Pylori Lipopolysaccharide Research Articles

Immunobiological effects of lipopolysaccharide derived from Helicobacter pylori and influence of a proton pump inhibitor lansoprazole on human polymorphonuclear leukocytes.

Helicobacter pylori colonizes the human gastric mucosa of more than half of the human population and has a unique lipopolysaccharide (LPS) structure. LPS is the most dominant and suitable pathogen-associated molecular pattern that is detected via pattern recognition receptors. Although the priming effect of H. pylori LPS on reactive oxygen species (ROS) production of PMNs is lower than that of Escherichia coli O111:B4 LPS, LPS released from H. pylori associated with antibiotics eradication therapy may activate PMNs and increase ROS production. In addition, we describe the effects of H. pylori and E. coli O111:B4 LPSs on gene expression and the anti-inflammatory effect of lansoprazole (LPZ) in human polymorphonuclear leukocytes. LPS isolated from H. pylori and E. coli O111:B4 alters toll-like receptor 2 (TLR) and TLR4 expressions similarly. However, LPS from E. coli O111:B4 and H. pylori caused a 1.8-fold and 1.5-fold increase, respectively, in CD14 expression. All LPS subtypes upregulated TNFα and IL6 expression in a concentration-dependent manner. Although E. coli O111:B4 LPS upregulated IL8R mRNA levels, H. pylori LPS did not (≦ 100ng/mL). Gene expression levels of ITGAM demonstrated no significant change on using both LPSs. These different effects on the gene expression in PMNs may depend on variations in LPS structural modifications related to the acquired immunomodulatory properties of H. pylori LPS. Proton pump inhibitors, i.e., LPZ, are used in combination with antibiotics for the eradication therapy of H. pylori. LPZ and its acid-activated sulphenamide form AG-2000 suppress ROS production of PMNs in a dose-dependent manner. These results suggest that LPZ combination with antibiotics for H. pylori eradication reduces gastric inflammation by suppressing ROS release from PMNs.

Galectin-2 Agglutinates Helicobacter pylori via Lipopolysaccharide Containing H Type I Under Weakly Acidic Conditions.

Galectins are β-galactoside-binding animal lectins involved in various biological functions, such as host defense. Galectin-2 and -3 are members of the galectin family that are expressed in the stomach, including the gastric mucosa and surface mucous cells. Galectin-3 exhibits aggregation and bactericidal activity against Helicobacter pylori in a β-galactoside-dependent manner. We previously reported that galectin-2 has the same activity under neutral pH conditions. In this study, the H. pylori aggregation activity of galectin-2 was examined under weakly acidic conditions, in which H. pylori survived. Galectin-2 agglutinated H. pylori even at pH 6.0, but not at pH 5.0, correlating with its structural stability, as determined using circular dichroism. Additionally, galectin-2 binding to the lipopolysaccharide (LPS) of H. pylori cultured under weakly acidic conditions was investigated using affinity chromatography and Western blotting. Galectin-2 could bind to H. pylori LPS containing H type I, a Lewis antigen, in a β-galactoside-dependent manner. In contrast, galectin-3 was structurally more stable than galectin-2 under acidic conditions and bound to H. pylori LPS containing H type I and Lewis X. In conclusion, galectin-2 and -3 might function cooperatively in the defense against H. pylori in the stomach under different pH conditions.

Masking of typical TLR4 and TLR5 ligands modulates inflammation and resolution by Helicobacter pylori.

