Alkylhydroperoxide reductase of Helicobacter pylori as a biomarker for gastric patients with different pathological manifestations

The development of various gastrointestinal diseases was suggested to be associated with chronic inflammation as a consequence of Helicobacter pylori (H. pylori) infection. Our previous studies showed that an antioxidant protein alkylhydroperoxide reductase (AhpC) is an abundant and important antioxidant protein present in H. pylori. In this study we have explored the potential of utilizing antibodies to AhpC for detection of patients who are at high risks of evolving into severe outcomes of gastric malignancies after H. pylori infection. The correlation between AhpC and extents of inflammatory damage in tissues was demonstrated by immunoblotting assays and endoscopic examinations. Oxidative stress-induced high-molecular-weight (HMW) AhpC with chaperone activity in vivo was further investigated by co-immunoprecipitation, 2-dimensional gel electrophoresis (2-DE) followed by nano-liquid chromatography coupled tandem mass spectrometry (nanoLC-MS/MS). We found AhpC was consistently expressed in higher amounts in H. pylori strains isolated from patients with gastric cancer (GC) than gastritis (GA). Immunological analysis of seropositivity for AhpC indicated that positive diagnostic rates for H. pylori-infected patients with GA, gastric ulcer (GU) and GC were 68% (15/22), 100% (50/50) and 100% (50/50), respectively. In great contrast to low-molecular-weight (LMW) AhpC, HMW AhpC with chaperone function was found to distribute inside of H. pylori cells. We also found that LMW forms of AhpC were recognized by serum antibodies from GA patients whereas HMW forms of AhpC reacted mainly with those from GU and GC patients. Based on the significant difference between AhpC isolated from strains of GC and GA, it is conceivable that AhpC of H. pylori may prove to be useful as a prognostic or diagnostic protein marker to monitor varied clinical manifestations of gastrointestinal patients infected with H. pylori.     

A novel Helicobacter pylori cell-surface polysaccharide

Helicobacter pylori bacteria colonize the gastric mucosa of more than half of the world's human population and its infection may instigate a wide spectrum of gastric diseases in the host. At the moment, there is no vaccine against H. pylori, a microorganism recognized as a category 1 human carcinogen, and treatment is limited to antibiotic management. Pioneering antigenic studies carried out by Penner and co-workers, which employed homologous H. pylori antisera specific for cell-surface lipopolysaccharide (LPS), revealed the presence of six distinct H. pylori serotypes (O1 to O6). Subsequent studies have shown that H. pylori serotype O1 expressed LPS with lengthy O-chain polysaccharide (PS) composed of Lewis blood-group structures ('Lewis O-chains'), serotype O3 LPS produced 'Lewis O-chains' attached to a heptoglycan domain, serotype O4 LPS possessed LPS with glucosylated 'Lewis O-chains' and serotype O6 LPS expressed the heptoglycan domain capped by a short 'Lewis O-chain'. These LPSs were terminated at the reducing-end by a core oligosaccharide and lipid A of conserved structures. With the intent of formulating a multivalent H. pylori LPS-based vaccine, we are studying the structural variability of H. pylori cell-surface glycans. Here, we describe the novel LPS structure produced by H. pylori serotype O2 that differed markedly from the typical H. pylori 'Lewis O-chain' structures, in that its main component was an elongated PS composed of alternating 2-, and 3-monosubstituted alpha-D-Glcp residues [-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]n. These findings revealed the bio-molecular basis for the observed serospecificity of H. pylori serotype O2, and that this unique bacterial PS must be included in the formulation of a multivalent LPS H. pylori vaccine.     

A model for the study of Helicobacter pylori interaction with human gastric acid secretion

We present a comprehensive mathematical model describing Helicobacter pylori interaction with the human gastric acid secretion system. We use the model to explore host and bacterial conditions that allow persistent infection to develop and be maintained. Our results show that upon colonization, there is a transient period (day 1-20 post-infection) prior to the establishment of persistence. During this period, changes to host gastric physiology occur including elevations in positive effectors of acid secretion (such as gastrin and histamine). This is promoted by reduced somatostatin levels, an inhibitor of acid release. We suggest that these changes comprise compensatory mechanisms aimed at restoring acid to pre-infection levels. We also show that ammonia produced by bacteria sufficiently buffers acid promoting bacteria survival and growth.     

