Differential Proteomics of Helicobacter pyloriAssociated ...

INTRODUCTIONAutoimmune gastritis (AG), also

known as autoimmune chronic atrophicgastritis or chronic type A gastritis, is anautosomal-dominant disease. AG is char-acterized by immune-mediated chronicinflammation, mucosal gland atrophy,with increased serum autoantibodies togastric parietal cells and/or intrinsic fac-tors, hypochlorhydria, vitamin B12 defi-ciency and, in some cases, neurologicalsymptoms and diffuse metaplasia. In thelate stages, patients show a higher riskfor developing both neuroendocrine (car-

cinoid) and glandular (adenocarcinoma)tumors (1–4). In the presence of atrophy,AG is called atrophic AG (AAG).

In Correa’s model of gastric carcino-genesis, Helicobacter pylori (HP) infectiontriggers the progressive sequence of gas-tric lesions from chronic gastritis, gastricatrophy, intestinal metaplasia, dysplasiaand finally gastric adenocarcinoma (5).

Metaplastic AAG demonstrates similarhistological and clinical findings as thoseof metaplastic AG related to HP (3). Ofnote, in the early phases of AG, HP infec-tion may induce autoantibodies to gas-

tric parietal cells, but later HP can spon-taneously disappear (3,4,6). Thus, theexact relation between HP and gastricautoimmunity remains controversial aswell as the question if HP may be thetrigger or a perpetuating hit to AAG (3).

HP is a gram-negative, microaerophilic,spiral-shape bacterium colonizing thehuman gastric mucosa of more than halfof the human world’s population. HPpreferentially colonizes the antrum of thestomach, for which pH is higher than inthe corpus, but during gastritis progres-sion, HP can invade the corpus. In mostcases, HP causes asymptomatic gastric in-fections, but in others, it may progress tosymptomatic chronic gastritis, gastric orpeptic duodenal ulcer (DU), gastric can-cer (GC) or mucosa-associated lymphoidtissue (MALT) lymphoma (7,8). For manyyears, the molecular cross-talk betweenHP and human gastric mucosa has beeninvestigated (9–12).

HP strains are extraordinarily numer-ous, with every individual harboring a

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Differential Proteomics of Helicobacter pylori Associated withAutoimmune Atrophic Gastritis

Ombretta Repetto,1 Stefania Zanussi,2 Mariateresa Casarotto,2 Vincenzo Canzonieri,3 Paolo De Paoli,1

Renato Cannizzaro,4 and Valli De Re1

1Facility of Bio-Proteomics, 2Microbiology-Immunology and Virology, 3Pathology Unit, and 4Gastroenterology Unit, Centro diRiferimento Oncologico (CRO), Aviano National Cancer Institute, Aviano, Italy

Atrophic autoimmune gastritis (AAG) is a condition of chronic inflammation and atrophy of stomach mucosa, for which develop-ment can be partially triggered by the bacterial pathogen Helicobacter pylori (HP). HP can cause a variety of gastric diseases, suchas duodenal ulcer (DU) or gastric cancer (GC). In this study, a comparative proteomic approach was used by two-dimensional fluo-rescence difference gel electrophoresis (DIGE) to identify differentially expressed proteins of HP strains isolated from patients with AAG,to identify markers of HP strain associated with AAG. Proteome profiles of HP isolated from GC or DU were used as a reference to com-pare proteomic levels. Proteomics analyses revealed 27 differentially expressed spots in AAG-associated HP in comparison with GC,whereas only 9 differential spots were found in AAG-associated HP profiles compared with DU. Proteins were identified after matrix-as-sisted laser desorption ionization (MALDI)-TOF and peptide mass fingerprinting. Some AAG-HP differential proteins were common be-tween DU- and GC-HP (peroxiredoxin, heat shock protein 70 [HSP70], adenosine 5′-triphosphate [ATP] synthase subunit α, flagellin A).Our results presented here may suggest that comparative proteomes of HP isolated from AAG and DU share more common proteinexpression than GC and provide subsets of putative AAG-specific upregulated or downregulated proteins that could be proposedas putative markers of AAG-associated HP. Other comparative studies by two-dimensional maps integrated with functional genomicsof candidate proteins will undoubtedly contribute to better decipher the biology of AAG-associated HP strains.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2013.00076

Address correspondence to Valli De Re, Facility of Bio-Proteomics, CRO Aviano National

Cancer Institute, Via F. Gallini, 2 33081 Aviano (PN), Italy. Phone: +39-0434-659672; Fax: +39-

0434-659799; E-mail: [email protected].

Submitted July 24, 2013; Accepted for publication December 23, 2013; Epub

(www.molmed.org) ahead of print December 24, 2013.

distinctive bacterial population withclonal variants. Furthermore, HP sub-clones may be isolated from the samebiopsy or biopsies from different stom-ach locations and HP disease–specificstrains may exist (13). Several geneticmarkers of pathogenicity characteristicsfor different HP strains have been exten-sively described; virulence factors associ-ated with gastric carcinogenesis havealso been identified, with the HP pathogenesis–related genes mostly resid-ing in the cytotoxin-associated gene (Cag)pathogenicity island (9,11,14).

To date, there exists no reliable diagnos-tics to predict HP-infected patients at riskfor developing HP-related pathologies, in-cluding AAG, DU and/or GC. Efforts fordeveloping predictive diagnostics havemostly focused on HP disease–relatedbiomarkers explored at gene level (14–18).However, the combination of the most ex-tensively studied factors of pathogenicitycagA, vacA and babA fails in segregating aparticular HP-virulent strain associatedwith a specific pathogenesis.

In this context, after the complete se-quencing of four HP strains (19) (strains26695, J99 and HPAG1: http://cmr.jcvi.org/ tigr-scripts/CMR/shared/Genomes.cgi; strain Shi470: NCBI, accession num-ber NC 010698), proteomic technologieshold promise for better disease-specificclassification of HP strains. Proteomicsallows bacteria to be characterized at theprotein level based on the expressionfrom active genes, thus encompassingthe limits of DNA level, where both ac-tive and inactive genes may be identifiedand the difference in protein expressioncannot been performed. The HP genomecontains about 1,600 open-readingframes, 200 of which are known to en-code expressed proteins (19,20). The HPproteome has been investigated for manyyears to characterize the intrinsic HP bi-ology, and two-dimensional maps of sev-eral standard strains of different origin(e.g., 26695 from gastritis, J99 from DU,and HP strain Sydney strain 1, CagAnegative [CagA– SS1]) are available (Sup-plementary Table S1). In parallel, pro-teomics identified some HP disease

markers associated with severe gastricpathologies (e.g., AG, GC and DU; Sup-plementary Table S2). In particular, pro-teome components of HP have been in-vestigated to identify functionally activegenes, subcellular or secreted/translo-cated proteins, disease-specific proteins,as well as immunoreactive ones (Supple-mentary Tables S1, S2).

The more diffuse model for the pro-gression from chronic AG to intestinalmetaplasia, dysplasia and finally GCtakes into consideration several steps;and it considers the HP infection as act-ing together with a variety of host ge-netic and environmental factors (21).Moreover, in patients with DU, moreoften mild AG occurs, and there is no in-creased risk of GC compared with risk inthe general population (22,23). At pres-ent, there is still a lack of proteomics datafor markers of patient tendency to de-velop AAG.

Proteome analysis represents a power-ful approach to resolve and identify pro-teins in complex biological samples. Pro-teome data supplement data fromgenome and transcriptome approaches.Only proteomics allows deciphering ofthe connections between the genetic in-formation and the phenotype-related re-sponse(s), even when the complete ge-nome of an organism is determined (24).

In this study, we used a targeted com-parative proteomic approach on the basisof two-dimensional fluorescence differ-ence gel electrophoresis (DIGE) to iden-tify differentially expressed proteins ofHP isolated from patients with AAG, tobe proposed as candidate markers forstrains associated with HP-related clinicaloutcomes of AAG. Proteome profiles ofHP isolated from GC or DU were used asa reference to compare AAG proteomiclevels. Proteomics analyses revealed 27differentially expressed proteins of AAG-associated HP in comparison with GC,whereas only nine differential proteinswere found for AAG-associated HP pro-files compared with DU. Our results maysuggest that HP proteomes of HP isolatedfrom AAG and DU share more commonproteins than in regards to GC and pro-

vide subsets of putative AAG-specific up-or downregulated proteins, which couldbe proposed as putative markers of AAG-associated HP.

MATERIALS AND METHODS

Patients and Autoimmune AtrophicGastritis Diagnosis

A total of 18 patients entering the diag-nostic criteria of AAG from 2004 to 2010were evaluated for the presence of past oractive HP infection by serum HP-IgG de-termination (HP-IgG ELISA Biohit, cutoff30 EIU/mL), histological examination andculture assays within the routine diagnos-tic workup for HP infection. Positive HPinfection was ascertained when at leasttwo parameters showed a positive resultat enrollment and/or in previous visits. Atotal of 10 of 18 (55.5%) patients had adocumented HP past or active infection.HP strains were isolated from antrumand/or corpus biopsies in 4 of 6 (66%) pa-tients with active infection (Table 1). Al-though without atrophy, patient 4 (Table 1)was included in the analyses because ofher high levels of antibodies anti-parietalcells (1:160), which increased to 1:1,2801 month later. Patient 4 is currently un-dergoing follow-up. This group of pa-tients was compared with both a group of8 DU-affected patients and another groupof 13 GC-affected patients. DU and GCpathologies were used as reference mapsfor comparative proteomics to individuateboth up- or downregulated putativeAAG-associated HP proteins, to find a po-tential marker (panel of markers) for theidentification of Hp-related AAG (that is,often asymptomatic but may be associ-ated with pernicious anemia and may beinvolved in GC development). All pa-tients have been notified of the purpose ofthe study, and an informed consent hasbeen obtained for all participants. The In-ternal Review Board of the CRO Instituteapproved the project as IRB-14-2013.

