Gastric epithelial cell death caused by Helicobacter suis and Helicobacter pylori γ‐glutamyl transpeptidase is mainly glutathione degradation‐dependent - Flahou - 2011 - Cellular Microbiology - Wiley Online Library Free Access Gastric epithelial cell death caused by Helicobacter suis and Helicobacter pylori γ-glutamyl transpeptidase is mainly glutathione degradation-dependent Bram Flahou, Corresponding Author Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineE-mail Bram.Flahou@UGent.be; Tel. (+32) 9 264 73 76; Fax (+32) 9 264 74 94.Search for more papers by this authorFreddy Haesebrouck, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorKoen Chiers, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorKim Van Deun, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorLina De Smet, Department of Biochemistry and Microbiology, Faculty of SciencesSearch for more papers by this authorBart Devreese, Department of Biochemistry and Microbiology, Faculty of SciencesSearch for more papers by this authorIsabel Vandenberghe, Department of Biochemistry and Microbiology, Faculty of SciencesSearch for more papers by this authorHerman Favoreel, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.Search for more papers by this authorAnnemieke Smet, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorFrank Pasmans, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorKatharina D\'Herde, Department of Basic Medical Sciences, Faculty of Medicine and Health Science, Ghent University, Ghent, Belgium Shared senior authorship.Search for more papers by this authorRichard Ducatelle, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine Shared senior authorship.Search for more papers by this author Bram Flahou, Corresponding Author Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineE-mail Bram.Flahou@UGent.be; Tel. (+32) 9 264 73 76; Fax (+32) 9 264 74 94.Search for more papers by this authorFreddy Haesebrouck, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorKoen Chiers, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorKim Van Deun, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorLina De Smet, Department of Biochemistry and Microbiology, Faculty of SciencesSearch for more papers by this authorBart Devreese, Department of Biochemistry and Microbiology, Faculty of SciencesSearch for more papers by this authorIsabel Vandenberghe, Department of Biochemistry and Microbiology, Faculty of SciencesSearch for more papers by this authorHerman Favoreel, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.Search for more papers by this authorAnnemieke Smet, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorFrank Pasmans, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary MedicineSearch for more papers by this authorKatharina D\'Herde, Department of Basic Medical Sciences, Faculty of Medicine and Health Science, Ghent University, Ghent, Belgium Shared senior authorship.Search for more papers by this authorRichard Ducatelle, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine Shared senior authorship.Search for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Helicobacter (H.) suis is the most prevalent non-H. pylori Helicobacter species colonizing the stomach of humans suffering from gastric disease. In the present study, we aimed to unravel the mechanism used by H. suis to induce gastric epithelial cell damage. H. suis lysate induced mainly apoptotic death of human gastric epithelial cells. Inhibition of γ-glutamyl transpeptidase (GGT) activity present in H. suis lysate and incubation of AGS cells with purified native and recombinant H. suis GGT showed that this enzyme was partly responsible for the observed apoptosis. Supplementation of H. suis or H. pylori GGT-treated cells with glutathione strongly enhanced the harmful effect of both enzymes and resulted in the induction of oncosis/necrosis, demonstrating that H. suis and H. pylori GGT-mediated degradation of glutathione and the resulting formation of glutathione degradation products play a direct and active role in the induction of gastric epithelial cell death. This was preceded by an increase of extracellular H2O2 concentrations, generated in a cell-independent manner and causing lipid peroxidation. In conclusion, H. suis and H. pylori GGT-mediated generation of pro-oxidant glutathione degradation products brings on cell damage and causes apoptosis or necrosis, dependent on the amount of extracellular glutathione available as a GGT substrate. Introduction Helicobacter (H.) pylori is considered to be the primary cause of gastritis, peptic ulcer disease as well as gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma in humans (Marshall and Warren, 1984; Parsonnet et al., 1991; Parsonnet et al., 1994; Kusters et al., 2006). In 0.2–6% of gastric biopsies, however, large spiral-shaped non-H. pylori helicobacters (NHPH) are found (Haesebrouck et al., 2009). Humans infected with the latter have been reported to suffer from gastritis (Stolte et al., 1997) sometimes accompanied by gastric ulcer, gastric MALT lymphoma and gastric adenocarcinoma (Morgner et al., 1995; 2000; Stolte et al., 1997; Debongnie et al., 1998). These bacteria have long been referred to as ‘H. heilmannii’. In reality, they represent a group of different bacterial species with similar spiral morphology, which are also highly prevalent in different animal species (Haesebrouck et al., 2009; 2011). The most prevalent NHPH in humans is H. suis (De Groote et al., 2005; Van den Bulck et al., 2005), hosted largely by pigs and only recently isolated in vitro (Baele et al., 2008). Until now, virtually nothing is known about possible H. suis virulence factors involved in human gastric pathology (Haesebrouck et al., 2009). Previously, we have shown that a H. suis infection induces loss of gastric epithelial, mainly parietal cells in mice and Mongolian gerbil models of human gastric disease (Flahou et al., 2010a). Gastric cell death is considered an important mechanism involved in gastric ulcer formation (Dixon, 2001), gastric atrophy and gastric cancer (Shirin and Moss, 1998). Therefore, in the present study, we investigated the ability of H. suis to directly cause gastric epithelial cell death in vitro. Additionally, we explored which mechanisms of H. suis are involved in this process. In theclosely related H. pylori, several factors have been shown to induce gastric epithelial cell death, including VacA (Cover et al., 2003), and a γ-glutamyl transpeptidase (GGT) (Shibayama et al., 2003; Kim et al., 2007a; Gong et al., 2010). For the latter, the exact mechanism involved in the induction of gastric epithelial cell apoptosis remains to be elucidated. Besides its role in gastric epithelial cell apoptosis, H. pylori GGT has also been shown to play a pivotal role in the upregulation of COX-2 and EGF-related peptide expression and the inhibition of T-cell proliferation (Busiello et al., 2004; Schmees et al., 2007). Membrane-associated GGT activity is often present in eukaryotic cells, where the enzyme plays an important role in the metabolism of glutathione (Orlowski and Meister, 1970), a free thiol maintaining an optimal intracellular redox environment (Circu and Aw, 2010). In this study, we demonstrate that H. suis actively causes apoptosis and necrosis of gastric epithelial cells. We identified H. suis ggt, encoding an active GGT, as an important factor involved in both apoptotic and necrotic death of these cells, with the type of cell death depending on the rate of GGT-induced glutathione disintegration into its degradation products. This actively causes an extracellular and cell-independent increase of the oxidative stress burden, leading to lipid peroxidation and finally resulting in gastric epithelial cell death. Finally, we revealed that H. pylori GGT acts in a very similar way to induce cell death. Viability of H. suis bacteria is drastically reduced, already after 1 h of incubation in various cell culture media, including Ham\'s F12 which is used for cultivation of AGS cells (human gastric adenocarcinoma cell line). Therefore, in order to investigate the effect of H. suis on the viability of gastric epithelial cells, AGS cells were incubated for 20–44 h with H. suis lysate (50–200 µg ml−1). After 20 h of incubation, flow cytometric analysis revealed an increase of the percentage of apoptotic cells, as shown by active caspase-3 staining or determination of the number of hypoploid cells (Fig. 1A). At this time point, no significant increase of the percentage of cells showing loss of plasma membrane integrity was observed, as detected by propidium iodide (PI) staining. Also after 44 h of incubation, an increase of the number of apoptotic cells was observed, although an equally high increase of the number of PI-positive cells was detected in AGS cultures treated with 200 µg ml−1 lysate (Fig. 1B–D). Possibly, these PI-positive cells could reflect late apoptotic cells, showing loss of plasma membrane integrity. Heat and trypsin treatment of H. suis lysate almost completely abolished the cell death-inducing capacity (data not shown), allocating one or more proteins as the inducer(s) of cell death. Death of AGS cells induced by incubation with H. suis lysate. A. Flow cytometric analysis of AGS cells treated for 20 h with 50 or 200 µg ml−1 whole bacterial cell lysate of H. suis (HS) strain 5 revealed a clear