Helicobacter pylori is a paradigm of chronic bacterial infection and is associated with peptic ulceration and malignancies. H. pylori uses specific masking mechanisms to avoid canonical ligands from activating Toll-like receptors (TLRs), such as lipopolysaccharide (LPS) modification and specific flagellin sequences that are not detected by TLR4 and TLR5, respectively. Thus, it was believed for a long time that H. pylori evades TLR recognition as a crucial strategy for immune escape and bacterial persistence. However, recent data indicate that multiple TLRs are activated by H. pylori and play a role in the pathology. Remarkably, H. pylori LPS, modified through changes in acylation and phosphorylation, is mainly sensed by other TLRs (TLR2 and TLR10) and induces both pro- and anti-inflammatory responses. In addition, two structural components of the cag pathogenicity island-encoded type IV secretion system (T4SS), CagL and CagY, were shown to contain TLR5-activating domains. These domains stimulate TLR5 and enhance immunity, while LPS-driven TLR10 signaling predominantly activates anti-inflammatory reactions. Here, we discuss the specific roles of these TLRs and masking mechanisms during infection. Masking of typical TLR ligands combined with evolutionary shifting to other TLRs is unique for H. pylori and has not yet been described for any other species in the bacterial kingdom. Finally, we highlight the unmasked T4SS-driven activation of TLR9 by H. pylori, which mainly triggers anti-inflammatory responses.

Helicobacter pylori Modulates Heptose Metabolite Biosynthesis and Heptose-Dependent Innate Immune Host Cell Activation by Multiple Mechanisms.

Heptose metabolites including ADP-d-glycero-β-d-manno-heptose (ADP-heptose) are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent proinflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites, which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question of how proinflammatory ADP-heptose biosynthesis can be regulated by bacteria. We have characterized the interstrain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori, which impact cell-autonomous proinflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains and was also variably expressed between strains. Amounts of gene transcripts in the hldE gene cluster displayed intrastrain and interstrain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori lipopolysaccharide (LPS) heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE, and GmhB proteins. On the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, the structures of major reaction products were identified as β-d-ADP-heptose and β-heptose-1-monophosphate. A proinflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed us to uncover additional strain variation in generating heptose metabolites. IMPORTANCE Bacterial heptose metabolites, intermediates of lipopolysaccharide (LPS) biosynthesis, are novel microbe-associated molecular patterns (MAMPs) that activate proinflammatory signaling. In the gastric pathogen Helicobacter pylori, heptoses are transferred into host cells by the Cag type IV secretion system, which is also involved in carcinogenesis. Little is known about how H. pylori, which is highly strain variable, regulates heptose biosynthesis and downstream host cell activation. We report here that the regulation of proinflammatory heptose production by H. pylori is strain specific. Heptose gene cluster activity is modulated by the presence of an active cag pathogenicity island (cagPAI), contact with human cells, and the carbon starvation regulator A. Reconstitution with purified biosynthesis enzymes and purified bacterial lysates allowed us to biochemically characterize heptose pathway products, identifying a heptose-monophosphate variant as a novel proinflammatory metabolite. These findings emphasize that the bacteria use heptose biosynthesis to fine-tune inflammation and also highlight opportunities to mine the heptose biosynthesis pathway as a potential therapeutic target against infection, inflammation, and cancer.

Chemical Synthesis of the Highly Sterically Hindered Core Undecasaccharide of Helicobacter pylori Lipopolysaccharide for Antigenicity Evaluation with Human Serum.

Helicobacter pylori, listed as a human carcinogen by the Department of Health and Human Services, colonizes the gastric mucosa of more than half of the world's population. The individuals infected with H. pylori have a high risk to develop chronic gastritis, peptic ulcers, and even gastric cancer. The conserved core structure of H. pylori lipopolysaccharide (LPS) has been regarded as a promising candidate structure for development of a glycoconjugate vaccine targeting multiple serotypes. Here, we report a total synthesis of the core undecasaccharide of H. pylori LPS and its subunit antigens. The match and mismatch between the glycosyl donor and acceptor caused by the inert hydroxyl groups were addressed by a judicious choice of orthogonal protection strategies and glycosylation conditions. A combination of acyl remote participation and solvent effects has been applied for selective formation of the five 1,2-cis-glucosidic bonds. The high steric hindrance induced by the high carbon sugars and trinacriform architecture required that the core undecasaccharide was synthesized through a finely tuned linear assembly [2 + (1 + (3 + (1 + (1 + 3))))] rather than convergent strategies. An antigenicity evaluation using glycan microarrays showed that an α-(1 → 6)-glucan trisaccharide is recognized by IgG antibodies in sera of H. pylori-infected patients. The phosphate group of the inner core trisaccharide key epitope is very important for IgG recognition. These findings are an important step toward designing carbohydrate-based vaccines against H. pylori.

