Kinase 1-Dependent Manner in a TLR4- and Apoptosis Signal

The Journal of Immunology

The Secreted Peptidyl Prolyl cis,trans-Isomerase HP0175 of Helicobacter pylori Induces Apoptosis of Gastric Epithelial Cells in a TLR4- and Apoptosis Signal-Regulating Kinase 1-Dependent Manner1

Chaitali Basak, Sushil Kumar Pathak, Asima Bhattacharyya, Shresh Pathak, Joyoti Basu, and Manikuntala Kundu2

Apoptosis contributes to the pathology of gastric epithelial cell damage that characterizes Helicobacter pylori infection. The secreted peptidyl prolyl cis, trans-isomerase of H. pylori, HP0175 executed apoptosis of the gastric epithelial cell line AGS in a dose- and time-dependent manner. The effect of HP0175 was conﬁrmed by generating an isogenic mutant of H. pylori disrupted in the HP0175 gene. The apoptosis-inducing ability of this mutant was impaired compared with that of the wild type. The effect of HP0175 was mediated through TLR4. Preincubation of the gastric epithelial cell line AGS with anti-TLR4 mAb inhibited apoptosis induced by HP0175. Downstream of TLR4, apoptosis signal-regulating kinase 1 activated MAPK p38, leading to the caspase 8-dependent cleavage of Bid, its translocation to the mitochondria, mitochondrial pore formation, cytochrome c release, and activation of caspases 9 and 3. We show for the ﬁrst time that a secreted bacterial Ag with peptidyl prolyl cis,trans-isomerase activity signals through TLR4, and that this Ag executes gastric epithelial cell apoptosis through a signaling pathway in which TLR4 and apoptosis signal-regulating kinase 1 are central players. The Journal of Immunology, 2005, 174: 5672–5680.

elicobacter pylori is a spiral, microaerophilic Gram-neg- and release of cytochrome c (cyt c)3 (19), apoptosis-inducing fac- ative bacterium that colonizes gastric epithelial cells. tor (20), and Smac (DIABLO) (21) from the mitochondria. Cyto- H Depending on the strain of H. pylori and the individual solic cyt c together with ATP/dATP induces a conformational it infects, the bacterium can cause peptic ulcer disease and gastric change in apoptotic protease-activating factor-1, leading to pepti- mucosa-associated lymphoid tissue lymphoma (1). Apoptosis is dyl prolyl cis,trans-isomerase oligomerization, binding of pro- involved in H. pylori-induced gastric epithelial cell damage (2–6). caspase 9 in a so-called apoptosome complex (22, 23), and pro- Apoptosis-related genes and proteins such as the Bcl-2 family, teolytic activation of procaspase 9 (24). Fas-Fas ligand, and p53 have been implicated in epithelial cell The MAPK cascade is one of the evolutionarily conserved phos- apoptosis (7–9). Recent data have shown that caspases 8, 3, and 9 phorylation-regulated protein kinase cascades that influences cell are activated in gastric epithelial cells during infection with H. survival or cell death. JNK and p38 mediate various types of pylori (10). This underscores the need to understand in detail the stress-induced apoptosis (25). Apoptosis signal-regulating kinase 1 nature of the factors responsible for H. pylori-infected gastric ep- (ASK1) is a member of the MAPK kinase kinase family that plays ithelial cell apoptosis. a role in stress-induced apoptosis, principally through activation of Apoptosis is an orchestrated suicide program in which the key the JNK or p38 MAPK signaling cascades (26–29). morphological alterations are mediated by a family of cysteine Secreted proteins of H. pylori, such as VacA (30), urease (31), ␥ proteases known as the caspases, which are activated by proteo- and -glutamyl transpeptidase (32), are apoptosis-inducing pro- lytic cleavage (11–13). In death receptor-mediated (or extrinsic) teins that contribute to gastric inflammation and epithelial cell cell death, procaspase 8 is recruited to the death-inducing signaling damage. However, apoptosis is likely to be regulated by a number complex through its N-terminal death effector domain (14, 15). of proteins. With this in view, we focused on the H. pylori reper- toire of secreted proteins identified by several groups in recent Activated caspase 8 can subsequently cleave downstream effector years (33, 34). We narrowed down to the protein HP0175, which caspases 3 and 7 (16–18). Death via the intrinsic pathway is char- is one of five Ags of H. pylori preferentially recognized by the Abs acterized by a decrease in mitochondrial transmembrane potential of patients with gastroduodenal ulcers rather than dyspepsia patients (35, 36). HP0175 is characterized by a C-terminal peptidyl prolyl cis,trans-isomerase (PPIase) core identified on the basis of sequence similarity. PPIases have been characterized as virulence Department of Chemistry, Bose Institute, Kolkata, India factors of Legionella pneumophila (37) and Trypanosoma Received for publication July 13, 2004. Accepted for publication February 8, 2005. cruzi (38). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the Indian Council of Medical Research, 3 Abbreviations used in this paper: cyt c, cytochrome c; ASK1, apoptosis signal- Government of India (to M.K.). S.K.P. was supported by a Junior Research Fellow- regulating kinase 1; ⌬␺ , mitochondrial membrane potential; DiOC , 3,3Ј-dihexy- ship from the Council of Scientific and Industrial Research, Government of India. m 6 loxacarbocyanine; FMK, fluoromethyl ketone; KO, knockout; MBP, myelin basic 2 Address correspondence and reprint requests to Dr. Manikuntala Kundu, Depart- protein; CCCP, carbonyl cyanide m-chlorophenylhydrazone; MPT, mitochondrial ment of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra, Road, Kolkata permeability transition; PARP, poly(ADP-ribose) polymerase; PPIase, peptidyl prolyl 700009, India. E-mail address: [email protected] cis,trans-isomerase; t-Bid, truncated Bid; WT, wild type.

In the present study we demonstrate that HP0175 is a proapop- Purification of VacA totic factor by two independent experimental approaches, namely, The vacA gene of H. pylori 26695 was amplified by PCR (30) and cloned histone ELISA and annexin V-fluos binding to phosphatidylserine in pET20bϩ (Novagen). The VacA construct was transformed into E. coli exposed on the cell surface. The contribution of HP0175 to H. strain BL21(DE3), and VacA was purified from the cell-free supernatant by ϩ pylori-induced cell death in AGS cells was confirmed by the ob- chromatography on Ni2 -NTA-agarose. servation that an isogenic mutant of H. pylori 26695 disrupted in Assay for LPS the HP0175 gene was impaired in its apoptosis-inducing ability. HP0175 appeared to signal through TLR4, because preincubation LPS contamination of the purified protein was determined by Limulus amebocyte lysate assay using the E-TOXATE kit (Sigma-Aldrich) with a sen- with a blocking anti-TLR4 Ab inhibited HP0175-mediated apo- sitivity limit of 0.1 UE/ml (39). ptosis in AGS cells. Also, HP0175 interacted directly with TLR4. ASK1 was activated downstream of TLR4, leading to the sequen- PPIase assay tial activation of p38 MAPK and caspase 8 linked to the cleavage PPIase activity was measured in a coupled assay with chymotrypsin, in of Bid. The translocation of Bid to the mitochondria led to the which the oligopeptide, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sig- disruption of mitochondrial membrane potential, cyt c release, and ma-Aldrich; 20 ␮M) in 100 mM sodium phosphate buffer (pH 7.8) was sequential activation of caspases 9 and 3, culminating in cell death. used as the substrate (40). After preincubation at 15°C for 2 min, the reaction was started by adding 10 ␮M chymotrypsin and was monitored HP0175 emerges as a novel pathogen-associated molecular pat- spectrophotometrically at 15°C for 5 min by recording the increase in A390 tern, which signals through TLR4 to execute cell death. To the best corresponding to the release of p-nitroanilide. The arbitrary unit of PPIase ϭ Ϫ of our knowledge, this is the first report of a secreted Ag of H. activity, Up, was calculated from Up (kp kn)/kn, where kp and kn are pylori capable of inducing apoptosis in a TLR4- and ASK1-de- the first-order rate constants for p-nitroanilide release in the presence and the absence of the protein respectively. pendent manner. Culture of the human gastric epithelial cell line AGS Materials and Methods The human gastric epithelial cell line AGS was obtained from the National Reagents Center of Cell Science and maintained in Ham’s F-12 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 Abs against total and phospho-specific MAPKs, poly(ADP-ribose) poly- ␮ g/ml streptomycin in a humidified atmosphere containing 5% CO2. merase (PARP), Bid, and cleaved caspases 3, 8, and 9 were purchased from Dishes were washed to remove nonadherent cells. Adherent cells were ␤ Cell Signaling Technology. Abs against ASK1, cyt c, -actin, and protein Ն95% viable as determined by trypan blue dye exclusion. A/G Plus agarose were purchased from Santa Cruz Biotechnology.

