Functional Properties of the p33 and p55 Domains of the Helicobacter pylori Vacuolating Cytotoxin
2005; Elsevier BV; Volume: 280; Issue: 22 Linguagem: Inglês
10.1074/jbc.m501042200
ISSN1083-351X
AutoresVictor J. Torres, Susan E. Ivie, Mark S. McClain, Timothy L. Cover,
Tópico(s)Galectins and Cancer Biology
ResumoHelicobacter pylori secretes an 88-kDa vacuolating cytotoxin (VacA) that may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. VacA cytotoxic activity requires assembly of VacA monomers into oligomeric structures, formation of anion-selective membrane channels, and entry of VacA into host cells. In this study, we analyzed the functional properties of recombinant VacA fragments corresponding to two putative VacA domains (designated p33 and p55). Immunoprecipitation experiments indicated that these two domains can interact with each other to form protein complexes. In comparison to the individual VacA domains, a mixture of the p33 and p55 proteins exhibited markedly enhanced binding to the plasma membrane of mammalian cells. Furthermore, internalization of the VacA domains was detected when cells were incubated with the p33/p55 mixture but not when the p33 and p55 proteins were tested individually. Incubation of cells with the p33/p55 mixture resulted in cell vacuolation, whereas the individual domains lacked detectable cytotoxic activity. Interestingly, sequential addition of p55 followed by p33 resulted in VacA internalization and cell vacuolation, whereas sequential addition in the reverse order was ineffective. These results indicate that both the p33 and p55 domains contribute to the binding and internalization of VacA and that both domains are required for vacuolating cytotoxic activity. Reconstitution of toxin activity from two separate domains, as described here for VacA, has rarely been described for pore-forming bacterial toxins, which suggests that VacA is a pore-forming toxin with unique structural properties. Helicobacter pylori secretes an 88-kDa vacuolating cytotoxin (VacA) that may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. VacA cytotoxic activity requires assembly of VacA monomers into oligomeric structures, formation of anion-selective membrane channels, and entry of VacA into host cells. In this study, we analyzed the functional properties of recombinant VacA fragments corresponding to two putative VacA domains (designated p33 and p55). Immunoprecipitation experiments indicated that these two domains can interact with each other to form protein complexes. In comparison to the individual VacA domains, a mixture of the p33 and p55 proteins exhibited markedly enhanced binding to the plasma membrane of mammalian cells. Furthermore, internalization of the VacA domains was detected when cells were incubated with the p33/p55 mixture but not when the p33 and p55 proteins were tested individually. Incubation of cells with the p33/p55 mixture resulted in cell vacuolation, whereas the individual domains lacked detectable cytotoxic activity. Interestingly, sequential addition of p55 followed by p33 resulted in VacA internalization and cell vacuolation, whereas sequential addition in the reverse order was ineffective. These results indicate that both the p33 and p55 domains contribute to the binding and internalization of VacA and that both domains are required for vacuolating cytotoxic activity. Reconstitution of toxin activity from two separate domains, as described here for VacA, has rarely been described for pore-forming bacterial toxins, which suggests that VacA is a pore-forming toxin with unique structural properties. Helicobacter pylori is a Gram-negative bacterium that chronically infects the human stomach (1Dunn B.E. Cohen H. Blaser M.J. Clin. Microbiol. Rev. 1997; 10: 720-741Crossref PubMed Google Scholar, 2Suerbaum S. Michetti P. N. Engl. J. Med. 2002; 347: 1175-1186Crossref PubMed Scopus (2162) Google Scholar, 3Marshall B.J. Warren J.R. Lancet. 