Chemoenzymatic synthesis of an α-1,6-glucan-based conjugate vaccine against Helicobacter pylori.

In this study, we investigated the utility of glycoconjugates based on a linear α-1,6-glucan chain synthesized using a recombinant α-1,6-glucosyltransferase from the 26695 strain of Helicobacter pylori. Capillary electrophoresis-mass spectrometry analysis confirmed the main product to contain 9-10 sequentially added α-1,6-linked glucose residues. This was consistent with a length of α-1,6-glucan structure present in the outer core region of H. pylori lipopolysaccharide (LPS) from strains 26695 and 26695 HP0826::Kan. The synthetic α-1,6-glucan was conjugated to either bovine serum albumin or tetanus toxoid and immunological properties of resultant glycoconjugates investigated. The conjugates were immunogenic in rabbits and mice and induced strong and specific IgG responses against purified LPS from typeable and nontypeable α-1,6-glucan-positive H. pylori strains. Furthermore, the post-immune sera from rabbits that received the conjugates were bactericidal and cross-reacted with selected clarithromycin-resistant and clarithromycin-susceptible clinical isolates of H. pylori. This technology offers a novel approach to the design of a synthetic carbohydrate-based vaccine against H. pylori.

Salvia cadmica extracts rich in polyphenols neutralize a deleterious effects of oxidative stress driven by Helicobacter pylori lipopolysaccharide in cell cultures of gastric epithelial cells or fibroblasts

IntroductionPlant ingredients are candidates for new therapeutic formulations against bacterial pathogens with high rate of antibiotic resistance and deleterious inflammation. Helicobacter pylori Gram-negative rods causing gastritis, gastric/duodenal ulcers and gastric cancer, which show high antibiotic resistance require new effective drugs. AimTo examine antibacterial activity of Salvia cadmica Boiss. extracts (roots and aerial parts) towards H. pylori strains resistant to clarithromycin and/or metronidazole. To determine whether extracts neutralize deleterious effects driven by H. pylori lipopolysaccharide (LPS) such as elevated oxidative stress and apoptosis, and promote cell regeneration of gastric epithelial cells or fibroblasts in vitro. Material and methodsHydromethanolic extracts (20:80 v/v) from roots and aerial parts of seed-derived plants of S. cadmica were used for biological study. Biocompatibility was tested according to the ISO norm 10993–5 in the MTT (3-(4,5-dimethylthiazol-2-yl)− 2,5-diphenyltetrazolium bromide) reduction assay. Antibacterial activity against H. pylori (two reference strains and five clinical isolates clarithromycin and/or metronidazole resistant) was tested in the broth microdilution assay as recommended by The European Committee on Antimicrobial Susceptibility (EUCAST). Cell cultures of guinea pig gastric epithelial cells or fibroblasts exposed to H. pylori LPS, and/or S. cadmica extracts, were tested for the rate of oxidative stress (3-nitrotyrosine-3NT), apoptosis (immunostaining of pro- or anti-apoptotic proteins, DNA degradation), phosphorylated extracellular signal-regulated kinase - pErk (immunostaining), cell migration (scratch assay), collagen I and interleukin (IL)− 33 (enzyme linked immunosorbent assay – ELISA). ResultsThe minimal inhibitory concentration (MIC) of root extract for H. pylori reference strains ranged from 39 to 78 µg/mL whereas minimal bactericidal concentration (MBC) was in the range of 78–310 µg/mL. The MIC for clinical H. pylori isolates ranged from 195 to 1560 µg/mL while MBC from 390 to 1560 µg/mL. Aerial part extract showed a weaker inhibitory and bactericidal effects towards H. pylori reference strains (MIC/MBC range 78 −156 µg/mL), and to clinical isolates (MIC/MBC range 390 −1560 µg/mL). S. cadmica extracts effectively diminished oxidative stress, cell apoptosis rate and phosphorylated extracellular signal-regulated kinase (pErk) level, which were driven by H. pylori lipopolysaccharide (LPS). ConclusionsThe hydromethanolic extracts from S. cadmica (root and aerial part) show anti-H. pylori activity, in conjunction with anti-oxidative/anti-apoptotic effects in cell cultures of gastric epithelial cells and fibroblasts. These effects are promising for the development of potentially therapeutic formulations.