SB203580, U0126, z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-FMK (z- Bacteria and infection DEVD-FMK), z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (z-LEHD-FMK), z-lle-Glu(OMe)-Thr-Asp(OMe)-FMK (z-IETD-FMK), and protease inhib- For AGS cell infections, H. pylori strains were grown under microaero- itors were purchased from EMD Biosciences. 3,3Ј-Dihexyloxacarbocya- philic conditions for 48 h on brain heart infusion agar plates containing 10% FBS and incubated with AGS cell monolayers at a multiplicity of nine (DiOC6) was obtained from Molecular Probes. Escherichia coli LPS and polymyxin B resin were purchased from Sigma-Aldrich. Annexin-fluos infection of 100 on culture plates in Ham’s F-12 medium supplemented was purchased from Roche. with 10% FBS. Plasmid constructs and transient transfections Cloning of the gene encoding HP0175 TLR4 was amplified from RNA isolated from THP-1 cells using the sense The gene encoding the protein HP0175 (accession no. P56112) was am- and antisense primers 5Ј-ATGGATCCGCATGGAGCTGAATTTCTA-3Ј plified by PCR using the genomic DNA of H. pylori (strain 26695) as and 5Ј-ATAAAGCTTCTAAGATGTTGCTTCCTG-3Ј, respectively, and template and the primers 5Ј-TAA GGA TCC GCT AGC ATG AAA AAA cloned between the BamHI and HindIII sites of pcDNA3.1. TLR4 AAT ATC TTA AAT-3Ј (sense) and 5Ј-TAG AAT TCT TACTTG TTG 1–643 (TLR4(dn)) encoding the first 643 aa of TLR4, was generated using the ATA ACA ATT TTA-3Ј (antisense) with BamHI and EcoRI sites (under- same sense primer and the antisense primer 5Ј-ATAAAGCTTCTACTGGCT lined) respectively, and cloned in pUC19. The cloned HP0175 gene was TGAGTAGAT-3Ј. Hemagglutinin-tagged wild-type ASK1 (ASK1(wt)) and a excised from pUC19 and cloned between NheI (indicated in bold) and ϩ catalytically inactive mutant (K709M) of ASK1 (ASK1(KM)) were obtained EcoRI sites in the vector pET28a (Novagen) for expression. The resulting from Dr. H. Ichijo (Graduate School of Pharmaceutical Sciences, University of plasmid is designated pCMK101. Tokyo, Tokyo, Japan). Flag-tagged p38 and its dominant negative mutant (p38(agf)) were obtained from Dr. R. Davis (University of Massachusetts Expression and purification of rHP0175 Medical School, Worcester, MA). pCMVdw caspase 9S was obtained from Induction of HP0175 from E. coli BL21 (DE3)/pCMK101 was conducted Dr. D. W. Seol (University of Pittsburgh School of Medicine, Pittsburgh, PA). ␮ at 37°C for 2 h with isopropyl thio-␤-D-galactoside (50 ␮M). Hexa-His- Cells were transfected with 2 g of plasmid (empty vectors or recombinants) tagged HP0175 was purified from the cell lysates by chromatography on using FuGene 6 (Roche) according to the manufacturer’s instructions. ␤-Ga- Ni2ϩ-NTA (Qiagen). Anti-HP0175 Abs were raised by Imgenex Biotech lactosidase reporter plasmid was used to normalize transfection efficiencies. by immunizing rabbits with purified HP0175. Cell death Production of the HP0175 gene-disrupted mutant strain For the detection of histones by ELISA, AGS cells were plated (6 ϫ 104 cells/plate) on 96-well plates. After treatments, cell death was detected The entire HP0175 gene region was amplified by PCR using genomic DNA with the cell death detection ELISA Plus kit (Roche) according to the of H. pylori strain 26695 as template and the primers 5Ј-CGG GGT ACC manufacturer’s protocol. AAT TTT TGA ATC TTC TTT-3Ј (sense; a) and 5Ј-TT GGA TCC GGG For measurements of annexin-fluos binding, 100 ␮l of cells in annexin- TAG CGT TCG CAC ATG G-3Ј(antisense; d) harboring KpnI and BamHI fluos binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM sites (underlined), respectively, and cloned in pBluescript (pBlue-NS). A CaCl ) were mixed with 2 ␮l of annexin-fluos. The mixture was incubated PstI site was created 450 bp downstream from the N terminus of the 2 for 15 min at room temperature, washed with binding buffer, and analyzed HP0175 gene by overlap extension PCR (pBlue-NPS). The kanamycin by flow cytometry. To assess necrosis, propidium iodide (1 ␮g/ml) was resistance cassette (aphA) from pUC4K (Amersham Biosciences) was added to the cell suspension just before analysis. cloned at the PstI site of pBlue-NPS. The resulting plasmid, pBlue-NPS- Km, was electroporated into H. pylori 26695. The transformants were Treatment of AGS with HP0175 and preparation of cell lysates plated on BHI agar plates with 10% FBS and 25 ␮g/ml kanamycin, and kanamycin-resistant clones (knockout (KO) strain, disrupted in HP0175) AGS cells were cultured in 24-well tissue culture plates at 4 ϫ 105 cells/ were selected. The insertion of the aphA gene within the HP0175 gene was well, treated with HP0175, and lysed with lysis buffer (20 mM Tris-HCl confirmed by PCR using genomic DNA from the KO strain as a template, (pH 7.4); 1% (v/v) Nonidet P-40; 10% (v/v) glycerol; 137 mM NaCl; 20 followed by restriction digestion of the PCR product with PstI to excise out mM sodium fluoride; 1 mM EDTA; 40 mM sodium-␤-glycerophosphate; 4 ␮ the kanamycin cassette. g/ml each of leupeptin, pepstatin, and aprotinin; 1 mM Na3VO4;1mM 5674 Helicobacter-MEDIATED GASTRIC EPITHELIAL CELL APOPTOSIS

