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A Dominant Negative Mutant of Helicobacter pyloriVacuolating Toxin (VacA) Inhibits VacA-induced Cell Vacuolation

1999; Elsevier BV; Volume: 274; Issue: 53 Linguagem: Inglês

10.1074/jbc.274.53.37736

1083-351X

Arlene D. Vinion-Dubiel, Mark S. McClain, Daniel M. Czajkowsky, Hideki Iwamoto, Dan Ye, Ping Cao, Wayne P. Schraw, Gábor Szabó, Steven R. Blanke, Zhifeng Shao, Timothy L. Cover,

Clostridium difficile and Clostridium perfringens research

Most Helicobacter pyloristrains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Δ6–27)) in which this hydrophobic segment was deleted. VacA-(Δ6–27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Δ6–27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Δ6–27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Δ6–27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Δ6–27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Δ6–27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity. Most Helicobacter pyloristrains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Δ6–27)) in which this hydrophobic segment was deleted. VacA-(Δ6–27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Δ6–27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Δ6–27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Δ6–27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Δ6–27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Δ6–27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity. enzyme-linked immunosorbent assay diethyl pyrocarbonate green fluorescent protein Helicobacter pylori are Gram-negative bacteria that persistently colonize the gastric mucosa of humans (1Dunn B.E. Cohen H. Blaser M.J. Clin. Microbiol. Rev. 1997; 10: 720-741Crossref PubMed Google Scholar). Colonization of the gastric mucosa by these bacteria results in mucosal inflammation and is a risk factor for the development of peptic ulcer disease, distal gastric adenocarcinoma, and gastric lymphoma (1Dunn B.E. Cohen H. Blaser M.J. Clin. Microbiol. Rev. 1997; 10: 720-741Crossref PubMed Google Scholar, 2AnonymousJ. Am. Med. Assoc. 1994; 272: 65-69Crossref PubMed Scopus (1063) Google Scholar, 3AnonymousIARC Monogr. Eval. Carcinog. Risks Hum. 1994; 61: 177-240PubMed Google Scholar, 4Fuchs C.S. Mayer J.J. N. Engl. J. Med. 1995; 333: 32-40Crossref PubMed Scopus (679) Google Scholar). Gastric adenocarcinoma is currently one of the most common causes of cancer deaths worldwide and is the only cancer that has been directly linked to a bacterial infection (3AnonymousIARC Monogr. Eval. Carcinog. Risks Hum. 1994; 61: 177-240PubMed Google Scholar). Most H. pylori strains secrete a toxin (VacA) that is unrelated to any other known bacterial protein toxin (5Cover T.L. Mol. Microbiol. 1996; 20: 241-246Crossref PubMed Scopus (259) Google Scholar, 6Montecucco C. Curr. Opin. Cell Biol. 1998; 10: 530-536Crossref PubMed Scopus (45) Google Scholar). When VacA is incubated with epithelial cells in vitro, the most prominent effect is the formation of large cytoplasmic vacuoles (5Cover T.L. Mol. Microbiol. 1996; 20: 241-246Crossref PubMed Scopus (259) Google Scholar). These vacuoles contain markers for both late endosomes and lysosomes and have an acidic intravacuolar pH (7Molinari M. Galli G. Norais N. Telford J.L. Rappuoli R. Luzio J.P. Montecucco C. J. Biol. Chem. 1997; 272: 25339-25344Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 8Papini E. Satin B. Bucci C. deBernard M. Telford J.L. Manetti R. Rappuoli R. Zerial M. Montecucco C. EMBO J. 1997; 16: 15-24Crossref PubMed Scopus (193) Google Scholar, 9Papini E. de Bernard M. Milia E. Bugnoli M. Zerial M. Rappuoli R. Montecucco C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9720-9724Crossref PubMed Scopus (201) Google Scholar). VacA-induced