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Originally published In Press as doi:10.1074/mcp.M600111-MCP200 on June 11, 2006.
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Molecular & Cellular Proteomics 5:1484-1496, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Comparative Immunoproteomics of Identification and Characterization of Virulence Factors from Helicobacter pylori Related to Gastric Cancer * ,S

Yu-Fen Lin{ddagger}, Ming-Shiang Wu§,¶, Chia-Che Chang||, Sheng-Wei Lin{ddagger}, Jaw-Town Lin§,¶, Yuh-Ju Sun**, Ding-Shinn Chen§ and Lu-Ping Chow{ddagger},{ddagger}{ddagger},§§

From the {ddagger} Graduate Institute of Biochemistry and Molecular Biology and Department of Primary Care Medicine, College of Medicine, National Taiwan University and § Departments of Internal Medicine and {ddagger}{ddagger} Medical Genetics, National Taiwan University Hospital, Taipei 100, Taiwan, || Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan, and ** Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Helicobacter pylori is an important risk factor of gastric cancer (GC). Although many H. pylori virulence factors have been reported, the pathogenic mechanism by which H. pylori infection causes GC remains unclear. The aims of this study were to identify GC-related antigens from H. pylori and characterize their roles in the development of GC. As GC and duodenal ulcer (DU) are considered clinically divergent, we compared two-dimensional immunoblots of an acid-glycine extract of H. pylori probed with serum samples from 15 patients with GC and 15 with DU to find GC-related antigens, which were subsequently identified by mass spectrometry. Many protein spots were recognized by more than one serum, and 24 of these were better recognized by GC sera. The proteins showing higher frequency of recognition in GC group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase {alpha} subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Of these proteins, GroES was identified as a dominant GC-related antigen with a much higher seropositivity of GC samples (64.2%, n = 95) compared with 30.9% for gastritis (n = 94) and 35.5% for DU (n = 124). GroES seropositivity was more commonly associated with antral GC than with non-antral GC (odds ratio = 2.7; 95% confidence interval, 1.1–6.7). In peripheral blood mononuclear cells, GroES stimulated production of interleukin (IL)-8, IL-6, granulocyte macrophage colony-stimulating factor, IL-1ß, tumor necrosis factor-{alpha}, cyclooxygenase-2, and prostaglandin E2. Moreover when incubated with gastric epithelial cells, GroES induced expression of IL-8, cell proliferation, and up-regulation of c-jun, c-fos, and cyclin D1 but caused down-regulation of p27Kip1. We conclude that GroES of H. pylori is a novel GC-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation.

Helicobacter pylori causes chronic active gastritis, gastric ulcer, and duodenal ulcer (DU)1 (1, 2) and is strongly associated with the development of gastric cancer (GC) (3, 4). Despite its decreasing incidence and mortality rate, GC is still the second most common cause of cancer-related deaths worldwide (5). In addition to host and environmental factors, chronic infection with H. pylori is regarded as a major cause of GC. Case-control studies have suggested a correlation between H. pylori seropositivity and GC. H. pylori seropositive patients have a 2.1–16.7-fold higher risk of developing GC than seronegative patients (3, 4), and H. pylori infection is found in the majority (more than 70%) of GC patients (6, 7).

Clinically DU and GC are considered to be divergent entities. Although acid production increases the risk of DU, it is reduced in patients with GC (8). Furthermore DU is associated with a lower risk of developing GC (6, 9); this finding may be attributed to the fact that DU patients have antral-predominant gastritis in contrast to the corpus-predominant atrophic gastritis characterized as a precursor of GC (10). Recently two studies reported the identification of candidate antigens of H. pylori associated with DU and GC by comparing the profiles of 2D immunoblots probed with DU and GC sera (11, 12). In both studies, differentially recognized antigens were determined by spot intensity, which might be biased by variations in the immune response in different diseases and in different individuals. Importantly the serological responses toward these proteins imply that these antigens are recognized, processed, or presented by human antigen-presenting cells for initiating immune response.

In addition to eliciting humoral immune responses, H. pylori infection strongly up-regulates cytokine production by monocytes/macrophages (13). These immune responses are principally associated with mucosal production of IL-8, IL-6, IL-1ß, and TNF-{alpha} (14, 15) and with IL-8 secretion by epithelial cells (16). Serum IL-6 and IL-1ß levels have been linked to the status of H. pylori-induced GC (17). IL-8 expression is associated with angiogenic events and is strongly correlated with vessel density in GC (18). Furthermore TNF-{alpha} and IL-1ß gene polymorphisms are associated with an increased risk of non-cardia GC (19). These cytokines are therefore proposed to be critical in the pathogenesis of H. pylori-associated GC (20, 21).

The host response to H. pylori infection induces multiple changes within the gastric mucosa leading to the formation of GC. The balance is altered toward decreasing in apoptosis and increasing in proliferation as H. pylori infection leads to adenocarcinoma. H. pylori infection alters expression of the cell cycle regulatory protein p27Kip1 that confers an apoptosis-resistant phenotype (22). Expression of proto-oncogenes c-jun and c-fos is induced by H. pylori infection (23). In addition, H. pylori also activates the expression of cyclin D1 gene in gastric epithelial cells (24). Importantly it should be noted that cytokine responses and molecular alterations to H. pylori infection depend on both host genetic background and microbial virulence. Identification of GC-associated virulence factors of H. pylori that potentially characterize pathogen-host interactions is therefore crucial for further elucidation of the pathogenesis of H. pylori-related gastroduodenal diseases.

