Helicobacter Pylori Lipopolysaccharide Research Articles

The influence of Salvia cadmica Boiss. extracts on the M1/M2 polarization of macrophages primed with Helicobacter pylori lipopolysaccharide in conjunction with NF-kappa B activation, production of cytokines, phagocytic activity and total DNA methylation.

The large number of secondary derivatives have been isolated from the genus Salvia with about 700 species, and used in the pharmacopoeia throughout the world. Various biological properties of Salvia formulations have been reported including as antioxidant, antimicrobial, hypotensive, anti-hyperglycemia, anti-hyperlipidemia, anti-cancer, and skin curative. Salvia cadmica Boiss. root and aerial part extracts enriched with polyphenols are bactericidal towards gastric pathogen Helicobacter pylori (Hp) and diminish deleterious effects induced by Hp lipopolysaccharide (LPS) towards gastric epithelial cells. To examine the influence of S. cadmica extracts on the M1/M2 polarization of macrophages primed with Hp LPS vs standard LPS Escherichia coli (Ec), and the macrophage cytokine as well as phagocytic activity, which are affected during Hp infection. Macrophages derived from THP-1 human monocytes primed with LPS Hp/Ec and/or S. cadmica extracts, were examined for the biomarkers of activation (surface, cytoplasmic or soluble), and phagocytic capacity. The bone marrow macrophages of Caviaporcellus were used to determine the engulfment of Hp. Priming of THP-1cells (24h) with LPS Hp/Ec resulted in polarization of M1 macrophages, activation of nuclear factor kappa B, secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1 beta, macrophage chemotactic protein (MCP)-1, immunoregulatory IL-10, and production of reactive oxygen species. These effects were diminished after restimulation of cells with S. cadmica extracts. THP-1 macrophages exposed to studied extracts showed an increased phagocytic capacity, in conjunction with elevated CD11b/CD11d expression and enhanced production of inducible nitric oxide synthase. They also increased Hp engulfment by bone marrow macrophages. These effects were not related to a global DNA methylation. S. cadmica extracts possess an immunomodulating activity, which might be useful in control of H. pylori LPS driven activity of macrophages.

Blueberry anthocyanin extracts protect against Helicobacter pylori-induced peptic epithelium injuries both in vitro and in vivo: the key role of MAPK/NF-κB pathway.

Anthocyanins are well-characterized by anti-oxidative and anti-inflammatory potentials. Peptic ulcers contribute to the development of severe gastric disorders. In the current study, the effects of blueberry anthocyanin extracts (BE) on the Helicobacter pylori lipopolysaccharide (LPS)-induced peptic epithelium injures were assessed and the associated mechanism driving the effects was explored by focusing on MAPK/NF-κB pathway. Peptic injures were induced in a mouse model using LPS plus ligation method and then the mice were treated with BE. Then changes in gastric histology, inflammatory response, and MAPK/NF-κB axis were detected. To reveal the role of MAPK/NF-κB axis in the effects of BE, human gastric epithelial cells (HGECs) were further subjected to co-treatment of BE, LPS, and MAPK activator. The assays of mouse model showed that BE attenuated gastric epithelial injuries by improving epithelial structure and suppressing gastric inflammatory response, which was associated with the inhibition of MAPK/NF-κB axis. In in vitro assays, BE suppressed viability and production of cytokines, and induced apoptosis in LPS-treated HGECs. The re-activation of MAPK pathway counteracted the effects of BE by re-inducing cell viability and suppressing cell apoptosis. The protective effects of BE against LPS-induced injuries in mouse stomach depended on the inhibition of both MAPK pathway and the downstream NF-κB signaling.

Trefoil Factor Family (TFF) Peptides and Their Diverse Molecular Functions in Mucus Barrier Protection and More: Changing the Paradigm.