Pefabloc; and 1 mM benzamidine) on ice for 15 min. Cell lysates were simultaneous treatment of cells with 10 ␮M carbonyl cyanide m-chloro- ϫ boiled for 5 min after the addition of 5 Laemmli sample buffer and phenylhydrazone (CCCP) and DiOC6. subjected to Western blotting. When performing Western blotting for detection of caspases, cells were pelleted and freeze-thawed three times in 20 Statistical analysis ␮ l of cell extraction buffer (50 mM PIPES/NaOH (pH 6.5), 2 mM EDTA, Ϯ 0.1% (w/v) CHAPS, 5 mM DTT, 20 ␮g/ml leupeptin, 10 ␮g/ml pepstatin, Data are represented as the mean SD of separate experiments. ANOVA t 10 ␮g/ml aprotinin, and 1 mM Pefabloc). The lysates were centrifuged at and Student’s test were performed to test statistical significance. A value p Ͻ 10,000 ϫ g for 5 min at 4°C, and the supernatants were collected for the of 0.05 was considered statistically significant. detection of caspases. Results Western blotting Purification of rHP0175 Proteins were separated on SDS-polyacrylamide gels, then transferred elec- HP0175 was expressed as an N-terminally hexa-His-tagged pro- trophoretically to polyvinylidene difluoride membranes. The blots were tein in E. coli BL21(DE3)/pCMK101 and was purified by affinity blocked with 5% nonfat dry milk and subsequently incubated overnight at 2ϩ 4°C with primary Abs in TBST (1%, v/v) with 5% (w/v) BSA. After chromatography on Ni -NTA agarose. It migrated as a single washing, the blots were incubated with HRP-conjugated goat anti-rabbit band of Mr 30 on SDS-PAGE (Fig. 1A). The catalytic efficiency Ϯ Ϫ1 Ϫ1 IgG (Cell Signaling Technology; or appropriate secondary Ab) in blocking (kcat/Km) of the enzyme was 181 20 mM s . Catalytic ac- bufferfor1hatroom temperature, followed by development with BM tivity confirmed that the rHis-tagged protein was biologically ac- chemiluminescence reagent (Roche) and exposed to x-ray film (Kodak Ͻ XAR5; Eastman Kodak). tive. A Limulus amebocyte lysate assay demonstrated that 0.05 ng of LPS was present per microgram of protein in the different ASK1 kinase assay preparations of HP0175 tested. AGS cells were cultured in 24-well tissue culture plates at 6 ϫ 105 cells/ well. After treatment, cells were lysed with lysis buffer containing 20 mM HP0175-induced cell death Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Taking into account previous reports that secreted proteins of H. Triton X-100, 1% sodium deoxycholate, 0.5 mM sodium pyrophosphate, 1 ␤ ␮ pylori induce apoptosis of gastric epithelial cells, we asked mM sodium -glycerophosphate, 1 mM Na3VO4, and 1 g/ml leupeptin. The supernatant (equivalent to 200 ␮g of protein) was incubated overnight whether HP0175 could be one of the apoptosis-inducing factors of at 4°C with rabbit polyclonal ASK1 Ab. Protein A/G Plus agarose was added and incubated at 4°C for an additional 3 h. The beads were washed twice with lysis buffer and twice with kinase buffer (25 mM Tris-HCl (pH ␤ 7.5), 5 mM sodium- -glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2). The pellet was washed once with kinase buffer without protease inhibitors. The beads were then incubated in 20 ␮l of kinase buffer in the presence of 0.5 ␮Ci of [␥-32P]ATP (sp. act., 6000 Ci/mmol) with 1 ␮g of myelin basic protein (MBP) as substrate at 30°C for 15 min. The reaction was stopped by adding protein gel denaturing buffer. After SDS- PAGE, gels were dried and subjected to autoradiography. Study of the interaction of HP0175 with TLR4 AGS cell (107/assay) lysates were incubated with Ni2ϩ-NTA or HP0175- bound Ni2ϩ-NTA agarose at 4°C for 2 h with shaking. The beads were washed and boiled in 2ϫ Laemmli buffer for 5 min, proteins were separated by 7.5% SDS-PAGE, transferred onto polyvinylidene difluoride, and immunoblotted with anti-TLR4 Ab to detect His-tagged protein-bound TLR4. In another set of experiments, cells were transfected with empty vector or TLR4 construct, followed by lysis and immunoprecipitation with anti-TLR4 Ab as described above. The pellet was washed, boiled with 2ϫ FIGURE 1. denaturing buffer, electrophoresed, and immunoblotted with anti-His Ab to Purification of HP0175 and HP0175-induced cell death of detect TLR4-bound His-tagged protein. AGS cells. A, Coomassie Blue-stained gels of uninduced (lane a), induced (lane b) E. coli cells expressing His-tagged HP0175, and purified HP0175 Isolation of cytosol and mitochondrial fractions for detection of (lane c). The arrow indicates the position of HP0175. AGS cells were cyt c and Bid incubated with HP0175 (100 ng/ml) for various periods of time (B) or with various concentrations of HP0175 or Vac A for 24 h (C). D, Cells were After treatments, 18 ϫ 106 ␮ cells were suspended in 400 l of suspension incubated with LPS or with equal amounts of His-tagged irrelevant protein buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2,1mM EDTA, 1 mM EGTA, 1 mM Pefabloc, 10 ␮g/ml leupeptin, 10 ␮g/ml or His-tagged HP0175. In separate experiments, HP0175 was subjected to aprotinin, and 150 mM sucrose) and broken with 20 passages through a boiling for1horwastreated with polymyxin B resin as indicated in the 26-gauge needle. The homogenate was centrifuged at 750 ϫ g for 10 min figure. For the latter, 100 ␮l of His-HP0175 was mixed with 100 ␮lof to remove nuclei and unbroken cells. The mitochondrial pellet was ob- polymyxin B bead slurry and incubated at 4°C for 1 h. Beads were re- tained by centrifugation at 10,000 ϫ g for 15 min and resuspended in 40 moved, and the supernatant was assayed for protein content and used for ␮l of resuspension buffer (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM apoptosis induction. In each case, the inducer was removed at the indicated Pefabloc, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin). The supernatant time point, cells were washed and lysed, and cell death was measured using ϫ obtained by centrifugation at 100,000 g for 60 min at 4°C was the the Cell Death ELISA kit (Roche) as described in Materials and Methods. cytosolic fraction. Before Western blotting, protein concentrations were Results are expressed as the fold increase in the release of histone com- assayed, and all samples were normalized to equal protein concentrations. Ϯ Blots were stripped and reprobed for ␤-actin expression. pared with that in the control (untreated AGS); values are the mean SD ,p Ͻ 0.05 vs His-HP0175 (by t test). E ,ء .of three different experiments Measurement of mitochondrial membrane potential Western blot analysis of HP0175. Purified HP0175 or cell extracts from wild-type H. pylori (WT) or an isogenic mutant inactivated in the HP0175 The induction of the mitochondrial permeability transition was determined gene (KO) were run on SDS-PAGE, electrotransferred onto a polyvinyli- in intact cells as the reduction in the accumulation of DiOC6 (41). Briefly, ϫ 5 dene difluoride membrane, and probed with anti-HP0175 Ab or Ab raised 5 10 AGS cells were loaded with DiOC6 (100 nM) during the last 30 min of treatment with HP0175, then lysed in deionized water, and fluo- against an irrelevant His-tagged protein (irr Ab). F, AGS cells were cocul- tured with H. pylori (WT or KO) at a multiplicity of infection of 50 for rescence of DiOC6 was read in a Hitachi F-4500 fluorescence spectropho- p Ͻ 0.05 vs WT ,ء .tometer at excitation and emission wavelengths of 488 and 500 nm, re- 24 h, and cell death was measured as described above spectively. Values for de-energized mitochondria were determined by (by t test). The Journal of Immunology 5675

H. pylori. His-HP0175 induced cell death of AGS cells in a time- the HP0175 gene led to a significant reduction in the induction of and dose-dependent manner (Fig. 1, B and C). HP0175 was a more apoptosis compared with the wild-type parent strain (Fig. 1F and potent apoptosis-inducing factor than Vac A (Fig. 1C), which was Table I). Apoptosis-inducing ability was not totally abolished in reported to be an inducer of apoptosis. Cell death was also assessed the KO. This was expected, because several other apoptosis-in- by measuring the binding of annexin-fluos to treated cells. This ducing factors, such as VacA, urease, and ␥-glutamyl transpepti- method also supported the view that HP0175 was an apoptosis- dase, also contribute to cell death. These results confirmed the inducing protein (Table I). Using propidium iodide staining, no involvement of HP0175 in the induction of apoptosis in the gastric necrosis was observed in the treated cells. epithelial cell line AGS.

HP0175-induced apoptosis is not due to His tag or LPS HP0175 signals through TLR4 To control for nonspecific effects of the His tag present in recom- TLR4 is expressed constitutively in AGS cells (42). AGS cell ly- binant HP0175, an irrelevant His-tagged protein was used in par- sates were incubated with HP0175 immobilized on Ni2ϩ-NTA allel. The irrelevant His-tagged protein failed to induce apoptosis agarose. Western blots probed with anti-TLR4 Ab showed that above the level observed in control cells (Fig. 1D and Table I), TLR4 is pulled down with immobilized HP0175 in the absence or ruling out the likelihood of the His tag contributing to the apop- the presence of polymyxin B (Fig. 2A), suggesting a direct inter- tosis-inducing ability. action between HP0175 and TLR4. TLR4 could not be pulled As shown in Fig. 1D and Table I, heat treatment inhibited the down in controls using Ni2ϩ-NTA agarose alone or when an ir- apoptosis-inducing ability of HP0175, whereas polymyxin B treat- relevant His-tagged protein was immobilized on Ni2ϩ-NTA aga- ment was without effect, ruling out the possibility of contaminating rose, confirming a specific interaction between HP0175 and TLR4. LPS being the factor responsible for induction of apoptosis. In addition, AGS cells were exposed to doses of E. coli LPS ranging from5ngto5␮g/ml up to 36 h, and apoptosis was assessed using histone ELISA. E. coli LPS was unable to induce apoptosis in AGS cells (Fig. 1D). These findings indicate that LPS contamination was not responsible for the apoptosis observed in cells exposed to HP0175. Moreover, the fact that heat treatment abrogated the apoptosis-inducing ability of HP0175 suggested that induction of apoptosis was a property specific to the protein.