In this study, we used a proteomic approach to identify GC-related antigens of H. pylori by comparing profiles of 2D immunoblots probed with DU and GC sera. Here we report the identification of a novel GC-related antigen, GroES. GroES enhanced the production by PBMC of proinflammatory cytokines associated with H. pylori-induced GC. Moreover treatment of KATO-III, a gastric carcinoma cell line, with GroES led to cell growth and up-regulation of marker proteins associated with cell proliferation. Taken together, these results suggest the promoting role of GroES in GC development. Furthermore our report presents a method for identifying novel GC-related H. pylori antigens that should help elucidate how these antigens contribute to the inflammation and neoplastic changes induced by this bacterium.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strain and Culture Conditions—
H. pylori strain HC5 was isolated from endoscopic biopsy sample from the stomach of a patient with GC at the National Taiwan University Hospital. The bacteria were cultured on a BBLTM StackerTM plate (BD Biosciences) at 37 °C under microaerobic conditions. Liquid cultures were grown in flasks containing Brucella broth (Difco) supplemented with 10% fetal bovine serum (FBS; Invitrogen), vancomycin (12.5 mg/liter; Sigma), and amphotericin B (2.5 mg/liter; Sigma) with constant agitation at 150 rpm for 48–72 h. The culture medium was centrifuged for 10 min at 1000 x g, and the supernatant was filtered through a 0.2-µm filter (Pall, Ann Arbor, MI) to eliminate intact bacterial cells.

Patients and Serum Samples—
Serum samples were prospectively collected from individuals who participated in a national project for the investigation of H. pylori and gastroduodenal disorders in Taiwan between December 1999 and December 2001. Our study protocol was approved by both the Institutional Research Board and the Department of Health, Executive Yuan, Taiwan. Patients with newly diagnosed GC (n = 95) who underwent curative gastrectomy at our institution were enrolled. For the non-cancer groups, we screened subjects from health examination at clinics; all received an upper gastrointestinal endoscopic examination and showed no GC lesions. Ninety-four patients with gastritis and 124 with DU were enrolled. H. pylori status was determined by culture and/or histological examination of gastric biopsy specimens. Tumors were histologically classified into intestinal and diffuse types based on Lauren’s (25) classification. Tumor stage and location were determined by a combined evaluation of a special report form completed by the patient’s doctor, the case record, and the pathology report. GC stage was categorized as early (tumor extent limited to the mucosa and submucosa) or advanced (tumor invasion beyond the muscularis propria), while tumor location was subdivided into antrum, body, and cardia. In addition, 32 subjects with a normal appearance of the gastric mucosa and no evidence of H. pylori infection were selected as controls. Fasting serum samples from all participants were collected, catalogued, aliquoted, and stored at –80 °C. Aliquots were only thawed once prior to analysis.

Two-dimensional Electrophoresis and Immunoblotting—
Cell surface proteins were extracted from H. pylori using an acid-glycine extraction procedure as described previously (26). The H. pylori acid-glycine extract was precipitated using TCA (20%), and the proteins were separated by two-dimensional electrophoresis as described previously (27). Briefly protein extract was incubated with 2D sample buffer (8 m urea, 2% Pharmalyte pH 3–10, 60 mm DTT, 4% CHAPS, bromphenol blue), the first dimension of the 2D gel was run on IPG strips (Immobiline DryStrip pH 3–10, 11 cm, Amersham Biosciences), and the second dimension was run on 12.5% SDS-polyacrylamide gels. For immunodetection, the proteins on the 2D gel were transferred to a PVDF membrane (Millipore, Bedford, MA), and then the membrane was blocked by incubation for 1 h at room temperature in blocking buffer (26 mm Tris-HCl, 150 mm NaCl, pH 7.5, 1% skimmed milk) and incubated with serum samples from GC patients or DU patients or pooled normal sera (1:1000 in 0.05% Tween 20 in blocking buffer). Horseradish peroxidase-conjugated goat anti-human IgG (Chemicon, Temecula, CA) was used as secondary antibody, and bound antibody was detected using 3-amino-9-ethylcarbazole (Sigma) as substrate.

Protein Identification—
The individual protein spots were excised and subjected separately to in-gel tryptic digestion. Briefly the spots were destained using 50 mm NH4HCO3 in 50% ACN and dried in a SpeedVac concentrator. The protein was then digested by incubation overnight at 37 °C with sequencing grade trypsin (Promega, Madison, WI) in 50 mm NH4HCO3, pH 7.8. The resulting peptides were extracted sequentially with 1% TFA and 0.1% TFA, 60% ACN. The combined extracts were lyophilized and analyzed using a QSTARTM XL Q-TOF mass spectrometer (Applied Biosystems, Framingham, MA) coupled to an UltiMateTM nano-LC system (Dionex/LC Packings, Amsterdam, Netherlands). Peak lists of MS/MS spectra were created using Mascot Search Version 1.6b4 in Analyst® QS 1.1 (Applied Biosystems). Then the peak lists were uploaded to Mascot MS/MS Ions Search program (Mascot Version 2.0) on the Matrix Science public web site, and protein identification was performed against the National Center for Biotechnology Information non-redundant (NCBInr) database (3,479,934 protein entries in it at time searched). Up to two missed cleavages were allowed. Cysteine carbamidomethylation, glutamine/asparagine deamidation, and methionine oxidation were set as possible modifications. The error windows for peptide and MS/MS fragment ion mass values were 0.3 and 0.5 Da, respectively. MH+ and MH22+ were selected as the precursor peptide charge states in the searching. The ions scores of more than 54 indicated a significant match. The individual score for the MS/MS spectrum of each peptide was >20. From the hit lists, the protein names and locus_tag in H. pylori 26695 strain were selected and are listed in Table I.


