Stimulation of adhesion molecules on vascular endothelium by capsular polysaccharide, lipopolysaccharide and components of lipopolysaccharide from Bacteroides thetaiotaomicron

The aim of this study was to assay the influence of capsular polysaccharide (CPS), lipopolysaccharide (LPS) and components of B. thetaiotaomicron lipopolysaccharide--polysaccharide part (PS) and lipid part (lipid A) on the expression of adhesion molecules associated with inflammation (ICAM-1, VCAM-1, E-selectin) on the surface of vascular endothelial cells. Capsular polysaccharide was isolated by the method of Poxton and Ip (1981). Lipopolysaccharides were extracted using the hot phenol-water method (Westphal and Jann, 1965). Components of LPS were prepared by mild acid hydrolysis of lipopolysaccharide. Experiments with bacterial compounds at concentrations 10, 1, 0.1 and 0.01 (mg/ml) were performed on HMEC-1 cell line (human dermal microvascular endothelial cells). Immunoenzymatic ELISA test with mouse monoclonal antibodies against human: ICAM-1, VCAM-1 and E-selectin was applied to determine adhesion molecules. Resting HMEC-1 and E. coli O55:B5 LPS were used as controls in each experiment. Lipopolysaccharides were the strongest stimulants of endothelial adhesion molecules. Capsular polysaccharide caused the expression of three adhesion molecules, but only at the highest concentration (10 mg/ml). The stimulatory activities of LPS lipid components were much higher than the activities of polysaccharide parts. PS preparations did not reveal the property of adhesion molecule stimulation or their activities were weak. The activity of B. thetaiotaomicron cell-surface antigens in the process of adhesion molecule stimulation on vascular endothelium was lower than the activity of E. coli LPS.     

A novel cell surface polysaccharide in Pseudomonas putida WCS358, which shares characteristics with Escherichia coli K antigens, is not involved in root colonization.

Previously we have shown that flagella and the O-specific polysaccharide of lipopolysaccharide play a role in colonization of the potato root by plant growth-promoting Pseudomonas strains WCS374 and WCS358. In this paper, we describe a novel cell surface-exposed structure in Pseudomonas putida WCS358 examined with a specific monoclonal antibody. This cell surface structure appeared to be a polysaccharide, which was accessible to the monoclonal antibody at the outer cell surface. Further study revealed that it does not contain 2-keto-3-deoxyoctonate, heptose, or lipid A, indicating that it is not a second type of lipopolysaccharide. Instead, the polysaccharide shared some characteristics with K antigen described for Escherichia coli. From a series of 49 different soil bacteria tested, only one other potato plant growth-promoting Pseudomonas strain reacted positively with the monoclonal antibody. Mutant cells lacking the novel antigen were efficiently isolated by an enrichment method involving magnetic antibodies. Mutant strains defective in the novel antigen contained normal lipopolysaccharide. One of these mutants was affected in neither its ability to adhere to sterile potato root pieces nor its ability to colonize potato roots. We conclude that the bacterial cell surface of P. putida WCS358 contains at least two different polysaccharide structures. These are the O-specific polysaccharide of lipopolysaccharide, which is relevant for potato root colonization, and the novel polysaccharide, which is not involved in adhesion to or colonization of the potato root.     

Adhesion of Desulfovibrio desulfuricans and Pseudomonas fluorescens to mild steel surfaces

The adhesion of micro-organisms to metal surfaces has been shown to be important in the corrosion process, but the cell surface structures participating in this adhesion have not previously been identified. Evidence is presented that a bacterial substance taking part in the initial adhesion of Pseudomonas fluorescens and Desulfovibrio desulfuricans (New Jersey) to mild steel is polysaccharide in nature. It is likely that this is present in the outer membrane of the bacterial cells as lipopolysaccharide.     

