Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.     

幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(UreB),研发基于UreB的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rUreB)基因的重组表达载体pET-rUreB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化重组尿素酶B亚基(rUreB),并进一步通过腹腔注射免疫BALB/c小鼠,研究rUreB的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体pET-rUreB及其重组菌株BL21(DE3)/pET-rUreB,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rUreB。重组蛋白rUreB辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和UreB的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。     

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。     

普通Taq DNA聚合酶扩增幽门螺杆菌尿素酶基因长片段

ＰＣＲ反应扩增较长的ＤＮＡ片段比较困难。本实验对幽门螺杆菌染色体ＤＮＡ上的２７Ｋｂ尿素酶基因用普通ＴａｑＤＮＡ聚合酶进行扩增,扩增结果以琼脂糖凝胶电泳证实,得到所需的２７Ｋｂ的片段。该实验为今后幽门螺杆菌工作的进一步研究提供了经济,有效,简捷的条件。     

Virulence genotypes and drug resistance of Helicobacter pylori from Vladivostok, Russia: another feature in the Far East

Helicobacter pylori in Vladivostok, Far Eastern Russia, was investigated during 2004 to 2009. The genotype cagA(+) vacA(+) (s1/m1 or m2) accounted for 74.7%, with cagA(-) vacA(+) (s2/m2) at 11.2%. The CagA EPIYA type was mainly Western ABC, with minor types (ABCCC and novel AAABC) or non-Western/non-East Asia type (AB). Regarding drug resistance, metronidazole resistance was the highest, with a marked decrease in 6 years (from 71.4% to 30.8%); in contrast, levofloxacin and clarithromycin resistance increased. The data indicate that in Vladivostok, H. pylori was mainly the Western (not East Asian) type and dynamic changes in drug resistance occurred during 6 years.     

Kinetic properties of Helicobacter pylori urease compared with jack bean urease

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.     

Mutational analysis of the Helicobacter pylori carbonic anhydrases

In the gastric microenvironment, Helicobacter pylori is exposed to bicarbonate, urea and acid. Here it is demonstrated that both H. pylori carbonic anhydrases (CAs) are required for maintaining urease activity and therefore influence H. pylori urea resistance at neutral pH. Furthermore, the beta-CA is required for acid resistance as indicated by a growth defect of the corresponding mutant at low pH. The alpha- and beta-CA mutants as well as the double mutant were more resistant to bicarbonate, indicating that both enzymes are involved in bicarbonate metabolism. These phenotypes support important CA-functions in H. pylori urea and bicarbonate metabolism and acid resistance. Thus, both CA enzymes might be required for survival in the gastric niche.     

A defined human gastrin sequence stimulates the growth of Helicobacter pylori

This study describes the interaction between gastrin and Helicobacter pylori. Human gastrin amino acids 4-17 were found to be the minimal growth-stimulating sequence. Gastrin from other mammals did not stimulate bacterial growth. When human serum was used to stimulate bacterial growth in brucella broth, gastrin was shown to be a necessary and sufficient growth-stimulating factor. Competition for the gastrin effect by pentagastrin and cholecystokinin (CCK-8) resulted in inhibition of bacterial growth. This effect was mediated by the four C-terminal amino acids which are shared by gastrin, CCK-8 and pentagastrin. In conclusion, the interaction between gastrin and H. pylori was shown to be specific, essential, and dependent on a defined gastrin sequence.     

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 .     

幽门螺杆菌在胃内定植的相关影响因素

胃内定植是引起幽门螺杆菌（Helicobacter pylori,H. pylori）感染的先决条件。H. pylori可穿过胃黏液层并与胃上皮细胞相互作用。这个定植过程主要受到H. pylori动力和尿素酶的影响。同时H. pylori形态、胃内pH、外膜蛋白及益生菌等也在其中扮演重要角色。该研究主要对H. pylori胃内定植过程中的相关影响因素进行综述。     

Single nucleotide polymorphisms of Helicobacter pylori dupA that lead to premature stop codons

Background: The detection of the putative disease‐specific Helicobacter pylori marker duodenal ulcer promoting gene A (dupA) is currently based on PCR detection of jhp0917 and jhp0918 that form the gene. However, mutations that lead to premature stop codons that split off the dupA leading to truncated products cannot be evaluated by PCR. Methods: We directly sequence the complete dupA of 75 dupA‐positive strains of H. pylori isolated from patients with gastritis (n = 26), duodenal ulcer (n = 29), and gastric carcinoma (n = 20), to search for frame‐shifting mutations that lead to stop codon. Results: Thirty‐four strains had single nucleotide mutations in dupA that lead to premature stop codon creating smaller products than the predicted 1839 bp product and, for this reason, were considered as dupA‐negative. Intact dupA was more frequently observed in strains isolated from duodenal ulcer patients (65.5%) than in patients with gastritis only (46.2%) or with gastric carcinoma (50%). In logistic analysis, the presence of the intact dupA independently associated with duodenal ulcer (OR = 5.06; 95% CI = 1.22–20.96, p = .02). Conclusion: We propose the primer walking methodology as a simple technique to sequence the gene. When we considered as dupA‐positive only those strains that carry dupA gene without premature stop codons, the gene was associated with duodenal ulcer and, therefore, can be used as a marker for this disease in our population.     