increase of the percentage of apoptotic cells, as determined by activated caspase-3 staining or determination of cell hypoploidy. No increase of the percentage of cells with loss of plasma membrane integrity (PI-positive) was observed. B. After 44 h of incubation, an increase of the number of apoptotic cells was observed, as well as an increase of the number of PI-positive cells, although only for cell cultures treated with 200 µg ml−1 lysate. C. Fluorescence microscopic images of activated caspase-3 (green) and nuclear Hoechst staining (blue) of AGS cells treated with HBSS for 44 h. Only one activated caspase-3-positive cell is observed, reflecting the basal level of apoptosis in normal AGS cell cultures. D. In AGS cell cultures treated with H. suis lysate for 44 h, an increased number (6) of cells positive for activated caspase-3 can be observed. Note that activated caspase-3-positive cells also reveal nuclear condensation and fragmentation, which can typically be observed in apoptotic cells. Shown are the mean values (± SD) of three independent experiments. An asterisk (*) depicts a statistically significant difference compared with HBSS-treated cells (P   0.05). HBSS, Hank\'s balanced salt solution; HS, Helicobacter suis; PI, propidium iodide. Inhibition of GGT activity in H. suis whole bacterial cell lysates strongly reduces H. suis-induced death of AGS cells Gamma-glutamyl transpeptidase activity was detected in the supernatant fluid of a 24-h-old biphasic culture of H. suis strain 5 (1.8–2.0 U l−1). No such activity could be detected in the supernatant fluid of the non-inoculated biphasic culture medium, showing that H. suis is responsible for the observed GGT activity. Moreover, GGT activity was detected in whole bacterial cell lysate of all nine H. suis strains available in our laboratory and ranged from 6 to 25 mU mg−1 total protein (Fig. 2A). Screening of the whole-genome sequence of H. suis strain 5 revealed the presence of a 1668 bp gene encoding a GGT (NCBI Accession No. on http://www.ncbi.nlm.nih.gov/: GU972556 and ADF28653.1). Based on the amino acid sequence, as predicted by SignalP 3.0 software (http://www.cbs.dtu.dk/services/SignalP/), the H. suis GGT was 72% identical to the GGT of H. pylori strain 26695 (NCBI Accession No.: NP_207909.1) and even 81% and 83% identical to the GGT of the closely related species H. bizzozeronii and H. felis respectively (NCBI Accession No.: CBZ41137.1 and CBY82772.1). Amino acid sequence alignment using clustal w software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) of several bacterial and mammalian GGTs revealed a strong conservation of amino acids in the H. suis GGT active site, especially compared with the GGT of other gastric helicobacters, including H. pylori, H. felis and H. bizzozeronii (Boanca et al., 2007) (Fig. 2B). GGT activity of H. suis and amino acid sequence analysis of H. suis GGT. A. Comparison of all nine available H. suis strains revealed a remarkable disparity of GGT activity detected in whole bacterial cell lysates. HS1–10, H. suis strains 1–10. B. Comparison of the amino acid sequence of H. suis GGT with several bacterial and mammalian GGTs revealed a high conservation of amino acids in the active site for all four gastric helicobacters displayed. Amino acids important for processing and substrate binding, based on the information available for the H. pylori GGT, are marked in black (Boanca et al., 2007). The arrow indicates the putative predicted signal sequence cleavage site of the H. suis GGT, as predicted by the SignalP 3.0 program (http://www.cbs.dtu.dk/services/SignalP/). The arrowhead indicates the site where putative cleavage of the pro-enzyme into its two subunits occurs. An asterisk (*) shows amino acids conserved for all aligned sequences. A colon (:) shows conserved amino acid substitutions, whereas a dot (.) shows semi-conserved amino acid substitutions. Hsuis: H. suis GGT strain 5 (ADF28653.1); Hfelis: H. felis GGT strain ATCC 49179 (CBY82772.1); Hbizzo: H. bizzozeronii GGT strain CCUG 35545 (CBZ41137.1); Hpylori: H. pylori GGT strain 26695 (NP_207909.1); Hbilis: H. bilis GGT strain ATCC 43879 (ZP_04580843.1); Ecoli: E. coli GGT strain K-12 (NP_417904.1); Human: GGT1 precursor Homo sapiens (NP_005256.2); Mouse: GGT1 precursor Mus musculus (NP_032142.1). This urged us to investigate the possible role of this enzyme in the induction of gastric epithelial cell death. Incubation of H. suis lysate with acivicin, a known GGT inhibitor (Schmees et al., 2007), completely abolished GGT activity (Fig. 3A) and reduced the H. suis-induced loss of plasma membrane integrity (Fig. 3B). These data suggest that H. suis GGT is involved in the induction of gastric epithelial cell death. Inhibition of GGT activity present in H. suis lysate and the effect on cell death induction. A. Pre-treatment of HS5 (H. suis strain 5) lysate with acivicin (50 µM) completely abolished its GGT activity. B. Inhibition of GGT activity present in H. suis (HS) lysate with the known GGT inhibitor acivicin affected its cell death-inducing capacity, as shown by the decrease of the number of PI-positive cells. Shown are the mean values (± SD) of one independent experiment. Asterisks (*) depict relevant statistically significant differences (P   0.05). HBSS: control cells treated with Hank\'s balanced salt solution. To confirm whether H. suis GGT is involved in the induction of gastric epithelial cell death, we first purified the native H. suis GGT for use in cell death experiments. After crude ultrafiltration fractionation, cation exchange chromatography, size exclusion chromatography and a second cation exchange chromatography, fraction F2 was obtained containing GGT activity (90 mU ml−1) and revealing protein bands of approximately 40 and 20 kDa (Fig. 4A). The 40 kDa band, putatively corresponding to the large subunit of H. suis GGT, was further analysed by mass spectrometry. Two of the peptides with the largest peak intensity and showing molecular masses of 1206.39 and 1240.58 were selected for MS/MS. The obtained MALDI-MS/MS spectra corresponded to peptides from H. suis GGT (NCBI Accession No.: ADF28653.1, coded by GU972556.1). Additionally, the observed mass of five peptides with a large peak intensity (919.4516; 1119.4056; 1174.4988; 1206.4401 and 1240.5835 Da) revealed by MALDI-MS peptide mass fingerprinting showed a good correlation with that of five theoretical peptide masses (919.5359; 1119.6044; 1174.6136; 1206.5538 and 1240.7008 Da) obtained after theoretical trypsin cleavage of H. suis GGT, as calculated with the PeptideMass tool. These data strongly suggest that the H. suis sonicate-derived fraction F2 indeed largely consists of native H. suis GGT. Purification of native H. suis GGT, recombinant H. suis and H. pylori GGT and processing of the enzymes. A. Proteins were visualized with Brilliant Blue G – Colloidal staining. Column M: Protein marker with size labelling in kilodalton (kDa) at the left. Column 1 shows an eluate after Ni-affinity chromatography, with a clear band of approximately 60 kDa, representing the unprocessed recombinant H. suis GGT (pro-enzyme). Columns 2 and 3 show recombinant H. suis and H. pylori GGT, respectively, after gel filtration chromatography, revealing the presence of three protein bands, reflecting the processing of the pro-enzymes (∼60 kDa) into a large (∼40 kDa) and small (∼20 kDa) subunit. Column 4 shows purified fraction F2, containing native H. suis GGT, with the putative large (full arrow) and small (dotted arrow) subunit indicated by the arrows. B. Processing at 37°C of both recombinant enzymes (rHSGGT, rHPGGT = recombinant H. suis and H. pylori GGT respectively) was monitored using the GGT activity assay. Maximum activity (and thus processing) for rHSGGT and rHPGGT was reached after 20 and 6 h respectively. Next, we expressed a recombinant 6xHis-tagged H. suis and H. pylori GGT in Escherichia coli. After nickel affinity chromatography, the enzymes were purified to homogeneity by gel filtration chromatography. SDS-PAGE analysis showed that recombinant H. suis GGT (rHSGGT) was mainly purified as a protein with a molecular weight of ∼60 kDa, although two discrete protein bands with a molecular weight of ∼40 and ∼20 kDa, respectively, could also be seen (Fig. 4A). This suggests that H. suis GGT is synthesized as a precursor enzyme with subsequent processing into a large and a small subunit, which has been described for other bacterial GGTs, such as the H. pylori and E. coli GGT (Suzuki and Kumagai, 2002; Boanca et al., 2006). Indeed, SDS-PAGE analysis of purified recombinant H. pylori GGT (rHPGGT) also revealed three protein bands of ∼60, ∼40 and ∼20 kDa (Fig. 4A), with the latter two being more dense compared with the rHSGGT. The identity of both recombinant enzymes was confirmed by mass spectrometry. After MALDI-MS peptide mass fingerprinting of the 40 kDa large subunits of the recombinant H. suis and H. pylori GGT, two peptides with high peak intensity were further characterized by MS/MS. These peptides showed molecular masses of 1206.3900/1240.5350 and 1648.6100/1481.5920 Da for rHSGGT and rHPGGT respectively. Protein database searches of the tandem mass spectral data showed a significant hit with H. suis GGT (GenBank Accession No.: ADF28653.1) and H. pylori GGT (GenBank Accession No.: NP_207909) respectively. Processing of both