Helicobacter pylori causes gastroduodenal disease, which is mediated in part by its outer membrane proteins [OMPs] . To identify OMPs of H . pylori strain 26695, we performed a proteomic analysis. A sarcosine-insoluble outer membrane fraction was resolved by two-dimensional electrophoresis with immobilized pH gradientstrips . Most of the protein spots, with molecular masses of10 to 100 kDa, were visible on the gel in the alkaline pI regions[6.0 to 10.0] . The proteome of the OMPs was analyzed by matrix-assistedlaser desorption ionization-time-of-flight mass spectrometry.Of the 80 protein spots processed, 62 spots were identified;they represented 35 genes, including 16 kinds of OMP . Moreover,we identified 9 immunoreactive proteins by immunoblot analysis.This study contributes to the characterization of the H . pyloristrain 26695 proteome and may help to further elucidate thebiological function of H . pylori OMPs and the pathogenesis ofH . pylori infection.

 
Helicobacter pylori is a spiral-shaped, microaerophilic gram-negative bacterium that causes acute and chronic gastritis, gastroduodenal ulcers, and gastric cancer [3, 7, 21, 40] . More than half ofthe world's population has suffered from H . pylori infection[4, 5, 46] . Surface proteins, including flagella, urease, andadhesin, are known to be involved in the pathogen-host relationshipbetween H . pylori and the human gastric mucosa . A correlationbetween the motility state of some H . pylori isolates, and theirability to colonize the gastric epithelium has been establishedin experiments with gnotobiotic piglets [18] . Urease enablesH . pylori to survive in the acidic environment of the stomach[13] and plays a key role in colonizing the gastric mucosa [17]. Adhesins, including BabA [25], AlpA/AlpB [42], HopZ [43], andSabA [26], are known to adhere to gastric epithelial cells.

The genomes of two H . pylori strains have been sequenced [2, 49] and extensively compared [1] . Of 64 theoretically predictedouter membrane proteins [OMPs], at least 8, including adhesinsand porins, have been confirmed experimentally . However, itis unclear whether all of the predicted OMPs are expressed.

Several methodological approaches have been applied to the identification of H . pylori surface proteins, including OMPs . Sarbarth et al. [48] selectively biotinylated intact H . pylori with the hydrophilicreagent sulfosuccinimidyl-6-[biotinamido]-hexanoate and purifiedthe labeled proteins by membrane isolation, solubilization,and affinity chromatography . Exner et al . [19] purified OMP fractions by sucrose gradient centrifugation and identified heat-mobile OMPs, which may be porins, by using two-dimensional [2-DE] gel electrophoresis . Doig et al . [15] identified six OMPs in a sarcosine-insoluble OMP fraction and by using monoclonal antibodies, demonstrated that these proteins are located within or are associated with the outer membrane . In addition, by comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] of outer membrane fractions isolated by different isolation procedures such as the use of sarcosine, a sucrose gradient ultracentrifuge, Triton X-100, and Triton X-114, eight majorprotein species with 6 to 10 minor proteins were identified.The outer membrane fraction prepared by sarcosine differentialsolubilization exhibited a higher level of these proteins thanthose of the other preparations . Moreover, it was demonstratedpreviously that the outer membrane fraction was insoluble insarcosine, whereas the cytoplasmic membrane was totally soluble[20].

2-DE analysis of bacterial OMPs has proven to be impractical because of technical difficulties associated with the solubilization of membrane proteins and with OMP preparation . Recent advancesin the solubilization of intractable proteins have promptedthe proteomic analysis of bacterial OMPs [37] . Specifically, proteomic analysis of Escherichia coli [37], Salmonella entericaserovar Typhimurium [38], Klebsiella pneumoniae [38], Caulobacter crescentus [38], and Leptospira interrogans serovar Lai [12]OMPs has been completed.

We sought here to identify the OMPs of H . pylori strain 26695 by using the sarcosine-insoluble outer membrane fraction . We identified 62 spots, including 16 OMPs, on 2-DE gels and identified9 immunogenic proteins by immunoblot analysis.

 
Bacterial strain and culture conditions. H . pylori strain 26695 was incubated on brucella agar platescontaining 10% bovine serum . The bacterial cells were cultivatedovernight at 37°C in an atmosphere of 10% CO₂and 100% humidity.