Bacterial Strains and CultureConditions

HP strains were isolated from endo-scopic biopsy samples from the stomach

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H E L I C O B A C T E R P Y L O R I P R O T E I N S O F A U T O I M M U N E G A S T R I T I S

(corpus and/or antrum). The biopsyspecimens were cultured in HP SelectiveMedium (Bio-Mèrieux, Rome, Italy), in-cubated at 37°C in a microaerophilic en-vironment (Campygen Oxoid, Bas-ingstoke, Hampshire, UK) for 3–4 d. Thecultured bacteria were identified as HPbased on gram-negative staining, curvedor spiral shape and positivity for catalase,oxidase and urease production. Identifi-cation was further confirmed by poly-merase chain reaction. Several sweeps ofcolonies, considered representative of thewhole HP population, were subculturedon Columbia sheep blood agar (Kima,Padua, Italy). After bacterial growth, asuspension was obtained and stored at–80°C in microbial storage medium (Mi-crobank; Pro-Lab Diagnostics, RichmondHill, ON, Canada). Strains were revital-ized after a median of 9 months (range2–98 months) in HP Selective Medium.After expansion in Columbia sheep bloodagar, an HP suspension was used for pro-teome extraction. Bacterial DNA extrac-tion and polymerase chain reaction onthe virulence factor genes CagA, CagEand VirB11, mapping into the Cag patho-genicity island (Cag PAI), Vac A, and HomA and B, were also performed as previ-ously described (25–27). As already re-ported, HP was likely isolated from gas-tric biopsy samples of AAG patientsbecause of an impairment in functionalgastric mucosa leading to increasinghypochlorhydria, saprofitic flora over-growth and progressive disappearance ofanti-HP antibodies (28,29). Indeed, most

of our patients showed a low incidence ofcurrent active infection (6 of 18).

Protein Isolation and Labeling withCyanine Dyes (CyDyes)

The AAG group of patients was com-pared with both a group of 8 DU-affectedpatients and another group of 13 GC- affected patients. DU and GC pathologieswere used as reference maps for compar-ative proteomics to individuate putativeup- or downregulated AAG-specific pro-teins for the identification of AAG.

The HP soluble as well as hydropho-bic proteins were methanol/chloroformextracted with DIGE lysis buffer (30 mmol/L Tris, 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate}). Proteins were recov-ered at the liquid interphase by threesubsequent steps: (a) methanol (MeOH)(4:1, v:v), (b) chloroform (1:1, v:v) and(c) Milli-Q H2O addition (3:1, v:v), fol-lowed by centrifugation (13,000g for5 min at 4°C). After removal of the aque-ous upper layer, proteins were MeOH-precipitated (3:1, v:v), and the pellet waswashed with ethanol and then resus-pended in rehydration buffer for two- dimensional electrophoresis analysis(7 mol/L urea, 2 mol/L thiourea, 4%CHAPS, 0.5% v/v pharmalytes). Proteinconcentration was determined by usingBio-Rad Bradford-based protein assay(Bio-Rad, Milan, Italy). Whatever thegastric disease, each individual proteinsample extracted from HP isolates of one

patient was always kept as a distinctsample. For DIGE labeling, the proteinlysates were labeled with CyDyes ac-cording to the manufacturer’s protocol(CyDye DIGE Fluor minimal dyes; GEHealthcare, Uppsala, Sweden). The sam-ple pairs were mixed with an internalCy2-labeled standard pool comprisingequal amounts of each protein sample,which was used to reduce inter-gel vari-ation. To minimize dye-specific labelingartifacts, Cy3- and Cy5-labeling patternswere swapped among the same group ofsamples (Supplementary Table S3).

Two-Dimensional DIGEProteins were first separated by isoelec-

trofocusing (IEF) on 11-cm immobilizedpH gradient dry strips (IPG) with a non-linear (NL) pH 3–10 gradient (Bio-Rad).For analytical gels, a pair of Cy3- andCy5-labeled samples (each 25 μg protein)and 25 μg Cy2-labeled internal standardwere pooled and filled up to 200 μL withrehydration buffer (7 mol/L urea, 2 mol/Lthiourea, 4% CHAPS, 2% dithiothreitol).Strips were passively rehydratedovernight with the rehydration buffersupplemented with 2% (v/v) IPG buffer,pH 3–10 NL, 50 mmol/L dithiothreitoland 0.1% bromophenol blue, and the pro-tein extracts at room temperature. IEF andsecond dimension were performed in Protean® IEF and Criterion™ Cells (Bio-Rad), respectively, as previously reported(30). After electrophoresis, analytical gelswere washed with Milli-Q H2O andscanned on a Typhoon Trio™ laser scan-

R E S E A R C H A R T I C L E


Table 1. Characteristics of the patients affected by AAG, from whom HP strains were isolated.

Patient number Age Sex PGI PGII PGI/PGII Gastrin 17 Ab anti-HP HP histology Ab anti-PC Atrophya Stomach locationb

1 70 F 22.3 17.4 1.28 103.8 87.9 – ND 1 Antrum2 37 F 25 7.26 3.44 40 6.5 – + 2 Corpus3 42 F 14.8 10.9 1.36 350.8 100.8 + + 3 Corpus4c 40 F 107.5 10.3 10.44 2.31 97.8 – + 0 Antrum and corpus

Ab anti-HP, antibodies against anti-HP; Ab anti-PC, antibodies against parietal cells; anti-HP, antibodies against HP; ND, not determined;PGI, serum pepsinogen I; PGII, serum pepsinogen II.aAtrophy scores according to operative link for gastritis assessment (OLGA) system (adapted considering only a simple sample of antral biopsy).bStomach location refers to the biopsies from which HP strains were isolated.cPatient at the first visit without atrophy, but with increasing anti-PC Ab (levels increasing from 1:160 to 1:1,280 after 1 month) and still underfollow-up.

ner (GE Healthcare) (30). For preparativegels, 360 μg unlabeled protein pooledfrom amounts of the 4 AAG protein ex-tracts was used, focused for 35 kilovolthours (kVh) and stained with CoomassieBrilliant Blue CBB G-250 (Bio-Rad). Gelimages were acquired on the TyphoonTrio 9400™ laser scanner (GE Healthcare)at 100 μm resolution by using a red excita-tion wavelength.

Image AnalysisImage analysis was performed by using

DeCyder™ software, version 6.5 (GEHealthcare), as previously described (30).Briefly, images were subjected to Differ-ence In-gel Analysis (DIA) to detect,quantify and normalize spots according tothe volume ratio of the correspondingspots detected in the Cy2 image of thepooled-sample internal standard, and theBiological Variation Analysis (BVA) mod-ule to allow matching of spots from mul-tiple gels, calculate average abundancechanges and statistically analyze the dif-ferential protein expression. The normal-ized spot quantities were collectively ana-lyzed as three independent groups: AA,GC and DU, which enabled matching ofmultiple gel images from different pa-tients to provide statistical data on aver-age abundance for each protein spotamong the DIGE gels. Statistical analysisof variance was performed to accuratelyassess protein expression changes occur-ring in biological replicates comparing thethree groups. Finally, the extended dataanalysis (EDA) module was used for mul-tivariate analysis of protein expressiondata, derived from the BVA module,through principal component analysis(PCA) pattern analysis and discriminantanalysis. Student t test was performed toassess the statistical significance of differ-entially expressed proteins. On the basisof average spot volume ratio, spots forwhich relative expression changed at least1.5-fold between AAG and GC/DU at95% confidence level (t test; p < 0.05) wereconsidered to be significant. The spotsidentified as differentially expressed weresubjected to matrix-assisted laser desorp-tion ionization (MALDI)-TOF analysis.

Protein Identification by MALDI-TOFPeptide Mass Fingerprinting

Protein spots of interest were excisedfrom the Coomassie-stained preparativegel using a spot cutter and destainedwith 25 mmol/L ammonium bicarbonatein 50% acetonitrile. After overnighttrypsin digestion, peptides were ex-tracted with 1% (trifluoroacetic acid)TFA, subjected to Zip Tip cleanup (Milli-pore, Milan, Italy) and eluted with 50%acetonitrile and 0.3% TFA. After mixingthe sample with α-cyano-4-hydroxycin-namic acid (CHCA) matrix solution (10g/L CHCA in 50% acetonitrile and 0.3%TFA) (1:1, v:v), peptides were spotted onthe MALDI target. The peptide mass fin-gerprinting measures were performed ona Voyager-DE PRO BiospectrometryWorkstation mass spectrometer (ABSciex, Framingham, MA, USA). After ex-ternal calibration with Peptide Mix4(Proteomix) 500–3,500 Da (LaserBio Labs,Sophia-Antipolis Cedex, France), MALDImass spectra were recorded, collectedand processed by using Data Explorer,version 5.1, software (AB Sciex), peaklists were obtained from the raw dataunder routine laboratory conditions (30)and mass spectra were finally processedusing Data Explorer, version 5.1, soft-ware (AB Sciex). Database searching wasdone with the MASCOT search engine,version 2.3 (Matrix Science, London,UK), against the National Center ForBiotechnology Information non-redundantprotein database (NCBInr) and Swiss-Prot databases, limiting the search tobacterial proteins, allowing for onetrypsin missed cleavage and a 0.5-Damass tolerance error. Protein localiza-tions were attributed according to eitherthe National Center for BiotechnologyInformation (NCBI) resource (http://www. ncbi.nlm.nih.gov/ protein) or, incase of lack of information in the NCBIdatabase, NCBI searches with the pub-licly available tool PSORTb, version 3.0.2,for bacterial localization predictionagainst gram-negative bacteria sequences(http://www.psort. org/ psortb). For eachprotein, biological processes and molecu-lar functions were reported according to

the Gene Ontology (GO) description inthe UniProtKN/ TrEMBL database(http://www. uniprot. org/ uniprot).