Structure of the O-specific, sialic acid containing polysaccharide chain and its linkage to the core region in lipopolysaccharide from Hafnia alvei strain 2 as elucidated by chemical methods, gas-liquid chromatography/mass spectrometry, and 1H NMR spectroscopy

Mild acid hydrolysis of Hafnia alvei strain 2 lipopolysaccharide released no O-specific polysaccharide but instead gave a monomeric octasaccharide repeating unit with N-acetylneuraminic acid as the reducing terminus. In addition, a dimer of the octasaccharide repeating unit, and also a decasaccharide composed of a fragment of the O-specific polysaccharide chain and the core region, were obtained in minute amounts. On the basis of the sugar and methylation analyses, periodate oxidation, and 1H NMR spectroscopy of the lipopolysaccharide hydrolytic products, the biological repeating unit of the O-specific polysaccharide was shown to be a branched octasaccharide: (Formula; see text) The linkage between the O-specific polysaccharide chain and core region has also been determined and has yield strong evidence that N-acetylneuraminic acid is an inherent lipopolysaccharide component. The lipopolysaccharide of H. alvei strain 2 is the first lipopolysaccharide reported to contain 4-substituted neuraminic acid in its O-specific polysaccharide region.     

Suppression of cytokine production and cell adhesion molecule expression in human monocytic cell line THP-1 by Tripterygium wilfordii polysaccharide moiety

Extracts of the vine-like plant Tripterygium wilfordii (TW) have been widely used in China as an immunosuppressant and anti-inflammatory drug for the treatments of rheumatoid arthritis, lupus erythematosus and other inflammatory disorders. In this study the molecular mechanisms of action of three TW extracts (ethanol, aqueous, polysaccharide) on the expression of inflammatory cytokines and adhesion molecules were investigated by RT-PCR and immunofluorescence binding techniques. The lipopolysaccharide (LPS)-mediated stimulatory effects of tumor necrosis factor-alpha (TNF-alpha) cytokine production and cell adhesion molecule (CD11c, CD18, CD14, CD54) expression in human monocytic THP-1 cells were modulated by treatments of the TW extracts or tacrolimus (FK506). The TW polysaccharide moiety exhibited more profound immunosuppressive properties than the aqueous and ethanol extracts. Biochemical characterization of the polysaccharide moiety revealed a major molecular weight of 22 kDa (viz. PSP22). The PSP22 was found to be a potential immunosuppressant that manifests the necessary immunomodulating properties.     

Pseudomonas fluorescens adhesion and transport through porous media are affected by lipopolysaccharide composition.

The objectives of this work were (i) to use transposon mutagenesis to produce mutants of Pseudomonas fluorescens that were altered in adhesion ability and transport through porous media and (ii) to identify the alterations in surface characteristics that were responsible for the changes in attachment. Mutants of P. fluorescens were generated with TnphoA, which enabled identification of mutants that were altered in surface proteins. Transposon mutants were screened for alterations in adhesion ability by attachment assays on hydrophobic polystyrene and water-wettable polystyrene. Four TnphoA mutants with increased adhesion to the hydrophobic surface and decreased adhesion to the water-wettable surface were obtained. Transport of the strains through porous media was evaluated by passing suspensions of each mutant and the parent through columns containing quartz sand and determining the number of cells retained in the columns. The mutants all demonstrated increased adhesion and retention in the columns. Southern analysis demonstrated two types of mutants with separate transposon insertion sites. Polyacrylamide gel electrophoresis of the strains demonstrated that the O antigen on the lipopolysaccharide was either attenuated or absent. Lack of this polysaccharide, and the consequent increased exposure of the lipid moiety of the lipopolysaccharide, is probably responsible for the increase in adhesion to the hydrophobic substrata and retention in the sand column. This work combined with previous studies of attachment of P. fluorescens demonstrates that more than one type of polymer can mediate the adhesion of this organism to nonbiological surfaces.     

Interaction of Pseudomonas Bacteriophage 2 with the Slime Polysaccharide and Lipopolysaccharide of Pseudomonas aeruginosa Strain BI

Purified slime polysaccharide B and lipopolysaccharide of Pseudomonas aeruginosa strain BI were shown to possess receptor-like properties in inactivating Pseudomonas phage 2, whereas lipoprotein and glycopeptide fractions were devoid of activity. On a weight basis, slime polysaccharide B was more effective than lipopolysaccharide in inactivating phage. The specificity of the reaction with slime polysaccharide B was indicated by the fact that slime polysaccharide A of P. aeruginosa strain EI failed to inactivate phage 2. Electron micrographs showed phage 2 in typical, tail-first position of attachment on intact cells of strain BI, slime polysaccharide B, and lipopolysaccharide. Tail fibers were discernible during phage attachment.     

