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PubMed1985

Energy dependence of lipopolysaccharide translocation in Salmonella typhimurium.

Energy inhibitors block translocation of pulse-labeled core lipopolysaccharide to outer membrane under conditions which allow maintenance of constant specific radioactivity of intracellular precursor pools throughout the chase period. Under the conditions used, approximately 75% of the total cellular label was membrane-bound at initiation of chase. Translocation of core lipopolysaccharide from inner to outer membrane showed apparent first order kinetics (t1/2 = 1.2 min, 32 degrees C). Translocation was blocked by arsenate (5-10 mM) under conditions where proton motive force was unchanged, while the uncouplers 2,4-dinitrophenol (0.1 mM to 0.8 mM) and carbonyl cyanide-m-chlorophenyl hydrazone (12-30 microM) inhibited translocation with no apparent effect on the ATP pool. Therefore, core lipopolysaccharide translocation appears to require maintenance of both proton motive force and high energy phosphate pools. Electron microscopic experiments show no gross disruption of zones of adhesion, the putative sites of lipopolysaccharide translocation, in the presence of arsenate or 2,4-dinitrophenol suggesting that energy is not required simply for maintenance of these structures.

2,4-Dinitrophenol↗

PubMed1982

Role of phosphatidylethanolamine in the biosynthesis of pyrophosphoethanolamine residues in the lipopolysaccharide of Escherichia coli.

The biosynthesis of pyrophosphoethanolamine residues linked to the core oligosaccharide region of the lipopolysaccharide of Escherichia coli K2 strain BB 26-36 has been investigated by means of isotope tracer experiments in living cells. Phosphoethanolamine was isolated from the pyrophosphoethanolamine residues after hydrolysis in 1 N HCl at 100 degrees C. The kinetics of labeling of the phosphoethanolamine from [3H]serine or sn-glycero-3-32P during pulse-chase experiments revealed that the biosynthetic precursor of the phosphoethanolamine must be a large, relatively stable pool, and not a small, rapidly metabolized pool such as that of free serine, or seryl-tRNA. Labeling of the pyrophosphoethanolamine residues of the lipopolysaccharide from the two isotopes was closely parallel, and the isotope ratio 3H/32P was closely similar to that in phosphatidylethanolamine at the same time intervals. These experiments offer strong evidence that phosphatidylethanolamine functions in the biosynthesis of pyrophosphoethanolamine residues in lipopolysaccharide in a reaction in which the phosphoethanolamine head-group of the phospholipid is transferred as a unit to a lipopolysaccharide acceptor.

Escherichia coli↗

PubMedDate not supplied

Purification of gonococcal R-type lipopolysaccharide not bearing the lactose-like antigen.

The gonococcal strain R-10 is shown to contain two antigenically different R-type lipopolysaccharides: one of these has the same properties described for the gonococcal lipopolysaccharide, while the other does not have any cross-reaction with it. The latter was purified from a mutated colony able to express only one type of lipopolysaccharide. By inhibition enzyme-linked immunosorbent assay it was shown that the difference between the two lipopolysaccharides is located in the core region which, when purified and analyzed by gel filtration and gas liquid chromatography, did not show any difference either in size or in chemical composition, leading to the conclusion that the two cores must differ just in the structure of the antigenic determinant.

Animals↗

PubMed1981

Phosphate localization in carbohydrates - a study on enterobacterial lipopolysaccharides.

The localization of the phosphate substituents in the core oligosaccharide of the lipopolysaccharides of Enterobacteriaceae has been reported for Salmonella minnesota and Escherichia coli B only. In these cases the localizations were done by a beta-elimination reaction in mild alkaline solution after periodate oxidation. We report now on a method generally applicable on carbohydrates. The localization of phosphate groups and the extent of substitution with phosphate residues in carbohydrates can be determined by the following reaction sequence: methylation, dephosphorylation, and reetherification (labelling) with C2H3J or C2H5J followed by derivatizing to partially methylated alditol acetates and analysis by combined gas liquid chromatography/mass spectrometry. The results presented here are obtained by application of this method to isolated core oligosaccharides of lipopolysaccharides from E. coli C23.1, E. coli C71, E. coli F2515, and P. mirabilis R4/O 28. Phosphate is localized at C-4 of the chain heptoses in the lipopolysaccharides of E. coli C and E. coli R4, and at C-7 of the branching heptose in the lipopolysaccharide of P. mirabilis R4/O 28.