recombinant enzymes at 37°C was monitored with the GGT assay. Recombinant H. pylori GGT reached a maximum activity already after 6 h of incubation, whereas the rHSGGT reached its maximum activity after 20 h of processing (Fig. 4B). In cell death assays comparing the effect of rHPGGT and rHSGGT, processed enzymes showing maximum activity were used. Native H. suis GGT and recombinant H. suis/H. pylori GGT cause gastric epithelial cell death AGS cells were treated for 44 h with the native H. suis GGT-containing fraction F2, resulting in a final GGT activity of approximately 4 mU ml−1 in each treated well, which equalled the amount of GGT activity in wells of AGS cells treated with 200 µg ml−1H. suis lysate. Compared with HBSS-treated cells, a clear increase of the percentage of apoptotic cells, as well as of the percentage of cells showing loss of plasma membrane integrity was observed, confirming that H. suis GGT plays a prominent role in the induction of gastric epithelial cell death (Fig. 5A). Gastric epithelial cell death induction by native H. suis GGT and recombinant H. suis or H. pylori GGT. A. As analysed by flow cytometry, incubation for 44 h of AGS cells with native H. suis GGT (fraction F2) causes an increase of both the percentage of apoptosis and the percentage of PI-positive cells, compared with HBSS-treated cells. B and C. (B) Incubation of AGS cells for 20 h with various concentrations of recombinant H. suis or H. pylori GGT resulted in an increase of apoptosis for concentrations ranging from 0.5–4 µg ml−1. However, no increase of the percentage of PI-positive cells could be observed in these cultures (C). For (A)–(C), the mean values (± SD) of one representative experiment or three independent experiments are shown. An asterisk (*) denotes a statistically significant different result compared with HBSS-treated cells. D. Light microscopic examination (magnification: 630×) of a toluidine blue-stained semithin section of adherent 20 h HBSS-treated cells, showing normal cell morphology. E. Light microscopic (magnification: 630×) examination of a toluidine blue-stained semithin section of adherent 20 h rHSGGT (2 µg ml−1)-treated AGS cells, showing blebbing of the plasma membrane and the formation of apoptotic bodies (arrows). F–H. Transmission electron microscopic (TEM) images of adherent AGS cultures treated for 20 h with 2 µg ml−1 rHSGGT (F, G) or rHPGGT (H), showing blebbing of the plasma membrane and the formation of apoptotic bodies (arrows). HBSS, Hank\'s balanced salt solution; rHSGGT, recombinant H. suis GGT; rHPGGT, recombinant H. pylori GGT; PI, propidium iodide. Incubation of AGS cells for 20 h with 0.5–4 µg ml−1 recombinant H. suis and H. pylori GGT caused a significant increase of the number of apoptotic cells (Fig. 5B). In contrast, no increase of the number of PI-positive cells could be observed (Fig. 5C). These findings were confirmed by light and transmission electron microscopic examination of the adherent population of treated cell cultures. Besides a sporadic apoptotic cell, the vast majority of cells in 20 h HBSS-treated control cultures showed no signs of cell death (Fig. 5D). A larger number of cells in rHSGGT- and rHPGGT-treated cell cultures showed blebbing of the plasma membrane and the presence of apoptotic bodies, of which some contained condensed chromatin (Fig. 5E–H). These are all morphological features of apoptosis (Krysko et al., 2008; Kroemer et al., 2009). After 44 h of incubation, a similar increase of apoptosis could be observed for rHSGGT and rHPGGT (Fig. 6A). Only the lowest concentration of 0.2 µg ml−1 did not yield a significant difference compared with control cells. When compared with control cells, a higher percentage of PI-positive cells could be observed for final rHSGGT and rHPGGT concentrations of 0.5–8 µg ml−1, although only incubation with 8 and 4 µg ml−1 rHSGGT yielded a statistically significant increase (Fig. 6B). These findings were confirmed by light and transmission electron microscopic examination of the adherent cell populations. In 44 h HBSS-treated control cultures, the vast majority of cells showed no signs of cell death (Fig. 6C and D), although approximately 3–4% of the cells showed blebbing of the plasma membrane and the presence of apoptotic bodies, compatible with apoptosis. In cultures treated with 2 µg ml−1 rHSGGT or rHPGGT, an increase of the number of apoptotic cells was observed, compatible with the results of flow cytometry. Besides apoptosis, however, cells appeared containing large, clear cytoplasmic vacuoles in cultures treated with higher concentrations of rHSGGT (4 and 8 µg l−1) (Fig. 6E–G), which is compatible with oncosis or primary necrosis (Krysko et al., 2008; Kroemer et al., 2009). Moreover, these cells did not show morphological signs of apoptosis. Possibly, the presence of these cells may account for the observed increase of PI-positive cells in these cultures. Induction of gastric epithelial cell death after 44 h incubation with recombinant H. suis or H. pylori GGT. A. Incubation of AGS cells for 44 h with various concentrations of recombinant H. suis or H. pylori GGT resulted in an increase of apoptosis for concentrations ranging from 0.5 to 4 µg ml−1, as determined by flow cytometry. B. In these cultures, an increase of the number of PI-positive cells was observed, but only statistically significant for rHSGGT concentrations of 4 and 8 µg ml−1. For (A) and (B), the mean values (± SD) of three independent experiments are shown. An asterisk (*) denotes relevant statistically significant differences when compared with HBSS-treated control cultures. C and D. Bright-field microscopic (magnification: 630×) and transmission electron microscopic (TEM) image, respectively, of an adherent control AGS culture treated for 44 h with HBSS, showing normal cells. E–G. Phase-contrast image of one adherent cell (E; magnification: 1000×) and TEM images (F, G) of one adherent cell, respectively, in an AGS culture treated for 44 h with 8 µg ml−1 rHSGGT. In these cultures, and to a lesser extent in those treated with 4 µg ml−1 rHSGGT, numerous cells appeared containing large, clear cytoplasmic vacuoles, compatible with oncosis or primary necrosis. Moreover, these cells did not show morphological signs of apoptosis. HBSS, Hank\'s balanced salt solution; rHSGGT, recombinant H. suis GGT; rHPGGT, recombinant H. pylori GGT; PI, propidium iodide. Pre-treatment of rHSGGT with 50 µM acivicin rendered GGT activity completely undetectable (Fig. 7A). Incubation of AGS cells for 44 h with 8 µg ml−1 of this inhibited rHSGGT, showed a complete loss of the cell death-inducing capacity of this enzyme (Fig. 7B). Inhibition of enzymatic activity of recombinant H. suis GGT with acivicin, a known GGT inhibitor. A. Pre-treatment of rHSGGT with acivicin (50 µM), completely abolished its GGT activity. B. rHSGGT-induced cell death could be abolished by acivicin-mediated inhibition of GGT activity, as shown by the decrease of the number of PI-positive cells. Shown are the results of one representative experiment. An asterisk (*) depicts relevant statistically significant differences (P   0.05). HBSS, Hank\'s balanced salt solution; rHSGGT, recombinant H. suis GGT; PI, propidium iodide. Recombinant H. suis and H. pylori GGT cause cell death of AGS cells through extracellular glutathione degradation Normal basal values of total glutathione in supernatants of an overnight culture of AGS cells varied between 30 and 140 µM. These concentrations did not alter significantly when AGS cells were incubated with 2 µg ml−1 rHSGGT for up to 44 h (not shown). To detect degradation of extracellular reduced glutathione (GSH) in the AGS model, concentrations of supplemented GSH were monitored in the supernatant of AGS cell cultures in the presence or absence of 2 µg ml−1 rHSGGT (Fig. 8A). Without the addition of rHSGGT, only9% of 5 mM supplemented GSH was degraded after 44 h of incubation, whereas 95% of supplemented GSH was degraded in the presence of rHSGGT. Pre-incubation of rHSGGT with 50 µM acivicin strongly reduced the capacity of the enzyme to degrade GSH. For comparison of the capacity of rHSGGT and rHPGGT to degrade GSH, the degradation of 5 mM supplemented GSH in AGS cell culture supernatants in the presence of 2 µg ml−1 fully processed rHSGGT or rHPGGT was monitored, revealing similar results for both enzymes (Fig. 8B). Recombinant H. suis GGT catalyses the degradation of reduced glutathione. A. Monitoring of the degradation of supplemented 5 mM GSH in AGS cell culture supernatant without rHSGGT, with 2 µg ml−1 rHSGGT and with 2 µg ml−1 acivicin-inhibited rHSGGT, showing rHSGGT-mediated degradation of GSH in the AGS model used. B. Similar rates of GSH degradation were assessed for rHSGGT and rHPGGT (both fully processed) at a concentration of 2 µg ml−1 in AGS cell culture supernatant. Shown are the mean (± SD) results of two independent experiments. An asterisk (*) depicts a relevant statistically significant difference. GSH, reduced glutathione; Ac, acivicin; rHSGGT, recombinant H. suis GGT; rHPGGT, recombinant H. pylori GGT. In AGS cell culture supernatant, 2 µg ml−1 rHSGGT reduced 1 mM GSH to only 55 µM after 20 h incubation, which equals normal basal GSH values present in AGS cell culture supernatants. For this reason, AGS cells were treated with 2 µg ml−1 rHSGGT or rHPGGT and 5 mM GSH (as