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Helicobacter pylori is a spiral-shaped, microaerophilic, gram-negative microorganism which colonizes human gastric epithelial cell surfaces and the overlying mucous layer. Infection with H. pylori, which affects approximately 50% of the world's population, causes chronic gastric inflammation, which in most cases remains asymptomatic. However, 10% of the H. pylori carriers develop severe gastric illness such as gastric or duodenal ulcer, atrophic gastritis, antral adenocarcinoma, or mucosa-associated lymphoid tissue (MALT) lymphoma. Therefore, infection by H. pylori causes a major health problem worldwide, especially in developing countries, where infection rates of >90% are encountered (13).

Several factors associated with the pathogenesis of H. pylori have been characterized so far, including flagella (18, 32); urease, which probably enables H. pylori to survive in the acidic environment of the stomach (9); an adhesin binding to the Lewis b blood group antigen (22); and the vacuolating cytotoxin VacA (3). In vitro VacA induces the formation of large acidic vacuoles in a number of eukaryotic cells (19). Furthermore, a 40-kb pathogenicity island (PAI) named cag has been identified in a subset of strains (1, 6). Based on the presence of the cag PAI, the H. pylori isolates are subdivided into two types. Type I strains, containing the cag PAI, exhibit increased virulence, since they are predominantly associated with severe gastric disease, while type II strains, lacking the cag PAI, are more frequently isolated from asymptomatic carriers. It has been demonstrated that some of the proteins encoded by the cag PAI trigger severe inflammatory responses in the host (6). However, the precise function of the gene products of the cag PAI and their role in virulence remain to be elucidated.

Pharmaceutical therapy to treat the H. pylori infection involves expensive combinations of various antibiotics, proton pump inhibitors, and bismuth compounds but shows only a limited efficacy (of approximately 80 to 90%) and does not prevent reinfection after successful eradication. In addition, H. pylori strains resistant to the most potent antibiotics used in the treatment of H. pylori infections, metronidazole and clarithromycin, are emerging rapidly (5). Considering further that the number of infected people worldwide requiring treatment is far beyond the reach of the antibiotic triple therapy, development of a vaccine seems to be the only suitable approach for the global control of H. pylori infection. It has been shown by various researchers that in animal models of infection protective immunity can be achieved by the coadministration of an appropriate mucosal adjuvant and various H. pylori antigens, either separately or in combination, via the orogastric route. The protective antigens identified include the urease; VacA; CagA, the immunodominant marker protein for the presence of the cag PAI; catalase; and HspA and HspB, the H. pylori homologs of the heat shock proteins GroES and GroEL (14, 24, 28, 30). In particular, the H. pylori urease gave rise to a high degree of protective immunity in vaccinated animals, and it was reported that 100% protection in H. pylori-challenged mice could be achieved by the administration of urease via a live carrier Salmonella strain expressing recombinant H. pylori subunits A and B (17). Furthermore, it has been demonstrated that therapeutic vaccination with recombinant VacA and CagA eradicates a chronic H. pylori infection in mice, demonstrating that the inability of the natural immune response to clear H. pylori infection can be overcome (16).

Considering the advantage of an efficacious vaccine, it is important to identify the H. pylori proteins which elicit a strong immune response in humans in order to analyze their capability to confer protective immunity. Furthermore, the identification and characterization of immunodominant proteins will contribute to the improvement of serological tests for detecting and monitoring H. pylori infections. Another important question is whether there exists a correlation between the presence of antibodies directed against specific H. pylori antigens and the particular H. pylori-associated gastroduodenal pathology from which a patient is suffering. In the present study, we used the proteome technology to identify common patterns of H. pylori antigens which are recognized by sera from patients showing various gastroduodenal pathologies.