Structure of an extracellular cross-reactive polysaccharide from Pseudomonas aeruginosa immunotype 4

A neutral small molecular mass (approximately 6.5 kDa) polysaccharide comprising a pentasaccharide repeat unit was isolated from culture supernatants of Pseudomonas aeruginosa immunotype 4. The polysaccharide had a pentasaccharide repeating unit as follows (formula; see text) where Rha is rhamnose. The structure was determined using acid hydrolysis, solvolysis with anhydrous hydrogen fluoride, methylation analysis, and 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments. The polysaccharide bound antibody raised to the lipopolysaccharide of the seven P. aeruginosa Fisher-Devlin immunotype strains. Inhibition assays demonstrated the presence of a serologically similar polysaccharide in supernatants of these strains. Affinity-purified antibody to the polysaccharide bound to lipopolysaccharide and whole cells of the immunotype strains of P. aeruginosa in a Western immunoblot and colony blot assay, respectively. This polysaccharide seems to contain an antigenic determinant present in the core of the P. aeruginosa lipopolysaccharide or may represent another minor polysaccharide substituent on the lipopolysaccharide in addition to the O side chain.     

Interaction of spinach leaf adenosine diphosphate glucose alpha-1,4-glucan alpha-4-glucosyl transferase and alpha-1,4-glucan, alpha-1,4-glucan-6-glycosyl transferase in synthesis of branched alpha-glucan

Salmonella newington lipopolysaccharide extracted from a cell paste grown up from a single smooth clone was fractionated by chromatography on DEAE-cellulose in the presence of 1% Triton X-100 into seven lipopolysaccharide fractions which differed in their degrees of polymerization of the repeating unit of the O-antigen side chain and in their substitution with ester phosphate. Several of the lipopolysaccharide fractions were hydrolyzed in 1% acetic acid at 100 °C to cleave the linkage between the polysaccharide and lipid A parts of the structure. The polysaccharide fractions from each of the purified lipopolysaccharides could be further fractionated on DEAE-cellulose columns to yield a number of peaks of polysaccharide having monosaccharide ratios quite distinct from those of the parent lipopolysaccharide. The results show a high degree of structural heterogeneity in the original lipopolysaccharide.     

Extracellular polysaccharide production by Klebsiella pneumoniae and its relationship to virulence

Klebsiella pneumoniae serotype 1 and serotype 2 and their capsular variants were examined for production of cell-associated capsular polysaccharides and extracellular capsular polysaccharides. The virulence of these organisms in experimental animals was examined via intraperitoneal injection in mice and transtracheal inoculation into the lungs of rats. It was found that the production of either polysaccharide component correlated with the observed virulence. The extracellular polysaccharides were purified by ethanol precipitation, electrodialysis, extraction with quaternary ammonium salts, and gel filtration. These purification steps allowed for the separation and purification of both the extracellular lipopolysaccharide and the extracellular capsular polysaccharide. Purified extracellular capsular polysaccharide and extracellular lipopolysaccharide were co-injected with K. pneumoniae intraperitoneally into mice to determine if either of these substances would produce an effect on the natural course of infection in these animals. These studies showed that only purified extracellular lipopolysaccharide enhanced the virulence of K. pneumoniae when co-injected into mice, and this virulence enhancement correlated with the content of extracellular lipopolysaccharide, but not extracellular capsular polysaccharide in mixtures of these polysaccharides. Saponification of K. pneumoniae serotype 1 extracellular polysaccharides significantly decreased their virulence-enhancing capabilities in mice, further suggesting that extracellular lipopolysaccharide may play a role in these infections.     

Somatic antigens of the Brucella genus. The structure of the O-specific polysaccharide chain of Brucella melitensis lipopolysaccharide

The phenol-phase soluble antigenic lipopolysaccharide was isolated from Brucella melitensis, strain 565, by the routine phenol/water procedure followed by chromatography on Sepharose 4B. After mild acid hydrolysis and chromatography on Sephadex G-50, the lipopolysaccharide yielded a linear O-specific polysaccharide built up from 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The structure of the polysaccharide was deduced mainly from the nuclear magnetic resonance and methylation analyses. The phenol-soluble lipopolysaccharide, isolated from commercial vaccine strain B. abortus 19-BA, on mild hydrolysis afforded material, 13C and 1H-NMR spectra of which were identical to those of the O-specific polysaccharide from B. melitensis 565.     

