Functional Divergence of Helicobacter pylori Related to Early Gastric

Nov 4, 2009 - cases, H. pylori may progress to symptomatic chronic gastritis, gastric or duodenal ulcers, or gastric cancer (reviewed in refs. 4r6). I...
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Functional Divergence of Helicobacter pylori Related to Early Gastric Cancer Kuvat T. Momynaliev,*,† Sergey V. Kashin,‡ Vera V. Chelysheva,† Oksana V. Selezneva,† Irina A. Demina,† Marya V. Serebryakova,† Dmitry Alexeev,§ Vladimir A. Ivanisenko,| Ewgeniya Aman,| and Vadim M. Govorun†,§ Research Institute for Physico-Chemical Medicine, Moscow, Russia, Endoscopy, Yaroslavl Regional Oncologic Hospital, Yaroslavl, Russia, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia, and Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia Received July 04, 2009

Helicobacter pylori is an extra macro- and microdiverse bacterial species, but where and when diversity arises is not well-understood. To test whether a new environment accelerates H. pylori genetic changes for quick adaptation, we have examined the genetic and phenotypic changes in H. pylori obtained from different locations of the stomach from patients with early gastric cancer (ECG) or chronic gastritis (CG). Macroarray analysis did not detect differences in genetic content among all of the isolates obtained from different locations within the same stomach of patients with EGC or CG. The extent and types of functional diversity of H. pylori isolates were characterized by 2-D difference gel electrophoresis (2D DIGE). Our analysis revealed 32 differentially expressed proteins in H. pylori related to EGC and 14 differentially expressed proteins in H. pylori related to CG. Most of the differentially expressed proteins belong to the antioxidant protein group (SodB, KatA, AphC/TsaA, TrxA, Pfr), tricarbon acid cycle proteins (Idh, FrdA, FrdB, FldA, AcnB) and heat shock proteins (GroEL and ClpB). We conclude that H. pylori protein expression variability is mostly associated with microorganism adaptation to morphologically different parts of the stomach, which has histological features and morphological changes due to pathological processes; gene loss or acquisition is not involved in the adaptation process. Keywords: 2D-DIGE • H. pylori • DNA-macroarray • proteomics • cancer

Introduction Helicobacter pylori is a Gram-negative, microaerophilic, helical-shaped bacterium that colonizes the human stomach of at least half of the world’s population.1-3 H. pylori preferentially colonizes the antrum of the stomach, where acidproducing parietal cells are not present, and the environmental pH is higher than in the corpus. In most cases, H. pylori can persist in the human stomach asymptomatically, but in some cases, H. pylori may progress to symptomatic chronic gastritis, gastric or duodenal ulcers, or gastric cancer (reviewed in refs 4-6). Inflammatory progression in the gastric lining depends on environmental factors, host state and H. pylori-specific virulence factors. H. pylori is an extra macro- and microdiverse bacterial species. The high level of macrodiversity is supported by the fact that up to 25% of H. pylori genes are dispensable in at least one strain.7-10 The most unusual characteristic of the * To whom correspondence should be addressed: Kuvat Momynaliev, Research Institute for Physico-Chemical Medicine, Malaya Pirogovskaya 1A, 119992, Moscow, Russia. Phone: ++7-095-247-0846. Fax: ++7-095-247-0846. E-mail: [email protected]. † Research Institute for Physico-Chemical Medicine. ‡ Yaroslavl Regional Oncologic Hospital. § Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry. | Institute of Cytology and Genetics SB RAS.

254 Journal of Proteome Research 2010, 9, 254–267 Published on Web 11/04/2009

nucleotide sequence diversity of H. pylori is very high number of unique sequences for a given gene across the different strains. Kansau et al. in 1996 showed that, in a group of 29 strains, the nucleotide sequence of ureC gene is unique for every isolate.11 This finding has been confirmed for numerous different genes: cagA, vacA, flaA, flaB, cysS, ureI, trpC, and so forth.12-16 Nucleotide sequence diversity ranges from 5 to 10%. Most of the determined nucleotide changes are synonymous. It is widely assumed that allelic diversity in H. pylori is the result of natural competence, high recombination frequency and mutational processes. Moreover, intrastrain recombination of identical repeats or recombination between different alleles in the genome was suggested to contribute to the high intrahost strain diversity.17,18 In fact, H. pylori is considered to be extraordinarily diverse bacteria, and every individual harbors a distinctive bacterial population with clonal variants. Furthermore, H. pylori subclones isolated from the same biopsy or biopsies from different locations of the stomach differ in genotype and phenotype. However, Lundin et al. found far less variation and slower genetic divergence (single base pair substitution per 6.0 kb) of H. pylori strains during long-term colonization (over 9 years) than expected.19 For example, Falush et al. estimated the H. pylori recombination rate and calculated that 25 000 bp of H. pylori should be replaced annually.20 Thus, there is a paradox: 10.1021/pr900586w

 2010 American Chemical Society

H. pylori Functional Diversity diversity of H. pylori is extraordinary, but where and when it arises is not well-understood. H. pylori transmission may be polyclonal, with different clonal variants initially colonizing the human stomach, and the final H. pylori clonal variant may be the result of population divergence over several generations within the host.19 On the other hand, it is possible that a single clone may rapidly diverge within the niches of different stomachs to adapt to the new environment, and the rate of change dramatically increases when the niches are occupied. To test the last assumption (i.e., new environments accelerate H. pylori genetic changes for quick adaptation), we examined the genetic and phenotypic changes in H. pylori obtained from different stomach locations in patients with early gastric cancer (EGC). We propose that the given model allows comparison of H. pylori in the new environmental conditions that have appeared as result of intestinal dysplasia in antrum with H. pylori isolated from an unaltered area of the same stomach (e.g., fundus and corpus). Tumors from stomach antra were localized in mucosal and submucosal layers, and they were limited to 1 cm (new environment). The unaltered corpus and fundus had no visible morphological changes (old environment). H. pylori obtained from different locations of the stomach of patient with chronic gastritis (CG) was used as reference to be able to compare the levels of heterogeneity in altered part against unaltered parts between two distinct states, namely, EGC and CG. The early gastric cancer concept was first defined in 1962 by the Japan Society of Gastroenterological Endoscopy as an adenocarcinoma confined to the mucosa or submucosa irrespective of lymph node involvement.21 The need for such a definition was based on the observation that gastric cancer of this type had a favorable prognosis. Indeed, Saeki (1938) reported that patients who had gastric cancer confined to the submucosa had a five-year survival greater than 90%. Additionally, the divergence of H. pylori in early gastric cancer has not been described earlier. In this study, we used proteomic and genomic approaches to identify genetic and phenotypic changes in H. pylori obtained from different locations of the stomach from patients with early gastric cancer; chronic gastritis was used as a reference to compare the levels of heterogeneity on genomic and proteomic levels. Here, we report no detectable differences in genetic content among all of the isolates obtained from different locations within the same stomach of patients with EGC or CG. Two-dimensional difference gel electrophoresis (2D DIGE) analysis revealed 32 differentially expressed proteins in H. pylori related to EGC and 14 differentially expressed proteins in H. pylori related to CG. Most of the differentially expressed proteins belong to antioxidant, tricarbon acid cycle and heat shock protein families. Our results suggest that protein expression variability is mostly associated with H. pylori adaptation to the stomach mucosa, while gene loss or acquisition is not involved in the adaptation process.

