Proteome Analysis of Alkylhydroxide Peroxidase-Deficient Isogenic Mutant of Helicobacter pylori 26695

In order to investigate the antioxidant effect of alkylhydroxide peroxidase (ahpC) of Helicobacter pylori (H. pylori) 26695, an ahpC-deficient mutant (H. pylori 26695 ahpC::cat) was generated. ahpC-deficient mutant was grown slowly at lower pressure of oxygen (5% oxygen) compared to the H. pylori 26695. Whole cell proteins isolated form H. pylori 26695 and H. pylori 26695 ahpC::cat were analyzed by MALDI-TOF and tandem-MS. The expression of 15 proteins, including Ppa, HypB, GrpE, Elp, RecA, GroES, Mda66, RibE, NapA, GlnA, BioB, TrxB, Tsf, FumC and Icd, was more than doubled in H. pylori 26695 ahpC::cat. Production of 10 proteins such as UreG, FabE, Adk, Pnp, OorC, AtpA, AtpD, Nqq3, Pfr, and TagD decreased below 50% in H. pylori 26695 ahpC::cat compared to the H. pylori 26695. In microarray analysis, 9 genes including sul1, amiE, frxA, fecA, hyuA, and katA increased in transcription level in H. pylori 26695 ahpC::cat compared to H. pylori 26695. A total of 24 genes, including flaB, protein kinase C inhibitor, cag16, pabC, and sabA, reduced in transcription. 27 genes, including HP0889, showed common expression changes in ahpC, katA, and sodB-deficient mutations. As a result of this study, there were not many genes whose expression was commonly changed by the deletion of each of the three major antioxidant enzymes of H. pylori. These results showed the functions and regulation of the three antioxidant enzymes were different in H. pylori.

Gene Expression Profiles of Th1-type Chemokines in Whole Blood of Mycobacterium avium subsp. paratuberculosis-Infected Cattle

Johne's disease (JD) is a chronic, debilitating disease of ruminants including cows, and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is not only important in animal husbandry, but also in public health as it is associated with the onset of Crohn's disease, a chronic inflammatory bowel disease in humans. JD, like other mycobacterial diseases including tuberculosis, is classified into different stages based on the progression of infection. In addition, development of diagnostic assays that can distinguish between subclinical and clinical stages of JD is essential to control mycobacterial infection by providing an effective treatment. For the development of novel diagnostic methods of JD, it is important to investigate and understand the mRNA expression of the various immune markers in individuals at each stage of infection. In this study, we measured the levels of Th1-type chemokines, CXCR3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11 in MAP-infected bovine blood by interferon (IFN)-γ release assay (IGRA) using IFN-γ as an alternative biomarker. The association of mRNA expression patterns of these chemokines with the MAP infection stages was analyzed and IFN-γ, CCL5, and CXCL10 were found to be significantly upregulated compared to IFN-γ, the biomarker used in IGRA. Our results further indicate that IFN-γ levels significantly increased in individuals with MAP-specific antibody, and CCL5 and CXCL10 levels significantly increased in those with MAP DNA. In particular, CCL5 was significantly upregulated in individuals, in which both MAP-specific antibody and MAP DNA were detected, but the expression of CXCL10 was specifically elevated in MAP DNA-detected individuals without MAP-specific antibody.

Characterization of Specific IgA Response to Antigenic Determinants of Helicobacter pylori Urease Encoded by ureA and ureB in Children

Helicobacter pylori (H. pylori), a causative agent of chronic gastritis and gastric cancer, has several virulent factors for own survival and progression toward gastric diseases in human stomach. Of those, H. pylori produces mainly urease (10~15% total protein weight) that neutralize the gastric acid for survival. Here, we identified the antigenic epitope of urease and then developed an ELISA using the antigen including the epitope of urease. We identified the antigenic epitope of urease that induces IgA antibodies in human using truncated mutants. Eight kinds of serially-truncated mutant of UreA and UreB were prepared and subjected to immunoblot using pooled sera of patients with gastric disorders. UreBEnd protein containing UreB epitope was produced and investigated its diagnostic value via ELISA in children. As a result, mutants having last 24 amino acid residues of UreB carboxyl terminus deleted did not show IgA-reactive band. The clones that contained the downstream of 448(th) amino acid in UreB showed IgA-reactive band. The serodiagnostic value of the UreBEnd recombinant protein including identified epitope was confirmed via IgA ELISA and shown to have 97% sensitivity and 100% specificity. These results demonstrated that carboxyl terminal region of UreB carries an antigenic epitope for IgA response in human. It may be useful for detecting H. pylori infection with improved test accuracy and minimum use of endoscopy.