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  		TABLE I Proteins of H. pylori showing higher frequency of recognition in GC group than in DU group identified by nano-LC-MS/MS analysis

 
Cloning and Purification of the Recombinant Proteins—
H. pylori was lysed followed by RNase treatment, and the genomic DNA was further purified using phenol-chloroform and precipitated with 70% ethanol. Primer pairs used to amplify the DNA fragment containing the H. pylori groES gene by PCR are listed in Supplemental Table I. PCR was performed using 35 cycles of 94 °C for 1 min, the annealing temperature for 1 min, and 72 °C for 2 min followed by a final extension at 72 °C for 15 min. The gene fragment was cloned into the expression vector pQE30 (Qiagen, Chatsworth, CA) and transformed into Escherichia coli strain M15. H. pylori FlaG clone (pQE30/SG13009) was kindly provided by Dr. Yuh-Ju Sun. For expression of recombinant proteins, cells were grown to an A600 value of 0.6, induced with 1 mm isopropyl ß-d-thiogalactoside, and harvested after 6 h at 25 °C (for GroES) or 3 h at 37 °C (for FlaG). The soluble recombinant proteins were purified on a Ni2+-chelating Sepharose column (Amersham Biosciences). To remove endotoxin from the recombinant proteins solutions, the resin was first washed in a centrifuge tube using binding buffer (20 mm Tris-HCl, 0.5 m NaCl, 5 mm imidazole, pH 7.9) containing 1% Triton X-114 (Sigma) and then loaded into a column and washed with binding buffer containing 0.1% Triton X-114 before elution of recombinant proteins. The purified recombinant proteins were dialyzed against PBS, and the endotoxin content was measured using a QCL-1000® kit (BioWhittakerr, Walkersville, MD). The final endotoxin content was about 36 endotoxin units/mg of protein.

Preparation of Polyclonal Anti-GroES Antibodies—
New Zealand White rabbits were injected intradermally with 500 µg of purified recombinant GroES (rGroES) in 1 ml of PBS emulsified with 1 ml of complete Freund’s adjuvant (Difco). Boosters of 500 µg in 1 ml of PBS emulsified with 1 ml of Freund’s incomplete adjuvant (Sigma) were given intradermally at weeks 3 and 6, then the rabbit was bled 10 days after the last boost, and the serum was used for immunoblotting experiments.

Serologic Study—
Serum samples from patients with GC, gastritis, or DU or normal controls diluted to 1:1000 were screened for reactivity with GroES by immunoblotting. Recombinant GroES was electrophoresed on a 15% SDS-polyacrylamide gel and transferred to a PVDF membrane. Immunoblotting was performed as described above.

Statistical Analysis—
Statistical analysis was performed using SPSS, Version 11.0. Categorical data were analyzed using the {chi}2 test. The odds ratio (OR) and 95% confidence interval (CI) were calculated by logistic regression. Comparisons between tests by ELISA or MTS assay were made using Student’s t test. A p value of <0.05 was considered statistically significant.

Cell Culture—
Heparinized venous blood was drawn from healthy volunteers, and mononuclear cells were isolated using Ficoll-Paque® Plus (Amersham Biosciences) density gradient centrifugation as recommended by the manufacturer. PBMC (1.8 x 106 cells/ml) were cultured in RPMI 1640 medium (Invitrogen) with 0.1% FBS at 37 °C in 5% CO2. A human gastric carcinoma cell line, KATO-III, was obtained from the Japan Cancer Research Bank and was maintained in RPMI 1640 medium with 10% FBS and 100 µg/ml streptomycin and penicillin at 37 °C in 5% CO2. KATO-III cells (7.3 x 104 cells/ml) were cultured in RPMI 1640 medium with rGroES to detect cytokines or incubated for 16–18 h in RPMI 1640 medium; following serum starvation, the KATO-III cells were incubated with rGroES in RPMI 1640 medium for Western blot analysis.

RT-PCR—
Cells were collected after 4- (PBMC) or 6-h (KATO-III) stimulation with rGroES, and mRNAs were isolated using a QuickPrepTM Micro mRNA purification kit (Amersham Biosciences) following the manufacturer’s recommendations. Reverse transcription reactions were performed according to the instruction manual for the SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen). The resulting cDNA was used as template for PCR amplification using the primer pairs and the annealing temperature conditions listed in Supplemental Table I. PCR was performed as described above. As a loading control, a parallel PCR was carried out using a primer pair for human GAPDH.

Measurement of Cytokines and PGE2—
Cells were incubated for 24 h with rGroES, and then the supernatants were collected and stored at –80 °C until assayed for cytokine production. Levels of cytokines and PGE2 in the culture supernatants were measured using Quantikine® ELISA assay kit (R&D Systems, Minneapolis, MN) for IL-8, IL-6, IL-1ß, TNF-{alpha}, and GM-CSF or a Direct Biotrak Assay ELISA kit (Amersham Biosciences) for PGE2 according to the manufacturer’s instructions. All experiments were performed in triplicate. Furthermore to verify that the cytokine release from cells was due to rGroES and not the contaminating LPS, rGroES and LPS were digested with proteinase K (PK/substrate molar ratio of 1:10) for 1 h at 37 °C, and then the PK was inactivated by heating at 100 °C for 10 min. PK-treated rGroES and LPS were then used to treat cells as described above.

