Abstract
The vacuolating cytotoxin (VacA) is an important virulence factor of Helicobacter pylori with pleiotropic effects on mammalian cells, including the ability to trigger mitochondria-dependent apoptosis. However, the mechanism by which VacA exerts its apoptotic function is unclear. Using a genetic approach, in this study we show that killing by VacA requires the proapoptotic Bcl-2 family members BAX and BAK at the mitochondrial level, but not adequate endoplasmic reticulum Ca2+ levels, similarly controlled by BAX and BAK. A combination of subcellular fractionation and imaging shows that wild-type VacA, but not mutants in its channel-forming region, induces the accumulation of BAX on endosomes and endosome–mitochondria juxtaposition that precedes the retrieval of active BAX on mitochondria. It is noteworthy that in Bax- and Bak-deficient cells, VacA is unable to cause endosome–mitochondria juxtaposition and is not retrieved in mitochondria. Thus, VacA causes BAX/BAK-dependent juxtaposition of endosomes and mitochondria early in the process of cell death, revealing a new function for these proapoptotic proteins in the regulation of relative position of organelles.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- Cyt:
-
cytosol
- CS:
-
culture supernatant
- DKO:
-
double knockout
- ER:
-
endoplasmic reticulum
- MEF:
-
mouse embryonic fibroblast
- Mt:
-
mitochondria
- mtBAX:
-
mitochondria-targeted BAX
- NP:
-
nuclear pellet
- PNS:
-
post nuclear supernatant
- SERCA:
-
sarco/endoplasmic reticulum Ca2+-ATPase
- VacA:
-
vacuolating cytotoxin A
- wt:
-
wild-type
- mtRFP:
-
red fluorescent protein targeted to mitochondria
- LBPA:
-
lysobisphosphatidic acid
- DMEM:
-
Dulbecco's modified Eagle's medium
- BSA:
-
bovine serum albumin
- FITC:
-
fluorescein isothiocyanate
- TRITC:
-
tetramethyl rhodamine isothiocyanate
- GFP:
-
green fluorescence protein
- IRES:
-
internal ribosomal entry site
References
Suerbaum S, Michetti P . Helicobacter pylori infection. N Engl J Med 2002; 347: 1175–1186.
Leunk RD, Johnson T, David BC, Kraft WG, Morgan DR . Cytotoxic activity in broth- culture filtrates of Campylobacter pylori. J Med Microbiol 1988; 26: 93–99.
D’Elios MM, Montecucco C, de Bernard M . VacA and HP-NAP, Ying and Yang of Helicobacter pylori-associated gastric inflammation. Clin Chim Acta 2007; 381: 32–38.
Cover TL, Blanke SR . Helicobacter pylori VacA, a paradigm for toxin multifunctionality. Nat Rev Microbiol 2005; 3: 320–332.
Tombola F, Carlesso C, Szabò I, de Bernard M, Reyrat JM, Telford JL et al. Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation. Biophys J 1999; 76: 1401–1409.
Ye D, Blanke SR . Mutational analysis of the Helicobacter pylori vacuolating toxin amino terminus: identification of amino acids essential for cellular vacuolation. Infect Immun 2000; 68: 4354–4357.
McClain MS, Iwamoto H, Cao P, Vinion-Dubiel AD, Li Y, Szabo G et al. Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin. J Biol Chem 2003; 278: 12101–12108.
Szabò I, Brutsche S, Tombola F, Moschioni M, Satin B, Telford JL et al. Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity. EMBO J 1999; 18: 5517–5527.
Molinari M, Galli C, Norais N, Telford JL, Rappuoli R, Luzio JP et al. Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers. J Biol Chem 1997; 272: 25339–25344.
Molinari M, Galli C, de Bernard M, Norais N, Ruysschaert JM, Rappuoli R et al. The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies. Biochem Biophys Res Commun 1998; 248: 334–340.
Gauthier NC, Monzo P, Gonzalez T, Doye A, Oldani A, Gounon P et al. Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin. J Cell Biol 2007; 177: 343–354.
Jones NL, Shannon PT, Cutz E, Yeger H, Sherman PM . Increase in proliferation and apoptosis of gastric epithelial cells early in the natural history of Helicobacter pylori infection. Am J Pathol 1997; 151: 1695–1703.