Anti-Helicobacter pylori Compounds from Maackia amurensis

Eight isoflavonoid compounds were isolated from the EtOAc fraction of Maackia amurensis which had shown the highest anti-Helicobacter pylori activity among the fractions, using medium pressure liquid chromatography and recrystallization. Based on the spectroscopic data including 1H-NMR, 13C-NMR, HMBC and MS data, the chemical structures of the isolates were determined to be (-)-medicarpin (1), afromosin (2), formononetin (3), tectorigenin (4), prunetin (5), wistin (6), tectoridin (7) and ononin (8). Anti-H. pylori activity of each compound was evaluated with broth dilution assay. As a result, (-)-medicarpin (1), tectorigenin (4) and wistin (6) showed anti-H. pylori activity. (-)-Medicarpin (1) exhibited the most potent growth inhibitory activity against H. pylori with the minimal inhibitory concentration (MIC)90 of 25 microM, and tectorigenin (4) with MIC90 of 100 microM ranked the second. This is the first study to show the anti-H. pylori activity of M. amurensis, and it is suggested that the stem bark of M. amurensis or the EtOAc fraction or the isolated compounds can be a new natural source for the treatment of H. pylori infection.

Proteome Analysis of a Catalase-deficient Isogenic Mutant of Helicobacter pylori 26695

Helicobacter pylori, a gram-negative bacterium, is a causative agent of gastroduodenal diseases of human. Human immune system produces harmful reactive oxygen species to kill this bacterium that locates the microaerophilic mucous layer. H. pylori harbors various antioxidant enzymes including SodB, KatA and AhpC to protect the oxygen toxicity. We removed the catalase gene (katA) from H. pylori 26695 genome, and the change of profile of the gene expression of the mutant was analyzed by high resolution 2-DE followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), tandem MS and microarray analysis. Eleven and 37 genes were upregulated and downregulated in the mutant respectively, either transcriptionally or translationally. Expression level of pfr and hp1588 that were decreased on protein level in the mutant was confirmed by RT-PCR analysis.

Gastric Autoantigenic Proteins in Helicobacter Pylori Infection

PURPOSE: This study tried to identify novel gastric autoimmune antigens that might be involved in aggravating the atrophic gastritis among patients with Helicobacter pylori infection using two-dimensional immunoblotting analysis. MATERIALS AND METHODS: Proteins from gastric mucosal antrectomy specimens and AGS cells (gastric adenocarcinoma cell lines derived from a Caucasian patient who had received no prior therapy) were 2-dimensionally immunoblotted separately with a pool of 300 sera from H. pylroi-infected patients at Gyeongsang National University Hospital. RESULTS: Thirty-eight autoantigenic proteins including alcohol dehydrogenase [NADP+], alpha enolase, gastrokine-1, gastric triacylglycerol lipase, heat shock 70 kDa protein 1, and peroxiredoxin-2 were identified in the gastric mucosal tissue. Fourteen autoantigenic proteins including programmed cell death 6-interacting protein, serum albumin and T-complex protein 1 subunit gamma were identified in the AGS cells. Albumin, alpha-enolase, annexin A3, cytoplasmic actin 1, heat shock cognate 71 kDa protein and leukocyte elastase inhibitor were commonly observed autoantigenic proteins in both gastric mucosal tissue and AGS cells. Alpha-enolase, glutathione S-transferase P, heat shock cognate 71 kDa protein, heat shock 70 kDa protein 1, human mitochondrial adenosine triphosphate synthase (ATP) subunit beta, mitochondrial 60 kDa heat shock protein, peroxiredoxin-2, 78 kDa glucose-regulated protein precursor, tyrosine-protein phosphatase non-receptor type 11 and Tryptophan-Aspartic acid (WD) repeat-containing protein 1 showed 60% or higher amino acid positivity. CONCLUSION: These newly identified gastric autoimmune antigens might be useful in the control and prevention of gastroduodenal disorders, and might be valuable in breaking the vicious circle that exists in gastroduodenal disorders if their pathophysiological roles could be understood in the progress of chronic atrophic gastritis, gastroduodenal ulcers, intestinal metaplasia, and gastric carcinogenesis.

