Helicobacter pylori γ-Glutamyl Transpeptidase Is a Pathogenic Factor in the Development of Peptic Ulcer Disease  Min Gong, Samantha Shi Min Ling, Sook Yin Lui, Khay Guan Yeoh, Bow Ho  Gastroenterology  Volume 139, Issue 2, Pages 564-573 (August 2010) DOI: 10.1053/j.gastro.2010.03.050 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 GGT activity of H pylori strains isolated from patients with different disease status. GGT activity of 98 H pylori isolates obtained from patients with NUD (n = 44), DU (n = 29), and GU (n =25). Values of GGT activity represent means ± SDs of triplicate measurements and are representative of 3 experiments. *P < .001 for comparison of NUD vs DU or GU, **P = .043 for comparison of DU vs GU. Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Purification of native H pylori GGT. M, low-molecular-weight protein marker; 1, cell sonicate before purification; protein fraction eluted from 2, first ion exchange chromatography (IEX); 3, gel filtration; 4, second IEX. Circles indicate the large and small subunit fragments of GGT protein of 38 and 22 kDa, respectively. Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 H pylori GGT induces H2O2 generation in AGS or primary human gastric epithelial cells. (A and B) H2O2 generation in AGS cells (5 × 104) with various treatments was examined by a fluorescence-based analysis. Data are means ± SDs of the H2O2 generated from AGS in each sample from 3 independent experiments. (C) H2O2 generation in primary gastric cells (5 × 104) with various treatments. NC, uninfected cells. Cells were treated with SBC, 10 mmol/L serine-borate complex; DFO, 50 μmol/L DFO; GSH, 0.1 mmol/L GSH, and 1 mmol/L glycyl-glycine; Fe3+, 20 μmol/L Fe3+; GGT, HP-nGGT (1.5 mU); BSA, 10 ng bovine serum albumin. The significant difference was compared with H pylori WT-treated cells (*P < .05), untreated cells (**P < .05), GGT-treated cells (***P < .01), and GGT + GSH–treated cells (****P < .01). Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 H pylori GGT induces NF-κB activation. (A) H pylori GGT–mediated I-κB degradation and NF-κB activation in which AGS cells (2 × 107) were treated with H pylori WT, various mutants, or HP-nGGT (500 mU). (B) AGS cells transfected with pNF-κB-MetLuc2-Reporters were cocultured with H pylori WT, various mutants, or HP-nGGT. Values represent the means ± SDs of luciferase activity relative to uninfected control from triplicate measurements and are representative of 3 experiments. *P < .05 for comparison of WT vs Δggt, **P < .05 for comparisons of HP-nGGT in the presence of various inhibitors vs HP-nGGT. (C) Preincubating AGS cells with inhibitors, NAC (10 mmol/L), ST638 (30 μmol/L), and MG132 (30 μmol/L) for 30 minutes blocked the H pylori GGT–mediated I-κB degradation and NF-κB activation. β-Actin was used as loading control. Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 H pylori GGT induces IL-8 production. Enzyme-linked immunoabsorbent assay of (A) IL-8 generation from AGS cells (5 × 105) after treatments with H pylori WT, various mutants, or HP-nGGT (15 mU) over a period of 24 hours. (B) IL-8 generation from primary gastric epithelial cells after treatment with H pylori WT, Δggt, and HP-nGGT for 4 hours. (C) Effects of inhibitors on H pylori GGT induced IL-8 production where AGS cells (5 × 105) were pretreated with or without the inhibitors NAC (10 mmol/L), ST638 (30 μmol/L), and MG132 (30 μmol/L) for 30 minutes. After which, the mixtures were cocultured with HP-nGGT for another 6 hours. (D) IL-8 generation from primary macrophages after treatment with H pylori WT, various mutants, or HP-nGGT for 4 hours. *P < .05 of HP-nGGT–treated cells vs negative control. **P < .05 preincubation of various inhibitors in HP-nGGT–treated cells vs HP-nGGT–treated cells alone. ***P < .05 for comparison of cells infected with WT vs Δggt. NC, uninfected cells culture served as negative control. (E) Real-time PCR analysis detecting IL-8 mRNA in AGS cells treated with H pylori WT, various mutants, HP-nGGT, or any one of the inhibitors. Values from the enzyme-linked immunoabsorbent assay test and real-time PCR represent the means ± SDs of triplicate measurements and are representative of 3 experiments. Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 H pylori GGT induces oxidative DNA damage in AGS. (A) AGS cells with oxidized DNA (8-OH-dG) as analyzed by flow cytometry. (B) DNA damage in AGS cells as examined with the comet assay. *P < .05 for comparison of WT vs Δggt; **P < .05 for comparisons of HP-nGGT in the presence of NAC vs HP-nGGT. Data are representative of 3 separate experiments. Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1A Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1B Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1C Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1D Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1E Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1F Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1G Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2A Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2B Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5A Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5B Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 6 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 7 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 8 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 9 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 10 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Table 1 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Table 2 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Table 3 Gastroenterology 2010 139, 564-573DOI: (10.1053/j.gastro.2010.03.050) Copyright © 2010 AGA Institute Terms and Conditions