Survival of Helicobacter pylori in well water

The mortality of a clinical Helicobacter pylori strain was assessed by inoculating it in untreated well water, filtered well water, and autoclaved well water. Two different temperatures (5 and 25 °C) were used during the experimental period. Because Escherichia coli is commonly used as indicator of faecal pollution of water, we compared the survival of H. pylori using E. coli as indicator of its persistence. H. pylori was not culturable 48 h after inoculation, whereas the population of E. coli, monitored at the same temperature, decreased slowly, especially in filtered water. In untreated water, both H. pylori and E. coli survived less well than in filtered and autoclaved water. In general the survival of H. pylori and E. coli was better in filtered water than in autoclaved water and the ability of H. pylori to survive several days in water at 5 °C is reported, supporting the observation that H. pylori survives better at 5 °C than at higher temperature. This suggests a possible faecal–oral transmission of H. pylori in the presence of a contaminated water.     

Aldobiouronic acid domains in Helicobacter pylori

Helicobacter pylori cell-surface glycans exert strong influences in host–microbe interplays and define the strain’s immunological signature. Envisaging the development of a carbohydrate-based vaccine against the gastroduodenal pathogen H. pylori, several clinical isolates are being screened for their cell-surface glycan profile. The present work concerns H. pylori clinical specimen PTAV79 that abundantly expressed amylose-like glycans. These polysaccharides were isolated in glycan-rich fractions resultant from phenol–water extractions and purified by Bio-Gel P2. Structural studies showed that the glycans are linked to glycerol and present aldobiouronic acid domains composed of [→3)-α-d-GlcA-(1→4)-α-d-Glc-(1→] repeating units. The amylose domains were constituted by an average of 19 Glc residues and the acidic moieties had an average number of 10 aldobiouronic acid repeating units. These polysaccharides were isolated in fractions that, although hydrophilic, were rich in stearic acid, strongly suggesting that they are present as glycerolipids anchored to cell-surface.     

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。     

Glyphosate-induced oxidative stress in rice leaves revealed by proteomic approach

Glyphosate is one of the most widely used herbicides in cereal-growing regions worldwide. In the present work, the protein expression profile of rice leaves exposed to glyphosate was analyzed in order to investigate the alternative effects of glyphosate on plants. Two-week-old rice leaves were subjected to glyphosate or a reactive oxygen species (ROS) inducing herbicide paraquat, and total soluble proteins were extracted and analyzed by two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. A total of 25 differentially expressed proteins were identified from the glyphosate treated sample, wherein 18 proteins were up-regulated and 7 proteins were down-regulated. These proteins had shown a parallel expression pattern in response to paraquat. Results from the 2-DE analysis, combined with immunoblotting, clearly revealed that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit was significantly decreased by the treatment of both herbicides. An increased accumulation of antioxidant enzymes including ascorbate peroxidase, glutathione S-transferase, thioredoxin h-type, nucleoside diphosphate kinase 1, peroxiredoxin and a superoxide dismutase [Cu–Zn] chloroplast precursor in the glyphosate-treated sample suggests that a glyphosate treatment possibly generates oxidative stress in plants. Moreover, a gene expression analysis of five antioxidant enzymes by Northern blot confirmed their mRNA levels in the rice leaves. A histo-cytochemical investigation with DAB (3,3-diaminobenzidine) to localize H₂O₂ and increases of the thiobarbituric acid reactive substances (TBARS) concentration revealed that the glyphosate application generates ROS, which resulted in the peroxidation and destruction of lipids in the rice leaves.     

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.     