Trefoil factor family peptides (TFF1, TFF2, TFF3) are typically co-secreted together with mucins. Tff1 represents a gastric tumor suppressor gene in mice. TFFs are also synthesized in minute amounts in the immune and central nervous systems. In mucous epithelia, they support rapid repair by enhancing cell migration (“restitution”) via their weak chemotactic and anti-apoptotic effects. For a long time, as a paradigm, this was considered as their major biological function. Within recent years, the formation of disulfide-linked heterodimers was documented for TFF1 and TFF3, e.g., with gastrokine-2 and IgG Fc binding protein (FCGBP). Furthermore, lectin activities were recognized as enabling binding to a lipopolysaccharide of Helicobacter pylori (TFF1, TFF3) or to a carbohydrate moiety of the mucin MUC6 (TFF2). Only recently, gastric TFF1 was demonstrated to occur predominantly in monomeric forms with an unusual free thiol group. Thus, a new picture emerged, pointing to diverse molecular functions for TFFs. Monomeric TFF1 might protect the gastric mucosa as a scavenger for extracellular reactive oxygen/nitrogen species. Whereas, the TFF2/MUC6 complex stabilizes the inner layer of the gastric mucus. In contrast, the TFF3–FCGBP heterodimer (and also TFF1–FCGBP) are likely part of the innate immune defense of mucous epithelia, preventing the infiltration of microorganisms.

Cyanidin-3-O-glucoside protects human gastric epithelial cells against Helicobacter pylori lipopolysaccharide-induced disorders by modulating TLR-mediated NF-κB pathway

Abstract Helicobacter pylori (HP) contributes to the development of gastrointestinal disorders. Cyanidin-3-O-glucoside (C3G) is a major anthocyanin in human diets. In the current study, the effects and associated mechanism of C3G on HP lipopolysaccharide (LPS)-induced symptoms on human gastric epithelial cell (HGEC) cells were explored. The cells were exposed to LPS and C3G, and cellular viability, apoptotic rate, inflammation level, and toll-like receptor (TLR)-mediated nuclear factor-kappa B (NF-κB) pathway activity were measured. C3G suppressed the abnormal DNA synthesis and induced apoptosis in LPS-treated HGEC cells. C3G also reduced inflammation in LPS-treated HGEC cells. Although C3G exposure reduced the expressions of TLR2 and TLR4, it had no influence on TLR5. C3G deactivated TLR-mediated NF-κB signaling. The effects of C3G were stronger than anthocyanin extract but weaker than curcumin. The protective effects of C3G against HP LPS-induced injuries in gastric epithelial cells were associated with the deactivation of TLR2- and TLR4-mediated NF-κB signaling.

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.

The redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains.

Helicobacter pylori lipopolysaccharide promotes chronic gastric colonisation through O-antigen host mimicry and resistance to mucosal antimicrobial peptides mediated primarily by modifications of the lipid A. The structural organisation of the core and O-antigen domains of H. pylori lipopolysaccharide remains unclear, as the O-antigen attachment site has still to be identified experimentally. Here, structural investigations of lipopolysaccharides purified from two wild-type strains and the O-antigen ligase mutant revealed that the H. pylori core-oligosaccharide domain is a short conserved hexasaccharide (Glc-Gal-DD-Hep-LD-Hep-LD-Hep-KDO) decorated with the O-antigen domain encompassing a conserved trisaccharide (-DD-Hep-Fuc-GlcNAc-) and variable glucan, heptan and Lewis antigens. Furthermore, the putative heptosyltransferase HP1284 was found to be required for the transfer of the third heptose residue to the core-oligosaccharide. Interestingly, mutation of HP1284 did not affect the ligation of the O-antigen and resulted in the attachment of the O-antigen onto an incomplete core-oligosaccharide missing the third heptose and the adjoining Glc-Gal residues. Mutants deficient in either HP1284 or O-antigen ligase displayed a moderate increase in susceptibility to polymyxin B but were unable to colonise the mouse gastric mucosa. Finally, mapping mutagenesis and colonisation data of previous studies onto the redefined organisation of H. pylori lipopolysaccharide revealed that only the conserved motifs were essential for colonisation. In conclusion, H. pylori lipopolysaccharide is missing the canonical inner and outer core organisation. Instead it displays a short core and a longer O-antigen encompassing residues previously assigned as the outer core domain. The redefinition of H. pylori lipopolysaccharide domains warrants future studies to dissect the role of each domain in host-pathogen interactions. Also enzymes involved in the assembly of the conserved core structure, such as HP1284, could be attractive targets for the design of new therapeutic agents for managing persistent H. pylori infection causing peptic ulcers and gastric cancer.

