Acid-induced Dissociation of VacA, the Helicobacter pylori Vacuolating Cytotoxin, Reveals Its Pattern of Assembly
Tóm tắt
In this study, we describe the ultrastructural changes associated with acid activation of Helicobacter pylori vacuolating cytotoxin (VacA). Purified VacA molecules imaged by deep-etch electron microscopy form ∼30-nm hexagonal “flowers,” each composed of an ∼15-nm central ring surrounded by six ∼6-nm globular “petals.” Upon exposure to acidic pH, these oligomeric flowers dissociate into collections of up to 12 teardrop-shaped subunits, each measuring ∼6 × 14 nm. Correspondingly, glycerol density gradient centrifugation shows that at neutral pH VacA sediments at ∼22 S, whereas at acidic pH it dissociates and sediments at ∼5 S. Immunoblot and EM analysis of the 5-S material demonstrates that it represents ∼90-kD monomers with 6 × 14–nm “teardrop” morphology. These data indicate that the intact VacA oligomer consists of 12 ∼90-kD subunits assembled into two interlocked six-membered arrays, overlap of which gives rise to the flower-like appearance. Support for this interpretation comes from EM identification of small numbers of relatively “flat” oligomers composed of six teardrop-shaped subunits, interpreted to be halves of the complete flower. These flat forms adsorb to mica in two different orientations, corresponding to hexameric surfaces that are either exposed or sandwiched inside the dodecamer, respectively. This view of VacA structure differs from a previous model in which the flowers were interpreted to be single layers of six monomers and the flat forms were thought to be proteolysed flowers. Since acidification has been shown to potentiate the cytotoxic effects of VacA, the present results suggest that physical disassembly of the VacA oligomer is an important feature of its activation.
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Atherton, 1995, Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori: association of specific vacA types with cytotoxin production and peptic ulceration, J Biol Chem, 270, 17771, 10.1074/jbc.270.30.17771
Austin, 1991, Macromolecular structure and aggregation states of Helicobacter pyloriurease, J Bacteriol, 173, 5663, 10.1128/jb.173.18.5663-5667.1991
Austin, 1992, Structural comparison of urease and a GroEL analog from Helicobacter pylori., J Bacteriol, 174, 7470, 10.1128/jb.174.22.7470-7473.1992
Boquet, 1982, Tetanus toxin fragment forms channels in lipid vesicles at low pH, Proc Natl Acad Sci USA, 79, 7614, 10.1073/pnas.79.24.7614
Cover, 1996, The vacuolating cytotoxin of Helicobacter pylori., Mol Microbiol, 20, 241, 10.1111/j.1365-2958.1996.tb02612.x
Cover, 1992, Purification and characterization of the vacuolating toxin from Helicobacter pylori., J Biol Chem, 267, 10570, 10.1016/S0021-9258(19)50054-0
Cover, 1996, Helicobacter pyloriinfection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention, Adv Intern Med, 41, 85
Cover, 1991, Effect of urease on HeLa cell vacuolation induced by Helicobacter pyloricytotoxin, Infect Immun, 59, 1264, 10.1128/iai.59.4.1264-1270.1991
Cover, 1994, Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pyloristrains, J Biol Chem, 269, 10566, 10.1016/S0021-9258(17)34097-8
de Bernard, 1995, Low pH activates the vacuolating toxin of Helicobacter pylori, which becomes acid and pepsin resistant, J Biol Chem, 270, 23937, 10.1074/jbc.270.41.23937
Domenighini, 1995, Identification of errors among database sequence entries and comparison of correct amino acid sequences for the heat-labile enterotoxins of Escherichia coli and Vibrio cholerae., Mol Microbiol, 15, 1165, 10.1111/j.1365-2958.1995.tb02289.x
Dumont, 1988, The pH-dependent conformational change of diphtheria toxin, J Biol Chem, 263, 2087, 10.1016/S0021-9258(19)77988-5
Farahbakhsh, 1987, Effect of low pH on the conformation of Pseudomonas exotoxinA, J Biol Chem, 262, 2256, 10.1016/S0021-9258(18)61647-3