opposed to 1 mM) to determine the possible role of a sustained, 44-h-lasting rHSGGT/rHPGGT-mediated GSH degradation in the induction of cell death. Supplementation of HBSS-treated AGS cells with 5 mM GSH did not alter the percentage of apoptosis observed after 20 h of incubation (Fig. 9A). When cells were treated with a combination of rHSGGT or rHPGGT and 5 mM GSH, often a decrease of the number of apoptotic cells could be observed compared with cells treated with enzyme alone, indicating that GSH protected these cells from GGT-induced apoptosis (Fig. 9A). No significant differences were observed for the percentage of PI-positivity in cell cultures treated for 20 h with a combination of rHSGGT or rHPGGT and GSH, compared with cultures treated only with rHSGGT or rHPGGT (Fig. 9B). Role of H. suis or H. pylori GGT-dependent glutathione degradation in the modulation of cell death. A and B. Incubation of AGS cells for 20 h with 5 mM GSH and 2–4 µg ml−1 rHSGGT or rHPGGT did not result in an increase of the percentage of apoptotic or PI-positive cells compared with AGS cells incubated with rHSGGT/rHPGGT alone. In contrast, rather a decrease of apoptosis was observed. C. This tendency of GSH to protect cells against apoptosis could also be observed after 44 h of incubation. D. In contrast, incubation of cells with a combination of GSH and rHSGGT/rHPGGT resulted in a vast increase of the percentage of PI-positive cells, compared to cells treated with rHSGGT/rHPGGT alone. In contrast, treatment of HBSS-treated cells with GSH resulted in a decrease of PI-positivity. The mean results (± SD) of one representative experiment are shown. An asterisk (*) depicts relevant statistically significant differences (P   0.05). HBSS, Hank\'s balanced salt solution; GSH, reduced glutathione; rHSGGT, recombinant H. suis GGT; rHPGGT, recombinant H. pylori GGT; PI, propidium iodide. Similar to the results of 20 h incubation, treatment of AGS cells for 44 h with a combination of recombinant H. suis or H. pylori GGT and 5 mM GSH, often resulted in a decrease of the number of apoptotic cells compared with cultures treated with rHSGGT or rHPGGT alone (Fig. 9C). Moreover, supplementing HBSS-treated cells with GSH resulted in a decrease of the number of PI-positive cells compared with cultures treated with HBSS alone. These findings again confirm that extracellular supplementation with GSH protects gastric epithelial cells from cell death (Fig. 9D). In contrast, however, incubation for 44 h with a combination of GSH on one hand and rHSGGT or rHPGGT on the other hand, resulted in a clear increase of the number of PI-positive cells (Fig. 9D). These results demonstrate that H. suis or H. pylori GGT-mediated degradation of glutathione can directly cause non-apoptotic death of AGS cells. Upon light microscopic examination of treated AGS cultures prior to cell death analysis, a much higher rate of cell detachment could be observed in cultures treated with a combination of rHSGGT or rHPGGT and 5 mM GSH, compared with all other treatments. So we decided to have a closer look at these floating non-adherent populations. First, the ratio floating/adherent cells was determined. The results indeed confirmed that treatment of cells for 44 h with a combination of GSH and rHSGGT or rHPGGT caused a marked increase of cell detachment (Fig. 10A). More detailed analysis of these floating populations of cultures treated with HBSS/GSH, rHSGGT/GSH and rHPGGT/GSH revealed that the majority of the cells treated with HBSS and GSH showed clear chromatin condensation near the nuclear envelope (Fig. 10B–D), one of the hallmarks of apoptosis (Krysko et al., 2008). In contrast, the majority of floating cells in cultures treated with a combination of GSH on one hand and rHSGGT or rHPGGT (2 µg ml−1) on the other hand did not show signs of apoptosis but did present as cells with loss of plasma membrane integrity, as shown by the high percentage of PI-positivity (Fig. 10B). Morphologically, they often revealed the presence of large cytoplasmic vacuoles, regularly accompanied by a clear loss of plasma membrane integrity (Fig. 10B and E–H). These are all typical features of oncosis/necrosis (Krysko et al., 2008). In conclusion, the type of cell death induced by H. suis or H. pylori GGT clearly depended on the amount of extracellular GSH, serving as a GGT substrate. Characterization of floating AGS cell populations. A. Treatment of AGS cells for 44 h with a combination of GSH and rHSGGT or rHPGGT resulted in a marked increase of cell detachment, as determined by flow cytometry, compared with cells treated with either HBSS, rHSGGT or rHPGGT. B. The majority of cells treated with HBSS and GSH showed chromatin condensation, typical for apoptosis. On the other hand, the majority of detached cells in cultures treated with rHSGGT/rHPGGT and GSH showed loss of plasma membrane integrity, as shown by the increase of PI positivity, and marked vacuolization. Shown in (A) and (B) are the mean (±SD) values of one representative experiment. An asterisk (*) depicts relevant statistically significant differences. C and D. Light microscopic (original magnification: 1000×) and TEM image of floating AGS cells in HBSS+GSH-treated cultures (44 h), showing clear chromatin condensation near the nuclear envelope (arrows) as a hallmark for apoptosis. E. Light microscopic image (original magnification: 1000×) of floating AGS cells in a 4 µg ml−1 rHSGGT+GSH-treated culture, showing cells with clear vacuolization (asterisk). Similar images were obtained for 2 µg ml−1 rHSGGT+GSH-treated cultures. F–H. TEM analysis of these cells showed the presence of large cytoplasmic vacuoles (asterisk) and loss of plasma membrane integrity (arrows), which are typical hallmarks of oncosis/necrosis. The two inserts show details of loss of plasma membrane integrity. HBSS, Hank\'s balanced salt solution; GSH, reduced glutathione; rHSGGT, recombinant H. suis GGT; rHPGGT, recombinant H. pylori GGT; PI, propidium iodide. H. suis and H. pylori GGT-mediated glutathione degradation plays an active role in the increase of the extracellular hydrogen peroxide concentration Because the cleavage of glutathione by plasma membrane GGT of eukaryotic cells has been associated with the extracellular production of hydrogen peroxide (Dominici et al., 1999), we sought to determine whether H. suis/H. pylori GGT-mediated glutathione degradation was correlated with an increase of extracellular hydrogen peroxide production. Incubation of AGS cells for 44 h with 2 µg ml−1 rHSGGT or rHPGGT was accompanied by a mild increase of the extracellular H2O2 concentration, as determined with the Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen) (Fig. 11A). However, when cells were incubated with a combination of 2 µg ml−1 rHSGGT or rHPGGT and 5 mM GSH, even much higher concentrations of extracellular H2O2 were observed (Fig. 11B). Shorter incubation for 20 h showed similar results, however less pronounced. As shown in Fig. 11A, incubation of AGS cells with 5 mM GSH alone, did not yield an increase in extracellular H2O2 concentrations, showing that the observed increase of the extracellular H2O2 concentration was due to recombinant H. suis/H. pylori GGT-mediated degradation of reduced glutathione. For H. suis GGT, the exact same experiments were performed in the same media, however without AGS cells. Comparable results were obtained (Fig. 11C), with equal or higher H2O2 concentrations in rHSGGT+GSH-containing wells without AGS cells compared with identical wells containing AGS cells. Moreover, acivicin-induced inhibition of the enzymatic activity of rHSGGT completely abolished the observed effect. These data show that the observed increase of the H2O2 concentration is an event taking place extracellularly. Finally, an increase of the extracellular H2O2 concentration was also observed after incubation of AGS cells for 1, 20 and 44 h with 200 µg ml−1H. suis lysate, when compared with HBSS-treated control wells (Fig. 11D). Determination of H2O2 concentrations in AGS cell culture supernatants. A. Incubation of AGS cells for 44 h with 2 µg ml−1 rHSGGT or rHPGGT resulted in higher H2O2 concentrations compared with values in supernatant of control cell cultures, supplemented with HBSS. B. When a combination of rHSGGT/rHPGGT and 5 mM GSH was used, this increase was even much higher, after both 20 and 44 h of incubation. C. Similar results were obtained in AGS culture medium in the absence of AGS cells. Additionally, pre-treatment of rHSGGT with acivicin completely abrogated the observed effect. D. Incubation of AGS cells for 1, 20 and 44 h with 200 µg ml−1H. suis lysate induced an increase of the extracellular H2O2 concentrations, compared with HBSS-treated cells. Shown are the mean values of three independent experiments or one representative experiment. An asterisk (*) depicts relevant statistically significant differences (P   0.05). HBSS, Hank\'s balanced salt solution; GSH, reduced glutathione; rHSGGT, recombinant H. suis GGT; rHPGGT, recombinant H. pylori GGT; Ac, acivicin; HS lysate, H. suis lysate. Glutathione degradation-dependent extracellular hydrogen peroxide generation causes lipid peroxidation prior to death of cells Because H. suis GGT-mediated degradation of glutathione resulted in an increase of the extracellular oxidative stress burden, we investigated if these