Intrafamilial spread of Helicobacter pylori: a genetic analysis

Background. A high incidence of Helicobacter pylori among family members of children with H. pylori gastritis has previously been documented on biopsy material. The main objective of this study was the genetic clarification of H. pylori strains involved in intrafamilial dispersion. Materials and Methods. Formalin‐fixed, paraffin‐embedded material of antral mucosa from 32 members of 11 families was studied for the presence of genetic homogeneity. To achieve this goal, the entire genome of H. pylori was studied by the polymerase chain reaction (PCR)‐based random amplified polymorphic DNA (RAPD) fingerprinting method. Furthermore, the Urease A gene was analyzed using a multiplex PCR‐assay and an alternative mutation detection method based on the Hydrolink? analysis. Results. RAPD fingerprinting confirmed that closely related H. pylori strains were involved in the intrafamilial dispersion. Mutations and small deletions in Urease A gene were found in 22 out of 32 individuals. Conclusions. The homology of the H. pylori genome in members of the same family strongly supports the hypothesis of transmission of H. pylori from person‐to‐person or from a common source.     

Amoxicillin resistance in Helicobacter pylori: studies from Tokyo, Japan from 1985 to 2003

BACKGROUND: Previous reports revealed no resistant strains of amoxicillin (AMPC), which is usually used in eradication therapy for H. pylori infection. However, the frequency and evolution of natural AMPC-resistant strains in the Japanese population remains unknown. AIM: To assess the prevalence of H. pylori resistance against AMPC in the Tokyo area, a collection of 648 H. pylori strains isolated from patients with GI diseases from 1985 to 2003 was tested for their sensitivity to AMPC. METHODS: The susceptibility of the strains was assessed by determination of the minimal inhibitory concentration (MIC) using the E-test and/or the Dry-plate method. The susceptibility breakpoints of AMPC for H. pylori were: sensitive (AMPC-S); MIC < 0.04 microg/ml, intermittent resistance (AMPC-I); 0.04-1, resistant (AMPC-R); > 1. RESULTS: No AMPC-R strains were detected in the strains isolated between 1985 and 1996, while the rate of resistance was determined to be 1.1%, 2.1%, 5.4%, 5.6%, 0%, 8.8%, and 1.5% every year, respectively, from 1997 to 2003. The percentage of AMPC-I strains increased from 2000 to 2003. The total eradication rate of H. pylori in the patients who received triple therapy containing AMPC was 81.4% (214/263). Classified as above, the rates of AMPC-S, AMPC-I, and AMPC-R were 84.6%, 77.8%, 25%, respectively. CONCLUSION: H. pylori resistance to AMPC is still rare in Japan, although the percentage of AMPC-I strains has increased over the last 4 years. The frequency of isolation of strains showing true resistance to AMPC may increase in the future, along with an increase in the frequency of isolation of AMPC-I strains.     

Antibiotic resistance of Helicobacter pylori in Mazandaran, North of Iran

Background: The aim of this study was to investigate the prevalence of resistances in Helicobacter pylori against commonly used antibiotics including metronidazole, clarithromycin, amoxicillin, and tetracycline in Iranian patients. Methods: H. pylori isolates were collected from gastric biopsies from patients referred for upper gastrointestinal endoscopy at Tooba Medical Center, Sari, Iran, from 2007 to 2010. None of them had been using antibiotics for at least 8 months. H. pylori was identified based on morphological shape and positive biochemical tests for catalase, oxidase, and urease activity. Antibiotic resistance for metronidazole, clarithromycin, amoxicillin, and tetracycline was investigated by using epsilometer test. Resistance was defined by minimal inhibitory concentration (MIC) > 0.5 mg/L for amoxicillin (AMX), >4 mg/L for tetracycline (TET), >8 mg/L for metronidazole (MTZ), and >1 mg/L for clarithromycin (CLR). Results: Strains were collected from 132 patients, mean age 45.8 years, 52 (39%) were women. Patients had diverse diagnoses: gastritis 42 (31.8%), duodenal ulcer 45 (34%), gastric cancer 15 (11.3%), or gastric ulcer 30 (22.7%). The prevalences of resistance of H. pylori strains isolated from the patients were 73.4% for metronidazole, 30% for clarithromycin, 6.8% for amoxicillin, and 9% for tetracycline. Twenty‐eight (21.2%) were double resistant to MTZ‐CLR, 16 (12.1%) showed triple resistance to MTZ‐CLR‐AMX, and 8 (6%) were resistant to all four tested antibiotics (MTZ‐CLR‐AMX‐TET). No associations were detected between multiple resistant strains and clinical manifestations (p > .05). Conclusions: The prevalence of H. pylori antibiotic resistance to metronidazole and clarithromycin was high in Iran consistent with the reported low success rates for H. pylori treatment in this country.     