Apoptosis-inducing ability of an isogenic mutant deﬁcient in HP 0175 An isogenic mutant disrupted in the HP0175 gene (KO) was con- structed. Successful disruption of the HP0175 gene in the KO strain was conﬁrmed by the absence of any band in cell extracts upon Western blotting and probing with anti-HP0175 Ab (Fig. 1E). The strain grew normally in vitro. However, inactivation of

Table I. TLR4-dependent HP0175-induced apoptosis in AGS cells FIGURE 2. HP0175 signals through TLR4 to induce apoptosis in AGS Annexin V-Fluos-Positive cells. A, The ability of HP0175 to interact directly with TLR4 was exam- Cells (%) ined by a pull-down assay. AGS cell lysate was incubated with HP0175 immobilized on Ni2ϩ-NTA agarose in the absence (lane b) or the presence Ϯ a Control 6 1 (lane e) of polymyxin B or with Ni2ϩ-NTA agarose alone (lane c)or His-HP0175 60 Ϯ 5 Ϯ combined with an irrelevant His-tagged protein (lane d) immobilized on Irrelevant His-protein 7 1 2ϩ Heat-treated His-HP0175 10 Ϯ 3 Ni -NTA agarose for 2 h; the agarose-bound proteins were separated on His-HP0175 ϩ anti-TLR4b 9 Ϯ 2c SDS-PAGE; and coprecipitated agarose-bound TLR4 was detected by His-HP0175 ϩ i/cb 58 Ϯ 7 Western blotting using anti-TLR4 Ab. Lane a represents immunoprecipi- His-HP0175 ϩ PBd 61 Ϯ 6 tated TLR4 only. The arrow indicates the band corresponding to TLR4. His-HP0175 ϩ SB203580 (5 ␮M) 7 Ϯ 1c The blot shown is representative of three separate experiments. B, AGS His-HP0175 ϩ U0126 (25 ␮M) 58 Ϯ 6 cells were transfected with pcDNA-TLR4 (lanes b, d, and e) or with empty HP(WT) 75 Ϯ 5 vector (lane c), Cell lysates from these cells were incubated with His- Ϯ e HP(KO) 35 2 HP0175 (lanes b, c, and e) or irrelevant His-tagged protein (lane d), fol- Vector control ϩ His-HP0175 60 Ϯ 2 lowed by immunoprecipitation with anti-TLR4 Ab (lanes b–d) or with ASK1 KM ϩ His-HP0175 20 Ϯ 4f p38 agf ϩ His-HP0175 21 Ϯ 5f anti-isotype IgG (lane e). Western blotting was performed with anti-His Ab Caspase 9s ϩ His-HP0175 22 Ϯ 4f to detect coprecipitated His-tagged protein. Lane a represents His-HP0175 IETD ϩ His-HP0175 6 Ϯ 1c only. The arrow indicates the band corresponding to HP0175. C, AGS cells DEVD ϩ His-HP0175 10 Ϯ 2c were transfected with empty vector or TLR4(dn), followed by incubation p Ͻ 0.05 vs ,ء .LEHD ϩ His-HP0175 9 Ϯ 3c without (Ⅺ) or with (f) HP0175 (100 ng/ml) for 24 h a Ϯ empty vector-transfected AGS challenged with HP0175 (by t test). D, AGS Results represent the means SD. ␮ b Cells were pretreated with anti-TLR4 mAb or isotype-matched Ab (i/c) before cells were incubated without or with 25 g/ml human TLR4-speciﬁc mAb challenge with His-HP0175. (HTA125) or an isotype-matched irrelevant Ab (i/c) for 1 h. Cells were c p Ͻ 0.001 vs His-HP0175. washed and incubated without or with HP0175 in fresh medium as de- d p Ͻ 0.005 vs HP0175-treated cells only (by ANOVA). C ,ء .His-HP0175 was incubated with polymyxin B resin as described in Fig. 1 before scribed in C incubation with AGS cells. e p Ͻ 0.05 vs WT. and D, Cell death was measured as described in Fig. 1. Values are the f p Ͻ 0.05 vs vector control ϩ His-HP0175. mean Ϯ SD of three different experiments. 5676 Helicobacter-MEDIATED GASTRIC EPITHELIAL CELL APOPTOSIS

No band appeared in Western blots performed using anti-CD14 or HP0175-induced cell death (Fig. 3B and Table I). ASK1 is a mem- anti-TLR2 Ab (data not shown), confirming that the band that was ber of the MAPK kinase kinase family that plays a role in stress- visible after pull-down could be attributed specifically to TLR4. induced apoptosis through activation of the stress-activated MAPK The interaction between HP0175 and TLR4 was also confirmed by signaling cascades (26–29). Catalytically inactive ASK1 the converse experiment. Cells were transfected with empty vector (ASK1(KM)) inhibited HP0175-induced cell death (Fig. 3B and or with TLR4 construct, cell lysates were incubated with HP0175, Table I), supporting the view that cell death was dependent immunoprecipitated with anti-TLR4 Ab, followed by Western on ASK1. analysis using anti-His Ab. HP0175 was specifically pulled down with TLR4 (Fig. 2B). No band was visible when immunoprecipi- Activation of ASK1 and p38 MAPK by HP0175 tation was performed using an isotype-matched Ab or when cells Incubation of AGS cells with HP0175 led to a time-dependent were transfected with empty vector or treated with an irrelevant increase in ASK1 kinase activity, as evidenced by in vitro phos- His-tagged protein. These results argued in favor of a specific in- phorylation of MBP (Fig. 4A). The activation of ASK1 was de- teraction between HP0175 and TLR4. TLRs recognize various pendent on TLR4, because transfection of cells with TLR4(dn) pathogen-derived molecular motifs (43). A recent study has shown before HP0175 challenge led to an inhibition of ASK1 activity that H. pylori infection leads to up-regulation of TLR4 and MD-2 (Fig. 4B). Activation of p38 MAPK was assessed by Western anal- expression in gastric mucosa (44). Transfection of AGS cells with ysis of the phospho form of the kinase. Although p38 MAPK was TLR4(dn) could inhibit HP0175-mediated cell death (Fig. 2C). At activated in cells transfected with wild-type ASK1 before chal- the same time, preincubation of AGS cells with blocking anti- lenge with HP0175, ASK1(KM) inhibited HP0175-induced acti- TLR4 mAb could also block HP0175-mediated cell death (Fig. 2D vation of p38 MAPK (Fig. 4C), suggesting that ASK1 was up- and Table I), whereas control isotype-matched Ab was without stream of p38 MAPK activation. effect. Taken together, these findings indicate that TLR4 signaling effects HP0175-induced cell death. Role of caspases in HP0175-mediated cell death Members of the caspase family are crucial mediators of apoptosis. HP0175-induced cell death involves MAPK The involvement of caspases 8, 9, and 3 was evident from the The stress-activated MAPK p38 is known to effect cell death under death inhibitory effects of z-IETD-FMK, z-LEHD-FMK, and z- a variety of conditions (25). The p38 MAPK inhibitor SB203580 DEVD-FMK, respectively (Fig. 5A and Table I). Caspase 9S is an could inhibit HP0175-induced cell death in a dose-dependent manner (Fig. 3A and Table I), whereas U0126, an inhibitor of ERK1/2 MAPK, was without effect. These results suggested that p38 MAPK was involved in mediating cell death effected by HP0175. This was also supported by our observations that transfection of cells with dominant negative p38 MAPK (p38(agf)) inhibited

