Helicobacter pylori infection generates genetic instability in gastric cells

The discovery that Helicobacter pylori is associated with gastric cancer has led to numerous studies that investigate the mechanisms by which H. pylori induces carcinogenesis. Gastric cancer shows genetic instability both in nuclear and mitochondrial DNA, besides impairment of important DNA repair pathways. As such, this review highlights the consequences of H. pylori infection on the integrity of DNA in the host cells. By down-regulating major DNA repair pathways, H. pylori infection has the potential to generate mutations. In addition, H. pylori infection can induce direct changes on the DNA of the host, such as oxidative damage, methylation, chromosomal instability, microsatellite instability, and mutations. Interestingly, H. pylori infection generates genetic instability in nuclear and mitochondrial DNA.     

Identification of cell-surface mannans in a virulent Helicobacter pylori strain

With the intent of contributing to a carbohydrate-based vaccine against the gastroduodenal pathogen, Helicobacter pylori, we report here the structure of cell-surface mannans obtained from a virulent strain. Unlike other wild-type strains, this strain was found to express in good quantities this polysaccharide in vitro. Structural analysis revealed a branched mannan formed by a backbone of α-(1→6)-linked mannopyranosyl residues with approximately 80% branching at the O-2 position. The branches were composed of O-2-linked Man residues in both α- and β-configurations:In addition, this strain also expressed cell-surface emblematic H. pylori lipopolysaccharides (LPS) containing partially fucosylated polyLacNAc O-chains. Affinity assays with polymyxin-B and concanavalin A revealed no association between the mannan and the LPS. The described mannans may be implicated in the mediation of host-microbial interactions and immunological modulation.     

Characterizing the polymeric status of Helicobacter pylori heat shock protein 60

Helicobacter pylori heat shock protein 60 (HpHsp60) was first identified as an adhesion molecule associated with H. pylori infection. Here we have analyzed the structure of HpHsp60 via amino acid BLAST, circular dichroism, and electrophoresis and the results indicate that most recombinant HpHsp60 molecules exist as dimers or tetramers, which is quite different from Escherichia coli Hsp60. Treatment of human monocytic cells THP-1 with HpHsp60 was found to up-regulate a panel of cytokines including IL-1α, IL-8, IL-10, IFN-γ, TNF-α, TGF-β, GRO, and RANTES. Carboxymethylated HpHsp60 molecules with a switched oligomeric status were able to further enhance NF-κB-mediated IL-8 and TNF-α secretion in THP-1 cells compared to unmodified HpHsp60 molecules. These results indicated that the oligomeric status of HpHsp60s might have an important role in regulating host inflammation and thus help facilitate H. pylori persistent infection.     

Glycosylated liposomes against Helicobacter pylori: Behavior in acidic conditions

Helicobacter pylori was isolated in 1982 and confirmed as a gastric pathogenic agent at the end of the 1980s. The present work deals with liposomes formulations in which are incorporated cholesteryl tetraethylene glycol oside as model ligands for H. pylori adhesins. This study is devoted to the behavior of liposomes in gastric conditions. The glycosylated vesicles are stable and the pH of the internal aqueous compartment remains close to 4 even through more acidic conditions are imposed to the external phase (pH 1.2-2). Such a pH gradient depends essentially on the nature of phospholipids used and is not extensively affected by the incorporation of the targeting agent. These aspects are particularly important to the development of liposome formulations against H. pylori, bacteria sensitive to antibiotics which are unstable in very acidic conditions.     

The CagA protein of Helicobacter pylori suppresses the functions of dendritic cell in mice

CagA protein is the most assessed effecter molecule of Helicobacter pylori. In this report, we demonstrate how CagA protein regulates the functions of dendritic cells (DC) against H. pylori infection. In addition, we found that CagA protein was tyrosine-phosphorylated in DC. The responses to cagA-positive H. pylori in DC were reduced in comparison to those induced by cagA-negative H. pylori. CagA-overexpressing DC also exhibited a decline in the responses against LPS stimulation and the differentiation of CD4⁺ T cells toward Th1 type cells compared to wild type DC. In addition, the level of phosphorylated IRF3 decreased in CagA-overexpressing DC stimulated with LPS, indicating that activated SHP-2 suppressed the enzymatic activity of TBK1 and consequently IRF3 phosphorylation. These data suggest that CagA protein negatively regulates the functions of DC via CagA phosphorylation and that cagA-positive H. pylori strains suppress host immune responses resulting in their chronic colonization of the stomach.     