Aegle marmelos fruit extract attenuates Helicobacter pylori Lipopolysaccharide induced oxidative stress in Sprague Dawley rats.

BackgroundBael (Aegle marmelos (L.) Corr.) has been widely used in indigenous systems of Indian medicine to exploit its medicinal properties including astringent, antidiarrheal, antidysenteric, demulcent, antipyretic, antiulcer, anti-inflammatory and anti cancer activities. The present study aims to evaluate the antioxidative and antiulcer effect of methanolic extract of unripe fruit of Aegle marmelos (MEAM) against Helicobacter pylori-Lipopolysaccharide (HP-LPS) induced gastric ulcer in Sprague Dawley (SD) rats.MethodsDose and duration of HP-LPS and MEAM were fixed based on ulcer index of gastric tissue of experimental animals. Various gastric secretory parameters such as volume of gastric juice, free and total acidity, acid output, pepsin concentration were analyzed. The activities of enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase), non-enzymatic antioxidants (reduced glutathione, vitamin C and vitamin E) and the levels of lipid peroxidation products were measured. Histological analysis was performed to evaluate the effect of Aegle marmelos on HP-LPS induced gastric ulcer.ResultsOral administration of HP-LPS (50 μg per animal) for four consecutive days resulted in induction of ulcer with the increase in gastric secretory parameters such as volume of gastric juice, free and total acidity, acid output, pepsin concentration. Oral administration of methanolic extract of Aegle marmelos fruit (MEAM) (25, 50, 100, 250 and 500 mg/kg) reduced the gastric ulcer by 2.8 %, 52.4 %, 73 %, 93 % and 93.98 %, respectively, compared to 89.2 % reduction by sucralfate (100 mg/kg). MEAM treatment significantly (p < 0.05) inhibited the increase in gastric secretory parameters in ulcerated rats, and it also prevented the reduction of enzymatic (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase) and non-enzymatic antioxidants (reduced glutathione, vitamin C and vitamin E) after HP-LPS induction. In addition, lipid peroxidation was inhibited by MEAM in HP-LPS induced rats. Results of histological analysis correlated well with biochemical parameters.ConclusionThese observations explored the antioxidant properties of MEAM contributing to the gastroprotective effect in HP-LPS induced gastric ulcer model.

Toll-Like Receptor 4 Wild Type Homozygozity of Polymorphisms +896 and +1196 Is Associated with High Gastrin Serum Levels and Peptic Ulcer Risk.

Toll-like receptor 4 is a part of the innate immune system and recognizes Helicobacter pylori lipopolysaccharide. The goal of this study was to analyze the role of Toll-like receptor 4 polymorphisms +896 (rs4986790) and +1196 (rs4986791) in the pathogenesis of Helicobacter pylori related gastroduodenal diseases in relation to gastric secretion and inflammation. Toll-like receptor 4 polymorphisms, serum gastrin-17 and pepsinogen I and II concentrations were determined, and gastroscopies with histopathological analyses were performed to 216 dyspeptic patients. As genotype controls, 179 controls and 61 gastric cancer patients were studied. In our study, the Toll-like receptor 4 +896 and +1196 polymorphisms were in total linkage disequilibrium. The homozygous wild types displayed higher gastrin-17 serum concentrations than the mutants (p = 0.001) and this effect was independent of Helicobacter pylori. The homozygous wild types also displayed an increased risk for peptic ulcers (OR: 4.390). Toll-like receptor 4 genotypes did not show any association with Helicobacter pylori positivity or the features of gastric inflammation. Toll-like receptor 4 expression was seen in gastrin and somatostatin expressing cells of antral mucosa by immunohistochemistry. Our results suggest a role for Toll-like receptor 4 in gastric acid regulation and that the Toll-like receptor 4 +896 and +1196 wild type homozygozity increases peptic ulcer risk via gastrin secretion.

Structural modifications of Helicobacter pylori lipopolysaccharide: an idea for how to live in peace.