Western Blot Analysis—
After treatment with rGroES for 12 or 24 h, cells were treated with lysis buffer (0.6% Nonidet P-40, 0.9% NaCl, 0.1% SDS, 1 mm EDTA, 10 mm Tris-HCl, pH 7.5) followed by centrifugation at 18,000 x g for 15 min at 4 °C to remove cell debris. Immunoblot analysis was performed as described above. The primary antibodies used were goat anti-COX-2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-cyclin D1 (1:500, Santa Cruz Biotechnology), mouse anti-p27Kip1 (1:1000, BD Biosciences Transduction Laboratories), and mouse anti-ß-actin (1:100,000, CashmereBiotech, Taipei Hsien, Taiwan). The secondary antibodies used were horseradish peroxidase-conjugated anti-mouse IgG antibody (BD Biosciences Pharmingen) or anti-goat IgG antibody (Sigma). Bound antibody was detected using ECLTM reagent (Amersham Biosciences) followed by exposure to x-ray film (Eastman Kodak Co.). ß-Actin was used as the loading control.

Cell Proliferation Assay—
KATO-III cells (8000 cells/well) were cultured in 100 µl of 0.1% FBS in RPMI 1640 medium with or without rGroES in a 96-well culture plate for 6, 24, 36, and 48 h. The number of viable cells was measured by MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). The assay was performed by adding 20 µl of the above reagent to each well, incubating at 37 °C for 1 h, and then measuring the absorbance at 490 nm. Results are presented as the percentage of nontreated cells after subtracting the blank values (medium only). The experiments were performed in triplicate.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Gastric Cancer-related Antigens of H. pylori by 2D Immunoblot Analysis—
To identify candidate H. pylori antigens associated with GC, we performed 2D SDS-PAGE on the bacterial proteins extracted with acidic glycine and compared the patterns of 2D immunoblots probed with sera from H. pylori-infected patients with either GC or DU. Silver staining revealed a complex protein profile of the acid-glycine extract (Fig. 1A). Probing with 15 GC sera and 15 DU sera gave unique and different patterns of reactivity. Two representative immunoblots are shown in Fig. 1B (GC) and Fig. 1C (DU). In general, the frequency of spot recognition was greater with GC sera than with DU sera. On the GC immunoblots, about 60 different reactive protein spots were detected with molecular masses ranging from 14 to 85 kDa and pI values ranging from 4.5 to 9.5. Some of these antigenic spots were recognized by an individual serum sample, but 49 spots were recognized by more than one. Comparing the antigenic protein profile of these 2D immunoblots, 24 spots/spot groups were more frequently recognized by GC sera. The spots with differential frequencies of recognition were subsequently identified by nano-LC-MS/MS ion search and are shown in Table I and Supplemental Table II. The proteins showing higher frequency of recognition in GC group (GC versus DU seropositivity ratio >2) are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase {alpha} subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Among the identified proteins, two forms of co-chaperonin GroES monomer and dimer, indicated in Fig. 2, exhibited the highest frequency of differential recognition by GC sera (66.7%) but was only recognized by one of the 15 (6.7%) DU sera (data not shown). Therefore, co-chaperonin GroES was considered as an important immunogenic protein.


Figure 1
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  		FIG. 1. 2D profiles of GC-related immunogenic proteins. An acid-glycine extract of cell surface proteins from H. pylori was separated by 2D electrophoresis using a linear pH 3–10 gradient in the first dimension and 12.5% SDS-PAGE in the second dimension. The separated proteins were detected by silver staining (A) or were transferred to a PVDF membrane and probed with serum from a patient with GC (B) or DU (C).

 

Figure 2
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  		FIG. 2. Human IgG binding analysis of H. pylori GroES in gastric cancer sera samples. An acid-glycine extract of cell surface proteins from H. pylori was separated by 2D electrophoresis. The portion of the silver-stained gel and immunoblots containing GroES isoforms are shown. The 2D immunoblots were analyzed by probing with 15 gastric cancer sera samples, respectively. The positions of GroES isoforms are indicated (arrowheads). mGroES denotes the monomeric form of GroES, and dGroES denotes the dimeric form of GroES. WB, Western blot

 
GroES Exists as Monomeric and Multimeric Protein and Can Be Secreted from H. pylori—
To investigate the biochemical features of GroES, we expressed recombinant His-tagged GroES fusion protein in E. coli M15 and used the purified rGroES to generate an anti-GroES antiserum in rabbits. Recombinant GroES with an apparent molecular mass of 17 kDa was successfully expressed in E. coli M15 (Fig. 3A, lane 1). The identity of the purified rGroES (Fig. 3A, lane 2) was confirmed by nano-LC-MS/MS. Furthermore the existence of monomeric and dimeric forms of rGroES was observed by immunoblot analysis using the anti-GroES antibodies (Fig. 3A, lane 3).


Figure 3
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  		FIG. 3. Characterization of native and recombinant GroES. A, purification of rGroES and reactivity with anti-rGroES antibodies. Proteins in the isopropyl ß-d-thiogalactoside-induced M15 cell lysate (lane 1) or the purified rGroES (lanes 2 and 3) were separated by 12.5% SDS-PAGE and then stained with Coomassie Blue (lanes 1 and 2) or immunoblotted with the anti-GroES polyclonal antibodies (lane 3). B, 2D immunoblots of acid-glycine extract from H. pylori probed with serum from a GC patient (left) or with anti-GroES antibodies (right). The monomeric form of GroES with a molecular mass ranging from 14 to 21 kDa is marked by the lower box, while the upper box indicates the dimeric form. C, Western blot analysis using anti-GroES antibodies showing the presence of secreted GroES in the culture medium of H. pylori collected after 48–72-h incubation (lane 2) but not in medium only (lane 1) (* and ** denote the monomeric and dimeric forms of GroES, respectively).