All supplementary materials are availableonline at www.molmed.org.

RESULTS

Proteoma of HP Strains Isolated fromPatients with Autoimmune AtrophicGastritis

In the HP strains successfully isolatedfrom biopsies of four AAG patients(Table 1), we hereby used DIGE to iden-tify differentially expressed proteins,compared with those isolated from DUand GC biopsies. Figure 1A represents aDIGE proteome map of HP proteins iso-lated from human biopsies of patient 4(corpus and antrum samples) to focus onand visualize AAG-associated HP cya-nine–labeled proteome maps. Around1,600 spots were clearly detected andsubsequently analyzed by using DeCyder software for differential proteinexpression. To investigate a possible pro-tein variation depending on the stomachlocation of HP isolation (corpus versusantrum), protein profiles of HP isolatedfrom corpus were compared with thosefrom antrum. Image analyses by DeCyder of HP differentially expressedproteins revealed that the stomach loca-tion was not a parameter significantly in-fluencing the pattern of HP protein ex-pression in our series. Indeed, proteinspot maps from antrum and corpus ofthe same patient analyzed with PCAwere close and thus not different; more-over, independently from the patient,within each group (AAG, GC or DU),spot maps from corpus and antro werenot placed in distinct subgroups (andthus not significantly different). There-fore, we further continued our analysesby comparing all proteome maps(AAG-HP maps versus DU- or GC-HPmaps) independently from the stomachlocation of HP isolation. All differentiallyexpressed proteins in AAG- associatedHP strains are listed in Tables 2 and 3.A total of 15 spots were upregulated in



AAG compared with GC (Table 2), ofwhich 4 (spots 57, 168, 161 and 166) werealso upregulated compared with DU(Table 3). Although a total of 12 spots re-sulted in downregulation in AAG com-pared with GC (Table 2), three of them(spots 37, 40 and 42) also downregulatedin DU. Two additional spots were down-regulated in only DU (spots 117 and 76)(Table 3).

Identification of HP DifferentiallyExpressed Proteins

These 29 spots, indicated with dots inFigure 1B, were excised from micro-preparative two-dimensional gels forprotein identification by tryptic in-geldigestion and MALDI-TOF analysis.After a Mascot peptide mass fingerprint-ing database search using the acquiredmass values, 20 spots were identified,corresponding to 15 distinct proteins.Details of protein identification, proteinscore, sequence coverage, theoretical iso-electric point (pI) value and molecularweight as well as GenBank® accessionnumber and average relative change(fold difference) are shown in Tables 2and 3. Some proteins were found inmore than one spot: (a) the probable per-oxiredoxin (gi|2507172; alternativenames: 26-kDa antigen or thioredoxin re-ductase) was detected in four spots (166,168, 244 and 248); (b) the urease β sub-unit (gi|57014163) was detected in twospots (33 and 34); and (c) the elongationfactor Tu (gi|2494256) was detected intwo spots (63 and 89).

Autoimmune Atrophic GastritisDifferentially Expressed ProteinInvolved in Various BiologicalProcesses

Globally, all the identified proteinswere involved in the following sevenclasses of biological processes: (a) proteinbiosynthesis/DNA translation/tRNAprocessing (ribosome-recycling or releas-ing factor, 50S ribosomal protein L30,elongation factor Tu and tRNApseudouridine synthase A); (b) proteinrefolding and stress response (60-kDachaperonin/ GroEL and chaperone pro-



Figure 1. Representative analytical DIGE proteome map of HP from AAG (A) and micro-preparative two-dimensional protein map of HP from AAG (B). (A) Proteins were resolved byIEF over the pI 3–10, followed by 8–16% gradient sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE) and overlaid by DeCyder. After extraction, proteins were la-beled with Cy3 and Cy5. In particular, this gel (number 19) refers to samples P102cy3

(antrum) and P103cy5 (corpus) extracted from AAG-associated HP strains. An internal stan-dard comprised of equal amounts of proteins from all samples (AAG, DU and GC) was la-beled with Cy2 and included in all gels. (B) Gel number 18 is shown as representative ofDIGE maps with proteins extracted from gastric cancer (cy5) comigrated with those ex-tracted from H. pylori associated with DU (cy3). (C) Numbered spots indicate the differen-tially expressed proteins in AAG-associated HP strains in regards to either GC or DU. Identi-fied spots are listed in Tables 2 and 3. Around 300 μg AAG-associated HP unlabeledprotein pooled from amounts of samples was resolved by IEF over the pI range 3–10 NL,followed by 8–16% gradient SDS-PAGE and stained with CBB G-250. MW, Bio-Rad two- dimensional molecular weight standards.



Tab

le 2

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late

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Pro

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tion

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ce

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nu

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(D

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un

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seq

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HP

pro

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s up

reg

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ted

in A

AG

co

mp

are

d w

ith G

C

1327

557/

9.68

gi|

2380

5773

1tR

NA

pse

ud

ou

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Cyt

op

lasm

/tR

NA

pro

ce

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P P

1232

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002

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10.1

93.

54E-

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syn

tha

se A

254

6644

/10.

96g

i|22

6703

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som

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301

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154.

50E-

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1)

pro

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the

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lod

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(str

ain

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1168

/

LMG

814

2/SP

-6)

206

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5.85

0.04

9

168

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88g

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ab

le p

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da

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82/0

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5.69

3.28

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244

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ity/

HP

75/0

.009

8/41

2.94

4.56

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(41–

43)

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n o

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thio

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2.5

0.02

5(2

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53,

pa

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ge

ne

sis/u

rea

ca

tab

olic

62

,75–

77)

pro

ce

ss/h

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lase

5767

136/

4.99

gi|

2267

3813

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pe

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365.

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pa

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62

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77)

pro

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2493

545

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106/

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-006

/30

2.21

1.76

E-03

(24,

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5,71

,

161

ca

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ce

ss/p

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/76

,78)

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8.87

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166

2233

5/5.

88g

i|25

0717

2P

rob

ab

le p

ero

xire

do

xin

or

Cyt

op

lasm

/pe

roxi

da

se a

ctiv

ity/

HP

97/6

e-0

05/4

82.

153.

10E-

03(4

1–43

)

26-k

Da

an

tige

n o

r o

xid

ore

du

cta

se

thio

red

oxi

n re

du

cta

se

177

n.i.

1.87

0.01

7

111

gi|

1706

274

Bifu

nc

tion

al e

nzy

me

Cyt

op

lasm

/GTP

ca

tab

olic

M

yco

ba

cte

rium

44

/18/

141.

77.

19E-

03n

.d.c

cys

N/c

ysC

pro

ce

ss/s

ulfa

te a

ssim

ilatio

ntu

be

rcu

losis

7774

074/

5.11

gi|

1223

0111

Fla

ge

llar h

oo

k–a

sso

cia

ted

Se

cre

ted

/ba

cte

rial f

lag

ellu

m/

HP

J99

39/4

1/10

1.6

0.02

3(7

9)

pro

tein

2c

ell

ad

he

sion

/fla

ge

llum

ass

em

bly

6520

180/

5.84

gi|

2084

3295

2Pe

ptid

og

lyc

an

-ass

oc

iate

d

Ou

ter m

em

bra

ne

/ba

cte

rial

HP

G27

41/9

.1e

+ 0

2/17

1.56

0.04

9(5

9)

lipo

pro

tein

en

velo

pe

inte

grit

y

Con

tiued

on

next

pag

e

tein dnaK/heat shock protein 70[HSP70]); (c) catabolic processes (urea:urease β; hydrogen peroxide: catalase),(d) metabolic processes (phosphate: inor-ganic pyrophosphatase; sulfate: bifunc-tional enzyme cysN/cysC); (e) energymetabolism (thioredoxin reductase/26-kDa antigen, adenosine 5′-triphosphate[ATP] synthase α chain); (f) flagellum assembly/motility and, indirectly, HPvirulence (flagellar hook–associated pro-tein 2 and flagellin A); and (g) bacterialenvelope integrity (peptidoglycan- associated lipoprotein) (Tables 2, 3). Inparticular, the differentially expressedproteins in AAG-HP proteomes belongedto all seven classes compared with theGC-HP proteome, whereas they werelimited to only four classes (b, d, e and f)if compared with the UD-HP proteome.

