The Expression of Iron-repressible Outer Membrane Proteins in Helicobacter pylori and Its Association with Iron Deficiency Anemia

Background: Helicobacter pylori infection is known to be a cause of iron deficiency anemia (IDA) that is unresponsive to iron supplements. H. pylori bind iron to a specific receptor by iron-repressible outer membrane proteins (IROMPs) under conditions of restricted iron.
Materials and Methods: We compared the expression of IROMPs from strains of H. pylori under both iron-restricted and iron-supplemented conditions to determine the difference between strains with and without IDA. One standard strain, two clinical strains, and three IDA strains were cultured; and then the IROMPs were extracted under iron-restricted and iron-supplemented conditions. We used SDS-PAGE to compare the expression of the IROMPs from each strain.
Results:  IROMPs were found in IDA strains under iron-restricted conditions and their molecular sizes were estimated to be 56, 48, 41, and 37 kDa. In the iron-repleted media, the IROMPs were no longer present.
Conclusion: In the iron-depleted state, specific H. pylori strains associated with IDA demonstrated an advantage in iron acquisition due to a higher expression of IROMPs. Our results can explain in part why some patients with H. pylori infection are more prone to develop clinical IDA under restricted iron conditions in the host.     

幽门螺杆菌外膜囊泡研究进展

幽门螺杆菌(Helicobacter pylori)被认为是引起人类胃部疾病的元凶之一。外膜囊泡(Outer Membrane Vesicles,OMVs)是由细菌外膜自发脱落而形成的囊泡状结构,其具有细菌外膜多数成分,包括外膜蛋白、多糖、脂质以及其他蛋白组分。越来越多的研究正在关注外膜囊泡在幽门螺杆菌感染、发生、发展过程中的作用。同时,研究表明幽门螺杆菌外膜囊泡作为疫苗,在防治幽门螺杆菌感染中也展现了良好的应用潜力。因此,本综述总结了目前关于幽门螺杆菌外膜囊泡组成成分的研究,并讨论了外膜囊泡在幽门螺杆菌存活和致病机制中的作用,以及外膜囊泡在幽门螺杆菌感染治疗中发挥的作用。     

Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells

Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N‐terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW‐GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW‐GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW‐GFP into OMVs through the increased OmpW‐GFP expression on the ΔnlpI cells. Finally, the amount of OmpW‐GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW‐GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:51–57, 2018     

NCTC11637外膜蛋白36ku基因的克隆及序列分析

目的:对人幽门螺杆菌(Helicobacter pylori,H.pylori)36ku(OMP36)外膜蛋白进行基因克隆表达,探索研制H.pylori疫苗的新途径。方法:培养H.pylori菌株NCTC11637,采用酚:氯仿抽提和纯化基因组DNA。设计上下游引物,并以该基因组为模板,采用聚合酶链反应(PCR)扩增目的基因片断。将目的基因和PET32a( )同时经HindⅢ和KpnⅠ双酶切,纯化,连接后,转化含有目的基因的重组载体,酶切鉴定后进行序列分析。结果:经酶切,测序分析表明,插入的基因片断为987bp,与GenBank公布的H.pylori 26695、ATCC43504及J99序列相比较,同源性高达94%-96%,推测的氨基酸序列同源性为97%-99%,GenBank登录号059968。结论:成功克隆了H.pylori 36ku的外膜蛋白的编码基因,其表达产物OMP36有望成为新的Hp疫苗候选分子,为H.pylori疫苗的研制和试剂盒的开发奠定了基础。     

Crystal structure of the secreted protein HP1454 from the human pathogen Helicobacter pylori

HP1454 is a protein of 303 amino acids found in the extracellular milieu of Helicobacter pylori. The protein structure, crystallized in the orthorhombic C222₁ space group with one molecule per asymmetric unit, has been determined using the single‐wavelength anomalous dispersion method. HP1454 exhibits an elongated bent shape, composed of three distinct domains. Each domain possesses a fold already present in other structures: Domain I contains a three‐strand antiparallel β‐barrel flanked by a long α‐helix, Domain II is an anti‐parallel three‐helix bundle, and Domain III a β‐sheet flanked by two α‐helices. The overall assembly of the protein does not bear any similarity with known structures. Proteins 2014; 82:2868–2873. © 2014 Wiley Periodicals, Inc.     

