Inhibition of Helicobacter pylori colonization by sulfated gastric mucin.

The role of human gastric mucin in mucosal protection against Helicobacter pylori colonization was investigated. H. pylori cells were incubated with purified intact mucin or its acidic fractions and then examined for their inhibitory capacity of H. pylori attachment to erythrocytes. Titration data established that the inhibitory activity of mucin was associated with its acidic component as the fraction enriched in sialic acid and sulfate showed 16-fold higher inhibitory titer than that of the intact mucin. While the inhibitory titer of acidic mucin fraction was not affected by the removal of sialic acid, the desulfation led to a complete loss of its inhibitory activity, thus pointing towards the importance of sulfate ester groups in this process. The results for the first time point towards the involvement of sulfomucins in the protection of gastric mucosa against colonization by H. pylori.     

Effectiveness of<Emphasis Type="Italic">Cyanothece</Emphasis> spp. and<Emphasis Type="Italic">Cyanospira capsulata</Emphasis> exocellular polysaccharides as antiadhesive agents for blocking attachment of<Emphasis Type="Italic">Helicobacter pylori</Emphasis> to human gastric cells

The effect of cyanobacterial polysaccharides (from Cyanothece spp. and Cyanospira capsulata) on the binding of Helicobacter pylori to gastric epithelial cells was evaluated. The antiadhesive action on Kato III and HeLa S3 human gastric cell lines was established.     

Pectin-like acidic polysaccharide from Panax ginseng with selective antiadhesive activity against pathogenic bacteria

Previous studies have revealed the inhibitory effects of an acidic polysaccharide purified from the root of Panax ginseng against the adhesion of Helicobacter pylori to gastric epithelial cells and the ability of Porphyromonas gingivalis to agglutinate erythrocytes. In this study, this acidic polysaccharide from P. ginseng, PG-F2, was investigated further, in order to characterize its antiadhesive effects against Actinobacillus actinomycetemcomitans, Propionibacterium acnes, and Staphylococcus aureus. The minimum inhibitory concentrations (MIC) were found to be in a range of 0.25-0.5mg/mL. However, results showed no inhibitory effects of PG-F2 against Lactobacillus acidophilus, Escherichia coli, or Staphylococcus epidermidis. PG-F2 is a pectin-type polysaccharide with a mean MW of 1.2 x 10(4) Da, and consists primarily of galacturonic and glucuronic acids along with rhamnose, arabinose, and galactose as minor components. The complete hydrolysis of PG-F2 via chemical or carbohydrolase enzyme treatment resulted in the abrogation of its antiadhesive activity, but limited hydrolysis via treatment with pectinase (EC. 3.2.1.15) yielded an oligosaccharide fraction, with activity comparable to the precursor PG-F2 (the MIC of ca. 0.01 mg/mL against H. pylori and P. gingivalis). Our results suggest that PG-F2 may exert a selective antiadhesive effect against pathogenic bacteria, while having no effects on beneficial and commensal bacteria.     

Almost all human gastric mucin O-glycans harbor blood group A, B or H antigens and are potential binding sites for Helicobacter pylori

Helicobacter pylori infects more than half of the world's population. Although most patients are asymptomatic, persistent infection may cause chronic gastritis and gastric cancer. Adhesion of the bacteria to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylori-related diseases and is mediated by mucin O-glycans. In order to define which glycans may be implicated in the binding of the bacteria to the gastric mucosa in humans, we have characterized the exact pattern of glycosylation of gastric mucins. We have identified that the major component was always a core 2-based glycan carrying two blood group H antigens, whatever was the blood group of individuals. We have also demonstrated that around 80% of O-glycans carried blood group A, B or H antigens, suggesting that the variation of gastric mucin glycosylation between individuals is partly due to the blood group status. This study will help better understanding the role of O-glycans in the physiology and homeostasis of gastric mucosa. Overall, the results reported here give us the necessary background information to begin studies to determine whether individuals who express certain carbohydrate epitopes on specific mucins are predisposed to certain gastric diseases.     