In this review, we discuss the findings and concepts underlying the "persistence mechanisms" of Helicobacter pylori (H. pylori), a spiral-shaped, Gram-negative rod bacterium that was discovered as a gastric pathogen by Marshall and Warren in 1984. H. pylori colonizes the gastric mucosa of nearly half of the human population. Infections appear in early childhood and, if not treated, persist for life. The presence or absence of symptoms and their severity depend on multiple bacterial components, host susceptibility and environmental factors, which allow H. pylori to switch between pathogenicity and commensalism. Many studies have shown that H. pylori components may facilitate the colonization process and the immune response of the host during the course of H. pylori infection. These H. pylori-driven interactions might result from positive or negative modulation. Among the negative immunomodulators, a prominent position is occupied by a vacuolating toxin A (VacA) and cytotoxin-associated gene A (CagA) protein. However, in light of the recent studies that are presented in this review, it is necessary to enrich this panel with H. pylori lipopolysaccharide (LPS). Together with CagA and VacA, LPS suppresses the elimination of H. pylori bacteria from the gastric mucosa by interfering with the activity of innate and adaptive immune cells, diminishing the inflammatory response, and affecting the adaptive T lymphocyte response, thus facilitating the development of chronic infections. The complex strategy of H. pylori bacteria for survival in the gastric mucosa of the host involves both structural modifications of LPS lipid A to diminish its endotoxic properties and the expression and variation of Lewis determinants, arranged in O-specific chains of H. pylori LPS. By mimicking host components, this phenomenon leaves these bacteria "invisible" to immune cells. Together, these mechanisms allow H. pylori to survive and live for many years within their hosts.

Implication of antigenic conversion of Helicobacter pylori lipopolysaccharides that involve interaction with surfactant protein D.

We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linked N-acetyl-D-glucosamine and β-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-β-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.

Defining Function of Lipopolysaccharide O-antigen Ligase WaaL Using Chemoenzymatically Synthesized Substrates

The WaaL-mediated ligation of O-antigen onto the core region of the lipid A-core block is an important step in the lipopolysaccharide (LPS) biosynthetic pathway. Although the LPS biosynthesis has been largely characterized, only a limited amount of in vitro biochemical evidence has been established for the ligation reaction. Such limitations have primarily resulted from the barriers in purifying WaaL homologues and obtaining chemically defined substrates. Accordingly, we describe herein a chemical biology approach that enabled the reconstitution of this ligation reaction. The O-antigen repeating unit (O-unit) of Escherichia coli O86 was first enzymatically assembled via sequential enzymatic glycosylation of a chemically synthesized GalNAc-pyrophosphate-undecaprenyl precursor. Subsequent expression of WaaL through use of a chaperone co-expression system then enabled the demonstration of the in vitro ligation between the synthesized donor (O-unit-pyrophosphate-undecaprenyl) and the isolated lipid A-core acceptor. The previously reported ATP and divalent metal cation dependence were not observed using this system. Further analyses of other donor substrates revealed that WaaL possesses a highly relaxed specificity toward both the lipid moiety and the glycan moiety of the donor. Lastly, three conserved amino acid residues identified by sequence alignment were found essential for the WaaL activity. Taken together, the present work represents an in vitro systematic investigation of the WaaL function using a chemical biology approach, providing a system that could facilitate the elucidation of the mechanism of WaaL-catalyzed ligation reaction.

Characterization and Functional Activity of Murine Monoclonal Antibodies Specific for α1,6-Glucan Chain of Helicobacter pylori Lipopolysaccharide

The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6-glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6-glucan and characterize their binding properties and functional activity. BALB/c mice were injected intraperitoneally with 10(8) formalin-fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti-α1,6-glucan-producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole-cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild-type and mutant strains of H. pylori. The generated anti-α1,6-glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6-linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD450 ≥ 0.2). Bactericidal activity was observed against selective wild-type and mutant H. pylori strains exhibiting OD450 values of ≥ 0.45 in WCE. Anti-α1,6-glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS-based vaccine against H. pylori infections.