Helicobacter pylori infection influences expression of genes related to angiogenesis and invasion in human gastric carcinoma cells

Infection with Helicobacter pylori (H. pylori) is considered a risk factor for gastric carcinoma. The purpose of this study was to clarify whether H. pylori infection plays a role in progression of gastric carcinoma. We examined the expression of genes encoding angiogenic factors and proteases by human gastric carcinoma cell lines (MKN-1 and TMK-1) co-cultured with or without H. pylori by cDNA microarray analysis. Co-culture with H. pylori increased expression of mRNAs encoding interleukin (IL)-8, vascular endothelial growth factor (VEGF), angiogenin, urokinase-type plasminogen activator (uPA), and metalloproteinase (MMP)-9 by gastric carcinoma cells. Up-regulation of these genes at the mRNA and protein levels was confirmed by Northern blot analysis, semi-quantitative RT-PCR analysis, and ELISA. In vitro angiogenic and collagenase activities of conditioned medium from the gastric carcinoma cells were also stimulated by co-culture with H. pylori. These results indicate that H. pylori infection may regulate angiogenesis and invasion of human gastric carcinoma.     

Gamma-glutamyltranspeptidase of Helicobacter pylori induces mitochondria-mediated apoptosis in AGS cells

Gamma-glutamyltranspeptidase (GGT) is a novel protein involved in the induction of Helicobacter pylori-mediated apoptosis; however, the signal pathway involved in GGT-induced apoptosis remains unclear. Using DNA recombination techniques, ggt was cloned into pET117b and transformed into Escherichia coli. Recombinant GGT was purified using nickel-affinity resin and was digested by thrombin. Recombinant GGT induced apoptosis in AGS cells in a time-dependent manner, which was confirmed by TUNEL staining, the MTT assay and immunoblot analysis for caspases-9, -3, Bax, Bcl-2, Bcl-xL and cytochrome c release. Activation of caspase-3 and -9 following exposure to GGT increased in a time-dependent manner and upregulation of proapoptotic Bax and a downregulation of antiapoptotic Bcl-2 and Bcl-xL was detected. Apoptotic signals also trigger changes in mitochondria, which lead to a release of cytochrome c into the cytosolic space. The GGT-deficient mutant was not as able to induce apoptosis as the wild-type strain. These results indicate that GGT of H. pylori induces apoptosis via a mitochondria-mediated pathway.     

DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.     

Inhibition of Helicobacter pylori growth and its cytotoxicity by 2-hydroxy 4-methoxy benzaldehyde of Decalepis hamiltonii (Wight & Arn); a new functional attribute

Helicobacter pylori mediated gastric ulcer and cancers are common global problems since it was found to colonize in ∼50% of gastric ulcer/cancer patients. Decalepis hamiltonii, (Asclepiadaceae family) extracts have been depicted with medicinal properties supporting the traditional knowledge of health beneficial attributes of D. hamiltonii. Previously we have shown that both aqueous as well as methanol extracts of D. hamiltonii containing abundant phenolics with predominant levels (20-40% of total phenolics) of 2-hydroxy-4-methoxy benzaldehyde (HMBA). Despite higher levels, HMBA contributed very little to the antioxidant activity (<10%) when compared to other phenolic compounds in the extract. In the current study we attempted to explore antimicrobial property, particularly anti-H. pylori activity, since traditional users document D. hamiltonii as a fighter of microbial infections. HMBA was isolated from the roots of D. hamiltonii by hydrodistillation and cold crystallization method; identified by HPLC and characterized using ESI-MS and confirmed by NMR studies as a compound of molecular mass 152 Da. Isolated HMBA was found to inhibit the growth of H. pylori, a potential ulcerogen in a dose dependent manner with MIC of ∼39 μg/mL as apposed to that of amoxicillin (MIC - 26 μg/mL) for which H. pylori is susceptible. Results were further substantiated by the lysis of H. pylori by electron microscopy and electrophoretic studies. Studies on the mechanism of action indicated the counteracting effect of vacuolating toxin (VacA) of H. pylori which otherwise would lead to host cell cytotoxicity. Further the increased binding ability of HMBA to DNA and protein offered an impact on DNA protectivity and bioavailability. Results for the first time provide a direct evidence for anti-microbial attribute of HMBA. Insignificant antioxidant attribute of HMBA also reveals the anti-H. pylori activity via mechanisms other than antioxidative routes.     

