Mechanism of action of low recurrence of gastritis caused by Helicobacter pylori with the type II urease B gene

Background. Low recurrence of gastritis is seen in patients infected with Helicobacter pylori carrying the type II urease B gene, compared with H. pylori carrying types I and III. The underlying mechanism has been studied in terms of the urease activity and interleukin (IL)‐8 production capacity of different strains of H. pylori. Materials and Methods. Forty‐five patients infected with different strains of H. pylori (type I; 15, type II; 15 and type III; 15) were enrolled in the study. H. pylori was isolated from gastric mucosa and cultured in the presence of urea at pH 5.5 to evaluate urease activity. The capacity of different strains of H. pylori to induce IL‐8 mRNA and IL‐8 from a human gastric cancer cell line and human peripheral blood mononuclear cells was evaluated. Results. The urease activity of type II H. pylori[523 ± 228 µg of ammonia/dl/10⁸ colony‐forming units (CFU)/ml] was significantly lower than that of type I (1355 ± 1369 µg of ammonia/dl/10⁸ CFU/ml) and type III (1442 ± 2229 µg of ammonia/dl/10⁸ CFU/ml) (p < .05). Gastric cancer cells cocultured with type II H. pylori produced lower levels of IL‐8 mRNA compared with type I and type III H. pylori. The levels of IL‐8 were also significantly lower in cultures induced by type II H. pylori compared with those induced by type I and type III H. pylori. Peripheral blood mononuclear cells also produced lower levels of IL‐8 when cocultured with type II compared with type I H. pylori. Conclusions. These results indicate that both the lower level of urease activity and the low IL‐8‐inducing capacity of type II H. pylori might underlie the lower recurrence rate of gastritis caused by type II H. pylori.     

Helicobacter pylori urease activates blood platelets through a lipoxygenase‐mediated pathway

The bacterium Helicobacter pylori causes peptic ulcers and gastric cancer in human beings by mechanisms yet not fully understood. H. pylori produces urease which neutralizes the acidic medium permitting its survival in the stomach. We have previously shown that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring platelet secretion, activation of calcium channels and lipoxygenase‐derived eicosanoids. We investigated whether H. pylori urease displays platelet‐activating properties and defined biochemical pathways involved in this phenomenon. For that the effects of purified recombinant H. pylori urease (HPU) added to rabbit platelets were assessed turbidimetrically. ATP secretion and production of lipoxygenase metabolites by activated platelets were measured. Fluorescein‐labelled HPU bound to platelets but not to erythrocytes. HPU induced aggregation of rabbit platelets (ED₅₀ 0.28 μM) accompanied by ATP secretion. No correlation was found between platelet activation and ureolytic activity of HPU. Platelet aggregation was blocked by esculetin (12‐lipoxygenase inhibitor) and enhanced ～3‐fold by indomethacin (cyclooxygenase inhibitor). A metabolite of 12‐lipoxygenase was produced by platelets exposed to HPU. Platelet responses to HPU did not involve platelet‐activating factor, but required activation of verapamil‐inhibitable calcium channels. Our data show that purified H. pylori urease activates blood platelets at submicromolar concentrations. This property seems to be common to ureases regardless of their source (plant or bacteria) or quaternary structure (single, di‐ or tri‐chain proteins). These properties of HPU could play an important role in pathogenesis of gastrointestinal and associated cardiovascular diseases caused by H. pylori.     

Identification of mixed genotypes in Helicobacter pylori from gastric biopsy tissue by analysis or urease gene polymorphisms

Abstract PCR-RFLP analysis of the urease A and B genes was used to investigate the frequency that Helicobacter pylori from gastric biopsy specimens comprised genotypically distinct strains. Seventy-five strains of H. pylori from 44 subjects from four geographically diverse locations were examined and 31 distinct urease gene profiles were identified. For most cultures, the totals of the RFLP sizes were within 4% of the 2.41-kb PCR product. Five strains were genomically more complex suggesting they contained a mixture of related genotypes. The validity of the test was confirmed by analysis of a known strain mixture and of single colony picks from biopsy cultures. Urease gene PCR-RFLP profiling was shown to be more sensitive than ribotyping for determining the genotypic homogeneity of H. pylori .     

