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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(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-
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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
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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-
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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.
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Tab
le 2
.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
GC
.
Pro
tein
d
esc
ribe
dG
en
Ban
k Lo
ca
liza
tion
/bio
log
ica
l pro
ce
ss/
Sco
re/e
xpe
ct/
Fold
p
revi
ou
slySp
ot
nu
mb
era
MW
(D
a)/
pI
ac
ce
ssio
n n
o.b
Pro
tein
an
no
tatio
nm
ole
cu
lar f
un
ctio
nO
rga
nism
seq
. co
vera
ge
diff
ere
nc
ep
(re
f.)
HP
pro
tein
s up
reg
ula
ted
in A
AG
co
mp
are
d w
ith G
C
1327
557/
9.68
gi|
2380
5773
1tR
NA
pse
ud
ou
ridin
e
Cyt
op
lasm
/tR
NA
pro
ce
ssin
gH
P P
1232
/1.9
e +
002
/26
10.1
93.
54E-
03n
.d.c
syn
tha
se A
254
6644
/10.
96g
i|22
6703
094
50S
ribo
som
al p
rote
in L
301
Cyt
op
lasm
/ D
NA
tra
nsla
tion
/Le
pto
thrix
37
/71/
307.
154.
50E-
03(4
1)
pro
tein
syn
the
sisc
ho
lod
nii
(str
ain
ATC
C 5
1168
/
LMG
814
2/SP
-6)
206
n.i.
5.85
0.04
9
168
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
82/0
.001
9/52
5.69
3.28
E-04
(41–
43)
26-k
Da
an
tige
n o
r o
xid
ore
du
cta
se
thio
red
oxi
n re
du
cta
se
244
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
75/0
.009
8/41
2.94
4.56
E-03
(41–
43)
26-k
Da
an
tige
n o
r o
xid
ore
du
cta
se
thio
red
oxi
n re
du
cta
se
3361
816/
5.64
gi|
5701
4163
Ure
ase
βsu
bu
nit
Sec
rete
d a
nd
cyt
oso
lic/
HP
J99
102/
2.1e
-005
/46
2.5
0.02
5(2
4,42
,43,
53,
pa
tho
ge
ne
sis/u
rea
ca
tab
olic
62
,75–
77)
pro
ce
ss/h
ydro
lase
5767
136/
4.99
gi|
2267
3813
6C
ha
pe
ron
e p
rote
in d
na
K
Cyt
op
lasm
/pro
tein
fold
ing
/str
ess
H
P (
stra
in S
hi4
70)
32/1
.8e
+ 0
0 2/
82.
440.
0115
(24,
41)
or h
ea
t sh
oc
k 70
-kD
a
resp
on
se
pro
tein
or H
SP70
3461
846/
5.64
gi|
5701
4163
Ure
ase
βsu
bu
nit
Sec
rete
d a
nd
cyt
oso
lic/
HP
(st
rain
P12
)75
/0.0
099/
372.
365.
45E-
03(2
4,41
–43,
53,
pa
tho
ge
ne
sis/u
rea
ca
tab
olic
62
,75–
77)
pro
ce
ss/h
ydro
lase
3958
706/
8.70
gi|
2493
545
Ca
tala
seC
yto
pla
sm/h
ydro
ge
n p
ero
xid
e
HP
106/
8.1e
-006
/30
2.21
1.76
E-03
(24,
32,5
5,71
,
161
ca
tab
olic
pro
ce
ss/p
ero
xid
ase
/76
,78)
oxi
do
red
uc
tase
n.i.
2.2
8.87
E-03
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
R E S E A R C H A R T I C L E
M O L M E D 2 0 : 5 7 - 7 1 , 2 0 1 4 | R E P E T T O E T A L . | 6 3
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.
6 4 | R E P E T T O E T A L . | M O L M E D 2 0 : 5 7 - 7 1 , 2 0 1 4
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
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).
R E S E A R C H A R T I C L E
M O L M E D 2 0 : 5 7 - 7 1 , 2 0 1 4 | R E P E T T O E T A L . | 6 5
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-
6 6 | R E P E T T O E T A L . | M O L M E D 2 0 : 5 7 - 7 1 , 2 0 1 4
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
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).
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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
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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.
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