FIGURE 4. HP0175 activates ASK1 and p38 MAPK. AGS cells were incubated with HP0175 (100 ng/ml) for various periods of time (A), or cells were transfected with empty vector or TLR4(dn), followed by treatment with HP0175 (100 ng/ml) for 60 min (B). Whole-cell lysates were prepared, ASK1 was immunoprecipitated, and in vitro ASK1 kinase activity was determined using MBP as substrate as described in Materials and FIGURE 3. HP0175-induced cell death depends on ASK1, MAPKs, Methods. The bottom panel is a representative Western blot with anti- and caspases. AGS cells were left untreated or were treated with the cell- ASK1 to show that the same amount of ASK1 was present in each sample. permeable MAPK inhibitors U0126 and SB203580 at the indicated con- The bar diagrams represent densitometric scans of the phospho-MBP bands centrations (A) for 60 min, followed by removal of the inhibitors and in- obtained in three different experiments (mean Ϯ SD). C, AGS cells were p Ͻ 0.001 vs transfected with ASK1(WT) or a catalytically inactive mutant of ,ء .cubation without or with HP0175 (100 ng/ml) for 24 h control treated with HP0175 (by ANOVA). B, AGS cells were transfected ASK1(KM), followed by incubation without or with HP0175 (100 ng/ml) with different constructs as indicated in the ﬁgure, followed by treatment for 2 h. Whole-cell lysates were prepared, followed by immunoblotting without or with HP0175 (100 ng/ml) for 60 min. Cell death was measured with anti-phospho-p38 MAPK Abs. The lower blots show that the same -p Ͻ 0.001 vs WT (by ANOVA). Values are the amount of p38 MAPK was present in each sample. The right panel rep ,ء .as described in Fig. 1 mean Ϯ SD of three different experiments. Ⅺ, Without HP0175; f, with resents the densitometric analysis of the phospho-p38 MAPK bands (error HP0175. bars represent the SD from three independent experiments). The Journal of Immunology 5677

Consistent with the effect of caspase inhibitors on HP0175-induced cell death, Western analysis using Abs specific for the cleaved form of caspase 3 showed that HP0175 challenge activated the executioner caspase 3 (Fig. 5C). The initiator caspase 9 (Fig. 5E) was also activated in a time-dependent manner, as evidenced by detection of the cleaved, activated form of the caspase by West- ern blotting. Caspase 3 activation was also supported by detection of the cleaved form of the caspase 3 substrate PARP in HP0175- challenged AGS cells (Fig. 5D). The generation of cleaved caspase 3 could be blocked by pretreatment with the caspase 3-specific inhibitor z-DEVD-FMK as well as the caspase 9-specific inhibitor z-LEHD-FMK (Fig. 5C). The latter result suggested that caspase 9 activation was necessary for the generation of cleaved caspase 3. Generation of cleaved caspase 9 could be blocked by transfection of cells with p38(agf) (Fig. 5F). Generation of cleaved caspase 9 could also be blocked by pretreating cells with the caspase 8-specific inhibitor z-IETD-FMK (Fig. 5E). These data supported the role of p38 MAPK as well as caspase 8 in the activation of caspase 9 in HP0175-challenged AGS cells. The role of caspase 8 in HP0175-induced death signaling was supported by Western analysis of active caspase 8. Cleaved caspase 8 was detected in HP0175-challenged AGS cells (Fig. 5G). This could be blocked in cells pretreated with the caspase 8-specific inhibitor z-IETD-FMK. HP0175-induced caspase 8 activation was also blocked in cells transfected with p38(agf) (Fig. 5H), suggesting a role for p38 in FIGURE 5. HP0175-induced caspase activation in AGS cells. AGS caspase 8 activation. cells were left untreated or were treated with the cell-permeable irrevers- ible caspase inhibitors (50 ␮M): z-IETD-FMK (for caspase 8), z-DEVD- Induction of mitochondrial permeability transition (MPT) FMK (for caspase 3), or z-LEHD-FMK (for caspase 9; A); or were transfected with different constructs as indicated in Fig. lB, followed by The MPT refers to the regulated opening of a large, nonspecific treatment without or with HP0175 (100 ng/ml) for 60 min. Cell death was pore in the inner mitochondrial membrane that causes loss of mi- measured as described in Fig. 1. Results are expressed as the fold increase ⌬␺ tochondrial membrane potential ( m) (46). The MPT reduces the in the release of histone compared with that in the control (untreated, non- accumulation of the fluorescent dye DiOC in the mitochondria as Ϯ 6 transfected AGS). Values are the mean SD of three different experi- a consequence of the loss of ⌬␺ . Loss of ⌬␺ is associated with p Ͻ 0.001 vs no m m ,ء :ments. Ⅺ, Without HP0175; f, with HP0175. A the release of cyt c, formation of the apoptosome, and activation of p Ͻ 0.05 vs empty vector-transfected AGS ,ء :inhibitor (by ANOVA). B cells treated with HP0175 (by t test). AGS cells were incubated with caspase 9. Treatment of AGS cells with HP0175 produced a time- HP0175 (100 ng/ml) for various periods of time (C and D) or were treated dependent decline in the retention of DiOC6. (Fig. 6A). Within 4 h, with z-DEVD-FMK (50 ␮M; lanes 5 and 6) or z-LEHD-FMK (50 ␮M; 25% of the dye was lost from the cells, and within 8 h, retention lanes 7 and 8) for 60 min (C) before incubation without (lanes 5 and 7)or of DiOC6 was reduced by 76%. Treatment of AGS cells with ⌬␺ with (lanes 6 and 8) HP0175 (100 ng/ml) for 14 h. Cell lysates were CCCP, a proton ionophore that dissipates m, resulted in a sim- prepared as described in Materials and Methods. Cleaved caspase 3 (17 ilar loss of DiOC6 fluorescence. Transfection of cells with kDa) and PARP (89 kDa) were detected by Western blotting using Abs ASK1(KM) or p38(dn) prevented the loss of DiOC6 fluorescence against cleaved caspase 3 (C) or PARP (D), respectively. E, AGS cells (Fig. 6B), suggesting that ASK1 and p38 are involved in HP0175- were incubated with HP0175 (100 ng/ml) for various periods of time or induced loss of ⌬␺ in AGS cells. were treated with z-IETD-FMK (50 ␮M) for 60 min (last two lanes) before m incubation without or with HP0175 (100 ng/ml) for 9 h. Cell lysates were prepared as described in Materials and Methods. F, AGS cells were trans- Generation of truncated Bid (t-Bid) and release of cyt c fected with p38(WT) or p38(agf), followed by incubation without or with Taking into consideration that caspase 8 was activated in HP0175- HP0175 (100 ng/ml) for 9 h. E and F, Cleaved caspase 9 (37 kDa) was treated cells, it appeared likely that caspase 8 activation was linked detected by Western blotting using Abs against caspase 9. G, AGS cells to the mitochondrial death pathway. Linkage of caspase 8 to mi- were treated with HP0175 (100 ng/ml) for various periods of time or were tochondrial death signaling occurs through the caspase 8-mediated treated with z-IETD-FMK (50 ␮M) for 60 min (last two lanes) before incubation without or with HP0175 (100 ng/ml) for 5 h. Cell lysates were cleavage of Bid and generation of t-Bid, which translocates to the prepared as described in Materials and Methods. H, AGS cells were trans- mitochondria and promotes the release of cyt c (47, 48). HP0175- fected with p38(WT) or p38(agf), followed by incubation without or with treated AGS cells showed the presence of t-Bid in the mitochon- HP0175 (100 ng/ml) for 5 h. Cleaved caspase 8 (20 kDa) was detected by drial fraction (Fig. 6C). Generation of t-Bid was abrogated in Western blotting using Abs against caspase 8 (G and H). Each blot is caspase 8 inhibitor-treated cells (Fig. 6C), suggesting that t-Bid representative of results obtained in three separate experiments. Equal pro- formation is dependent on caspase 8 activation. tein loading was verified in all cases by analyzing samples for ␤-actin Concomitant with the detection of t-Bid in the mitochondria, cyt expression. c was detected in the cytosol in a time-dependent manner, although the content of actin remained the same (Fig. 6D). Pretreatment with the caspase 8 inhibitor z-IETD-FMK (Fig. 6D) or transfection inactive mutant of caspase 9 missing most of the large subunit of with p38(agf) or ASK1(KM) (Fig. 6E) abrogated HP0175-induced caspase 9, including the catalytic site (45). The role of caspase 9 cyt c release, suggesting a role for ASK1/p38 MAPK signaling in was confirmed by our observations that caspase 9S inhibited mitochondrial cyt c release in HP0175-challenged gastric epithe- HP0175-induced cell death (Fig. 5B and Table I). lial cells. 5678 Helicobacter-MEDIATED GASTRIC EPITHELIAL CELL APOPTOSIS