1984; 1: 1311-1315Abstract PubMed Scopus (4208) Google Scholar). A major secreted protein produced by H. pylori is an 88-kDa cytotoxin, known as VacA (4Leunk R.D. Johnson P.T. David B.C. Kraft W.G. Morgan D.R. J. Med. Microbiol. 1988; 26: 93-99Crossref PubMed Scopus (535) Google Scholar, 5Cover T.L. Blaser M.J. J. Biol. Chem. 1992; 267: 10570-10575Abstract Full Text PDF PubMed Google Scholar, 6Telford 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 (523) Google Scholar, 7Cover T.L. Blanke S.R. Nat. Rev. Microbiol. 2005; 3: 320-332Crossref PubMed Scopus (412) Google Scholar). VacA is considered an important virulence factor in the pathogenesis of peptic ulceration and gastric cancer (6Telford 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. 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VacA is expressed in H. pylori as a 140-kDa protoxin that undergoes amino- and carboxyl-terminal processing, yielding a mature 88-kDa secreted VacA toxin (5Cover T.L. Blaser M.J. J. Biol. Chem. 1992; 267: 10570-10575Abstract Full Text PDF PubMed Google Scholar, 6Telford 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 (523) Google Scholar, 25Cover 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, 26Schmitt W. Haas R. Mol. Microbiol. 1994; 12: 307-319Crossref PubMed Scopus (282) Google Scholar, 27Nguyen V.Q. Caprioli R.M. Cover T.L. Infect. Immun. 2001; 69: 543-546Crossref PubMed Scopus (68) Google Scholar). The 88-kDa VacA monomers can assemble into large water-soluble flower-shaped structures comprising 6–14 monomers (28Cover T.L. Hanson P.I. Heuser J.E. J. Cell Biol. 1997; 138: 759-769Crossref PubMed Scopus (179) Google Scholar, 29Lupetti P. Heuser J.E. 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, 30Adrian M. Cover T.L. Dubochet J. Heuser J.E. J. Mol. Biol. 2002; 318: 121-133Crossref PubMed Scopus (45) Google Scholar). The assembly of VacA into oligomeric structures is likely to be required for membrane channel formation, as well as for many effects of VacA on mammalian cells (31Torres V.J. McClain M.S. Cover T.L. J. Biol. Chem. 2004; 279: 2324-2331Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Several cell-surface receptors for VacA have been reported, including two receptor protein tyrosine phosphatases (RPTP-α and -β) (16Fujikawa A. Shirasaka D. Yamamoto S. Ota H. Yahiro K. Fukada M. Shintani T. Wada A. Aoyama N. Hirayama T. Fukamachi H. Noda M. Nat. Genet. 2003; 33: 375-381Crossref PubMed Scopus (219) Google Scholar, 32Yahiro K. Niidome T. Kimura M. Hatakeyama T. Aoyagi H. Kurazono H. Imagawa K. Wada A. Moss J. Hirayama T. J. Biol. Chem. 1999; 274: 36693-36699Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 33Padilla P.I. Wada A. Yahiro K. Kimura M. Niidome T. Aoyagi H. Kumatori A. Anami M. Hayashi T. Fujisawa J. Saito H. Moss J. Hirayama T. J. Biol. Chem. 2000; 275: 15200-15206Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 34Yahiro K. Wada A. Nakayama M. Kimura T. Ogushi K. Niidome T. Aoyagi H. Yoshino K. Yonezawa K. Moss J. Hirayama T. J. Biol. Chem. 2003; 278: 19183-19189Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), the epidermal growth factor receptor (35Seto K. Hayashi-Kuwabara Y. Yoneta T. Suda H. Tamaki H. FEBS Lett. 1998; 431: 347-350Crossref PubMed Scopus (76) Google Scholar), and heparan sulfate (36Utt M. Danielsson B. Wadstrom T. FEMS Immunol. Med. Microbiol. 2001; 30: 109-113Crossref PubMed Google Scholar). VacA also localizes to lipid raft domains on the surface of mammalian cells (12Schraw W. Li Y. McClain M.S. van der Goot F.G. Cover T.L. J. Biol. Chem. 2002; 277: 34642-34650Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 37Patel H.K. Willhite D.C. Patel R.M. Ye D. Williams C.L. Torres E.M. Marty K.B. MacDonald R.A. Blanke S.R. Infect. Immun. 2002; 70: 4112-4123Crossref PubMed Scopus (69) Google Scholar, 38Geisse N.A. Cover T.L. Henderson R.M. Edwardson J.M. Biochem. J. 2004; 381: 911-917Crossref PubMed Scopus (39) Google Scholar). Following binding of VacA to the plasma membrane, the toxin is internalized by cells (39Garner J.A. Cover T.L. Infect. Immun. 