Concerted evolution between duplicated genetic elements in Helicobacter pylori

The Helicobacter pylori genome includes a family of outer membrane proteins (OMPs) with substantial N and C-terminal identity. To better understand their evolution, the nucleotide sequences for two members, babA and babB, were determined from a worldwide group of 23 strains. The geographic origin of each strain was found to be the major determinant of phylogenetic structure, with strains of Eastern and Western origin showing greatest divergence. For strains 96-10 (Japan) and 96-74 (USA), the 5' regions of babB are replaced with babA sequences, demonstrating that recombination occurs between the two loci. babA and babB have nearly equivalent variation in nucleotide and amino acid identity, and frequencies of synonymous and non-synonymous substitutions. Both genes have segmental conservation but within the 3' segment, substitution patterns are nearly identical. Although babA and babB 5' and midregion segment phylogenies show strong interstrain similarity, the 3' segments show strong intrastrain similarity, indicative of concerted evolution. Within these 3' segments, the lower intrastrain than interstrain frequencies of nucleotide substitutions, which are below mean background H. pylori substitution frequencies, indicate selection against intrastrain diversification. Since babA/babB gene conversions likely underlie the concerted evolution of the 3' segments, in an experimental system, we demonstrate that gene conversions can frequently (10(-3)) occur in H. pylori. That these events are recA-dependent and DNase-resistant indicates their likely cause is intragenomic recombination.     

Structure of the neutrophil-activating protein from Helicobacter pylori

Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes.     

Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells

Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P ≤ 0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.     

Quantitation of Helicobacter pylori ureC gene and its comparison with different diagnostic techniques and gastric histopathology

Numerous diagnostic assays for Helicobacter pylori detection are available. However, these techniques have their own advantages as well as limitations. Here we tried to develop a real-time quantitative (Q) PCR assay to measure ureC copy number to detect H. pylori, based on the fact that there is only one copy of the ureC gene per bacterium. We enrolled 120 adult patients [non-ulcer dyspepsia (NUD) 60, peptic ulcer disease (PUD) 20, gastric cancer (GC) 40] undergoing upper gastrointestinal endoscopies. During each endoscopic examination, antral biopsies from normal region of the antrum were obtained and subjected to the following tests: RUT, culture, histopathology, H. pylori-specific ureC PCR and ureC Q-PCR. Calculation of H. pylori copy number was based on the standard curve generated using 10-fold dilutions of DNA extracted from the H. pylori control strain varying from 10⁵ to 10¹ copies. The prevalence of H. pylori infection in our study population was 54% with no significant difference among disease and control population. The sensitivity of Q-PCR was found to be 100% which was highest among all diagnostic tests. The established Q-PCR is around 10 times more sensitive than the conventional PCR method. The copy number of H. pylori DNA was significantly increased when overall gastritis, H. pylori density, chronic inflammation and intestinal metaplasia were present. In summary, we developed a rapid and sensitive Q-PCR method for detecting H. pylori. This technique offers a significant improvement over other available methods for detecting H. pylori in clinical and research samples.     

Expression cloning of cholesterol alpha-glucosyltransferase, a unique enzyme that can be inhibited by natural antibiotic gastric mucin O-glycans, from Helicobacter pylori

Helicobacter pylori infects over half the world's population, but only 3% of those infected develop peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. In H. pylori, alpha-glucosyl cholesterol constitutes more than 25% of cell wall lipids, and it has been suggested that alpha-glucosyl cholesterol is essential for H. pylori viability. Here, we identified cholesterol alpha-glucosyltransferase (CHLalphaGcT) using an expression cloning strategy and showed that this enzyme is distinctively inhibited by mucin-type O-glycans similar to those present in deeper portions of the gastric mucosa. Moreover, inactivation of CHLalphaGcT by homologous recombination led to H. pylori lethality. These results indicate that H. pylori CHLalphaGcT is a unique enzyme targeted by a natural antibiotic mucin and constitutes an excellent therapeutic target to prevent H. pylori-induced peptic ulcer, gastric carcinoma, and MALT lymphoma.     

