Phosphonate metabolism in Helicobacter pylori

Helicobacter pylori has been shown to degrade two phosphonates, N-phosphonoacetyl-l-aspartate and phosphonoacetate; however, the bacterium does not have any genes homologous to those of the known phosphonate metabolism pathways suggesting that H. pylori may have a novel phosphonate metabolism pathway. Growth of H. pylori on phosphonates was studied and the catabolism of these compounds was measured employing ¹H-nuclear magnetic resonance spectroscopy. The specificity of the catabolic enzymes was elucidated by assaying the degradation of several phosphonates and through substrate competition studies. H. pylori was able to utilise phenylphosphonate as a sole source of phosphate for growth. Three strains of H. pylori showed sigmoidal enzyme kinetics of phenylphosphonate catabolism. Allosteric kinetics were removed when lysates were fractionated into cytosolic and membrane fractions. Catabolic rates increased with the addition of DTT, Mg²⁺ and phosphate and decreased with the addition of EDTA. The physiological properties of H. pylori phosphonate metabolism were characterised and the presence of at least two novel phosphonate catabolism pathways that do not require phosphate starvation growth conditions for activity has been established.     

Helicobacter pylori and gastric autoimmunity

Host specific T-cell response is critical for the outcome of Helicobacter pylori infection. In genetically susceptible individuals, H. pylori can activate gastric CD4⁺ Th1 cells that recognize cross-reactive epitopes shared by H. pylori proteins and self H⁺, K⁺-ATPase, leading to gastric autoimmunity via molecular mimicry.     

幽门螺杆菌基因组研究

幽门螺杆菌Helicobacter pylori(Hp)是引起人慢性活动性胃炎和消化性溃疡的病原菌,同时与胃癌的发生密切相关.近年来Hp感染过程和其致病性的分子机理研究受到广泛的关注.根据其全基因组测序及基因功能解析结果阐述了Hp基因组中重要的结构特征,基因的表达调控方式,以及毒力相关基因在Hp感染中的作用等.     

幽门螺杆菌适应性定植蒙古沙鼠前、后的蛋白质组学研究

为比较研究幽门螺杆菌(Hp)适应性定植蒙古沙鼠前后的蛋白表达谱变化,将Hp临床分离株感染沙鼠,并体内连续传代,驯化一株高适应性菌株,然后采用双向电泳和质谱技术对适应性变化前后两株菌的差异蛋白进行鉴定。结果表明,随着Hp临床株在沙鼠体内的连续传代,感染率逐渐升高,第10代后,感染率稳定在90%以上。适应性定植后,Hp蛋白谱发生了变化。在选择的5个候选差异蛋白点中,共鉴定出4个蛋白,分别为Hp菌的功能未知的HP0318编码蛋白、氢化酶表达/形成蛋白(hypB)、异柠檬酸脱氢酶(icd)、ADP-L-D-甘露庚糖表异构酶(rfaD)。上述鉴定蛋白可能与Hp适应性定植具有很大的相关性。     

幽门螺杆菌分子伴侣GroEL相互作用蛋白分析

【目的】获得幽门螺杆菌(Helicobacter pylori,HP) GroEL结合蛋白质组构成谱,为进一步探究GroEL及其与相互作用蛋白在HP致病机制中的作用提供新思路。【方法】在构建HP GroEL原核表达重组大肠杆菌(Escherichia coli) BL21(DE3)⁽pET-28a(+)-groEL)基础上,纯化带有His标签的GroEL蛋白,与HP全菌蛋白提取液共孵育后,利用Protein G磁珠和抗His标签抗体免疫沉淀法对复合物进行捕获,然后对复合物中GroEL及其结合的蛋白质进行质谱法鉴定,根据主要功能对其进行分类,并完成蛋白质相互关系网络分析。【结果】对GroEL蛋白捕获成分进行分析,共鉴定出59种可能与GroEL结合的蛋白质,其中包括19种代谢酶类(KatA、GltA和AhpC等参与氧化还原相关酶类7种,PepA、RocF和HtrA等肽酶5种,以及2种参与脂肪代谢酶、2种参与ATP合成酶、2种尿素酶和HP17＿08079蛋白等)、15种外膜蛋白(黏附素BabA、SabA、HapA及其他膜蛋白等)、8种转录翻译相关蛋白(Tuf、RpoBC...     