Experimental Procedures Patients and Early Gastric Cancer Diagnosis. In total, six samples from two patients (men aged 63 and 67 years) undergoing gastroscopy in Yaroslavl Regional Oncologic Hospital were included in the study. The study protocol was approved by the medical ethics committee of the Research Institute for Physico-Chemical Medicine, and informed consent was obtained from participating patients. Patients fasted as per the routine upper gastrointestinal endoscopy protocol to clarify

research articles diagnosis. The first patient was suspected to have early cancer, and the second patient was suspected to have gastritis. The procedures were performed using Olympus endoscopes (GIF Q160Z) under conscious sedation with intravenous midazolam (2.5-5 mg), local anesthesia (6 mL of 2% oral xylocaine and 10% spray solution) and cardiopulmonary monitoring. We used indigocarmine (0.1% solution) to diagnose minute mucosal changes. Two biopsies from each sitesantrum [2 cm from pylorus (anterior and posterior walls)], corpus and fundusswere taken from each patient using Olympus biopsy forceps. The depth of involvement and the histological types of gastric cancer were determined histologically. EGC was defined as IIa + IIc type (Figure 1). It should be noted that fundus and corpus of stomach of the cancer patient have histological changes corresponding to “atrophic gastritis”. Bacterial Strain and Culture Conditions. H. pylori strains were isolated from endoscopic biopsy samples from different locations (fundus, corpus and antrum) of the stomach. The bacteria were cultured on a BBL Stacker plate (BD Biosciences) at 37 °C under microaerobic conditions. Liquid cultures were grown in flasks containing Brucella broth (Difco) supplemented with 10% fetal bovine serum (FBS; Invitrogen), vancomycin (12.5 mg/L; Sigma), and amphotericin B (2.5 mg/L; Sigma) with constant agitation at 150 rpm for 48-72 h. The culture medium was centrifuged for 10 min at 1000g, and the supernatant was filtered through a 0.2 µm filter (Pall; Ann Arbor, MI) to eliminate intact bacterial cells. Construction of the H. pylori DNA Macroarray. Primer sets for amplifying H. pylori ORFs were purchased from Eurogentec (Belgium). The primers were designed to amplify each ORF beginning at the start codon and ending at the stop codon of H. pylori 26695 strain. The PCR reactions were performed using 96-well plates with a 100 µL reaction volume. To control the yield and the specificity of the amplified ORFs, all PCR reactions were analyzed by electrophoresis on a 1% agarose gel. The 1571 PCR products successfully obtained (96.8%) were printed robotically (Qpix, Genetix, England) in 4 × 4 double offset configuration onto two 22 cm × 22 cm positively charged nylon membranes (Amersham) in duplicate. Before printing, 30 µL of each PCR product was diluted with 5.3 µL of 20× SSC. Thereafter, membranes were alkali-denatured by incubating on 3M paper (Whatman) soaked with 0.4 N NaOH and 3 M NaCl for 5 min, followed by neutralization on paper soaked with 6× SSC for 5 min and fixation by baking at 80 °C for 60 min. The membranes were stored dry at room temperature or used immediately for hybridization. All arrays included spots with different concentrations of fragmented genomic DNA of the H. pylori 26695 reference strain as a control. Preparation and Hybridization of Genomic DNA Probes. Genomic DNAs were labeled radioactively with [R-33P]dATP (3000 Ci/mmol) by random priming using the Megaprime DNA labeling system (Amersham Pharmacia Biotech). Reactions were carried out at 37 °C for 1 h and stopped by the addition of 0.05 M EDTA. Unincorporated nucleotides were removed by Sephadex chromatography (Sephadex G-50, Amersham). Prior to hybridization, arrays were prehybridized for 2 h at 55 °C with 30 mL of 1× hybridization buffer (5× SSC, pH 7.5, 5× Denhardt’s solution, 0.5% SDS). Hybridization was carried out in 5 mL of 1× hybridization buffer containing 5.5 × 106 cpm/ mL DNA for 20 h at 55 °C. Posthybridization, the DNA macroarrays were washed in 2× SSC containing 0.1% SDS, then incubated in 2× SSC containing 0.1% SDS for 20 min at 55 °C, and finally incubated in 0.2× SSC containing 0.1% SDS for 1 h Journal of Proteome Research • Vol. 9, No. 1, 2010 255

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Figure 1. Chromoendoscopy and histology of isolated biopsies from the stomach of a patient with EGC. To diagnose minute mucosal changes, indigocarmine 0.1% solution was used. Biopsies were taken from EGC and mucosa close to the tumor area using Olympus biopsy forceps. Two biopsies from each sitesthe antrum [2 cm from pylorus (anterior and posterior walls)], the corpus and the fundusswere taken from each patient. The depth of involvement and the histological types of gastric cancer were determined histologically. EGC was defined as IIa + IIc type. (A) Before dying (contrast method, indigocarmine 0.2%). (B) After dying. Photomicrograph of stomach (C) Hematoxylin and Eosin ×100, and (D) Hematoxylin and Eosin ×200.

at 55 °C. The arrays were exposed to storage phosphor screens (Molecular Dynamics; Sunnyvale, CA) for 48 h and scanned with a Storm 820 PhosphorImager (Molecular Dynamics) at a 50 µm/pixel resolution and a color depth of 16 bits. For each clinical isolate, genomic DNAs were prepared from two independent cultivations and then used for two independent genomic DNA labelings and DNA array hybridizations. Data Analysis. The image files were analyzed using ArrayVision software (Imaging Research, St. Catharines, Ontario, Canada). A template that contains the spot layout of the array was overlaid on the phosphorimage, and the pixel intensity of each spot on the array was determined. The backgroundcorrected pixel intensities for each spot were exported from ArrayVision into ArrayStat (Imaging Research) for statistical analysis. These values were logarithmically transformed, the common error was determined, and outliers were removed. The values were then normalized by the mean across conditions by an iterative process. The z test for two independent conditions using the false-discovery rate method (nominal alpha, P e 0.01) was used as the basis for computing confidence intervals. The cutoff for absence of a gene in an H. pylori strain 256

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was defined as normalized ratio of strain 26695/experiment >2.0. With this cutoff, we obtained 2% false positives and 3% false negatives. The cutoff for missing genes was determined empirically based on PCR scanning of absent genes in H. pylori clinical isolates. For PCR analysis, primers were designed to amplify conservative regions of genes based on two available genomes: strains 26695 and J99. The validation experiments confirmed the predicted absence in all cases. For cluster analysis, the results of genome scanning were presented as a binary score corresponding to the presence or absence of a gene, analyzed by average hierarchical clustering using the Cluster program, and displayed with Treeview (http://www.microarrays.org/software.html).22 The complete data set used is available at http://www.ripcm.org.ru/2_1/2/2_5/index.php. Multilocus Sequence Typing (MLST) of H. pylori Isolates. Intragenic regions of seven genes (atpA, efp, mutY, ppa, trpC, ureI and yhpC) were targeted and sequenced as previously described.16 Phylogenetic analyses were conducted using MEGA version 3.1.23