Helicobacter pylori-Mediated Immunity and Signaling Transduction in Gastric Cancer.

Helicobacter pylori infection is a leading cause of gastric cancer, which is the second-most common cancer-related death in the world. The chronic inflammatory environment in the gastric mucosal epithelia during H. pylori infection stimulates intracellular signaling pathways, namely inflammatory signals, which may lead to the promotion and progression of cancer cells. We herein report two important signal transduction pathways, the LPS-TLR4 and CagA-MET pathways. Upon H. pylori stimulation, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) mainly on macrophages and gastric epithelial cells. This induces an inflammatory response in the gastric epithelia to upregulate transcription factors, such as NF-κB, AP-1, and IRFs, all of which contribute to the initiation and progression of gastric cancer cells. Compared with other bacterial LPSs, H. pylori LPS has a unique function of inhibiting the mononuclear cell (MNC)-based production of IL-12 and IFN-γ. While this mechanism reduces the degree of inflammatory reaction of immune cells, it also promotes the survival of gastric cancer cells. The HGF/SF-MET signaling plays a major role in promoting cellular proliferation, motility, migration, survival, and angiogenesis, all of which are essential factors for cancer progression. H. pylori infection may facilitate MET downstream signaling in gastric cancer cells through its CagA protein via phosphorylation-dependent and/or phosphorylation-independent pathways. Other signaling pathways involved in H. pylori infection include EGFR, FAK, and Wnt/β-Catenin. These pathways function in the inflammatory process of gastric epithelial mucosa, as well as the progression of gastric cancer cells. Thus, H. pylori infection-mediated chronic inflammation plays an important role in the development and progression of gastric cancer.

Helicobacter pylori lipopolysaccharide structural domains and their recognition by immune proteins revealed with carbohydrate microarrays

The structural diversity of the lipopolysaccharides (LPSs) from Helicobacter pylori poses a challenge to establish accurate and strain-specific structure-function relationships in interactions with the host. Here, LPS structural domains from five clinical isolates were obtained and compared with the reference strain 26695. This was achieved combining information from structural analysis (GC-MS and ESI-MSn) with binding data after interrogation of a LPS-derived carbohydrate microarray with sequence-specific proteins. All LPSs expressed Lewisx/y and N-acetyllactosamine determinants. Ribans were also detected in LPSs from all clinical isolates, allowing their distinction from the 26695 LPS. There was evidence for 1,3-d-galactans and blood group H-type 2 sequences in two of the clinical isolates, the latter not yet described for H. pylori LPS. Furthermore, carbohydrate microarray analyses showed a strain-associated LPS recognition by the immune lectins DC-SIGN and galectin-3 and revealed distinctive LPS binding patterns by IgG antibodies in the serum from H. pylori-infected patients.

Chemical Synthesis and Immunological Evaluation of Helicobacter pylori Serotype O6 Tridecasaccharide O-Antigen Containing a dd-Heptoglycan.

The development of glycoconjugate vaccines against Helicobacter pylori is challenging. An exact epitope of the H. pylori lipo-polysaccharide (LPS) O-antigens that contain Lewis determinant oligosaccharides and unique dd-heptoglycans has not yet been identified. Reported here is the first total synthesis of H. pylori serotype O6 tridecasaccharide O-antigen containing a terminal Ley tetrasaccharide, a unique α-(1→3)-, α-(1→6)-, and α-(1→2)-linked heptoglycan, and a β-d-galactose connector, by an [(2×1)+(3+8)] assembly sequence. Seven oligosaccharides covering different portions of the entire O-antigen were prepared for immunological investigations with a particular focus on elucidation of the roles of the dd-heptoglycan and Ley tetrasaccharide. Glycan microarray analysis of sera from rabbits immunized with isolated serotype O6 LPS revealed a humoral immune response to the α-(1→3)-linked heptoglycan, a key motif for designing glycoconjugate vaccines for H. pylori serotype O6.