Mannick EE, Bravo LE, Zarama G, Realpe JL, Zhang XJ, Ruiz B et al. Inducible nitric oxide synthase, nitrotyrosine, and apoptosis in Helicobacter pylori gastritis: effect of antibiotics and antioxidants. Cancer Res 1996; 56: 3238–3243.
Moss SF, Calam J, Agarwal B, Wang S, Holt PR . Induction of gastric epithelial apoptosis by Helicobacter pylori. Gut 1996; 38: 498–501.
Rudi J, Kuck D, Strand S, von Herbay A, Mariani SM, Krammer PH et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand system in Helicobacter pylori-induced gastric epithelial apoptosis. J Clin Invest 1998; 102: 1506–1514.
Correa P, Houghton J . Carcinogenesis of Helicobacter pylori. Gastroenterology 2007; 133: 659–672.
Peek Jr RM, Vaezi MF, Falk GW, Goldblum JR, Perez-Perez GI, Richter JE et al. Role of Helicobacter pylori cagA(+) strains and specific host immune responses on the development of premalignant and malignant lesions in the gastric cardia. Int J Cancer 1999; 82: 520–524.
Kawahara T, Teshima S, Kuwano Y, Oka A, Kishi K, Rokutan K . Helicobacter pylori lipopolysaccharide induces apoptosis of cultured guinea pig gastric mucosal cells. Am J Physiol Gastrointest Liver Physiol 2001; 281: G726–G734.
Cover TL, Krishna US, Israel DA, Peek Jr RM . Induction of gastric epithelial cell apoptosis by Helicobacter pylori vacuolating cytotoxin. Cancer Res 2003; 63: 951–957.
Boquet P, Ricci V, Galmiche A, Gauthier NC . Gastric cell apoptosis and H. pylori: has the main function of VacA finally been identified?. Trends Microbiol 2003; 11: 410–413.
Danial NN, Korsmeyer SJ . Cell death: critical control points. Cell 2004; 116: 205–219.
Kimura M, Goto S, Wada A, Yahiro K, Niidome T, Hatakeyama T et al. Vacuolating cytotoxin purified from Helicobacter pylori causes mitochondrial damage in human gastric cells. Microb Pathos 1999; 26: 45–52.
Galmiche A, Rassow J, Doye A, Cagnol S, Chambard JC, Contamin S et al. The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release. EMBO J 2000; 19: 6361–6370.
Willhite DC, Blanke SR . Helicobacter pylori vacuolating cytotoxin enters cells, localizes to the mitochondria, and induces mitochondrial membrane permeability changes correlated to toxin channel activity. Cell Microbiol 2004; 6: 143–154.
Willhite DC, Cover TL, Blanke SR . Cellular vacuolation and mitochondrial cytochrome c release are independent outcomes of Helicobacter pylori vacuolating cytotoxin activity that are each dependent on membrane channel formation. J Biol Chem 2003; 278: 48204–48209.
Blanke SR . Micro-managing the executioner: pathogen targeting of mitochondria. Trends Microbiol 2005; 13: 64–71.
Yamasaki E, Wada A, Kumatori A, Nakagawa I, Funao J, Nakayama M et al. Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, independent of vacuolation. J Biol Chem 2006; 281: 11205–11209.
Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 2001; 292: 727–730.
Genisset C, Puhar A, Calore F, de Bernard M, Dell’Antone P, Montecucco C . The concerted action of the Helicobacter pylori cytotoxin VacA and of the v-ATPase proton pump induces swelling of isolated endosomes. Cell Microbiol 2007; 9: 1481–1490.
Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T et al. BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 2003; 300: 135–139.
Lytton J, Zarain-Herzberg A, Periasamy M, MacLennan DH . Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. J Biol Chem 1989; 264: 7059–7065.
Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ . Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 1997; 139: 1281–1292.
Goping IS, Gross A, Lavoie JN, Nguyen M, Jemmerson R, Roth K et al. Regulated targeting of BAX to mitochondria. J Cell Biol 1998; 143: 207–215.
Nechushtan A, Smith CL, Hsu YT, Youle RJ . Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J 1999; 18: 2330–2341.
Ouasti S, Matarrese P, Paddon R, Khosravi-Far R, Sorice M, Tinari A et al. Death receptor ligation triggers membrane scrambling between Golgi and mitochondria. Cell Death Differ 2007; 14: 453–461.
Terebiznik MR, Raju D, Vázquez CL, Torbricki K, Kulkarni R, Blanke SR et al. Effect of Helicobacter pylori's vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells. Autophagy 2009; 5: 370–379.