extracellular changes had an impact on the intracellular redox balance, since H2O2 is known to be able to permeate the eukaryotic plasma membrane (Bienert et al., 2006; 2007), with subsequent actions inside the cell, possibly leading to necrotic cell death (Kim et al., 2007b). When AGS cells were treated for 44 h with rHSGGT, no change of intracellular concentrations of total glutathione was observed (Fig. 12A). However, after 44 h of incubation, an increase of membrane lipid peroxidation was detected in AGS cells treated with 2 µg ml−1 rHSGGT and 5 mM GSH, compared with HBSS-treated cells and cells treated with rHSGGT alone (Fig. 12B–D). In cells treated with GSH alone, no increased lipid peroxidation was observed, compared with HBSS-treated control cells, whereas treatment of cells with rHSGGT alone caused a slight increase of lipid peroxidation, although statistically not significant. These data indicate that this effect is largely caused by rHSGGT-mediated degradation of reduced glutathione. Interestingly, comparable but less pronounced effects were observed after 20 h of incubation, a point in time at which no increased cell death could yet be observed. Cellular effects of increased extracellular H2O2 concentrations. A. When AGS cells were treated for 44 h with rHSGGT, no change of intracellular glutathione concentrations was observed. Subsequently, cells were stained with BODIPY 581/591 C11. Upon peroxidation, fluorescence of this dye shifts from red to green. B and C. Example of the analysis of the red (FL-2) and green (FL-1) fluorescence intensities of a cell population treated for 44 h with HBSS (B) and a combination of 2 µg ml−1 rHSGGT and 5 mM GSH (C). Note the marked decrease of the red fluorescence (less cells in Q1 quadrant) accompanied by an increased green fluorescence (more cells in Q4 quadrant) after treatment with 2 µg ml−1 rHSGGT and 5 mM GSH, indicating peroxidation of cellular lipids. D. Already after 20 h of incubation with a combination of rHSGGT and GSH, an increase of the percentage of cells showing a fluorescence shift was observed. This lipid peroxidation was even more pronounced after 44 h of incubation. Shown are the mean results of three independent experiments. Incubation for 30 min with 500 µM H2O2 was included as a positive control. Asterisks (*) depict relevant statistically significant differences. HBSS, Hank\'s balanced salt solution; GSH, reduced glutathione; rHSGGT, recombinant H. suis GGT. Mitochondrial respiration is considered one of the main sources of intracellular ROS (Orrenius, 2007; Poyton et al., 2009; Pourova et al., 2010). Therefore, the possibility that this mitochondria-derived ROS could serve as a source of lipid peroxidation was investigated. Already after 24 h of incubation of AGS cells with a combination of rHSGGT and GSH, lower levels of mitochondrial polarization were observed, as shown by the decrease in the red/green fluorescence intensity ratio of the cationic dye JC-1 (Fig. 13). This loss of mitochondrial membrane potential rather indicates a decrease of mitochondrial ROS generation (Starkov and Fiskum, 2003; Brookes et al., 2004). Measurement of mitochondrial membrane potential (ΔΨm) in treated AGS cultures. Cells were stained with JC-1, a ΔΨm-sensitive probe. A decrease in the red/green fluorescence intensity ratio of this probe reflects mitochondrial depolarization. After 24 h of incubation of AGS cells with rHSGGT and GSH, a decrease of the mitochondrial membrane potential could be observed. Shown are the mean values of three experiments. Asterisks (*) depict relevant statistically significant differences. HBSS, Hank\'s balanced salt solution; GSH, reduced glutathione; rHSGGT, recombinant H. suis GGT. Until now, very little is known about H. suis virulence genes (Haesebrouck et al., 2009). In the present study, we showed that H. suis is actively involved in the induction of gastric epithelial cell death in vitro. These data support previous findings of H. suis-induced necrosis of gastric epithelial, mainly parietal cells in mice and Mongolian gerbils (Flahou et al., 2010a). This cell death may have important implications for the development of various gastric pathologies, such as gastric erosion and/or ulcer formation (Dixon, 2001), gastric atrophy and even gastric cancer (Shirin and Moss, 1998). These lesions all have been observed in humans infected with NHPH (Morgner et al., 1995; Stolte et al., 1997; Debongnie et al., 1998) and are very often accompanied by gastritis (Stolte et al., 1997). Similarly, H. suis elicits a strong inflammatory response in experimentally infected mice and Mongolian gerbils (Flahou et al., 2010a), possibly caused by direct effects of H. suis, but most likely also driven indirectly by necrosis of gastric epithelial cells. Cell necrosis results in the release of cellular contents, including molecules involved in the promotion of inflammation (Fink and Cookson, 2005; Vanlangenakker et al., 2008). Whole-genome screening of H. suis strain 5 has revealed the presence of a gene homologous to the H. pylori ggt, but also the absence of other virulence factors (Vermoote et al., 2011) involved in H. pylori-induced cell death, such as VacA (Cover et al., 2003). Inhibition of GGT activity present in H. suis lysate and the use of native and recombinant H. suis GGT revealed an important role for this enzyme in the induction of gastric epithelial cell death in the present study. Similarly, the H. pylori GGT has also been identified as a cell death-inducing enzyme (Shibayama et al., 2003); however, the exact mechanism behind this effect remained unknown. Enzymatic GGT activity typically catalyses the release and transpeptidation of a γ-glutamyl group. This enzyme, also present on the membrane of mammalian cells, plays a role in the degradation and thus metabolism of extracellular glutathione (Orlowski and Meister, 1970). The tripeptide glutathione is an important antioxidant which degrades reactive oxygen species (ROS), including O2-derived free radicals, as well as O2-derived non-radical species such as hydrogen peroxide (H2O2) (Circu and Aw, 2010). In the present study, we describe the H. suis GGT-mediated degradation of reduced glutathione. A comparative study showed that the capacity of H. suis GGT to degrade reduced glutathione is very similar to that of H. pylori GGT. Supplementation of the extracellular medium with GSH enhanced H. suis or H. pylori GGT-induced cell death, suggesting that metabolites of glutathione degradation are directly involved in this process. The recently published genomes of the closely related species H. felis and H. bizzozeronii, both also of zoonotic importance (Haesebrouck et al., 2009), reveal the presence of a GGT homologue with strong conservation of amino acids in their active sites (Arnold et al., 2011; Schott et al., 2011). This suggests that GGTs from highly related gastric helicobacters can damage the gastric epithelium in a very similar way. In the present study, approximately 2 U l−1 GGT activity was detected in the supernatant of a 24-h-old H. suis culture, containing a majority of live and motile bacteria. Native or recombinant H. suis GGT was added to AGS cells to reach similar GGT activity levels in AGS cell culture supernatant. It is reasonable to assume that similar levels of GGT activity are present in the region of H. suis colonization in the human or porcine stomach. Conflicting reports have been made about the localization of GGT in the highly related H. pylori. Some authors suggest a periplasmic localization (Chevalier et al., 1999; Shibayama et al., 2007). In this case, GGT could have free access to the surrounding glutathione, both with or without autolysis of the bacteria, which has been shown to be an event taking place frequently in H. pylori (Marcus and Scott, 2001; Fujita et al., 2005). Other authors suggest, with strong argumentation, that H. pylori GGT is in fact a secreted protein (Bumann et al., 2002; Busiello et al., 2004; Schmees et al., 2007). In any case, the conclusion is that H. pylori GGT, and by extension most likely also GGTs from other gastric helicobacters, have free access to glutathione, either as a secreted or as a periplasmic enzyme. In the present study, normal basal concentrations of extracellular glutathione were in the micromolar range, which is in accordance with the results of previous studies (Yang et al., 1997). Interestingly, no differences in extracellular concentrations of total glutathione (combined fractions of reduced and oxidized glutathione) were observed between control and H. suis GGT-treated cells (results not shown), suggesting that de novo synthesis of glutathione and subsequent transport out of the cells takes place. Indeed, it is well known that glutathione is synthesized intracellularly and subsequently translocated towards the extracellular space (Griffith and Meister, 1979). By this means, epithelial cells could provide H. suis GGT with a continuous supply of its main substrate. Supplementation of AGS cell cultures with 5 mM GSH, strengthened the observed H. suis or H. pylori GGT-mediated increase of extracellular H2O2 concentrations and subsequent cell death. In order to produce similar effects in vivo, GGT from gastric helicobacters must have access to relatively high concentrations of extracellular glutathione, which is indeed most likely provided by several