Inhibition of T-Cell Proliferation by Helicobacter pylori γ-Glutamyl Transpeptidase
2007; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.02.031
ISSN1528-0012
AutoresChristian Schmees, Christian Prinz, Tilman Treptau, Roland Rad, Ludger Hengst, Petra Voland, Stefan Bauer, Lena Brenner, Roland M. Schmid, Markus Gerhard,
Tópico(s)Cancer-related gene regulation
ResumoBackground & Aims: Helicobacter pylori colonizes the human gastric mucosa of >50% of the world’s population. Most of the patients have no overt clinical symptoms. However, the infection is invariably associated with the development of active chronic gastritis, leading in some cases to the development of peptic ulcer disease, distal gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. In contrast to most other pathogens, infection with H pylori persists lifelong, but reasons for the persistence remain obscure. CD4-positive T cells are crucial for bacterial elimination but are inhibited by H pylori. We aimed to identify the factor responsible for suppression of T-cell response and characterize this inhibitory effect on a cellular and molecular level. Methods: Using size-exclusion chromatography, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and a spectrophotometric enzyme assay, we identified the secreted γ-glutamyl transpeptidase of H pylori (HPGGT) as the factor responsible for inhibition of T-cell proliferation. Results: Mutagenesis of HPGGT in different H pylori strains completely abrogated this inhibitory effect. Recombinantly expressed HPGGT protein showed full antiproliferative activity. Site-directed mutagenesis and application of the GGT inhibitor acivicin revealed that inhibition of T cells depends on catalytic activity of HPGGT. Cell cycle analysis of human T cells indicated that HPGGT was necessary and sufficient to induce G1 arrest. Reduced levels of c-Myc and phosphorylated c-Raf protein suggest the disruption of Ras-dependent signaling by HPGGT. Conclusions: GGT is a novel immunosuppressive factor of H pylori inhibiting T-cell proliferation by induction of a cell cycle arrest in the G1 phase. Background & Aims: Helicobacter pylori colonizes the human gastric mucosa of >50% of the world’s population. Most of the patients have no overt clinical symptoms. However, the infection is invariably associated with the development of active chronic gastritis, leading in some cases to the development of peptic ulcer disease, distal gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. In contrast to most other pathogens, infection with H pylori persists lifelong, but reasons for the persistence remain obscure. CD4-positive T cells are crucial for bacterial elimination but are inhibited by H pylori. We aimed to identify the factor responsible for suppression of T-cell response and characterize this inhibitory effect on a cellular and molecular level. Methods: Using size-exclusion chromatography, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and a spectrophotometric enzyme assay, we identified the secreted γ-glutamyl transpeptidase of H pylori (HPGGT) as the factor responsible for inhibition of T-cell proliferation. Results: Mutagenesis of HPGGT in different H pylori strains completely abrogated this inhibitory effect. Recombinantly expressed HPGGT protein showed full antiproliferative activity. Site-directed mutagenesis and application of the GGT inhibitor acivicin revealed that inhibition of T cells depends on catalytic activity of HPGGT. Cell cycle analysis of human T cells indicated that HPGGT was necessary and sufficient to induce G1 arrest. Reduced levels of c-Myc and phosphorylated c-Raf protein suggest the disruption of Ras-dependent signaling by HPGGT. Conclusions: GGT is a novel immunosuppressive factor of H pylori inhibiting T-cell proliferation by induction of a cell cycle arrest in the G1 phase. For more than 20 years, the role of Helicobacter pylori in the human stomach has been investigated in detail; nevertheless, the reasons for the persistence of the gastric pathogen have remained obscure.1Blaser M.J. Atherton J.C. Helicobacter pylori persistence: biology and disease.J Clin Invest. 