vacuoles are thought to represent novel intracellular compartments that form as a result of heterotypic fusion events (7Molinari M. Galli G. Norais N. Telford J.L. Rappuoli R. Luzio J.P. Montecucco C. J. Biol. Chem. 1997; 272: 25339-25344Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 8Papini E. Satin B. Bucci C. deBernard M. Telford J.L. Manetti R. Rappuoli R. Zerial M. Montecucco C. EMBO J. 1997; 16: 15-24Crossref PubMed Scopus (193) Google Scholar, 9Papini E. de Bernard M. Milia E. Bugnoli M. Zerial M. Rappuoli R. Montecucco C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9720-9724Crossref PubMed Scopus (201) Google Scholar). In addition to altering the morphology of cells, VacA causes multiple functional changes, including alterations in the intracellular trafficking and processing of procathepsin D and epidermal growth factor (10Satin B. Norais N. Telford J.L. Rappuoli R. Murgia M. Montecucco C. Papini E. J. Biol. Chem. 1997; 272: 25022-25028Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). When added to polarized epithelial cell monolayers, VacA induces an increase in monolayer permeability for various ions and small uncharged molecules (11Papini E. Satin B. Norais N. deBernard M. Telford J.L. Rappuoli R. Montecucco C. J. Clin. Invest. 1998; 102: 813-820Crossref PubMed Scopus (211) Google Scholar). VacA also interferes with the process of antigen presentation, which may be one mechanism by which H. pylori resists immune clearance (12Molinari M. Salio M. Galli C. Norais N. Rappuoli R. Lanzavecchia A. Montecucco C. J. Exp. Med. 1998; 187: 135-140Crossref PubMed Scopus (241) Google Scholar). The H. pylori vacA gene is translated as a 140-kDa protoxin, which undergoes amino- and carboxyl-terminal processing to yield a mature secreted toxin of about 87 kDa (13Cover T.L. Blaser M.J. J. Biol. Chem. 1992; 267: 10570-10575Abstract Full Text PDF PubMed Google Scholar, 14Cover T.L. Tummuru M.K.R. Cao P. Thompson S.A. Blaser M.J. J. Biol. Chem. 1994; 269: 10566-10573Abstract Full Text PDF PubMed Google Scholar, 15Schmitt W. Haas R. Mol Microbiol. 1994; 12: 307-319Crossref PubMed Scopus (283) Google Scholar, 16Telford J.L. Ghiara P. Dell'Orco M. Comanducci M. Burroni D. Bugnoli M. Tecce M.F. Censini S. Covacci A. Xiang Z. Papini E. Montecucco C. Parente L. Rappuoli R. J. Exp. Med. 1994; 179: 1653-1658Crossref PubMed Scopus (524) Google Scholar). Secretion of VacA probably occurs via a mechanism analogous to that used for secretion ofNeisseria gonorrhoeae IgA protease (14Cover T.L. Tummuru M.K.R. Cao P. Thompson S.A. Blaser M.J. J. Biol. Chem. 1994; 269: 10566-10573Abstract Full Text PDF PubMed Google Scholar, 15Schmitt W. Haas R. Mol Microbiol. 1994; 12: 307-319Crossref PubMed Scopus (283) Google Scholar). Mature 87-kDa VacA monomers assemble into complex water-soluble oligomers typically comprised of 12 or 14 subunits (17Cover T.L. Hanson P.I. Heuser J.E. J. Cell Biol. 1997; 138: 759-769Crossref PubMed Scopus (179) Google Scholar, 18Lupetti P. Heuser J. Manetti R. Massari P. Lanzavecchia S. Bellon P.L. Dallai R. Rappuoli R. Telford J.L. J. Cell Biol. 1996; 133: 801-807Crossref PubMed Scopus (158) Google Scholar). Upon exposure to acidic pH, these oligomers disassemble into monomeric components (17Cover T.L. Hanson P.I. Heuser J.E. J. Cell Biol. 1997; 138: 759-769Crossref PubMed Scopus (179) Google Scholar). Acidification of VacA enhances its cytotoxic activity and permits the toxin to insert into lipid membranes to form anion-conductive channels (19Czajkowsky D.M. Iwamoto H. Cover T.L. Shao Z. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2001-2006Crossref PubMed Scopus (191) Google Scholar, 20Iwamoto H. Czajkowsky D.M. Cover T.L. Szabo G. Shao Z. FEBS Lett. 1999; 450: 101-104Crossref PubMed Scopus (111) Google Scholar, 21de Bernard M. Papini E. de Filippis V. Gottardi E. Telford J.L. Manetti R. Fontana A. Rappuoli R. Montecucco C. J. Biol. Chem. 1995; 270: 23937-23940Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 22Molinari M. Galli C. deBernard M. Norais N. Ruysschaert J.-M. Rappuoli R. Montecucco C. Biochem. Biophys. Res. Commun. 1998; 248: 334-340Crossref PubMed Scopus (80) Google Scholar, 23Tombola F. Carlesso C. Szabo I. deBernard M. Reyrat J.-R. Telford J.L. Rappuoli R. Montecucco C. Papini E. Zoratti M. Biophys. J. 1999; 76: 1401-1409Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The mechanisms by which VacA causes alterations in cellular morphology and function are not yet well understood. Transfection of HeLa cells with plasmids expressing VacA results in cell vacuolation, which suggests that VacA has an intracellular site of action (24deBernard M. Arico B. Papini E. Rizzuto R. Grandi G. Rappuoli R. Montecucco C. Mol. Microbiol. 1997; 26: 665-674Crossref PubMed Scopus (106) Google Scholar, 25deBernard M. Burroni D. Papini E. Rappuoli R. Telford J.L. Montecucco C. Infect. Immun. 1998; 66: 6014-6016Crossref PubMed Google Scholar, 26Ye D. Willhite D.C. Blanke S.R. J. Biol. Chem. 1999; 274: 9277-9282Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Garner J.A. Cover T.L. Infect. Immun. 1996; 64: 4197-4203Crossref PubMed Google Scholar). Nearly all bacterial toxins that act intracellularly have an enzymatic activity, but thus far, no enzymatic activity of VacA has been identified. The formation of membrane channels by VacA also may contribute to cytotoxic effects, perhaps analogous to the mechanism by which aerolysin causes vacuolation of cells (28Abrami L. Fivaz M. Glauser P.-E. Parton R.G. van der Goot F.G. J. Cell Biol. 1998; 140: 525-540Crossref PubMed Scopus (196) Google Scholar). Structure-function analysis of VacA may be helpful in deciphering how this toxin exerts its cytotoxic effects. However, the construction of VacA mutants has been hindered by the inability to express a functional form of recombinant toxin in Escherichia coli (29Manetti R. Massari P. Burroni D. deBernard M. Marchini A. Olivieri R. Papini E. Montecucco C. Rappuoli R. Telford J.L. Infect. Immun. 1995; 63: 4476-4480Crossref PubMed Google Scholar). In this study, we utilized a recently developed mutagenesis method (30Copass M. Grandi G. Rappuoli R. Infect. Immun. 1997; 65: 1949-1952Crossref PubMed Google Scholar) to analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus. We report that a VacA mutant protein (VacA-(Δ6–27)) lacking this amino-terminal hydrophobic segment is indistinguishable from wild-type VacA in its secretion, assembly into oligomeric structures, and uptake by HeLa cells. However, this mutant protein is markedly altered in its capacity to form ion-conductive channels, lacks cytotoxic activity, and completely inhibits the vacuolating activity of the wild-type toxin. H. pylori 60190 (ATCC 49503) was the parent strain used for construction of all mutants in this study. Characteristics of the vacA gene and the secreted VacA protein from this strain have been reported previously (13Cover T.L. Blaser M.J. J. Biol. Chem. 1992; 267: 10570-10575Abstract Full Text PDF PubMed Google Scholar, 14Cover T.L. Tummuru M.K.R. Cao P. Thompson S.A. Blaser M.J. J. Biol. Chem. 1994; 269: 10566-10573Abstract Full Text PDF PubMed Google Scholar,17Cover T.L. Hanson P.I. Heuser J.E. J. Cell Biol. 1997; 138: 759-769Crossref PubMed Scopus (179) Google Scholar, 19Czajkowsky D.M. Iwamoto H. Cover T.L. Shao Z. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2001-2006Crossref PubMed Scopus (191) Google Scholar). H. pylori strains were grown routinely on trypticase soy agar plates containing 5% sheep blood in room air containing 6% CO2 at 37 °C.