Chemical Phenomena↗

PubMed1981

[Further characterization of lipopolysaccharides from Pasteurella multocida].

The biological properties, sedimentation behaviour, and electron-optically visualised structures of lipopolysaccharides from Pasteurella multocida were found to be identical with lipopolysaccharides from Enterobacteriaceae. Hard evidence to the effect that lipopolysaccharides from Pasteurella multocida are R-lipopolysaccharides were produced by the composition of monosaccharides together with experiments on the basis of disc electrophoresis.

Animals↗

PubMed1975

[Chemical and serological characterization of Salmonella lipopolysaccharides from different phases of growth (author's transl)].

Chemical and serological investigations were carried out on lipopolysaccharides of 4 Salmonella S-forms and of 1 SR-mutant, extracted from bacteria at different ages of culture (early exponential to stationary growth phase). The results show that the fatty acid composition of Lipid A (lauric-, myristic-, palmitic-, and beta-hydroxy-myristic acids) does not undergo any significant change during the growth of the cultures. However, there are differences in the molar ratios of the fatty acids from strain to strain. In all phases of growth Lipid A is substituted by basaloligosaccharide, to the same extent, as can be seen from the constant ratios of beta-hydroxy-myristic acid: heptose. Serological experiments (haemagglutination inhibition tests, absorption of antibodies by LPS-coated erythrocytes) showed that in no case the basaloligosaccharide is completely substituted by O-specific chains and that basaloligosaccharide exhibits free R-antigen structures which are mainly of chemotypes Ra, Rb and Rc, for the SR-mutant only of types Ra and Rb. There is no demonstrable dependence upon the phases of growth. In the O-specific polysaccharide chains the sugars of the main chain and the side bound dideoxy sugars (abequose and tyvelose) show a constant 1:1 molar ratio in all phases. In the case of S. typhimurium, antigen factors 1, 4 and 12(2), the biosynthesis of which is controlled by modifying oaf genes and/or by a lysogenic phage, are of a somewhat weaker expression in the exponential phase than in the latter phases of growth. In the SR-mutant, lipopolysaccarides with (low) serological O1 and O12(2) activity are only extractable by the phenol/water method, but not by the PCP method. In three out of four S-forms, changes occur in the length of the O-specific polysaccharide chains, whereas the number of repeating units of the fourth strain remains almost unchanged. The lipopolysaccharides of the SR-mutant contain in all phases of growth about one repeating unit. In all strains the covering of the cell surface by lipopolysaccharide molecules changes during the course of growth, as can be seen by comparing the relative cell surface and the content of Lipid A fatty acids of the bacteria. Lipid A synthesis in the 4 S-forms is reduced in the exponential phase and/or in the phase of delayed growth acceleration. The extent of biosynthesis of the carbohydrate moiety of lipopolysaccharides is independent of that of Lipoid A. In the SR-mutant, Lipoid A and Polysaccharide are formed in increased amounts in the exponential growth phase.

Cell Wall↗

PubMed1993

Prostaglandin E2 mediates lipopolysaccharide-induced macrophage procoagulant activity by a cyclic adenosine monophosphate-dependent pathway.