1996; 64: 4197-4203Crossref PubMed Google Scholar, 40McClain M.S. Schraw W. Ricci V. Boquet P. Cover T.L. Mol. Microbiol. 2000; 37: 433-442Crossref PubMed Scopus (82) Google Scholar). Internalized VacA can localize to late endocytic compartments (from which vacuoles arise) as well as mitochondria (24Willhite D.C. Blanke S.R. Cell Microbiol. 2004; 6: 143-154Crossref PubMed Scopus (128) Google Scholar, 41Li Y. Wandinger-Ness A. Goldenring J.R. Cover T.L. Mol. Biol. Cell. 2004; 15: 1946-1959Crossref PubMed Scopus (58) Google Scholar). It has been proposed that VacA causes cell vacuolation by forming anion-selective channels in the membrane of endocytic compartments (11Szabo I. Brutsche S. Tombola F. Moschioni M. Satin B. Telford J.L. Rappuoli R. Montecucco C. Papini E. Zoratti M. EMBO. J. 1999; 18: 5517-5527Crossref PubMed Scopus (235) Google Scholar, 41Li Y. Wandinger-Ness A. Goldenring J.R. Cover T.L. Mol. Biol. Cell. 2004; 15: 1946-1959Crossref PubMed Scopus (58) Google Scholar) and that it increases mitochondrial membrane permeability by forming channels in the inner membrane of mitochondria (24Willhite D.C. Blanke S.R. Cell Microbiol. 2004; 6: 143-154Crossref PubMed Scopus (128) Google Scholar). Partial proteolytic digestion of the mature secreted 88-kDa VacA toxin yields two fragments that are ∼33 and 55 kDa in mass (p33 and p55, respectively) (see Fig. 1A). This proteolytic cleavage occurs primarily between amino acids 311 and 312 of the mature secreted toxin from H. pylori strain 60190 (and possibly several adjacent sites) (27Nguyen V.Q. Caprioli R.M. Cover T.L. Infect. Immun. 2001; 69: 543-546Crossref PubMed Scopus (68) Google Scholar), which are predicted to comprise a hydrophilic surface-exposed loop of VacA. It has been suggested that the p33 and p55 fragments represent two domains or subunits of VacA (6Telford 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 (523) Google Scholar, 28Cover T.L. Hanson P.I. Heuser J.E. J. 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Immun. 2000; 68: 4354-4357Crossref PubMed Scopus (39) Google Scholar, 44McClain M.S. Cao P. Cover T.L. Infect. Immun. 2001; 69: 1181-1184Crossref PubMed Scopus (51) Google Scholar), and that the p55 domain is responsible for VacA binding to mammalian cells (39Garner J.A. Cover T.L. Infect. Immun. 1996; 64: 4197-4203Crossref PubMed Google Scholar, 45Reyrat J.M. Lanzavecchia S. Lupetti P. de Bernard M. Pagliaccia C. Pelicic V. Charrel M. Ulivieri C. Norais N. Ji X. Cabiaux V. Papini E. Rappuoli R. Telford J.L. J. Mol. Biol. 1999; 290: 459-470Crossref PubMed Scopus (70) Google Scholar, 46Wang W.C. Wang H.J. Kuo C.H. Biochemistry. 2001; 40: 11887-11896Crossref PubMed Scopus (32) Google Scholar, 47Wang H.J. Wang W.C. Biochem. Biophys. Res. Commun. 2000; 278: 449-454Crossref PubMed Scopus (36) Google Scholar). However, detailed analysis of the functional roles of p33 and p55 domains has not been performed. In this work, we describe the expression and functional analysis of recombinant p33 and p55 domains. Our data indicate that neither the p33 nor the p55 recombinant VacA domain exhibits detectable vacuolating cytotoxic activity when added individually to the surface of mammalian cells. In contrast, we demonstrate that when combined, the p33 and p55 domains form p33·p55 protein complexes and complement each other to permit VacA internalization into cells and reconstitution of vacuolating cytotoxic activity. Bacterial Strains and Growth Conditions—Escherichia coli DH5α was used for plasmid propagation and was grown in Luria-Bertani (LB) broth or on LB agar at 37 °C. For expression of recombinant proteins, expression plasmids were transformed into E. coli strain JM109 (DE3) (Promega), which encodes an isopropyl-β-d-thiogalactopyranoside-inducible copy of the RNA polymerase gene from bacteriophage T7. Transformants were grown in Terrific broth (Invitrogen) supplemented with 25 μg of kanamycin/ml (TB-KAN). H. pylori wild-type strain 60190 (American Type Culture Collection 49503; Manassas, VA) and strain VT330 (encoding a c-Myc-tagged VacA protein) (48McClain M.S. Cao P. Iwamoto H. Vinion-Dubiel A.D. Szabo G. Shao Z. Cover T.L. J. Bacteriol. 