Inverse nickel-responsive regulation of two urease enzymes in the gastric pathogen Helicobacter mustelae

The acidic gastric environment of mammals can be chronically colonized by pathogenic Helicobacter species, which use the nickel-dependent urea-degrading enzyme urease to confer acid resistance. Nickel availability in the mammal host is low, being mostly restricted to vegetarian dietary sources, and thus Helicobacter species colonizing carnivores may be subjected to episodes of nickel deficiency and associated acid sensitivity. The aim of this study was to investigate how these Helicobacter species have adapted to the nickel-restricted diet of their carnivorous host. Three carnivore-colonizing Helicobacter species express a second functional urea-degrading urease enzyme (UreA2B2), which functions as adaptation to nickel deficiency. UreA2B2 was not detected in seven other Helicobacter species, and is in Helicobacter mustelae only expressed in nickel-restricted conditions, and its expression was higher in iron-rich conditions. In contrast to the standard urease UreAB, UreA2B2 does not require activation by urease or hydrogenase accessory proteins, which mediate nickel incorporation into these enzymes. Activity of either UreAB or UreA2B2 urease allowed survival of a severe acid shock in the presence of urea, demonstrating a functional role for UreA2B2 in acid resistance. Pathogens often express colonization factors which are adapted to their host. The UreA2B2 urease could represent an example of pathogen adaptation to the specifics of the diet of their carnivorous host, rather than to the host itself.     

Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process

In this study, we examined the anti-Helicobactor pylori effects of the main protoberberine-type alkaloids in Rhizoma Coptidis. Coptisine exerted varying antibacterial and bactericidal effects against three standard H. pylori strains and eleven clinical isolates, including four drug-resistant strains, with minimum inhibitory concentrations ranging from 25 to 50?μg/mL and minimal bactericidal concentrations ranging from 37.5 to 125?μg/mL. Coptisine’s anti-H. pylori effects derived from specific inhibition of urease in vivo. In vitro, coptisine inactivated urease in a concentration-dependent manner through slow-binding inhibition and involved binding to the urease active site sulfhydryl group. Coptisine inhibition of H. pylori urease (HPU) was mixed type, while inhibition of jack bean urease was non-competitive. Importantly, coptisine also inhibited HPU by binding to its nickel metallocentre. Besides, coptisine interfered with urease maturation by inhibiting activity of prototypical urease accessory protein UreG and formation of UreG dimers and by promoting dissociation of nickel from UreG dimers. These findings demonstrate that coptisine inhibits urease activity by targeting its active site and inhibiting its maturation, thereby effectively inhibiting H. pylori. Coptisine may thus be an effective anti-H. pylori agent.     

高效表达幽门螺杆菌UreB重组蛋白工程菌的构建

克隆表达幽门螺杆菌(Hp)的尿素酶B亚单位(UreB)重组蛋白,可为Hp疫苗开发和快速诊断试剂盒的研究奠定基础。用PCR方法由幽门螺杆菌染色体DNA扩增UreB基因片段,将其融合插入原核表达载体pQE30中,并在M15大肠杆菌表达。经酶切、测序分析,包括部分融合载体基因在内的重组UreB基因片段由1773bp组成。为编码591个氨基酸残基的多肽。SDS-PAGE分析显示重组表达的目的蛋白相对分子量约为66kD,表达量点菌体总蛋白的23．5％,并经免疫印迹分析证实被幽门螺杆菌感染的阳性血清可与纯化UreB重组蛋白发生特异性的结合反应。UreB重组蛋白具有良好的抗原性,将有可能成为一种有效蛋白质疫苗以及快速诊断试剂盒用于Hp感染的防治和检测。     

Development and evaluation of a DNA vaccine based on Helicobacter pylori urease B: failure to prevent experimental infection in the mouse model

BACKGROUND: The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene (ureB). METHODS: The ureB gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, intramuscular, intrarectal (40 micro g each) and intranasal (16 micro g) route, three doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. Four weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. RESULTS: Despite in vitro confirmation of successful cloning and functionality of the ureB gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo. Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. CONCLUSION: A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations.     