BACKGROUND: Multiple organ failure syndrome (MOFS) and adult respiratory distress syndrome (ARDS) continue to be significant clinical problems. Microvascular thrombosis and intraalveolar fibrin deposition play an integral role in the pathogenesis of MOFS and ARDS. The macrophage participates in these processes by expressing procoagulant activity (PCA) after exposure to endotoxin. One potential method to ameliorate organ dysfunction in ARDS and MOFS is to prevent macrophage activation of the coagulation cascade. Because inhibitors of arachidonic acid metabolism attenuate inflammatory lung injury, we investigated the role of eicosanoids in endotoxin-induced alveolar macrophage PCA. METHODS: Rabbit alveolar macrophages were incubated with selective inhibitors of arachidonic acid metabolism. PCA was determined in cell lysates. PCA was also assessed in cultures treated with cyclooxygenase inhibitor that had exogenous prostaglandin E2 (PGE2) added. Intracellular cyclic adenosine monophosphate (cAMP) was examined after treatment with lipopolysaccharide, ibuprofen, PGE2, and forskolin. RESULTS: Ibuprofen significantly reduces lipopolysaccharide-stimulated PCA by alveolar macrophages. 5-Lipoxygenase and thromboxane synthetase inhibitors had no effect on PCA. Inhibition of PCA by ibuprofen is reversed by adding exogenous PGE2. Decreased intracellular cAMP is associated with attenuated lipopolysaccharide-stimulated PCA elaboration. CONCLUSIONS: Endotoxin-stimulated alveolar macrophage PCA is prostanoid dependent, with cAMP acting as a second messenger. Although expression of PCA is prostanoid-cAMP dependent, neither prostanoids nor agents that directly increase cAMP are sufficient to elicit PCA in the absence of lipopolysaccharide.

1-Methyl-3-isobutylxanthine↗

PubMedDate not supplied

[Characteristics of Pseudomonas fluorescens lipopolysaccharide].

A lipopolysaccharide was isolated from Pseudomonas fluorescens IMV 1152 (biovar I). The fractions of the structural moieties of lipopolysaccharide macromolecule were extracted and studied separately. 3-hydroxydecanoic, 3-hydroxydodecanoic and 2-hydroxydodecanoic fatty acids were identified in lipid A, total quantity of those was more than 80%. In acid hydrolysate of lipid A the glucosamine and phosphoethanolamine were found. The rhamnose, arabinose, glucose, glucosamine and phosphorus were identified as the components of core oligosaccharide fractions. A trisaccharide repeating unit of the O-cpecific polysaccharide chain consists of the residues of aminosugars: 4-acet-amido-4,6-dideoxy-D-galactose, 2-acetamido-2,6-dideoxy-D-glucose and 2-acetamido-2,6-dideoxy-L-glucose. The studied lipopolysaccharide resembled the structure of lipid A and core oligosaccharide with lipopolysaccharide from type strain of Pseudomonas fluorescens IMV 4125 (ATCC 13525), whereas their O-chains were different.

Carbohydrate Conformation↗

PubMed1994

Distribution of lipid A species between long and short chain lipopolysaccharides isolated from Salmonella, Yersinia, and Escherichia as seen by 252Cf plasma desorption mass spectrometry.

Smooth type endotoxins of Salmonella, Yersinia, and Escherichia were fractionated into long and short chain lipopolysaccharides by silica gel chromatography. Lipid A was prepared from the fractions and analyzed by plasma desorption mass spectrometry. Both Yersinia and Salmonella endotoxins had a large proportion of aminoarabinose-containing lipopolysaccharide molecular species that were found to be concentrated in the long chain fraction. In the Escherichia endotoxin, hypoacylated lipopolysaccharides (lacking the tetradecanoate and one of the four hydroxytetradecanoates) were found mostly in the short chain fraction. Possible implications of these results for the lipopolysaccharide biosynthetic pathway and for studies on the influence of sugar chain length on the biological effects of endotoxins are discussed.

Californium↗

PubMed1994

Cell-bound albumin is the 70-kDa peptidoglycan-, lipopolysaccharide-, and lipoteichoic acid-binding protein on lymphocytes and macrophages.