a property specific to HP0175. Appreciable reduction in the induction of apoptosis by the isogenic strain lacking HP0175 also confirmed the apoptosis-inducing ability of HP0175. The signaling pathway downstream of HP0175 challenge was then dissected. HP0175 was observed to interact directly with TLR4, and HP0175-induced cell death was dependent on TLR4, because preincubation of cells with blocking anti-TLR4 Ab could block HP0175-mediated cell death. Not surprisingly, we observed that caspases 3, 8, and 9 are activated after challenge of macrophages with HP0175, and that these are all required for HP0175-induced death of AGS cells, because inhibition of any one of these caspases with caspase-specific peptide inhibitors was sufficient to block cell death. Previous reports have shown that H. pylori induces activa- FIGURE 6. HP0175-induced loss of mitochondrial membrane perme- tion of caspase 8 and 3 and subsequent cleavage of PARP, leading ability transition, cleavage of Bid, and release of cyt c from the mitochon- to an increased level of apoptosis in gastric epithelial cells both in dria. AGS cells were treated with HP0175 for various periods of time (A). vivo and in vitro (53). Caspase 3 activation also occurs when In separate experiments, AGS cells were transfected with p38(WT), NF-␬B signaling is suppressed in H. pylori-challenged gastric ep- p38(agf), ASK1(WT), or ASK1(KM) before incubation without or with ithelial cell lines as well as primary gastric epithelial cells (54). HP0175 (100 ng/ml) for8h(B). Cells were washed, and the fluorescence H. pylori is known to activate MAPKs (55), which are mediators of DiOC6 was determined at excitation and emission wavelengths of 488 and 500 nm, respectively. Values for de-energized mitochondria were de- of stress-induced apoptosis. Choi et al. (56) have reported that the termined by simultaneous treatment of cells with 10 ␮M CCCP and DiOC6 inhibition of p38 MAPK by SB203580 decreased H. pylori-medi- (A). f, Without HP0175; Ⅺ, with HP0175. Values are the mean Ϯ SD of ated apoptosis of epithelial cells. Inhibition of HP0175-mediated three different experiments. Cells were left untreated or were treated with cell death in cells transfected with a dominant negative mutant of the caspase 8 inhibitor z-IETD-FMK (50 ␮M; C and D) for 1 h before p38 MAPK suggested that p38 MAPK is required to trigger treatment with HP0175 (100 ng/ml) for6h(C) or for various periods of HP0175-mediated cell death. ASK1 is a MAPK kinase kinase ki- time (D). In a separate set of experiments, cells were transfected with nase that effects stress-activated cell death by activation of p38 in p38(WT), p38(agf), ASK1(WT), or ASK1(KM) before incubation without many cell types. We observed that HP0175 challenge activated or with HP0175 (100 ng/ml) for8h(E). Cells were processed to obtain ASK1 in a TLR4-dependent manner. mitochondrial (C) or cytosolic (D and E) fractions as described in Mate- rials and Methods. Truncated Bid (15 kDa; C) or cyt c (12 kDa) and actin The link between ASK1-p38 MAPK signaling and caspase ac- (D and E) were detected by SDS-PAGE, followed by Western blotting. tivation was evident from the observation that a catalytically in- Blots are representative of results obtained from three separate active mutant of ASK1 could prevent activation of caspases 8, 9, experiments. and 3, suggesting that ASK1 plays a central role in HP0175-induced cell death. The role of the mitochondrial death pathway appeared likely, because inactive caspase 9 blocked death. The loss of retention of the probe DiOC from cells challenged with Discussion 6 HP0175, concomitant with the release of cyt c, confirmed the role Apoptosis is an orchestrated suicide program, and the key morphological alterations of apoptosis are mediated by caspases. H. of the mitochondrial pathway in HP0175-induced cell death. p38 MAPK regulated this pathway, because loss of DiOC retention pylori induces gastric epithelial cell apoptosis (49). Apoptosis con- 6 tributes to the pathological outcome of the infection by disturbing could be inhibited in cells transfected with p38(dn) or ASK1(KM). the balance between the rate of new cell production and the rate of The release of cyt c from the mitochondria could be blocked by cell loss by apoptosis (50, 51). Atrophic gastritis and gastric dys- caspase 8 inhibitor, suggesting that the death signal triggered by plasia after H. pylori infection are associated with accelerated ap- caspase 8 activation is amplified by the mitochondrial release of optosis of the gastric epithelium (52). It is therefore of consider- cyt c. cyt c release could also be blocked in p38(dn)-transfected able importance to understand the nature of the apoptosis-inducing cells, suggesting that ASK1/p38 MAPK signaling plays a crucial factors of H. pylori. With this in view, we narrowed down to the role not only in activation of caspase 8, but also in downstream secreted protein HP0175, a putative PPIase to evaluate its effects events leading to the release of cyt c and activation of caspases 9 on gastric epithelial cells. HP0175 was expressed in E. coli as a and 3 (Fig. 6). The use of inhibitors as well as the kinetics of hexahistidine-tagged protein. It was found to possess PPIase ac- caspase activation and cyt c release suggested sequential activation tivity, indicating that the recombinant protein was biologically ac- of caspases 8, 9, and 3, as depicted in the model proposed in Fig. tive. The gastric epithelial cell line AGS was challenged with 7. Caspase 3 inhibition did not affect the activation of caspase 8, HP0175 to evaluate its effects on gastric epithelium. The apopto- ruling out caspase 3 as the mediator of caspase 8 activation. sis-inducing ability was tested using two independent approaches, Death signals are in several instances amplified through the mi- namely measurement of histone release by ELISA and measure- tochondrial pathway by the caspase 8-mediated cleavage of Bid ment of annexin binding to cells using annexin-fluos. HP0175- and its translocation to the mitochondria. Bid-deficient mice are challenged cells underwent apoptosis in a time- and dose-depen- resistant to hepatocellular apoptosis (57). We analyzed the status dent manner, suggesting that HP0175 is one of a number of of Bid in the mitochondrial fractions of HP0175-challenged mac- secreted apoptosis-inducing factors of H. pylori. The apoptosis- rophages. Western blotting showed an increase of t-Bid in the mi- inducing property of HP0175 was not due to LPS contamination, tochondria as a function of time of challenge with HP0175, because LPS (up to a concentration of 5 ␮g/ml) was not able to whereas caspase 8 inhibitor could block the generation of t-Bid in induce apoptosis in AGS cells, and treatment of HP0175 with the mitochondrial fraction, suggesting that caspase 8-mediated Bid polymyxin B resin did not affect its ability to induce apoptosis. The cleavage activates the mitochondrial death pathway in this case. inability of irrelevant His-tagged recombinant protein to induce This was supported by the concomitant detection of cyt c in the apoptosis clearly suggested that the apoptosis-inducing ability was cytosolic fraction. The Journal of Immunology 5679