2001; 183: 6499-6508Crossref PubMed Scopus (95) Google Scholar) were grown on Trypticase soy agar plates containing 5% sheep blood at 37 °C in ambient air containing 5% CO2. H. pylori liquid cultures were grown in sulfite-free brucella broth supplemented with 0.5% activated charcoal (49Hawrylik S.J. Wasilko D.J. Haskell S.L. Gootz T.D. Lee S.E. J. Clin. Microbiol. 1994; 32: 790-792Crossref PubMed Google Scholar). Purification of VacA from H. pylori—VacA was purified in an oligomeric form from culture supernatants of H. pylori, as described previously (28Cover T.L. Hanson P.I. Heuser J.E. J. Cell Biol. 1997; 138: 759-769Crossref PubMed Scopus (179) Google Scholar). In all experiments, purified VacA preparations from H. pylori were acid-activated before the addition of VacA to the cells, as described previously (40McClain M.S. Schraw W. Ricci V. Boquet P. Cover T.L. Mol. Microbiol. 2000; 37: 433-442Crossref PubMed Scopus (82) Google Scholar, 50de Bernard M. Papini E. de Filippis V. Gottardi E. Telford J. Manetti R. Fontana A. Rappuoli R. Montecucco C. J. Biol. Chem. 1995; 270: 23937-23940Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Plasmid Construction—VacA-expressing plasmids were constructed by cloning vacA sequences from H. pylori strain 60190 into pET-41b (Novagen) using procedures similar to those described previously (51McClain M.S. Cover T.L. Infect. Immun. 2003; 71: 2266-2271Crossref PubMed Scopus (17) Google Scholar). A vacA sequence encoding the VacA p33 domain (amino acids 1–312 of the mature, secreted H. pylori VacA toxin) with a His6 tag at the carboxyl terminus (p33His) was PCR-amplified from the pMM592 plasmid (51McClain M.S. Cover T.L. Infect. Immun. 2003; 71: 2266-2271Crossref PubMed Scopus (17) Google Scholar) using primers BA9146, 5′-CCCACTAGTAAGAGGAGACGCCATGTTTTTTACAACCGTG-3′, and OP6228, 5′-CCCCTGCAGCT AGTGATGGTGATGGTGATGTTTAGCACCACTTTGAGAAGG-3′. The PCR product was digested with SpeI and SalI and ligated into XbaI- and SalI-digested pET-41b (conferring kanamycin resistance) (Novagen). We also generated a plasmid that encoded a VacA p33 domain with two tags (c-Myc and His6), each at the carboxyl terminus of the protein (p33Myc-His). A vacA sequence encoding the p55 domain (amino acids 312–821 of the mature, secreted H. pylori VacA toxin) with a c-Myc tag (p55Myc) at the amino terminus was PCR-amplified from H. pylori VT330 genomic DNA using primers OP6229, 5′-CCCACTAGTAAGAGGAGACGCCATGGCAAACGCCGCACAGG-3′ and AND515a, 5′-CCCCGTCGACTTAAGCGTAGCTAGCGAAACGCG-3′. Also, a vacA sequence encoding the p55 domain with a His6 tag at the amino terminus was generated using primers AND7265, 5′-CCCACTAGTAAGAGGAGACGCCATGCATCACCATCACCATCACAAAAACGACAAACAAGAGAGC-3′ and the AND515a primer. PCR products were digested and cloned into pET-41b, as described above. The use of these primers resulted in a modification of the ribosomal binding site of pET-41b and encoded a methionine at the amino terminus of each VacA protein. The entire vacA fragment in each plasmid was analyzed by nucleotide sequence analysis to verify that no unintended mutations had been introduced. Expression of Recombinant VacA Proteins—VacA expression plasmids were transformed into the E. coli expression strain JM109 (DE3), and transformants were then inoculated into TB-KAN and grown at 37 °C overnight with shaking. These cultures were diluted 1:100 into TB-KAN and incubated at 37 °C until they reached an absorbance (A600) of 0.5. Cultures were then induced with a final isopropyl-β-d-thiogalactopyranoside concentration of 0.5 mm and incubated at 25 °C for 16–18 h (p55 proteins) or at 37 °C for 2 h (p33 proteins). These varying conditions for isopropyl-β-d-thiogalactopyranoside induction were selected to optimize expression and activity of the two different VacA domains. E. coli soluble extracts were generated as described previously with minor modifications (51McClain M.S. Cover T.L. Infect. Immun. 