Pathogenesis and host response of Helicobacter pylori

The 5th International Workshop on Pathogenesis and Host Response in Helicobacter Infections was held in Elsinore, Denmark, 4–7 July, 2002.     

复方嗜酸乳杆菌片治疗幽门螺杆菌感染的Meta分析

系统评价复方嗜酸乳杆菌片治疗幽门螺杆菌感染的有效性、安全性。

以“复方嗜酸乳杆菌片”“幽门螺杆菌感染”“Compound Eosinophil-Lactobacillus Tablets”“Helicobacter pylori infection”为主题词, 系统检索The Cochrane Library、PubMed、Springer、ProQuest、中国知网、万方和维普等数据库(建库-2020年5月)的随机对照试验文献。利用RevMan 5.3软件对复方嗜酸乳杆菌片的有效性、安全性进行Meta分析。

依据严格的纳入排除标准, 最终纳入18篇随机对照试验文献。Meta分析结果显示: 复方嗜酸乳杆菌片与一线治疗方案联合用药治疗幽门螺杆菌感染临床疗效更优[RR=0.84, 95%CI(0.80, 0.87), P < 0.000 01], 安全性更好[OR=2.83, 95%CI(2.30, 3.49), P < 0.000 01]。进一步亚组分析表明, 复方嗜酸乳杆菌片与四联疗法联合用药治疗幽门螺杆菌感染与四联疗法相比, 临床疗效更优[RR=0.88, 95%CI(0.83, 0.93), P < 0.000 01], 安全性更优[OR=2.58, 95%CI(1.92, 3.48), P < 0.000 01];复方嗜酸乳杆菌片联合三联疗法治疗幽门螺杆菌感染较三联疗法临床疗效更优[RR=0.81, 95%CI(0.76, 0.85), P < 0.000 01], 且安全性好[OR=3.09, 95%CI(2.31, 4.14), P < 0.000 01]。

基于现有文献, 在治疗幽门螺杆菌感染时, 复方嗜酸乳杆菌片与四联疗法或三联疗法联合用药具有一定的临床优势, 能提高幽门螺杆菌根除率, 降低不良反应发生率, 提高安全性。

     

益生菌抑制幽门螺杆菌的作用机制及研究进展

幽门螺杆菌在胃部疾病的发病过程中起着重要作用,是导致胃炎、胃溃疡,甚至胃癌的关键因素之一。随着胃部疾病患者幽门螺杆菌阳性检出率的不断升高,人们对于胃病和幽门螺杆菌的相关性研究也有了一定进展。如今,对于幽门螺杆菌阳性患者根除治疗的必要性,以及抗生素治疗耐药性等问题已引起广泛关注。在这种情况下,益生菌作为相对安全的天然微生物,在抑制幽门螺杆菌并促进胃部健康的益生功能方面具有重要的研究潜力。本综述对幽门螺杆菌的致病机理、不同基因分型的致病程度等方面进行了总结,并对益生菌抑制幽门螺杆菌的机制进行了探讨。建议在治疗幽门螺杆菌感染时,应与常规的治疗手段结合应用,不仅会增加幽门螺杆菌的根除率,还能减少治疗相关的副作用。     

Mutation as an origin of genetic variability in Helicobacter pylori

The availability of two complete Helicobacter pylori genome sequences and recent studies of its population genetics have provided a detailed picture of genetic diversity in this important human gastric pathogen. It is believed that, in addition to genetic recombination, de novo mutation could have a role in generating the high level of genetic variation in H. pylori.     