Bacterial Localization and Secretionof Selected Proteins

Among the 15 unique identified pro-teins, the majority has a cytoplasmatic lo-calization (60%), with two proteins beingmembrane-associated (peptidoglycan- associated lipoprotein and ATP synthasesubunit α) and three were secreted (ureaseβ subunit, flagellar hook–associated pro-tein 2 and flagellin A). Several proteinsare already considered important HP virulence factors, namely urease β, fla-gellin A, catalase and chaperone GroEL(Tables 2, 3). With the exception of spots13 and 111 (tRNA pseudouridine synthaseA and bifunctional enzyme cysN/cysC),all the identified proteins were previouslydescribed in works about HP proteomes(Tables 2, 3). However, among them, onlynine were reported to be related to spe-cific gastric disease(s) (Table 4).

Proteome of HP Isolated from Patientswith Autoimmune Atrophic Gastritisand from Patients with DU Showed aGreater Similarity than ThoseObtained from Patients with GastricCancer

A PCA analysis indicated that AAG- associated HP can be discriminated fromDU and GC on the basis of the proteomecharacterization. The most important



Tab

le 2

. Con

tinue

d.

HP

pro

tein

s d

ow

nre

gul

ate

d in

AA

G c

om

pa

red

with

GC

3558

321/

5.44

gi|

2267

0413

660

-kD

a c

ha

pe

ron

in o

r C

yto

pla

sm/p

rote

in re

fold

ing

/ H

P (

stra

in P

12)

68/0

.057

/23

–6.5

82.

84E-

03(2

4,32

,41–

43,

Gro

ELst

ress

resp

on

se/A

TP b

ind

ing

53,5

9,62

,75,

77,8

0)

248

2233

5/5.

88g

i|25

0717

2P

rob

ab

le p

ero

xire

do

xin

or

Cyt

op

lasm

/pe

roxi

da

se a

ctiv

ity/

HP

44/1

3/34

–5.3

12.

45E-

03(4

1–43

)

26-k

Da

an

tige

n o

r o

xid

ore

du

cta

se

thio

red

oxi

n re

du

cta

se

37n

.i.–4

.96

9.54

E-06

8943

734/

5.17

gi|

2494

256

Elo

ng

atio

n fa

cto

r Tu

Cyt

op

lasm

/GTP

ca

tab

olic

H

P90

/0.0

0035

/33

–2.9

11.

22E-

05(4

1,43

,62,

75)

141

n.i.

pro

ce

ss/p

rote

in b

iosy

nth

esis

–2.6

6.38

E-03

4055

280/

5.29

gi|

2267

3989

3A

TP s

ynth

ase

su

bu

nit α

Inn

er m

em

bra

ne

/pla

sma

H

P14

0/3.

2e-0

09/3

2–2

.56

5.52

E-05

(24,

62,7

5,77

)

or F

-ATP

ase

su

bu

nit α

me

mb

ran

e A

TP s

ynth

esis

–

hyd

roly

sis c

ou

ple

d p

roto

n

tra

nsp

ort

4253

252/

6.04

gi|

6039

2282

Fla

ge

llin

ASe

cre

ted

/ba

cte

rial f

lag

ellu

m/

HP

J99

88/0

.000

46/3

0–2

.45

0.02

5(2

4,31

,32,

43,

flag

ellu

m m

otil

ity a

nd

viru

len

ce

60)

165

n.i.

–1.9

90.

034

92n

.i.–1

.99

1.66

E-04

163

2058

4/5.

20g

i|25

4809

526

Rib

oso

me

-re

cyc

ling

or

Cyt

op

lasm

/pro

tein

bio

syn

the

sisR

ho

do

co

cc

us

32/2

.2e

+ 0

02/1

5–1

.93

0.03

6(4

2)

rele

asin

g fa

cto

ro

pa

cu

s

(str

ain

B4)

135

n.i.

–1.8

66.

50E-

04

6343

734/

5.17

gi|

2494

256

Elo

ng

atio

n fa

cto

r Tu

Cyt

op

lasm

/GTP

ca

tab

olic

H

P79

/0.0

037/

33–1

.73

1.14

E-04

(42,

62,7

7)

pro

ce

ss/p

rote

in b

iosy

nth

esis

asp

ot

nr.,

sp

ot

nu

mb

ers

refe

r to

Fig

ure

1.

bn

.i., n

ot

ide

ntif

ied

.cn

.d.,

no

t d

esc

ribe

d.

principal component, PC1, explains thevariation and discriminates against thebiological samples according to group(AAG, DU and GC) (Figure 2). PC2 iscorrelated with intragroup variability. In the score plot visualization mode(Figure 2A), each full black circle insidethe ellipse is a significantly expressedprotein, which contributes to discrimi-nate spot maps (Figure 2B). In the load-ing plot, we see that there is more vari-ability in GC- associated HP spot mapsthen in both DU- and AAG-associatedones. In particular, AAG-related HP pro-teome maps are positioned between DUand GC but are closer to DU-relatedmaps (Figure 2B).

Peroxiredoxin, HSP70, F-ATPase andFlagellin A Are the HP proteins MoreSpecifically Associated withAutoimmune Atrophic Gastritis

Among the all–AAG-HP differentialproteins, we focused on three upregu-lated spots (168, 166: peroxiredoxin; 57:HSP70) and two downregulated spots(40: F-ATPase; 42: flagellin A), which wefound of particular interest because oftheir common variation in level in AAGversus both GC and DU. We analyzedtheir contents in each AAG patient aslog standard volume, as calculated bythe DeCyder software, and we evalu-ated the presence of a possible associa-tion between protein up-/downregula-tion and the HP strain characterizationat the levels of CagA, CagE, VirB11, VacAand Hom genes and the available clinicaldata of autoimmunity and atrophy (Fig-ure 3A, Table 1). In our analyzed HPisolates associated with AAG, the geno-typing results on the three virulencegenes within the Cag PAI (CagA, CagEand VirB11), on VacA polymorphismsand on Hom selection are shown in Fig-ure 3B. Patient 4 represents successfulHP isolation from both antrum and cor-pus. Furthermore, for this patient, we il-lustrate in Figure 3A that there is no sig-nificant difference in protein logstandard abundance between corpusand antrum, even if there is an apparentdifference in spot 42.



Tab

le 3

.Diff

ere

ntia

lly e

xpre

sse

d p

rote

ins

of

HP

iso

late

s re

late

d t

o A

AG

co

mp

are

d w

ith t

ho

se o

f H

P is

ola

tes

rela

ted

to

DU

.

Pro

tein

G

en

Ban

k d

esc

ribe

da

cc

ess

ion

Lo

ca

liza

tion

/bio

log

ica

l pro

ce

ss/

Sco

re/e

xpe

ct/

Fold

pre

vio

usly

Sp

ot

nu

mb

era

MW

(D

a)/

pI

no

.bP

rote

in a

nn

ota

tion

mo

lec

ula

r fu

nc

tion

Org

an

ismSe

q. c

ove

rag

ed

iffe

ren

ce

p(r

ef.)

Pro

tein

s u

pre

gu

late

d

by

AA

G c

om

pa

red

w

ith D

U57

6713

6/4.

99g

i|22

6738

136

Ch

ap

ero

ne

pro

tein

C

yto

pla

sm/p

rote

in fo

ldin

g/

HP

(st

rain

32

/1.8

e +

002

/84.

237.

50E-

04(2

4,41

)d

na

K o

r HSP

70st

ress

resp

on

seSh

i470

)16

822

335/

5.88

gi|

2507

172

Pro

ba

ble

pe

roxi

red

oxi

n

Cyt

op

lasm

/pe

roxi

da

se a

ctiv

ity/

HP

82/0

.001

9/52

2.15

0.02

77(4

1–43

)o

r 26-

kDa

an

tige

n

oxi

do

red

uc

tase

or t

hio

red

oxi

n

red

uc

tase

161

n.i.

2.09

0.02

7716

622

335/

5.88

P21

762

Pro

ba

ble

pe

roxi

red

oxi

n

Cyt

op

lasm

/pe

roxi

da

se a

ctiv

ity/

HP

97/6

e-0

05/4

81.

690.

0467

(41–

43)

or 2

6-kD

a a

ntig

en

o

xid

ore

du

cta

seo

r th

iore

do

xin

re

du

cta

sePr

ote

ins

do

wn

reg

ula

ted

by

AA

G in

co

mp

aris

on

w

ith D

U37

n.i.

–7.3

61.

09E-

0411

7n

.i.–2

.30.

039

7619

317/

5.01

gi|

2500

043

Ino

rga

nic

C

yto

pla

sm/p

ho

sph

ate

-co

nta

inin

gH

P30

/2.9

E +

002

/12

–2.1

20.

037

(13,

24,6

2,77

)p

yro

ph

osp

ha

tase

co

mp

oun

d m

eta

bo

lic p

roc

ess

/m

ag

ne

sium

ion

bin

din

g40

5528

0/5.

29g

i|22

6739

893

ATP

syn

tha

se s

ubun

it α

Me

mb

ran

e/A

TP h

ydro

lysis

co

uple

d

HP

140/

3.2e

-009

/32

–2.0

37.

36E-

03(1

3,24

,62,

75)

or F

-ATP

ase

su

bu

nit α

pro

ton

tra

nsp

ort

/pla

sma

m

em

bra

ne

ATP

syn

the

sis c

oup

led

p

roto

n tr

an

spo

rt42

5325

2/6.