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。     

Molecular diagnosis of Helicobacter pylori antibiotic resistance in the Taif region,Saudi Arabia

Success in eradication of Helicobacter pylori is declining globally because H. pylori has developed resistance against most of the antibiotics proposed for eradication regimens, mainly through point mutations. The present study included 200 patients with dyspepsia attending Taif Hospital. Gastric biopsies were obtained during gastroscopy and subjected to rapid urease testing. Molecular methods were used to confirm diagnoses of H. pylori infection and to identify resistance gene variants of four antibiotics; namely, clarithromycin, metronidazole, fluoroquinolones and tetracycline (23S rRNA, gyrA, rdxA and 16S rRNA respectively). Of all investigated patients, Molecular diagnoses were made in 143 of all investigated patients; thus, the prevalence was .5%. The overall rate of resistance to clarithromycin among the H. pylori‐positive patients was high (39.9%) and the rate of resistance significantly greater (48.2%) among the secondary resistance group, secondary resistance being defined as resistance as a result of previous exposure to the relevant antibiotic. The rate of resistance to fluoroquinolones was considered moderate; the difference in rate of resistance between the primary and secondary resistance groups (8.4% and 9.5%, respectively) was not significant Also, there was a low prevalence of both primary and the secondary tetracycline resistance in the study cohort. In contrast, the prevalence of metronidazole resistance was considered high with no significant difference between the two resistance groups. H. pylori showed an increased prevalence of resistance to all four of the commonly used therapeutic agents. Thus, eradication therapy should be based on the regional results of susceptibility testing. Moreover, treatment tailored according to individually determined H. pylori susceptibility may be a reasonable future goal.     

Monocyte and Macrophage Killing of Helicobacter pylori: Relationship to Bacterial Virulence Factors

Background:  Helicobacter pylori infection is an important health problem, as it involves approximately 50% of the world's population, causes chronic inflammatory disease and increases the risk of gastric cancer development. H. pylori infection elicits a vigorous immune response, but this does not usually result in bacterial clearance. We have investigated whether the persistence of H. pylori in the host could be partly due to an inability of macrophages to kill this bacterium.
Materials and Methods:  Monocytes and macrophages isolated from the peripheral blood of normal human controls were infected in vitro with five H. pylori isolates. The isolates were characterized for known H. pylori virulence factors; vacuolating cytotoxin (VacA), the cag pathogenicity island ( cag PAI), urease, and catalase by Western blot and polymerase chain reaction analysis. The ability of primary human monocytes and macrophages to kill each of these H. pylori strains was then defined at various time points after cellular infection.
Results:  The five H. pylori strains showed contrasting patterns of the virulence factors. There were different rates of killing for the bacterial strains. Macrophages had less capacity than monocytes to kill three H. pylori strains. There appeared to be no correlation between the virulence factors studied and differential killing in monocytes.
Conclusions:  Primary human monocytes had a higher capacity to kill certain strains of H. pylori when compared to macrophages. The VacA, cag PAI, urease, and catalase virulence factors were not predictive of the capacity to avoid monocyte and macrophage killing, suggesting that other factors may be important in H. pylori intracellular pathogenicity.     

Investigation of Helicobacter pylori ascorbic acid oxidating activity

Abstract Helicobacter pylori sonicate was shown to oxidize ascorbic acid. Ascorbic acid oxidation was determined by chromatography combined with electrochemical detection. Water soluble ascorbic acid oxidase activity was rather independent of pH with a pH optimum around 2. By gel filtration the oxidizing activity co-eluted with an absorbency peak at 408 nm. The relative molecular mass ( M _r) was approximately 14000. It is suggested that this oxidating activity was caused by a cytochrome c -like molecule. Ascorbic acid oxidating activity could also be extracted from bacterial membranes by detergents. Gel filtration showed several forms, the major one with a M _r= 19000. pH optimum was 6–7. Other oxidase-positive bacterial strains like Campylobacter coli, Enterobacter cloacae and Pseudomonas aeruginosa could degrade ascorbic acid. Since ascorbic acid oxidation by Helicobacter pylori whole bacterial lysates has a pH optimum in the acidic range corresponding to pH in gastric fluid, the activity of the cytochrome c -like water soluble oxidant of Helicobacter pylori seems to be primarily important for the destruction of ascorbic acid in the gastric juice of infected patients.     

Differentiation between isolates of Helicobacter pylori by PCR-RFLP analysis of urease A and B genes and comparison with ribosomal RNA gene patterns

Abstract Genetic diversity amongst 21 human gastric isolates of Helicobacter pylori was investigated by polymerase chain reaction amplification and Hae III digest (restriction fragment length polymorphism) analysis of an internal 2.4-kb segment of the urease A and urease B genes. H. pylori from 11 independent individuals yielded nine distinct restriction fragment patterns but only one pattern was common to H. pylori from two individuals. By contrast, multiple isolate sets of H. pylori from two patients each had common urease gene patterns. Most strains with the same urease gene patterns were distinguishable in their ribosomal RNA gene patterns. The study demonstrated diversity amongst H. pylori and established that PCR analysis of urease genes provided a novel method of identifying isolates. The profiles were reproducible and convenient to obtain and analyse, and were almost as discriminatory as Hae III ribopatterns.