sources. When cells die by means of necrosis, intracellular contents are released into the surroundings (Vanlangenakker et al., 2008), including large amounts of intracellular glutathione. In the stomach, intracellular glutathione concentrations are high (up to 10 mM), compared with most other tissues (Body et al., 1979; Meister and Anderson, 1983; Mårtensson et al., 1990). Moreover, foods like asparagus, cooked ham and orange juice contain high concentrations of glutathione (Valencia et al., 2001; Kuśmierek and Bald, 2008), resulting in daily uptakes of up to more than 100 mg in adult humans (Flagg et al., 1994). Bile reflux could also serve as an important source of extracellular glutathione, since bile contains high levels (up to 6 mM) of GSH (Meister and Anderson, 1983). Several studies suggest that GSH can freely diffuse through the gastric mucus (Mårtensson et al., 1990; Ovrebøet al., 1997), in this way also reaching the fraction of H. suis or H. pylori bacteria residing in the mucus layer. Interestingly, Farinati et al. (1996) have described an increase of both the reduced and oxidized glutathione content of gastric mucosa of H. pylori-infected humans suffering from chronic non-atrophic gastritis, possibly providing a greater source for H. pylori GGT-mediated degradation of glutathione. Similar results have been described for total (sum of reduced and oxidized) glutathione contents in the stomach of H. pylori-infected Mongolian gerbils (Suzuki et al., 1999). However, these findings are in contrast with the results from another study, describing lower levels of reduced glutathione in H. pylori-infected human patients compared with H. pylori-negative individuals (Jung et al., 2001). Deprivation of glutathione is one of the suggested mechanisms of H. pylori GGT-mediated cell death (Shibayama et al., 2007). However, in our attempt to reduce the H. suis/H. pylori GGT-induced cell death by supplementing cell culture supernatants with reduced glutathione, we observed the opposite effect, a higher number of dead cells. An increase of the extracellular H2O2 concentration was observed in the supernatant of AGS cells treated with H. suis or H. pylori GGT. The relevance of this finding was confirmed by the observation that H. suis lysate also generated higher extracellular H2O2 concentrations, compared with HBSS treatment of cells, despite the presence in H. suis of enzymes involved in the neutralization of various ROS, including catalase and superoxide dismutase (Vermoote et al., 2011). The increase of the extracellular H2O2 concentration generated by H. suis or H. pylori GGT was even much higher when cells were co-incubated with H. suis or H. pylori GGT and reduced glutathione. Interestingly, this vast increase was also observed in the absence of AGS cells, showing that the hydrogen peroxide was mainly generated in a cell-independent manner, which contrasts with recently published results describing the H. pylori GGT-mediated production of H2O2 by AGS cells (Gong et al., 2010). In the present study, we did not examine the exact mechanism by which H2O2 was generated. Several studies have described that plasma membrane-bound GGT of mammalian cells initiates pro-oxidant reactions through the catabolism of GSH (Dominici et al., 1999; Maellaro et al., 2000). Transpeptidation of the γ-glutamyl group generates the more reactive thiol cysteinyl-glycine, leading to the production of H2O2 through the reduction of Fe3+ and subsequent production of thiyl radicals and the superoxide anion (O2•−) (Dominici et al., 1999). Moreover, H2O2 can generate, through Haber-Weiss and Fenton reactions, the highly reactive hydroxyl radical (Vanlangenakker et al., 2008). So probably, the increased H2O2 concentrations also reflect the presence of these other ROS or thiyl radicals, responsible for the observed increase of cell death. Anyhow, the observed H2O2 concentrations in the present study were relatively low compared with cell death-inducing amounts (supplemented on one single occasion to cells) described in other studies (Hampton and Orrenius, 1997; Zhuang et al., 2008). In these studies, however, these higher H2O2 concentrations induced cell death already after 2–6 h, so longer incubation with lower H2O2 concentrations, as in the present study, most likely explain the induced epithelial cell death. Interestingly, the type of cell death (apoptosis versus oncosis/necrosis) caused by H. suis or H. pylori GGT was shown to be related to the amount of extracellular ROS generated, which was directly linked to the availability of extracellular GSH as a GGT substrate. In vitro, the type of H2O2-induced cell death has indeed been associated with variations in the concentration of this ROS, with the higher concentrations inducing necrosis rather than apoptosis (Hampton and Orrenius, 1997; Krysko et al., 2008). Additionally, both apoptosis and necrosis have been observed in the same cell culture, dependent on a sustained c-Jun N-terminal kinase (JNK) activation due to an enhanced production of ROS (Kamata et al., 2005). In any case, it is well established that increased concentrations of ROS often result in necrotic cell death (Fiers et al., 1999; Duprez et al., 2009; Kroemer et al., 2009). Twenty hours of incubation of AGS cells with the combination of rHSGGT or rHPGGT and GSH did not result in a higher number of dead cells compared with cells treated with rHSGGT or rHPGGT alone. At this point in time, however, increased concentrations of extracellular H2O2 as well as peroxidation of cellular lipids could be observed. Lipid peroxidation in the plasma membrane often leads to loss of membrane integrity (Vanlangenakker et al., 2008), which is observed in the present study at later points in time compared with the detection of lipid peroxidation. Additionally, in mitochondria, reactive aldehydes derived from lipid peroxidation can affect oxidative phosphorylation and mitochondrial membrane potential (Vanlangenakker et al., 2008), which was also observed in the present study. In conclusion, for the first time a H. suis virulence factor was identified, supporting earlier findings that also NHPH can cause human gastric disease. We have shown that H. suis directly causes gastric epithelial cell death in vitro, confirming the results of previous in vivo studies (Flahou et al., 2010a). We have also shown that H. suis GGT is the main cause of H. suis-induced cell death of human gastric epithelial cells in vitro. Additionally, as briefly summarized in Fig. 14, we revealed the mechanism by which H. suis and H. pylori GGT cause an increase of gastric epithelial cell death. Both the observed capacity to cause cell death and the type of cell death brought about were shown to depend upon H. suis or H. pylori GGT-mediated degradation of reduced glutathione, resulting in an increase of extracellular concentrations of H2O2, generated by glutathione degradation products in a cell-independent manner and resulting in cellular lipid peroxidation. This oxidative cell damage finally results in cell death. Model summarizing the effects of H. suis GGT on gastric epithelial cells. H. suis GGT is an important H. suis virulence factor causing death of human gastric epithelial cells. The observed capacity to cause cell death was shown to depend upon H. suis (or H. pylori) GGT-mediated degradation of reduced glutathione, resulting in an increase of extracellular concentrations of ROS (including H2O2), generated by glutathione degradation products in a cell-independent manner. This results in oxidative cell damage on different levels, finally leading to different types of cell death, depending on the amount of extracellular ROS generated. GSH, reduced glutathione; HsGGT, H. suis GGT; Glu, glutamate; CysGly, cysteinyl glycine; ROS, reactive oxygen species; conc., concentration. Helicobacter suis strains HS1-9, isolated from the gastric mucosa of pigs (Baele et al., 2008), were grown under biphasic culture conditions as described previously (Flahou et al., 2010b). H. pylori strain 26695 was obtained from the CCUG (Culture Collection, University of Göteborg) and grown on BHI agar (Oxoid, Basingstoke, UK) plates supplemented with 10% horse blood, 5 mg ml−1 amphotericin B (Fungizone; Bristol-Myers Squibb, Epernon, France), Campylobacter selective supplement (Skirrow, Oxoid; containing 10 mg l−1 vancomycin, 5 mg ml−1 trimethoprim lactate and 2500 U l−1 polymyxin B) and Vitox supplement (Oxoid). Bacterial genomic DNA of H. suis strain 5 and H. pylori strain 26695 was isolated as described by Wilson (1994) and used for expression of recombinant H. suis and H. pylori GGT (rHSGGT and rHPGGT respectively). For the whole bacterial cell lysate of H. suis, bacteria were harvested by centrifugation, washed two times with HBSS and resuspended in HBSS. The bacterial suspension was sonicated eight times for 30 s and centrifuged (15 000 g, 5 min, 4°C) to remove cellular debris. The supernatant was filtered through a 0.22 µm pore filter (Schleicher and Schuell, Gent, Belgium) and stored at −80°C. The resulting protein concentration was determined with the RC DC Protein Assay (Bio-Rad, Hercules, CA, USA). In order to remove most of the proteins  50 kDa, H. suis strain 5 whole bacterial cell lysate was subjected to four centrifugation/dilution cycles using a VIVASPIN 20 ultrafiltration column (50000 MWCO; Sartorius stedim