2004; 113: 321-333Google Scholar It has been shown that CD4-positive T cells are crucial for bacterial elimination2Ermak T.H. Giannasca P.J. Nichols R. Myers G.A. Nedrud J. Weltzin R. Lee C.K. Kleanthous H. Monath T.P. Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses.J Exp Med. 1998; 188: 2277-2288Google Scholar but are inhibited in their proliferation by H pylori.3Gerhard M. Schmees C. Voland P. Endres N. Sander M. Reindl W. Rad R. Oelsner M. Decker T. Mempel M. Hengst L. Prinz C. A secreted low-molecular-weight protein from Helicobacter pylori induces cell-cycle arrest of T cells.Gastroenterology. 2005; 128: 1327-1339Abstract Full Text Full Text PDF Scopus (69) Google Scholar In this context, several groups have investigated the immunosuppressive effects of proteins from H pylori. Knipp et al partially purified a so-called “proliferation-inhibiting protein,” which reduced the proliferation of lymphocytes and monocytes independently of the virulence factors CagA (cytotoxin-associated gene A) and VacA (vacuolating cytotoxin A).4Knipp U. Birkholz S. Kaup W. Opferkuch W. Partial characterization of a cell proliferation-inhibiting protein produced by Helicobacter pylori.Infect Immun. 1996; 64: 3491-3496Google Scholar In contrast, 2 groups recently reported that lymphocyte proliferation was suppressed in the presence of high concentrations of purified VacA.5Gebert B. Fischer W. Weiss E. Hoffmann R. Haas R. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation.Science. 2003; 301: 1099-1102Google Scholar, 6Sundrud M.S. Torres V.J. Unutmaz D. Cover T.L. Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion.Proc Natl Acad Sci U S A. 2004; 101: 7727-7732Google Scholar Paradoxically, however, VacA-deficient H pylori mutants had no defect regarding their proliferation-inhibiting properties.5Gebert B. Fischer W. Weiss E. Hoffmann R. Haas R. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation.Science. 2003; 301: 1099-1102Google Scholar In addition, it has been recognized that gastric inflammation is not altered or even increased in patients infected with VacA-expressing Helicobacter strains.7Atherton J.C. Peek Jr, R.M. Tham K.T. Cover T.L. Blaser M.J. Clinical and pathological importance of heterogeneity in VacA, the vacuolating cytotoxin gene of Helicobacter pylori.Gastroenterology. 1997; 112: 92-99Google Scholar, 8Rad R. Gerhard M. Lang R. Schoniger M. Rosch T. Schepp W. Becker I. Wagner H. Prinz C. The Helicobacter pylori blood group antigen-binding adhesin facilitates bacterial colonization and augments a nonspecific immune response.J Immunol. 2002; 168: 3033-3041Google Scholar In a previous study, we showed that secreted products of H pylori inhibited T-lymphocyte proliferation by inducing a cell cycle arrest in G1 phase.3Gerhard M. Schmees C. Voland P. Endres N. Sander M. Reindl W. Rad R. Oelsner M. Decker T. Mempel M. Hengst L. Prinz C. A secreted low-molecular-weight protein from Helicobacter pylori induces cell-cycle arrest of T cells.Gastroenterology. 2005; 128: 1327-1339Abstract Full Text Full Text PDF Scopus (69) Google Scholar This effect was independent of known virulence factors, including the proteins VacA and CagA. Thus, another factor of H pylori with an estimated molecular weight of 29–66 kilodaltons accounted for the attenuated proliferation of T lymphocytes during infection. The current study aimed to identify this immunosuppressive factor in H pylori culture supernatants. In the system used here, we identified γ-glutamyl transpeptidase (GGT) as a novel mediator of H pylori, which is sufficient to impair the function of T cells at low doses. Mutant bacteria deficient in GGT lost the ability to inhibit proliferation of lymphocytes, which was restored by recombinant γ-glutamyl transpeptidase of H pylori (HPGGT). Analysis of its effects on signal transduction in T cells suggests disruption of Ras-dependent signaling leading to induction of a G1 cell cycle arrest. The H pylori wild-type strain G27 WT (vacA+ cagA+) used in this study was obtained from A. Covacci (IRIS, Siena, Italy). The bacteria were cultured on Wilkins–Chalgren or Brain Heart Infusion plates supplemented with Dent supplement antibiotic mix (Oxoid, Wesel, Germany) as previously described.9Gerhard M. Lehn N. Neumayer N. Boren T. Rad R. Schepp W. Miehlke S. Classen M. Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.Proc Natl Acad Sci U S A. 1999; 96: 12778-12783Google Scholar Liquid culture of H pylori was performed in Brain Heart Infusion broth supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Taufkirchen, Germany) and 1% Dent supplement. For production of H pylori supernatants, the bacteria were grown on plates for 48 hours, washed 3 times in phosphate-buffered saline, and adjusted to OD600nm of 1 (corresponding to approximately 2 × 108 bacteria/mL). The bacteria were incubated in phosphate-buffered saline for 2 hours under microaerophilic conditions with vigorous shaking and pelleted by subsequent centrifugation steps at 3000g and 10,000g to remove bacteria and cellular debris. Subsequently, supernatants were concentrated using ultrafiltration (Amicon Ultra MWCO 10kDa; Millipore, Schwalbach, Germany). The total protein content of the supernatants was measured by Bradford assay (BioRad Laboratories, Richmond, VA) with bovine serum albumin as standard, and proteins were stored at −80°C. Escherichia coli were cultured on Luria broth agar plates (USB, Cleveland, OH) and in Luria broth (USB) with relevant antibiotics for liquid culture. Supernatants from H pylori wild-type strain G27 were prepared as described previously. Size-exclusion chromatography was performed as described previously.3Gerhard M. Schmees C. Voland P. Endres N. Sander M. Reindl W. Rad R. Oelsner M. Decker T. Mempel M. Hengst L. Prinz C. A secreted low-molecular-weight protein from Helicobacter pylori induces cell-cycle arrest of T cells.Gastroenterology. 2005; 128: 1327-1339Abstract Full Text Full Text PDF Scopus (69) Google Scholar Briefly, 500 μg of protein was loaded on a Superdex 200 10/300 column (GE Healthcare, Munich, Germany) and eluted with degassed phosphate-buffered saline at 4°C. Standard proteins α-amylase (200 kilodaltons), alcohol dehydrogenase (150 kilodaltons), bovine serum albumin (66 kilodaltons), and carbonic anhydrase (29 kilodaltons) were used for molecular weight estimation of eluted proteins. Each fraction was tested for proliferation inhibition and GGT activity as described in the following text. The GGT k.o. plasmid (kindly provided by K. Shibayama) was transformed into H pylori strain G27 by natural transformation. Transformants were incubated on agar plates containing 25 μg/mL kanamycin (Sigma-Aldrich). After 3 days, clones were picked and spread on fresh agar plates with kanamycin. Insertion of the plasmid was verified by polymerase chain reaction (primers: sense, 5′-AAACGATTGGCTTGGGTGTGATAG-3′; antisense, 5′-GACCGGCTTAGTAACGATTTGATAG-3′) of bacterial DNA and Western blotting of proteins from H pylori ΔGGT supernatants. Isolation of peripheral blood mononuclear cells (PBMCs) was performed as described previously.3Gerhard M. Schmees C. Voland P. Endres N. Sander M. Reindl W. Rad R. Oelsner M. Decker T. Mempel M. Hengst L. Prinz C. A secreted low-molecular-weight protein from Helicobacter pylori induces cell-cycle arrest of T cells.Gastroenterology. 2005; 128: 1327-1339Abstract Full Text Full Text PDF Scopus (69) Google Scholar All cells were incubated at 37°C with 5% CO2 Jurkat T cells and PBMCs were cultured in RPMI 1640 (Invitrogen, Karlsruhe, Germany) with 10% FCS (Sigma). EL-4 T cells were cultured in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% horse serum (Cambrex, Verviers, Belgium). Primary human T cells were isolated from buffy coats or heparinized peripheral venous blood from H pylori–infected and –uninfected volunteers by negative selection using the Pan T cell Isolation Kit II (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Cells (105 PBMCs, purified primary T cells, or 104 Jurkat/EL-4 cells/well) were cultured in 96-well flat-bottom plates in complete medium. PBMCs were stimulated in triplicate with phorbol myristate acetate (PMA) (20 ng/mL; Sigma) and ionomycin (100 ng/mL; Sigma), and cells were incubated with or without indicated total protein concentrations of H pylori supernatants or recombinant proteins. Primary human T cells were stimulated with either PMA/ionomycin as described previously or with anti-CD3/CD28 beads (Invitrogen) at 1 bead per T cell. Cellular proliferation was determined after 48 hours by methyl-[3H]-thymidine (GE Healthcare, Freiburg, Germany) incorporation using a Packard Direct Beta Counter Matrix 9600 (Packard Instruments Co, Downer’s Grove, IL). The GGT protein of H pylori was expressed as 6xHis-tagged protein according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The coding region of the GGT protein from H pylori was amplified by polymerase chain reaction (primers: sense, 5′-TGAAAGGAAAACCCATGGGACGGAG-3′; antisense, 5′-CAAAGGTACCAAATTCTTTCCTTGG-3′). The polymerase chain reaction product was separated by agarose gel electrophoresis and purified by gel extraction (Qiagen). It was then restricted with NcoI and KpnI (New England Biolabs, Ipswich, MA), followed by ligation into the pQE-Tri System vector (Qiagen) after repurification. The resulting vector was transformed into E coli strain M15. Luria broth supplemented with 100 μg/mL ampicillin (Sigma) and 25 μg/mL kanamycin was inoculated with an overnight culture of transformed bacteria and grown at 37°C with vigorous shaking until OD600 reached 0.6. Expression of recombinant HPGGT was induced by adding isopropyl β-d-1-thiogalactopyranoside (Applichem, Darmstadt, Germany) at a final concentration of 1 mmol/L and was performed for 4 hours at 25°C to minimize the amount of inclusion bodies. Afterward, the whole culture was centrifuged (5000g) for 10 minutes at 4°C. For lysis under native conditions, pellets were solubilized in ice-cold binding buffer (20 mmol/L Tris-HCl, 500 mmol/L NaCl, 20 mmol/L imidazole [Sigma], pH 7.4) containing protease inhibitors (protease inhibitor cocktail for His-tagged proteins; Sigma). Cells were then lysed by 2 freeze-and-thaw cycles in liquid N2 and subsequent sonication (2 × 1 minute with a 5-minute break) on ice. After centrifugation (17,500g at 4°C) for 10 minutes, the supernatant was submitted to DNA and RNA digestion. After a further centrifugation step (22,000g for 10 minutes at 4°C), supernatants were prepared for purification. In the first purification step, 5 mL HisTrapHP columns (GE Healthcare) were used. Purification was performed at room temperature, and samples were kept on ice throughout. Lysate of E coli was loaded on an Ni-sepharose column at 1 mL/min, and flow through was collected. After sample loading, the column was washed with 10 column volumes binding buffer, 10 column volumes wash buffer (20 mmol/L Tris-HCl, 900 mmol/L NaCl, 20 mmol/L imidazole, pH 7.4), and another 10 column volumes binding buffer. Bound protein was eluted with elution buffer (20 mmol/L Tris-HCl, 500 mmol/L NaCl, 100–1000 mmol/L imidazole, pH 7.4) using a stepwise imidazole gradient (100-mmol/L steps). Eluates were collected in one fraction per step of gradient. Each fraction was then tested for GGT enzyme activity and processed to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. For further purification of recombinant HPGGT, enzymatically active fractions from Ni-Sepharose affinity chromatography were pooled, dialyzed for 1 hour against 20 mmol/L Tris-HCl, pH 7.5, at 4°C, and processed to the second purification step. The dialyzed sample was loaded on an Affi-Gel Blue (BioRad Laboratories) column (column volume, 12.3 mL). The column was washed with 2 column volumes of binding buffer, and bound protein was eluted with elution buffer (20 mmol/L Tris-HCl, 50–1000 mmol/L NaCl, pH 7.5) using a stepwise NaCl gradient (50-mmol/L steps). All collected fractions were analyzed by immunoblotting using anti-GGT antibody (see following text) and by GGT enzyme activity assay (see following text) for presence of recombinant HPGGT. Active fractions were pooled, dialyzed against 20 mmol/L Tris-HCl, pH 7.5, for 90 minutes at 4°C, aliquoted, and stored at –80°C until further use. Native VacA (kindly provided by Patrice Boquet, Nice, France) was purified as described by Manetti et al10Manetti 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; 63: 4476-4480Crossref Google Scholar and stored on melting ice until use. Before use, VacA was acid activated for 10 minutes by titration to pH 2 followed by neutralization. Similarly treated buffer was used as control. Site-directed