04g

i|60

3922

82Fl

ag

elli

n A

Sec

rete

d/b

ac

teria

l fla

ge

llum

/H

P (

stra

in

88/0

.000

46/3

0–2

.45

0.08

97(1

3,24

,31,

43)

flag

ellu

m m

otil

ity a

nd

viru

len

ce

J99)

aSp

ot

nu

mb

ers

refe

r to

Fig

ure

1.

bn

.i., N

ot

ide

ntif

ied

.

DISCUSSIONWith the exception of two works

(31,32), which were based on a DIGE ap-proach, our study is the first to analyzethe HP protein isolated from patientswith different gastric diseases by two- dimensional DIGE approaches. More-over, to our knowledge, at present, thereis a lack of comparative proteomics in-formation among maps of clinical HPstrains isolated from patients affected byAAG and those of clinical HP strains iso-lated from patients affected by DU orGC. Identification and characterization ofthe HP-related proteins isolated fromAAG patients is important, since it isknown that AAG related to HP infectionmay be a risk factor for further diseasedevelopment into GC (33,34).

In this study, we focused on the com-parative proteome of HP associated withAAG versus those of HP associated withDU or GC by using the DIGE approach(35). Protein profiles of HP isolated fromAAG patients (corpus or antrum) werecompared with reference maps of HP as-sociated with corpus or antrum proteinmaps of DU or GC patients (Supplemen-tary Table S3). The stomach location didnot significantly influence the pattern ofHP protein expression in our series. More-over, protein profiles analyzed by PCA(Figure 2) succeeded in discriminatingAAG-related HP from those associatedwith both DU and GC. Interestingly, evenpatient 4 without atrophy was included inthe PCA-evidenced AAG group.

A total of 29 distinct spots were differ-entially regulated, of which 20 wereidentified by MALDI-TOF MS and data-base searches as 15 distinct proteins. It isinteresting to note that all 15 proteinswere already identified in HP proteome,with the exception of two (the tRNApseudouridine synthase A, spot 13, andthe bifunctional enzyme cysN/cysC, spot111), but only 9 were previously de-scribed in the HP proteome associatedwith a gastric disease (urease β subunit,catalase, peptidoglycan-associated pro-tein, HSP70, GroEL, elongation factor Tu,inorganic pyrophosphatase, ATP syn-thase subunit α and flagellin A).



Tab

le 4

.List

of

diff

ere

ntia

l pro

tein

s fo

un

d in

HP

ass

oc

iate

d w

ith A

AG

an

d p

revi

ou

sly re

po

rte

d a

s H

P p

rote

ins

ass

oc

iate

d w

ith g

ast

ric d

isea

se(s

).

Ou

r fin

din

gs

Pro

tein

Ga

stric

dise

ase

Up

reg

ula

ted

in A

AG

HP

vers

us

on

ly G

CH

PU

rea

se β

sub

un

itC

G, G

C a

nd

DU

(24

); G

C o

r CG

>D

U (

62)

Ca

tala

seEa

rly G

C (

32);

CG

, GC

an

d D

U (

24);

CG

, GC

an

d D

U (

55)

Pep

tido

gly

ca

n-a

sso

cia

ted

C

G a

nd

DU

(59

)lip

op

rote

in

Up

reg

ula

ted

in A

AG

HP

vers

us

bo

th G

CH

Pa

nd

UD

HP

Ch

ap

ero

ne

pro

tein

dn

aK

G

C a

nd

DU

(24

)o

r HSP

70

Pero

xire

do

xin

a

Do

wn

reg

ula

ted

in A

AG

HP

vers

us

on

ly G

CH

P60

-kD

a c

ha

pe

ron

in o

r Gro

ELEa

rly G

C (

32);

CG

, GC

an

d D

U (

24);

GC

>D

U>

CG

(62

); C

G a

nd

DU

(59

)

Elo

ng

atio

n fa

cto

r Tu

CG

(32

); G

C>

CG

>D

U (

62)

Do

wn

reg

ula

ted

in A

AG

HP

vers

us

on

ly U

DH

PIn

org

an

ic p

yro

ph

osp

ha

tase

GC

an

d D

U (

24);

CG

, GC

an

d D

U (

62);

CG

an

d G

C (

13)

Do

wn

reg

ula

ted

in A

AG

HP

vers

us

bo

th G

CH

Pa

nd

UD

HP

ATP

syn

tha

se s

ub

un

it α

or

CG

, GC

an

d D

U (

24);

CG

, GC

an

d D

U (

62)

F-A

TPa

se s

ub

un

it α

Fla

ge

llin

AEa

rly G

C (

32);

CG

, GC

an

d D

U (

24)

AA

GH

P, H

P a

sso

cia

ted

with

au

toim

mu

ne

atr

op

hic

ga

strit

is; D

UH

P, H

P a

sso

cia

ted

with

DU

; GC

HP, H

P a

sso

cia

ted

with

ga

stric

ca

nc

er.

aU

nfo

un

d a

sso

cia

tion

till

th

e p

rese

nt.

A total of 15 spots were upregulated inAAG compared with GC, of which fourspots (57, 161, 166 and 168) were also up-regulated compared with DU; these fourspots were thus considered as proteins ofthe hallmarks characterizing HP strainsassociated with the AAG disease. Thefour spots were involved in the biologi-cal processes of “protein folding/stressresponse” and “oxidoreductase,” andthey corresponded to a probable perox-iredoxin (or 26-kDa antigen or thiore-doxin reductase; spots 166, 168) and achaperone protein dnaK (or heat shock70-kDa protein or HSP70, spot 57), withspot 161 not being identified.

Among these two specifically upregu-lated proteins of AAG, the peroxiredoxinis HP-specific (36). In Escherichia coli, inaddition to its protein disulfide iso-merase activity, the peroxiredoxin may

interact with unfolded/denatured pro-teins similarly to molecular chaperones,and it can also promote the functionalfolding of citrate synthase after urea de-naturation (37). Overall, the members ofthe peroxiredoxin family (PRX) are con-sidered thiol-specific antioxidant pro-teins, which confer a protective role incells. It has been demonstrated that HP-associated gastric inflammation causeepithelial cell damage by induction ofoxidative and nitrosative stress, whichmoreover plays an important role in gas-tric carcinogenesis (38). Recently, Betting-ton and Brown (1) clearly evidenced howthe inflammation of autoimmune gastri-tis displayed more eosinophil and lym-phocyte infiltrations of the basal epithe-lium than other forms of chronicgastritis. To ensure its pathogenesis andpersistence, HP has evolved a wide

range of mechanisms of reactive oxida-tive species detoxification, including per-oxiredoxin production (39,40). In thiscontext, it is tempting to suggest thatduring AAG HP may produce more anti-oxidant molecules than in DU and GC.In our work, interestingly, this proteinwas identified in four distinct differentialspots (166, 168, 244 and 248), three ofthem (spots 166, 168 and 244) upregu-lated with regard to GC and, moreover,spots 166 and 168 were also upregulatedwith regard to DU. The occurrence ofperoxiredoxin in HP proteomes was pre-viously described (41–43), but to ourknowledge, this is the first time that itsupregulation was associated with anAAG disease state.

In parallel, together with peroxire-doxin protein, we found the chaperoneprotein dnaK hallmark upregulated pro-



Figure 2. Principal component analysis of HP proteins isolated from AAG, DU and GC patients. (A) Score plot showing an overview of theproteins. Each circle represents a protein. The ellipse represents a 95% significance level. Proteins outliers can either be very strongly differen-tially expressed proteins or mismatched spots. (B) Loading plot showing an overview of the spot maps from the three groups AAG, DU andGC. Each circle represents a spot map. The ellipse groups the five spot maps from AAG, which can be separated from DU and GC onesand only one DU sample (4Cgel8). AAG-associated HP spot maps are displayed in black; those from GC and DU are displayed in red andblue, respectively. For each spot map, abbreviations indicate the identifier of the patients reported in Table 3 and include the diagnosis(AAG, GC, DU), the stomach location of HP isolation (a, antrum; c, corpus), and a superscript gel number in the Deyder work flow.

A B

tein in AAG. The chaperone dnaK familyis ubiquitous in bacteria and eukaryotes,and it is usually associated with the co-chaperones GrpE and HSP40 (dnaJ). Thechaperone protein dnaK is a major sur-face-exposed HP antigen with significanthomology with other bacterial dnaKs(41,44,45) and, similarly to the peroxire-doxin, it was implicated in protein fold-ing and stress response (41,46). More-over, at the surface of HP, the chaperoneprotein dnaK may act as a stress-inducedsurface adhesin capable of mediating therecognition of sulfatide glycolipid recep-tors on gastric epithelial cells (47). Datahave suggested that high levels of thisprotein may allow a more abundant col-

onization of HP into the host cells andthus favor the development of a gastriclesion (48). Interesting, chaperone proteindnaK, even from different HP strains,has been previously found immunoreac-tive toward more than one gastric cancerserum (24), suggesting its potentiality asa marker for GC. To date, this is the firstreport of HP-related chaperone dnaK ex-pression in the AAG state.

The other seven proteins upregulatedin AAG-associated HP proteomes com-pared with the only GC-associated oneswere identified as follows: a tRNApseudouridine synthase A, a functionalenzyme cysN/cysC, a 50S ribosomal pro-tein L30, a urease β subunit, a catalase, a

flagellar hook–associated protein 2 and apeptidoglycan-associated lipoprotein.