HP0175-TLR4 interaction are under investigation. ASK1 plays a central role in this pathway by activating p38 MAPK (Fig. 7). Signaling downstream of p38 leads to the sequential activation of caspase 8, truncation of Bid, its translocation to the mitochondria, and loss of mitochondrial membrane potential initiating the release of cyt c and the activation of caspases 9 and 3. It has been reported that H. pylori-mediated apoptosis of gastric epithelial cells involves translocation of Bax, followed by mitochondrial depolar- ization and subsequent activation of caspase 3 (58), and that reduced levels of Bcl-2 are associated with H. pylori-mediated apoptosis of gastric epithelial cells (59). Our studies strengthen the view that the mitochondrial pathway plays a critical role in H. pylori-mediated apoptosis in gastric epithelial cells. Our present investigation provides insight into the pathways through which a secreted protein of H. pylori with PPIase activity signals to induce apoptosis. HP0175 may be regarded an addition to the growing list of molecules that effect TLR-mediated apoptotic cell death (60). HP0175 may therefore contribute to the pathology of gastric cancer by inducing hyperproliferation of the gastric epithelium. These studies provide new insights into H. pylori pathogenicity and reveal for the first time a novel characteristic of a secreted protein with PPIase activity. Acknowledgments We thank all the scientists who gifted constructs used in this study, and Dr. T. Ramamurthy (National Institute of Cholera and Enteric Diseases, Kolkata, India) for help in culturing H. pylori. Disclosures The authors have no financial conflict of interest. References 1. Peek, R. M., Jr., and M. J. Blaser. 2002. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat. Rev. Cancer 2:28. 2. Kohda, K., K. Tanaka, Y. Aiba, M. Yasudo, T. Mina, and Y. Koga. 1999. Role of apoptosis induced by Helicobacter pylori infection in the development of duodenal ulcer. Gut 44:456. 3. Moss, S. F., J. Calam, B. Agarwal, S. Wang, and P. R. Holt. 1996. Induction of gastric epithelial apoptosis by Helicobacter pylori. Gut 38:398. 4. Rudi, J., D. Kuck, S. Strand, A. von Herbay, S. M. Mariani, P. H. Krammer, P. R. Galle, and W. Stremmel. 1998. Involvement of the CD95 (APO-1/Fas) receptor and ligand system in Helicobacter pylori-induced gastric epithelial apoptosis. J. Clin. Invest. 1028:1506. 5. Wagner, S., W. Beil, J. Westermann, R. P. Logan, C. Trautwein, J.S. Bleck, and M. P. Manns. 1997. Regulation of gastric epithelial cell growth by Helicobacter pylori: evidence for a major role of apoptosis. Gastroenterology 113:1836. 6. Mannick, E. E., L. E. Bravo, G. Zarama, J. L. Realpe, X. J. Zhang, B. Ruiz, E. T. Fontham, R. Mera, M. J. Miller, and P. Correa. 1996. Inducible nitric oxide synthase, nitrotyrosine, and apoptosis in Helicobacter pylori gastritis: effect of antibiotics and antioxidants. Cancer Res. 56:3283. 7. Nagasako, T., T. Sugiyama, T. Mizushima, Y. Miura, M. Kato, and M. Asaka. 2003. Up-regulated Smad5 mediates apoptosis of gastric epithelial cells induced by Helicobacter pylori infection. J. Biol. Chem. 278:4821. 8. Konturek, P. C., P. Pierzchalski, S. J. Konturek, H. Meixner, G. Faller, T. Kirchner, and E. G. Hahn. 1999. Helicobacter pylori induces apoptosis in gastric mucosa through an upregulation of Bax expression in humans. Scand. J. Gastroenterol. 34:375. 9. Nardone, G., S. Staibano, A. Rocco, E. Mezza, F. P. D’Armiento, Z. Insabato, FIGURE 7. Proposed model for HP0175-induced death of AGS cells. A. Coppola, G. Salvatore, A. Lucariello, N. Figura, et al. 1999. Effect of Heli- cobacter pylori infection and its eradication on cell proliferation, DNA status, and oncogene expression in patients with chronic gastritis. Gut 44:789. 10. Potthoff, A., S. Ledig, J. Martin, O. Jandl, M. Cornberg, B. Obst, W. Beil, In summary, we provide evidence for the role of a novel apop- M. P. Manns, and S. Wagner. 2002. Significance of the caspase family in Heli- cobacter pylori induced gastric epithelial apoptosis. Helicobacter 7:367. tosis-inducing secreted PPIase of H. pylori in gastric epithelial cell 11. Cohen, G. M. 1997. Caspases: the executioners of apoptosis. Biochem. J. 326:1. apoptosis. We have observed basal expression of TLR4 in AGS 12. Shi, Y. 2002. Mechanisms of caspase activation and inhibition during apoptosis. cells (data not shown) as reported previously by Smith et al. (42). Mol. Cell 9:459. 13. Thornberry, N. A., and Y. Lazebnik. 1998. Caspases: enemies within. Science We have identified TLR4 as a critical molecule that controls sig- 281:1312. naling events associated with HP0175-induced cell death. Ishihara 14. Kischkel, F. C., S. Hellbardt, I. Behrmann, M. Germer, M. Pawlita, et al. (44) have recently reported low cell surface expression of P. H. Krammer, and M. E. Peter. 1995. Cytotoxicity-dependent APO-1 (Fas/ CD95)-associated proteins form a death-inducing signaling complex (DISC) with TLR4, probably due to low constitutive levels of MD-2 in AGS. the receptor. EMBO J. 14:5579. Whether HP0175 modulates surface expression of TLR4 or 15. Salvesen, G. S., and V. M. Dixit. 1999. Caspase activation: the induced-proximity model. Proc. Natl. Acad. Sci. USA 96:10964. whether HP0175-induced apoptosis of AGS cells is independent of 16. Muzio, M., G. Salvesen, and V. Dixit. 1997. FLICE induced apoptosis in a cell- cell surface expression of TLR4, and the detailed mechanism of free system: cleavage of caspase zymogens. J. Biol. Chem. 272:2952. 5680 Helicobacter-MEDIATED GASTRIC EPITHELIAL CELL APOPTOSIS

