重组幽门螺杆菌ureA,ureB低温表达、纯化及抗原鉴定

目的：建立经济有效的方法,从大肠杆菌表达,纯化重组幽门螺杆菌尿素酶A、B亚单位。方法：含重组表达质粒pGEX-2T/ureA,pGEX-2T/ureB大肠杆菌分别在不同浓度IPTG、不同温度条件下作诱导培养,以SDS-PAGE分析表达产物,采用谷胱甘肽-Sepharose 4B亲和层分离纯化表达产物,以Western-blotting和ELISA对纯化产物作抗原性分析鉴定。结果：IPTG诱导浓度为0.1mmol/L;诱导温度为28℃,含重组质粒大肠杆菌表达可溶性融合蛋白GST-ureA、GST-ureB各约占总表达产物的78%和87%,经亲和层析,得纯度90%以上的GST-ureA约为14mg/L。GST-ureB约为18mg/L;融合蛋白呈ureA、ureB抗原性反应。结论：建立了低温诱导及纯化高纯度ureA、ureB基因表达产物的方法,所得纯化产物具有ureA、ureB抗原活性,为进一步的应用研究打下基础。     

Acidic conditions enhance bactericidal effects of sodium bisulfite on Helicobacter pylori

BACKGROUND: The Brucella broth medium, which is often used for the cultivation of microaerobic bacteria including Helicobacter pylori. It contains sodium bisulfite to decrease oxygen content in the medium. The growth of H. pylori, however, is inhibited by sodium bisulfite. In this study, the effect of sodium bisulfite was compared with several antioxidants and quantified under acidic conditions, mimicking the gastric environment. METHODS: Growth of H. pylori in the presence of several antioxidants was evaluated at OD655 nm. Effect of sodium bisulfite on H. pylori under acidic conditions was evaluated by measuring colony forming units (cfu). RESULTS: Under neutral conditions, sodium bisulfite was a more potent suppressor of H. pylori. Resveratrol, a polyphenol found in wine, exhibited the most potent inhibitory activity. To quantify the effect of sodium bisulfite on H. pylori under acidic conditions, the bacteria were grown at 37 degrees C for 30 minutes in 0.15 mol/l HCl/KCl (pH 2.0) with or without urea and sodium bisulfite. Sodium bisulfite (0.5 mmol/l) did not affect the viability at neutral pH 7.0, however, it killed H. pylori under acidic conditions, even if urea, the key substance enabling H. pylori to survive under acidic conditions, was present. The bacteria, which had been incubated under acidic conditions in the presence of urea, could survive a subsequent 30 minute-incubation at pH 2.0 without urea. Presence of sodium bisulfite, however, in the subsequent 30 minute-incubation, killed the bacteria. CONCLUSIONS: The bactericidal effect of sodium bisulfite on H. pylori was greater under acidic conditions and independent of urease activity.     

幽门螺杆菌尿素酶B单克隆抗体的制备与鉴定

目的:制备稳定分泌抗幽门螺杆菌尿素酶B单克隆抗体(mAb)的杂交瘤细胞系,并对其分泌的mAb进行鉴定。方法:用初步纯化的重组幽门螺杆菌尿素酶B免疫BALB/c小鼠,利用杂交瘤技术制备抗尿素酶B的mAb,用间接ELISA检测mAb的特异性和亲和力,检测mAb腹水效价,鉴定Ig亚类并测定其抗原决定簇。结果:获得8株能稳定分泌抗尿素酶B的mAb杂交瘤细胞系,这8株单抗与能产生尿素酶的小肠结肠耶尔森氏菌、肺炎克雷伯氏菌和普通变形杆菌均无交叉反应,相对亲和力为1.13×10-8～4.66×10-10,腹水mAb效价可达2×104～3.2×105。其中2株单抗属IgG1亚类,3株单抗属IgG2a亚类。8株单抗分属于3种不同的抗原决定簇。结论:获得了IgG1和IgG2a类型的针对3种不同抗原决定簇的特异性幽门螺杆菌尿素酶B的mAb,为进一步用于幽门螺杆菌的临床诊断和实验研究创造了条件。     