biotech, Goettingen, Germany). The fraction containing proteins  50 kDa, including H. suis GGT, was subjected to several purification steps. First, it was diluted fivefold in 0.1 M phosphate buffer (pH 7.0) and subjected to a first cation exchange chromatography. Briefly, the GGT-containing fraction was transferred to an Econo Column® (Bio-Rad), containing SP sepharose Fast Flow (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). After one washing step, elution was performed with 0.1 M phosphate buffer containing increasing salt concentrations (200–500 mM NaCl). For further purification, three 300 mM NaCl-eluted fractions containing the highest GGT activity were pooled and loaded on a Superdex™ 200 gel filtration column (GE Healthcare Bio-Sciences AB), eluted with HBSS and collected as 1 ml fractions. Four consecutive fractions containing the highest GGT activity were then pooled and subjected to a second cation exchange chromatography as described above. Three 300 mM NaCl-eluted fractions containing the highest GGT activity were pooled and concentrated to 300 µl by ultrafiltration (VIVASPIN 20, 50000 MWCO; Sartorius stedim biotech). This fraction containing purified native H. suis GGT was named F2 and subjected to SDS-PAGE with subsequent Brilliant Blue G – Colloidal (Sigma-Aldrich, St. Louis, MO, USA) staining and identification by mass spectrometry. The enzymes were expressed in the E. coli Expression System with Gateway® Technology (Invitrogen, Carlsbad, CA, USA) as follows. The coding region of the H. suis strain 5 GGT, without the predicted 18 aa signal sequence, was amplified by PCR (forward primer: 5′-CACCATGGCCACTTTGCCTCCTATTAAAGGC-3′; reverse primer: 5′-TTAAAATTCCTTGCGTGGATCTTGAGC-3′) using Pwo polymerase with proofreading activity (Roche Applied Science, Mannheim, Germany) according to the guidelines for this enzyme. The same was done for the coding region of the H. pylori strain 26695 GGT, without the predicted 26 aa signal sequence and using the following primers: forward primer: 5′-CACCATGGCGAGTTACCCCCCCATTAAAAACAC-3′; reverse primer: 5′-TTAAAATTCTTTCCTTGGATCCGTTGAACCATAG-3′. The resulting PCR products were cloned into the pENTR™/SD/D-TOPO® vector and transferred into the pDEST™17 destination vector. The resulting expression clones were transformed into the chemically competent E. coli strain BL21-AI™. A fresh culture was allowed to grow until the OD600 reached 0.4, after which 0.2% l-arabinose was added to induce the expression of recombinant H. suis or H. pylori GGT (rHSGGT or rHPGGT). After incubation at 37°C for 3 h with shaking, bacteria were harvested by centrifugation (4500 g for 20 min). N-terminal 6xHis-tagged rHSGGT and rHPGGT were purified on a Ni-sepharose column (His GraviTrap; GE Healthcare Bio-Sciences AB) according to the manufacturer\'s instructions. Bound protein was eluted with 3 ml of elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4) and collected in 17 ml of HBSS, to prevent precipitation due to high imidazole concentrations. The eluates were concentrated to a final volume of 1.5 ml by ultrafiltration (VIVASPIN 20, 5000 MWCO; Sartorius stedim biotech), analysed in the GGT activity assay and by SDS-PAGE with subsequent Brilliant Blue G – Colloidal (Sigma-Aldrich) staining. For further purification, the concentrated eluates were loaded on a Superdex™ 75 gel filtration column (GE Healthcare Bio-Sciences AB), eluted with HBSS and collected as 1 ml fractions. For both recombinant enzymes, the purity and GGT activity of three to five peak fractions were determined by SDS-PAGE and the GGT activity assay (see below) respectively. Since all tested fractions were pure on SDS-PAGE and showed GGT activity, they were pooled and stored at −80°C until further use. Protein concentration was determined with the RC DC Protein Assay (Bio-Rad). The 40 kDa band of purified fraction F2, putatively corresponding to the large subunit of H. suis GGT, was cut from the gel and subjected to reduction, alkylation and in-gel protein digestion with trypsin (Devreese et al., 2002). Subsequently, peptides were extracted from the gel with 0.1% HCOOH in 60% acetonitrile, dried in a SpeedVac system and resuspended in 0.1% HCOOH. After mixing 1 µl of the peptide mixture with an equal volume of α-cyano-4-hydroxycinnamic acid (5 mg ml−1, 50% ACN, 10 mM ammoniumcitrate, 0.1% TFA), 0.75 µl was spotted onto the MALDI target plate and analysed with the 4800 plus MALDI TOF/TOF Analyser (Applied Biosystems, Foster City, CA, USA). After peptide mass fingerprinting by MALDI-MS, tandem mass spectrometry (MS/MS) was performed on a selection of peptides with the largest peak intensity. The obtained MALDI-MS/MS spectra were searched against SwissProt and NCBI non-redundant protein databases using an in-house MASCOT server (Matrixscience, London, UK). As a control, the large (∼40 kDa) subunits of both the recombinant H. suis and H. pylori GGT were treated and analysed similarly. For the H. suis GGT, theoretical masses of peptides generated by trypsin cleavage were predicted using the PeptideMass tool at the ExPASy proteomics server. All in vitro cell experiments were performed with AGS cells (human gastric adenocarcinoma cell line; ATCC: CRL-1739). The cells were cultured in Ham\'s F12 (Invitrogen; containing 1 mM glutamine) supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT, USA), penicillin and streptomycin (Invitrogen). AGS cells were seeded at 2 × 104 cells for each well of a 24-well flat-bottom cell-culture plate or 1 × 105 cells for each well of a six-well flat-bottom cell-culture plate (Greiner Bio One, Frickenhausen, Germany). Cells were allowed to adhere for 6 h and serum-starved overnight. Prior to each incubation experiment, cells were washed two times with HBSS. During the experiments, cells were incubated in Ham\'s F12 supplemented with penicillin and streptomycin and FCS at a final concentration of 5%. Cell medium was supplemented with H. suis strain 5 whole bacterial cell lysate at a final total protein concentration of 50–200 µg ml−1 for 44 h at 37°C, unless stated otherwise, corresponding to a calculated theoretical multiplicity of infection (moi) of approximately 50–200 respectively. To determine whether the cell death-inducing agent was of protein nature, the bacterial lysate was pre-treated with heat (95°C, 1 h) or trypsin (1 mg ml−1; 2 h, 37°C; Invitrogen), which in turn was inactivated by the addition of 1 mg ml−1 soybean trypsin inhibitor (Sigma-Aldrich) and 5% FCS prior to incubation with AGS cells. Alternatively, cell medium was supplemented with native H. suis GGT to reach a similar final GGT activity to that in 200 µg ml−1 lysate-treated cell cultures. For recombinant H. suis or H. pylori GGT, AGS cells were treated with final protein concentrations ranging from 0.2–8 µg ml−1 and incubation was performed for 20 and 44 h at 37°C, unless stated otherwise. For inhibition of GGT activity in both whole bacterial cell lysates and rHSGGT, pre-incubation with 50 µM acivicin (Enzo life Sciences, Farmingdale, NY, USA), a known GGT inhibitor, was performed for 1 h at 37°C (Schmees et al., 2007). After this incubation, unbound acivicin was removed from the lysate and rHSGGT by repeated ultrafiltration (VIVASPIN 500, 5000 MWCO; Sartorius stedim biotech). In additional experiments, the effect on AGS cells of supplementation with reduced glutathione (GSH) in the presence of recombinant H. suis or H. pylori GGT was determined. Therefore, GSH (Sigma-Aldrich) was added to the cell medium at a final concentration of 5 mM. For each experiment, negative control cells were incubated with HBSS which was treated in the same way as H. suis lysate or recombinant GGT. For most cell death experiments, the combined fractions of floating and adherent AGS cells were analysed by flow fluorocytometry using different staining protocols. All analyses were performed on a BD FACSCanto II flow cytometer and processed using FACSDiva software (Becton Dickinson, Erembodegem, Belgium). After an initial centrifugation step (1700 g, 5 min), cells were resuspended and divided into two identical subpopulations. One half was used for the assessment of loss of plasma membrane integrity as a marker for necrosis (Fig. 15A) (Fink and Cookson, 2005; Galluzzi et al., 2009). Briefly, after an initial washing step in HBSS, cell pellets were incubated for 15 min on ice with 1 µg ml−1 PI in HBSS. Triton X-100 (0.1%)-treated cells served as a positive control. The second cell subpopulation was used for the detection of caspase-3 activation as a marker for apoptosis (Fink and Cookson, 2005; De Bock et al., 2006; Galluzzi et al., 2009). Briefly, these cells were fixed with 4% paraformaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 in HBSS for 2 min. Cells were incubated with a primary rabbit antibody directed against activated caspase-3 (R&D Systems Europe) for 1 h at 37°C, followed by an Alexa Fluor 488-conjugated goat anti-rabbit antibody (Invitrogen). Cells treated for 20 h with 0.5 µM staurosporine (Sigma-Aldrich) served as a positive control. Additionally or alternatively to this staining for activated caspase-3, cell populations were analysed for the presence of a hypoploid DNA content. This method relies on the fact that apoptotic cells show DNA fragmentation and even partial DNA loss due to formation of apoptotic bodies, resulting in a decrease of