Changes in Anti-Group A Rotavirus Antibody Seroprevalence and Levels in the Western Gyeongnam Province of Korea Over 16 Years

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- > or = 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.

Comparison of Gene Expression Patterns between Helicobacter pylor 26695 and its Superoxide Dismutase Isogenic Mutant

Helicobacter pylori, a causative agent of gastroduodenal diseases, is a Gram-negative microaerophilic bacterium. Although H. pylori locates in the microaerophilic mucous layer, the bacteria would come into contact harmful reactive oxygen species generated by host immune system. It has been reported that H. pylori harbors various defense mechanisms which can protect bacterial cells from oxygen exposure. The change of the gene expression profile of sodB-negative isogenic mutant of H. pylori 26695 was analyzed by high resolution 2-DE followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS and microarray analysis. Eighteen genes and 41 genes were upregulated and downregulated respectively, either transcriptionally or translationally. Expression levels of three genes including trxB, yxjE and ribE that were changed both on a mRNA level and on a protein level were confirmed by RT-PCR analysis. However, change of expression levels of other major antioxidants such as KatA, AhpC and NapA were not detected, which means Sod is regulated by different way from that of KatA and AhpC. Mutant study of other antioxidant proteins may give us better understanding for the regulation of stress response in H. pylori.

Comparison of the Antibiotic Resistance of Helicobacter pylori Isolated in Jinju Over a 15-year Period

The aims of this study were to investigate the changing pattern of Helicobacter pylori antibiotic resistance in Jinju over a 15-year period. H. pylori strains were isolated from 170 adults living in Jinju from 1985-1989, 1990-1994 and 1995-1999, and from 23 adults living in Cheongju from 1995 to 1999. Susceptibility to erythromycin, clarithromycin, azithromycin, amoxicillin, tetracycline, metronidazole, furazolidone, levofloxacin, ciprofloxacin, moxifloxacin, and rifabutin was tested using the serial two-fold agar dilution method. Moxifloxacin resistance significantly increased in Jinju from 1985-1989 (0%) to 1995-1999 (14.9%) (p < 0.0001). Resistance to amoxicillin was increasesed trend to decreased trend from 1985 to 1999 (p = 0.033), whereas metronidazole resistance decreased from 37.5% to 21.3%. Resistance to furazolidone was greater from 1985-1989 (9.4%) than in 1995-1999 (2.1%). In comparing Jinju and Cheongju, minimal inhibitory concentrations (MICs) of tetracycline and levofloxacin among H. pylori isolated from Jinju were lower than for isolates from Cheonju (p < 0.05). The levofloxacin resistance rate was higher in Cheongju than in Jinju (p = 0.02). No macrolide resistance was observed in Cheongju. Overall, we did not observe any remarkable antimicrobial resistance increase of H. pylori strains isolated from Jinju over 15 years. The MIC distributions of antimicrobials and antimicrobial resistant rates were time- and region-specific among different strains. Future anti-H. pylori eradication regimens should be designed based on the changing patterns of antimicrobial resistance according to the resident area.

Proteomic Analysis of Thiol-active Proteins of Helicobacter pylori 26695

Helicobacter pylori are a capnophilic bacterium, which colonize gastric mucosa and are resistant to acidic and oxidative damage. Thiol-active proteins subserve redox functions in tolerating oxidative stress and environmental toxicants, such as hydrogen peroxide and hypochlorous acid. We analyzed disulfide-containing proteins of H. pylori strain 26695. Active disulfide-containing proteins were separated by thiol-affinity chromatography, displayed with two-dimensional electrophoresis (2-DE), and identified by MALDI-TOF-MS. Thirty-five putative disulfide proteins, including AhpC (HP1563), GroEL (HP0011), and FrdB (HP0191), were identified in this study. In addition, 4 disulfide proteins of HypB, FusA, TufB, and AhpC showed enhanced intensities in the periplasmic space when compared with the pellet, suggesting that these proteins might play roles in the first redox system against environmental oxidative stresses. Disulfide-containing proteins identified in this study will provide the standard landscape for constructing the proteome components responsible for redox regulation of H. pylori.

