共查询到20条相似文献,搜索用时 15 毫秒
1.
Logan SM Altman E Mykytczuk O Brisson JR Chandan V Schur MJ St Michael F Masson A Leclerc S Hiratsuka K Smirnova N Li J Wu Y Wakarchuk WW 《Glycobiology》2005,15(7):721-733
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. 相似文献
2.
Zanotti G Papinutto E Dundon W Battistutta R Seveso M Giudice G Rappuoli R Montecucco C 《Journal of molecular biology》2002,323(1):125-130
Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes. 相似文献
3.
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. 相似文献
4.
The O-antigen chain from the lipopolysaccharide of Helicobacter pylori
strain UA861 was determined to be composed of an elongated type 2 N -
acetyllactosamine backbone,
-[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1- ]n-->, with
approximately half of the GlcNAc units carrying a terminal alpha-d-Glc
residue at the O -6 position. The O-chain of H.pylori UA861 was terminated
by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D- GlcNAc] (LacNAc)
epitope and did not express terminal Lewis X or Lewis Y blood-group
determinants as previously found in other H.pylori strains. The absence of
terminal Lewis X and Lewis Y blood-group epitopes and the replacement of
Fuc by Glc as a side chain in the O- chain of H.pylori UA861 represents yet
another type of lipopolysaccharide structure from H.pylori species. These
structural differences in H.pylori lipopolysaccharide molecules carry
implications with regard to possible different pathogenic events between
strains and respective hosts.
相似文献
5.
Rudnicka W Jarosinska A Bak-Romaniszyn L Moran A Planeta-Malecka I Wadstrom T Chmiela M 《FEMS immunology and medical microbiology》2003,36(3):141-145
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. 相似文献
6.
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. 相似文献
7.
Sun HY Lin SW Ko TP Pan JF Liu CL Lin CN Wang AH Lin CH 《The Journal of biological chemistry》2007,282(13):9973-9982
Helicobacter pylori alpha1,3-fucosyltransferase (FucT) is involved in catalysis to produce the Lewis x trisaccharide, the major component of the bacteria's lipopolysaccharides, which has been suggested to mimic the surface sugars in gastric epithelium to escape host immune surveillance. We report here three x-ray crystal structures of FucT, including the FucT.GDP-fucose and FucT.GDP complexes. The protein structure is typical of the glycosyltransferase-B family despite little sequence homology. We identified a number of catalytically important residues, including Glu-95, which serves as the general base, and Glu-249, which stabilizes the developing oxonium ion during catalysis. The residues Arg-195, Tyr-246, Glu-249, and Lys-250 serve to interact with the donor substrate, GDP-fucose. Variations in the protein and ligand conformations, as well as a possible FucT dimer, were also observed. We propose a catalytic mechanism and a model of polysaccharide binding not only to explain the observed variations in H. pylori lipopolysaccharides, but also to facilitate the development of potent inhibitors. 相似文献
8.
Christopher M. Stead Jinshi Zhao Christian R. H. Raetz M. Stephen Trent 《Molecular microbiology》2010,78(4):837-852
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. 相似文献
9.
Lebrun AH Wunder C Hildebrand J Churin Y Zähringer U Lindner B Meyer TF Heinz E Warnecke D 《The Journal of biological chemistry》2006,281(38):27765-27772
O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003-1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol-alpha-glucosyltransferase. 相似文献
10.
Inhibition of gastric mucosal laminin receptor by Helicobacter pylori lipopolysaccharide. 总被引:4,自引:0,他引:4
B L Slomiany J Piotrowski S Sengupta A Slomiany 《Biochemical and biophysical research communications》1991,175(3):963-970
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. 相似文献
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Hiratsuka K Logan SM Conlan JW Chandan V Aubry A Smirnova N Ulrichsen H Chan KH Griffith DW Harrison BA Li J Altman E 《Journal of bacteriology》2005,187(15):5156-5165
We have identified a Helicobacter pylori d-glycero-d-manno-heptosyltransferase gene, HP0479, which is involved in the biosynthesis of the outer core region of H. pylori lipopolysaccharide (LPS). Insertional inactivation of HP0479 resulted in formation of a truncated LPS molecule lacking an alpha-1,6-glucan-, dd-heptose-containing outer core region and O-chain polysaccharide. Detailed structural analysis of purified LPS from HP0479 mutants of strains SS1, 26695, O:3, and PJ1 by a combination of chemical and mass spectrometric methods showed that HP0479 likely encodes alpha-1,2-d-glycero-d-manno-heptosyltransferase, which adds a d-glycero-d-manno-heptose residue (DDHepII) to a distal dd-heptose of the core oligosaccharide backbone of H. pylori LPS. When the wild-type HP0479 gene was reintegrated into the chromosome of strain 26695 by using an "antibiotic cassette swapping" method, the complete LPS structure was restored. Introduction of the HP0479 mutation into the H. pylori mouse-colonizing Sydney (SS1) strain and the clinical isolate PJ1, which expresses dd-heptoglycan, resulted in the loss of colonization in a mouse model. This indicates that H. pylori expressing a deeply truncated LPS is unable to successfully colonize the murine stomach and provides evidence for a critical role of the outer core region of H. pylori LPS in colonization. 相似文献
13.