Regions of the lipopolysaccharide of Pseudomonas aeruginosa essential for antitumor and interferon-inducing activities.

Resistance against ascites tumor development and interferon-inducing activity were demonstrated in lipopolysaccharide derived from the protein-lipopolysaccharide complex obtained from an autolysate of Pseudomonas aeruginosa. Lipid A obtained from the lipopolysaccharide was sufficient to induce interferon in vitro but no antitumor activity was found if lipid A or the polysaccharide derived from lipopolysaccharide was injected into the animal. Chemical modification of the polysaccharide portion or deacylation of the lipopolysaccharide also diminished antitumor activity. In contrast, interferon was induced by these incomplete lipopolysaccharides. These results indicate that both the lipid A portion and covalently linked polysaccharide are necessary for the inhibition of ascites tumor development, whereas incomplete lipid A with amide-linked fatty acids is sufficient to induce interferon in vitro.     

Somatic antigens of Shigella and Escherichia coli. Determination of the structure of O-specific polysaccharide from Shigella boydii type 12 and its serological properties compared with the O-specific polysaccharide from Escherichia coli

The structure of the O-specific polysaccharide of the somatic antigen (lipopolysaccharide) of Shigella boydii, type 12, was established by 1H- and 13C-NMR, methylation analysis and partial acid hydrolysis methods. The polysaccharide consists of pentasaccharide repeating units of the following structure: (formula; see text) The amount of O-acetyl groups was far less than stoichiometric, only about 2 for 3-4 repeating units. Nevertheless, the results of serological studies revealed 3-O-acetyl-alpha-L-rhamnose residue to be the major immunodominant group. In spite of the presence of similar trisaccharide fragments, the lipopolysaccharide and polysaccharide from Shigella boydii type 12 gave no crossreaction with lipopolysaccharide and polysaccharide from Escherichia coli 07. The possible reasons of the absence of serological relatedness between the Sh. boydii, type 12, and E. coli 07 cells were discussed.     

Ammonia enhanced dark respiration in Chlorella vulgaris is related to collapse of a transmembrane pH gradient

Abstract We obtained, by different methods, isogenic lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen) mutants from Klebsiella pneumoniae strains able to induce experimental infections (cytitis and pyelonephritis) in rats. We compared the induction of experimental infections in rats by wild-type strains and the lipopolysaccharide and capsular polysaccharide mutants. The high-molecular mass lipopolysaccharide of K. pneumoniae is clearly implicated in the infection process of the rat urinary tract, whilst the capsular polysaccharide seems not to be involved to the same extent.     

Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the polysaccharide chain of Ps. aeruginosa O-serogroup 7 (Lanyi) lipopolysacharide

A loosely bound lipopolysaccharide-protein complex was extracted from cells of Pseudomonas aeruginosa strain 170015 (O:7ab; Lanyi classification) by saline solution and purified from contaminant nucleic acid by Cetavlon treatment followed by precipitation in an ultracentrifuge. The saline-treated cells were re-extracted with hot aqueous phenol to give firmly bound lipopolysaccharide which was isolated from the phenol layer and purified by ultracentrifugaiton. The identity of both lipopolysaccharide preparations was proved by serological and chemical evidence. Mild acid degradation of the lipopolysaccharide resulted in the splitting off of a lipid component and led to polysaccharide which was purified by gel-filtration on a Sephadex G-50 column. The polysaccharide consisted of N-acetyl-D-fucosamine, N-acetyl-L-fucosamine and D-glucose in the ratio 1:1:1. On the basis of nuclear magnetic resonance spectra, results of methylation analysis and two sequential Smith degradations, the following structure can be assigned to the repeating unit of the polysaccharide: -3)LFucNAc(alpha 1-3)DFucNAc(beta 1-2)DGlc(beta 1-. The polysaccharide did not show serological activity whereas alkali-treated lipopolysaccharide readily sensitised sheep erythrocytes and inhibited the passive haemagglutination reaction with anti-(O:7a,b)serum. Evidence is presented that the oligosaccharide repeating units of the polysaccharide and alkali-treated lipopolysaccharide are indistinguishable. Ps. aeruginosa strain 170016 (O:7a,c) was shown to have the O-specific lipopolysaccharide identical with that from strain 170015. The presented data show that subfactors 7b and 7c in the Lanyi classification of Ps. aeruginosa O-antigens seem to relate to components of the bacterial surface other than lipopolysaccharides.     