A potential role for Helicobacter pylori heat shock protein 60 in gastric tumorigenesis

Helicobacter pylori has been found to promote the malignant process leading to gastric cancer. Heat shock protein 60 of H. pylori (HpHSP60) was previously been identified as a potent immunogene. This study investigates the role of HpHSP60 in gastric cancer carcinogenesis. The effect of HpHSP60 on cell proliferation, anti-death activity, angiogenesis and cell migration were explored. The results showed that HpHSP60 enhanced migration by gastric cancer cells and promoted tube formation by umbilical vein endothelial cells (HUVECs); however, HpHSP60 did not increase cell proliferation nor was this protein able to rescue gastric cancer cells from death. Moreover, the results also indicated HpHSP60 had different effects on AGS gastric cancer cells or THP-1 monocytic cells in terms of their expression of pro-inflammatory cytokines, which are known to be important to cancer development. We propose that HpHSP60 may trigger the initiation of carcinogenesis by inducing pro-inflammatory cytokine release and by promoting angiogenesis and metastasis. Thus, this extracellular pathogen-derived HSP60 is potentially a vigorous virulence factor that can act as a carcinogen during gastric tumorigenesis.     

Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 10⁷ M^− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)₃₅] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.     

Rapid paper disk test for identification of Helicobacter pylori in mixed cultures of gerbil gastric homogenates

A method denominated rapid paper disk test (RPDT) was developed to identify H. pylori colonies in complex cultures obtained from gerbil gastric homogenates. Identification is based on a characteristic reaction pattern (RP) for H. pylori colonies given by the combination of the urease-oxidase activities on a paper disk. Compared to the RPs obtained from gerbil's intestinal tract isolated bacteria, H. pylori RP is completely distinguishable, even from those of bacteria that share one or both activities as are Aerococcus urinae, Bacillus sphaericus, Bacillus brevis, Corynebacterium pseudogenitalium, and Staphylococcus simulans, as well as from those produced by collection strains Proteus vulgaris and Pseudomonas aeruginosa. This method allows the practical quantification of H. pylori colonies in highly contaminated plates. RPDT has the following advantages over other methodologies that use indicators in the medium: it employs two of the three routinely used H. pylori biochemical identification tests, the reagents do not interfere with bacterial viability, there are no restrictions in relation to the medium used, and it is a simple, fast, and low-cost method.     

Helicobacter pylori causes runx3 gene methylation and its loss of expression in gastric epithelial cells, which is mediated by nitric oxide produced by macrophages

Previous reports have indicated that Helicobacter pylori (H. pylori) causes epigenetic changes of certain genes such as cancer suppression genes, which may be associated with carcinogenesis. However, the mechanism by which it causes epigenetic changes in certain genes and not in others is unclear. Presently, we focused on a cancer suppression gene, runx3, and demonstrated the following: (1) H. pylori induces nitric oxide (NO) production in macrophages. (2) NO causes methylation of runx3 in epithelial cells. (3) H. pylori induces the methylation of epithelial cells in the presence of macrophages, which is reversed by an NO-specific inhibitor. These results indicate that H. pylori-induced methylation is mediated by NO, and suggest that NO may be a key to the mechanism of how H. pylori causes epigenetic changes in certain genes. Additionally, we demonstrated that lipopolysaccharide, as well as H. pylori, induces NO-mediated methylation, indicating that other inflammation inducers beside H. pylori might induce aberrant methylation of runx3.     

Functional characterization of Helicobacter pylori TlyA: Pore-forming hemolytic activity and cytotoxic property of the protein

Helicobacter pylori is a human specific gastric pathogen. H. pylori pathogenesis process involves a number of well-studied virulence factors that include the ‘vacuolating cytotoxin’ and the ‘cytotoxin associated gene A’. Analysis of the H. pylori genome, however, indicates presence of additional virulence factors that are yet to be characterized in molecular detail. For example, H. pylori genome harbors a gene that has potential to encode a protein with sequence similarity to those of the TlyA-like proteins of several pathogenic bacteria. Earlier studies have indicated potential association of this H. pylori tlyA gene in the virulence mechanism of the organism. Despite such notions, however, the TlyA-like protein of H. pylori has not been studied previously in molecular detail. In particular, purified form of H. pylori TlyA has never been studied before toward exploring its functional properties. Here, we report characterization of the H. pylori TlyA protein purified from the recombinant over-expression system in Escherichia coli. Purified form of the recombinant TlyA exhibits prominent hemolytic activity against human erythrocytes, presumably via formation of pores of specific diameter in the cell membrane. Purified TlyA also triggers prominent cytotoxic responses in human gastric adenocarcinoma cells. Altogether, our study establishes H. pylori TlyA as a potential virulence factor of the organism.     

Integrated microfluidic magnetic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori

In this paper, we have developed and characterized a microfluidic magnetic immunosensor coupled to a gold electrode for the rapid and sensitive quantification of human serum IgG antibodies to Helicobacter pylori. This microorganism cause peptic ulcers and chronic gastritis, affecting around the 10% of the world population. The sensor was completely automated and the antibodies detection in serum samples was carried out using a non-competitive immunoassay based on the use of purified H. pylori antigens that are immobilized on magnetic microspheres 3-aminopropyl-modified. The magnetic microbeads were injected into microchannel devices and manipulated for an external removable magnet. The IgG antibodies in human serum sample are allowed to react immunologically with the immobilized antigens, and the bounded antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP and an electroactive product was detected on gold layer electrode at 0.250 V. The response current obtained from the product of enzymatic reaction is directly proportional to the activity of the enzyme and, consequently, to the amount of IgG antibodies to H. pylori in serum samples. The electrochemical detection can be done within 1 min and total assay time was 25 min. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.37 and 2.1 U mL⁻¹, respectively, and the within- and between-assay coefficients of variation were below 5%. Our results indicate the potential usefulness of our fabricated microbiochip for the early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.     