Role of futC slipped strand mispairing in Helicobacter pylori Lewis phase variation

The O antigen of the Helicobacter pylori lipopolysaccharide is composed of repeating units of fucosylated Lewis (Le) antigens. The α(1,2)-fucosyltransferase (futC) of H. pylori, which catalyzes the conversion of Le^x to Le^y by addition of fucose, is subject to slipped-strand mispairing involving a homonucleotide (poly-C) tract. To explore the distribution of Le phenotypes within H. pylori cells grown in vitro, 379 single colonies of strain J166 were examined for Le expression. Two major populations with reciprocal Le^x/Le^y phenotypes were identified. Phenotypes correlated with futC frame status, suggesting that strain J166 represents a mixed population with respect to futC poly-C tract length, which was confirmed by a translational reporter. After hundreds of generations in vitro, phenotypes did not change significantly, indicating that the observed J166 Le diversity reflects the founding population. Since slipped-strand mispairing in the futC poly-C tract was postulated to explain the Le^y phenotypic change observed in J166 derivative strain 98–169 isolated 10 months after rhesus monkey challenge, in trans complementation with in-frame futC was performed. Le^y synthesis was restored and Le^x expression was reciprocally lowered. From these studies, we confirmed the principal role of futC slipped-strand mispairing in Le antigenic variation in vitro and in vivo.     

Crystal structure and biophysical characterisation of Helicobacter pylori phosphopantetheine adenylyltransferase

Helicobacter pylori is a bacterium that causes chronic active gastritis and peptic ulcers. Drugs targeting H. pylori phosphopantetheine adenylyltransferase (HpPPAT), which is involved in CoA biosynthesis, may be useful. Herein, we report the expression in Escherichia coli and purification of recombinant HpPPAT and describe a crystal structure for an HpPPAT/CoA complex. As is the case for E. coli PPAT (EcPPAT), HpPPAT is hexameric in solution and as a crystal. Each protomer has a well-packed dinucleotide-binding fold in which CoA binds. Structural characterisation demonstrated that CoA derived from the E. coli expression system bound tightly to HpPPAT, presumably to initiate feedback inhibition. However, the interactions between the active-site residues of HpPPAT and CoA are not identical to those of other PPATs. Finally, CoA binding affects HpPPAT thermal denaturation.     

Antibacterial and immuno-modulatory activity of ethanol extracts from Lespedeza sp. during Helicobacter pylori infections

Chemical therapeutics targeted against H. pylori may lead to host toxicity and pathogen eradication failures. In this study, ethanolic extracts from five Lespedeza sp. plants were shown to inhibit the gastric-pathogen H. pylori and to modulate cytokine production. Disc agar diffusion assays showed that Lespedeza sp. ethanol extracts possess potent anti-H. pylori activity. Among the five plant extracts, the extracts from L. cyrtobotrya demonstrated the highest anti-H. pylori effect. The growth inhibitory effect against H. pylori was initiated after six h of treatment with plant extracts and the effect remained for a continuous period of 48 h. Incubation of the gastric cells infected with H. pylori with 1.25 to 50 mg/mL of sp. plant extracts resulted in a reduction of the production of cytokine IL-8. The plant ethanol extracts generally had little influence on AGS cell viability, indicating their safety for the treatment of bacterial infections. Three active fractions of L. cyrtobotrya also demonstrated similar anti-H. pylori and immuno-modulatory effects. Taken together, these results provide evidence that Lespedeza sp. plant extracts might be potential sources of new host friendly anti-H. pylori agents.     

Antibacterial and ulcer healing effects of organoselenium compounds in naproxen induced and Helicobacter pylori infected Wistar rat model