Helicobacter pylori Infection and Extraintestinal Manifestations in Children / 대한소아소화기영양학회지

Helicobacter pylori (H. pylori) may be one of the most common pathogen in gastrointestinal tract. Although several recent articles have reported a decline in the prevalence of H. pylori infection in both children and adults over the last several years, H. pylori infection usually occurs early in life and persists for a long time. The role of H. pylori in some digestive diseases, such as gastritis, ulcer and gastric cancer has been well established. And the possible role of H. pylori as a trigger for some extraintestinal diseases in children and adults has been considered in the last year. H. pylori infection might be associated with refractory iron deficiency anemia, idiopathic thrombocytopenic purpura, growth retardation and obesity etc, directly or indirectly. Most of the studies are classified as epidemiological, clinical researches about effects on extraintestinal manifestations after eradication, or case reports. This review describes the possibility of association with several extraintestinal diseases and H. pylori infection and their possible mechanisms based on reported studies in the world and our several studies, even though there are still many conflicting results about that.

Purification and Characterization of Helicobacter pylori gamma-Glutamyltranspeptidase

Gamma-glutamyltranspeptidase (GGT) was purified to electrophoretic homogeneity from the cell extract of H. pylori. The purified enzyme consisted of heavy and light subunits with molecular weights of 38 kDa and 21 kDa, respectively. N-terminal amino acid sequence of heavy and light subunits revealed that H. pylori GGT was processed into 3 parts for a signal peptide of 27 amino acid residues, a heavy subunit of 352 residues, and a light subunit of 188 residues during translation. The reaction rate for hydrolysis of gamma-GpNA was 84.4 micromol/min per milligram of protein, and that for the gamma-glutamyl transfer from gamma-GpNA to gly-gly was 23.8 micromol/min per milligram of protein. The apparent Km values of H. pylori GGT for gamma-glutamyl compounds were on the order of 10-3 to 10-4 M and those for acceptor peptides and amino acids were on the order of 10-1 to 10-2 M. The GGT protein kept approximately 80% of the initial enzymatic activity on incubation at 60degrees C for 15 min. The optimum temperature and pH for reactions of both hydrolysis and transpeptidation were 40degrees C and 9.0, respectively. The transpeptidation and hydrolysis reactions catalyzed by H. pylori GGT were strongly inhibited by L-Gln and moderately inhibited by L-Ala, L-Ser, beta-chloro-L-Ala, and L-Glu. These results demonstrated that the biochemical properties of H. pylori GGT are different from those of other bacterial GGTs. Further, H. pylori GGT might degrade glutathione in the gastric mucous layer of humans if the enzyme could be secreted in the bacterial niches.

Comparison of Proteome Components of Helicobacter pylori Before and After Mouse Passage

The mouse model is alleged to be a useful tool for understanding of pathophysiological roles of Helicobacter pylori in the development of gastric disorders. However, it has been observed that H. pylori strains significantly differed in their fitness in mice and even mouse strains differed in their susceptibilities to a H. pylori strain. Bacterial components of H. pylori which could affect on its fitness in mice have to be elucidated for the establishment of the mouse model for H. pylori infections. In the comparison of colonization ability between two H. pylori Korean isolates, 51 (isolated from a patient with duodenal ulcer) and 52 (isolated from a patient with gastric cancer), 52 could colonize better than 51 on the gastric mucosa of mouse. Proteome components of H. pylori 52, as a good colonizer and H. pylori 51, as a poor one were quantitatively compared each other. Five bacterial proteins including catalase, urease subunit alpha/beta, enolase and ferritin, were up-regulated in 52. In addition, the respective proteome components of the two strains were also compared with their mouse-passaged homologous strains. Seven and five proteins, which included catalase, flagellin A/B in common, were up-regulated in mouse-adapted 51 and 52, respectively. Among the fourteen identified proteins, urease subunit alpha/beta, flagellin A/B, catalase, ferritin, superoxide dismutase and neutrophil-activation protein have been previously known to be necessary to gastric colonization of H. pylori in animal models. The other up-regulated proteins including enolase, elongation factor Tu and fructose-bisphosphate aldolase have been reported to be associated with acid tolerance of H. pylori. These data provide confirmatory evidence for the importance of those proteins in the development of H. pylori-associated gastric disorders.