The 70-kDa protein that is present on the surface of lymphocytes and macrophages and that binds peptidoglycan, lipopolysaccharide, lipoteichoic acid, heparin, and sulfated heparinoids was identified as cell-bound albumin. It originated from the tissue culture medium or from the serum in vivo and was not produced by the cells. The following results supported this conclusion: (a) mouse and human cell lines grown in serum-free and albumin-free medium did not have this 70-kDa protein and did not bind peptidoglycan and lipopolysaccharide in photoaffinity cross-linking assay; (b) the appearance of the 70-kDa protein in these cells and the ligand binding were restored by 30-min incubation with serum or purified albumin; (c) soluble albumin bound peptidoglycan and lipopolysaccharide, and this binding was inhibited by the same competitive inhibitors that inhibited the binding to the 70-kDa cellular protein; (d) albumin co-migrated with the 70-kDa cellular protein in two-dimensional polyacrylamide gel electrophoresis; (e) peptide maps of albumin and the 70-kDa cellular protein digested with four proteases were identical; (f) the only protein recognized by anti-albumin monoclonal and polyclonal antibodies on Western immunoblots was the 70-kDa protein that exactly matched with the cellular peptidoglycan/lipopolysaccharide-binding protein; (g) anti-albumin monoclonal antibodies immunoprecipitated the 70-kDa cellular protein; and (h) species-specific anti-bovine, anti-human, and anti-mouse albumin antibodies recognized the 70-kDa protein on mouse and human cells according to the species of albumin that was present in the culture medium or in the serum in vivo, but not according to the species of the cells. The cell-bound albumin was not required for cell activation, because macrophage cell lines that were grown in albumin-free medium and did not have the cell-bound albumin (the 70-kDa protein) fully responded to the stimulation by peptidoglycan and lipopolysaccharide with production of tumor necrosis factor-alpha.

Affinity Labels↗

PubMed1994

Structural studies of the cell envelope lipopolysaccharides from Haemophilus ducreyi strains ITM 2665 and ITM 4747.

The structures of the lipopolysaccharides from Haemophilus ducreyi strains ITM 2665 and ITM 4747 have been investigated. Oligosaccharides were obtained from phenol/water-extracted lipopolysaccharide by mild acid hydrolysis and were studied with methylation analysis, fast atom bombardment-mass spectrometry, and NMR spectroscopy. The major oligosaccharide obtained from strain 2665 is a nonasaccharide with the following structure: beta-D-Galp-1-->4-beta-D-GlcNAcp-1-->3-beta-D-Galp-1-->4-D-alpha-D -Hepp- 1-->6-beta-D-Glcp-1-->(L-alpha-D-Hepp-1-->2-L-alpha-D-Hepp-1 -->3)-4-L-alpha- D-Hepp-Kdo, where the reducing terminal 3-deoxy-D-manno-octulosonic acid (or Kdo) exists in reduced anhydro forms. The proposed structure complements the preliminary structure described for Haemophilus ducreyi strain 35000 (Melaugh, W., Phillips, N. J., Campagnari, A. A., Karalus, R., and Gibson, B. W. (1992) J. Biol. Chem. 267, 13434-13439) with the missing anomeric configurations. The saccharide isolated from strain 4747 is a markedly simpler hexasaccharide with the following structure: beta-D-Galp-1-->4-beta-D-Glcp-1-->(L-alpha-D-Hepp-1-->2-L-alpha-D- Hepp- 1-->3)4-L-alpha-D-Hepp-Kdo. Apart from a different phosphorylation of the inner core region the proposed structure is identical to the structure of lipopolysaccharide from an only distantly related bacterium, viz. Haemophilus influenzae nontypable strain 2019 (Phillips, N. J., Apicella, M. A., Griffiss, J. M., and Gibson, B.W. (1992) Biochemistry 31, 4515-4526). The implications of these findings as regards the role of lipopolysaccharide as a virulence factor are discussed.

Carbohydrate Conformation↗

PubMed1994

Lipopolysaccharide-neutralizing antibody reduces hepatocyte injury from acute hepatotoxin administration.