Sarcosine preparation of H . pylori OMPs. The sarcosine-insoluble outer membrane fraction of H . pyloriwas prepared as described previously [15] with minor modification.H . pylori cells were harvested by centrifugation [12,000 x g,20 min, 4°C] and washed three times with 20 mM Tris-HCl[pH 7.5] . The cells were suspended in 20 mM Tris-HCl [pH 7.5]and then disrupted with an ultrasonicator [Sonics & Materials,Inc., Danbury, Conn.] . DNase and RNase [20 µg/ml each]were added to the cell suspension, and the mixture was incubatedat room temperature for 30 min . The unbroken cells were removedby centrifugation [12,000 x g, 20 min, 4°C], and the supernatantwas retained . Total membrane proteins were then collected bycentrifugation [40,000 x g, 30 min, 4°C], resuspended in20 mM Tris-HCl [pH 7.5] containing 2.0% [wt/vol] sodium laurylsarcosine, and incubated at room temperature for 30 min . OMPswere collected by centrifugation [40,000 x g, 30 min, 4°C]and washed three times with distilled water . The pellet wasresuspended in distilled water, divided into aliquots, and storedat -20°C until use.

Protein quantification. Protein concentrations were determined by the Bradford method[8], with bovine serum albumin as a standard.

2-DE electrophoresis. Isoelectric focusing [IEF] was performed by using IPG strips[Bio-Rad, Hercules, Calif.] [50] . Portions [300 µg] ofthe OMPs were applied to strips of pH ranges of 3.0 to 10.0and 6.0 to 11.0 . The samples were diluted by incubation in arehydration solution containing 7 M urea, 2 M thiourea, 2 mM tributyl phosphine [Sigma-Aldrich, St . Louis, Mo.], 40 mM Tris base, 1% Triton X-100, and 0.5% ampholyte [pH 3.0 to 10.0 [Bio-Rad] and pH 6.0 to 11.0 [Amersham]] overnight in a reswelling tray [Bio-Rad] . The strips were rehydrated under the following passive conditions: 0 V, 20°C, and a 14- to 16-h reaction time ina Protean IEF cell [Bio-Rad] . Three preset programs were executedwith slight modifications such that focusing conditions comprisedthe conditioning step, voltage ramping, and final focusing.The purpose of the conditioning step [250 V for 15 min] wasto remove salt ions and charged contaminants, and this was followedby linear voltage ramping for 3 h at 10,000 V . In the finalfocusing step, the maximum voltage of the ramp step was maintained[up to 80,000 V · h], and the current did not exceed50 µA/strip . After IEF, the strips were equilibrated in0.375 M Tris buffer [pH 8.8] containing 6 M urea, 2% SDS, 20%glycerol, 2% dithiothreitol, and 0.01% bromophenol blue, followedby the addition of the same buffer supplemented with 2.5% iodoacetamide.SDS-PAGE was performed according to the Laemmli method [32]with a 12.5% resolving polyacrylamide gel [20 by 30 cm] withouta stacking gel . Separation in the second dimension was carriedout at 30 mA/gel at 4°C until the running dye reached the bottom.

Silver staining and gel drying. Proteins resolved on gels were visualized by using a silverstaining method [25, 27 [see also http://prospector.ucsf.edu]]with slight modifications . Briefly, the gel was fixed in a solutioncontaining 50% methanol, 12% acetic acid, and 0.5 ml of 37%formaldehyde for 1.5 h . All incubations were performed in ashaker with gentle shaking . After a fixing step, the gel waswashed with 50% ethanol twice for 20 min and then washed againwith double-distilled water [dDW] for 20 min . The gel was pretreatedwith Na₂S₂ · 5H₂O [0.1 g/liter] for 1 min and washedagain with dDW . The gel was impregnated with silver by incubationin AgNO₃[2 g/liter] and 0.75 ml of 37% formic acid for 30 min,and it was then rinsed with dDW three times for 20 s each time.A developing solution consisting of Na₂CO₃[60 g/liter], Na₂S₂· 5H₂O [4 mg/liter], and 0.5 ml of 37% formic acid wasprepared ahead of time and preserved in ice slurry . The visualizationwas performed by incubating the gel in the developing solutionuntil a clear image was observed . When clear spots appeared,the gel was washed with dDW twice for 20 s each time, afterwhich the reaction was stopped by adding 50% methanol and 12%acetic acid for 10 min . The visualized gel was dried with cellophanepaper.