Bacterial factors that mediate colonization of the stomach and virulence of Helicobacter pylori

Helicobacter pylori is a Gram-negative microaerophilic organism that colonizes the gastric mucosa of humans. Helicobacter pylori is one of the most common infections in humans and results in the development of gastritis in all infected individuals, although the majority of people are asymptomatic. A subset of infected people develop serious disease including duodenal ulceration and gastric cancer. Helicobacter pylori exhibits many striking characteristics. It lives in the hostile environment of the stomach and displays a very strict host and tissue tropism. Despite a vigorous immune response, infection persists for the lifetime of the host unless eradicated with antimicrobials. Why H. pylori is so pathogenic in some individuals and not in others is unknown but is thought to be due to a variety of host, environmental and bacterial factors. In this review, some of the bacterial factors that mediate colonization of the gastric mucosa and play a role in the pathogenesis of this organism have been considered.     

Potential antigen candidates for subunit vaccine development against Helicobacter pylori infection

Helicobacter pylori (H. pylori) is a resident bacterium in the stomach that accounts for 75% cases of gastric cancer. In this review, we comprehensively studied published papers on H. pylori vaccines using Google Scholar and NCBI databases to gather information about vaccines against H. pylori. Considering the pivotal roles of the enzyme urease (in production of NH₃ and neutralization of the acidic medium of the stomach), cytotoxin-associated gene A, and vacuolating cytotoxin A proteins in H. pylori infection, they could be the best candidates for the construction of recombinant vaccines. The outer membrane porins (Hop), blood group antigen-binding adhesin (BabA), sialic acid-binding adhesin (SabA), and outer inflammatory protein A, play significant roles in binding of bacterium to human gastric tissues, and because binding is the first step in bacterial fixation and colonization, these antigens also can be considered as suitable candidates for designing vaccines. Likely, other significant bacterial antigens, such as NapA (chemotactic factor for recruitment of human neutrophils and monocytes to the site of infection), duodenal ulcer promoting protein A (to promote duodenal ulcer), and Hsp60 (as a molecular chaperon for activation of urease enzyme), can be used in the construction of subunit vaccines. New vaccines in use currently, such as DNA vaccines and subunit vaccines, can efficiently replace the dead and attenuated vaccines. Nonetheless, the results show that urease enzyme is most used compared with bacterial components in the designing and construction of recombinant vaccines. The BabA and SabA antigens belong to the outer membrane porins family in H. pylori and are required for binding and fixation of the bacterium to the human gastric tissues.     

Contribution of dppA to urease activity in Helicobacter pylori 26695

BACKGROUND: The gastric pathogen Helicobacter pylori produces urease in amounts up to 10% of its cell protein. This enzyme, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide, protects the bacterium from gastric acid. Urease, a nickel metalloenzyme, requires active uptake of nickel ions from the environment to maintain its activity. NixA is a nickel transport protein that resides in the cytoplasmic membrane. Mutation of nixA significantly reduces but does not abolish urease activity, strongly suggesting the presence of a second transporter. We postulated that the dipeptide permease (dpp) genes that are homologous to the nik operon of Escherichia coli could be a second nickel transporter. The predicted Dpp polypeptides DppA, DppC, and DppD of H. pylori share approximately 40%, 53%, and 56% amino acid sequence identity with their respective E. coli homologs. METHODS: A mutation in dppA, constructed by insertional inactivation with a chloramphenicol resistance cassette, was introduced by allelic exchange into H. pylori strain 26695. RESULTS: When compared to the parental strain, urease activity was not decreased in a dppA mutant. CONCLUSIONS: DppA does not contribute to the synthesis of catalytically active urease in H. pylori 26695 and is likely not a nickel importer in 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.     

