montecucco C. Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers. J Biol Chem. 1997 Oct 3;272(40):25339–25344. doi: 10.1074/jbc.272.40.25339. [DOI] [PubMed] [Google Scholar]
  18. Molinari M., Galli C., de Bernard M., Norais N., Ruysschaert J. M., Rappuoli R., Montecucco C. The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies. Biochem Biophys Res Commun. 1998 Jul 20;248(2):334–340. doi: 10.1006/bbrc.1998.8808. [DOI] [PubMed] [Google Scholar]
  19. Molinari M., Salio M., Galli C., Norais N., Rappuoli R., Lanzavecchia A., Montecucco C. Selective inhibition of Ii-dependent antigen presentation by Helicobacter pylori toxin VacA. J Exp Med. 1998 Jan 5;187(1):135–140. doi: 10.1084/jem.187.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moll G., Papini E., Colonna R., Burroni D., Telford J., Rappuoli R., Montecucco C. Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori cytotoxin. Eur J Biochem. 1995 Dec 15;234(3):947–952. doi: 10.1111/j.1432-1033.1995.947_a.x. [DOI] [PubMed] [Google Scholar]
  21. Montecucco C., Papini E., Schiavo G., Padovan E., Rossetto O. Ion channel and membrane translocation of diphtheria toxin. FEMS Microbiol Immunol. 1992 Sep;5(1-3):101–111. doi: 10.1111/j.1574-6968.1992.tb05892.x. [DOI] [PubMed] [Google Scholar]
  22. Morré D. J., Minnifield N., Mollenhauer H. H. Kinetics of monensin-induced swelling of Golgi apparatus cisternae of H-2 hepatoma cells. Eur J Cell Biol. 1985 May;37:107–110. [PubMed] [Google Scholar]
  23. Nelson N. Organellar proton-ATPases. Curr Opin Cell Biol. 1992 Aug;4(4):654–660. doi: 10.1016/0955-0674(92)90086-r. [DOI] [PubMed] [Google Scholar]
  24. Papini E., Bugnoli M., De Bernard M., Figura N., Rappuoli R., Montecucco C. Bafilomycin A1 inhibits Helicobacter pylori-induced vacuolization of HeLa cells. Mol Microbiol. 1993 Jan;7(2):323–327. doi: 10.1111/j.1365-2958.1993.tb01123.x. [DOI] [PubMed] [Google Scholar]
  25. Papini E., Satin B., Bucci C., de Bernard M., Telford J. L., Manetti R., Rappuoli R., Zerial M., Montecucco C. The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin. EMBO J. 1997 Jan 2;16(1):15–24. doi: 10.1093/emboj/16.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Papini E., Satin B., Norais N., de Bernard M., Telford J. L., Rappuoli R., Montecucco C. Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin. J Clin Invest. 1998 Aug 15;102(4):813–820. doi: 10.1172/JCI2764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Papini E., de Bernard M., Milia E., Bugnoli M., Zerial M., Rappuoli R., Montecucco C. Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9720–9724. doi: 10.1073/pnas.91.21.9720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Parsonnet J., Hansen S., Rodriguez L., Gelb A. B., Warnke R. A., Jellum E., Orentreich N., Vogelman J. H., Friedman G. D. Helicobacter pylori infection and gastric lymphoma. N Engl J Med. 1994 May 5;330(18):1267–1271. doi: 10.1056/NEJM199405053301803. [DOI] [PubMed] [Google Scholar]
  29. Reyrat J. M., Charrel M., Pagliaccia C., Burroni D., Lupetti P., de Bernard M., Ji X., Norais N., Papini E., Dallai R. Characterisation of a monoclonal antibody and its use to purify the cytotoxin of Helicobacter pylori. FEMS Microbiol Lett. 1998 Aug 1;165(1):79–84. doi: 10.1111/j.1574-6968.1998.tb13130.x. [DOI] [PubMed] [Google Scholar]
  30. Ricci V., Sommi P., Fiocca R., Romano M., Solcia E., Ventura U. Helicobacter pylori vacuolating toxin accumulates within the endosomal-vacuolar compartment of cultured gastric cells and potentiates the vacuolating activity of ammonia. J Pathol. 1997 Dec;183(4):453–459. doi: 10.1002/(SICI)1096-9896(199712)183:4<453::AID-PATH950>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  31. Satin B., Norais N., Telford J., Rappuoli R., Murgia M., Montecucco C., Papini E. Effect of helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and on epidermal growth factor degradation. J Biol Chem. 1997 Oct 3;272(40):25022–25028. doi: 10.1074/jbc.272.40.25022. [DOI] [PubMed] [Google Scholar]
  32. Szabò I., Bàthori G., Tombola F., Coppola A., Schmehl I., Brini M., Ghazi A., De Pinto V., Zoratti M. Double-stranded DNA can be translocated across a planar membrane containing purified mitochondrial porin. FASEB J. 1998 Apr;12(6):495–502. doi: 10.1096/fasebj.12.6.495. [DOI] [PubMed] [Google Scholar]
  33. Telford J. L., Ghiara P., Dell'Orco M., Comanducci M., Burroni D., Bugnoli M., Tecce M. F., Censini S., Covacci A., Xiang Z. Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease. J Exp Med. 1994 May 1;179(5):1653–1658. doi: 10.1084/jem.179.5.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983 Jun 4;1(8336):1273–1275. [PubMed] [Google Scholar]
  35. Van Dyke R. W. Anion inhibition of the proton pump in rat liver multivesicular bodies. J Biol Chem. 1986 Dec 5;261(34):15941–15948. [PubMed] [Google Scholar]
  36. de Bernard M., Arico B., Papini E., Rizzuto R., Grandi G., Rappuoli R., Montecucco C. Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol. Mol Microbiol. 1997 Nov;26(4):665–674. doi: 10.1046/j.1365-2958.1997.5881952.x. [DOI] [PubMed] [Google Scholar]
  37. de Bernard M., Papini E., de Filippis V., Gottardi E., Telford J., Manetti R., Fontana A., Rappuoli R., Montecucco C. Low pH activates the vacuolating toxin of Helicobacter pylori, which becomes acid and pepsin resistant. J Biol Chem. 1995 Oct 13;270(41):23937–23940. doi: 10.1074/jbc.270.41.23937. [DOI] [PubMed] [Google Scholar]

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