Lewis epitopes on outer membrane vesicles of relevance to Helicobacter pylori pathogenesis

BACKGROUND: Helicobacter pylori extrudes protein- and lipopolysaccharide-enriched outer membrane vesicles from its cell surface which have been postulated to act to deliver virulence factors to the host. Lewis antigen expression by lipopolysaccharide of H. pylori cells has been implicated in a number of pathogenic roles. The aim of this study was to further characterize the expression of lipopolysaccharide on the surface of these outer membrane vesicles and, in particular, expression of Lewis antigens and their association with antibody production in the host. MATERIALS AND METHODS: H. pylori strains were examined for outer membrane vesicle production using transmission electron microscopy and Lewis antigen expression probed using immunoelectron microscopy. Sera from patients were analyzed for cross-reacting anti-Lewis antibodies and, subsequently, absorbed using outer membrane vesicle preparations to remove the cross-reacting antibodies. RESULTS: The formation of outer membrane vesicles by H. pylori was observed in both in vitro and in vivo samples. Furthermore, vesicles were produced following culture in either liquid or solid medium by all strains examined. Moreover, we observed the presence of Lewis epitopes on outer membrane vesicles using immunoelectron microscopy and immunoblotting. Circulating anti-Lewis antibodies were found in the sera of gastric cancer patients but not in the sera of H. pylori-negative control subjects. Absorption of patient sera with outer membrane vesicles decreased the levels of anti-Lewis autoantibodies. CONCLUSIONS: Our results demonstrate the ability of H. pylori to generate outer membrane vesicles bearing serologically recognizable Lewis antigens on lipopolysaccharide molecules which may contribute to the chronic immune stimulation of the host. The ability of these vesicles to absorb anti-Lewis autoantibodies indicates that they may, in part, play a role in putative autoimmune aspects of H. pylori pathogenesis.     

Composition and function of Helicobacter pylori outer membrane vesicles

The gastric pathogen Helicobacter pylori sheds outer membrane vesicles (OMV) that possess many of the surface elements of the bacterium. Here we review current knowledge on the composition of H. pylori OMV and discuss evidence for their potential roles in bacterial survival and pathogenesis.     

Vaccine-induced immunity against Helicobacter pylori in the absence of IL-17A

Background: Helicobacter pylori (H. pylori) is a gram negative bacterium that can cause diseases such as peptic ulcers and gastric cancer. IL‐17A, a proinflammatory cytokine that can induce the production of CXC chemokines for neutrophil recruitment, has recently been shown to be elevated in both H. pylori‐infected patients and mice. Furthermore, studies in mouse models of vaccination have reported levels significantly increased over infected, unimmunized mice and blocking of IL‐17A during the challenge phase in immunized mice reduces protective immunity. Because many aspects of immunity had redundant or compensatory mechanisms, we investigated whether mice could be protectively immunized when IL‐17A function is absent during the entire immune response using IL‐17A and IL‐17A receptor knockout (KO) mice immunized against H. pylori. Materials and Methods: Gastric biopsies were harvested from naïve, unimmunized/challenged, and immunized/challenged wild type (WT) and KO mice and analyzed for inflammation, neutrophil, and bacterial levels. Groups of IL‐17A KO mice were also treated with anti‐IFNγ or control antibodies. Results: Surprisingly, all groups of immunized KO mice reduced their bacterial loads comparably to WT mice. The gastric neutrophil counts did not vary significantly between IL‐17A KO and WT mice, whereas IL‐17RA KO mice had on average a four‐fold decrease compared to WT. Additionally, we performed an immunization study with CXCR2 KO mice and observed significant gastric neutrophils and reduction in bacterial load. Conclusion: These data suggest that there are compensatory mechanisms for protection against H. pylori and for neutrophil recruitment in the absence of an IL‐17A‐CXC chemokine pathway.     

Release of the Outer Membrane Vesicles from Vibrio cholerae and Vibrio parahaemolyticus

We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze-substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti-O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron-dense mass.     

The role of interleukin-17 in the Helicobacter pylori induced infection and immunity

Background: Helicobacter pylori infection is regarded as the major cause of various gastric diseases and induces the production of several cytokines including interleukin‐17 (IL‐17) recently recognized as an important player in the mammalian immune system. Objective: This review deals with the role of IL‐17 on the H. pylori‐induced infection and immunity in humans and experimental animals. Results: H. pylori infection increases IL‐17 in the gastric mucosa of humans and experimental animals. In humans, IL‐17 induces the secretion of IL‐8 by activating the ERK 1/2 MAP kinase pathway and the released IL‐8 attracts neutrophils promoting inflammation. IL‐23 is increased in patients with H. pylori‐related gastritis and regulates IL‐17 secretion via STAT3 pathway. Studies in H. pylori‐infected mice indicate that IL‐17 is primarily associated with gastric inflammation. The early events in the immune response of immunized and challenged mice include the recruitment of T cells and the production of IL‐17. Neutrophil attracting chemokines are released, and the bacterial load is considerably reduced. IL‐17 plays a dual role in infection and vaccination. In infection, T regulatory cells (Tregs) suppress the inflammatory reaction driven by IL‐17 thereby favoring bacterial persistence. Immunization produces Helicobacter‐specific memory T‐helper cells that can possibly alter the ratio between T‐helper 17 and Treg responses so that the IL‐17‐driven inflammatory reaction can overcome the Treg response leading to bacterial clearance. Conclusion: IL‐17 plays an important role in H. pylori‐related gastritis and in the reduction of Helicobacter infection in mice following immunization.     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

