Structure of a D-glycero -D-manno-heptan from the lipopolysaccharide of Helicobacter pylori

A lipopolysaccharide (LPS) was isolated by hot phenol-water extraction from Helicobacter pylori strain D4 and found to contain no fucosylated poly-N-acetyllactosamine chain typical of most H. pylori strains studied but a homopolymer of D-glycero-D-manno-heptose (DD-Hep). The heptan attached to a core oligosaccharide was released by mild acid degradation of the LPS, and the following structure of the trisaccharide-repeating unit was established by chemical methods and 1H and 13C NMR spectroscopy: --> 2)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 -->. 1H NMR spectroscopy performed on small amounts of the intact LPS revealed the presence of the same polysaccharide in LPS of H. pylori strains D2 and D5, but not strain D10.     

Structure and heterogeneity of the O-antigen chain of Salmonella Agona lipopolysaccharide

Lipopolysaccharide of Salmonella Agona smooth-type cells was obtained from bacteria by a hot phenol-water extraction procedure. Mild acid hydrolysis of lipopolysaccharide, followed by gel filtration, yielded the pure O-polysaccharide. Abequose, rhamnose, mannose, galactose and glucose in the molar ratio 0.8 : 1.0 : 1.0 : 1.1 : 0.5 were detected, and their linkages were established. Sugar configurations were determined by gas chromatography. Two repeating units, namely -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-alpha-d-Galp-(1-->and -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-[alpha-d-Glcp-(1-->4)-]-alpha-d-Galp-(1-->, were deduced from nuclear magnetic resonance studies. The effort to separate them was unsuccessful. An immunochemical test performed by means of Western blotting with anti O12 serum demonstrated that glucose was present in the longer lipopolysaccharide chains, at some distance from the core region.     

Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori

Activity screening and insertional inactivation of lipopolysaccharide (LPS) biosynthetic genes in Helicobacter pylori have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (rfaJ) family of glycosyltransferases. Activity screening of a genomic library led to the identification of the enzyme involved in the biosynthesis of the type 2 N-acetyl-lactosamine O-chain backbone, the beta-1,3-N-acetyl-glucosaminyl transferase. In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the alpha-1,6-glucan polymer. This alpha-1,6-glucosyltransferase protein is encoded by the HP0159 ORF. Both enzymes play an integral part in the biosynthesis of LPS, and insertional inactivation leads to the production of a truncated LPS molecule on the bacterial cell surface. The LPS structures were determined by mass spectrometry and chemical analyses. The linkage specificity of each glycosyltransferase was determined by nuclear magnetic resonance (NMR) analysis of model compounds synthesized in vitro. A cryogenic probe was used to structurally characterize nanomole amounts of the product of the HP1105 (JHP1032) enzyme. In contrast to the HP0159 enzyme, which displays the GT-8-predicted retaining stereochemistry for the reaction product, HP1105 (JHP1032) is the first member of this GT-8 family to have been shown to have an inverting stereochemistry in its reaction products.     

A reinvestigation of the lipopolysaccharide structure of Helicobacter pylori strain Sydney (SS1)

In this study, we describe a reinvestigation of the lipopolysaccharide (LPS) structure of Helicobacter pylori strain Sydney (SS1) based on the NMR analysis of oligosaccharides obtained through the use of various degradations of the LPS as well as capillary electrophoresis-MS data. The results of the analysis indicated that the core region of a major H. pylori SS1 LPS glycoform consists of a backbone core oligosaccharide substituted at the D-glycero-D-manno-heptose (DD-Hep) residue by a linear chain composed of a trisaccharide fragment α-ddHep-3-α-L-Fuc-3-β-GlcNAc, as previously demonstrated for H. pylori strain 26695, further elongated by consecutively added α-Glc and β-Gal residues, and terminating in a novel linear chain consisting of 1,2-linked β-ribofuranosyl residues, where the last β-ribofuranosyl residue provides a point of attachment for the O-chain polysaccharide: [Formula: see text] where [2-β-Ribf-](n) is a short (three to five residues) oligomer of 1,2-linked β-ribofuranose (riban), and PS is a polysaccharide chain consisting of N-acetyllactosamine, substituted with α-Fuc to form Lewis (Le)-type structures. In addition to the previously identified LacNAc, Le(y) and Le(x) components, the O-chain polysaccharide of H. pylori SS1 LPS was found to contain a novel LacNAc unit carrying a phosphoethanolamine substituent at the O-6 position of β-GlcNAc residues.     