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H. pylori Functional Diversity Two-Dimensional Difference Gel Electrophoresis (2D DIGE). Cell pellets (10 µL) were dissolved in buffer: 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM TrisHCl, pH 8.5. Protein concentration was determined using Quick-Start Bradford dye reagent (Bio-Rad) and standardized to 2 mg/mL. A total of 50 µg of the sample proteins was labeled with 400 pmol of either Cy3 or Cy5 CyDye DIGE Fluor minimal dyes (Amersham Biosciences, Austria) according to the manufacturer’s instructions. After protein equalization, Cy3 and Cy5 labeled samples were mixed, and 100 mM DTT (Bio-Rad) and 2% (v/v) Ampholine 3-10 (Bio-Rad) were added. Isoelectric focusing IEF was performed using tube gels (20 cm × 1.5 mm) containing carrier ampholytes and applying a voltage gradient in an IEFchamber produced in house.24 After IEF, the ejected tube gels were incubated in equilibration buffer (125 mM TrisHCl, 40% (w/v) glycerol, 3% (w/v) SDS, 65 mM DTT, pH 6.8) for 10 min. The tube gels were placed on 20 cm × 18 cm × 1.5 mm polyacrylamide gels (9-16%) (Protean II Multi-Cell, Bio-Rad) and were fixed using 1.0% (w/v) agarose containing 0.01% (w/ v) bromphenol blue. Electrophoresis was carried out for 12-14 h, and the gels were scanned using a Typhoon Trio Imager at 200 dpi resolution (Amersham Biosciences, Austria). The gels were subsequently fixed and silver stained as described by Shevchenko et al.25 Image analysis was performed using PDQest software (Bio-Rad). Two independent experiments were performed for each experimental setup. Spot quantities were normalized to remove nonexpression-related variations in spot intensity. Differential expression of a certain protein between the two subsets was defined as at least a 2-fold change in spot optical density between the two matched sets in duplicates. All these spots were selected for MALDI- MS analysis. Trypsin Digestion and Mass Spectrometry. The protein spots were excised from the gel and digested with trypsin. The silver-stained protein spots (1-2 mm3) were destained with 20 µL of 15 mM potassium ferricyanide and 50 mM sodium thiosulfate solution and were washed twice with 100 µL of Millipore-Q water. The gel pieces were dehydrated with 40 µL acetonitrile (ACN) and rehydrated with 2 µL of digestion solution containing 20 mM ammonium bicarbonate and 10 ng/ µL sequencing grade trypsin (Promega, Madison, WI). Digestion was carried out overnight at 37 °C. Peptides were extracted with 4 µL of 0.5% trifluoroacetic acid (TFA) solution. To get the peptide mass fingerprint, 2 µL of extract was mixed with 0.5 µL of 2,5-dihydroxybenzoic acid saturated solution in 20% ACN and 0.5% TFA on the stainless steel MALDI sample target plate. Mass spectra were recorded on an Ultraflex II MALDI-TOF/ TOF mass spectrometer (Bruker Daltonics, DE) equipped with an Nd laser (354 nm). The MH+ molecular ions were detected in reflecto-mode in the mass range 700-4000 m/z. The accuracy of mass peak measurement after internal calibration with the peaks of trypsin autolysis was 0.01%. Peaklist generation was performed with flexAnalysis 2.4 software package from Bruker Daltonik. Protein identification was performed with the help of Mascot software release version 2.1.0 (Matrix Science, London, U.K.) and the full genome database of H. pylori 26695 (www.tigr.org). Partial oxidation of methionine and propionamidomethylation of cysteine residues was assumed for all peptide mass fingerprint searches. Protein scores greater than 44 were considered as significant (p < 0.05). All proteins were manually validated (Supplementary Table S1). Real-Time PCR. Real-time PCR was used to verify data obtained by 2D DIGE. Total RNA from H. pylori strains was extracted using a commercial kit (RNAqueous_4PCR purifica-

tion kit; Ambion). The quantity and quality of the extracted RNA were checked by microcapillary electrophoresis using a Bioanalyzer 2100 (Agilent Technologies; Wilmington, DE). The reverse transcription reaction was carried out as follows. RNA (1 µg) was initially denatured at 94 °C for 30 s in 10 µL of a mixture containing NTPs (0.5 mM each) and 150 pmol of random hexamer primers. For primer annealing, the mixture was incubated in ice for 30 min. After this incubation, 20 U RNase inhibitor (Promega), 200 U MMLV reverse transcriptase (Promega), and reverse transcription buffer were added. The resultant mixture was incubated at 37 °C for 30 min. PCR was carried out in a 50 µL volume containing 10 pmol of primers (Supplementary Table S2), 0.003% SybrGreen, 25 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2, 50 mM KCl, a mixture of four types of dNTP (0.2 mM each), and 2 U Taq-polymerase. PCR products were amplified and detected using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) and MicroAmp Optical 96-well reaction plate with optical caps (Applied Biosystems) in the following mode: denaturation at 94 °C for 2 min followed by 40 cycles of 93 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s. Amplification results were treated using Sequence Detection System software version 1.6 (Applied Biosystems). Reconstructing Molecular-Genetic Interaction Networks for H. pylori Differentially Expressed Proteins. Molecular-genetic interactions were reconstructed using the AND (Associative Network Discovery) computer system.26

Results Multilocus Sequence Typing (MLST) H. pylori Isolates. To determine whether the same strain persisted in the stomach of the patient with either EGC or CG, we obtained five colony isolates from distinct biopsy sites (i.e., antrum, corpus, fundus). For identification of H. pylori isolates, we used MLST. DNA sequence comparison of seven housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI and yhpC) showed 100% identity of all H. pylori isolates from one stomach (Supplementary Figure S1). The nucleotide sequence of housekeeping genes also differed for H. pylori isolates from patients with EGC and CG. Because H. pylori isolates from one stomach represent a closely related population, we chose three clones for each of six H. pylori strains for further exploration. Differential Protein Expression of HPEGC and HPCG Isolates. To identify differentially expressed proteins in H. pylori isolates, 2-D DIGE was used (Figure 2). To exclude nonbiological variation, cytoplasmic proteins were extracted from three clones from each of the six strains. Single protein expression was considered significant if the fluorescence intensity for individual spots differed more than 2-fold compared to the corresponding spot on other gels. Analysis of HPCG strains (HPCG-A, antrum strain H. pylori; HPCG-F, fundus strain H. pylori and HPCG-C, corpus strain H. pylori) revealed 14 differentially expressed proteins, 12 of which were identified (Table 1). In HPEGC (HPEGC-A, antrum strain H. pylori; HPEGC-F, fundus strain H. pylori and HPEGCC, corpus strain H. pylori) strains, we found 32 differentially expressed proteins and identified 18 of them (Table 2). In total, we identified 28 variable proteins in all the H. pylori isolates. Because the protein concentrations for all samples were equal (2 mg/mL), comparative analysis for HPCG and HPEGC strains was carried out. Statistically significant (p < 0.05) differences in spot intensity were found for PepA, KatA, FrdA, ClpB, HylB, TrxA, FusA UreG, HP0204 and HP1037 (Figure 2). Journal of Proteome Research • Vol. 9, No. 1, 2010 257