The production of neuraminidase and fucosidase by Helicobacter pylori: Their possible relationship to pathogenicity

Abstract The pathogenicity of enterobacteria often correlates with their production of neuraminidase (sialidase). Forty-nine Helicobacter pylori isolates have therefore been examined for their production of neuraminidase and other glycosidases. All 49 isolates produced considerable neuraminidase (median 228 IU/μgmg protein, interquartile range 121–370), pH optimum 7.5. Nine of the 49 also produced fucosidase (median 23 IU/μgmg protein, interquartile range 12–39), pH optimum 7.0. Production of these enzymes did not correlate with bacterial Cag A expression or duodenal ulceration. Neutrophils exposed to neuraminidase show increased adherence to endothelium so the neuraminidase production by H. pylori could partly explain the predominant neutrophil inflammatory infiltrate seen in H. pylori -associated gastritis. Inhibition of this enzyme by use of neuraminidase-inhibitors could be a useful therapeutic approach.     

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 %。     

乳杆菌治疗幽门螺杆菌感染疗效的Meta分析

目的 系统评价乳杆菌治疗幽门螺杆菌（H. pylori）感染的有效性。方法 计算机检索PubMed、Cochrane Central Register of Controlled Trials、Clinicatrials.gov、中国临床试验注册中心（http://www.chictr.org.cn/）、中国知网（CNKI）、万方数据库。检索时间从建库至2016年6月30日。此外追索已纳入文献和综述的参考文献。经两位研究者独立筛选文献、提取资料和评价质量,交叉核对后,采用Stata 12.0进行Meta分析。结果 最终纳入17个RCT,共1 758例患者。Meta分析结果：乳杆菌联合常规三联疗法能提高H. pylori根除率[OR=2.762,95%CI(2.163,3.526),P<0.001],降低不良反应发生率[OR=0.334,95%CI(0.242,0.461),P<0.001],但乳杆菌单独应用对H. pylori根除率结果与对照组比较差异无统计学意义。根据乳杆菌给药疗程的亚组分析结果,0~14（含14）d乳杆菌联合给药方案显著提高了H. pylori根除率[OR=2.893,95%CI(2.187,3.827),P<0.05],14~28（含28）d给药疗程也显著提高了H. pylori根除率[OR=2.619,95%CI(1.652,4.153),P<0.05]。乳杆菌联合三联疗法在欧洲人群及亚洲人群的亚组分析中,均提高了H. pylori根除率。结论 乳杆菌联合三联疗法有利于提高H. pylori根除率,并能降低总不良反应的发生,在H. pylori感染相关疾病的治疗中具有一定意义。     

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.     

幽门螺杆菌空泡毒素研究进展

幽门螺杆菌空泡毒素是该菌产生的已知其它细菌毒素无明显源性的唯一蛋白毒素。该毒素是幽门螺杆菌重要的毒力致病因子,它的产生与感染胃肠上皮损伤和溃疡形成密切相关。本就幽门螺杆菌空泡毒素的结构与功能研究进展以及在未来免疫预防与免疫治疗中的作用进行了阐述。     

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.     

A novel synthetic peptide from a tomato defensin exhibits antibacterial activities against Helicobacter pylori

Defensins are a class of cysteine‐rich proteins, which exert broad spectrum antimicrobial activity. In this work, we used a bioinformatic approach to identify putative defensins in the tomato genome. Fifteen proteins had a mature peptide that includes the well‐conserved tetradisulfide array. We selected a representative member of the tomato defensin family; we chemically synthesized its γ‐motif and tested its antimicrobial activity. Here, we demonstrate that the synthetic peptide exhibits potent antibacterial activity against Gram‐positive bacteria, such as Staphylococcus aureus A170, Staphylococcus epidermidis, and Listeria monocytogenes, and Gram‐negative bacteria, including Salmonella enterica serovar Paratyphi, Escherichia coli, and Helicobacter pylori. In addition, the synthetic peptide shows minimal (<5%) hemolytic activity and absence of cytotoxic effects against THP‐1 cells. Finally, SolyC exerts an anti‐inflammatory activity in vitro, as it downregulates the level of the proinflammatory cytokines TNF‐α and IFN‐γ. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Epitope mapping and features of the epitope for monoclonal antibodies inhibiting enzymatic activity of Helicobacter pylori urease