Aim of the present study was to evaluate in vitro toxicity and in vivo antibacterial, anti-inflammatory, antiulcer, and antioxidant activities of two organoselenium compounds, selenocystine (SeCys) and ebselen (Ebs). The study was conducted in experimentally induced ulcers in rodent model infected with Helicobacter pylori (H. pylori). In vitro toxicological studies on normal spleenic lymphocytes revealed that SeCys and Ebs were non-toxic to the cells even at 100 μM concentration. Antibacterial activity was observed at 500 μg/mL concentration of either of the compounds against H. pylori. In vivo studies after treatment with SeCys and Ebs (500 μg/kg/day) resulted in significant reduction in ROS production and inhibition of lipid peroxidation in gastric tissue. The antioxidant and anti-inflammatory activities of both the compounds were also confirmed by their ability to lower GSH reduction, to induce the expression of antioxidant genes such as GPx-4, and MnSOD and to suppress inflammatory genes namely COX-2, TNF-α and TGF-β. In addition, the immunomodulatory activity of both the compounds was evident by enhance of the CD4 levels and maintenance of the IgG, IL-6 and IL-10 levels. Persistent treatment (500 μg/kg, for 28 days) with both the compounds showed considerable (p < 0.05) ulcer healing property supporting its role in gastro protection. In conclusion, the results of our study suggest that both SeCys and Ebs possess broad spectrum of activities without any potential toxicity.     

Characterization of a Helicobacter pylori vaccine candidate by proteome techniques

In a previous two-dimensional (2D) gel electrophoretic study of protein antigens of the gastric pathogen, Helicobacter pylori recognized by human sera, one of the highly and consistently reactive antigens, a protein with M_r of approximately 30?000 (Spot 15) seemed to be of special interest because of low yields on N-terminal protein sequencing. This suggested possible N-terminal modification, as the N-terminal sequence analysis of this 30?000 protein (Spot 15) did not provide a definitive match within the H. pylori genomic database. This protein was isolated by 2D polyacrylamide gel electrophoresis, evaluated by liquid chromatography–mass spectrometry, and found to consist of two related species of approximately 28?100 and 26?500. In parallel, the proteins within this spot were digested in situ with the endoprotease Lys-C. Analysis of the Lys-C digest by matrix-assisted laser desorption time-of-flight mass spectrometry, peptide mapping, and sequence analysis was conducted. Comparison of the mass and sequence of the Lys-C peptides with those derived from a H. pylori genomic library identified an open reading frame of approximately 300 base pairs as the source of the Spot 15 protein. This corresponded to HP0175 in the recently reported H. pylori genome sequence, an open reading frame with some homology to Campylobacter jejeuni cell binding protein 2. Mass spectral and sequence analysis indicated that Spot 15 was a processed product generated by proteolytic cleavage at both the carboxy and amino termini of the 34 open reading frame precursor.     

重组酿酒酵母表达幽门螺杆菌VacA蛋白及其免疫原性分析*

目的:利用酿酒酵母表面展示技术筛选幽门螺杆菌候选疫苗,并分析其免疫原性。方法:以幽门螺杆菌的空泡型细胞毒素A(vacA)基因作为研究对象,构建重组S.cerevisiae EBY100/pYD1-VacA,通过Western blot、免疫荧光标记和流式细胞仪对S.cerevisiae EBY100/pYD1-VacA进行体外表达分析。以PBS和S.cerevisiae EBY100/pYD1为对照组,S.cerevisiae EBY100/pYD1-VacA为实验组,口服免疫SPF级BALB/c小鼠。通过ELISA分析检测口服免疫后小鼠抗VacA特异性IgG及分泌型IgA效价。结果:VacA抗原蛋白被成功地展示在S.cerevisiae EBY100表面。小鼠经口服免疫S.cerevisiae EBY100/pYD1-VacA后可诱导产生较高的VacA特异性抗体。结论:表面展示型酿酒酵母可以作为幽门螺杆菌候选疫苗的递送载体,与此同时,这也为开发其他细菌或病毒疫苗提供新思路。     

Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori

Background  

Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism.     