the cellular DNA content (Krysko et al., 2008). Briefly, PI was added to cells in a round-bottom FCM tube at a concentration of 20 µg ml−1. Subsequently, cells were permeabilized by a brief freeze/thaw cycle using liquid nitrogen and analysed by flow cytometry (Fig. 15B). A very good correlation was observed between the latter method and activated caspase-3 staining (Fig. 15C). Different methods of flow fluorocytometric determination of cell death. A. Example of the flow cytometric detection (in channel FL-2) of loss of plasma membrane integrity, using PI (propidium iodide) as fluorescent marker. The gate showing PI-positive cells is delineated. B. Example of the flow cytometric detection of hypoploid apoptotic cells (in channel FL-3) by means of quantification of DNA content, bound by PI, after cell permeabilization. The gate showing hypoploid cells is delineated. C. A good correlation between staining for activated caspase-3 and assessment of hypoploidy was seen. Shown are the trend lines (with equations) of two independent experiments. To confirm active caspase-3 staining of H. suis lysate-treated apoptotic cells, some cell suspensions were additionally analysed by fluorescence microscopy after counterstaining cell nuclei with Hoechst (100 µM, 15 min, RT). Images were captured using a Cell*M imaging workstation connected to an IX81 fluorescence microscope (Olympus). Except for brightness/contrast adjustments applied to the entire images, images were not digitally manipulated. Washing steps in HBSS were included at appropriate points in time during all experimental protocols. To further characterize H. suis or H. pylori GGT-induced cell death, treated cells were examined by light and transmission electron microscopy. Adherent cells were fixed in 4% formaldehyde in a buffer (pH 7.4) containing 0.121 M Na-cacodylate and 1% CaCl2. After post-fixation in 1% osmium tetroxide, cultures were dehydrated in a graded series of ethanol and embedded in LX resin. Semithin sections of 2 µM were stained with toluidine blue and examined by light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate solutions before examining under a Jeol EX II transmission electron microscope at 80 kV. In some experiments, floating cell populations were analysed. Briefly, cell culture supernatant, containing detached cells, was collected and centrifuged (1000 g, 10 min). The cell pellet was resuspended in 2% glutaraldehyde, supplemented with bovine serum albumin (BSA) to a final concentration of 8% and centrifuged (1000 g, 10 min). The resulting pellet, containing precipitated BSA and cells, was collected and subjected to post-fixation and subsequent processing as described above. Semithin sections were used for counting the number of cells showing clear chromatin condensation and vacuolization in at least three separate microscopic fields (630×). For determination of the floating/adherent cell ratio, cell culture supernatant (1 ml) was carefully mixed and aspirated. The adherent cell population was briefly trypsinized and collected in AGS culture medium containing 5% FCS to obtain a final volume of 1 ml. PI was added to both populations at a concentration of 2.5 µg ml−1. Cells of both populations, each in a total volume of 1 ml, were acquired using a BD FACSCanto II flow cytometer at the highest acquisition rate. The number of cells acquired during 100 s was determined and used to determine the proportion of floating versus adherent cells. Detection of GGT activity of H. suis whole bacterial cell lysate, H. suis culture supernatant or recombinant H. suis and H. pylori GGT was based on the method described by Orlowski and Meister (1963). Briefly, reaction buffer consisted of 2.9 mM l-glutamic acid 5-(3-carboxy-4-nitroanilide) (Sigma-Aldrich) as a donor substrate and 100 mM glycyl-glycine (Sigma-Aldrich) as an acceptor in 100 mM Tris buffer (pH 8.25). Bacterial supernatants, whole bacterial cell lysate of H. suis or the recombinant H. suis or H. pylori GGT were added and the reaction mixture was incubated at 37°C for 5–60 min. The release of p-nitroaniline was determined by measuring the absorbance at 405 nm. Activity was expressed as U l−1 or mU mg−1 protein and one unit was defined as the amount of enzyme that catalyses the formation of 1 µmole of p-nitroaniline per minute under specified conditions. To test the effect of H. suis and H. pylori GGT on reduced glutathione (GSH), AGS culture supernatants were supplemented with GSH to a final concentration of 5 or 1 mM GSH. Recombinant H. suis or H. pylori GGT was added at a final concentration of 2 µg ml−1 and incubation was done at 37°C for up to 44 h. At the appropriate time points, an aliquot was removed to determine the concentration of GSH with the Glutathione Assay Kit (Sigma-Aldrich), according to the manufacturer\'s instructions. This assay is based on the continuous reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to TNB, which is measured spectrophotometrically at 412 nm. This same Glutathione Assay Kit was used for monitoring basal concentrations of total glutathione in the supernatants of AGS cell cultures. Total glutathione comprises both reduced glutathione (GSH), which is by far the most abundant form (Meister and Anderson, 1983), and oxidized glutathione disulfide (GSSG). Catabolism of both forms has been shown to be mediated by eukaryotic membrane-bound GGT (Meister and Anderson, 1983; Franco et al., 2007). To determine if H. suis GGT activity also influences intracellular levels of total glutathione, the combined fractions of floating and adherent AGS cells were used. After counting cells in a Bürker counting chamber, the cells were pelleted by centrifugation (1700 g, 7 min) and lysed by three freeze/thaw cycles in a 5% 5-sulfosalicylic acid solution. Precipitated proteins were removed by centrifugation (10 000 g, 10 min) and the supernatant was used for glutathione determination using the Glutathione Assay Kit. The amount of total glutathione was expressed as pmol per cell. To detect extracellular H2O2 production in supernatants of AGS cell cultures treated or non-treated with H. suis whole bacterial cell lysate, rHSGGT/rHPGGT or the combination of one of both enzymes and 5 mM reduced glutathione, 25 µl of the cell supernatants were collected for processing with the Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen). This assay is based on the horseradish peroxidase-catalysed oxidation of Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine), a non-fluorescent reagent that becomes highly fluorescent after oxidation by H2O2 (Maellaro et al., 2000). The possible involvement of previously described treatments in lipid peroxidation was determined by using 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591 C11; Invitrogen), a probe which is distributed heterogeneously throughout the cell with predominant staining in the perinuclear region (Drummen et al., 2002). On excitation at 488 nm, this dye yields red emission with a 595 nm maximum. Upon oxidation, its red fluorescence is lost, coinciding with a shift of the emission maximum from 595 nm to 520 nm. This results in an increase of green fluorescence (Drummen et al., 2002; MacDonald et al., 2007). After incubation of AGS cells for 15 or 39 h with rHSGGT or a combination of rHSGGT and GSH, cell culture plates were briefly centrifuged (1700 g, 2 min) to collect both floating and adherent cell populations at the bottom of the wells. The cell supernatant was carefully aspired and kept at 37°C until further use. Cells were detached by brief trypsinization and resuspended in Ham\'s F12 supplemented with 10% fetal calf serum. After centrifugation (1700 g, 5 min), the cell pellet was resuspended in 200 µl of HBSS containing 5 µM of the BODIPY 581/591 C11 probe. After incubation for 30 min at 37°C, cells were centrifuged (1700 g, 5 min), followed by two washing steps in HBSS. Subsequently, the pellet was resuspended in the original cell culture supernatant and incubated for another 5 h at 37°C, with occasional shaking to keep the cells in suspension. Finally, cells were washed twice in HBSS and resuspended in HBSS for analysis of the red and green fluorescence intensity in the FL-2 and FL-1 channel of a BD FACSCanto II flow cytometer. Only cells with unchanged FSC (forward scatter) and SSC (side scatter) values were analysed. Some control cells were treated with 500 µM H2O2 (37°C; 30 min) prior to analysis and served as a positive control. To investigate the possible role of mitochondria in the generation of ROS in the present study, a ΔΨm-sensitive probe (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; JC-1; Invitrogen) was used, since mitochondrial ROS generation is dependent on ΔΨm (Starkov and Fiskum, 2003; Brookes et al., 2004; Galluzzi et al., 2009). Briefly, AGS cells were treated with HBSS or 2 µg ml−1 rHSGGT in the presence or absence of 5 mM reduced glutathione. After 24 h of incubation, the combined fractions of floating and adherent cells were pooled and stained with 10 µg ml−1 JC-1 in HBSS (37°C, 15 min). Cells were centrifuged (1700 g, 5 min), washed twice with HBSS and resuspended in HBSS prior to analysis with a BD FACSCanto II flow cytometer. Upon mitochondrial depolarization, a decrease in the red/green fluorescence intensity ratio of this probe can be observed. 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