Adherence protects the binding sites of Helicobacter pylori urease from acid-induced damage

Colonization by Helicobacter pylori partly depends on acid-dependent adherence by urease to gastric mucin. To further verify the relevance of urease adherence to colonization, the influence of acidity on the binding sites of H. pylori urease was investigated. When enzyme-based in vitro ligand capture assays were used, the effect of acidity on the binding site of H. pylori urease was determined against a backdrop medium consisting of acidic buffers simulating the luminal side of gastric mucus. A high degree of stability was exhibited by adherent urease, suggesting a pivotal role by the denatured enzyme in the persistence of the bacterium within the acidified compartment of gastric mucus.     

Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells

Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.     

Characterization of monospecies biofilm formation by Helicobacter pylori

As all bacteria studied to date, the gastric pathogen Helicobacter pylori has an alternate lifestyle as a biofilm. H. pylori forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral H. pylori to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed H. pylori arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the H. pylori life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic H. pylori while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach, H. pylori planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene, luxS, and the cagE type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by H. pylori and the selective enhancement of planktonic growth in the presence of gastric mucin.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

Identification and characterization of binding properties of Helicobacter pylori by glycoconjugate arrays

The microaerophilic bacterium Helicobacter pylori is well established for its role in development of different gastric diseases. Bacterial adhesins and corresponding binding sites on the epithelial surface allow H. pylori to colonize the gastric tissue. In this investigation, the adhesion of H. pylori to dot blot arrays of natural glycoproteins and neoglycoproteins was studied. Adhesion was detected by overlay with fluorescence-labeled bacteria on immobilized (neo)glycoproteins. The results confirmed the interaction between the adhesin BabA and the H-1-, Lewis b-, and related fucose-containing antigens. In addition, H. pylori bound to terminal alpha2-3-linked sialic acids as previously described. The use of a sabA mutant and sialidase treatment of glycoconjugate arrays showed that the adherence of H. pylori to laminin is mediated by the sialic acid-binding adhesin, SabA. The adhesion to salivary mucin MUC5B is mainly associated with the BabA adhesin and to a lesser extent with the SabA adhesin. This agrees with reports, that MUC5B carries both fucosylated blood group antigens and alpha2-3-linked sialic acids. The adhesion of H. pylori to fibronectin and lactoferrin persisted in the babA/sabA double mutant. Because binding to these molecules was abolished by denaturation rather than by deglycosylation, it was suggested to depend on the recognition of unknown receptor moieties by an additional unknown bacterial surface component. The results demonstrate that the bacterial overlay method on glycoconjugate arrays is a useful tool for exploration and the characterization of unknown adhesin specificities of H. pylori and other bacteria.     

An extract of Pelargonium sidoides (EPs 7630) inhibits in situ adhesion of Helicobacter pylori to human stomach

Root extract from Pelargonium sidoides DC is used therapeutically as antimicrobial agent against infections of the respiratory system. In order to elucidate possible modes of actions we investigated the influence of P. sidoides root extract on microbial adhesion with Helicobacter pylori as model microorganism, a germ with a strong adherence to human stomach tissue. In an in-situ anti-adhesion assay intact human stomach tissue from patient resectates was incubated with fluorescent-labelled bacteria. Epithelial adhesion occurred in untreated samples and was quantified by fluorescent microscopy. Pre-treatment of the bacteria with Pelargonium extract showed good anti-adhesive activity. The antiadhesive effect was clearly dose-dependent in a range from 0.001 to 10 mg/ml. Within agar diffusion-test the extract had no direct cytotoxicity against H. pylori. The results show that the root extract from Pelargonium sidoides is a potent anti-adhesive agent against H. pylori and could therefore be a useful choice to avoid the first step of a bacterial infection.     