Antimicrobial effects of antioxidants with and without clarithromycin on Helicobacter pylori

Increasing resistance to currently used antimicrobials has resulted in the evaluation of other agents that have antimicrobial activity against Helicobacter pylori. H. pylori American Type Culture Collection (ATCC) strain 49503 (a toxin-producing strain known to be associated with gastric cancer) was grown, a cell suspension prepared in 2 mL PBS and diluted 10-fold. One hundred L of this cell suspension was added to vitamin C 0.5%, vitamin E 0.5%, garcinol 100 g/mL, Protykin® (containing 50% rans-resveratrol) 100 g/mL and garcinol + Protykin® 100 g/mL in Lennox broth, and incubated for 16 h under microaerophilic conditions. Three replicates of 10 L from each 10^–7 dilution tube were plated, colonies were counted after 16 h, and growth of H. pylori was confirmed by the CLO® test. These colony counts were compared to control cultures without the addition of any antioxidants. The experiments were then repeated with the addition of 15 g/mL of clarithromycin to experimental and control samples. Enhanced killing of H. pylori by 37.6% was noted when vitamin C was added, which increased to 66% when clarithromycin was added, compared to controls (p < 0.05). With garcinol and Protykin® alone there was 91.4 and 87% killing of H. pylori, respectively, while a combination of garcinol + Protykin® resulted in 90.8% killing compared to controls (p < 0.05). When clarithromycin was added, there was 76.3% increased killing with garcinol alone, 55.3% with Protykin® alone, and 73.7% with garcinol + Protykin® compared to controls (containing clarithromycin) (p < 0.05). Vitamin E had no effect on H. pylori growth compared to controls. We conclude from this study that some antioxidants such as vitamin C, garcinol and Protykin®, but not vitamin E, may have potential as antimicrobial agents against H. pylori. (Mol Cell Biochem 270: 125–130, 2005)     

Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air–liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2–3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.     

Essential role of magnesium ion in water for colonization of Helicobacter pylori in 2-week-old miniature pigs

This study was designed to determine whether magnesium ion in water would influence the colonization of Helicobacter pylori in 2-week-old miniature pigs. Groups A (2 pigs) and B (1 pig) were both fed a milk diet dissolved in drinking water, Group C (2 pigs) was fed a milk diet dissolved in deionized distilled water (DDW), and Group D (1 pig) was fed a milk diet dissolved in DDW supplemented with MgCl2. Groups B, C, and D were all challenged with H. pylori, and Group A was not. Necropsy was performed on the pigs on postinfection Day 5, and biopsy specimens were taken from 16 sites of the stomach. H. pylori were recovered from 11 of 16 sites in Group B, 1 of 32 sites in Group C, and 13 of 16 sites in Group D. On the other hand, the degree of lymphocyte infiltration increased in the order of Group A < Group B < Group C < Group D. These observations suggest that magnesium ion in drinking water is essential for the colonization of H. pylori in the pig stomach. Possible mechanisms for the lymphocyte infiltration are discussed.     

Comparative Genome Analysis of Helicobacter pylori Strains

DNA macroarrays were used to characterize 17 Helicobacter pylori strains isolated in four geographic regions of Russia (Moscow, St. Petersburg, Kazan, and Novosibirsk). Of all genes, 1272 (81%) proved to occur in all strains and to constitute a functional core of the genome, and 293 (18.7%) were strain-specific and greatly varied among the H. pylori strains. Most (71%) of the latter had unknown functions; the remainder included restriction–modification genes (3–9%), transposition genes (2–4%), and genes coding for outer membrane proteins (2–4%). The Russian H. pylori strains did not differ in genome organization or in the number and distribution of strain-specific genes from strains isolated in other countries.     