幽门螺杆菌尿素酶B亚单位的克隆和序列分析

幽门螺杆菌是消化道疾病的主要致病菌。其有效抗原成份尿素酶B亚单位(UreB)可刺激机体产生保护性的免疫反应。用高保真PCR扩增系统扩增出UreB基因片段,将其克隆至质粒pUC19中,对其全序列进行了测定。克隆的UreB基因序列与报道的序列完全一致。这一研究获得了序列正确的UreB基因,为将来以UreB分子为抗原的疫苗研制工作打下了良好的基础。     

幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺的研究

通过不同培养基、不同葡萄糖浓度、不同溶氧条件、补料与非补料对幽门螺杆菌尿素酶B亚单位(UreB)基因工程菌的菌体生长与外源蛋白表达量的影响的比较 ,建立了稳定、适宜的幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺。多批实验结果证明 ,菌体单产可达 86 g/L ,目的蛋白的表达率为 38.2 %。     

Helicobacter pylori antibodies and gastric cancer: a gender-related difference

Helicobacter pylori has been proposed as a causative agent of gastric cancer. The aim of this study was to define serum antibodies response against different H. pylori antigens in patients with gastric cancer. Serum samples were collected from 115 Lithuanian patients with non-cardia gastric cancer and 110 age- and sex-matched controls without cancer. Heat-stable, low-molecular-mass, and outer membrane proteins were used as antigens to analyze serum IgG antibody response against H. pylori by enzyme-linked immunosorbent assay. Seroprevalence of H. pylori using low-molecular-mass antigen was significantly higher in gastric cancer patients, compared to controls (77% versus 57%, p<0.05). Significant differences in the prevalence of H. pylori infection between gastric cancer patients and controls were found in females using all three studied antigens: heat-stable (98% versus 84%, p<0.05), low-molecular-mass (88% versus 48%, p<0.05) and outer membrane proteins (78% versus 57%, p<0.05). In males, no significant differences were revealed between gastric cancer patients and controls. There may be other cofactors in addition to H. pylori that are important for the development of gastric cancer. H. pylori seems, however, to be a more important for development of gastric cancer in females than in males or males may have more confounding risk factors for gastric cancer than females.     

Helicobacter felis does not stimulate human neutrophil oxidative burst in contrast to 'Gastrospirillum hominis' and Helicobacter pylori

Helicobacter pylori is a human pathogen, whereas the natural hosts for 'Gastrospirillum hominis' and Helicobacter felis are animals. 'G. hominis' is occasionally found to cause infection in humans, whereas H. felis only rarely infects humans. The pathogenesis of H. pylori infection is not completely understood and in order to reveal differences in immune response to the three Helicobacter species, the upregulation of adherence molecule CD11b/CD18, chemotactic activity and oxidative burst response of neutrophils after stimulation with H. pylori, 'G. hominis' and H. felis sonicates, were compared. Like H. pylori, 'G. hominis' and H. felis induced upregulation of CD11b/CD18 and chemotaxis of neutrophils. 'G. hominis' demonstrated a more pronounced upregulation of CD11b/CD18, whereas H. felis was the strongest stimulant of neutrophil chemotaxis. H. felis was unable to stimulate neutrophils to oxidative burst response, whereas 'G. hominis' activated neutrophils in a dose-dependent way similar to H. pylori. 'G. hominis' and H. felis were both able to prime neutrophils for oxidative burst response similar to H. pylori. In conclusion, we observed clear differences in neutrophil responses to different Helicobacter species, which indicates that bacterial virulence factors may be important for the diversity in the pathogenetic outcome of Helicobacter infections.     