Analysis of Low Molecular Weight Proteome from H. pylori Cell Extract Using the High Performance Liquid Chromatography

Low molecular proteins (LMPs) which are smaller than 20 kDa are difficult to visible on a standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-D SDS-PAGE) map. LMPs must be enriched appropriately to be analyzed. We isolated LMPs of Helicobacter pylori 26695 from 1-D polyacrylamide gel and digested by pepsin. Pepsin-digested LMPs were separated by HPLC and each fraction was analyzed by hybrid tandem mass spectrometer. Seventy nine peptides, representing 27 genes, including copper ion binding protein (CopP, 7 kDa), thioredoxin (TrxA, 11.9 kDa) and ribosomal protein L23 (Rpl23, 10.5 kDa) were identified. Some proteins larger than 40 kDa including Omp2, Omp21, Omp27, Omp30, Omp32, catalase and HP1083 were also identified. This work may give researchers a useful way to analyse the expressed LMPs which could not be identified on the conventional 2-D SDS-PAGE.

Usefulness of Escherichia coli-expressed Recombinant VP6 Proteins of Group A Rotavirus in Serodiagosis of Rotavirus Infection / 대한소아소화기영양학회지

PURPOSE: The serologic diagnosis of rotaviral infections is not commonly used in clinical practice, but is used in seroepidemiologic studies. In this study, the usefulness of Escherichia coli-expressed recombinant VP6 proteins of group A rotavirus in the serodiagnosis of rotavirus infections by ELISA was evaluated. METHODS: The recombinant VP6 proteins of group A rotavirus expressed in E. coli Rosetta II strain were purified and identified. One hundred sera from 22 children (4 healthy neonates, 13 healthy children, and 5 immunocompromised children) who had serial sera samples prior to and after rotavirus infections were provided by the Gyeongsang National University Hospital, a member of the National Biobank of Korea. IgG, IgA, and IgM antibodies against rVP6 were analyzed by ELISA in all of the patients and Western blot analysis in 4 neonates. RESULTS: ELISA tests using rVP6 proteins of group A rotavirus as antigen revealed that IgG, IgA, and IgM antibodies increased after rotaviral infections in most neonates and healthy children. IgG antibodies also increased after rotaviral infections in most immunocompromised children without an adequate increase in IgM or IgA antibodies. Western blot analysis in four neonates revealed very early IgM antibody responses, even in the sera with low optical densities in ELISA tests. CONCLUSION: Our study showed that ELISA using rVP6 as an antigen is a valid diagnostic tool for seroepidemiologic studies of rotavirus infections and Western blot analysis is a sensitive test in detecting IgG, IgA, and and IgM antibodies in patients with rotavirus infections.

Usefulness of Escherichia coli-expressed Recombinant VP6 Proteins of Group A Rotavirus in Serodiagosis of Rotavirus Infection / 대한소아소화기영양학회지

PURPOSE: The serologic diagnosis of rotaviral infections is not commonly used in clinical practice, but is used in seroepidemiologic studies. In this study, the usefulness of Escherichia coli-expressed recombinant VP6 proteins of group A rotavirus in the serodiagnosis of rotavirus infections by ELISA was evaluated. METHODS: The recombinant VP6 proteins of group A rotavirus expressed in E. coli Rosetta II strain were purified and identified. One hundred sera from 22 children (4 healthy neonates, 13 healthy children, and 5 immunocompromised children) who had serial sera samples prior to and after rotavirus infections were provided by the Gyeongsang National University Hospital, a member of the National Biobank of Korea. IgG, IgA, and IgM antibodies against rVP6 were analyzed by ELISA in all of the patients and Western blot analysis in 4 neonates. RESULTS: ELISA tests using rVP6 proteins of group A rotavirus as antigen revealed that IgG, IgA, and IgM antibodies increased after rotaviral infections in most neonates and healthy children. IgG antibodies also increased after rotaviral infections in most immunocompromised children without an adequate increase in IgM or IgA antibodies. Western blot analysis in four neonates revealed very early IgM antibody responses, even in the sera with low optical densities in ELISA tests. CONCLUSION: Our study showed that ELISA using rVP6 as an antigen is a valid diagnostic tool for seroepidemiologic studies of rotavirus infections and Western blot analysis is a sensitive test in detecting IgG, IgA, and and IgM antibodies in patients with rotavirus infections.