 
We further characterized the native GroES of H. pylori by immunoblot analysis of the 2D map of acid-glycine-extracted proteins using GC sera (Fig. 3B, left) and the anti-GroES antibodies (Fig. 3B, right). As with rGroES, we detected the presence of multimeric forms of native GroES in the H. pylori cell extract in addition to the monomeric and dimeric forms originally identified as the GC-related antigenic spots. Using patients’ sera, the dimeric form of native GroES appeared to be more prevalent than the monomeric and trimeric forms (Fig. 3B, left). Furthermore although mainly found in the H. pylori extract, GroES was also detected in the filtered medium from H. pylori cultures, suggesting that GroES is secreted out of H. pylori (Fig. 3C).

GroES Seropositivity Is Related to Gastric Cancer—
To examine the clinicopathological significance of GroES seropositivity in H. pylori-infected patients, a GroES immunoblot assay was performed on a series of clinical samples. A serum was defined as GroES seropositive if rGroES was recognized by serum IgG. No seropositivity was seen with any serum sample from 32 persons without H. pylori infection (controls). We then examined the serum IgG response to GroES in 313 H. pylori-infected patients with GC (95 patients), gastritis (94 patients), or DU (124 patients). Overall 42.8% of the H. pylori-infected patients gave a positive response. GroES seropositivity was related to patient age, increasing from 18.8% in patients aged less than 30 years to 40.2% in patients aged 30–49 years (OR, 2.9; 95% CI, 0.8–10.9; p = 0.1) and to 46.2% in patients aged more than 50 years (OR, 3.7; 95% CI, 1.0–13.4; p = 0.04) (Supplemental Table III). Furthermore the prevalence of GroES seropositivity in patients with GC, gastritis, or DU was 64.2, 30.9, and 35.5%, respectively. After adjustment for age difference, the GroES seropositivity in GC patients was significantly higher than that in gastritis patients (OR, 3.9; 95% CI, 2.1–7.4; p < 0.001) or DU patients (OR, 2.7; 95% CI, 1.5–4.9; p < 0.001). There was also a statistically significant difference in GroES seropositivity between controls and H. pylori-infected subjects but not between patients with DU or gastritis (Table II). To further characterize the relationship between GC and GroES seropositivity, 95 GC patients were classified into several subtypes by gender, stage, histological type, and tumor location for statistical analysis of GroES positivity; the results are listed in Table III. Importantly although gender, stage, and histological subtype had no significant effect on GroES seropositivity, GC located in the antrum exhibited a significantly higher rate of GroES seropositivity than those in a non-antrum location (71.9 versus 48.4%; OR, 2.7; 95% CI, 1.1–6.7; p = 0.03).


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  		TABLE II Serum IgG GroES positivity in various upper gastrointestinal diseases

 

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  		TABLE III Characteristics of gastric cancer analyzed by anti-GroES antibody status

For gastric cancer serum samples, n = 95.

 
Induction of Proinflammatory Cytokine Production and COX-2 Expression in PBMC Stimulated with GroES—
Previous results demonstrated close association of GroES with GC, a cancer known to result from chronic inflammation caused by H. pylori infection. Moreover GroES is a secreted protein and in direct contact with host may mediate an important interaction between H. pylori and host. We therefore investigated the effect of GroES on the inflammatory responses of mononuclear cells. PBMC were incubated with rGroES, and then mRNA levels for seven cytokines were determined by RT-PCR. As shown in Fig. 4A, rGroES stimulation caused a marked increase in IL-8, IL-6, IL-1ß, and TNF-{alpha}, cytokines commonly found in H. pylori-infected patients. In addition, GM-CSF was slightly increased by rGroES (Fig. 4A), whereas interferon-{gamma} and IL-12 were not changed (data not shown). Furthermore mRNA levels of COX-2, an enzyme crucial for inflammatory responses, were also greatly enhanced after rGroES stimulation (Fig. 4A). These data showed that H. pylori GroES causes up-regulation of the expression of proinflammatory cytokines and COX-2 at the transcriptional level.


Figure 4
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  		FIG. 4. GroES stimulates inflammatory responses in PBMC. A, PBMC were treated with rGroES (5 µg/ml) for 4 h, and then RT-PCR was used to detect mRNAs for IL-8, IL-6, GM-CSF, IL-1ß, TNF-{alpha}, COX-2, and GAPDH (loading control). PBMC were incubated with various concentrations of rGroES for 24 h, and then protein levels of IL-8 (B and G), IL-6 (C), GM-CSF (D), IL-1ß (E), or TNF-{alpha} (F) in the culture supernatant were quantified by ELISA. G, rGroES and LPS were first digested with PK, the PK was inactivated, then the mixtures were incubated with PBMC as described above (rGroES and LPS at 5 and 1 µg/ml, respectively), and IL-8 was measured in the culture supernatant. H, Western blot analysis of COX-2 protein expression. PBMC were incubated with rGroES for 24 h, and then the cell lysate was examined for COX-2 and ß-actin (loading control) by Western blotting. I, PGE2 secretion into the culture medium of PBMC treated for 24 h with rGroES. All ELISA experiments were carried out in triplicates; the results are shown as mean ± S.D. Student’s t test was used for the statistical evaluation (*, p < 0.05; **, p < 0.01 versus control).