Inhibition of Helicobacter pylori haemogglutination activity by human salivary mucins

Abstract Thirty isolates of Helicobacter pylori from gastric biopsies agglutinated human erthyrocyte suspensions. Crude mucin preparation derived from saliva of 20 different donors were examined for their ability to inhibit haemagglutination. All mucin preparations exhibited strong inhibitory activity. Removal of sialic residues from mucin preparations by treatment with neuraminidase resulted in a substantial reduction of their inhibitory activity. The mucin prepations had no bactericidal or aggregation activity for H. pylori . These results are discussed in the context of the role of mucins in colonization of the gastric mucosa by H. pylori     

Inhibition of Helicobacter pylori haemagglutination activity by human salivary mucins

Thirty isolates of Helicobacter pylori from gastric biopsies agglutinated human erythrocyte suspensions. Crude mucin preparations derived from saliva of 20 different donors were examined for their ability to inhibit haemagglutination. All mucin preparations exhibited strong inhibitory activity. Removal of sialic residues from mucin preparations by treatment with neuraminidase resulted in a substantial reduction of their inhibitory activity. The mucin preparations had no bactericidal or aggregation activity for H. pylori. These results are discussed in the context of the role of mucins in colonization of the gastric mucosa by H. pylori.     

The effect of the cag pathogenicity island on binding of Helicobacter pylori to gastric epithelial cells and the subsequent induction of apoptosis

BACKGROUND: Helicobacter pylori infection leads to gastritis, peptic ulcer, and gastric cancer, in part due to epithelial damage following bacteria binding to the epithelium. Infection with cag pathogenicity island (PAI) bearing strains of H. pylori is associated with increased gastric inflammation and a higher incidence of gastroduodenal diseases. It is now known that various effector molecules are injected into host epithelial cells via a type IV secretion apparatus, resulting in cytoskeletal changes and chemokine secretion. Whether binding of bacteria and subsequent apoptosis of gastric epithelial cells are altered by cag PAI status was examined in this study. METHODS: AGS, Kato III, and N87 human gastric epithelial cell lines were incubated with cag PAI-positive or cag PAI-negative strains of H. pylori in the presence or absence of clarithromycin. Binding was evaluated by flow cytometry and scanning electron microscopy. Apoptosis was assessed by detection of DNA degradation and ELISA detection of exposed histone residues. RESULTS: cag PAI-negative strains bound to gastric epithelial cells to the same extent as cag PAI-positive strains. Both cag PAI-positive and cag PAI-negative strains induced apoptosis. However, cag PAI-positive strains induced higher levels of DNA degradation. Incubation with clarithromycin inactivated H. pylori but did not affect binding. However, pretreatment with clarithromycin decreased infection-induced apoptosis. CONCLUSIONS: cag PAI status did not affect binding of bacteria to gastric epithelial cells but cag PAI-positive H. pylori induced apoptosis more rapidly than cag PAI-negative mutant strains, suggesting that H. pylori binding and subsequent apoptosis are differentially regulated with regard to bacterial properties.     

Essential components of antimicrobial gastrointestinal epithelial barrier: specific interaction of mucin with an integral apical membrane protein of gastric mucosa

BACKGROUND: The gastric mucosal protective barrier consists of two essential elements: mucus glycoprotein, mucin, secreted by gastric mucosal cells, and the mucin binding protein (MBP), an integral component of the apical epithelial membrane. The studies described here provide evidence on the structure of MBP, its interaction with mucin, and the susceptibility to phospholipase C (PLC) and Helicobacter pylori protease. MATERIAL AND METHODS: The rat gastric mucosa was used to isolate mucin and the apical epithelial membranes. A buffered saline extract of the mucosal cells was used for the isolation of mucin and the 1% Triton X-100-insoluble gastric apical membranes for the preparation of MBP. RESULTS: The studies on MBP, the mucosal mucin receptor revealed that the protein is anchored in apical membrane through glycosylphosphatidylinositol (GPI). The deamination of MBP with nitrous acid afforded phosphatidylinositols (PIs) and a water soluble, 97 kDa glycosylated protein. The in situ studies with untreated rat gastric mucosa and the mucosa depleted of mucin showed that MBP without mucin was susceptible to the proteolytic degradation with pepsin and H. pylori proteases, but was not released from the apical membrane by the treatment with bacterial PLC. CONCLUSION: The study of carbohydrate ligands for MBP revealed binding of octa- and decasaccharides of gastric mucin. The severe impairment in mucin adhesion to MBP, induced by the diet containing ethanol, supports the conclusion that specific carbohydrate determinants participate in mucin attachment to MBP and epigenetic control of the processes that coordinates its interaction with apical mucosal epithelium in the formation of innate protective barrier.     