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Figure 2. 2-D DIGE analysis of H. pylori isolates. Protein concentration was determined using Quick-Start Bradford dye reagent (Bio-Rad) and standardized to 2 mg/mL. A total of 50 µg of sample proteins was labeled with 400 pmol of either Cy3 or Cy5 CyDye DIGE Fluor minimal dyes (Amersham Biosciences, Austria). IEF was performed using tube gels (20 cm × 1.5 mm) containing carrier ampholytes and applying a voltage gradient in an IEF-chamber produced in house. After IEF, the ejected tube gels were incubated in equilibration buffer [125 mM TrisHCl, 40% (w/v) glycerol, 3% (w/v) SDS, 65 mM DTT, pH 6.8] for 10 min. The tube gels were placed onto the 20 cm × 18 cm × 1.5 mm polyacrylamide gels (9-16%) (Protean II Multi-Cell, Bio-Rad) and fixed using 1.0% (w/v) agarose containing 0.01% (w/v) bromphenol blue. Electrophoresis was carried out for 12-14 h, and the gels were scanned using a Typhoon Trio Imager at 200 dpi resolutions (Amersham Biosciences, Austria). Image analysis was performed using PDQest software (Bio-Rad). Two independent experiments were performed for each experimental setup. All the spots for differentially expressed proteins were selected for MALDI-MS analysis. H. pylori isolates related to early gastric cancer were obtained from different locations within the stomach, (A) fundus (Cy5) and antrum (Cy3) strains; (B) corpus (Cy5) and antrum (Cy3) strains; (C) corpus (Cy5) and fundus (Cy3) strains; and H. pylori isolates related to gastritis, (D) fundus (Cy3) and antrum (Cy5) strains; (E) corpus (Cy3) and antrum (Cy5) strains; and (F) corpus (Cy5) and fundus (Cy3) strains 258

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H. pylori Functional Diversity Table 1. Differentially Expressed Proteins of H. pylori Isolates Related to CG no.

1 2 3 4 5 6 7 8 9 10 11 12

a

protein name

locus_tag

molecular mass (Da)

pI

score

ratio HPCG-F/HPCG-A

Chaperone and heat shock protein (GroEL) Heat shock protein (ClpB) Hypothetical protein Putative neuraminyllactose-binding hemagglutinin homologue (HpaA) Hemolysin secretion protein precursor, (HylB) Flagellin A (FlaA) Nonheme iron-containing ferritin (Pfr) Trigger factor (Tig) Conserved hypothetical protein Translation elongation factor EF-G (FusA) Translation elongation factor EF-Tu (TufB) Alkyl hydroperoxide reductase (TsaA)

HP0010 HP0264 HP0318 HP0410

58228 96670 28489 28331

5.55 6.01 7.27 7.88

135 120 134 88

3.2

HP0599 HP0601 HP0653 HP0795 HP1037 HP1195 HP1205 HP1563

48331 53252 19274 51956 40771 76972 43620 22221

5.85 6.04 5.4 5.33 5.82 5.25 5.17 5.88

97 67 104 109 38 100 104 63

6.3 5.5

2

8.7 2.2 2 2.1

ratio HPCG-C/HPCG-A

0.3 3.1 0.3 4.4 3.4 0.5 6.5 2.2 0.5 2.7

a

Protein signal intensities of H. pylori from corpus and fundus were normalized to the signal intensities of corresponding proteins from H. pylori from antrum.

Table 2. Differentially Expressed Proteins of H. pylori Isolates Related to EGCa no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

protein name

locus_tag

molecular mass (Da)

pI

Alkyl hydroperoxide reductase (TsaA) Fumarate reductase, iron-sulfur subunit (FrdB) S-adenosylmethionine synthetase (MetX) Fumarate reductase flavoprotein subunit (FrdA) Nonheme iron-containing ferritin (Pfr) Hypothetical protein Superoxide dismutase (SodB) Conserved hypothetical protein Chaperone and heat shock protein (GroEL) Isocitrate dehydrogenase (Icd) Cinnamyl-alcohol dehydrogenase ELI3-2 (Cad) GTP-binding protein, fusA-homologue (YihK) Translation elongation factor EF-G (FusA) Heat shock protein (ClpB) Aconitate hydratase 2 (Citrate hydro-lyase 2) (AcnB) NADH dehydrogenase gamma subunit (NQO3) Flavodoxin (FldA) Ribosomal protein S1 (RpsA)

HP1563 HP0191

22221 27620

5.88 5.34

43 61

HP0197 HP0192 HP0653 HP0318 HP0389 HP1037 HP0010 HP0027 HP1104 HP0480 HP1195 HP0264 HP0779 HP1266

42330 80070 19274 28489 24602 40771 58228 47501 38621 66634 76972 96670 92604 94169

6.04 6.87 5.4 7.27 5.77 5.82 5.55 7.6 6.96 5.3 5.25 6.01 6.17 5.32

70 100 104 134 115 38 63 145 102 108 100 120 134 105

3.3 0.2 5.1 4.2 2.1

HP1161 HP0399

17482 62788

4.45 6.33

56 105

2.1 3.2

score

ratio HPEGC-F/HPac-A

ratio HPEGC-C/HPac-A

vv 0.2

2.6 2 3.6 2.3

0.2 2.1

0.4 2.5

0.5 3 2.9 3.1

a Protein signal intensities of H. pylori from corpus and fundus were normalized to the signal intensities of corresponding proteins of H. pylori from antrum.

Intensities of UreG, ClpB, HylB and HP1037 were higher in HPEGC isolates compared to HPCG. Proteins Expressed Only in HPCG or HPEGC Isolates. Idh, FrdB, MetX, SodB and Cad protein expression was identified only in HPEGC strains (Figure 3). FlaA expression was found only in HPCG isolates. Moreover, AhpC/TsaA spots were not found in HPEGC-A or HPEGC-F isolates, but the spot intensity was significant in HPEGC-C (spot intensity -3371). To test whether this difference is caused by pI shift due to amino acid sequence differences, we sequenced icd, sod, cad and tsaA genes in all given isolates (Supplementary Table S3). Nucleotide sequences of four genes were identical in both HPEGC and HPCG isolates. Comparing the amino acid sequences of Icd, Sod, Cad and TsaA proteins in HPEGC and HPCG revealed that isocitrate dehydrogenase and alkyl hydroperoxide reductase differ only in one amino acid substitution, which does not affect the charge. Therefore, the absence of the corresponding spots on 2-DE gels in HPCG isolates is not induced by amino acid polymorphisms. Amino acid substitutions in Sod and Cad resulted in a charge shift, and this possibly

led to an absence of these spots in the corresponding positions on 2-DE gels of HPCG when compared to HPEGC isolates (Figure 3). Using gene-specific PCR, we showed that genes coding Icd, FrdB, MetX, SodB, FlaA and Cad are present in both HPEGC and HPCG isolates. Real-time PCR showed (Figure 4) an absence of mRNA only for the icd gene, while it was present for all other genes. FrdB, MetX, SodB, FlaA and Cad proteins may be subject to additional post-translational modifications in HPCG isolates, which may be the reason for the shift on 2-DE gels. Expression of H. pylori Proteins from Different Stomach Locations. To estimate the protein expression level for H. pylori, strains isolated from different stomach locations from patients with EGC or CG, protein signal intensities of H. pylori from the corpus and fundus were normalized to the signal intensities of corresponding proteins from the antrum. We found increased expression of six proteins in both HPCG-F and HPCG-C isolates (Table 1). The ClpB, HpaA, Pfr and FusA expression decreases in HPCG-C isolates only. For HPEGC isolates (Table Journal of Proteome Research • Vol. 9, No. 1, 2010 259

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Figure 3. Magnified images of corresponding zones on 2-DE gels of HPEGC (left) compared to HPCG (right). (A) absence of FlaA in HPEGC; (B) absence of SodB in HPCG.