Helicobacter pylori Recombinant CagA Regulates Th1/Th2 Balance in a BALB/c Murine Model

Purpose: Helicobacter pylori is recognized as one of the prevalent causes of human gastricinfection. In the present study, the role of mixed immunization with H. pylori lipopolysaccharide(LPS) and recombinant cytotoxin-associated gene A (rCagA) as a stimulator of host immuneresponses was determined. Methods: BALB/c mice were immunized with different formulations by the systemic administrationat 14-day intervals. The effects of the formulations plus CpG adjuvants were assessed before andpost-immunization in separated studies. Moreover, the expression of Th1/Th2 cytokines wasquantified in sera of immunized mice using reverse transcription polymerase chain reaction (RTPCR)test and the protein levels confirmed with enzyme linked immunosorbent assay (ELISA).Finally, the specific antibody levels in sera were studied by ELISA and the tendency of cellularresponse was examined by IgG1/IgG2a ratio. Results: Data of Western blotting verified the presence of constructed protein. Analysisof lymphocyte proliferation showed that CpG-conjugated rCagA increases lymphocytesproliferation compared to the control group. Also, it was shown that formulations containing LPSand rCagA promote a Th1 response indicated by interferon-gamma expression and induced Th1/Th2 balance. Additionally, the specific IgG1, total IgG and IgG2a levels elevated in response toall treatments. Ultimately, the IgG2a/IgG1 ratio in the mice immunized with rCagA-containingformulations increased. Conclusion: These results indicated that rCagA protein carried with CpG adjuvant not onlymaintained its antigenicity throughout the experiment but also induced robust Th1-biasedimmune responses. Therefore, it holds promise for the production of an efficient vaccine against H. pylori infection.

Variations in the Expression of Terminal Oligosaccharide Units and Glycosylation of Poly(N-acetyllactosamine) Chain in the Helicobacter pylori Lipopolysaccharide upon Colonization of Rhesus Macaques.

Helicobacter pylori is an important human pathogen that causes gastritis, gastric and duodenal ulcers, and gastric cancer. O-polysaccharides of H. pylori lipopolysaccharide (LPS) are composed of (β1→3)-poly(N-acetyllactosamine) (polyLacNAc) decorated with multiple α-L-fucose residues. In many strains, their terminal LacNAc units are mono- or di-fucosylated to mimic Lewis X (Lex) and/or Lewis Y (Ley) oligosaccharides. The studies in rhesus macaques as a model of human infection by H. pylori showed that this bacterium adapts to the host during colonization by expressing host Lewis antigens. Here, we characterized LPS from H. pylori strains used in the previous study, including the parental J166 strain and the three derivatives (98-149, 98-169, and 98-181) isolated from rhesus macaques after long-term colonization. Chemical and NMR spectroscopic analyses of the LPS showed that the parent strain expressed Lex, Ley, and H type 1 terminal oligosaccharide units. The daughter strains were similar to the parental one in the presence of the same LPS core and fucosylated polyLacNAc chain of the same length but differed in the terminal oligosaccharide units. These were Lex in the isolates 98-149 and 98-169, which corresponded to the Lea phenotype of the host animals, and Ley was found in the 98-181 isolate from the macaque characterized by the Leb phenotype. As Lea and Leb are isomers of Lex and Ley, respectively, the observed correlation confirmed adaptation of the expression of terminal oligosaccharide units in H. pylori strains to the properties of the host gastric mucosa. The 98-181 strain also acquired glucosylation of the polyLacNAc chain and was distinguished by a lower expression of fucosylated internal LacNAc units (internal Lex) as a result of decoration of polyLacNAc with β-glucopyranose, which may also play a role in the bacterial adaptation.

