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.     

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。     

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。     

13C-urea breath test results in pigs challenged with Helicobacter pylori or other urease producing bacteria

The gastric bacterial flora and its influence on the 13C-urea breath test (UBT) for detection of Helicobacter pylori infection was studied in a pig model. Seven SPF minipigs were used. H. pylori or a mix of other urease positive bacteria were administered orally. UBT, serum and biopsies for histology and culture were collected. Our results show that UBT is not specific for H. pylori in pigs as the gastric bacterial flora is responsible for the high UBT values observed. Furthermore, the Ellegaard G?ttingen SPF minipigs are not useful in an animal model for H. pylori studies.     

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.     

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.     

幽门螺杆菌尿素酶单克隆抗体的研制及鉴定

应用杂交瘤技术 ,建立稳定分泌抗幽门螺杆菌尿素酶单克隆抗体 (HPU McAb)的细胞系。用纯化幽门螺杆菌尿素酶 (HPU)抗原加福氏佐剂免疫BALB/c小鼠 ,按常规方法进行细胞融合 ,以纯化HPU抗原包被 ,间接ELISA方法筛选 ,并经多次有限稀释法克隆。获得 6株抗HPU的杂交瘤 ,腹水效价达 1∶6 .4× 10 4～ 1∶2 .5 6× 10 5,特异性专一 ,并对其进行体内外连续传代 3个月 ,分泌抗体能力稳定。IgG亚类分型主要为IgG1型。该细胞系能稳定分泌幽门螺杆菌尿素酶单抗。     

幽门螺杆菌尿素酶的纯化及其特性

本文利用Ｓｅｐｈａｃｒｙ１ Ｓ－２００和Ｑ－Ｓｅｐｈａｒｏｓｅ两步层析,从ＨＰ蒸馏水浸液中提取纯化尿素酶,并对其特性进行了测定,证明ＨＰ尿素酶各含一个６６ｋＤ和２９．５ｋＤ的亚单位,并以６个分子聚合成６２５ｋＤ大分子蛋白,有很好的抗原性,并显示特异的血清学反应。用ＨＰ超声浸液抗原和尿素酶,加入２μｇ霍乱毒素粘膜佐剂,给ＳＰＦ ＢＡＬＢ／Ｃ小鼠口服免疫,可使８０％小鼠抵抗ＨＰ活菌的攻击,证明尿素酶是一种抗ＨＰ的保护性抗原。     

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。     

Animal models for gastric Helicobacter immunology and vaccine studies

Over the last decade animal models have been used extensively to investigate disease processes and therapy for Helicobacter pylori infections. The H. pylori animal models which have been used in pathogenesis and vaccine studies include the gnotobiotic pig, non-human primates, cats, dogs, and several species of rodents including mice, rats, gerbils and guinea pigs. H. felis infection of mice and H. mustelae infection of ferrets have also been used. Recently, investigators have begun using transgenic mice and gene-targeted 'knock-out' mice to investigate Helicobacter infections. Each of these animal models has distinct advantages and disadvantages which are discussed in this minireview. The choice of an animal model is dictated by factors such as cost and an understanding of how each model will or will not allow fulfillment of experimental objectives.     

The urease structural gene ureA in Rhizobium meliloti is preceded by an open reading frame necessary for urease activity

Abstract An open reading frame (ORF1) located upstream of the urease structural gene ureA in Rhizobium meliloti strain AK631 was cloned and characterized by DNA sequencing. Comparison of the amino acid sequence revealed partial homology with the urease accessory gene ureD of Klebsiella aerogenes and Proteus mirabilis . Mutational analysis of ORF1 showed that the gene is necessary for urease activity. Its function is still unknown.     

Urea uptake and urease activity in Corynebacterium glutamicum

When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 × 10^–7 cm s^–1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a K _m for urea of 9 μM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate (K _m approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2–3.5 nmol min^–1 (mg dry wt.)^–1]. Received: 11 August 1997 / Accepted: 2 December 1997     

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.     

Salivary specific IgG is a sensitive indicator of the humoral immune response to Helicobacter pylori

Abstract In humans, salivary antibodies are secreted during humoral immune response. Helicobacter pylori infection is associated with systemic humoral immune response reflected by raised serum levels of specific IgG. The present study was aimed at exploring whether salivary concentrations of specific H. pylori IgG are a reliable indicator of H. pylori infection. Serum and salivary samples were obtained from 291 subjects attending the GI clinic and tested for H. pylori -specific IgG by a direct ELISA (94% sensitivity, 95% specificity for serum determinations) using a crude H. pylori sonicate as antigen. Data are given as optical density (mean±S.D.). Levels of salivary H. pylori IgG paralleled those of circulating specific IgG in the 291 subjects studied (0.981±0.431 vs. 0.777±0.682, respectively). A significant positive correlation was found between specific H. pylori IgG in sera and saliva samples (r = 0.981, P < 0.0001). An overall concordance between circulating and salivary H. pylori IgG was observed in 238 out of the 291 (81.7%) subjects. Salivary H. pylori IgG represent a sensitive marker of specific humoral immune response and they may substitute circulating H. pylori IgG measurement when sera samples are not available.     

枸橼酸铋雷尼替丁+克拉霉素7天根除幽门螺旋杆菌疗效观察

目的观察枸橼酸铋雷尼替丁(RBC) 克拉霉素根除幽门螺旋杆菌疗效。方法RBC 350Mg 克拉霉素500mg口服,每天2次,共7d。结果治疗后Hp的根除率为86．5％。结论RBC 克拉霉素7d根除Hp效果满意。     

Recombinant antigen from Helicobacter pylori urease as vaccine against H. pylori-associated disease

It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit. An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H. pylori urease and by a homology-based study of the urease of other bacteria and plants. The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST). Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen. The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease. About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease. Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa. The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H. pylori. Moreover, the antiserum suppressed the enzymatic activity of H. pylori urease, while the purified H. pylori urease could not induce such an antiserum.     

N-monoarylacetothioureas as potent urease inhibitors: synthesis,SAR, and biological evaluation

Abstract

A urease inhibitor with good in vivo profile is considered as an alternative agent for treating infections caused by urease-producing bacteria such as Helicobacter pylori. Here, we report a series of N-monosubstituted thioureas, which act as effective urease inhibitors with very low cytotoxicity. One compound (b19) was evaluated in detail and shows promising features for further development as an agent to treat H. pylori caused diseases. Excellent values for the inhibition of b19 against both extracted urease and urease in intact cell were observed, which shows IC₅₀ values of 0.16?±?0.05 and 3.86?±?0.10?µM, being 170- and 44-fold more potent than the clinically used drug AHA, respectively. Docking simulations suggested that the monosubstituted thiourea moiety penetrates urea binding site. In addition, b19 is a rapid and reversible urease inhibitor, and displays nM affinity to urease with very slow dissociation (k _off=1.60?×?10^?3 s^?1) from the catalytic domain.