Peran Lipopolisakarida Helicobacter pylori terhadap Aktivitas Neutrofil pada Penderita Infark Miokard Akut melalui Degradasi Kolagen Tipe IV

Matrix metalloproteases (MMPs) are proteolytic enzymes that play a critical role in decreasing the stability of atherosclerotic plaque causing Acute Myocardial Infarction (AMI). In this process, degradation of type IV collagen as an important component of endothelial membrane is thought to be important. Several microorganisms and their products including Lipopolysaccharide (LPS) have been suggested to be able to activate MMPs. The aim of this study is to investigate the role of Helicobacter pylori LPS in type IV collagen degradation (in vitro). We used two stages in this study: MMP production stage and degradation of type IV collagen. MMP derived from the neutrophils of AMI and non- AMI patients that had been stimulated with LPS of Helicobacter pylori were used to digest type IV collagen. Analysis of type IV collagen degradation was performed with SDS-PAGE and Western blot. This study showed MMP isolated from neutrophil of AMI patients is an active MMP-9 at Molecular weight of 72 kDa. It can degrade type IV collagen at MW 78.6 kDa, 60 kDa, 50 kDa, 47.8 kDa and 43.8 kDa. Whereas MMP isolated from neutrophil of non-AMI have MW of 91.2 kDa and can only degrade collagen IVat merely 60 kDa. In conclusion, Helicobacter pylori LPS is able to induce n eutrophil of AMIpatients to produce an active MMP-9 at MW 72 kDa and the enzyme may play strategic role in the degradation of type IV collagen.

S1640 Can Helicobacter pylori Lipopolysaccharide Lipid a Composition Affect Its Ability to Induce an Inflammatory Response Through Toll Like Receptor 4

Introduction H pylori recognition by the host is poorly understood, although there is evidence that lipid A recognition occurs via Toll-like receptor 4. H pylori produces two lipid A species, a minor E coli -like hexa-acylated species and a major tetra-acylated species. The latter is thought to be important for immune evasion, with many laboratory strains thought to lack the minor more immunogenic species which is present in clinical strains. Aim To assess whether changes in lipid A alter H pylori9s ability to induce an inflammatory response. Methods H pylori strains were repeatedly subcultured on blood agar and structural changes in the lipopolysaccharide were analysed using Deoxycholate Polyacrylamide Gel Electrophoresis (DOC-PAGE) and Gas Chromatography Mass Spectrometry (GC-MS). Additional ultracentrifugation removed contaminating lipoproteins and capsular polysaccharides. A monocyte/LPS challenge system was used to assess pro-inflammatory cytokine production by the different H pylori LPS preparations. Prediction of the orientations adopted by H pylori LPS structures within TLR4 receptor complex interactions were generated using the automated docking program AutoDock v4. Results H pylori LPS was not altered by subculturing, although purified laboratory and clinical strains were shown to have compositional differences. Fatty acids of carbon length 14 and 16 (indicative of hexa-acylated lipid A) were absent from laboratory strain H pylori lipid A following purification, but remained in the lipid A purified from clinical strains. Unpurified laboratory strain LPS induced greatest levels of cytokine production in monocyte/LPS challenge experiments followed by purified clinical strain LPS, with purified laboratory strain LPS inducing the lowest levels (Abstract 053). Modelling analysis confirmed that the tetra-acylated species was less effective than the hexa-acylated species at interacting with the TLR4 signalling complex. Conclusion Purification of H pylori LPS had a dramatic effect on inflammatory cytokine production indicating that contaminating lipoprotein and or capsular polysaccharide can impact the immune response. However, LPS derived from clinical isolates is significantly more immunogenic than laboratory strains. This further strengthens the evidence that TLR4-mediated immune responses are important in H pylori infections.

Helicobacter pylori Lipopolysaccharide Is Synthesized via a Novel Pathway with an Evolutionary Connection to Protein N-Glycosylation

Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation.