Cytokine expression due to Helicobacter pylori in a tissue culture model

Helicobacter pylori, in recent years, has been recognized as the major causative agent in chronic gastritis and peptic ulcer disease in humans. H. pylori is a ubiquitous organism, with at least half of the world’s population infected. Of those individuals with peptic ulcer disease, it is estimated that 90% of cases are caused by H. pylori. Currently, the efficacy of therapies is starting to decline due to increasing resistance rates, especially towards clarithromycin. Due to this, new therapies are needed to combat this bacterium. It is hypothesized that cytokine release (especially interleukin-1β, -6, -8, and TNF-α) due to H. pylori infection and the subsequent influx of inflammatory cells causes a massive release of reactive oxygen species (ROS) during the inflammatory reaction. The ROS then cause the pathologic changes seen in the infected tissues. In this study, human gastric adenocarcinoma cell line ATCC 1739 (a cell line not previously evaluated) was examined for its production of interleukin-1β, -6, -8, and TNF-α when cocultured in a ratio of 10:1 H. pylori to adenocarcinoma cells, to determine its value as a model to demonstrate the inflammatory response. Results from this study indicated that ATCC 1739 cells only reliably produced IL-8 when cocultured with H. pylori and stimulated with TNF-α. The production of IL-1β, IL-6, and TNF-α by the ATCC 1739 cells was no different in H. pylori-exposed cells than non-exposed cells. It was concluded that the ATCC 1739 cell line is not suitable to study the effects of coculture with H. pylori on cytokine production.     

Oxidative stress by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> causes apoptosis through mitochondrial pathway in gastric epithelial cells

Helicobacter pylori is a gram negative bacterium that infects the human stomach of approximately half of the world’s population. It produces oxidative stress, and mitochondria are one of the possible targets and the major intracellular source of free radicals. The present study was aimed at determining mitochondrial alterations in H. pylori-infected gastric epithelial cells and its relationship with oxidative stress, one of the recognized causes of apoptotic processes. Cells were treated with a strain of H. pylori for 24 h. Cellular oxidative burst, antioxidant defense analysis, mitochondrial alterations and apoptosis-related processes were measured. Our data provide evidence on how superoxide acts on mitochondria to initiate apoptotic pathways, with these changes occurring in the presence of mitochondrial depolarization and other morphological and functional changes. Treatment of infected cells with Vitamin E prevented increases in intracellular ROS and mitochondrial damage consistent with H. pylori inducing a mitochondrial ROS mediated programmed cell death pathway.     

Cloning, expression, and purification of Helicobacter pylori L-asparaginase

Asparaginase from Helicobacter pylori (HpA) has been cloned and expressed in E. coli cells. The recombinant strain stably expressed catalytically active HpA. Optimization of culturing and expression conditions resulted in the expression level of the recombinant enzyme amounting up to 6% of total protein of the producer strain. A method developed for HpA purification included a single chromatographic stage and provided more than 60%-yield of the active enzyme. Specific asparaginase activity was 92 U/mg of protein, whereas the rate of glutamine hydrolysis was just 8.3 × 10^?3 U/mg, respectively. Data obtained indicate that due to low glutaminase specificity HpA may be employed as a non-toxic enzyme preparation for treatment of leukemia.     

Helicobacter pylori infection promotes methylation of WWOX gene in human gastric cancer

WW domain-containing oxidoreductase (WWOX), a tumor suppressor gene, was reported to be downregulated in gastric cancer and other tumors. However, the mechanism by which WWOX is inactivated remains unclear. In our study, methylation status of WWOX was determined by MSP and sequencing. Our results showed that WWOX hypermethylation was frequently detected in gastric cancer, and also significantly correlated with Helicobacter pylori (H. pylori) infection. Promoter methylation of WWOX was induced in BCG823 and AGS cells co-cultured with H. pylori. Finally, we found that expression of DNMT1 and DNMT3A were enhanced when cells were co-cultured with H. pylori. Our study indicated that H. pylori infection promoted methylation of WWOX gene in gastric cancer.     