Image analysis. A gel image was obtained by scanning the silver stained gelswith the Fluor-S MultiImager [Bio-Rad] . The image was documentedthrough the PDQUEST 2-D gel analysis software [version 6; Bio-Rad].

Destaining and in-gel digestion of proteins. Silver-stained spots were excised from 2-DE gels and transferredinto microcentrifuge tubes . Silver-stained proteins were destainedwith chemical reducers as described previously [22] . The chemical reducer mixture was freshly prepared and comprised a 1:1 ratio of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate.A portion [100 µl] of the mixture was added to the microcentrifuge tube, and this was vortexed occasionally until the brownishcolor disappeared . Gel pieces were rinsed three times with DWto stop the reaction, and 500 µl of 200 mM ammonium bicarbonatewas added to cover the gel for 20 min and then discarded . Gelpieces were dehydrated with 100 µl of acetonitrile anddried in a vacuum centrifuge . An in-gel digestion was carriedout by the method of O'Connell and Stalts [41] . Gel pieces containing proteins were rehydrated by adding a digestion buffer containing12.5 ng of trypsin/ml for 45 min on ice . The enzyme solutionwas removed and replaced with 20 µl of the buffer withoutthe enzyme, such that the gel pieces were kept wet overnightat 37°C . The gel pieces were subjected to vigorous vortexingfor 30 min, after which 20 µl of the digested solutionwas transferred into a clean microcentrifuge tube and driedunder vacuum . The resulting samples were dissolved in 2 µl of 0.1% trifluoroacetic acid.

Peptide mass fingerprinting. A matrix solution composed of -cyano-4-hydroxy cinnamic acid[40 mg/ml] in 50% acetonitrile and 0.1% trifluoroacetic acidwas prepared for peptide mass fingerprinting . The, 2 µl each of the matrix solution and sample solution were mixed, applied to the target well, rapidly dried, and washed with deionized water . The solution mixture was dried for 10 min at room temperature and subjected to a matrix-assisted laser desorption ionization-time-of-flight mass spectrometry [MALDI-TOF-MS] operation by using the Voyager Biospectrometry Workstation [PE Biosystems] with the following parameters: 20-kV accelerating voltage, 75% grid voltage, 0.02%guide wire voltage, 70-ns delay, and a mass gate of 800 to 2,500.

Identification of proteins. Peptide mass fingerprints were analyzed by using the MS-FITProteinProspecter program developed by the UCSF Mass SpectrometryFaculty [http://prospector.ucsf.edu]. Helicobacter proteinsin the NCBI database were searched to identify proteins . Monoisotopicpeptide masses were used to search the database, allowing amolecular mass range for 2-DE analyses of ±15%, a peptidemass accuracy of 50 ppm, and one partial cleavage . If matchedproteins were absent, the molecular mass window was extended. Pyroglutamic acid modification of N-terminal glutamine, oxidation of methionine, and acrylamide modification of cysteine were considered . Matches were defined by the number of homologouspeptides and the percentage of total translated ORF sequencecovered by those peptides, in comparison to other database entries.Identified proteins were deemed identical if they produced thesame results from the same site spots of more than five independent2-DE gels.

Immunoblot analysis. OMPs were transferred from the 2-DE gels onto a nitrocellulosemembrane [PROTRAN; Schleicher & Schuell] with a blottingbuffer containing 39 mM glycine, 48 mM Tris base, 20% methanol,and 0.037% SDS and running conditions of 15 V constant voltagefor 2 h . The membrane was blocked with 1% bovine serum albuminin Tris-buffered saline containing 0.05% Tween 20 [TBST] for 1 h at room temperature . A pool of 300 sera obtained from seropositive patients in Gyeongsang National University Hospital, Jinju, Korea, was used as an antibody source, and a pool of 13 serafrom H . pylori-uninfected persons was used as a negative control;in each case the presence or absence of antibodies had beenconfirmed previously by Western blot [3, 51] . The membrane wasincubated for 30 min at room temperature with the pooled sera,which were diluted 10-fold in TBST . After the membrane was washedwith TBST, an alkaline phosphatase-conjugated rabbit anti-humanimmunoglobulin A [IgA], IgG, IgM [diluted by 1/1,000; Dako]was added, and the membrane was incubated for 1 h at room temperature.After a wash with TBST, the bound antibody was detected by usingBCIP [5-bromo-4-chloro-3-indolylphosphate] and nitroblue tetrazoliuum[ImmunoPure; Pierce].