Identification of immunogenic proteins of H. pylori by the proteome technology. H. pylori G27 (36) was grown on Columbia agar plates containing 5% horse blood and 0.2% cyclodextrin as described previously (4). The bacteria were harvested from the plates, washed with phosphate-buffered saline, and lysed by incubation in lysis buffer (35 mM Tris, 9 M urea, 65 mM dithiothreitol, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS]) for 10 min at room temperature. Two-dimensional (2D) gel electrophoresis was performed by the method of O'Farrell (27), modified by Hochstrasser et al. (20, 21). Protein samples containing up to 200 µg of protein were subjected to isoelectric focusing (IEF) in a pH gradient ranging from pH 4 to pH 8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with pairs of identical IEF samples, and the gels were further processed in parallel by silver staining or immunoblotting using either control sera derived from five individuals identified H. pylori negative by serological tests (23) or the sera from 16 H. pylori-infected persons suffering from different gastroduodenal pathologies. These sera were collected at the Universitätsklinik Würzburg, Würzburg, Germany, the Hospital Calderon Guardia, San Jose, Costa Rica, and the Hospital Max Peralta, Cartago, Costa Rica, and were taken from patients identified as H. pylori positive by endoscopy and suffering from gastritis (AR, KE, BB, EE, MM, and SR), gastric or duodenal ulcer (CR3/4, CR6/10, CR7, CR9/11, and CR5/8), gastric cancer (18129, CR15, CR16, and CR19) or MALT lymphoma (H6031). Immunogenic H. pylori proteins identified in this way were eluted from preparative gels stained with colloidal Coomassie blue (26) and analyzed by digestion with trypsin followed by LC-mass spectrometry (MS). Briefly, the Coomassie blue-stained protein bands were precisely excised from the acrylamide gel, cut into small cubes, and rinsed several times with water (100 µl) for 15 to 30 min each. The gel pieces were washed three times with 100 µl of acetonitrile-water (1:1) for 10 to 20 min. To shrink the gel and extract residual water, pure acetonitrile was added for 10 min. The acetonitrile was removed, and 30 to 50 µl of digestion buffer (50 mM N-methylmorpholine [pH 8.1]) as well as trypsin (0.5 µg) were added. Digestion was performed at 37°C for 6 to 12 h. The supernatant containing the resulting peptides was recovered, and the gel pieces were extracted twice with 0.1% trifluoroacetic acid (20 to 30 min). The volume of the combined extracts was reduced to 5 µl in a Speed-Vac concentrator. LC-MS and collision-induced fragmentation (CID) spectra were recorded on a Finnigan LCQ ion trap mass spectrometer equipped with an electrospray ionization source. Grouping of fragment ion (CID) spectra originated from the same precursor ion, and cross-correlation analysis of the data was performed by using the Sequest program (10). The Sequest algorithm compares the measured fragment ion spectra of all selected peptides to the predicted spectra of tryptic peptides contained in the database and exhibiting the same molecular weight. Identification of multiple peptides derived from the same protein and evaluation of their cross-correlation scores results in unambiguous identification of the protein. The 2,591 database entries for peptide searches were created by selecting all annotated and predicted H. pylori proteins from the composite OWL protein sequence database (version 30.2).

View larger version (32K): [in this window] [in a new window]   FIG. 1.   2D map (pH 4 to 8) of a whole-cell lysate of H. pylori G27 and identification of H. pylori antigens by immunoblot analysis. A 100-µg portion of a whole-cell lysate of H. pylori G27 was loaded onto the IEF gels. Identified proteins are indicated by the spot numbers given in Table 1. The positions of molecular weight (MW) standards are indicated on the right. (A) Silver stain of a typical 2D gel. (B) Western blot of a duplicate 2D gel hybridized with serum AR, which is derived from an H. pylori-infected individual suffering from gastritis. Western blots were developed using the enhanced chemiluminescence detection system (Amersham). (C) Western blot of a duplicate 2D gel hybridized with a control serum from an H. pylori-negative individual (N1).

View larger version (110K): [in this window] [in a new window]   FIG. 2.   Silver-stained 2D map of a whole-cell lysate of H. pylori G27 showing the positions of the 29 identified immunogenic proteins listed in Table 1. The positions of molecular weight (MW) standards are indicated on the right. Isoelectric points are indicated at the bottom of the figure and were obtained by running a 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis standard under conditions identical to those applied for sample separation.

Comparison of antigenic patterns of H. pylori lysates obtained with different patient sera. The H. pylori-positive sera used in this study were taken from patients suffering from chronic gastritis (six sera), peptic ulcer (five sera), gastric cancer (four sera), or MALT lymphoma (one serum). The antigenic patterns which were obtained when the different sera were hybridized to whole-cell protein lysates of H. pylori G27 in individual immunoblot experiments were compared with each other for the presence of a subset of 20 of the previously analyzed 29 spots which could be easily identified according to the electrophoretic mobility of the corresponding proteins. The results of this comparison are listed in Table 2 and demonstrate a highly variable humoral immune response in the 16 individuals under investigation. A single antigen, the flagellin A antigen, was recognized by all the sera tested, while antibodies against a second component of the flagella, flagellar hook-associated protein 2, and the chaperone GroEL were present in 14 sera. Several other antigens, i.e., urease subunit B, trigger factor, neutrophil-activating protein, alkyl hydroperoxide reductase, pyruvate ferredoxin oxidoreductase, EF-Tu, ribosomal protein L7/L12, Omp18, and the DnaK protein, reacted with about two-thirds of the analyzed sera, while EF-Ts, isocitrate dehydrogenase, and aspartate ammonia-lyase were recognized less frequently. However, it should be noted that FlaA, UreB, GroEL, EF-Tu, ribosomal protein L7/L12, and Omp18 also reacted with a low frequency with sera from H. pylori-negative individuals (Table 2).