反流性食管炎患者口腔菌群变化及其与幽门螺杆菌感染的关系

探讨反流性食管炎（RE）患者口腔菌群变化及其与幽门螺杆菌（H. pylori）感染的关系,为该类患者的治疗提供参考。

选择南京同仁医院2019年5月至2021年5月收治的140例RE患者为研究对象,将A、B级RE患者纳入AB组,C、D级纳入CD组;同时将患者分为合并H. pylori感染组和未合并H. pylori感染组。以一次性无菌收集管收集患者唾液标本,使用Illumina Hiseq 2500高通量测序平台对合格文库进行双端测序。

AB组患者H. pylori阳性率（16.67%）低于CD组（45.45%）。AB组患者口腔菌群Chao 1指数和Shannon指数均高于CD组,Simpson指数低于CD组（均P＜0.05）。未合并H. pylori感染组患者口腔菌群Chao 1指数和Simpson指数高于合并H. pylori感染组,Shannon指数低于合并H. pylori感染组（均P＜0.05）。未合并H. pylori感染组患者口腔放线菌门、放线菌属相对丰度高于合并H. pylori感染组,拟杆菌门相对丰度低于合并H. pylori感染组（均P＜0.05）。

RE病情较重者和合并H. pylori感染者表现为口腔菌群α多样性降低和门、属水平菌群相对丰度改变,合并H. pylori感染对RE患者病情和口腔菌群会产生一定影响。

     

双歧杆菌乳杆菌三联活菌片对幽门螺杆菌根治后消化道不良反应的改善作用

探讨加用双歧杆菌乳杆菌三联活菌片对临床四联疗法根除幽门螺杆菌(H.pylori)过程中患者消化道不良反应的改善作用。

选择2019年1月至2020年1月在昆明医科大学第二附属医院门诊就诊的100例H.pylori感染患者, 分为益生菌组和正常组, 各50例。全部患者通过四联疗法根除H.pylori后, 益生菌组患者继续加用双歧杆菌乳杆菌三联活菌片治疗, 对结果进行分析。

2组患者使用含有铋剂的四联疗法根除H.pylori后效果满意。益生菌组患者不良反应发生率低于正常组(25.5% vs 47.5%;χ²=11.023, P=0.001)。

益生菌可减轻四联疗法根除H.pylori后消化道不良反应, 根除H.pylori过程中加用益生菌的时机尚待进一步探讨。

     

幽门螺杆菌多价表位疫苗CWAE的表达及其免疫学性质的研究 *

目的 利用生物信息学软件评价幽门螺杆菌(Helicobacter pylori)多价表位疫苗CWAE的抗原结构,经原核表达获得高纯度CWAE蛋白,进而鉴定多价表位疫苗CWAE的免疫学性质。方法 通过生物信息学软件分析H. pylori多价表位疫苗CWAE的抗原结构;用人工合成的H. pylori多价表位肽融合基因WAE替换重组质粒pET28a-CUE中的UE基因,构建重组质粒pET28a-CWAE。然后,将pET28a-CWAE转入大肠杆菌BL21(DE3)中,经IPTG诱导表达,并通过Ni-NTP镍离子亲和层析纯化抗原蛋白CWAE;利用GM1-ELISA鉴定CWAE中CTB组分的黏膜免疫佐剂活性。最后,通过ELISA和小鼠脾脏淋巴细胞增殖实验检测CWAE激发BALB/c小鼠产生抗H. pylori抗体体液免疫和淋巴细胞免疫应答的能力。结果 通过生物信息学软件证实H. pylori多价表位疫苗CWAE具有科学合理的结构;重组表达质粒pET28a-CWAE经PCR、双酶切和基因测序鉴定,融合基因CWAE与设计序列完全一致;重组基因工程菌株pET28a-CWAE/BL21(DE3)经IPTG诱导表达,抗原蛋白CWAE主要以包涵体形式存在,经Ni-NTP镍离子亲和层析纯化,纯度约达93.2%;GM1-ELISA实验证实,CWAE中CTB组分依旧保持有较好的黏膜佐剂活性;ELISA结果证实CWAE能够激发BALB/c小鼠产生H. pylori特异性抗体,而小鼠脾脏淋巴细胞增殖实验进一步证实CWAE能够激发针对H. pylori多种致病因子的淋巴细胞免疫反应。结论 H. pylori多价表位疫苗CWAE具有科学合理的抗原结构,经原核表达可获得高纯度抗原蛋白,能够激发BALB/c小鼠产生H. pylori特异性抗体体液免疫和淋巴细胞免疫应答。为研发防治H. pylori感染的多价表位疫苗奠定实验基础。