Urea, fluorofamide, and omeprazole treatments alter helicobacter colonization in the mouse gastric mucosa

BACKGROUND: Helicobacter pylori is a causative agent of gastric and duodenal ulcers and gastric cancer. Its urease enzyme allows survival in acid conditions and drives bacterial intracellular metabolism. We aimed to investigate the role of urease in determining the intragastric distribution of Helicobacter species in vivo. MATERIALS AND METHODS: The C57BL/6 mouse model of gastritis was used for infection with Helicobacter felis (CS1) or H. pylori (SS1). Urease-modulating compounds urea and/or fluorofamide (urease inhibitor) were administered to mice over 7 days. Concurrent gastric acid inhibition by omeprazole was also examined. Bacterial distribution in the antrum, body, antrum/body, and body/cardia transitional zones was graded "blindly" by histologic evaluation. Bacterial colony counts on corresponding tissue were also conducted. RESULTS: Urease inhibition by fluorofamide decreased H. pylori survival in most gastric regions (p < .05); however, there were no marked changes to H. felis colonization after this treatment. There was a consistent trend for decreased antral colonization, and an increase in antrum/body transitional zone and body colonization with excess 5% or 6% (w/v) urea treatment. Significant reductions of both Helicobacter species were observed with the co-treatment of urea and fluorofamide (p < .05). Collateral treatment with omeprazole did not alter H. pylori colonization patterns caused by urea/fluorofamide. CONCLUSIONS: Urease perturbations affect colonization patterns of Helicobacter species. Combined urea and fluorofamide treatment reduced the density of both Helicobacter species in our infection model.     

Prevention and suppression of Helicobacter felis infection in mice using colostral preparation with specific antibodies

Background. Specific antibodies against Helicobacter were enriched from the colostra of hyperimmunized cows. Efficacies of colostral control preparation and immune preparation containing specific antibodies against Helicobacter felis were studied in the prevention and treatment of experimental H. felis infection in mice. Materials and Methods. H. felis‐infected mice were given either immune or control preparation with or without complement or amoxicillin orally in four different trials. H. felis status was assessed on the basis of bacterial stainings, gastric histology and serum antibodies. Results. Immune, but not control preparation, prevented H. felis infection (p > 0.01), the efficacy being dependent on the presence of specific antibodies. In the trial on infected Balb/c mice treatment with immune preparation (p = 0.029) but not control preparation decreased the colonization of gastric antrum by H. felis. In the further trials with infected SJL‐mice, treatments with colostral preparations did not decrease colonization. Amoxicillin treatment decreased the colonization with trend‐setting significance (p = 0.056; infected mice as controls), whereas amoxicillin combined with immune preparation had a significant effect (p < 0.0005). Conclusions. Specific colostral antibodies were useful in the prevention of Helicobacter infection in a mouse model. The results of the treatment trials were controversial but a similar colostral immune preparation against H. pylori could be effective and useful in preventing infections in humans and during antibiotic treatment.     

Cloning of Helicobacter pylori urease subunit B gene and its expression in tobacco (Nicotiana tabacum L.)

Vaccines produced by transgenic plants would have the potential to change the traditional means of production and inoculation of vaccines, and to reduce the cost of vaccine production. In the present study, an UreB antigen gene from a new Helicobacter pylori strain ZJC02 was cloned into the binary vector pBI121 which contains a CaMV35S promoter and a kanamycin resistance gene, and then transformed UreB into tobacco leaf-disc by Agrobacterium-mediated method. A total of 50 regenerated plants with kanamycin resistance were obtained in the selection media. The 35 putative transgenic individuals were tested and verified the presence and integration of the UreB into the nuclear genome of tobacco plants by PCR, PCR-southern, and Southern analyses. Expression of UreB gene in the tobacco plants was confirmed by RT-PCR and Western Blot analysis using polyclonal human antiserum. To our knowledge, this is the first report of the expression of Helicobacter pylori UreB antigen gene in a plant system, suggesting a major step in the production of plant-based vaccines for Helicobacter pylori.