2003; 71: 2266-2271Crossref PubMed Scopus (17) Google Scholar). Briefly, 50 ml of isopropyl-β-d-thiogalactopyranoside-induced cultures were pelleted, washed in 0.9% NaCl, and resuspended in a solution (1 ml) that contained 10 mm Tris (pH 7.5), 100 mm NaCl, 1 mm EDTA, protease inhibitors (Complete Mini; Roche Applied Science), and 20,000 units of ReadyLyse lysozyme (Epicenter)/ml. Bacterial cells were incubated on ice for 30 min with periodic mixing, after which a solution (3 ml) containing 50 mm Tris (pH 8.0), 2.67 mm MgCl2, and 74 units of Omnicleave Nuclease (Epicenter)/ml was added. The samples were then mixed briefly, subjected to four successive rounds of freezing (in a dry ice methanol bath) and thawing at 37 °C, and then the insoluble debris was pelleted. The E. coli soluble extracts containing the VacA proteins were collected and stored at –20 °C until use. Immunoblot Analysis of Recombinant VacA—Proteins in E. coli soluble extracts were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and immunoblotted using a polyclonal anti-His antibody (Santa Cruz Biotechnology), a monoclonal anti-c-Myc (9E10) antibody, or a polyclonal anti-VacA serum (958) (12Schraw W. Li Y. McClain M.S. van der Goot F.G. Cover T.L. J. Biol. Chem. 2002; 277: 34642-34650Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), followed by secondary antibodies conjugated with horseradish peroxidase (Bio-Rad). Signals were generated by the enhanced chemiluminescence reaction (Amersham Biosciences) and detected using x-ray film. In experiments requiring the use of multiple recombinant VacA proteins, the relative concentrations of recombinant VacA in different E. coli soluble extracts were analyzed by immunoblotting with an anti-His antibody, and the extracts were then normalized such that the relative molar concentrations of VacA in different preparations were approximately equivalent. Cell Culture and Vacuolating Assay—HeLa cells were grown in minimal essential medium (modified Eagle's medium containing Earle's salts) supplemented with 10% fetal bovine serum in a 5% CO2 atmosphere at 37 °C. AZ-521 cells, a human gastric adenocarcinoma cell line (Culture Collection of Health Science Research Resources Bank, Japan Health Sciences Foundation) were grown in minimal essential medium supplemented with 10% fetal bovine serum and 1 mm non-essential amino acids (Invitrogen). AGS human gastric epithelial cells (American Type Culture Collection CRL 1739) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. For vacuolating assays, the cells were seeded at 1.2 × 104 cells/well into 96-well plates 24 h prior to each experiment. E. coli soluble extracts, containing recombinant VacA proteins, were normalized as described above and were added to fetal bovine serum-free tissue culture medium overlying the cells (supplemented with 50 μg/ml gentamicin and 10 mm ammonium chloride) for 1 h at 37 °C. The cells were washed two times with phosphate-buffered saline and then incubated in fetal bovine serum-free tissue culture medium, containing 10 mm ammonium chloride and gentamicin, for 4–6 h at 37 °C. For the p33/p55 complementation assays, E. coli soluble extracts were mixed and incubated for 1 h at 25 °C prior to addition to the medium overlying the cells. Purified VacA from H. pylori culture supernatant was routinely acid-activated prior to testing in cell culture assays (40McClain M.S. Schraw W. Ricci V. Boquet P. Cover T.L. Mol. Microbiol. 