Figura, 1989, Cytotoxin production by Campylobacter pyloristrains isolated from patients with peptic ulcers and from patients with chronic gastritis only, J Clin Microbiol, 27, 225, 10.1128/jcm.27.1.225-226.1989
Garner, 1996, Binding and internalization of Helicobacter pyloricytotoxin by epithelial cells, Infect Immun, 64, 4197, 10.1128/iai.64.10.4197-4203.1996
Hardy, 1988, Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro, Proc Natl Acad Sci USA, 85, 7109, 10.1073/pnas.85.19.7109
Hawrylik, 1994, Bisulfite or sulfite inhibits growth of Helicobacter pylori., J Clin Microbiol, 32, 790, 10.1128/jcm.32.3.790-792.1994
Heuser, 1983, Procedure for freeze-drying molecules adsorbed to mica flakes, J Mol Biol, 169, 155, 10.1016/S0022-2836(83)80179-X
Heuser, 1989, Development of the quick-freeze, deep-etch, rotary-replication technique of sample preparation for 3-D electron microscopy, Prog Clin Biol Res, 295, 71
Heuser, 1993, Proton pumps populate the contractile vacuoles of Dictyostelium amoebae., J Cell Biol, 121, 1311, 10.1083/jcb.121.6.1311
Hoch, 1985, Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes, Proc Natl Acad Sci USA, 82, 1692, 10.1073/pnas.82.6.1692
Hu, 1990, Purification and NH2-terminal analysis of urease from Helicobacter pylori., Infect Immun, 58, 992, 10.1128/iai.58.4.992-998.1990
Lupetti, 1996, Oligomeric and subunit structure of the Helicobacter pylorivacuolating cytotoxin, J Cell Biol, 133, 801, 10.1083/jcb.133.4.801
Manetti, 1995, The Helicobacter pyloricytotoxin: importance of native conformation for induction of neutralizing antibodies, Infect Immun, 63, 4476, 10.1128/iai.63.11.4476-4480.1995
Marchetti, 1995, Development of a mouse model of Helicobacter pyloriinfection that mimics human disease, Science (Wash DC), 267, 1655, 10.1126/science.7886456
Martin, 1961, A method for determining the sedimentation behavior of enzymes: application to protein mixtures, J Biol Chem, 236, 1372, 10.1016/S0021-9258(18)64180-8
Milne, 1993, pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen, Mol Microbiol, 10, 647, 10.1111/j.1365-2958.1993.tb00936.x
Milne, 1994, Anthrax protective antigen forms oligomers during intoxication of mammalian cells, J Biol Chem, 269, 20607, 10.1016/S0021-9258(17)32036-7
Moll, 1995, Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pyloricytotoxin, Eur J Biochem, 234, 947, 10.1111/j.1432-1033.1995.947_a.x
Montecucco, 1994, Bacterial protein toxins penetrate cells via a four-step mechanism, FEBS Lett, 346, 92, 10.1016/0014-5793(94)00449-8
Narhi, 1996, Induction of α-helix in the β-sheet protein tumor necrosis factor-α: acid-induced denaturation, Biochemistry, 35, 11454, 10.1021/bi952767n
Papini, 1988, On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells, EMBO (Eur Mol Biol Organ) J, 7, 3353, 10.1002/j.1460-2075.1988.tb03207.x
Papini, 1994, Cellular vacuoles induced by Helicobacter pylorioriginate from late endosomal compartments, Proc Natl Acad Sci USA, 91, 9720, 10.1073/pnas.91.21.9720
Papini, 1997, The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pyloricytotoxin, EMBO (Eur Mol Biol Organ) J, 16, 15, 10.1093/emboj/16.1.15
Quigley, 1987, pH of the microclimate lining human gastric and duodenal mucosa in vivo: studies in control subjects and in duodenal ulcer patients, Gastroenterology, 92, 1876, 10.1016/0016-5085(87)90619-6
Ruddock, 1995, Kinetics of acid-mediated disassembly of the B subunit pentamer of Escherichia coliheat-labile enterotoxin: molecular basis of pH stability, J Biol Chem, 270, 29953, 10.1074/jbc.270.50.29953
Sixma, 1993, Refined structure of Escherichia coliheat-labile enterotoxin, a close relative of cholera toxin, J Mol Biol, 230, 890, 10.1006/jmbi.1993.1209
Telford, 1994, Gene structure of the Helicobacter pyloricytotoxin and evidence of its key role in gastric disease, J Exp Med, 179, 1653, 10.1084/jem.179.5.1653
Telford, 1994, Unravelling the pathogenic role of Helicobacter pyloriin peptic ulcer: potential new therapies and vaccines, Trends Biotechnol, 12, 420, 10.1016/0167-7799(94)90031-0