Structure and biological functions of Helicobacter pylori lipopolysaccharide

Helicobacter pylori (H.pylori) infection is one of the most common chronic infectious diseases in humans. It is the leading cause of chronic gastritis and peptic ulcer, and also the most important risk factor for gastric cancer. H. pylori lipopolysaccharide has unique chemical structures that are critical to the persistent infection and pathogenesis of H. pylori in human gastric mucosa. Recent studies have elucidated the H. pylori lipid A constitutive modification pathway, redefined the core-oligosaccharide and O-antigen domains, discovered that the heptan commonly present in the lipopolysaccharide structure of European strains is absent in East Asian strains and more importantly, the identification of ADP-heptose (the donor of heptose residues in lipopolysaccharide structure) as the newly discovered pathogen-associated molecular pattern, which is dependent on the type Ⅳ secretion system of H. pylori to induce inflammatory responses in host cells. This paper reviewed the resent research progress in the structure and biological functions of H. pylori lipopolysaccharide. Key words: Helicobacter pylori; Lipopolysaccharide; Heptose; ADP-heptose

The Interaction of Helicobacter pylori with TFF1 and Its Role in Mediating the Tropism of the Bacteria Within the Stomach.

Helicobacter pylori colonises the human stomach and has tropism for the gastric mucin, MUC5AC. The majority of organisms live in the adherent mucus layer within their preferred location, close to the epithelial surface where the pH is near neutral. Trefoil factor 1 (TFF1) is a small trefoil protein co-expressed with the gastric mucin MUC5AC in surface foveolar cells and co-secreted with MUC5AC into gastric mucus. Helicobacter pylori binds with greater avidity to TFF1 dimer, which is present in gastric mucus, than to TFF1 monomer. Binding of H. pylori to TFF1 is mediated by the core oligosaccharide subunit of H. pylori lipopolysaccharide at pH 5.0–6.0. Treatment of H. pylori lipopolysaccharide with mannosidase or glucosidase inhibits its interaction with TFF1. Both TFF1 and H. pylori have a propensity for binding to mucins with terminal non-reducing α- or β-linked N-acetyl-d-glucosamine or α-(2,3) linked sialic acid or Gal-3-SO42−. These findings are strong evidence that TFF1 has carbohydrate-binding properties that may involve a conserved patch of aromatic hydrophobic residues on the surface of its trefoil domain. The pH-dependent lectin properties of TFF1 may serve to locate H. pylori deep in the gastric mucus layer close to the epithelium rather than at the epithelial surface. This restricted localisation could limit the interaction of H. pylori with epithelial cells and the subsequent host signalling events that promote inflammation.

Ebselen suppresses inflammation induced by Helicobacterpylori lipopolysaccharide via the p38 mitogen-activated protein kinase signaling pathway.

Ebselen is a seleno-organic compound that has been demonstrated to have antioxidant and anti-inflammatory properties. A previous study determined that ebselen inhibits airway inflammation induced by inhalational lipopolysaccharide (LPS), however, the underlying molecular mechanism remains to be elucidated. The present study investigated the effect of ebselen on the glutathione peroxidase (GPX)‑reactive oxygen species (ROS) pathway and interleukin‑8 (IL‑8) expression induced by Helicobacterpylori LPS in gastric cancer (GC) cells. Cells were treated with 200ng/ml H. pylori‑LPS in the presence or absence of ebselen for various durations and concentrations (µmol/l). The expression of toll‑like receptor 4 (TLR4), GPX2, GPX4, p38 mitogen‑activated protein kinase (p38 MAPK), phosphorylated‑p38 MAPK, ROS production and IL‑8 expression were detected with western blotting or ELISA. The present study revealed that TLR4 expression was upregulated; however, GPX2 and GPX4 expression was reduced following treatment with H. pylori LPS, which led to increased ROS production, subsequently altering the IL‑8 expression level in GC cells. Additionally, it was determined that ebselen prevented the reduction in GPX2/4 levels induced by H. pylori LPS, however, TLR4 expression was not affected. Ebselen may also block the expression of IL‑8 by inhibiting phosphorylation of p38 MAPK. These data suggest ebselen may inhibit ROS production triggered by H. pylori LPS treatment via GPX2/4 instead of TLR4 signaling and reduce phosphorylation of p38 MAPK, resulting in altered production of IL‑8. Ebselen may, therefore, be a potential therapeutic agent to mediate H. pylori LPS-induced cell damage.

Store-operated Ca2+ Entry Facilitates the Lipopolysaccharide-induced Cyclooxygenase-2 Expression in Gastric Cancer Cells

Helicobacter pylori has been identified as one of the major causes of chronic gastritis, gastric and duodenal ulcers, and gastric cancer. Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria, and H. pylori LPS might play an exclusively important role in activating inflammatory pathways in monocytes and macrophages. To study the role of LPS in the underlying mechanism of inflammatory responses, we established an in vitro model using the human AGS gastric cancer cell line. We found that LPS mediates inflammation through setting off a cascade of events: activation of the store-operated calcium (SOC) channel, initiation of downstream NF-κB signaling, and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Phosphorylated ERK1/2 promotes the nuclear translocation of NF-κB, and eventually elevates the expression level of COX-2, a major inflammatory gene.

Role of LPS-elicited signaling in triggering gastric mucosal inflammatory responses to H. pylori: modulatory effect of ghrelin.

Infection with Helicobacter pylori is a primary culprit in the etiology of gastric disease, and its cell-wall lipopolysaccharide (LPS) is recognized as a potent endotoxin responsible for triggering a pattern of the mucosal inflammatory responses. The engagement by the LPS of gastric mucosal Toll-like receptor 4 (TLR4) leads to initiation of signal transduction events characterized by the activation of mitogen-activated protein kinase (MAPK) cascade, induction of phosphoinositide-specific phospholipase C (PLC)/protein kinase C (PKC)/phosphatidylinositol 3-kinase (PI3K) pathway, and up-regulation in Src/Akt. These signaling events in turn exert their influence over H. pylori-elicited excessive generation of NO and PGE2 caused by the disturbances in nitric oxide synthase and cyclooxygenase isozyme systems, increase in epidermal growth factor receptor transactivation, and the induction in matrix metalloproteinase-9 (MMP-9) release. Interestingly, the extent of gastric mucosal inflammatory response to H. pylori is influenced by a peptide hormone, ghrelin, the action of which relays on the growth hormone secretagogue receptor type 1a (GHS-R1a)-mediated mobilization of G-protein dependent transduction pathways. Yet, the signals triggered by TLR-4 activation as well as those arising through GHS-R1a stimulation converge at MAPK and PLC/PKC/PI3K pathways that form a key integration node for proinflammatory signals generated by H. pylori LPS as well as for those involved in modulation of inflammation by ghrelin. Hence, therapeutic targeting these signals' convergence and integration node could provide a novel and attractive opportunities for developing more effective treatments of H. pylori-related gastric disease.

Insights from the redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains.

H. pylori is a Gram-negative extracellular bacterium, first discovered by the Australian physicians Barry Marshall and Robin Warren in 1982, that colonises the human stomach mucosa. It is the leading cause of peptic ulcer and commonly infects humans worldwide with prevalence as high as 90% in some countries. H. pylori infection usually results in asymptomatic chronic gastritis, however 10-15% of cases develop duodenal or gastric ulcers and 1-3% develop stomach cancer. Infection is generally acquired during childhood and persists for life in the absence of antibiotic treatment. H. pylori has had a long period of co-evolution with humans, going back to human migration out of Africa. This prolonged relationship is likely to have shaped the overall host-pathogen interactions and repertoire of virulence strategies which H. pylori employs to establish robust colonisation, escape immune responses and persist in the gastric niche. In this regard, H. pylori lipopolysaccharide (LPS) is a key surface determinant in establishing colonisation and persistence via host mimicry and resistance to cationic antimicrobial peptides. Thus, elucidation of the H. pylori LPS structure and corresponding biosynthetic pathway represents an important step towards better understanding of H. pylori pathogenesis and the development of novel therapeutic interventions.