Identification of Helicobacter pylori Strain 51 Major Outer Membrane Proteins by Quadrupole Time of Flight Mass Spectrometry

As part of an initial inquiry into the function of the outer membrane proteins (OMPs) of Helicobacter pylori Korean strain 51, we have conducted an extensive proteome analysis via quadrupole time of flight (Q-TOF) mass spectrometry (MS). Fifty one OMPs of H. pylori were purified using sarcosine and resolved via two-dimensional electrophoresis with immobilized pH gradient strips. The most abundant proteins were observed in the alkaline pI regions (6.0~11.0) at molecular masses between 10~100 KDa. Here, 15 spots were identified, representing 9 types of genes (KHP0852, KHP0853, KHP1353, KHP1017, KHP0172, KHP0076, KHP0617, KHP1069, KHP0614) from the sarcosin-insoluble fraction of H. pylori 51. These may be employed in the characterization of the OMPs of H. pylori 51, which will help to identify new potential target proteins for vaccine development and drug therapy.

Proteomic analysis of Helicobacter pylori J99 Outer Membrane Protein by Tandem Mass Spectrometry

The protein identity of sarcosine-insoluble outer membrane proteins (OMPs) of Helicobacter pylori J99 was determined with the basic study of understanding the function of proteins. A sarcosine-insoluble OMPs was resolved by two-dimensional electrophoresis with immobilized pH gradient strips. The most abundant proteins were shown in the alkaline pI regions (6.0~11.0) with molecular masses of 10 to 100 kDa. We have performed an extensive proteome analysis by quadrupole time of flight (Q-TOF) mass spectrometry (MS). Here, of 50 spots processed, 42 spots were identified, which represented 16 genes and we newly detected 8 kinds of proteins (JHP0119, JHP0388, JHP1046, JHP1405, JHP0073, JHP0551, JHP1382, JHP0552) from the sarcosin-insoluble fraction of H. pylori J99. Those may be used to elucidate the characterization of the OMPs of H. pylori J99, which will help identify new potential target proteins for vaccine development and drug therapy.

Proteomic Analysis of Helicobacter pylori Whole Cell Proteins using the Narrow Range IPG Strips

It has been reported that most of Helicobacter pylori proteome components appear so crowded in the region of pH 4.5~8.0 that a lot of them were inseparable in 2-DE using the broad range IPG strip. Therefore, inseparable protein spots in 2-DE profiles have to be apart from each other for improving the protein identification. Here, we attempt to examine the usability of the narrow range IPG strips for separating close spots in the broad range IPG strip at proteomic analysis of H. pylori. The whole cell proteins of H. pylori strain 26695 were separated by narrow range IPG strips (pI 3.9~5.1, 4.7~5.9, 5.5~6.7, and 6.3~8.3, respectively), followed by SDS-PAGE, and visualized by silver staining, showing that the distances between spots were widened and the total number of detectable spots was increased. Resolved protein spots were identified by the peptide fingerprinting using MALDI-TOF-MS. As a result, 87 expressed proteins were identified by the peptide fingerprinting. Of them, 23 proteins, including hydrogenase expression/formation protein, purine-binding chemotaxis protein, and ribosomal protein S6, have not been reported in the previous proteome studies of H. pylori. Thus, these results demonstrate that the high complexity proteome components could be effectively separated using the narrow range IPG strips, which might be helpful to strengthen the contents of the master protein map of the H. pylori reference strain.

Comparison of Proteome Component of Helicobacter pylori in Different Atmospheric CO2 Concentration

Helicobacter pylori is a spiral, slow growing gram-negative microaerophilic bacterium. It has been shown to be the etiological agent of gastroduodenal diseases, such as chronic gastritis, gastric and duodenal ulcers, and gastric cancer. General culture condition of H. pylori is 5% O2, 10% CO2 and 100% humid atmosphere. We have compared proliferation protein expression profile of H. pylori incubated under normal microaerophilic (10% CO2) and environment stress (4% CO2, 18% CO2) conditions. H. pylori cultured under environment stress displayed coccoid morphology and timedependent decrease in proliferation. We have further compared the protein expression profiles of H. pylori under normal growing and environment stress conditions by a global proteomic analysis, which includes high-resolution 2-DE followed by matrix-assisted laser desorption/ionization time of flight and nanoelectrospray/tandem mass spectrometry. In total, 42 protein spots were found to be up- or down-regulated by more than 2-fold under environment stress conditions. Of the 42 protein spots processed, 27 spots were identified; they represented 19 genes, including 2 kinds of hypothetical proteins.