T1636 Pellino1 and Tribbles3 - Novel Regulators of Toll-Like Receptor Signaling in Response to Helicobacter pylori Lipopolysaccharide

T1636 Pellino1 and Tribbles3 - Novel Regulators of Toll-Like Receptor Signaling in Response to Helicobacter pylori Lipopolysaccharide

Effect of the HP0159 ORF mutation on the lipopolysaccharide structure and colonizing ability ofHelicobacter pylori

The outer core region of Helicobacter pylori lipopolysaccharide of the majority of isolates contains an alpha-1,6-glucan polymer synthesized by the product of the HP0159 ORF. Structural studies carried out on HP0159 lipopolysaccharide mutants by a combination of chemical methods, mass spectrometry and nuclear magnetic resonance spectroscopy confirmed that insertional inactivation of HP0159 gene in H. pylori strains 26695 and SS1 resulted in formation of a truncated lipopolysaccharide molecule characterized by the presence of a terminal dd-heptose residue in the side-chain outer core fragment and maintaining an inner core backbone structure compared with the wild-type Lewis antigen-expressing strains. Colonization studies with HP0159 mutants of two mouse-colonizing strains, SS1 and M6, confirmed their inability to successfully colonize the murine stomach.

Role of futC slipped strand mispairing in Helicobacter pylori Lewis y phase variation

The O antigen of the Helicobacter pylori lipopolysaccharide is composed of repeating units of fucosylated Lewis (Le) antigens. The α(1,2)-fucosyltransferase ( futC) of H. pylori, which catalyzes the conversion of Le x to Le y by addition of fucose, is subject to slipped-strand mispairing involving a homonucleotide (poly-C) tract. To explore the distribution of Le phenotypes within H. pylori cells grown in vitro, 379 single colonies of strain J166 were examined for Le expression. Two major populations with reciprocal Le x/Le y phenotypes were identified. Phenotypes correlated with futC frame status, suggesting that strain J166 represents a mixed population with respect to futC poly-C tract length, which was confirmed by a translational reporter. After hundreds of generations in vitro, phenotypes did not change significantly, indicating that the observed J166 Le diversity reflects the founding population. Since slipped-strand mispairing in the futC poly-C tract was postulated to explain the Le y phenotypic change observed in J166 derivative strain 98–169 isolated 10 months after rhesus monkey challenge, in trans complementation with in-frame futC was performed. Le y synthesis was restored and Le x expression was reciprocally lowered. From these studies, we confirmed the principal role of futC slipped-strand mispairing in Le antigenic variation in vitro and in vivo.

Synthesis of immunoregulatory Helicobacter pylori lipopolysaccharide partial structures

The synthesis of immunoregulatory glycoconjugates, namely the active entity of lipopolysaccharide (LPS) from Helicobacter pylori was achieved. The results of biological activities of the LPS partial structures provide the structural basis for the immunobiological activity, especially for the immune inhibitory activity of H. pylori LPS.

Immune Activation in Patients With Irritable Bowel Syndrome

We set out to test the hypothesis that irritable bowel syndrome (IBS) is characterized by an augmented cellular immune response with enhanced production of proinflammatory cytokines. We further aimed to explore whether symptoms and psychiatric comorbidity in IBS are linked to the release of proinflammatory cytokines. We characterized basal and Escherichia coli lipopolysaccharide (LPS)-induced cytokine production in peripheral blood mononuclear cells (PBMCs) from 55 IBS patients (18 mixed-, 17 constipation-, 20 diarrhea-predominant) and 36 healthy controls (HCs). PBMCs were isolated by density gradient centrifugation and cultured for 24 hours with or without (1 ng/mL) LPS. Cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, and IL-6) was measured by enzyme-linked immunosorbent assay. Abdominal symptoms and psychiatric comorbidities were assessed by using the validated Bowel Disease Questionnaire and the Hospital Anxiety and Depression Scale. IBS patients showed significantly (P < .017) higher baseline TNF-alpha, IL-1beta, IL-6, and LPS-induced IL-6 levels compared with HCs. Analyzing IBS subgroups, all cytokine levels were significantly (P < .05) higher in diarrhea-predominant IBS (D-IBS) patients, whereas constipation-predominant IBS patients showed increased LPS-induced IL-1beta levels compared with HCs. Baseline TNF-alpha and LPS-induced TNF-alpha and IL-6 levels were significantly higher in patients reporting more than 3 bowel movements per day, urgency, watery stools, and pain associated with diarrhea compared with patients without these symptoms (all P < .05). LPS-induced TNF-alpha production was associated significantly (r = 0.59, P < .001) with anxiety in patients with IBS. Patients with D-IBS display enhanced proinflammatory cytokine release, and this may be associated with symptoms and anxiety.