2), we found only two proteins that significantly changed in expression level in two (HPEGC-F and HPEGC-C) isolates simultaneously. In addition, a majority (12 of 18) of highly expressed proteins were found only in HPEGC-F isolates in contrast to HPCG isolates. Genomic Differences between HPEGC and HPCG Isolates. For each H. pylori isolate, four hybridizations were performed (two independent cultivations and two independent labelings). These data were compared to a whole-genome DNA macroarray hybridization of H. pylori 26695. A signal ratio of 1 indicated the presence of gene in H. pylori strains. A signal ratio 26695/(EGC or CG) strains >2.0 was interpreted as evidence of loss of the respective gene. Whole-genome genotyping with our H. pylori DNA macroarray revealed 1394 genes that were present in all of the isolated H. pylori from both patients. The number of strain-specific genes for H. pylori strains related to gastritis and EGC were 18 and 80, respectively. Genes present only in HPEGC strains, which we defined as EGC-related (EGC), are given in Supplementary Table S4 (41 hypothetical genes are not shown). Cag PAI genes are present in all HPEGC strains regardless of location within the stomach. Additionally, in HPEGC strains, we found genes coding transposon-related functions (six genes) and restriction-modification system components (four genes). The genes coding transposon-related functions are not unique for the HPEGC strains because they were also found in HPCG isolates (IS 200 insertion sequence from SARA17, tnpA, tnpB, PS3IS). Additionally, two adeninespecific DNA methyltransferases (HP0260 and HP0481) were found in HPEGC strains. DNA hybridization revealed several differences in the genetic content of HPEGC-C, HPEGC-F and HPEGC-A isolates. We identified four variable genes: three hypothetical (HP0442, HP0704, HP0968) and mobA (molybdopterin-guanine dinucle260

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otide biosynthesis protein A). PCR analysis of above genes confirmed that the HPEGC-C isolate lacks HP0442 genes, the HPEGC-A isolate lacks HP0704, HP0968, mobA, and all four genes present in HPEGC-HP. Five variable genes were found in HPCG-C, HPCG-F and HPCG-A strains: methyl-accepting chemotaxis transducer (tlpC) and four hypothetical (HP0008, HP0061, HP0086 and HP0429). All five genes are present in the HPCG-F strain, three (tlpC, HP0061, HP0086) in HPCG-A, and two (HP0008, HP0429) in HPCG-C. Molecular-Genetic Interaction Networks of H. pylori Differentially Expressed Proteins. Molecular-genetic networks of the H. pylori differentially expressed proteins from patients with EGC and CG were reconstructed using the AND (Associative Network Discovery) computer system.26 The core of associative network of differentially expressed proteins from HPCG isolates (Figure 5A) is formed by the molecular chaperones (GroES, GroEL, DnaK, TiG, ClpB). They constitute a closely related group with proteins and genes involved in replication (SsB), transcription (RpoBC), export (SecA), proteolysis (HP0264) and other processes. A separate node is composed of proteins interacting with thioreoxine reductase (TsaA) and thioredoxine (ThiO). This cluster is bound to the chaperone core by enzymes (malatdehydrogenase and citrate synthase). An additional cluster in networks is formed by elongation factor proteins and genes (EftU, EfG, EftS, Ef2(HP1195). A subnetwork of flagellin A (FlaA), flagellar adhesin (HpaA) and ClpB chaperon also includes CagA (cytotoxine associated protein), FeoB (ferrous iron transporter), UvrA (component of UvrABC system, responsible for DNA reparation), RpoBC (DNA-dependent RNA polymerase), Rpl15 (50s ribosomal protein) and VacA (cytotoxin). The associative network for differentially expressed proteins for HPEGC isolates comprises most of the proteins, genes and

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Figure 4. Real-time PCR. cDNA encoding periplasmic carbonic anhydrase, fumarate reductase, iron-sulfur subunit, S-adenosylmethionine synthetase, isocitrate dehydrogenase, superoxide dismutase and flagellin A (flaA) from typical experiments of the proteome analysis of H. pylori strains related to EGC and gastritis were subjected to PCR amplification. The lines represent amplifications from cDNAs generated from the RNAs that were isolated from HPEGC and HPCG strains.

associations from gastritis associative network; however, there are few new proteins (Figure 5B), such as fumarate reductase (FrdA), aconitase (AcoN2), isocitrate dehydratase (Idh), superoxiddismutase (SodF) and methionine adenylyltransferase (MetK). Thus, the associative network of differentially expressed proteins and genes for early cancer includes group of chaperons from gastritis network on the one hand and, on the other hand, contains enzymes taking part in the citric acid cycle, antioxidant defenses and amino acid metabolism.

Discussion H. pylori is considered to be extra diverse bacteria, and almost every individual harbors a distinctive bacterial popula-

tion with clonal variants of unprecedented genetic diversity.17 One of the suggested scenarios for the variability generation in H. pylori is that a single H. pylori clone may rapidly diverge within the new niches of stomach to adapt to the new environments. When the niches are occupied, the rate of change dramatically decreases.19 Because H. pylori is acquired during childhood by intrafamilial transmission, monitoring H. pylori changes over a prolonged period and regular H. pylori sampling is not ethically feasible.27 To explore the issue that a new environment accelerates H. pylori genetic changes for quick adaptation, we have examined the genetic and phenotypic changes in H. pylori obtained from different locations of the stomach from patients with early gastric cancer. Although Journal of Proteome Research • Vol. 9, No. 1, 2010 261

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Figure 5. The associative network of differentially expressed proteins of H. pylori from patients with CG (A) and EGC (B). Molecular-genetic networks were reconstructed using the AND (Associative Network Discovery) computer system.26 Different types of objects are marked by different icons: cylinders, gene; circles, protein; octagons, association.

early experiments had shown that H. pylori transmission to a new host did not induce rapid genetic changes in the bacterial population,27 this model may not be adequate because a laboratory strain of H. pylori BCS-100 (ATCC BAA-945) was used for experimental human infection. BCS-100 probably already has a restricted diversity after the first strain isolation and host selection. On the contrary, our given model allows us to estimate the degree of H. pylori functional and structural divergence inside the given stomach (without experimental infection) during pathological changes. In this work, we 262

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characterized H. pylori in the new environmental conditions that have appeared as a result of intestinal dysplasia in the antrum and isolated H. pylori from unaltered area of the same stomach (i.e., fundus and corpus). Macroarray experiments with the HPEGC and HPCG strains showed that only HPEGC isolates contain cag PAI, a major genetic determinant of H. pylori virulence.28 The cag PAI encodes a type IV secretion system and an immunodominant antigen, CagA, which is translocated into gastric epithelial cells. In comparison to infection with cag PAI-negative H. pylori

H. pylori Functional Diversity strains, infection with cag PAI-positive strains is associated with an increased severity of gastric mucosal inflammation, an increased risk for development of peptic ulceration, and an increased risk of gastric cancer.28 Moreover, HPEGC isolates contained intact cag PAI genes, which are associated with the development of more severe pathology than the partially deleted cag PAI.29 Strains lacking the cagT (HP0532) gene had a defective ‘molecular syringe’ encoded by the PAI,30 reflecting the inability of this Type IV system to eject the cagA protein. The cagE (HP0544) gene, on the other hand, is known to induce NF-κB activation and IL-8 secretion31 in addition to mediating host-cell cytokine rearrangements in infected epithelial cells. Additionally, we found the iceA gene (genotype iceA1) in EGCrelated H. pylori isolates, the expression of which is upregulated by contact with epithelial cells.32 In some, but not all, populations,32,24 the iceA1 genotype is associated with gastric ulcers. DNA analysis indicates that iceA1 has strong homology to nlaIIIr,35,36 which encodes a CATG-specific endonuclease. Among a set of genes specific to EGC-related H. pylori isolates, our analysis revealed adenine-specific DNA methyltransferases (HP0260 and HP0481). Bacterial DNA adenine methylase regulates a variety of functions, including chromosome replication, transcription and DNA repair.37 Recently, DNA adenine methylase (Dam), which methylates the N-6 of adenine in GATC sequences, has been reported to play a key role in virulence regulation of several bacterial pathogens. Dam- Salmonella typhimurium displays reduced M-cell cytotoxicity and reduced invasion in tissue culture.38 In an animal model, these mutants colonize Peyer’s patches but fail to invade enterocytes or cause disease. It has also been reported that Dam is required for expression of virulence genes in Yersinia pseudotuberculosis and Vibrio cholerae. Adenine-specific DNA methyl-transferases in HPEGC strains may be associated with EGC. According to Israel et al., there is no specificity of gene loss in the H. pylori J99 strain obtained from different gastric locations.40 The total number of ORFs lost by at least one of the recent J99 isolates represents 2.3% of the J99 ORFs assayed, and each individual isolate had lost between 0.28 and 1.52% of ORFs. Our macroarray analysis showed that the number of variable genes in H. pylori isolates obtained from distinct biopsy sites in one patient was not more then five in both clinical cases. In addition, the sequence of genes displaying different intensities on DNA macroarrays showed that all H. pylori isolates from one stomach are identical (data not shown). Slow nucleotide divergence was also mentioned by Lundin et al.19 It is unlikely that the detected genetic macro-diversity of H. pylori isolates reflects the adaptation to local environmental changes during inflammation or carcinogenesis of the intestinal mucosa. In this study, we characterized the extent and types of functional diversity of H. pylori isolates that appear during EGC of the human stomach. Fine quantitation is a critical step in identifying the proteins associated with or responsible for the biological phenotype. Systematic variability of traditional 2-DE is significant due to the lack of reproducibility between gels, which makes it difficult to distinguish between system variation and induced biological change. To minimize protein variations due to system variation, we used 2D DIGE (reviewed in ref 41). The most important advantage of 2D DIGE is the ability to run multiple prelabeled samples on the same 2D gel with a high dynamic range. This eliminates the integral variability, common to standard 2D electrophoresis, thereby increasing the repro-

research articles ducibility and accuracy of results. 2D DIGE also allows normalization of spot intensities in one gel, thereby minimizing gel-specific variations. In our work, individual protein expression in a given pair samples was considered significantly changed if fluorescence intensity differed more than 2-fold. Furthermore, we used three clones from each of three H. pylori isolates (obtained from antrum, corpus and fundus) to exclude nonbiological variations. Gene expression of H. pylori during in vitro culture is dependent on the growth phase.42,43 While 80% genes of H. pylori were either constitutively expressed or were not expressed at all during batch culture, the expression of 325 of 1590 genes was significantly changed (e.g., more then 2-fold). Analysis of the expression profiles highlighted a major switch in gene expression during the late log-to-stationary phase transition.42 Among the genes that were significantly induced or repressed during the log-to-stationary phase transition, many of these genes were related to virulence; these include the cag PAI genes (cagA and cag1), the neutrophilactivating protein napA, the major flagellin flaA, and OMP genes (omp5, omp29, omp11, and hopA). With regard to these data, 2D DIGE experiments were performed only with H. pylori strains from the middle of the log phase (36 h) after the fifth passage on solid media after biopsy isolation. 2D DIGE analysis revealed 32 differentially expressed proteins in H. pylori related to EGC and 14 differentially expressed proteins in H. pylori related to CG (Figure 2). The degree of functional diversity may reflect the state of the stomach mucosa and submucosa. The functional diversity in both cases was found in the context of nucleotide homogenity of H. pylori isolates identity within the same stomach. We assume two processes of microorganism adaptation: (1) H. pylori adaptation to morphologically different parts of the stomach antrum, corpus and fundus and (2) H. pylori adaptation to pathological processes (neoplastic process vs chronic inflammation) in the stomach mucosa. To examine these assumptions, we compared dependency of the expression profiles from H. pylori localization within the stomach. The fundus and corpus, which are hardly histologically separable, contain large amounts of parietal cells that produce gastric acid (hydrochloric acid) and peptic cells that release pepsinogen, gastric lipase and rennin. The antrum of the stomach does not contain acid-producing parietal cells, which results in a less acidic environment than in the corpus and fundus. Our analysis indicated that correlation of expression profiles for H. pylori obtained from corpus and fundus (r ) 0.89 for EGC and r ) 0.99 for CG) is somewhat higher than antrum to fundus and corpus isolates (r ) 0.85 for EGC and r ) 0.90 for CG). Moreover, the expression profile of HPEGC-A strain is more similar to HPCG-A (r ) 0.89) than to H. pylori isolates from the same stomach: HPEGC-F and HPEGC-C (r < 0.79). These data may provide evidence that stomach histological features slightly predetermine H. pylori phenotype variability. The examination of protein expression level for H. pylori strains isolated from different locations of the stomach from patients with EGC and CG showed a simultaneous increase in six of 12 differentially expressed proteins in both HPCG-F and HPCG-C isolates (Table 1). Analysis of the EGC-related isolates revealed only two (from 18 differentially expressed) proteins whose expression significantly changed simultaneously in two HPEGC-F and HPEGC-C isolates. Most of the highly expressed proteins (12 of 18) were found only in HPEGC-F isolates in contrast to HPCG isolates. Thus, phenotypic changes of H. pylori may reflect the microorganism adaptation to morphoJournal of Proteome Research • Vol. 9, No. 1, 2010 263