Helicobacter pylori lipopolysaccharide in the IL-2 milieu activates lymphocytes from dyspeptic children

In this study, we assessed the proliferative response of peripheral blood mononuclear leukocytes (PBML) from 33 children/young adolescents with chronic dyspepsia, to H. pylori LPS in the presence and absence of IL-2 as a T cell growth factor. A rapid urease test (RUT) and a presence of Helicobacter-like organisms (HLO) in the biopsy specimens allowed us to distinguish RUT/HLO-positive (17/33) and -negative (16/33) patients. H. pylori LPS alone induced a proliferation of PBML from 4 out of 33 dyspeptic patients. IL-2 increased the prevalence of the response to LPS to 59% and 74% of RUT/HLO-positive and -negative patients, respectively. PBML from RUT/HLO-positive patients responded significantly less intensively to H. pylori LPS in the presence of IL-2, to IL-2 alone and to H. pylori LPS+IL-2. However, there was no difference in PHA-driven proliferation of PBML from the patients of those two groups. A negative correlation between the responsiveness to H. pylori LPS of PBML and occurrence of type B inflammation in gastric mucosa was demonstrated. The results suggest a contribution of H. pylori LPS to an outcome of H. pylori infection. It is speculated that H. pylori LPS by an activation of immunocompetent cells may reduce gastric inflammation, decrease bacterial load and prolong H. pylori infection.     

Intra-strain variation in expression of lipopolysaccharide by Helicobacter pylori

Intra-strain variation in the expression of lipopolysaccharide (LPS) by two clinical isolates of Helicobacter pylori was examined. Lipopolysaccharide was prepared from successive cultures of individual colonies from each strain, separated by SDS-PAGE, and detected by silver staining and by immunoblotting. The genetic 'relatedness' of the colonies was investigated using PCR-RFLP analysis of the urease and vacuolating cytotoxin genes. Although individual colonies of each of the two strains examined appeared to have the same genetic origins, variation in the expression of their long-chain LPS was observed. The same LPS profiles were maintained by individual colonies over four subcultures on solid media containing 10% (v/v) defibrinated horse blood.     

The pepsinogen releasing effect of Helicobacter pylori lipopolysaccharide

Background. Helicobacter pylori lipopolysaccharide (LPS) affects pepsinogen release by a nontoxic mechanism. We hypothesized that this effect was characteristic of the organism and related to the clinical status of the strain. Materials and methods. LPS was isolated from 11 H. pylori strains whose pathogenic profile was known and four other nongastric bacteria. The effects of luminal LPS on guinea pig gastric mucosal pepsinogen release was evaluated using the Ussing chamber technique. CCK‐8 (10^?9M) was used as a positive control. Results. H. pylori LPS dose‐dependently stimulated pepsinogen release with a maximal stimulation at 250 µg/ml (~4500%; p < .001 vs. control). LPS from other Helicobacter or Campylobacter species had no effect on pepsinogen release. ANOVA demonstrated significant differences in the efficacies of pepsinogen release between the 11 clinical H. pylori strains (p < .0001) despite the fact that they were all cagA⁺ and 90% had the cytotoxic vacA subtype s1. Physical and chemical disruption of the LPS suggested that both the structure and the carbohydrate composition of this molecule may play a critical role in pepsinogen release. Polymyxin B partly (p < .03) inhibited and dephosphorylation completely inhibited (p = .0002) LPS‐stimulated pepsinogen release. Conclusion. Pepsinogen release is an innate property of all cagA⁺H. pylori LPS. The structure of the molecule and composition of side‐chains are important in this response which appears to be partially lipid A driven.     

Genome of Helicobacter pylori strain 908

Helicobacter pylori is a genetically diverse and coevolved pathogen inhabiting human gastric niches and leading to a spectrum of gastric diseases in susceptible populations. We describe the genome sequence of H. pylori 908, which was originally isolated from an African patient living in France who suffered with recrudescent duodenal ulcer disease. The strain was found to be phylogenetically related to H. pylori J99, and its comparative analysis revealed several specific genome features and novel insertion-deletion and substitution events. The genome sequence revealed several strain-specific deletions and/or gain of genes exclusively present in HP908 compared with different sequenced genomes already available in the public domain. Comparative and functional genomics of HP908 and its subclones will be important in understanding genomic plasticity and the capacity to colonize and persist in a changing host environment.     

Characterization of the respiratory chain of Helicobacter pylori

The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium.     