17. Stennicke, H. R., J. M. Jurgensmeier, H. Shin, Q. Deveraux, B. B. Wolf, X. Yang, 39. Yin, E. T., C. Galanos, S. Kinsky, R.A. Bradshaw, S. Wessler, O. Luderity, and Q. Zhou, H. M. Ellerby, L. M. Ellerby, D. Bredesen, et al. 1998. Pro-caspase-3 M. F. Sarimento. 1972. Picogram-senstivie assay for endotoxin: gelation of Limu- is a major physiologic target of caspase-8. J. Biol. Chem. 273:27084. lus polyphemus blood cell lysates induced by purified lipopolysaccharides and 18. Srinivasula, S. M., M. Ahmad, T. Fernandez-Alnemri, G. Litwack, and lipid A from Gram-negative bacteria. Biochim. Biophys. Acta 261:284. E. S. Alnemri. 1996. Molecular ordering of the Fas-apoptotic pathway: the Fas/ 40. Takahashi, N., T. Hayano, and M. Suzuki. 1989. Peptidyl-prolyl cis-trans isomer- APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple ase is the cyclosporin A-binding protein cyclophilin. Nature 337:473. Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. USA 93:14486. 41. Krippner, A., A. Matsuno-Yagi, R. A. Gottlieb, and B. M. Babior. 1996. Loss of 19. Liu, X., C. N. Kim, J. Yang, R. Jemmerson, and X. Wang. 1996. Induction of function of cytochrome c in Jurkat cells undergoing Fas-mediated apoptosis. apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. J. Biol. Chem. 271:21629. Cell 86:147. 42. Smith, M. F., A. Mitchell, G. Li, S. Dong, A. M. Fitzmaurice, K. Ryan, S. Crowe, 20. Susin, S. A., H. K. Lorenzo, N. Zamzami, I. Marzo, B. E. Snow, G. M. Brothers, and J. B. Goldberg. 2003. Toll-like receptor (TLR) 2 and TLR5, but not TLR4, J. Mangion, E. Jacotot, P. Costantini, M. Loeffler, et al. 1999. Molecular char- are required for Helicobacter pylori-induced NF-␬B activation and chemokine acterization of mitochondrial apoptosis-inducing factor. Nature 397:441. expression by epithelial cells. J. Biol. Chem. 278:32552. 21. Verhagen, A. M., P. G. Ekert, M. Pakusch, J. Silke, L. M. Connolly, G. E. Reid, 43. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Im- R. L. Moritz, R. J. Simpson, and D. L. Vauz. 2000. Identification of DIABLO, a munol. 21:336. mammalian protein that promotes apoptosis by binding to and antagonizing IAP 44. Ishihara, S., M. A. K. Rumi, Y. Kadowaki, C. F. Ortega-Cava, T. Yuki, proteins. Cell 102:43. N. Yoshino, Y. Miyaoka, H. Kazumori, N. Ishimura, Y. Amano, et al. 2004. 22. Zou H., W. J. Henzel, X. Liu, A. Lutschg, and X. Wang. 1997. Apaf-1, a human Essential role of MD-2 in TLR 4-dependent signaling during Helicobacter pylori- protein homologous to C. elegans CED-4, participates in cytochrome c-dependent associated gastritis. J. Immunol. 173:1406. activation of caspase-3. Cell 90:405. 45. Seol, D. W., and T. R. Billiar. 1999. A caspase-9 variant missing the catalytic site 23. Hu, Y., M. A. Benedict, L. Ding, and G. Nunez. 1999. Role of cytochrome c and is an endogenous inhibitor of apoptosis. J. Biol. Chem. 274:2072. dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis. 46. Hengartner, M. O. 2000. The biochemistry of apoptosis. Nature 407:770. EMBO J. 18:3586. 47. Gross, A., X. M. Yin, K. Wang, M. C. Wei, J. Jockel, C. Milliman, 24. Srinivasula, S. M., M. Ahmad, T. Fernandes-Alnemri, and E. S. Alnemri. 1998. H. Erdjument-Bromage, P. Tempst, and S. J. Korsmeyer. 1999. Caspase cleaved Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell BID targets mitochondria and is required for cytochrome c release, while Bcl-xL 1:949. prevents this release but not tumor necrosis factor-R1/Fas death. J. Biol. Chem. 25. Tibbles, L. A., and J. R. Woodgett. 1999. The stress-activated protein kinase 274:1156. pathways. Mol. Life Sci. 55:1230. 48. Wang, K., X. M. Yin, D. T. Chao, C. L. Milliman, and S. J. Korsmeyer. 1996. 26. Ichijo, H., E. Nishida, N. Irie, P. den Dijke, M. Saitoh, T. Moriguchi, M. Takagi, BID: a novel BH3 domain-only death agonist. Genes Dev. 10:2859. K. Matsumoto, K. Miyazono, and Y. Gotoh. 1997. Induction of apoptosis by 49. Jones, N. L., P. T. Shannon, E. Cut, H. Yeger, and P. M. Sherman. 1997. Increase ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling in proliferation and apoptosis of gastric epithelial cells early in the natural history pathways. Science 275:90. of Helicobacter pylori infection. Am. J. Pathol. 151:1695. 27. Nishitoh, H., M. Saitoh, Y. Mochida, K. Takeda, H. Nakano, M. Rothe, 50. Jones, N. L., A. S. Day, H. A. Jennings, and P. M. Sherman. 1999. Helicobacter K. Miyazono, and H. Ichijo. 1998. ASK1 is essential for JNK/SAPK activation pylori induces gastric epithelial cell apoptosis in association with increased Fas by TRAF2. Mol. Cell 2:389. receptor expression. Infect. Immun. 67:4237. 28. Tobiume, K., A. Matsuzawa, T. Takahashi, H. Nishitoh, K.-I. Morita, K. Takeda, O. Minowa, K. Miyazono, T. Noda, and H. Ichijo. 2001. ASK1 is required for 51. Correa, P., and M. J. Miller. 1998. Carcinogenesis, apoptosis and cell prolifera- Br. Med. Bull. 54:151 sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep. tion. . 21:222. 52. Xia, H. H., and N. J. Talley. 2001. Apoptosis in gastric epithelium induced 29. Matsuzawa, A., and H. Ichijo. 2001. Molecular mechanisms of the decision be- by Helicobacter pylori infection: implications in gastric carcinogenesis. tween life and death: regulation of apoptosis by apoptosis signal-regulating ki- Am. J. Gastroenterol. 96:16. nase 1. J. Biochem. 130:1. 53. Ashktorab, H., M. Neapolitano, C. Bomma, C. Allen, A. Ahmed, A. Dubois, 30. Kuck, D., B. Kolmerer, C. Konert, P.H. Krammer, W. Stremmel, and J. Rudi. T. Naab, and D. T. Smoot. 2002. In vivo and in vitro activation of caspase 8 and 2001. Vacuolating cytotoxin of Helicobacter pylori induces apoptosis in the hu- -3 associated with Helicobacter pylori infection. Microbes Infect. 7:713. man gastric epithelial cell line AGS. Infect. Immun. 69:5080. 54. Kim, J. M., J. S. Kim, H. C. Jung, Y. K. Oh, H. Y. Chung, C. H. Lee and ␬ 31. Fan, X., H. Gunasena, Z. Cheng, R. Espejo, S. E. Crowe, P. B. Ernst, and I. S. Song. 2003. Helicobacter pylori infection activates NF- B signaling path- V. E. Reyes. 2000. Helicobacter pylori urease binds to class II MHC on gastric way to induce i-NOS and protect human gastric epithelial cells from apoptosis. epithelial cells and induces their apoptosis. J. Immunol. 165:1918. Am. J. Physiol. 285:G1171. 32. Shibayama, K., K. Kamachi, N. Nagata, T. Yagi, T. Nada, Y. Doi, N. Shibata, 55. Keates, S., A. C. Keates, M. Warny, R. M. Peek, Jr., P. G. Murray, and C. P. Kelly. 1999. Differential activation of mitogen-activated protein kinases in K. Yokoyama, K. Yamane, H. Kato, et al. 2003. A novel apoptosis-inducing ϩ Ϫ protein from Helicobacter pylori. Mol. Microbiol. 47:443. AGS gastric epithelial cells by cag and cag Helicobacter pylori. J. Immunol. 33. Kim, N., D. L. Weeks, J. M. Shin, D. R. Scott, M. K. Young, and G. Sachs. 2002. 163:5552. Proteins released by Helicobacter pylori in vitro. J. Bacteriol. 184:6155. 56. Choi, I. J., J. S. Kim, J. M. Kim, H. C. Jung, and S. Song. 2003. Effect of 34. Bumann, D., S. Aksu, M. Wendland, K. Janek, U. Zimny-Arndt, N. Sabarth, inhibition of extracellular-signal regulated kinase 1 and 2 pathway in apoptosis T. F. Meyer, and P. R. Jungblut. 2002. Proteome analysis of secreted proteins of and Bcl 2 expression in Helicobacter pylori infected AGS cells. Infect. Immun. the gastric pathogen Helicobacter pylori. Infect. Immun. 70:3396. 71:830. 35. Atanassov, C., L. Pezennec, J. d’Alayer, G. Grollier, B. Picard, and J.-L. Fau- 57. Yin, X.-M., K. Wang, A. Gross, Y. Zhao, S. Zindel, B. Klocke, K. A. Roth, and chere. 2002. Novel antigens of Helicobacter pylori correspond to ulcer-related S. J. Korsmeyer. 1999. Bid-deficient mice are resistant to Fas-induced hepato- antibody. J. Clin. Microbiol. 40:547. cellular apoptosis. Nature 400:886. 36. McAtee, C. P., K. E. Fry, and D. E. Berg. 1998. Identification of potential di- 58. Ashktorab, H., S. Frank, A. R. Khaled, S. K. Durum, B. Kifle, and D. T. Smoot. agnostic and vaccine candidates of Helicobacter pylori by “proteome” technol- 2004. Bax translocation and mitochondrial fragmentation induced by Helicobac- ogies. Helicobacter 3:163. ter pylori. Gut 53:805. 37. Fischer, G., H. Bang, B. Ludwig, K. Mann, and J. Hacker. 1992. Mip protein of 59. Chu, S. H., J. W. Lim, K. H. Kim, and H. Kim. 2003. NF-␬B and Bcl-2 Heli- Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) ac- cobacter pylori-induced apoptosis in gastric epithelial cells. Ann. NY Acad. Sci. tivity. Mol. Microbiol. 6:1375. 1010:568. 38. Pereira, P. J., M. C. Vega, E. Gonzalez-Rey, R. Fernandez-Carazo, 60. Into, T., K. Kiura, M. Yasuda, H. Kataoka, N. Inoue, A. Hasebe, K. Takeda, S. Macedo-Ribeiro, F. X. Gomis-Ruth, A. Gonzalez, and M. Coll. 2002. Trypano- S. Akira, and K. I. Shibata. 2004. Stimulation of human Toll-like receptor (TLR)2 soma cruzi macrophage infectivity potentiator has a rotamase core and a highly and TLR6 with membrane lipoproteins of Mycoplasma fermentans induce apo- exposed ␣-helix. EMBO Rep. 3:88. ptotic cell death after NF-␬B activation. Cell. Microbiol. 6:187.