 
To correlate the aforementioned increase in mRNA levels with induction of cytokine secretion, we analyzed cytokine protein levels in culture supernatants of PBMC stimulated with rGroES. As shown in Fig. 4, B–F, rGroES induced a dose-dependent increase in the levels of secreted IL-8, IL-6, GM-CSF, IL-1ß, and TNF-{alpha}. Induction of cytokine release was seen at concentrations of rGroES as low as 0.1 µg/ml. Stimulation of IL-6 production was almost maximal at 5 µg/ml rGroES, whereas secretion of the other cytokines was greatly increasing at this concentration.

To exclude the possibility that the increase in cytokine release induced by rGroES was caused by contaminating LPS, rGroES was digested with PK before treatment of PBMC, and complete digestion was confirmed by the absence of rGroES on silver-stained SDS-PAGE (data not shown). As shown in Fig. 4G, digested materials only caused basal levels of IL-8 production, whereas LPS-induced IL-8 production by PBMC was not affected by PK digestion. These data confirmed that the cytokine production indeed resulted from stimulation by rGroES instead of LPS.

We also examined the ability of rGroES to induce COX-2 expression at the protein level. As with cytokine production, rGroES induced a dose-dependent increase in COX-2 protein levels in PBMC (Fig. 4H). To confirm this, we examined rGroES-treated PBMC for secretion of PGE2, whose production depends on COX-2 and is crucial for inflammatory processes. We found that rGroES greatly stimulated PGE2 release in a dose-dependent manner (Fig. 4I). The level of PGE2 production was almost saturated at 5 µg/ml rGroES. Overall these results showed that rGroES increases the expression of proinflammatory cytokines, COX-2, and PGE2 at both the transcriptional and translational levels, suggesting that GroES plays a promoting role in the inflammation triggered by H. pylori infection.

GroES Causes an Increase in Proinflammatory Cytokine mRNA Levels in Gastric Epithelial Cells—
To test whether GroES exerted a direct effect on gastric epithelial cells, KATO-III cells, a gastric carcinoma cell line, were treated with rGroES followed by RT-PCR to determine proinflammatory cytokine production. As shown in Fig. 5A, IL-8, GM-CSF, IL-1ß, and TNF-{alpha} mRNA levels were all increased in rGroES-treated KATO-III cells, whereas IL-6 mRNA levels were unchanged. Of the four cytokines showing increased expression at the transcriptional level, only IL-8 showed a dose-dependent increase in protein secretion (Fig. 5B).


Figure 5
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  		FIG. 5. GroES causes potential neoplastic changes in KATO-III cells. rGroES induces expression of proinflammatory cytokine genes and production of IL-8 protein. A, cells were treated with rGroES (5 µg/ml) for 6 h, and then RT-PCR was used to examine levels of mRNAs for IL-8, IL-6, GM-CSF, IL-1ß, TNF-{alpha}, and GAPDH. B, cells were treated with rGroES for 24 h, and then IL-8 protein in the culture supernatant was measured by ELISA. C, rGroES stimulates cell growth. Cells were treated with rGroES for 24 h, and then the number of viable cells was measured by MTS assay. ELISA and cell proliferation experiments were carried out in triplicates; the results are shown as the mean ± S.D. Student’s t test was used for statistical evaluation (*, p < 0.05; **, p < 0.01 versus control). D, expression of the proto-oncogenes c-jun and c-fos is induced by rGroES. Cells were treated with rGroES (5 µg/ml) for 6 h, and then RT-PCR was used to detect mRNAs for c-jun, c-fos, and GAPDH. E, GroES induces expression of cell cycle-related molecules favoring cell proliferation. Cells were treated with rGroES (5 µg/ml) for 12 h, and the protein levels of cyclin D1, p27Kip1, and ß-actin were examined by Western blotting.

 
GroES Stimulates KATO-III Cell Proliferation—
In addition to its promoting role in inflammation, GroES might contribute to GC development by supporting cell proliferation. To test this hypothesis, KATO-III cell proliferation was determined by MTS assay after rGroES stimulation. When treated with 5 µg/ml rGroES, KATO-III cells significantly increased the number of viable cells up to about 1.2-fold compared with untreated control (Fig. 5C).

GroES Stimulates the Expression of Proto-oncogenes c-jun and c-fos in KATO-III Cells—
Next we used RT-PCR to evaluate the expression of c-jun or c-fos in gastric epithelial cells after rGroES treatment. As shown in Fig. 5D, although c-jun mRNA was absent and c-fos mRNA was very low in untreated KATO-III cells, the expression of both proto-oncogenes was dramatically increased after rGroES stimulation.

GroES Modulates the Expression of Cell Cycle-related Molecules—
We further examined the supporting role of GroES in GC development by analyzing the protein levels of marker molecules associated with cell cycle regulation. Protein expression of cyclin D1 was up-regulated by rGroES (Fig. 5E). Notably aberrant expression of cyclin D1 has been reported in GC (28). Moreover we found that p27Kip1 protein expression was down-regulated by rGroES (Fig. 5E); importantly reduced expression of p27Kip1 is seen in H. pylori-associated intestinal metaplasia (29). Overall the effect of H. pylori GroES on these cell cycle-related molecules closely matched those documented in clinical investigations of precancerous gastric lesions and GC.