Characterization of an Helicobacter pylori environmental strain

Aims:  To investigate the main genotypic virulence markers and the phenotypic features of an environmental Helicobacter pylori strain, named MDC1.
Methods and Results:  The H. pylori MDC1 genotypic status was evaluated by PCR amplification. The mosaicism in vac A alleles was expressed by the s1m1 allelic combination, as found in strains which are strong vacuolating cytotoxin producers; the number of cag A variable EPIYA motifs displayed P1P2P3P3 pattern and the ice A1 was recorded between the ice A allelic types and the bab A2 gene found in strains causing more severe disease. The biofilm formation was evaluated on a polystyrene surface in static conditions by scanning electron microscopy and confocal scanning laser microscopy. Helicobacter pylori MDC1 displayed a dense mature biofilm with cells in a coccoid morphology persistent in time in which the expression of the lux S gene, related to the quorum-sensing signalling, was always detected.
Conclusions:  Helicobacter pylori MDC1 strain had the main virulence markers closely related to gastric pathogenesis and displayed a well-structured biofilm which allowed this bacterium to be more protected in the environment.
Significance and Impact of the Study:  The persistence of the environmental virulent H. pylori strain in a clustered state suggests a long-term survival of this bacterial community outside of the host, enabling the bacterial transmission with important clinical repercussions.     

Structural characterization of closely related O-antigen lipopolysaccharide (LPS) chain length regulators

The surface O-antigen polymers of Gram-negative bacteria exhibit a modal length distribution that depends on dedicated chain length regulator periplasmic proteins (polysaccharide co-polymerases, PCPs) anchored in the inner membrane by two transmembrane helices. In an attempt to determine whether structural changes underlie the O-antigen modal length specification, we have determined the crystal structures of several closely related PCPs, namely two chimeric PCP-1 family members solved at 1.6 and 2.8 Å and a wild-type PCP-1 from Shigella flexneri solved at 2.8 Å. The chimeric proteins form circular octamers, whereas the wild-type WzzB from S. flexneri was found to be an open trimer. We also present the structure of a Wzz^FepE mutant, which exhibits severe attenuation in its ability to produce very long O-antigen polymers. Our findings suggest that the differences in the modal length distribution depend primarily on the surface-exposed amino acids in specific regions rather than on the differences in the oligomeric state of the PCP protomers.     

Mechanism of O-antigen distribution in lipopolysaccharide.

O-antigen units are nonuniformly distributed among lipid A-core molecules in lipopolysaccharide (LPS) from gram-negative bacteria, as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the actual distribution patterns are complex, multimodal, and strain specific. Although the basic biochemical steps involved in synthesis and polymerization of O-antigen monomers and their subsequent attachment to lipid A-core are known, the mechanism by which specific multimodal distribution patterns are attained in mature LPS has not been previously considered theoretically or experimentally. We have developed probability equations which completely describe O-antigen distribution among lipid A-core molecules in terms of the probability of finding a nascent polymer (O antigen linked to carrier lipid) of length k (Tk) and the probability that a nascent polymer of length k will be extended to k + 1 by polymerase (pk) or transferred to lipid A-core by ligase (qk). These equations were used to show that multimodal distribution patterns in mature LPS cannot be produced if all pk are equal to p and all qk are equal to q, conditions which indicate a lack of selectivity of polymerase and ligase, respectively, for nascent O-antigen chain lengths. A completely stochastic model (pk = p, qk = q) of O-antigen polymerization and transfer to lipid A-core was also inconsistent with observed effects of mutations which resulted in partial inhibition of O-antigen monomer synthesis, lipid A-core synthesis, or ligase activity. The simplest explanation compatible with experimental observations is that polymerase or ligase, or perhaps both, have specificity for certain O-antigen chain lengths during biosynthesis of LPS. Our mathematical model indicates selectively probably was associated with the polymerase reaction. Although one may argue for a multimodal distribution pattern based on a kinetic mechanism i.e., varying reaction parameters in space or in time during cell growth, such a model requires complex sensory and regulatory mechanisms to explain the mutant data and mechanisms for sequestering specific components of LPS biosynthesis to explain the distribution pattern in normal cells. We favor the simple alternative of enzyme specificity and present generalized equations which should be useful in analysis of other analogous biochemical systems.     

Removal of the outer Kdo from Helicobacter pylori lipopolysaccharide and its impact on the bacterial surface

Helicobacter pylori produces a unique surface lipopolysaccharide (LPS) characterized by strikingly low endotoxicity that is thought to aid the organism in evading the host immune response. This reduction in endotoxicity is predicted to arise from the modification of the Kdo–lipid A domain of Helicobacter LPS by a series of membrane bound enzymes including a Kdo (3‐deoxy‐d ‐manno‐octulosonic acid) hydrolase responsible for the modification of the core oligosaccharide. Here, we report that Kdo hydrolase activity is dependent upon a putative two‐protein complex composed of proteins Hp0579 and Hp0580. Inactivation of Kdo hydrolase activity produced two phenotypes associated with cationic antimicrobial peptide resistance and O‐antigen expression. Kdo hydrolase mutants were highly sensitive to polymyxin B, which could be attributed to a defect in downstream modifications to the lipid A 4′‐phosphate group. Production of a fully extended O‐antigen was also diminished in a Kdo hydrolase mutant, with a consequent increase in core–lipid A. Finally, expression of O‐antigen Lewis X and Y epitopes, known to mimic glycoconjugates found on human tissues, was also affected. Taken together, we have demonstrated that loss of Kdo hydrolase activity affects all three domains of H. pylori LPS, thus highlighting its role in the maintenance of the bacterial surface.     