Duplicate 2-DE gels were simultaneously prepared under identical conditions, one for Ponceau-S staining and immunoblotting andthe other for silver staining . The spots profile of immunoblotmembrane was compared to that of the silver-stained gel andPonceau-S-stained membrane after electrotransfer by using PDQUESTsoftware for further identification of immunoreactive spots.

 
We analyzed the OMP proteome of the H . pylori 26695 strain by using the sarcosine-insoluble outer membrane fraction . Thisfraction was loaded onto precast IPG strips with a pH gradientof 3.0 to 10.0 for separation in the first dimension . The stripswere then loaded onto a 12.5% acrylamide gel of 20 by 30 cmfor electrophoretic separation in the second dimension, andseparated spots were visualized by silver staining . Sarcosine-treatedproteins were enriched in the alkaline pI regions, and theirmolecular masses were between approximately 10 and 100 kDa [Fig.1A] . Because the spots within alkaline pI regions tended tobe located in close proximity, it was difficult to identifythem with a pH 3.0 to 10.0 IPG strip since the alkaline regionof that strip was too small to separate alkaline proteins withgood resolution . Thus, to resolve these protein spots the sarcosine-insolublefraction was applied to an IPG strip with a narrower pH rangeof 6.0 to 11.0 . Using this approach, more than 80 protein spotswere visualized on the corresponding silver-stained 2-DE gel[Fig . 1B] . These spots were numbered, excised, destained, andthen digested in the gel with trypsin for peptide fingerprinting[Fig . 1] . The mass of the resulting peptide mixtures was measuredby MALDI-TOF-MS . The theoretical or observed pI values of themajority of the H . pylori OMPs identified in the present studywere higher than 8.0 [Fig . 2], which is higher than that of other bacterial OMPs [12, 37, 38] . It has been reported thatthe pI ranges of OMPs of E . coli [37], Salmonella enterica serovarTyphimurium [38], Klebsiella pneumoniae [38], Caulobacter crescentus[38], and Leptospira interrogans serovar Lai [12] are pI 4 to7, which is consistent with the theoretical pI values predicted from their genome databases . In contrast to these bacterial OMPs, H . pylori OMPs were detected in the alkaline pI region of the 2-DE gel . This may reflect evolutionary pressure forhigh alkaline proteins because of the acidic environment ofthe organism.

 
In the present study, we identified 62 protein spots that represented 35 genes [Table 1] . Of these 35 proteins, several have already been predicted to be surface-exposed in H . pylori based on results from various independent methods . Antibody staining indicated that UreA, UreB, catalase, and a homologue of HP0410 are presenton the cell surface [16, 33, 44] . Neutrophil-activating protein[NapA], phosphoglycerate dehydrogenase [SerA], glutamine synthetase[GlnA], and alkyl hydroperoxide reductase [TsaA] have previouslybeen identified in whole-cell extracts of H . pylori [10] . In addition, surface localization of NapA has been demonstrated[39] . Glutamine sythetase [GlnA] from Azotobacter vinelandiiis attached to the plasma membrane [ca . 30%], while the mainfraction is found in the cytosol [31], as is the case for H. pylori urease [44] . Even though these proteins may be cytoplasmicproteins, they did appear in the sarcosine-insoluble fraction.These results suggested that the surface properties of H . pyloricould promote adsorption of cytoplasmic proteins.

 
We identified five hypothetical proteins [HP0205, HP1349, HP0052, HP1173, and HP0139] . These proteins are not theoretically OMPs,and their localization is unclear . HP1173 is secreted into the extracellular medium [9], but its functions are not yet known. Of the other proteins we found, the flagellar basal-body L-ring protein [HP0325] is encoded by the flgH gene and is locatedin the outer membrane [1, 11] . The iron ABC transporter [CeuE]is likely to be localized in the periplasm based on data fromCampylobacter coli [47] and other ABC transporters, such asthe amino acid ABC transporter, glutamine ABC transporter, andiron[III] ABC transporter are theoretically predicted to beperiplasmic binding proteins [49] . [3R]-Hydroxymyristol-[acylcarrier protein] dehydratase [FabZ] is involved in fatty acidsynthesis such that it efficiently catalyzed the dehydrationof short-chain ß-hydroxyacyl-acyl carrier proteinsand unsaturated ß-hydroxyacyl-acyl carrier proteins in E . coli [36] . However, the localization of H . pylori FabZhas not been reported yet . MtrC is an outer membrane decahaemc-type cytochrome that appears to be required for the activityof the terminal Fe[III] reductase from Shewanella putrefaciens[6] . Although cellular localization of H . pylori MtrC has notbeen reported, it may be localized to the outer membrane dueto its functional similarity to S . putrefaciens MtrC.

It has been well documented that cytoplasmic and periplasmic components and inner membrane proteins are present as contaminantsin the outer membrane preparation . This partially reflects theirsurface localization [e.g., urease, catalase, and neutrophil-activating protein], as well as the tight association between the innerand outer membranes [15] . The present study focused on whether or not theoretically predicted OMPs were expressed and exposed on the H . pylori surface . A total of 16 OMPs were identified in the present study, whereas previously 33 ORFs have been assigned as putative OMPs in the H . pylori 26695 genome [49] . Five horizontallyaligned spots [69 kDa each] were identified as Omp27 . The horizontalseparation may be due to posttranslational modifications thatresult in differentially charged side chains on the amino acidsresidues of one species of protein . Maguire et al . [34] reportedthat horizontally aligned spots may represent the same proteinisoforms in different phosphorylation states in the phosphotyrosineproteome from thrombin-activated platelets . Omp4, Omp15, Omp20,Omp21, Omp31, and Omp32 showed similar horizontal arrays ofspots.

The functions of at least six OMPs have been already predicted. Omp19 is a homologue of BabB, which is a Lewis B binding adhesin[25] . Omp6, Omp21, Omp20, Omp2, and Omp15 have previously beendesignated HopA, HopB, HopC, HopD, HopE, respectively, and functionas porins [14, 19] . In particular, Omp20 [HopC] has been reported to be expressed in this strain [35] . Omp20 and Omp21, which have been reported to be enriched in the supernatant when H. pylori was grown in the absence of nalidixic acid [29], werealso identified in the present study . It was reported that Omp4 might be associated with bacterial adherence due to its sequence similarity to established adhesins although this has not been proven . The expression level of Omp4 was reduced in both the virB11 mutant and the fliI mutant, revealing that Omp4 transport was dependent on a flagellum export apparatus and virulence factor export [45] . The functions of the other identified outermembrane proteins remain to be elucidated . Therefore, our resultsmay serve as a first step toward further functional characterizationof H . pylori OMPs.

After 2-DE within a pH range of 6.0 to 11.0 IPG, proteins were transferred to nitrocellulose membranes for immunoblotting.We used a pool of 300 sera obtained from H . pylori-infectedpatients for immunoblotting, and the antibody reactivities withthe sarcosine-insoluble fraction are shown in Fig . 3 . The pooled sera from the infected patients bound to at least 10 spots.Of these, nine proteins were identified: catalase, Omp11, HP1173,UreA, a putative neuraminyllactose-binding hemagglutinin homolog[HP0410], [3R]-hydroxymyristoyl-[acyl carrier protein] dehydratase [FabZ], Omp14, Omp20, and Omp21 [Table 2] . The presence of catalase,UreA, a putative neuraminyllactose-binding hemagglutinin homolog[HP0410], and Omp20 have previously been demonstrated by immunoblotanalysis [23, 35] . Therefore, in the present study, we identifiedfive new immunoreactive proteins: hypothetical protein [HP1173],[3R]-hydroxymyristoyl-[acyl carrier protein] dehydratase [FabZ],Omp11, Omp14, and Omp21 . The pooled sera from uninfected personsthat was used as a negative control did not exhibit any immunoreactivity[data not shown].

 
Our results contribute to the characterization of H . pylori OMPs and may help to identify new target proteins for vaccine development and drug therapy.

 
This study was supported by grant 02-PJ1-PG10-20201-005 fromthe Ministry of Health and Welfare of Korea . J.-Y.S., K.-M.K.,S.-M.S., D.-S.K., J.-S.J., S.-G.L., and J.-U.P . were supportedby the Brain Korea 21 program from the Ministry of Educationof Korea . J.-U.P . was supported by a Korea Research FoundationGrant [KRF-2002-050-E00002].

 
* Corresponding author . Mailing address: Department of Microbiology, Gyeongsang National University College of Medicine, Chiram-dong 90, Jinju, Gyeong-nam 660-751, Republic of Korea . Phone: 82-55-751-8745 . Fax: 82-55-759-1588 . E-mail: khrhee@gaechuk.gsnu.ac.kr .

S.-C.B . and K.-M.K . contributed equally to this study.

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