2000; 37: 433-442Crossref PubMed Scopus (82) Google Scholar, 50de Bernard M. Papini E. de Filippis V. Gottardi E. Telford J. Manetti R. Fontana A. Rappuoli R. Montecucco C. J. Biol. Chem. 1995; 270: 23937-23940Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), whereas E. coli soluble extracts were not acid-activated (51McClain M.S. Cover T.L. Infect. Immun. 2003; 71: 2266-2271Crossref PubMed Scopus (17) Google Scholar). After incubation, cell vacuolation was examined by inverted light microscopy and quantified by a neutral red uptake assay (52Cover T.L. Puryear W. Pérez-Pérez G.I. Blaser M.J. Infect. Immun. 1991; 59: 1264-1270Crossref PubMed Google Scholar). Neutral red uptake data are presented as A540 values (mean ± S.D.). Immunoprecipitation of VacA Complexes—E. coli soluble extracts containing different recombinant VacA proteins were normalized as described above. Normalized soluble extracts were then mixed and incubated for 1 h at 25 °C. VacA complexes were immunoprecipitated with an anti-c-Myc monoclonal antibody (2 μg/ml antibody 9E10) and protein G-coated beads (Zymed Laboratories Inc.) (48McClain M.S. Cao P. Iwamoto H. Vinion-Dubiel A.D. Szabo G. Shao Z. Cover T.L. J. Bacteriol. 2001; 183: 6499-6508Crossref PubMed Scopus (95) Google Scholar). To analyze interactions between p33, p55, and p33/p55 mixture with full-length VacA, normalized soluble extracts were mixed with acid-activated c-Myc-tagged VacA (Myc-VacA) purified from H. pylori culture supernatant (2 μg/ml) for 1 h at 25 °C, and the proteins were immunoprecipitated as described above. Immunoprecipitated proteins were analyzed by immunoblotting with an anti-His (Santa Cruz Biotechnology) antibody or anti-c-Myc monoclonal antibody (9E10), followed by secondary antibodies conjugated with horseradish peroxidase (Bio-Rad), as described above. Analysis of VacA Binding and Internalization into Mammalian Cells—To analyze interactions of VacA with the surface of cells, E. coli soluble extracts, containing recombinant VacA proteins, were added to HeLa cells grown on cover glasses in 6-well plates for 1 h at 4 °C or 37 °C. VacA interactions with mammalian cells were then analyzed by indirect immunofluorescence (12Schraw W. Li Y. McClain M.S. van der Goot F.G. Cover T.L. J. Biol. Chem. 2002; 277: 34642-34650Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 41Li Y. Wandinger-Ness A. Goldenring J.R. Cover T.L. Mol. Biol. Cell. 2004; 15: 1946-1959Crossref PubMed Scopus (58) Google Scholar). Briefly, the cells were washed with Tris-buffered saline (TBS) (10 mm Tris, 150 mm NaCl, pH 7.5) and fixed with 3.7% formaldehyde. Fixed cells were incubated with an anti-c-Myc antibody (1:500) or with an anti-VacA polyclonal antiserum that recognizes the p55 domain for 1 h at 25 °C. The cells were then washed and incubated with a Cy3-conjugated secondary antibody (1: 500) for 1 h at 25 °C. To analyze VacA internalization into host cells, E. coli soluble extracts containing single recombinant VacA proteins or mixtures of recombinant VacA proteins were incubated with HeLa cells for 1 h at 37 °C. Afterward, medium containing unbound proteins was removed, and the cells were incubated in fresh tissue culture medium (without fetal bovine serum or ammonium chloride) for 16 h at 37 °C. The cells were then washed with TBS, fixed with 3.7% formaldehyde, and permeabilized with 100% methanol for 30 min at –20 °C (41Li Y. Wandinger-Ness A. Goldenring J.R. Cover T.L. Mol. Biol. Cell. 2004; 15: 1946-1959Crossref PubMed Scopus (58) Google Scholar). The cells were incubated with the anti-VacA polyclonal antiserum or the anti-c-Myc antibody, followed by a Cy3-conjugated secondary antibody. After immunolabeling, cover glasses were washed with phosphate-buffered saline, mounted on slides with Aqua-Polymount (Polysciences, Warrington, PA), and viewed with a LSM 510 confocal laser scanning inverted microscope (Carl Zeiss). For immunoblot analysis of VacA interactions with cells, HeLa cells were seeded into a 96-well plate and incubated with E. coli soluble extracts, as describ
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