The functional status of the Helicobacter pylori sabB adhesin gene as a putative marker for disease outcome

Background. Helicobacter pylori factors that contribute to disease outcome are largely unknown, but intimate contact with host cells mediated by outer membrane proteins is thought to play an important role. Expression of the outer membrane proteins OipA, HopZ, SabA, and SabB is regulated by phase‐variable dinucleotide repeats in the coding regions of the respective genes. We have evaluated the correlation between the expression status of these four genes and disease outcome of H. pylori infection in a Dutch patient population. Materials and Methods. H. pylori strains, isolated from 96 Dutch patients with gastritis (n = 29), duodenal ulcer (n = 28), gastric ulcer (n = 21), gastric carcinoma (n = 9), and lymphoma (n = 9), were analyzed for the ‘on/off’ expression status of the H. pylori genes oipA, hopZ, sabA, and sabB by direct DNA sequence analysis of amplified fragments. Results. The off‐status of sabB was significantly associated with duodenal ulcer (p = .036), but not with gastric ulcer. In contrast, the expression status of oipA, hopZ, and sabA did not correlate with disease outcome. Furthermore, lymphoma strains appeared to express a significantly smaller amount of putative adhesins when compared to gastritis, gastric ulcer, duodenal ulcer and gastric carcinoma strains (p < .02 for all groups tested). Conclusion. The off‐status of sabB was found to be associated with duodenal ulcer disease, and thus represents a putative marker for disease outcome. Assuming that SabB is involved in bacterial adhesion, this association suggests that adherent H. pylori are more prone to elimination by the host immune system.     

幽门螺杆菌Lpp20融合蛋白的表达、标签切除及鉴定

目的:利用GST融合基因表达系统表达Lpp20-GST融合蛋白,并利用凝血酶切除GST标签.方法:IPTG诱导重组质粒Lpp20/pGEX-4T -1在大肠杆菌BL21 (DE3)中表达,菌体经反复冻融、溶菌酶裂解及超声破菌后,发现Lpp20-GST融合蛋白以部分可溶性的形式表达.采用GST蛋白纯化系统对其纯化,得到Lpp20- GST融合蛋白,再用凝血酶进行GST标签的切除,所得产物进行Western Blot鉴定.结果:高效表达出Lpp20-GST融合蛋白的相对分子质量约4.5kDa,凝血酶成功切除了GST标签,Western Blot证实Lpp20蛋白能被鼠抗Lpp20单克隆抗体识别.结论:成功表达和纯化了重组Lpp20蛋白,为深入研究Lpp20的功能奠定了基础.     

Cloning, Expression, Purification and Toxicity Evaluation of Helicobacter pylori Outer Inflammatory Protein A

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni–NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.     

立氏立克次体外膜蛋白H基因片段的克隆表达与免疫原性分析

目的：克隆表达立氏立克次体（Rickettsia rickettsii）外膜蛋白H基因（ompH）片段并对其进行免疫原性分析。方法：采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果：获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论：pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

立氏立克次体外膜蛋白H 基因片段的克隆表达与免疫原性分析

目的:克隆表达立氏立克次体(Rickettsia rickettsii)外膜蛋白H基因(ompH)片段并对其进行免疫原性分析。方法:采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果:获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论:pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

溶藻弧菌铁载体合成及外膜蛋白表达的研究

初步研究了海洋动物病原菌溶藻弧菌的铁摄取机制。溶藻弧菌能够在高浓度铁螯合剂2-2二联吡啶的培养基中存活。在限铁环境中,溶藻弧菌生长受到抑制,补加铁可以消除这种抑制作用。通过铁载体定量检测,发现分离于发病鱼体的溶藻弧菌MVP01产铁载体量大于分离于海水的菌株No·1·1587。互补实验证明溶藻弧菌的铁载体粗提物能够被铁载体合成缺陷的大肠杆菌突变株AN93利用。在铁限制培养环境中,溶藻弧菌合成了约80kD铁调控外膜蛋白。铁摄取系统在溶藻弧菌的生存和致病性方面,都有重要的作用。