Immune Evasion by Helicobacter pylori: Gastric Spiral Bacteria Lack Surface Immunoglobulin Deposition and Reactivity with Homologous Antibodies

Background. Helicobacter pylori infection persists in the presence of potent serum and gastric mucosal anti-body responses against bacterial antigens. The aim of this article is to report on a study determine whether there is antibody deposition on H. pylori in vivo in the stomach of infected patients and whether gastric and cultured forms of H. pylori differ in their antibody reactivity.
Materials and Methods. Serum, gastric biopsies, and antral brushings were obtained from 10 patients having endoscopy. H. pylori was cultured from gastric biopsies. Bacterial samples were stained directly for immunoglobulin deposition and indirectly using rabbit antiurease serum or patient serum. Samples were examined by immunofluorescence microscopy and flow cytometry.
Results. Although spiral bacteria could be identified easily by acridine orange staining and antiurease staining of gastric brushings from H. pylori infected patients, gastric bacteria did not have detectable IgG or IgA present, and only one of five samples could be stained for IgG and IgA indirectly using patient serum. In contrast, cultured bacteria could be stained readily with homologous serum for IgG and IgA in the majority of cases. Low pH inhibited immunoglobulin reactivity with cultured H. pylori.
Conclusions. Gastric H. pylori may evade humoral defense owing to poor deposition of immunoglobulin in the gastric environment or failure to express surface antigens that are present on cultured forms of H. pylori.     

Helicobacter pylori CagA-dependent macrophage migration inhibitory factor produced by gastric epithelial cells binds to CD74 and stimulates procarcinogenic events

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has recently been implicated in carcinogenesis. Helicobacter pylori, which is closely linked to gastric cancer, induces the gastric epithelium to produce proinflammatory cytokines, including MIF. MIF can bind to CD74, which we have previously shown to be highly expressed on the surface of gastric epithelial cells (GEC) during H. pylori infection. In this study, we sought to investigate the role of the H. pylori-induced MIF on epithelial proliferation and procarcinogenic events. Upon establishing a role for the H. pylori CagA virulence factor in MIF production, MIF binding to CD74 on GEC was confirmed. rMIF and H. pylori were shown to increase GEC proliferation, which was decreased when cagA- strains were used and when CD74 was blocked by mAbs. Apoptosis was also decreased by MIF, but increased by cagA- strains that induced much lower amounts of MIF than the wild-type bacteria. Furthermore, MIF binding to CD74 was also shown to decrease p53 phosphorylation and up-regulate Bcl-2 expression. This data describes a novel system in which an H. pylori virulence factor contributes to the production of a host factor that in turn up-regulates procarcinogenic events by the gastric epithelium.     

The sialic acid binding SabA adhesin of Helicobacter pylori is essential for nonopsonic activation of human neutrophils

Infiltration of neutrophils and monocytes into the gastric mucosa is a hallmark of chronic gastritis caused by Helicobacter pylori. Certain H. pylori strains nonopsonized stimulate neutrophils to production of reactive oxygen species causing oxidative damage of the gastric epithelium. Here, the contribution of some H. pylori virulence factors, the blood group antigen-binding adhesin BabA, the sialic acid-binding adhesin SabA, the neutrophil-activating protein HP-NAP, and the vacuolating cytotoxin VacA, to the activation of human neutrophils in terms of adherence, phagocytosis, and oxidative burst was investigated. Neutrophils were challenged with wild type bacteria and isogenic mutants lacking BabA, SabA, HP-NAP, or VacA. Mutant and wild type strains lacking SabA had no neutrophil-activating capacity, demonstrating that binding of H. pylori to sialylated neutrophil receptors plays a pivotal initial role in the adherence and phagocytosis of the bacteria and the induction of the oxidative burst. The link between receptor binding and oxidative burst involves a G-protein-linked signaling pathway and downstream activation of phosphatidylinositol 3-kinase as shown by experiments using signal transduction inhibitors. Collectively our data suggest that the sialic acid-binding SabA adhesin is a prerequisite for the nonopsonic activation of human neutrophils and, thus, is a virulence factor important for the pathogenesis of H. pylori infection.     