Functional characterization of Helicobacter pylori 26695 sedoheptulose 7-phosphate isomerase encoded by hp0857 and its association with lipopolysaccharide biosynthesis and adhesion

Helicobacter pylori is a notorious human pathogen and the appearance of antibiotic resistance of this bacterium has posed a serious threat to human health. Lipopolysaccharide (LPS) is a key virulence factor and plays important roles in pathogenesis of H. pylori infection. Sedoheptulose 7-phosphate isomerase (GmhA), as an enzyme participating in the first step of heptose biosynthesis, is indispensable for the formation of inner core oligosaccharide of LPS. In this study, we cloned one putative gmhA ortholog, hp0857, from H. pylori 26695 and overexpressed it in Eschericha coli. Based on the results of molecular weight determination, the recombinant HP0857 is likely a homodimer. Analysis of enzymatic kinetic properties of this protein confirmed that hp0857 is indeed encoded a phosphoheptose isomerase which can utilize sedoheptulose 7-phosphate as the substrate in the ADP-L-glycero-D-manno-heptose (ADP- L,D-Hep) biosynthesis pathway. We also generated an HP0857 knockout mutant and explored its phenotypic changes. This mutant exhibited a decreased growth rate and displayed a “deep rough” type of LPS structure. In addition, it also had a slight decrease in its motility and was more susceptible to hydrophobic antibiotic novobiocin and detergents Triton X-100 and SDS. Furthermore, the adhesive capacity of the HP0857 knockout mutant to AGS cells was reduced significantly, and most of the infected cells didn’t show a classic hummingbird phenotype. However, complementation of the HP0857 knockout mutation restored most of these phenotypic changes. In conclusion, we demonstrated that HP0857 protein is essential for inner core biosynthesis of H. pylori LPS and is a potential target for developing new antimicrobial agents against H. pylori infection.

Helicobacter pylori antigens, acetylsalicylic acid, LDL and 7-ketocholesterol - their potential role in destabilizing the gastric epithelial cell barrier. An in vitro model of Kato III cells.

Colonization of gastric tissue in humans by H. pylori Gram-negative bacteria initiates gastric and duodenal ulcers and even gastric cancers. Infections promote inflammation and damage to gastric epithelium which might be followed by the impairment of its barrier function. The role of H. pylori components in these processes has not been specified. H. pylori cytotoxicity may potentially increase in the milieu of anti-inflammatory drugs including acetylsalicylic acid (ASA). The lipid transport-associated molecule such as low density lipoprotein (LDL), which is a classic risk factor of coronary heart disease (CHD) and 7-ketocholesterol (7-kCh) a product of cholesterol oxidation, which may occur during the oxidative stress in LDL could also be considered as pro-inflammatory. The aim of this study was to evaluate the cytotoxicity of H. pylori antigens, ASA, LDL and 7-kCh towards Kato III gastric epithelial cells, on the basis of the cell ability to reduce tetrazolium salt (MTT) and morphology of cell nuclei assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Kato III cells were stimulated for 24 h, at 37°C and 5% CO2, with H. pylori antigens: cytotoxin associated gene A (CagA) protein, the urease A subunit (UreA), lipopolysaccharide (LPS) and ASA, LDL or 7-kCh. H. pylori LPS, ASA, LDL and 7-kCh, but not H. pylori glycine acid extract (GE), demonstrated cytotoxicity against Kato III cells, which was related to a diminished percentage of MTT reducing cells and to an increased cell population with the signs of DNA damage. The results suggest that damage to gastric epithelial cells can be induced independently by H. pylori antigens, ASA and endogenous lipid transport-associated molecules. During H. pylori infection in vivo, especially in CHD patients, synergistic or antagonistic interactions between these factors might possibly influence the disease course. Further study is necessary to explain these potential effects.