H. pylori GroES and FlaG Exhibit Different Effects on Inflammatory Responses and Cell Proliferation—
To elucidate the significance of H. pylori GroES in inflammation and cell proliferation, we compared the effects of GroES with the additional H. pylori protein FlaG (HP0751). FlaG, a polar flagellin, had similar molecular weight to GroES and reacted with low frequency with sera from GC and DU groups (3.1%, n = 95 and 12.5%, n = 124, respectively). Recombinant FlaG (rFlaG) were also purified and endotoxin-depleted for the treatment of PBMC and KATO-III cells.

As shown in Fig. 6A, protein levels of IL-8, IL-6, GM-CSF, IL-1ß, TNF-{alpha}, and PGE2 were highly enhanced by the treatment of rGroES in PBMC. In contrast, rFlaG slightly induced the production of IL-8 in PBMC but not the other cytokines and PGE2. In KATO-III cells, IL-8 production was induced much more by rGroES, whereas rFlaG had no effect on IL-8 production at all (Fig. 6B). We next evaluated the effects of these recombinant proteins on the cell proliferation in KATO-III cells. As shown in Fig. 6C, cell number was significantly increased after incubation with rGroES for 24–36 h. In contrast, rFlaG had no effect on cell proliferation.


Figure 6
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  		FIG. 6. Comparing the effects on PBMC and KATO-III cells between GroES and FlaG. PBMC (A) and KATO-III cells (B) were treated with a 5 µg/ml concentration of each recombinant protein for 24 h, respectively. Protein levels of IL-8, IL-6, GM-CSF, IL-1ß, TNF-{alpha}, and PGE2 in the culture supernatant were measured by ELISA. C, KATO-III cells were treated with a 5 µg/ml concentration of each recombinant protein for 6–48 h, and then the number of viable cells was measured by MTS assay. ELISA and cell proliferation experiments were carried out in triplicates; the results are shown as the mean ± S.D. Student’s t test was used for statistical evaluation (*, p < 0.05; **, p < 0.01 versus control).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The Gram-negative bacterium H. pylori is closely linked to gastric carcinogenesis. To characterize candidate H. pylori antigens associated with GC, we used a proteomic approach for the global detection of H. pylori antigens recognized by serum IgG from GC patients. It led us to identify GroES, which was recognized at a significantly higher frequency by serum samples from GC patients than by those from DU patients. A serological study showed that 64.2% of GC sera reacted with H. pylori GroES compared with 30.9% of gastritis samples and 35.5% of DU samples and that there was no significant difference in GroES seropositivity between the early and advanced stages of GC. Notably our results of the prevalence survey were different from those reported by three other groups, who found that GroES seropositivity among H. pylori-infected adults increased gradually with age in developed countries and in a developing country, Mexico (30–32). In addition, Pérez-Pérez et al. (30) reported that the incidence of GroES seropositivity is high in adenocarcinoma of the cardia, a lesion not associated with H. pylori infection, whereas Ng et al. (31) showed that GroES antigenicity is not related to the clinical outcome of H. pylori infection. In contrast to their findings, our study demonstrated that GroES seropositivity was closely associated with antral GC, a non-cardia cancer associated with H. pylori infection (3). Antral GC may develop from H. pylori-induced pangastritis or corpus-predominant gastritis, whereas antral-predominant gastritis usually leads to duodenal ulceration (8). Therefore, it may be possible that GroES seropositivity is associated with pangastritis or corpus-predominant gastritis, but this remains to be clarified. However, there are still one-third of the GC patients that have no immune reaction to GroES. The reasons may be the different level of GroES expression in different H. pylori strains that infect patients or the genetic differences in the host immune responses to H. pylori infection. Moreover multiple factors should be involved in the development of gastric diseases, and it is the coordination and/or competition of these factors that determine the eventually clinical outcomes. Thus this may be the reason why 35.5% of DU patients have immune reaction to GroES and clinically belong to a group divergent from GC.

A previous study on the physiological function of GroES indicated that it serves as an assisting heptameric ring for chaperonin GroEL to help in proper protein folding (33). It is well known that a variety of stress stimuli induce a marked increase in chaperone synthesis and production (34). Importantly prokaryotic molecular chaperones have been shown to modulate host immune reactions by interacting with host cells responsible for specific and nonspecific immune responses (35, 36). Several studies have indicated that it is the immunogenicity of these prokaryotic molecular chaperones that is critical for the development of human diseases (37, 38). To date, only the GroESs of Mycobacterium tuberculosis and Mycobacterium leprae have been shown to modulate immune responses associated with pathogenesis caused by these bacteria (39). In contrast, E. coli GroES is unable to induce expression of proinflammatory cytokines in human keratinocytes, monocytes, and endothelial cells (40, 41). Thus, it is likely that GroES from different bacterial strains may contribute differently to the development of human diseases.