Four modes of adhesion are used during Helicobacter pylori binding to human mucins in the oral and gastric niches

Background:  Helicobacter pylori causes peptic ulcer disease and gastric cancer, and the oral cavity is likely to serve as a reservoir for this pathogen. We investigated the binding of H. pylori to the mucins covering the mucosal surfaces in the niches along the oral to gastric infection route and during gastric disease and modeled the outcome of these interactions.
Materials and Methods:  A panel of seven H. pylori strains with defined binding properties was used to identify binding to human mucins from saliva, gastric juice, cardia, corpus, and antrum of healthy stomachs and of stomachs affected by gastritis at pH 7.4 and 3.0 using a microtiter-based method.
Results:  H. pylori binding to mucins differed substantially with the anatomic site, mucin type, pH, gastritis status, and H. pylori strain all having effect on binding. Mucins from saliva and gastric juice displayed the most diverse binding patterns, involving four modes of H. pylori adhesion and the MUC5B, MUC7, and MUC5AC mucins as well as the salivary agglutinin. Binding occurred via the blood-group antigen-binding adhesin (BabA), the sialic acid-binding adhesin (SabA), a charge/low pH-dependent mechanism, and a novel saliva-binding adhesin. In the healthy gastric mucus layer only BabA and acid/charge affect binding to the mucins, whereas in gastritis, the BabA/Le^b-dependent binding to MUC5AC remained, and SabA and low pH binding increased.
Conclusions:  The four H. pylori adhesion modes binding to mucins are likely to play different roles during colonization of the oral to gastric niches and during long-term infection.     

Identification of glycolipid receptors for Helicobacter pylori by TLC-immunostaining

Helicobacter pylori has been identified as a causative agent in active chronic gastritis. The receptor for this bacteria, however, is not known. It is likely that the receptor molecules may be glycosphingolipids as shown in the cases of other bacteria. We explored this possibility by a thin-layer chromatography (TLC)-immunostaining method. Among glycosphingolipids extracted from human gastric mucosa, intact Helicobacter pylori specifically bound to I3SO3-GalCer and II3NeuAc-LacCer, whereas no specific binding to neutral glycosphingolipids, which share the same ceramide moiety with I3SO3-GalCer or II3NeuAc-LacCer, was demonstrated. Sonicated bacteria could still bind to II3NeuAc-LacCer with comparable affinity. In contrast, the binding of bacteria to I3SO3-GalCer was greatly diminished upon sonication. These results suggest that each of the oligosaccharide moieties of II3NeuAc-LacCer and I3SO3-GalCer may be specifically recognized by different ligand molecules of Helicobacter pylori.     

Use of a three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria

A mixed culture of human fecal bacteria was grown for 120 days in a three-stage continuous culture system. To reproduce some of the nutritional and pH characteristics of the large gut, each vessel had a different operating volume (0.3, 0.5, and 0.8 liter) and pH (6.0, 6.5, and 7.0). A mixture of polysaccharides and proteins was used as carbon and nitrogen sources. Measurements of H2, CH4, S2-, sulfate reduction rates, sulfate-reducing bacteria (SRB), and volatile fatty acids were made throughout the experiment. After 48 days of running, porcine gastric mucin (5.8 g/day) was independently fed to vessel 1 of the multichamber system. The mucin was extensively degraded as evidenced by the stimulation of volatile fatty acid production. In the absence of mucin, sulfate-reducing activity was comparatively insignificant and methanogenesis was the major route for the disposal of electrons. The reverse occurred upon the addition of mucin; sulfate reduction predominated and methanogenesis was completely inhibited. This was attributed to release of sulfate from the mucin which enabled SRB to outcompete methanogenic bacteria for H2. SRB stimulated by mucin were acetate-utilizing Desulfobacter spp., lactate- and H2-utilizing Desulfovibrio spp., and propionate-utilizing Desulfobulbus spp. When the mucin pump was switched off, the multichamber system reverted to a state close to its original equilibrium. These data provide further evidence that sulfated polysaccharides such as mucin may be a source of sulfate for SRB in the human large gut.