Composition and function of Helicobacter pylori outer membrane vesicles

The gastric pathogen Helicobacter pylori sheds outer membrane vesicles (OMV) that possess many of the surface elements of the bacterium. Here we review current knowledge on the composition of H. pylori OMV and discuss evidence for their potential roles in bacterial survival and pathogenesis.     

Free-soluble and outer membrane vesicle-associated VacA from Helicobacter pylori: Two forms of release, a different activity

Helicobacter pylori releases VacA both as free-soluble and as outer membrane vesicle (OMV)-associated toxin. In this study, we investigated the amount of VacA released in each of the two forms and the role of each form in VacA-induced cell vacuolation in vitro. We found that: (1) free-soluble toxin accounted for about 75% of released VacA, while the remaining 25% was OMV-associated; (2) although OMV-associated VacA caused a statistically significant vacuolation, virtually all the vacuolating activity of a H. pylori broth culture filtrate was due to free-soluble VacA. While it is widely accepted that OMVs may represent an important vehicle for delivering virulence factors to the gastric mucosa, our results suggest that OMV-associated VacA could play a pathobiological role different from that of free-soluble toxin. This conclusion fits with mounting evidence that VacA exerts a large pattern of pathobiological effects among which cell vacuolation might not be the main one.     

Spectroscopic and kinetic studies of Nor1, a cytochrome P450 nitric oxide reductase from the fungal pathogen Histoplasma capsulatum

The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. Although macrophage production of the antimicrobial effector nitric oxide (NO) restricts H. capsulatum growth, the pathogen is able to establish a persistent infection. H. capsulatum contains a P450 nitric oxide reductase homologue (NOR1) that may be important for detoxifying NO during infection. To characterize the activity of this putative P450 enzyme, a 404 amino acid fragment of Nor1p was expressed in Escherichia coli and purified to homogeneity. Spectral characterization of Nor1p indicated that it was similar to other fungal P450 nitric oxide reductases. Nor1p catalyzed the reduction of NO to N₂O using NADH as the direct reductant. The K_M for NO was determined to be 20 μM and the k_cat to be 5000 min⁻¹. Together, these results provide evidence for a protective role of a P450 nitric oxide reductase against macrophage-derived NO.     

Involvement of nitric oxide in the gastroprotective effects of an aqueous extract of Pfaffia glomerata (Spreng) Pedersen, Amaranthaceae, in rats

The plants belonging to Pfaffia genus are used in folk medicine to treat gastric disturbances. This study examined the effects of an aqueous extract of Pfaffia glomerata (Spreng) Pedersen (AEP) on the gastrointestinal tract. Wistar rats were pretreated orally (p.o.) with the AEP (125, 250, 500 and 1000 mg.kg(-1)) before induction of ulcers by hypothermic restraint stress (HRS, 3 h restraint stress at 4 degrees C), ethanol (ET, 70%; 0.5 ml/animal; p.o.) or indomethacin (IND, 20 mg.kg(-1); s.c.). Control animals received water (C) or ranitidine (60 mg.kg(-1)) p.o. The AEP protected rats against HRS and ET-induced ulcers, but was not able to protect the gastric mucosa against IND-induced ulcers. When injected into the duodenal lumen, the AEP reduced total acidity and both basal and histamine-stimulated acid secretion in pylorus-ligated rats. In addition, gastric secretion from AEP-treated animals exhibited increased concentrations of nitrite and nitrate. Treatment of animals with L-NAME (120 mg.kg(-1), p.o.) prevented both the reduction of total acidity and the increase in NOx levels promoted by AEP treatment. In conclusion, AEP effectively protected the gastric mucosa and inhibited gastric acid secretion in rats, probably by involving the histaminergic pathway and an enhanced production of nitric oxide in the stomach.     