The tRNA pseudouridine synthase A isthought to play a role in the initiation oftranslation (49). Recently, it was discov-ered that this protein can be induced bystress, thus proposing a regulatory role insurvival, and that, by mediating a non-sense-to-sense codon conversion, it mayrepresent new means of generating cod-ing or protein diversity (50). This protein(spot 13) has not been identified previ-ously in HP proteome, like the spot 111and spot 254, which may be involved inthe synthesis of activated sulfate upon ox-idative stress and the structure of the 50Sribosomal subunit, respectively (51,52).



Figure 3. Protein contents of selected HP spots and HP gene characterization in the analyzed atrophic autoimmune gastritis-affected pa-tients. (A) Protein content, expressed as log standard volume, as calculated by the DeCyder software, is represented for spots 168, 166(peroxiredoxin), 57 (HSP70), 40 (F-ATPase subunit) and 42 (flagellin A) in the four patients of Table 3, with “4a” and “4c” above being forantrum and corpus, respectively. (B) The presence (+) or absence (–) of the CagA, CagE and VirB11 genes, together with the polymor-phisms for VacA and Hom genes are shown for each patient. *Patient at the first visit without atrophy, but with increasing anti-PC anti-body (levels increasing form 1:160 to 1:1,280 after 1 month) and still under follow-up.

Two spots (33 and 34) were identifiedas urease β subunit. The HP urease isknown to be critical for bacterial survivalin the highly acidic environment such asin human gastric mucosa by the hydroly-sis of urea to yield ammonia and carbondioxide, thereby buffering HP periplasmaand cytoplasm. Because of its effectiverole for HP survival, urease is one of themost abundant proteins synthesized byHP (41). This protein has been alreadydescribed in HP proteomes obtainedfrom different gastric diseases (24,53). Inparticular, Pyndiah et al. (53) showed ahigher intensity of urease B (UreB) in HPisolates from patients with chronic gastri-tis or GC compared with isolates frompatients with DU. Of note, Kobayashi etal. (54) showed that HP urease can stimu-late in vitro innate B-cells to secrete anumber of autoantibodies and suggest anew paradigm of innate-dependent B-cell initiation of autoimmunity relatedto the urease protein. Accordantly, wespeculate that higher content of UreBfounded in AAG-associated HP proteinprofiles may thus be associated with amore favorable status for an autoim-mune development pathognomic ofAGG condition.

Similarly to peroxiredoxin, HP cata-lase is known to allow HP to resistagainst the highly oxidative stress com-ing from the H2O2 generation at the in-fection sites in gastric epithelial cells(40). HP catalase acts synergistically to-gether with other decomposing proteinsto detoxify the cell from aggressive oxy-gen metabolites, thus preventing themisfolding or unfolding of proteinsunder long-term stress conditions. Forthese reasons, HP catalase is includedamong the HP virulence factors, and ithas been reported as an important en-zyme in different HP-related disorderssuch as gastritis, GC and DU (24,32,55).Probably the overexpressed catalasefound in AAG compared with GC in ourseries is associated with either a moreaccentuated oxidative stress occurringduring AAG state with respect to the GCones, or, alternatively, an environmentalselection of bacteria with a higher capa-

bility to counteract oxidative stress byenzyme overexpression. Accordantly, Ni et al. (56) reported a higher expres-sion of human 8-oxoguanine DNAN-glycosylase 1 (hOOG1), which pre-vents the oxidative DNA damage inAAG compared with the GC condition.

Flagellar hook–associated protein 2 is aprotein required for the morphogenesisand elongation of the flagellar filament,and it is considered as essential to colo-nize and establish infection in gastricmucosa because of its essential role inHP motility (57). While the peptidogly-can-associated lipoprotein has been de-scribed as essential for bacterial survivaland pathogenesis, its exact role in viru-lence has not been clearly defined (58).Its presence in HP proteome has been re-ported in both CG and DU (59), but atpresent, there is a lack of data about itsdifferential expression in AAG-relatedHP. It may be tempting to hypothesizethat the HP envelope may change itsprotein contents/composition dependingon the particular host physiology/ disease status.

A total of 12 spots were downregu-lated in AAG compared with GC, ofwhich 3 spots (37, 40 and 42) were alsodownregulated as compared with DU.They were identified as follows: ATPsynthase subunit α (or F-ATPase subunitα; spot 40) and flagellin A (3 spots puttogether into spot 42), with spot 37 notbeing identified. These downregulatedproteins may come from alterations inmetabolic processes more specific ofAAG than both DU and GC.

Flagellin A is the subunit protein that,together with flagellin B, polymerizes toform the bacterial filaments. The proteinwas reported in HP proteome by severalworks (31,43,60), as well as in the pro-teomes of some gastric disease–associ-ated HP strains (32). The important roleof flagellin A in both bacterial motilityand virulence is well documented (61).Since flagellin A was downregulated inthe only AAG-associated HP, we mayspeculate that a decrease in the virulenceof AAG-HP strains may partly be relatedto a decrease in motility.

The ATP synthase subunit α (or F-ATPase subunit α) is a regulatory sub-unit of a protein producing ATP fromadenosine 5′-diphosphate (ADP) in thepresence of a proton gradient across themembrane. Its occurrence in HP pro-teomes is also documented in some bac-terial strains associated with CG, GC andDU (24,62). In the pH range of 3.5–5.0,HP is known to maintain the proton mo-tive force (PMF) across its periplasmicmembrane, ensuring a continued supplyof energy through ATP synthesis (63). Inthe presence of a high local acid concen-tration, the protective mechanism fails tokeep up with hydrogen ion influx, ATPsynthesis declines and the bacteriumdies, or at least loses virulence (64,65).The lower content of F-ATPase found inAAG-related HP strains may be associ-ated with a decrease in their overall sur-vival rates.

These data all together seem to evi-dence that AAG-associated HP strainsmay be less motile and less able to main-tain their proton motive force than bothDU-HP and GC-HP. However, the nega-tive effect for HP survival may be com-pensated for by a higher capacity to neu-tralize the high local hydrogenconcentration found in HP isolated frompatients with AAG compared with con-centrations isolated from patients withDU or GC.

The remaining spots resulted in down-regulation in HP proteome associatedwith AAG compared with only the HPproteome of GC and corresponded to a60-kDa chaperonin or GroEL, an elonga-tion factor Tu, a ribosome-recyclingor -releasing factor, a probable peroxire-doxin or 26-kDa antigen or thioredoxinreductase, and four spots not identified.

The 60-kDa chaperonin or GroEL (orHSPB) is known as a highly abundantheat shock protein (66), which is also in-volved in inflammatory responses andautoimmunity (66) through the stimula-tion of inflammatory and gastric cellswith the production of interleukin (IL)-8cytokine (67).

Inflammation and IL-8 productionwere found to be higher in GC than in



normal gastric tissue (68) and directlycorrelate with angiogenesis (69). Thus,the down-expression of GroEL found inAAG might have a protective role in GCdevelopment by reducing inflammationand IL-8 cytokine production. In agree-ment with our data showing downregu-lation of GroEL in AAG-associated HPproteomes, Park et al. (62) reported thelowest contents of GroEL in patients withchronic gastritis, followed by DU andGC, with the highest levels being in HPfrom GC patients.

The elongation factor thermo unstable(EF-Tu) plays a central role during theselection of the correct amino acidsthroughout the elongation phase oftranslation (70). An additional propertyin interaction with the extracellular ma-trix of infected host cells was also pro-posed by Backert et al. (71). High levelsof EF-Tu expression in HP isolates fromGC patients have been reported (32,62).In our work, the lowest content of EF-Tuin the HP proteome associated with AAGwas accompanied by a low content of another protein involved in proteinbiosynthesis: the ribosome-recycling or -releasing factor, responsible for therelease of ribosomes from mRNA at thetermination of translation (72). This is thefirst time that this protein was found inthe proteome of HP associated with gas-tric diseases. Both downregulation of EF-Tu and releasing factor proteins sug-gest an overall lower level of proteinsynthesis of HP associated with AAGwith respect to HP isolated from GC.

Finally, one protein was specificallydownregulated in the HP proteome iso-lated from an AAG patient comparedwith the DU-isolated patient: the inor-ganic pyrophosphatase (spot 76). Thisenzyme converts one molecule of inor-ganic pyrophosphate into two phosphateions by a highly exergonic reaction. Thisprotein was identified in HP proteomeisolated from chronic gastritis, DU andGC (13,24,62), with a higher expressionin GC with respect to the proteome ofHP from patients with gastritis (73).

A pivotal role in HP-induced patho-genesis is played by the virulence fac-

tors included in the Cag PAI: CagA, thecytotoxin-associated protein translo-cated in gastric epithelial cells; CagE,the protein involved in IL-8 expression;the type IV secretion system, VirB11 (9);the vacuolating cytotoxin, VacA; and theouter-membrane protein, Hom (74). Inour patients, the contents of the proteinsof interest (spots 168, 166, 57, 40 and 42)did not seem to correlate overall withthe HP virulence or the atrophy grade.However, patient 4 (negative for all theCag PAI virulence genes and without at-rophy) appeared to behave differentlyfrom others for the downregulated spot40; this finding was hypothesized to bein some way related to the absence ofatrophy.