Pyruvate metabolism in Helicobacter pylori

The metabolism of pyruvate by Helicobacter pylori was investigated employing one- and two-dimensional ¹H and ¹³C nuclear magnetic resonance spectroscopy. Generation of pyruvate from l-serine in incubations with whole cell lysates indicated the presence of serine dehydratase activity in the bacterium. Pyruvate was formed also in cell suspensions and lysates from phosphoenol pyruvate. Metabolically competent cells incubated aerobically with pyruvate yielded alanine, lactate, acetate, formate, and succinate. The production of alanine and lactate indicated the presence of alanine transaminase and lactate dehydrogenase activities, respectively. Accumulation of acetate and formate as metabolic products provided evidence for the existence of a mixed-acid fermentation pathway in the microorganism. Formation of succinate suggested the incorporation of the pyruvate carbon skeleton into the Kreb's cycle. Addition of pyruvate to various liquid culture media did not affect bacterial growth or loss of viability. The variety of products formed using pyruvate as the sole substrate showed the important role of this metabolite in the energy metabolism of H. pylori.     

The changes of proteomes components of Helicobacter pylori in response to acid stress without urea

Acid stress is the most obvious challenge Helicobacter pylori encounters in human stomach. The urease system is the basic process used to maintain periplasmic and cytoplasmic pH near neutrality when H. pylori is exposed to acidic condition. However, since the urea concentration in gastric juice is approximately 1 mM, considered possibly insufficient to ensure the survival of H. pylori, it is postulated that additional mechanisms of pH homeostasis may contribute to the acid adaptation in H. pylori. In order to identify the acid-related proteins other than the urease system we have compared the proteome profiles of H. pylori strain 26695 exposed to different levels of external pH (7.4, 6.0, 5.0, 4.0, 3.0, and 2.0) for 30 min in the absence of urea using 2-DE. Differentially expressed proteins were identified by MALDI-TOF-TOF-MS analysis, which turned out to be 36 different proteins. The functions of these proteins included ammonia production, molecular chaperones, energy metabolism, cell envelope, response regulator and some proteins with unknown function. SOM analysis indicated that H. pylori responds to acid stress through multi-mechanisms involving many proteins, which depend on the levels of acidity the cells encounter.     

Development of an enzyme-linked glycosorbent method to monitor the inhibition of sialic acid-dependent Helicobacter pylori adhesion

Horseradish peroxidase conjugation with fetuin, which expresses sialic acid-dependent binding specificity to Helicobacter pylori, was used to develop an enzyme-linked glycosorbent method. This method yielded results that were consistent with those from a hemagglutination assay using a microscope and allowed the quantitative analysis of inhibitors of sialic acid-dependent Helicobacter pylori adhesion to host cells. The results of inhibitor screening with carbohydrates, including commercially available polysaccharides and extracted from various sources, displayed not only the relative inhibition potencies among carbohydrates, but also their respective concentration-dependencies.     

Protein Evolutionary Rates Correlate with Expression Independently of Synonymous Substitutions in Helicobacter pylori

In free-living microorganisms, such as Escherichia coli and Saccharomyces cerevisiae, both synonymous and nonsynonymous substitution frequencies correlate with expression levels. Here, we have tested the hypothesis that the correlation between amino acid substitution rates and expression is a by-product of selection for codon bias and translational efficiency in highly expressed genes. To this end, we have examined the correlation between protein evolutionary rates and expression in the human gastric pathogen Helicobacter pylori, where the absence of selection on synonymous sites enables the two types of substitutions to be uncoupled. The results revealed a statistically significant negative correlation between expression levels and nonsynonymous substitutions in both H. pylori and E. coli. We also found that neighboring genes located on the same, but not on opposite strands, evolve at significantly more similar rates than random gene pairs, as expected by co-expression of genes located in the same operon. However, the two species differ in that synonymous substitutions show a strand-specific pattern in E. coli, whereas the weak similarity in synonymous substitutions for neighbors in H. pylori is independent of gene orientation. These results suggest a direct influence of expression levels on nonsynonymous substitution frequencies independent of codon bias and selective constraints on synonymous sites. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Expression screening of integral membrane proteins from Helicobacter pylori 26695

The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins < or =40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions.