H. Pylori Neutrophil-activating Protein Research Articles

Structures and metal-binding properties of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center.

Helicobacter pylori causes severe diseases, such as chronic gastritis, peptic ulcers, and stomach cancers. H. pylori neutrophil-activating protein (HP-NAP) is an iron storage protein that forms a dodecameric shell, promotes the adhesion of neutrophils to endothelial cells, and induces the production of reactive oxygen radicals. HP-NAP belongs to the DNA-protecting proteins under starved conditions (Dps) family, which has significant structural similarities to the dodecameric ferritin family. The crystal structures of the apo form and metal-ion bound forms, such as iron, zinc, and cadmium, of HP-NAP have been determined. This review focused on the structures and metal-binding properties of HP-NAP. These metal ions bind at the di-nuclear ferroxidase center (FOC) by different coordinating patterns. In comparison with the apo structure, metal loading causes a series of conformational changes in conserved residues among HP-NAP and Dps proteins (Trp26, Asp52, and Glu56) at the FOC. HP-NAP forms a spherical dodecamer with 23 symmetry including two kinds of pores. Metal ions have been identified around one of the pores; therefore, the negatively-charged pore is suitable for the passage of metal ions.

Helicobacter pylori neutrophil-activating protein: from molecular pathogenesis to clinical applications.

Helicobacter pylori (H. pylori) neutrophil-activating protein (HP-NAP) was originally identified as a virulence factor of H. pylori for its ability to activate neutrophils to generate respiratory burst by releasing reactive oxygen species. Later on, HP-NAP was also found to be involved in the protection of H. pylori from DNA damage, supporting the survival of H. pylori under oxidative stress. This protein is highly conserved and expressed by virtually all clinical isolates of H. pylori. The majority of patients infected with H. pylori produced antibodies specific for HP-NAP, suggesting its important role in immunity. In addition to acting as a pathogenic factor by activating the innate immunity through a wide range of human leukocytes, including neutrophils, monocytes, and mast cells, HP-NAP also mediates adaptive immunity through the induction of T helper cell type I responses. The pro-inflammatory and immunomodulatory properties of HP-NAP not only make it play an important role in disease pathogenesis but also make it a potential candidate for clinical use. Even though there is no convincing evidence to link HP-NAP to a disease outcome, recent findings supporting the pathogenic role of HP-NAP will be reviewed. In addition, the potential clinical applications of HP-NAP in vaccine development, clinical diagnosis, and drug development will be discussed.

Detection and evaluation of antibodies against neutrophil-activating protein of Helicobacter pylori in patients with gastric cancer

To detect and evaluate the antibodies against Helicobacter pylori (H pylori) neutrophil-activating protein (HP-NAP) in patients with gastric cancer and other gastroduodenal diseases. Recombinant HP-NAP was prepared from a prokaryotic expression system in Escherichia coli. Serum positivity and level of HP-NAP-specific antibodies in sera from 43 patients with gastric cancer, 28 with chronic gastritis, 28 with peptic ulcer, and 89 healthy controls were measured by rHP-NAP-based ELISA. rHP-NAP-stimulated production of interleukin-8 (IL-8) and growth-related oncogene (GRO(alpha)) cytokines in the culture supernatant of SGC7901 gastric epithelial cells was also detected. The serum positivity and mean absorbance value of HP-NAP-specific antibodies in the gastric cancer group (97.7% and 1.01 +/- 0.24) were significantly higher than those in the chronic gastritis group (85.7% and 0.89 +/- 0.14, P < 0.005) and healthy control group (27.7% and 0.65 +/- 0.18, P < 0.001). The sensitivity and specificity of ELISA for the detection of HP-NAP-specific antibodies were 95.5% and 91.5%, respectively. HP-NAP could slightly up-regulate IL-8 production in gastric epithelial cell lines but had no effect on GRO(alpha) production. Infection with virulent H pylori strains secreting HP-NAP is associated with severe gastroduodenal diseases, and HP-NAP may play a role in the development of gastric carcinoma. rHP-NAP-based ELISA can be used as a new method to detect H pylori infection. The direct effect of HP-NAP on gastric epithelial cells may be limited, but HP-NAP may contribute to inflammatory response or carcinogenesis by activating neutrophils.

Helicobacter pylori neutrophil-activating protein promotes myeloperoxidase release from human neutrophils

Helicobacter pylori infection induces acute and chronic inflammation and plays a key role in gastric mucosal diseases. H. pylori neutrophil-activating protein (HP-NAP), one of its virulence factors, induces not only chemotactic but also oxidative burst responses of neutrophils. Activated neutrophils use myeloperoxidase (MPO) to generate many cytotoxic oxidants, which might result in gastric mucosal injury. In this study, we evaluated whether HP-NAP could promote MPO release from human neutrophils. Recombinant HP-NAP expressed in Escherichia coli was purified by two sequential gel filtration chromatographies and then subjected to syringe filtration for endotoxin removal. The purified recombinant HP-NAP assembles into a multimer with a secondary structure of the typical α-helix. In addition to stimulating the production of reactive oxygen species, HP-NAP is able to induce the secretion of MPO in human neutrophils. The increased MPO release from neutrophils induced by HP-NAP may be related to the pathogenesis of H. pylori-associated gastritis.

Helicobacter pylori neutrophil activating protein's potential as tool in therapeutic immune modulation

Helicobacter pylori is one of the most common human pathogens and is associated with chronic gastritis, peptic ulcers and gastric carcinoma. H. pylori establishes a persistent infection, suggesting that the host response is ineffective in clearing the infection. H. pylori employs multiple mechanisms to undermine host responses. For example, CD4+ T cells are recruited to the gastric mucosa of infected individuals, but these cells are predominantly T helper 1 (TH1) cells, which do not provide protection from the extracellular pathogen. H. pylori neutrophil-activating protein (HP-NAP) promotes the TH1 polarized response. HP-NAP induces macrophages and dendritic cells to produce IL-12 and IL-23, which could explain the effect on T cells. These properties of HP-NAP make it a potential immunotherapeutic tool to redirect harmful TH2 cell responses, as seen in atopic allergy, to TH1 cell responses and also in IL-12 induction in cancer therapy.

The Neutrophil-Activating Protein of Helicobacter pylori Crosses Endothelia to Promote Neutrophil Adhesion In Vivo

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of beta(2) integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection.

Construction of an oral recombinant DNA vaccine fromH pylorineutrophil activating protein and its immunogenicity

To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity. By genetic engineering methods, the genomic DNA of H pylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction (PCR) and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments. A 435 bp product was cloned using high homology with HP-NAP gene in GenBank (more than 98%). With identification by PCR and restriction enzyme digestion, a recombinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with H pylorii whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 (pIRES-NAP) also induced a specific immune response. The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against H pylori infection.

Helicobacter pylori induce neutrophil transendothelial migration: Role of the bacterial HP-NAP

Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.

A low molecular weight factor is a significant mediator of non-opsonic neutrophil activation by Helicobacter pylori.

Helicobacter pylori is believed to trigger neutrophil activation through several factors, including the H. pylori neutrophil-activating protein (HpNAP). The aim of this study was to characterise the factors within H. pylori cell-free extracts that stimulate neutrophil activation. Neutrophil activation was found to be dose-dependent and exhibited considerable variation between different clinical isolates. Activity was attributable to more than one protein factor. A low mol. wt fraction of <3 kDa was found to contribute a large proportion of the neutrophil-stimulating activity within H. pylori cell-free extract. Additional activity was provided by a high mol. wt fraction, possibly representing HpNAP. An inhibition ELISA and neutralisation experiments failed to identify or exclude formyl methionyl leucyl phenylalanine as the active factor within the low mol. wt fraction. The importance of the putative, low mol. wt neutrophil-activating factor may have been overlooked by those studies that have used concentrated H. pylori extracts.