mutagenesis of HPGGT was performed with a QuikChange site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands) according to the manufacturer’s protocol. Primer sequences were as follows: S451/452A sense, 5′-CCAATAAGCGCCCTTTAGCCGCCATGTCGCCTACGATTGTG-3′; S451/452A antisense, 5′-CACAATCGTAGGCGACATGGCGGCTAAAGGGCGCTTATTGG-3′. Successful mutagenesis was confirmed by sequencing. For immunoblot analysis, 107 Jurkat T cells or PBMCs were used. Before the experiment, Jurkat cells were serum starved for 18 hours in medium containing 0.2% FCS. Afterward, cells were released with 10% FCS and treated as depicted. At the indicated time points, the cells were harvested, washed once with ice-cold phosphate-buffered saline, resuspended in 1× lysis buffer (Cell Signaling Technology, Danvers, MA) containing protease inhibitors (2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 μg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride; Sigma), and sonicated with a microtip using a Branson Sonifier 250 (F. Schultheiss, Munich, Germany) on ice for 30 seconds. Lysates were centrifuged at 10,000g for 10 minutes at 4°C, and supernatants were used for immunoblotting. Equal amounts of protein (determined by Bradford assay; BioRad) were separated by Tricine/SDS-PAGE and electrotransferred onto nitrocellulose membranes (Whatman GmbH, Dassel, Germany). For detection, membranes were probed with primary antibodies anti-p27, anti–cyclin D3, anti–cyclin E, anti–c-Myc (Dianova, Hamburg, Germany), anti-Cdk2 (Santa Cruz Biotechnology, Heidelberg, Germany), anti–phospho-AKT (Ser 473), anti–phospho-c-Raf (Ser 338), anti–phospho-p70S6K (Thr 389; Cell Signaling, Beverly, MA), anti–phospho-FKHRL1/Foxo3 (Thr 32; Upstate, Lake Placid, NY), anti-actin (Sigma), and anti-VacA (Austral Biologicals, San Ramon, CA). Binding of primary antibodies was revealed using appropriate peroxidase-conjugated secondary antibodies (Dianova) and chemiluminescent reagents (Perbio Science, Bonn, Germany). For detection of the large subunit of the HPGGT protein, a polyclonal rabbit anti-GGT antibody raised against a synthesized peptide IQPDTVTPSSQIKPGM including amino acid residues 356–371 of the H pylori 1118 gene product (Charles River, Kisslegg, Germany) was used. For detection of HPGGT specific antibodies in human sera, 0.1 μg of purified recombinant HPGGT protein was separated by SDS-PAGE and transferred onto nitrocellulose membranes as described previously. The membrane was stained with Ponceau S solution (0.2% Ponceau S, 3% trichloroacetic acid in H2O) and cut into strips. After blocking (1× Tris-buffered saline + 5% low-fat dry milk), each strip was incubated with serum (diluted 1:20,000 in blocking buffer) of H pylori–infected and –uninfected patients, respectively, at 4°C with agitation overnight. After washing, membrane strips were incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibody (Dianova; dilution 1:10,000) for 1 hour and washed, and binding of serum antibodies to HPGGT protein was revealed by chemiluminescence reaction. Patients’ status of H pylori infection was assessed using conventional H pylori immunoglobulin G enzyme-linked immunosorbent assay. Before analysis, Jurkat T cells (5 × 106 cells/analysis) were serum starved for 18 hours in medium containing 0.2% FCS. After release with 10% FCS and treatment of cells with indicated supernatants of H pylori strains for 24 hours, cell cycle analysis was performed by bromodeoxyuridine-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Sigma) staining according to the manufacturer’s protocol using an FITC-conjugated anti-bromodeoxyuridine antibody (BD Biosciences, Heidelberg, Germany). During subsequent fluorescence-activated cell sorter (FACS) analysis, using a Becton Dickinson FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany), 10,000 events were acquired. Data were analyzed using the Cell Quest software package (BD Biosciences). The assay for GGT activity was adapted from the method of Meister et al.11Meister A. Tate S.S. Griffith O.W. Gamma-glutamyl transpeptidase.Methods Enzymol. 