Neutrophil-activating Protein Polymorphism of Helicobacter pylori Determines the Host Risk of Dyspepsia.
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ABSTRACT:
SUBMITTER: Matsuzaki J
PROVIDER: S-EPMC6718361 | biostudies-literature | 2019
REPOSITORIES: biostudies-literature
Publications
Neutrophil-activating Protein Polymorphism of Helicobacter pylori Determines the Host Risk of Dyspepsia.
Matsuzaki Juntaro J Tsugawa Hitoshi H Kashiwazaki Yuki Y Mori Hideki H Yamamoto Yuta Y Kameyama Hisako H Masaoka Tatsuhiro T Kanai Takanori T Suzuki Hidekazu H
Cellular and molecular gastroenterology and hepatology 20190518 2
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Project description:Helicobacter pylori-associated gastritis is mainly an inflammatory cell response. In earlier work we showed that activation of human neutrophils by a cell-free water extract of H. pylori is characterized by increased expression of neutrophil CD11b/CD18 and increased adhesiveness to endothelial cells. The work reported here indicates that the neutrophil-activating factor is a 150,000-molecular-weight protein (150K protein). Neutrophil proadhesive activity copurified with this protein, which is a polymer of identical 15K subunits. Specific antibody, prepared against the purified 15K subunit, neutralized the proadhesive activity of the pure protein and of water extracts obtained from different strains of H. pylori. The gene (napA) for this protein (termed HP-NAP, for H. pylori neutrophil-activating protein) was detected, by PCR amplification, in all of the H. pylori isolates tested; however, there was considerable strain variation in the level of expression of HP-NAP activity in vitro. HP-NAP could play an important role in the gastric inflammatory response to H. pylori infection.
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Project description:AimTo produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori (H pylori) neutrophil-activating protein (NAP) and E. coli maltose-binding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.MethodsNeutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis. Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment, Western blotting with human H pylori anti-sera.ResultsE.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG. The molecular weight of rBMP-NAP was about 57 kD, accounting for 37.55% of the total protein in the sonicated supernatant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture. The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.ConclusionRecombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.
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Project description:The ultimate aim of the immune system is to eliminate pathogens without being harmful to the host. But what if eliminating the pathogen in itself is discomforting for the host? One such emerging case is of Helicobacter pylori. Modern medicine, infantile vaccination, and ultra-hygienic conditions have led to progressive disappearance of H. pylori in different parts of the world. However, the adversities caused by H. pylori's absence are much larger than those caused by its presence. Asthma is rising as an epidemic in last few decades and several reports suggest an inverse-relationship between H. pylori's persistence and early-life onset asthma. Regulatory T cells play an important role in both the cases. This is further supported by experiments on mouse-models. Hence, need of the hour is to discern the relationship between H. pylori and its host and eliminating its negative impacts without disturbing our indigenous microbiota. To resolve whether H. pylori is a pathogen or an amphibiont is another important side. This review explores the biological basis of H. pylori-induced priming of immune system offering resistance to childhood-onset asthma. HP-NAP-Tregs interaction has been predicted using molecular docking and dynamic simulation.
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Project description:A new crystal lattice structure of Helicobacter pylori neutrophil-activating protein (HP-NAP) has been determined in two forms: the native state (Apo) at 2.20 Å resolution and an iron-loaded form (Fe-load) at 2.50 Å resolution. The highly solvated packing of the dodecameric shell is suitable for crystallographic study of the metal ion-uptake pathway. Like other bacterioferritins, HP-NAP forms a spherical dodecamer with 23 symmetry including two kinds of channels. Iron loading causes a series of conformational changes of amino-acid residues (Trp26, Asp52 and Glu56) at the ferroxidase centre.
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Project description:BackgroundHelicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-induced gastric inflammation. Due to its immunogenic and immunomodulatory properties, HP-NAP has been used for developing vaccines against H. pylori infection and new drugs for cancer therapy.ResultsHere, we provide a simple process for high-yield production of HP-NAP by applying one-step negative chromatography to purify recombinant HP-NAP expressed in Escherichia coli (E. coli). In our E. coli expression system, recombinant HP-NAP constitutes nearly 70% of the total protein. Overexpressed recombinant HP-NAP is almost completely soluble upon cell lysis at pH 9.5. Under the optimal condition at pH 8.0, recombinant HP-NAP with purity higher than 95% can be obtained from E. coli by collecting the unbound fraction using diethylaminoethyl (DEAE) Sephadex resin in batch mode. The overall yield of HP-NAP from a 50-ml E. coli culture is ~19 mg. The purified HP-NAP folds into a multimer with a secondary structure of α-helix and is able to trigger the production of reactive oxygen species by neutrophils.ConclusionsPurification of recombinant HP-NAP overexpressed in E. coli using DEAE Sephadex negative mode batch chromatography is an efficient method for high-yield production of highly pure HP-NAP in its native state. The purified HP-NAP is useful for various clinical applications including vaccine development, diagnosis, and new drug development.
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Project description:BackgroundGastric cancer arises, mainly, on an inflammatory background. Helicobacter pylori neutrophil activating (HP-NAP) protein functions as a potent pro-inflammatory mediator. Similarly, IL-4 plays a critical role in the inflammation pathway, the levels of which are altered by C to T transition at position -590 in its promoter region. Here, we have aimed to assess the risk of gastritis and gastric cancer in the co-presence of these two inflammation modulating mediators.MethodsGastritis (n=58) and gastric cancer (n=31) patients were evaluated and compared with H. pylori-positive asymptomatic controls (n=46), for serum antibodies against recombinant HP-NAP and IL-4 C-590T single nucleotide polymorphism using immunoblotting and PCR-RFLP, respectively. Multivariable logistic regression, adjusting for age, gender and ethnicity, was used for data analysis.ResultsIn terms of susceptibility to gastritis, seropositivity to HP-NAP projected a risk impact of 4.62 fold (OR=4.62, 95% CI=1.50-14.22), which when present in IL-4 -590 T carriers augmented the risk up to 9.7 fold (OR=9.70, 95% CI=2.06-45.69). A similar pattern, but of a stronger magnitude, occurred for the risk of gastric cancer, which was estimated at 9.07 fold (OR=9.07, 95% CI=1.99-42.0) for HP-NAP-seropositive subjects and was drastically amplified (OR=33.64, 95% CI=2.06-548.68), when double-positive (HP-NAP seropositive/IL-4 -590 T carrier) subjects were examined against double negatives (HP-NAP seronegative/IL-4 -590 CC).ConclusionOur preliminary data indicate that serum antibodies against HP-NAP represent a state of risk, which is further exacerbated in IL-4 -590 T carriers. These biomarkers, if validated in larger prospective studies, can be used to screen for gastric cancer susceptibility.
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Project description:Neutrophil-activating protein (NAP) is a major virulence factor expressed by Helicobacter pylori isolates associated with severe chronic gastroduodenal inflammation and peptic ulcers. NAP is one of the main protective antigens and a target for vaccine development against Helicobacter infection. In addition, NAP is a potent immune stimulator with potential application as a general vaccine adjuvant and in treatment of allergic diseases and cancer immunotherapy. NAP-specific immunoassays are needed for both H. pylori diagnostics and characterization of NAP-based vaccines and immunomodulatory preparations. We generated a panel of NAP-specific monoclonal antibodies (MAbs) by immunization of BALB/c mice with synthetic NAP peptides. The antibody reactivity against recombinant or native NAP antigen was characterized by enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence. A sensitive capture ELISA was developed using MAbs 23C8 and 16F4 (directed against different NAP epitopes) for detection of native or measles virus (MV) vector-expressed recombinant NAP in a concentration range of 4 ng/ml to 2000 ng/ml. MAb 23C8 antigen-binding depends on Tyr101 in a variable amino acid sequence of the NAP molecule, indicating the existence of antigenic variants among H. pylori strains. MAb 16F4 reacted with NAP from different H. pylori strains and was a sensitive tool for detection of small amounts of isolated NAP antigen or whole bacteria by immunoblotting or immunofluorescence. In conclusion, MAb-based immunoassays are highly specific and sensitive for detection of native NAP antigen and recombinant NAP immunostimulatory transgenes expressed by replication competent virus vectors.
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