Characterization and inactivation of an agmatine deiminase from Helicobacter pylori

Helicobacter pylori encodes a potential virulence factor, agmatine deiminase (HpAgD), which catalyzes the conversion of agmatine to N-carbamoyl putrescine (NCP) and ammonia - agmatine is decarboxylated arginine. Agmatine is an endogenous human cell signaling molecule that triggers the innate immune response in humans. Unlike H. pylori, humans do not encode an AgD; it is hypothesized that inhibition of this enzyme would increase the levels of agmatine, and thereby enhance the innate immune response. Taken together, these facts suggest that HpAgD is a potential drug target. Herein we describe the optimized expression, isolation, and purification of HpAgD (10-30 mg/L media). The initial kinetic characterization of this enzyme has also been performed. Additionally, the crystal structure of wild-type HpAgD has been determined at 2.1 Å resolution. This structure provides a molecular basis for the preferential deimination of agmatine, and identifies Asp198 as a key residue responsible for agmatine recognition, which has been confirmed experimentally. Information gathered from these studies led to the development and characterization of a novel class of haloacetamidine-based HpAgD inactivators. These compounds are the most potent AgD inhibitors ever described.     

Helicobacter pylori-derived Heat shock protein 60 enhances angiogenesis via a CXCR2-mediated signaling pathway

Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently, H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cells and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLCβ2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLCβ2/Ca2+ signal transduction in endothelial cells.     

The Crystal Structure of Ferritin from Helicobacter pylori Reveals Unusual Conformational Changes for Iron Uptake

The crystal structure of recombinant ferritin from Helicobacter pylori has been determined in its apo, low-iron-bound, intermediate, and high-iron-bound states. Similar to other members of the ferritin family, the bacterial ferritin assembles as a spherical protein shell of 24 subunits, each of which folds into a four-α-helix bundle. Significant conformational changes were observed at the BC loop and the entrance of the 4-fold symmetry channel in the intermediate and high-iron-bound states, whereas no change was found in the apo and low-iron-bound states. The imidazole rings of His149 at the channel entrance undergo conformational changes that bear resemblance to heme configuration and are directly coupled to axial translocation of Fe ions through the 4-fold channel. Our results provide the first structural evidence of the translocation of Fe ions through the 4-fold channel in prokaryotes and the transition from a protein-dominated process to a mineral-surface-dominated process during biomineralization.     

Up-regulation of neutrophil activating protein in Helicobacter pylori under high-salt stress: Structural and phylogenetic comparison with bacterial iron-binding ferritins

It is generally accepted that most gastrointestinal diseases are probably caused by the bacterial pathogen Helicobacter pylori (H. pylori). In this study we have focused on the comparison of protein expression profiles of H. pylori grown under normal and high-salt conditions by a proteomics approach. We have identified about 190 proteins whose expression levels changed after growth at high salt concentration. Among these proteins, neutrophil-activating protein (NapA) was found to be consistently up-regulated under osmotic stress brought by high salts. We have investigated the effect of high salt on secondary and tertiary structures of NapA by circular dichroism spectroscopy followed by analytical ultracentrifugation to monitor the change of quaternary structure of recombinant NapA with increasing salt concentration. The loss of iron-binding activity of NapA coupled with noticeable energetic variation in protein association of NapA as revealed by isothermal titration calorimetry was found under high salt condition. The phylogenetic tree analysis based on sequence comparison of 16 protein sequences encompassing NapA proteins and ferritin of H. pylori and other prokaryotic organisms pointed to the fact that all H. pylori NapA proteins of human origin are more homologous to NapA of Helicobacter genus than to other bacterial NapA. Based on computer modeling, NapA proteins from H. pylori of human isolates are found more similar to ferritin from H. pylori than to NapA from other species of bacteria. Taken together, these results suggested that divergent evolution of NapA and ferritin possessing dissimilar and diverse sequences follows a path distinct from that of convergent evolution of NapA and ferritin with similar dual functionality of iron-binding and ferroxidase activities.     

Chromone and chromanone glucosides from Hypericumsikokumontanum and their anti-Helicobacter pylori activities

Chromone glucosides, takanechromones A-C (1, 2 and 5) and chromanone glucosides, named takanechromanones A and B (3 and 4), were isolated from the methanolic extracts of Hypericumsikokumontanum together with 27 known compounds. Their structures were established based on spectroscopic evidence. The isolated compounds and some chromone derivatives were assayed for antimicrobial activity against Helicobacter pylori and cytotoxicity against human cancer cell lines.     

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.