Endogenous lipopolysaccharide has been implicated as a cofactor in the hepatocellular injury and death resulting from toxic liver injury. To prevent this lipopolysaccharide-induced injury and to further understand the mechanism of this effect, an anti-lipopolysaccharide antibody was administered to rats in which toxic hepatocellular injury was induced. Rats were given the hepatotoxin galactosamine together with an isotypic control antibody B55 or the anti-lipopolysaccharide antibody E5. E5 treatment resulted in reductions of serum AST levels of 43% at 36 hr (p < 0.02) and 60% at 48 hr (NS) after galactosamine administration. These decreases in AST values were accompanied by diminished histological evidence of injury and inflammation. In carbon tetrachloride-induced liver injury, E5 similarly reduced serum AST levels at 36 and 48 hr by 47% (p < 0.04) and 54% (p < 0.03), respectively. E5 treatment was equally effective in reducing AST levels 48 hr after administration of carbon tetrachloride, whether the initial dose of antibody was given 1 hr before or 3 or 6 hr after the administration of this toxin. To understand the mechanism of this E5 effect, the activation of the toxic cytokine tumor necrosis factor-alpha and the chemotactic cytokine monocyte chemoattractant protein 1 was examined by Northern-blot analysis of RNA from rat livers after galactosamine-induced injury and treatment with B55 or E5. Despite E5's efficacy in reducing hepatocellular damage, E5 treatment did not affect the timing or magnitude of tumor necrosis factor-alpha or monocyte chemoattractant protein 1 activation during galactosamine-induced injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1978

Serologic examination of the Westphal-type lipopolysaccharides of Pasteurella multocida.

The serologic specificities of the Westphal-type lipopolysaccharides from 16 serotypes of Pasteurella multocida were compared with those reported for the heat-stable typing antigens in the gel diffusion precipitin test. Like the heat-stable typing antigen, the Westphal-type lipopolysaccharide from each of the serotypes reacted only with its homologous antiserum in 14 of the 16 serotypes. The lipopolysaccharide from type strains representing serotypes 2 and 5 reacted with either of the corresponding typing sera, as did field isolates of these serotypes to a lesser extent. Although differences among the properties of these antigens were minor, the lipopolysaccharide appeared to be a major component of the heat-stable antigen responsible for the type specificity.

Cross Reactions↗

PubMed1999

Interaction of peptides and proteins with bacterial surface glycolipids: a comparison of glycosphingolipids and lipopolysaccharides.

The bacterial cell wall of Gram-negative bacteria consists, in addition to the cytoplasmic membrane, of another permeability barrier, the outer membrane. The lipid distribution between both sides of this membrane is strictly asymmetric. The outer leaflet is made up of glycolipids, usually lipopolysaccharides. In Sphingomonas spp glycosphingolipids were found to substitute for lipopolysaccharides. In this review, it is shown by an electrophysiological approach that glycosphingolipid can replace lipopolysaccharide with respect to its function as antigenic surface structure as well as to its contribution to the diffusion barrier properties of the outer membrane. This review is focused on: (i) the function of porins, as examples of transmembrane proteins, in the different glycolipid environments; (ii) the interaction of polymyxin B with the outer membrane, as an example of polycationic antibacterial peptides; and (iii) the activation of the human complement system by lipopolysaccharides and glycosphingolipids.

Journal Article↗

PubMed1970

The purification and chemical composition of the lipopolysaccharide of Pseudomonas alcaligenes.

1. A method for obtaining lipopolysaccharide free from glycosaminopeptide from isolated cell walls of Pseudomonas alcaligenes is discussed. 2. About 70-75% of the lipopolysaccharide and 86-90% of the isolated lipid A have been accounted for in terms of identifiable components. 3. Hydrolysates of lipid A contain mainly inorganic phosphate, glucosamine, O-phosphorylglucosamine and fatty acids (dodecanoic acid, dodec-2-enoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid), of which the last is the main N-acylating acid of the glucosamine backbone. 4. Material corresponding to the polysaccharide moiety of the lipopolysaccharide is extensively degraded. 5. Solubilization of the lipopolysaccharide by using sodium deoxycholate appreciably affects the chemical composition of the material.

Alcaligenes↗

PubMed1998

The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides.