CONCLUSIONWe have successfully performed DIGE

differential proteomics analysis of HPstrains isolated from AAG patients andidentified some proteins that had notbeen characterized in AAG-HP before.The presence of some common versusdifferential proteins in AAG-HP versusDU- and/or GC-HP shows a certainlevel of different physiology of HP de-pending on the gastric disease. A higherantioxidant activity was found in AAG-associated HP strains, which were hy-pothesized to be less motile/virulentand able to neutralize the high local hy-drogen concentration, as well as to ac-complish protein biosynthesis and re-lated processes, in comparison with DU-or GC-associated HP. Some of the identi-fied proteins may provide some new in-formation on understanding the mecha-nism of the differential HP behavior inhuman stomach disease(s) and indicatepotential protein markers for the specificdetection of AAG-related HP. In particu-lar, it may be interesting to screen someof these found AAG-associated HP anti-gens (e.g., peroxiredoxin, chaperone pro-tein dnaK) from HP-affected patientsthrough cheap and fast technical ap-proaches based on noninvasive samples(e.g., feces) (study in course in our labo-ratory). Finally, further studies shouldbe conducted to confirm a specific func-

tional role of some identified proteins ofinterest in HP strains associated withAAG disease.

ACKNOWLEDGMENTSThis work was supported by Associ-

azione Italiana per la Ricerca sul Cancro(AIRC) grant #10266 (to V De Re); AIRCgrant #12214 (to R Cannizzaro); and theBio- Proteomics Core Facility, CRO Scien-tific Direction. We thank Bruno Bacherfor the use of DeCyder.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molec-ular Medicine, or other interests thatmight be perceived to influence the re-sults and discussion reported in thispaper.

REFERENCES1. Bettington M, Brown I. (2013) Autoimmune gas-

tritis: novel clues to histological diagnosis.Pathology. 45:145–49.

2. Miceli E, et al. (2012) Common features of pa-tients with autoimmune atrophic gastritis. Clin.Gastroenterol. Hepatol. 10:812–14.

3. Erdoðan A, Yilmaz U. (2011) Is there a relation-ship between Helicobacter pylori and gastric auto-immunity? Turk. J. Gastroenterol. 22:134–8.

4. Bergman MP, et al. (2005) The story so far: Heli-cobacter pylori and gastric autoimmunity. Int. Rev.Immunol. 24:63–91.

5. Correa P. (1996) Helicobacter pylori and gastriccancer: state of the art. Cancer Epidemiol. Biomark-ers Prev. 5:477–81.

6. Presotto F, et al. (2003) Helicobacter pylori infectionand gastric autoimmune diseases: is there a link?Helicobacter. 8:578–84.

7. Ferreira AC, et al. Helicobacter and gastric malig-nancies. Helicobacter. 1:28–34.

8. Blaser MJ, Atherton JC. (2004) Helicobacter pylori:persistence: biology and disease. J. Clin. Invest.113:321–33.

9. Ricci V, Romano M, Boquet P. (2011) Molecularcross-talk between Helicobacter pylori and humangastric mucosa. World J. Gastroenterol. 17:1383–99.

10. Costa AC, Figueiredo C, Touati E. (2009) Patho-genesis of Helicobacter pylori infection. Helicobac-ter. 14 (Suppl. 1):15–20.

11. Wen S, Moss SF. (2009) Helicobacter pylori viru-lence factors in gastric carcinogenesis. CancerLett. 282:1–8.

12. Rieder G, Fischer W, Haas R. (2005) Interaction ofHelicobacter pylori with host cells: function of se-creted and translocated molecules. Curr. Opin.Microbiol. 8:67–73.

13. Enroth H, Akerlund T, Sillén A, Engstrand L.



(2000) Clustering of clinical strains of Helicobacterpylori analyzed by two-dimensional gel elec-trophoresis. Clin. Diagn. Lab. Immuno. 7:301–6.

14. Proença-Modena JL, Acrani GO, Brocchi M.(2009) Helicobacter pylori: phenotypes, genotypesand virulence genes. Future Microbiol. 4:223–40.

15. Duncan SS, et al. (2013) Comparative genomicanalysis of east Asian and non-Asian Helicobacterpylori strains identifies rapidly evolving genes.PLoS One. 8:e55120.

16. Dong QJ, et al. (2012) Relatedness of Helicobacterpylori populations to gastric carcinogenesis.World J. Gastroenterol. 18:6571–6.

17. Dong QJ, Wang Q, Xin YN, Li N, Xuan SY. (2009)Comparative genomics of Helicobacter pylori.World J. Gastroenterol. 15:3984–91.

18. McClain MS, Shaffer CL, Israel DA, Peek RM Jr,Cover TL. (2009) Genome sequence analysis ofHelicobacter pylori strains associated with gastriculceration and gastric cancer. BMC Genomics.10:3.

19. Tomb JF, et al. (1997) The complete genome se-quence of the gastric pathogen. Helicobacter py-lori. Nature. 388:539–47.

20. Alm RA, et al. (1999) Genomic-sequence com-parison of two unrelated isolates of the humangastric pathogen Helicobacter pylori. Nature.397:176–80.

21. Gomceli I, Demiriz B, Tez M. (2012) Gastric car-cinogenesis. World J. Gastroenterol. 18:5164–70.

22. Gashi Z, Zekaj S, Haziri A, Bakalli A. (2011) Theinfluence of the type of ulcers in the degree of at-rophic gastritis. Medicinski Arhiv. 65:20–2.

23. Zhang Z. (2007) The risk of gastric cancer in pa-tients with duodenal and gastric ulcer: researchprogresses and clinical implications. J. Gastroin-test. Cancer. 38:38–45.

24. Mini R, et al. (2006) Comparative proteomics andimmunoproteomics of Helicobacter pylori relatedto different gastric pathologies. J. Chromatogr. BAnalyt. Technol. Biomed. Life Sci. 833:63–79.

25. Oleastro M, Monteiro L, Lehours P, Megraud F,Menard A. (2006) Identification of markers del-l’Helicobacter pylori strains isolated from childrenwith peptic ulcer disease by suppressive subtrac-tive hybridization. Infect. Immun. 74:4064–74.

26. Schmidt HM, et al. (2010) The cag PAI is intactand functional but HP0521 varies significantly inHelicobacter pylori isolates from Malaysia andSingapore. Eur. J. Clin. Microbiol. Infect. Dis.29:439–51.

27. Tomasini ML, et al. (2003) Heterogeneity of caggenotypes in Helicobacter pylori isolates fromhuman biopsy specimens. J. Clin. Microbiol.41:976–80.

28. Karnes WE Jr, et al. (1991) Positive serum anti-body and negative tissue staining for Helicobac-ter pylori in subjects with atrophic body gastritis.Gastroenterology. 101:167–74.

29. Kokkola A, et al. (2003) Spontaneous disappear-ance of Helicobacter pylori antibodies in patientswith advanced atrophic corpus gastritis. APMIS.111:619–24.

30. Simula MP, et al. (2010) PPAR signaling pathway

and cancer-related proteins are involved in celiacdisease-associated tissue damage. Mol. Med.16:199–209.

31. Franco AT, et al. (2009) Delineation of a carcino-genic Helicobacter pylori proteome. Mol. Cell. Pro-teomics. 8:1947–58.

32. Momynaliev KT, et al. (2010) Functional diver-gence of Helicobacter pylori related to early gastriccancer. J. Proteome Res. 9:254–67.

33. Oh JD, Kling-Bäckhed H, Giannakis M, EngstrandLG, Gordon JI. (2006) Interactions between gastricepithelial stem cells and Helicobacter pylori in thesetting of chronic atrophic gastritis. Curr. Opin.Microbiol. 9:21–7.

34. Ohata H, et al. (2004) Progression of chronic at-rophic gastritis associated with Helicobacter pyloriinfection increases risk of gastric cancer. Int. J.Cancer. 109:138–43.

35. McNamara LE, Dalby MJ, Riehle MO, BurchmoreR. (2010) Fluorescence two-dimensional differ-ence gel electrophoresis for biomaterial applica-tions. J R Soc. Interface. 6:S107–18.

36. O’Toole PW, Logan SM, Kostrzynska M, Wad-ström T, Trust TJ. (1991) Isolation and biochemi-cal and molecular analyses of a species-specificprotein antigen from the gastric pathogen Heli-cobacter pylori. J. Bacteriol. 173:505–13.

37. Kern R, Malki A, Holmgren A, Richarme G.(2003) Chaperone properties of Escherichia colithioredoxin and thioredoxin reductase. Biochem.J. 371:965–72.

38. Federico A, Morgillo F, Tuccillo C, Ciardiello F,Loguercio C. (2007) Chronic inflammation andoxidative stress in human carcinogenesis. Int. J.Cancer. 121:2381–6.

39. Stent A, Every AL, Sutton P. (2012) Helicobacterpylori defense against oxidative attack. Am. J.Physiol. Gastr. Liver Physiol. 302:G579–87.

40. Wang G, Alamuri P, Maier RJ. (2006) The diverseantioxidant systems of Helicobacter pylori. Mol.Microbiol. 61:847–60.

41. Backert S, et al. (2005) Subproteomes of solubleand structure-bound HP proteins analyzed bytwo-dimensional gel electrophoresis and massspectrometry. Proteomics. 5:1331–45.