research articles logically different regions of the stomach as well as microorganism adaptation to pathological processes in the stomach mucosa. Most of the differentially expressed proteins belong to antioxidant (SodB, KatA, AphC/TsaA, TrxA, Pfr), tricarbon acid cycle (Idh, FrdA, FrdB, FldA AcnB) and heat shock (GroEL and ClpB) protein families. Additionally, we found that the expression of UreG, MetX, HpaA, YihK, PepA, HylB AtpA, HylB and Tig also varies in H. pylori isolates. Idh (45 kDa, pI 7.6), FrdB (27.6 kDa, pI 5.34), MetX (42 kDa, pI 6.04), SodB (24.6 kDa, pI 5.77) and Cad (38.6 kDa, pI 6.96) proteins were identified only in H. pylori isolates related to EGC. Spots corresponding to Idh, FrdB, MetX, SodB and Cad from H. pylori isolates related to CG were not found on the proteomic map, possibly due to a pI shift caused by amino acid substitutions. A previous proteome analysis of H. pylori indicated that amino acid exchanges D f A; G f E; Q f K; E f G; and I f V in SodB from H. pylori 99 shifted the pI from 5.77 to 6.04 when compared to H. pylori 26695.44 We found amino acid substitution only in Superoxide dismutase and Cad, leading to a pI shift and subsequent spot shifts on the 2D DIGE map of H. pylori isolates related to CG compared to H. pylori isolates related to EGC (Supplementary Table S3). In HPEGC and in HPCG isolates, isocitrate dehydrogenase and alkyl hydroperoxide reductase differed only in one amino acid, which did not affect the charge or presence of the spots in the corresponding position on the HPCG 2-DE gels. Gene-specific and RT-PCR data suggested that FrdB, MetX, SodB, FlaA and Cad were subject to additional post-translational modifications in HPCG isolates, which could shift positions on 2-DE gels. Differential expression of antioxidant proteins (SodB, KatA, AphC/TsaA, TrxA, Pfr) in H. pylori indicates that ROS (reactive oxygen species) are involved in inflammation, caused by microorganism. Large amounts of ROS are produced by phagocytes, mainly neutrophils and macrophages that infiltrate the gastric lamina propria of gastric patients.45 H. pylori stimulated gastric hyperproliferation, which is a necessary step in the preliminary stages of gastric carcinoma development.46 H. pylori infection induces the expression of proto-oncogenes, such as c-fos and c-jun, and cyclo-oxygenase-2 in the gastric epithelial cells.47 The H. pylori-induced expression of inflammatory genes, oncogenes and cell-cycle regulators may be mediated by the ROS-induced activation of oxidant-sensitive transcription factors in the gastric epithelial cells. In HPEGC-A and HPEGC-F isolates, we failed to find AhpC/ TsaA, but expression was considerable (mean spot intensity 3371) in HPEGC-C. Sequencing ahpC/tsaA from H. pylori EGC isolates showed complete nucleotide identity between isolates. The absence of corresponding spots for AhpC/TsaA in HPEGC-A and HPEGC-F is not the result of amino acid substitution. It was previously demonstrated that AhpC/TsaA, a member of the thiol-dependent 2-Cys peroxiredoxin family, is the most abundant antioxidant protein in H. pylori.48,49 The AhpC of H. pylori is phylogenetically closer to eukaryotic Prx than to other prokaryotic AhpC,50 and H. pylori AhpC also acts as a molecular chaperone like yeast Prx. Yeast Prxs can switch from an enzymatic activity to a molecular chaperone function under oxidative stresses.51 Under long-term oxidative stress, all AhpC/ TsaA that are expressed in H. pylori may switch to molecular chaperoning to salvage unfolded proteins.50 The protein structure of AhpC/TsaA could shift from low-molecular weight oligomers with reductase activity under normal microaerobic conditions to high-molecular weight complexes (>699 kDa) 264

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Momynaliev et al. after severe long-term oxidative stress. The absence of AhpC/ TsaA in HPEGC-A and HPEGC-F isolates in its predicted position on proteomic maps reflects the modification caused by oxidative stress. In a group of proteins that were more highly expressed in CG, we found FldA, an electron acceptor of the pyruvateoxidoreductase enzyme complex that catalyzes the oxidative decarboxylation of pyruvate.52 H. pylori isolates expressing FldA have been reported to be closely associated with gastric MALT lymphoma.53,54 The intensity of FldA was significantly higher in H. pylori isolates from patients with chronic gastritis than in isolates from patients with gastric ulcer and gastric cancer.55 H. pylori FldA might be one of the potent bacterial factors that cause chronic gastritis and is strongly associated with the development of MALT lymphoma. We also found that the intensity of ClpB (stress response protein), but not the GroEL chaperone is higher in H. pylori isolates related to EGC than in H. pylori related to CG. It has been previously demonstrated that GroEL expression in H. pylori isolates from patients with gastric cancers is higher than in isolates from patients with ulcer and gastritis.55 However, these findings were not confirmed by two-dimensional immunoblots of serum samples from patients with gastric cancer.56 To reconstruct possible molecular genetic interaction between differentially expressed proteins of H. pylori isolates related to EGC and CG, we used an Associative Network Discovery (AND) algorithm,26 which is based on the ANDCell knowledge database. This database contains over 5 billion described cases of molecular-genetic interactions, chemical enzyme reactions, and genetic regulation that are automatically extracted from PubMed abstract texts and molecular genetic databases. We also used the ANDVisio program, which provides access to knowledge databases and representation of results as associative semantic networks. Network members are molecular genetic objects (i.e., genes, proteins, microRNAs, low molecular products), biological processes and diseases. Relationships between these members are characterized by physical interactions (e.g., complexes formation), chemical transformations, enzyme reactions, proteolysis, coexpression, positive and negative regulation of protein activity, gene expression, transport of various molecules and protein structure stability. The associative network core for H. pylori differentially expressed genes in CG consists of GroES, GroEL, DnaK, TiG and clpB chaperones. A separate node is formed by proteins interacting with tioreoxine reductase (TsaA) and tioredoxine (ThiO). This cluster is associated with chaperones through malate dehydrogenase and citrate synthetase. GroEl and GroES can prevent their aggregation under heat shock conditions by binding these enzymes.57-60 In Escherichia coli, thioredoxin reductase and thioredoxin interact with unfolded and denatured proteins in a manner similar to molecular chaperones that are involved in protein folding and renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase after urea denaturation.61 Thioredoxin is also able to interact with malate dehydrogenase.62 It was suggested that thioredoxin system, in addition to its protein disulfide isomerase activity, possesses chaperonelike properties. This thioredoxin reductase component plays a major role in this function. An additional network cluster is formed by elongation factor proteins and genes (Ef-Tu, Ef-G, Ef-Ts, EF-2 (HP1195)). Recently, it has been reported that the E. coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection

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H. pylori Functional Diversity 63,64

against protein thermal denaturation. Other elongation factors may also possess the same activity. A subnetwork of flagellin A (FlaA), flagellar adhesin (HpaA) and ClpB also includes CagA (cytotoxin associated protein), FeoB (ferrous iron transporter), UvrA (component of UvrABC system that is responsible for DNA reparation), RpoBC (DNA-dependent RNA polymerase), Rpl15 (50s ribosomal protein) and VacA (vacuolating cytotoxin). These proteins are associated through physical interactions, but the functional significance of these interactions is unknown. The associative network for differentially expressed proteins for HPEGC isolates contains most of the genes, proteins and associations from the gastritis associative network, and some new objects, such as fumarate reductase (FfdA), akonitase (AcnoN2), isocitrate dehydratase (Ihd), superoxiddismutase (SodF) and methionine adenylyltransferase (MetK). Most of these enzyme interactions with the chaperone core were found with two-hybrid systems, and their functional significance is unknown. Our interaction network for genes and proteins, the expression of which changes in EGC, includes chaperones, while the gastritis network contains enzymes that are involved in the tricarbon acid cycle, antioxidant defense and amino acid metabolism. Expression changes of these proteins may be caused by direct modulation of pathogen metabolism by cancer cells, which are characterized by higher metabolic rates than noncancer cells. Cancer cells are generally more active than normal cells in metabolic ROS generation and are constantly under oxidative stress.65-67 For example, cancer cells consume 10-30 times more glucose from the media than normal cells.68,69 H. pylori, which are located close to cancer cells, grow under glucose deficiency with an excess of di- and tricarbon acids in the environment, the intermediate products of protein, fat and carbohydrate metabolism in cancer cells. We presume that expression of several enzymes (FrdA, FrdB, Icd, FldA, AcnB) from the tricarbon acid cycle in gastric cancer is directly induced by neoplastic processes. In addition, local accumulation of toxic products in the stomach of patients with EGS stimulates H. pylori to upregulate CadA, which catalyzes benzaldehyde dismutation into benzyl alcohol and benzoic acid, thus providing a mechanism to reduce aldehyde concentration within the cell.70 The differentially regulated proteins of H. pylori that show significant differences in gastric disease (e.g., chronic gastritis, gastric cancers and gastric/duodenal ulcers) are poor prognostic markers. Park et al.55 have shown that CagA, UreB, GroEL, EF-Tu, EF-P, TagD and FldA were significantly different between isolates from patients with different gastric diseases. Statistically significant differences were found for UreB, EFTu and EP-P between isolates from patients with gastric/ duodenal ulcers (DU) and those with gastric cancer (GC). However, Lin et al.,56 who used a proteomic approach to identify GC-related antigens of H. pylori by comparing profiles of 2D immunoblots probed with DU and GC sera, did not find any association between the immunogenicity of these proteins and gastric cancer or gastric/duodenal ulcers (i.e., none of those identified by Park et al.). Differentially expressed proteins in H. pylori induced an immune response. Our data indicate that, even within the same stomach, protein expression level of H. pylori may differ significantly (i.e., more then 2-fold, Tables 1 and 2) in the absence of genetic diversity. We suggest that H. pylori protein expression variability is mostly associated with microorganism adaptation to stomach mucosa, which has histological features and changes due to pathological processes.

In conclusion, we report a comparison of expression profiles for H. pylori isolates that were obtained from different locations within the same stomach from patients with EGC and CG. Most of the H. pylori differentially expressed proteins are antioxidants, tricarbon cycle enzymes and heat shock proteins. The variable expression of H. pylori proteins is associated with adaptation to the stomach, while loss or acquisition of genes is not involved in the adaptation process. Abbreviations: PAI, pathogenicity island; EGC, early gastric cancer; CG, chronic gastritis; HPCG, H. pylori isolates from a patient with gastritis; HPEGC, H. pylori isolates from a patient with EGC; 2D DIGE, 2-D difference gel electrophoresis; ORF, open reading frames.

Supporting Information Available: Tables of protein validation, primers used for real-time PCR, comparison of four assigned spots in strains HPEGC and HPCG, and genes that were present only in H. pylori isolates related to EGC. Cluster of H. pylori multi-locus haplotypes. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Poundeyr, R. E.; Ng, D. The prevalence of Helicobacter pylori infection in different countries. Aliment. Pharmacol. Ther. 1995, 9, S33–S39. (2) Suerbaum, S.; Michetti, P. Helicobacter pylori infection. N. Engl. J. Med. 2002, 347, 1175–1186. (3) Covacci, A.; Telford, J. L.; Del Giudice, G.; Parsonnet, J.; Rappuoli, R. Helicobacter pylori virulence and genetic geography. Science 1999, 284, 1328–1333. (4) Blaser, M. J.; Atherton, J. C. Helicobacter pylori: persistence: biology and disease. J. Clin. Invest. 2004, 113, 321–333. (5) Kusters, J. G.; van Vliet, A. H.; Kuipers, E. J. Pathogenesis of Helicobacter pylori infection. Clin. Microbiol. Rev. 2006, 19, 449– 490. (6) Algood, H. M.; Cover, T. L. Helicobacter pylori persistence: an overview of interactions between H. pylori and host immune defenses. Clin. Microbiol. Rev. 2006, 19, 597–613. (7) Gressmann, H.; Linz, B.; Ghai, R.; Pleissner, K. P.; Schlapbach, R.; Yamaoka, Y.; Kraft, C.; Suerbaum, S.; Meyer, T. F.; Achtman, M. Gain and loss of multiple genes during the evolution of Helicobacter pylori. PLoS Genet. 2005, 1, e43. (8) Salama, N.; Guillemin, K.; McDaniel, T. K.; Sherlock, G.; Tompkins, L.; Falkow, S. A. whole-genome microarray reveals genetic diversity among Helicobacter pylori strains. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14668–14673. (9) Han, Y. H.; Liu, W. Z.; Shi, Y. Z.; Lu, L. Q.; Xiao, S.; Zhang, Q. H.; Zhao, G. P. Comparative genomics profiling of clinical isolates of Helicobacter pylori in Chinese populations using DNA microarray. J. Microbiol. 2007, 45, 21–28. (10) Momynaliev, K. T.; Smirnova, O. V.; Kudriavtseva, L. V.; Govorun, V. M. Comparative genome analysis of Helicobacter pylori strains. Mol. Biol. (Moscow) 2003, 37, 625–633. (11) Kansau, I.; Raymond, J.; Bingen, E.; Courcoux, P.; Kalach, N.; Bergeret, M.; Braimi, N.; Dupont, C.; Labigne, A. Genotyping of Helicobacter pylori isolates by sequencing of PCR products and comparison with the RAPD technique. Res. Microbiol. 1996, 147, 661–669. (12) Hook-Nikanne, J.; Berg, D. E.; Peek, R. M., Jr.; Kersulyte, D.; Tummuru, M. K.; Blaser, M. J. DNA sequence conservation and diversity in transposable element IS605 of Helicobacter pylori. Helicobacter 1998, 3, 79–85. (13) Suerbaum, S.; Smith, J. M.; Bapumia, K.; Morelli, G.; Smith, N. H.; Kunstmann, E.; Dyrek, I.; Achtman, M. Free recombination within Helicobacter pylori. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12619– 12624. (14) Garner, J. A.; Cover, T. L. Analysis of genetic diversity in cytotoxinproducing and non-cytotoxin-producing Helicobacter pylori strains. J. Infect. Dis. 1995, 172, 290–293. (15) Evans, D. J., Jr; Queiroz, D. M.; Mendes, E, N.; Evans, D. G. Diversity in the variable region of Helicobacter pylori cagA gene involves more than simple repetition of a 102-nucleotide sequence. Biochem. Biophys. Res. Commun. 1998, 245, 780–784. (16) Achtman, M.; Azuma, T.; Berg, D. E.; Ito, Y.; Morelli, G.; Pan, Z. J.; Suerbaum, S.; Thompson, S. A.; van der Ende, A.; van Doorn, L. J.

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