The major core oligosaccharide biosynthesis operons from prototype Escherichia coli strains displaying R1 and R4 lipopolysaccharide core types were polymerase chain reaction-amplified and analyzed. Comparison of deduced products of the open reading frames between the two regions indicate that all but two share total similarities of 94% or greater. Core oligosaccharide structures resulting from nonpolar insertion mutations in each gene of the core OS biosynthesis operon in the R1 strain allowed assignment of all of the glycosyltransferase enzymes required for outer core assembly. The difference between the R1 and R4 core oligosaccharides results from the specificity of the WaaV protein (a beta1, 3-glucosyltransferase) in R1 and WaaX (a beta1, 4-galactosyltransferase) in R4. Complementation of the waaV mutant of the R1 prototype strain with the waaX gene of the R4 strain converted the core oligosaccharide from an R1- to an R4-type lipopolysaccharide core molecule. Aside from generating core oligosaccharide specificity, the unique beta-linked glucopyranosyl residue of the R1 core plays a crucial role in organization of the lipopolysaccharide. This residue provides a novel attachment site for lipid A-core-linked polysaccharides and distinguishes the R1-type LPS from existing models for enterobacterial lipopolysaccharides.

Amino Acid Sequence↗

PubMed1994

Chemical structure of the lipid A component of lipopolysaccharides of the genus Pectinatus.

The chemical structure of the lipid A components of smooth-type lipopolysaccharides isolated from the type strains of strictly anaerobic beer-spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were analyzed. The hydrophilic backbone of lipid A was shown, by controlled degradation of lipopolysaccharide combined with chemical assays and 31P-NMR spectroscopy, to consist of the common beta 1-6-linked disaccharide of pyranosidic 2-deoxy-glucosamine (GlcN), phosphorylated at the glycosidic position and at position 4'. In de-O-acylated lipopolysaccharide, the latter phosphate was shown to be quantitatively substituted with 4-amino-4-deoxyarabinose, whereas the glycosidically linked phosphate was present as a monoester. Laser-desorption mass spectrometry of free dephosphorylated lipid A revealed that the distal (non-reducing) GlcN was substituted at positions 2' and 3' with (R)-3-(undecanoyloxy)tridecanoic acid, whereas the reducing GlcN carried two unsubstituted (R)-3-hydroxytetradecanoic acids at positions 2 and 3. The lipid A of both Pectinatus species were thus of the asymmetric hexaacyl type. The linkage of lipid A to polysaccharide in the lipopolysaccharide was relatively resistant to acid-catalyzed hydrolysis, enabling the preparation of a dephosphorylated and deacylated saccharide backbone. Methylation analysis of the backbone revealed that position 6' of the distal GlcN of lipid A was the attachment site of the polysaccharide. Despite the quantitative substitution of the lipid A 4'-phosphate by 4-amino-4-deoxyarabinose, which theoretically should render the bacteria resistant to polymyxin, P. cerevisiiphilus was shown to be susceptible to this antibiotic. P. cerevisiiphilus was, however, also susceptibile to vancomycin and bacitracin, indicating that the outer membrane of this bacterium does not act as an effective permeability barrier.

Amino Sugars↗

PubMedDate not supplied

[The use of liposomes for detection of the surface lipopolysaccharide antigen, Vibrio cholerae cells, and antibodies against them].

A test system for determination of Vibrio cholerae cells, surface O-antigen, and antibodies against them was developed on the basis of complement-dependent lysis of liposomes sensitized by the lipopolysaccharide-dependent antigen from Vibrio cholerae 569B. The factors that affect the function of the liposomal reagent were studied, and the conditions for detecting antibodies and antigenic material were optimized. This system is highly specific and sensitive to be used for the determination of anticholeraic antibodies (30-50 times as effective as agglutination tests), lipopolysaccharide antigen (100 ng/ml, which corresponded to 3.0 ng of lipopolysaccharide in the sample studied), and Vibrio cholerae cells (3.3 x 10(7) m.b./ml, which corresponded to 10(6) m.b. in sample). It takes 30-40 min to detect the lipopolysaccharide antigen and 90 min to detect V. cholerae cells.

Antibodies, Bacterial↗