42. Lock RA, Cordwell SJ, Coombs GW, Walsh BJ,Forbes GM. (2001) Proteome analysis of Heli-cobacter pylori: major proteins of type strainNCTC 11637. Pathology. 33:365–74.

43. Jungblut PR, et al. (2000) Comparative proteomeanalysis of H. pylori. Mol Microbiol . 36:710–25.

44. Cao P, McClain MS, Forsyth MH, Cover TL.(1998) Extracellular release of antigenic proteinsby Helicobacter pylori. Infect. Immun. 66:2984–6.

45. McAtee CP, et al. (1998) Identification of potentialdiagnostic and vaccine candidates of Helicobacterpylori by two-dimensional gel electrophoresis, se-quence analysis, and serum profiling. Clin. Diagn.Lab. Immunol. 5:537–42.

46. el Yaagoubi A, Kohiyama M, Richarme G. (1994)Localization of DnaK (chaperone 70) from Es-cherichia coli in an osmotic-shock-sensitive com-partment of the cytoplasm. J. Bacteriol. 176:7074–8.

47. Hoffman PS, Garduno RA. (1999) Surface- associated heat shock proteins of Legionella pneumophila and Helicobacter pylori: roles inpathogenesis and immunity. Infect. Dis Obstet.Gynecol. 7:58–63.

48. Osawa H, et al. (2001) Comparative analysis ofcolonization of Helicobacter pylori and glycol-ipids receptor density in Mongolian gerbils andmice. Dig. Dis. Sci. 46:69–74.

49. Hamma T, Adrian R, Ferré-D’Amaré AR. (2006)Pseudouridine synthases. Chem. Biol. 13:1125–35.

50. Ge J, Yu YT. (2013) RNA pseudouridylation: newinsights into an old modification. Trends Biochem.Sci. 338:210–8.

51. Shajani Z, Sykes MT, Williamson JR. (2011) As-sembly of bacterial ribosomes. Annu. Rev. Biochem.80:501–26.

52. Dunn BE, et al. (1997) Localization of Helicobacterpylori urease and heat shock protein in humangastric biopsies. Infect. Immun. 65:1181–8.

53. Pyndiah S, et al. (2007) Two-dimensional blue native/SDS gel electrophoresis of multiproteincomplexes from Helicobacter pylori. Mol Cell Pro-teomics. 6:193–206.

54. Kobayashi F, et al. (2011) Production of autoanti-bodies by murine B-1a cells stimulated with Heli-cobacter pylori urease through toll-like receptor 2signaling. Infect. Immun. 79:4791–801.

55. Huang CH, Chiou SH. (2011) Proteomic analysisof upregulated proteins in Helicobacter pyloriunder oxidative stress induced by hydrogen per-oxide. Kaohsiung J. Med. Sci. 27:544–53.

56. Ni J, Mei M, Sun L. (2012) Oxidative DNA dam-age and repair in chronic atrophic gastritis andgastric cancer. Hepatogastroenterology. 59:671–5.

57. Yonekura K, Maki-Yonekura S, Namba K. (2002)Growth mechanism of the bacterial flagellar fila-ment. Res. Microbiol. 153:191–7.

58. Godlewska R, Wisniewska K, Pietras Z, Jagusztyn-Krynicka EK. (2009) Peptidoglycan-associatedlipoprotein (Pal) of gram-negative bacteria: func-tion, structure, role in pathogenesis and potentialapplication in immunoprophylaxis. FEMS Micro-biol. Lett. 298:1–11.

59. Govorun VM, et al. (2003) Comparative analysisof proteome maps of Helicobacter pylori clinicalisolates. Biochemistry. 68:42–9.

60. Lee HW, Choe YH, Kim DK, Jung SY, Lee NG.(2004) Proteomic analysis of a ferric uptake regu-lator mutant of Helicobacter pylori: regulation ofHelicobacter pylori gene expression by ferric up-take regulator and iron. Proteomics. 4:2014–27.

61. Hopf PS, et al. (2011) Protein glycosylation in He-licobacter pylori: beyond the flagellins? PLoS One.6:e25722.

62. Park JW, et al. (2006) Quantitative analysis of rep-resentative proteome components and clusteringof Helicobacter pylori clinical strains. Helicobacter.11:533–43.

63. Meyer-Roseberg K, Scott DR, Melchers K, SachsG. (1996) The effect of environmental pH on theproton motive force of H. pylori. Gastroenterology.111:886–900.



64. Dixon MF. (2001) Pathology of gastritis and pep-tic ulceration. In: Helicobacter pylori: Physiologyand Genetics. Mobley HLT, Mendz GL, Hazell SL,Eds. Washington, DC: ASM Press.

65. Ferrero RL, Lee A. (1991) The importance of urease in acid protection for the gastric-colonis-ing bacteria H. pylori and H. felis sp. nov. Microb.Ecol. Health. Dis. 4:121–34.

66. Kansau I, Labigne A. (1996) Heat shock proteinsof Helicobacter pylori. Aliment Pharm. Ther. 1:51–6.

67. Yamaguchi H, et al. (1999) Induction of secretionof interleukin 8 from human gastric epithelialcells by heat-shock protein 60 homologue of Heli-cobacter pylori. J. Med. Microbiol. 48:927–33.

68. Yamaoka Y, et al. (2001) Relation between cy-tokines and Helicobacter pylori in gastric cancer.Helicobacter. 6:116–24.

69. Kitadai Y, et al. (1999) Transfection of interleukin-8 increases angiogenesis and tumorigenesis ofhuman gastric carcinoma cells in nude mice. Br.J. Cancer. 81:647–53.

70. Kavaliauskas D, Nissen P, Knudsen CR. (2012)The busiest of all ribosomal assistants: elongationfactor Tu. Biochemistry. 51:2642–51.

71. Backert S, et al. (2005) Gene expression and pro-tein profiling of AGS gastric epithelial cells uponinfection with Helicobacter pylori. Proteomics.5:3902–18.

72. Janosi L, Shimizu I, Kaji A. (1994) Ribosome re-cycling factor (ribosome releasing factor) is es-sential for bacterial growth. Proc. Natl. Acad. Sci.U. S. A. 91:4249–53.

73. Liu LN, et al. (2011) A comparison of proteomicanalysis of Helicobacter pylori in patients withgastritis and gastric cancer between areas of highand low incidence of gastric cancer. Beijing DaXue Xue Bao. 43:827–32.

74. Jung SW, Sugimoto M, Graham DY, Yamaoka Y.(2009) homB status of Helicobacter pylori as anovel marker to distinguish gastric cancer fromduodenal ulcer. J. Clin. Microbiol. 47:3241–5.

75. Park JW, et al. (2008) Proteomic analysis of Heli-cobacter pylori cellular proteins fractionated byammonium sulfate precipitation. Electrophoresis.29:2891–903.

76. Baik SC, et al. (2004) Proteomic analysis of the sar-cosine-insoluble outer membrane fraction of Heli-cobacter pylori strain 26695. J. Bacteriol. 186:949–55.

77. Cho MJ, et al. (2002) Identifying the major pro-teome components of Helicobacter pylori strain26695. Electrophoresis. 23:1161–73.

78. Sabarth N, et al. (2002) Identification of surfaceproteins of Helicobacter pylori by selective biotiny-lation, affinity purification, and two-dimensionalgel electrophoresis. J. Biol. Chem. 277:27896–902.

79. Bumann D, et al. (2002) Proteome analysis of se-creted proteins of the gastric pathogen Helicobac-ter pylori. Infect. Immun. 70:3396–403.

80. Krah A, et al. (2003) Analysis of automaticallygenerated peptide mass fingerprints of cellularproteins and antigens from Helicobacter pylori26695 separated by two-dimensional elec-trophoresis. Mol Cell Proteomics. 2:1271–83.

81. Yonezawa H, et al. (2011) Analysis of outer mem-brane vesicle protein involved in biofilm forma-tion of Helicobacter pylori. Anaerobe. 117:388–90.

82. Jungblut PR, et al. (2010) Helicobacter pylori pro-teomics by 2-DE/MS, 1-DE-LC/MS and func-tional data mining. Proteomics. 10:182–93.

83. Hynes SO, McGuire J, Wadström T. (2003) Poten-tial for proteomic profiling of Helicobacter pyloriand other Helicobacter spp. using a ProteinChiparray. FEMS Immunol. Med. Microbiol. 36:151–8.

84. Nilsson CL, Larsson T, Gustafsson E, KarlssonKA, Davidsson P. (2000) Identification of proteinvaccine candidates from Helicobacter pylori usinga preparative two-dimensional electrophoreticprocedure and mass spectrometry. Anal. Chem.72:2148–53.

85. Zhang YN, et al. (2011) Comparative proteomeanalysis of Helicobacter pylori clinical strains bytwo-dimensional gel electrophoresis. J. ZhejiangUniv. Sci. B. 12:820–27.

86. Khoder G, Yamaoka Y, Fauchère JL, Burucoa C,Atanassov C. (2009) Proteomic Helicobacter pyloribiomarkers discriminating between duodenalulcer and gastric cancer. J. Chromatogr. B. Analyt.Technol. Biomed. Life Sci. 877:1193–9.

87. Yamaoka Y, et al. (2006) Helicobacter pylori outermembrane proteins and gastroduodenal disease.Gut. 55:775–81.



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