1981; 77: 237-253Google Scholar Briefly, reaction buffer consisting of 20 mmol/L glycyl-glycine (Sigma) as acceptor, 2.5 mmol/L l-γ-glutamyl-p-nitroanilide (Calbiochem, Schwalbach, Germany) as donor substrate, and 100 mmol/L Tris-HCl (pH 8.0) was prepared. In some experiments, pH of assay buffer was titrated between 2 and 10. Supernatants of different H pylori strains, purified recombinant HPGGT, or equine kidney GGT (Sigma) were added, and the reaction proceeded at 37°C for 30 minutes. The release of p-nitroanilide was monitored by spectrophotometry at 405 nm. One unit of activity was defined as the quantity of enzyme that released 1 μmol of p-nitroanilide per minute and per milligram of protein at 37°C. PBMCs (5 × 105 each) were treated for 24 hours as depicted. At indicated time points, cells were removed by centrifugation and supernatants were analyzed for amounts of interleukin-2 (eBioscience, San Diego, CA) and interferon gamma (Biosource, Solingen, Germany) by enzyme-linked immunosorbent assay according to the manufacturer’s instructions. The lower limits of detection were 4 pg/mL. A total of 5 × 105 Jurkat T cells were treated as indicated. After 24 hours, cells were harvested by centrifugation, washed, resuspended in 500 μL Annexin V binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2), and stained for 10 minutes each with 5 μL recombinant Annexin V-FITC (Caltag, Burlingame, CA) and 0.5 μg/ml PI at room temperature in the dark. Apoptotic cells were measured by FACS analysis (see previous text). Data were analyzed using Cell Quest software. Data are presented as mean ± SD. For statistical analysis, Student t test was used. P values less than .05 were considered significant. We have previously shown that a secreted low-molecular-weight protein from H pylori inhibits proliferation of T lymphocytes.3Gerhard M. Schmees C. Voland P. Endres N. Sander M. Reindl W. Rad R. Oelsner M. Decker T. Mempel M. Hengst L. Prinz C. A secreted low-molecular-weight protein from Helicobacter pylori induces cell-cycle arrest of T cells.Gastroenterology. 2005; 128: 1327-1339Abstract Full Text Full Text PDF Scopus (69) Google Scholar To identify the immunosuppressive factor, we performed size-exclusion chromatography with supernatants from H pylori strain G27. In line with our previous work, only fractions eluting with a molecular weight between 30 and 66 kilodaltons inhibited proliferation of lymphocytes, whereas all the other fractions did not (data not shown). Two independent groups previously performed a systematic analysis of secreted H pylori proteins by different proteomics techniques.12Kim N. Weeks D.L. Shin J.M. Scott D.R. Young M.K. Sachs G. Proteins released by Helicobacter pylori in vitro.J Bacteriol. 2002; 184: 6155-6162Google Scholar, 13Bumann D. Aksu S. Wendland M. Janek K. Zimny-Arndt U. Sabarth N. Meyer T.F. Jungblut P.R. Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori.Infect Immun. 2002; 70: 3396-3403Google Scholar Using these data, we assembled all secreted H pylori proteins with a molecular weight between 30 and 66 kilodaltons (Figure 1A). Proteins of obtained chromatographic fractions were further analyzed by SDS-PAGE and silver staining (Figure 1B). We found 4 potential candidates with a size between 30 and 66 kilodaltons (Figure 1B; indicated by arrows) that displayed an elution profile matching the inhibitory activity profile of the fractions. All other protein bands in the inhibiting fractions were also present in the noninhibiting fractions and could therefore not be responsible for inhibition of T-cell proliferation. The molecular weights of 2 of the 4 candidate proteins (Figure 1B) corresponded to fragments of the secreted H pylori protein GGT (HP1118). The first band at 60 kilodaltons might represent the GGT pro-form (mol wt, 61 kilodaltons), with the second at 38 kilodaltons the large subunit of the GGT.14Chevalier C. Thiberge J.M. Ferrero R.L. Labigne A. Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice.Mol Microbiol. 1999; 31: 1359-1372Google Scholar To investigate the presence of catalytically active HPGGT in these supernatant fractions, a photometric GGT activity assay was performed. Figure 1C shows that only fractions inhibiting lymphocyte proliferation (b–f) also display GGT activity. To determine whether GGT was responsible for the observed inhibition of lymphocyte proliferation, isogenic GGT knockout mutants of H pylori were generated. The mutants grew normally in vitro as described by other groups, indicating that GGT is not essential for survival and growth of H pylori.14Chevalier C. Thiberge J.M. Ferrero R.L. Labigne A. Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice.Mol Microbiol. 1999; 31: 1359-1372Google Scholar, 15Shibayama K. Kamachi K. Nagata N. Yagi T. Nada T. Doi Y. Shibata N. Yokoyama K. Yamane K. Kato H. Iinuma Y. Arakawa Y. A novel apoptosis-inducing protein from Helicobacter pylori.Mol Microbiol. 2003; 47: 443-451Google Scholar, 16McGovern K.J. Blanchard T.G. Gutierrez J.A. Czinn S.J. Krakowka S. Youngman P. Gamma-Glutamyltransferase is a Helicobacter pylori virulence factor but is not essential for colonization.Infect Immun. 2001; 69: 4168-4173Google Scholar Supernatants of these mutants were tested for their proliferation-inhibiting activity toward isolated human T cells and PBMCs, stimulated with anti-CD3/CD28 or PMA/ionomycin, in comparison to the corresponding wild-type strain (Figure 2A and C). Here, the inhibitory potential of supernatants from ΔGGT bacteria toward primary human T cells and PBMCs was completely abrogated. To exclude spontaneous recombination and reactivation of the GGT, supernatants from GGT-deficient bacteria were verified by measuring enzyme activity and by immunoblotting using a polyclonal antibody that we raised against the large subunit of HPGGT (Figure 2B). The loading control shows the presence of secreted VacA protein in supernatants from wild-type and GGT-deficient bacteria (Figure 2B). Thus, GGT is responsible for inhibition of T-cell proliferation by H pylori in our system, while VacA had no effect at the concentrations of bacterial supernatants used. To further show that the observed inhibition was mediated solely by the HPGGT, we expressed a recombinant His-tagged HPGGT protein in E coli. The protein was purified to homogeneity by chromatography as described in Materials and Methods. SDS-PAGE and silver staining as well as immunoblotting indicated that the recombinant HPGGT was synthesized as a pro-form and subsequently processed into a large and small subunit with molecular weights of ∼38 and ∼20 kilodaltons, respectively (Figure 3A and B). The recombinant protein showed strong GGT activity (Figure 3C) and efficiently inhibited PBMC proliferation (Figure 3D). To exactly determine the inhibitory activity of the recombinant GGT, a dose-response curve was generated by incubating serial dilutions of recombinant HPGGT protein or purified VacA with PMA/ionomycin-stimulated human PBMCs, yielding a median inhibitory concentration value for GGT of approximately 0.5 μg/mL (Figure 3D). Concentrations as low as 1 μg/mL induced complete inhibition of T-cell proliferation. In comparison, purified, acid-activated VacA protein (kindly provided by P. Boquet) inhibited T-cell proliferation at concentrations of 5–10 μg/mL. Acid-treated buffer used as a control did not inhibit PBMC proliferation (not shown). Thus, the inhibitory activity of HPGGT in our study was observed at 10–20 times lower concentrations compared with VacA. Similar differences in activity were observed using bacterial supernatants (not shown). Because the amount of these proteins could differ in bacterial supernatants (reflecting differences in level of secretion or protein stability), we compared the amount of HPGGT and VacA in bacterial supernatants with defined quantities of recombinant proteins by semiquantitative comparison of signals using immunoblotting. Here, we observed no major differences in the amount of these proteins. Using 10 μg of total bacterial supernatants, the intensity of HPGGT or VacA bands in the supernatants correlated with that of 50–100 ng of recombinant proteins (data not shown), excluding major differences in protein availability. Therefore, at low concentrations of bacterial supernatants that we used in our assay system, the inhibitory effect is dependent on HPGGT and independent of VacA protein. Further, we exposed recombinant GGT to acidic conditions to assess its functional activity at lo
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