In this study, we provided evidence to link another GroES to human disease by demonstrating that H. pylori GroES, a secreted protein, is a virulence factor associated with induction of inflammation. This notion is supported by the finding that H. pylori GroES can induce the production of proinflammatory cytokines, including IL-8, IL-6, GM-CSF, IL-1ß, and TNF-{alpha}, in human PBMC and trigger IL-8 production in gastric epithelial cells. It is well recognized that release of these proinflammatory cytokines is closely linked to the pathogenesis of H. pylori-associated GC (20, 21). Specifically IL-6 is a multifunctional cytokine that functions as a growth and differentiation factor for tumor cells (42). IL-8 has been proposed to act as a promoter of tumor growth through its angiogenic properties (43). GM-CSF and IL-1ß are also potent growth factors for gastric epithelial cells (44). TNF-{alpha} and IL-1ß gene polymorphisms have been shown to confer a 5-fold increase of risk to develop non-cardia GC (19). Furthermore IL-1ß gene polymorphisms proposed to enhance its production are associated with the hypochlorhydria induced by H. pylori and with GC (20). Moreover IL-1ß and TNF-{alpha} are powerful inhibitors of gastric acid secretion (45). It is known that reduced acid secretion leads to increased levels of gastrin and thus provides continuous proliferating stimuli to gastric epithelial cells (46), and the subsequent atrophic changes may lead to an increased risk of non-cardia carcinogenesis (8). Our data also argue that, aside from up-regulating the production of proinflammatory cytokines, GroES might promote inflammation by enhancing COX-2 expression in PBMC, leading to the production of PGE2, which is known to participate in the inflammatory process, inhibition of apoptosis, angiogenesis, and tumorigenesis (47–50). Taken together, our findings demonstrate that GroES plays an important role in the induction of proinflammatory cytokine expression, which leads to the chronic inflammation status essential for progression from tissue damage to gastric cancer.

A growing body of evidence has indicated that long standing inflammation may potentiate or promote tumor development, growth, and progression in a number of human malignancies, including gastric cancer (51, 52). H. pylori infection is the most important etiologic factor of gastric inflammation, and increasing in vitro and in vivo data suggest that H. pylori-induced persistent and uncontrolled inflammation can lead to increased cellular turnover and participates in the neoplastic process of GC (52). However, the disease phenotypes of H. pylori infection include asymptomatic gastritis and peptic ulcer, and only a fraction of infected individuals have gastric malignancies. It is now becoming clear that differences in host immune responses and magnitude of gastritis would dictate the variable outcomes after exposure to H. pylori. Thus, identification of the factors of H. pylori that trigger or modulate gastric inflammation, such as GroES in this study, is of value not only for a better understanding of gastric carcinogenesis but also for prevention and therapy of H. pylori-associated diseases.

In addition, our studies also suggested a positive effect of H. pylori GroES on the growth of gastric epithelial cells by enhancing the expression of proteins associated with cell proliferation. We found that GroES induced expression of c-jun, c-fos, and cyclin D1, whereas p27Kip1 protein was down-regulated. Importantly this expression profile is very similar to that derived from immunohistochemical studies on precancerous lesions and GC (28, 29), indicating a promoting effect of GroES on the proliferation of gastric epithelial cells. In particular, the proto-oncogenes c-jun and c-fos are required for cell proliferation, and overexpression of these genes causes neoplastic transformation (53, 54). Aberrant expression of cyclin D1 promotes cell cycle progression and is known to play a pivotal role in the development of several human cancers (55). In contrast, a decrease in p27Kip1 levels is predicted to confer an apoptosis-resistant phenotype and increased susceptibility to GC (56). Overall the changes of gene expression pattern induced by H. pylori GroES are in favor of cell proliferation and may therefore contribute to the increased risk for GC observed in humans chronically exposed to H. pylori.

To evaluate the importance of H. pylori GroES in promoting inflammation and neoplastic changes, the additional H. pylori protein FlaG, which reacted with low frequency with sera, was included in our analysis. Undoubtedly H. pylori GroES showed promoting effects on the production of proinflammatory cytokines as well as PGE2 and cell proliferation. In contrast, H. pylori FlaG barely induced proinflammatory cytokine production and had no effects on cell proliferation. Taken together, our results indicate that H. pylori GroES exhibited distinct effects on promoting inflammation and proliferation. Although this study suggests that H. pylori GroES is one of the virulence factors that contribute to inflammation and carcinogenesis triggered by H. pylori infection, there should be other factors that remain to be identified.

In conclusion, we report here a comparison of responses of serum antibodies from GC and DU patients to the H. pylori proteome that led to the identification of GroES as a dominant GC-associated antigen of H. pylori. We further demonstrate that GroES seropositivity is highly associated with antral GC, suggesting its value as a prediction marker for GC. Moreover a novel role for H. pylori GroES in the development of GC is established that appears to involve the inflammation induced by H. pylori infection and the promotion of molecular changes favoring cell proliferation.


  FOOTNOTES
 
Received, April 3, 2006, and in revised form, June 5, 2006.

Published, MCP Papers in Press, June 11, 2006, DOI 10.1074/mcp.M600111-MCP200

1 The abbreviations used are: DU, duodenal ulcer; 2D, two-dimensional; CI, confidence interval; FBS, fetal bovine serum; GC, gastric cancer; GM-CSF, granulocyte macrophage colony-stimulating factor; LPS, lipopolysaccharide; OR, odds ratio; PBMC, peripheral blood mononuclear cells; PK, proteinase K; rGroES, recombinant GroES; IL, interleukin; TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGE2, prostaglandin E2; COX, cyclo-oxygenase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; rFlaG, recombinant FlaG. Back

* This work was supported in part by the Program for Excellence Research Teams from the Ministry of Education and Grant NSC 94-3112-B-002-010 from the National Science Council of the Republic of China. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

§§ To whom correspondence should be addressed: Graduate Inst. of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd, Taipei 100, Taiwan. Tel.: 886-2-23123456 (ext. 8212); Fax: 886-2-23958814; E-mail: lupin{at}ha.mc.ntu.edu.tw


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