Two characteristic monoclonal antibodies (HpU-2 and -18) out of 26 monoclonal antibodies (HpU-1 approximately 26) produced against Helicobacter pylori (H. pylori) urease showed a strong inhibitory effect against the enzymatic activity of the urease. Epitope mapping about some monoclonal antibodies of the HpU-series inhibiting enzymatic activity was performed by using a surface plasmon resonance apparatus and by digesting H. pylori urease with trypsin, followed by mass spectroscopy. The sequences of the epitopes recognized by HpU-2 and -18 were SVELIDIGGNRRIFGFNALVDR (22 mer) and IFGFNALVDR (10 mer), respectively. The former sequence is present as a part of a loop structure at a position close to the C-terminal of the alpha-subunit of H. pylori urease, although it has been suggested that the active site of the urease resides in the beta-subunit. The above peptide (22 mer) was chemically synthesized in a linear and cyclic form, and its conjugate with BSA was immunized in rabbits. The resultant serum induced by the linear form could specifically bind to H. pylori infecting human gastric mucosa. These results suggest that the above sequence (22 mer) must be an important epitope, although it locates in the alpha-subunit but not in the beta-subunit.     

益生菌与幽门螺杆菌对黏膜屏障的影响及其机制的研究进展

消化系黏膜屏障对致病菌有防御功能,但是在根除幽门螺杆菌(H. pylori)过程中会导致黏膜屏障的破坏。大量研究表明,益生菌通过修复上皮细胞以及细胞间连接、减少黏液分泌和炎症反应以及缓解消化道菌群紊乱等方式修复黏膜屏障。本文主要综述幽门螺杆菌对消化系黏膜屏障的损害及其机制,以及益生菌对黏膜屏障损伤的保护及修复机制研究的进展。     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

酪酸梭菌辅助根除幽门螺杆菌感染的Meta分析

目的 系统评价酪酸梭菌辅助根除幽门螺杆菌（Helicobacter pylori,H. pylori）感染的疗效和安全性。方法 计算机检索PubMed、CENTRAL（2016年7期）、CBM、CNKI、万方数据库、维普数据库,同时追溯纳入文献和相关综述的参考文献,收集关于酪酸梭菌辅助根除H. pylori感染的随机对照试验（RCT）,检索时间均从建库至2016年7月30日。由两名评价员根据纳入排除标准筛选文献、提取资料、评价纳入研究的偏倚风险,采取RevMan 5.3进行Meta分析。结果 最终纳入13个RCT,共1 372例患者（其中试验组742例,对照组630例）。Meta分析结果显示：与标准根除方案相比,酪酸梭菌辅助治疗组有利于提高H. pylori的根除率（RR=1.26,95%CI：1.18~1.34,P＜0.00001）,并且降低总不良反应（RR=0.41,95%CI：0.32~0.52,P＜0.00001）、腹泻（RR=0.31,95%CI：0.16~0.58,P=0.0003）、味觉紊乱的发生率（RR=0.35,95%CI：0.17~0.73,P=0.005）;然而,在腹胀、恶心的发生率上,酪酸梭菌辅助治疗组虽低于标准方案组,但差异无统计学意义。结论 当前证据显示酪酸梭菌联合标准方案不仅可提高H. pylori的根除率,还能降低总不良反应、腹泻及味觉紊乱的发生。受纳入研究的质量和数量的限制,上述结论有待更多高质量的研究予以验证。