Y H Liau R A Lopez A Slomiany B L Slomiany 《Biochemical and biophysical research communications》1992,184(3):1411-1417
The effect of H. pylori lipopolysaccharide on the synthesis and secretion of sulfated mucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]Na2SO4. The lipopolysaccharide, while showing no discernible effect on the apomucin synthesis was found to inhibit the process of mucin glycosylation and sulfation, which at 100 micrograms/ml lipopolysaccharide reached the optimal inhibition of 65%. The analysis of mucin secretory responses revealed that the lipopolysaccharide by first 15 min caused a 57% stimulation in sulfomucin secretion followed thereafter by inhibition, which reached maximum of 32% by 45 min. The results suggest that colonization of gastric mucosa by H. pylori may be detrimental to the process of gastric sulfomucin synthesis and secretion. 相似文献
14.
Platelet-activating factor modulates gastric mucosal inflammatory responses to Helicobacter pylori lipopolysaccharide 总被引:4,自引:0,他引:4
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. 相似文献
15.
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. 相似文献
16.
Hong Li Tingting Liao Aleksandra W. Debowski Hong Tang Hans‐Olof Nilsson Keith A. Stubbs Barry J. Marshall Mohammed Benghezal 《Helicobacter》2016,21(6):445-461
This review covers the current knowledge and gaps in Helicobacter pylori lipopolysaccharide (LPS) structure and biosynthesis. H. pylori is a Gram‐negative bacterium which colonizes the luminal surface of the human gastric epithelium. Both a constitutive alteration of the lipid A preventing TLR4 elicitation and host mimicry of the Lewis antigen decorated O‐antigen of H. pylori LPS promote immune escape and chronic infection. To date, the complete structure of H. pylori LPS is not available, and the proposed model is a linear arrangement composed of the inner core defined as the hexa‐saccharide (Kdo‐LD‐Hep‐LD‐Hep‐DD‐Hep‐Gal‐Glc), the outer core composed of a conserved trisaccharide (‐GlcNAc‐Fuc‐DD‐Hep‐) linked to the third heptose of the inner core, the glucan, the heptan and a variable O‐antigen, generally consisting of a poly‐LacNAc decorated with Lewis antigens. Although the glycosyltransferases (GTs) responsible for the biosynthesis of the H. pylori O‐antigen chains have been identified and characterized, there are many gaps in regard to the biosynthesis of the core LPS. These limitations warrant additional mutagenesis and structural studies to obtain the complete LPS structure and corresponding biosynthetic pathway of this important gastric bacterium. 相似文献
17.
Kawahara T Teshima S Kuwano Y Oka A Kishi K Rokutan K 《American journal of physiology. Gastrointestinal and liver physiology》2001,281(3):G726-G734
Helicobacter pylori lipopolysaccharide (LPS) is generally accepted as a low-toxicity virulence. Primary cultures of guinea pig gastric mucosal cells expressed the Toll-like receptor 4 and were sensitive to H. pylori LPS as well as Escherichia coli LPS. H. pylori LPS stimulated phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and c-Jun NH(2)-terminal kinase (JNK) 2. H. pylori LPS at >2.1 endotoxin unit/ml (>1 ng/ml) activated caspase-8, stimulated cytochrome c release from mitochondria, and subsequently activated caspases-9 and -3, leading to apoptosis. Epidermal growth factor blocked all of these apoptotic processes and inhibited apoptosis, whereas it did not modify the phosphorylation of TAK1, TAB1, and JNK2. A comparatively specific inhibitor of caspase-8 or -9 blocked apoptosis, whereas cytochrome c release was prevented only with a caspase-8-like inhibitor. Our results suggest that caspase-8 and mitochondria may play crucial roles in H. pylori LPS-induced apoptosis and that this accelerated apoptosis may be involved in abnormal cell turnover of H. pylori-infected gastric mucosa. 相似文献
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Partha Krishnamurthy Mary H. Parlow John Schneider Stephanie Burroughs Catherine Wickland Nimish B. Vakil Bruce E. Dunn Suhas H. Phadnis 《Journal of bacteriology》1999,181(16):5107-5110
The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth. 相似文献