García-Ruiz C, Colell A, Morales A, Calvo M, Enrich C, Fernández-Checa JC . Trafficking of ganglioside GD3 to mitochondria by tumor necrosis factor-alpha. J Biol Chem 2002; 277: 36443–36448.
Sheftel AD, Zhang AS, Brown C, Shirihai OS, Ponka P . Direct interorganellar transfer of iron from endosome to mitochondrion. Blood 2007; 110: 125–132.
Aslan JE, Thomas G . Death by committee: organellar trafficking and communication in apoptosis. Traffic 2009; 10: 1390–1404.
Motyl T, Gajkowska B, Płoszaj T, Wareski P, Skierski J, Zimowska W . Expression and subcellular redistribution of Bax during TGF-beta1-induced programmed cell death of HC11 mouse mammary epithelial cells. Cell Mol Biol (Noisy-le-grand) 2000; 46: 175–185.
Lovell JF, Billen LP, Bindner S, Shamas-Din A, Fradin C, Leber B et al. Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell 2008; 135: 1074–1084.
Marchetti M, Aricò B, Burroni D, Figura N, Rappuoli R, Ghiara P . Development of a mouse model of Helicobacter pylori infection that mimics human disease. Science 1995; 267: 1655–1658.
Skibinski DA, Genisset C, Barone S, Telford JL . The cell-specific phenotype of the polymorphic vacA midregion is independent of the appearance of the cell surface receptor protein tyrosine phosphatase beta. Infect Immun 2006; 74: 49–55.
Cover TL, Blaser MJ . Purification and characterization of the vacuolating toxin from Helicobacter pylori. J Biol Chem 1992; 267: 10570–10575.
de Bernard M, Aricò B, Papini E, Rizzuto R, Grandi G, Rappuoli R et al. Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol. Mol Microbiol 1997; 26: 665–674.
De Brito O, Scorrano L . Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 2008; 456: 605–610.
Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254.
Sawada M, Sun W, Hayes P, Leskov K, Boothman DA, Matsuyama S . Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nat Cell Biol 2003; 5: 320–329.
Komada M, Masaki R, Yamamoto A, Kitamura N . Hrs, a tyrosine kinase substrate with a conserved double Zinc finger domain is localized to the cytoplasmic surface on early endosomes. J Biol Chem 1997; 272: 20538–20544.
Tokuyasu KT . A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 1973; 57: 551–565.
Slot JW, Geuze HJ . Cryosectioning and immunolabeling. Nat Protoc 2007; 2: 2480–2491.
Acknowledgements
This work was supported by the Italian Ministry of University and Research, Progetto di Eccellenza Fondazione Cassa di Risparmio di Padova e Rovigo, research grant by University of Padova (CPDA074121/07), Fondazione Berlucchi, Associazione Italiana per la Ricerca sul Cancro grant regionale (to MDB). FC was supported by the Graduate Academy of the EuroPathoGenomics Network of the Sixth Framework Programme of the European Community. LS is a senior telethon scientist of the Dulbecco-Telethon Institute and research in his lab is supported by Telethon Italy, Compagnia di San Paolo Italy, AIRC Italy. EM studies were performed at the Telethon Facility for Electron Microscopy, Genova, Italy (Grant GTF07002). We thank Tullio Pozzan for his critical reading and helpful suggestions.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Edited by G Melino
Supplementary Information accompanies the paper on Cell Death and Differentiation website
Rights and permissions
About this article
Cite this article
Calore, F., Genisset, C., Casellato, A. et al. Endosome–mitochondria juxtaposition during apoptosis induced by H. pylori VacA. Cell Death Differ 17, 1707–1716 (2010). https://doi.org/10.1038/cdd.2010.42
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cdd.2010.42
Keywords
This article is cited by
-
Infiltration to infection: key virulence players of Helicobacter pylori pathogenicity
Infection (2024)
-
Chronic in vivo exposure to Helicobacter pylori VacA: Assessing the efficacy of automated and long-term intragastric toxin infusion
Scientific Reports (2020)
-
Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs
Cell Death & Differentiation (2018)
-
Natural history of Helicobacter pylori VacA toxin in human gastric epithelium in vivo: vacuoles and beyond
Scientific Reports (2017)
-
Helicobacter pylori targets mitochondrial import and components of mitochondrial DNA replication machinery through an alternative VacA-dependent and a VacA-independent mechanisms
Scientific Reports (2017)