Structural studies of the O-antigen of the lipopolysaccharide from an avirulent strain (M4S) of Pseudomonas solanacearum

The structure of the O-antigen of the lipopolysaccharide from an avirulent strain (M4S) of Pseudomonas solanacearum has been investigated by methylation analysis, n.m.r. spectroscopy, and N-deacetylation-deamination, followed by analysis and controlled Smith-degradation of the product. These studies demonstrate that the O-antigen is composed of a tetrasaccharide repeating-unit having the following structure: ----3)-alpha-D-GlcpNAc-(1----2)-alpha-L-Rhap-(1----2)-alpha- L-Rhap-(1----3)- alpha-L-Rhap-(1----.     

Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.     

Inhibition of gastric mucosal laminin receptor by Helicobacter pylori lipopolysaccharide.

Laminin receptor was isolated from gastric epithelial cell membrane by the procedure involving membrane solubilization with octylglucoside followed by affinity chromatography on laminin-coupled Sepharose. The receptor protein, eluted from the matrix with cation-free EDTA buffer, yielded on SDS-PAGE a single 67kDa band. After radioiodination, the protein was incorporated into liposomes which displayed specific affinity toward the laminin-coated surface. The binding of liposomal receptor to the laminin-coated surface was inhibited by lipopolysaccharide from H.pylori. The inhibitory effect was proportional to the concentration of lipopolysaccharide up to 50 micrograms/ml at which point a 96% decrease in the receptor binding occurred. It is suggested that a similar process may account for the loss of mucosal integrity in the pathogenesis of H. pylori associated gastric disease.     

Platelet-activating factor modulates gastric mucosal inflammatory responses to Helicobacter pylori lipopolysaccharide

Platelet-activating factor (PAF) is a phospholipid messenger implicated in mediation of inflammatory events associated with the resolution of inflammation. We applied the animal model of Helicobacter pylori LPS-induced gastritis in conjunction with prophylactic and therapeutic administration of a specific PAF antagonist, BN52020, to investigate the role of PAF in gastric mucosal responses to H. pylori infection. Prophylactic BN52020 administration produced up to 73.6% reduction in the severity of the LPS-induced inflammatory changes, whereas up to 38.4% increase in the severity of mucosal involvement occurred with BN52020 administered therapeutically. The prophylactic effects of BN52020 were accompanied by a drop in apoptosis and the expression of TNF-alpha and NOS-2, while BN52020 administered therapeutically caused a marked upregulation in apoptosis, TNF-alpha, and NOS-2. The untoward therapeutic effects of BN52020, moreover, were potentiated further in the presence of COX-2 inhibitor, whereas NOS-2 inhibitor caused a reduction in the extent of inflammatory changes. Our findings point to PAF as a key mediator of gastric mucosal inflammatory responses to H. pylori and suggest its modulatory role in the expression of COX-2 derived anti-inflammatory prostaglandins that are involved in controlling the extent of NOS-2 induction.     

Platelet-activating factor mediates Helicobacter pylori lipopolysaccharide interference with gastric mucin synthesis

Platelet-activating factor (PAF) is now recognized as the most proximal mediator of cellular events triggered by bacterial infection. In this study, we report that a specific PAF antagonist, BN52020, impedes the reduction in mucin synthesis evoked in gastric mucosal cells by H. pylori LPS. The impedance by BN52020 of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (P13K), which also obviated the inhibitory effect of BN52020 on the LPS-induced upregulation in apoptosis, TNF-alpha, and NO generation. A reduction in the impedance by BN52020 of the LPS detrimental effect on mucin synthesis was also attained with cNOS inhibitor, L-NNA, whereas NOS-2 inhibitor, 1400W caused a potentiation in the impedance effect of BN52020. However, while 1400W and BN52020 countered the potentiating effect of wortmannin on the LPS-induced decrease in mucin synthesis, a further exacerbation of the potentiating effect of wortmannin was attained in the presence of cNOS inhibitor, L-NNA. Our findings suggest that PAF, through the interference with PI3K-dependent cNOS activation, plays a critical role in influencing the extent of pathological consequences of H. pylori infection on the synthesis of gastric mucin.