Insights from the molecular docking of curcumin to the virulent factors of Helicobacter pylori

The domains of virulent (Ureα/β, VacA-p55, and CagA) factors of Helicobacter pylori play a pivotal role in developmentalprocesses of numerous diseases including gastric cancer. The pharmacological role of curcumin indicates that it could regulate thesignaling of virulent factors by interacting with active domains. However, the controlling mechanism of the curcumin interactionsand the binding diversity on structural basis of virulent (Ureα/β, VacA-p55, and CagA) factors are unknown. Curcumin astherapeutic agent was filtered by using Lipinski rule׳s five and the druglikeness property for assessment of pharmacologicalproperties. Here outcome of molecular docking presented the 3-D structure of curcumin complex, that interacted with especiallyconserved residues of target domains. The structure revealed that the curcumin complexation with domains of these proteinsprovided structural insight into the diverse nature of proteins (Ureα/β, VacA-p55, and CagA) recognition. In silico studyelucidated that the broad specificity of curcumin was achieved by multiple binding mode mechanisms such as distinct hydrogenand hydrophobic interactions with involvement of binding energy. The higher score of curcumin in complexation with bothsubunits Ureα/β showed the stable binding, and less stability with VacA-p55 complexation with lower score. Curcumin exhibitedgood interaction with these targeted virulent factors, although extensive interactions of curcumin with Ureα/β subunits could havean important implication to prevent survival and colonisation of H. pylori in stomach.     

幽门螺杆菌检测技术研究进展及展望

幽门螺杆菌是(Helicobacter pylori,H. pylori)是导致人类多种胃肠疾病的主要病因,通过多种毒力因子导致各种疾病的发生和发展。鉴于目前幽门螺杆菌缺少商业化疫苗且多重耐药性问题日益严重,临床上对其根除效果不佳,因此,精准的检测技术是预防幽门螺杆菌感染的关键,也是评估感染后治疗效果的重要手段。幽门螺杆菌的检测方法主要包括尿素呼气试验、快速尿素酶试验、粪便抗原检测、血清学检查、内镜检查、组织学病理检查、聚合酶链式反应及细菌培养,每种方法在临床上皆存在优点与局限性。目前,对于幽门螺杆菌检测的“金标准”尚无统一定论,因此本文重点综述当前应用的幽门螺杆菌检测技术,分析其优点和局限性,并指明精准、快速且便捷的检测技术在流行病学调查等方面具备的优势,旨在为临床和研究提供科学的参考依据。     

Cytochrome bo(3) from Escherichia coli: the binding and turnover of nitric oxide

The reaction of nitric oxide (NO) with fast and reduced cytochrome bo(3)(cyt bo(3)) from Escherichia coli has been investigated. The stoichiometry of NO binding to cyt bo(3) was determined using an NO electrode in the [NO] range 1-14 microM. Under reducing conditions, the initial decrease in [NO] following the addition of cyt bo(3) corresponded to binding of 1 NO molecule per cyt bo(3) functional unit. After this "rapid" NO binding phase, there was a slow, but significant rate of NO consumption ( approximately 0.3molNOmol bo(3)(-1)min(-1)), indicating that cyt bo(3) possesses a low level of NO reductase activity. The binding of NO to fast pulsed enzyme was also investigated. The results show that in the [NO] range used (1-14 microM) both fast and pulsed oxidised cyt bo(3) bind NO with a stoichiometry of 1:1 with an observed dissociation constant of K(d)=5.6+/-0.6 microM and that NO binding was inhibited by the presence of Cl(-). The binding of nitrite to the binuclear centre causes spectral changes similar to those observed upon NO binding to fast cyt bo(3). These results are discussed in relation to the model proposed by Wilson and co-workers [FEBS Lett. 414 (1997) 281] where the binding of NO to Cu(B)(II) results in the formation of the nitrosonium (Cu(B)(I)-NO(+)) complex. NO(+) then reacts with OH(-), a Cu(B) ligand, to form nitrite, which can bind at the binuclear centre. This work suggests for the first time that the binding of NO to oxidised cyt bo(3) does result in the reduction of Cu(B).