Antigenic bacterial polysaccharides. 14. Structure of the O-specific polysaccharide chain of a lipopolysaccharide from Pseudomonas aeruginosa (Lányi)

The lipopolysaccharide from Pseudomonas aeruginosa O12 (Lányi classification) gave on mild acid hydrolysis an O-specific polysaccharide built of D-ribose and N-acetyl-D-galactosamine. The disaccharide structure----4)-alpha-GalNAcp-(1----2)-beta-Ribf-(1----for the repeating unit of the polysaccharide was established by nondestructive way involving full interpretation of its 1H- and 13C-NMR-spectra, using homonuclear and selective heteronuclear 13C[1H] double resonances.     

Interferon Production in Mice by Cell Wall Mutants of Salmonella typhimurium

The interferon response elicited by Salmonella typhimurium mutants in mice is not dependent on the presence of a complete cell wall lipopolysaccharide. In fact, a mutant (G30/C21) which has lost all the polysaccharide side chains and sugars of the O antigen and contains only 2-keto-3-deoxyoctonate and lipid is indistinguishable in its interferon-stimulating ability from the wild type which possesses a complete O antigen with polysaccharide side chains.     

NMR and MS evidences for a random assembled O-specific chain structure in the LPS of the bacterium Xanthomonas campestris pv. Vitians. A case of unsystematic biosynthetic polymerization.

Xanthomonas campestris pv. vitians is a Gram-negative plant-associated bacterium that acts as causative agent of bacterial leaf spot and headrot in lettuce. The lipopolysaccharide of this bacterium is suspected to be an important molecule for adhesion to and infection of the plants. The lipopolysaccharide has been isolated from the phenol phase and the O-specific chain characterized by compositional analysis, high field NMR and MALDI-TOF MS. It consists of a nonrepetitive branched polysaccharide with a rhamnan backbone to which Fuc3NAc is linked. The NMR and MS approach led to the characterization of the fine structure of the polymer, which is randomly assembled. The rhamnan backbone is built up of beta-Rhap and alpha-Rhap, this last is present in one, two or three adjacent units and branched by an alpha-Fucp3NAc unit. This is a real case of a random constituted O-specific chain, therefore biosynthetic studies towards the comprehension of this irregular biosynthesis are needed.     

Studies of the polysaccharide fraction from the lipopolysaccharide of Pseudomonas alcaligenes

Studies of the lipopolysaccharide of Pseudomonas alcaligenes strain BR 1/2 were extended to the polysaccharide moiety. The crude polysaccharide, obtained by mild acid hydrolysis of the lipopolysaccharide, was fractionated by gel filtration. The major fraction was the phosphorylated polysaccharide, for which the approximate proportions of residues were; glucose (2), rhamnose (0.7), heptose (2-3), galactosamine (1), alanine (1), 3-deoxy-2-octulonic acid (1), phosphorus (5-6). The heptose was l-glycero-d-manno-heptose. The minor fractions from gel filtration contained free 3-deoxy-2-octulonic acid, P(i) and PP(i). The purified polysaccharide was studied by periodate oxidation, methylation analysis, partial hydrolysis, and dephosphorylation. All the rhamnose and part of the glucose and heptose occur as non-reducing terminal residues. Other glucose residues are 3-substituted, and most heptose residues are esterified with condensed phosphate residues, possibly in the C-4 position. Free heptose and a heptosylglucose were isolated from a partial hydrolysate of the polysaccharide. The location of galactosamine in the polysaccharide was not established, but either the C-3 or C-4 position appears to be substituted and a linkage to alanine was indicated. In its composition, the polysaccharide from Ps. alcaligenes resembles core polysaccharides from other pseudomonads: no possible side-chain polysaccharide was detected.