Search

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.

A pentaacyl precursor of lipid A biosynthesis, termed precursor Ib, and a structural isomer have been chemically synthesized. These compounds were, in comparison to synthetic Escherichia-coli type lipid A or lipopolysaccharide, analyzed for their activity in typical endotoxin test systems. It was found that both precursor I b and the isomer exhibited similar or only slightly lower pyrogenic, lethal and Shwartzman-phenomenon-inducing activity than lipid A. All preparations were comparable in their B-lymphocyte mitogenicity, macrophage- activating capacity and immunoreactivity towards lipid A antisera. The proton nuclear magnetic resonance spectra of the 1-dephospho derivative of synthetic and bacterial precursor Ib were indistinguishable proving that the previously proposed structure for precursor Ib is correct. [ABSTRACT FROM AUTHOR]

After mild acid hydrolysis, a disaccharide of 3-deoxy-D-manno-octulosonic acid (dOclA) was obtained from Re-mutant lipopolysaccharide of Salmonella minnesota, Salmonella godesberg, Proteus mirabilis, and Escherichia coli, and from the lipopolysaccharide of an S. minnesota Rb2 mutant. Combined gas-liquid chromatography/ mass spectrometry of the reduced and permethylated derivatives indicated that the disaccharide is interlinked by a 2→4-glycosidic bond in all lipopolysaccharides tested. In addition, it was shown by gas-liquid chromatography of appropriate synthetic standards and a previously characterized α2→4-linked dOclA disaccharide (derived from lipopolysaccharide of S. godesberg) that the non-reducing dOclA residue possesses the α configuration. In the case of lipopolysaccharide of S. minnesota Rb2 mutant, this result, together with earlier findings, suggests that it contains a linear dOclA trisaccharide of the sequence doclA(α2-4)dOclA(α2-4)dOclA. The results show that a dOclA(α2-4)dOclA disaccharide represents a common architectural principle in enterobacterial lipopolysaccharides. [ABSTRACT FROM AUTHOR]

The absolute configuration of 3-hydroxy fatty acids has been studied, which are present in the lipopolysaccharides of the following bacteria: Rhodopseudomonas gelatinosa, Rh. virdis. Rhodospirillure tenue, Chromobacterium riolaceum, Pseudomonas aeruginosa. Bordetella pertussis, Vibrio metchnikovii, Vibrio cholerae, Sathnonella spp., Escherichia coli, Shigella flexneri, Proteus mirabilis, Yersinia enterocolitica and Fusobacterium nucleatum. The 3-hydroxy acids were liberated by strong alkaline hydrolysis, converted to 3-methoxy acid L-phenylethylamides and analyzed by gas-liquid chromatography. With the aid of authentic D-3-hydroxy fatty acids it was shown for all lipopolysaccharides that the 3-hydroxy acids, regardless of chain lengths, branching. 3-O-substitution or type of linkage, possess the D-configuration. 2-Hydroxydodecanoic acid, which is present in some lipopolysaccharides, was analyzed in an analogous way and shown to possess the L-configuration. [ABSTRACT FROM AUTHOR]

A degradation procedure of lipopolysaccharides was worked out which allows the isolation of the reduced backbone of lipid A in a total yield of between 20 and 30%. This procedure was applied to lipopolysaccharides of S forms (Salmonella minnesota, Shigellaflexneri 5b, Escherichia coli 086, E. coli 0111, Xanthomonas sinensis, Rhodopseudomonas gelatinosa) and R mutants (Salmonella minnesota, Shigella flexneri 5b, E. coli BB9 and E. coli EH 100). Chemical analysis, reaction with β-N-acetyl-glucosaminidase and application of methylation analysis revealed that the lipid A backbone of all strains contains β1′,6-1inked glucosamine disaccharides carrying two phosphate groups, one in glycosidic and one in ester linkage, a structure, identified previously in the Salmonella minnesota Re mutant. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gramnegative bacteria, may assume various three-dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small-angle X-ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia-coli-type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (> 90%) at different temperatures (20, 37, and 55 °C) and at different lipid/divalent cation molar ratios (20:1 to 1:1). We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20°C, i.e. below the β↔α acyl-chain-melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55 °C), 4.60 nm for Re lipopolysaccharide (at 37 °C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37°C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20°C. At higher concentrations of divalent cations (e. g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1:√2, 1:√3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides of different wild-type and mutant gram-negative bacteria, as well as synthetic and bacterial free lipid A, were studied for their ability to activate arachidonic acid metabolism in mouse peritoneal macrophages in vitro. It was found that lipopolysaccharides of deep-rough mutants of Salmonella minnesota and Escherichia coli (Re to Rc chemotypes) stimulated macrophages to release significant amounts of leukotriene C4 (LTC4) and prostaglandin E2 (PGE2). Lipopolysaccharides of wild-type strains (S. abortus equi, S. friedenau) only induced PGE2 and not LTC4 formation. Unexpectedly, free bacterial and synthetic E. coli lipid A were only weak inducers of LTC4 and PGE2 production. Deacylated Re-mutant lipopolysaccharide preparations were inactive. However, co-incubation of macrophages with both deacylated lipopolysaccharide and lipid A lead to the release of significant amounts of LTC4 and PGE2, similar to those obtained with Re-mutant lipopolysaccharide. The significance of the lipid A portion of lipopolysaccharide for the induction of LTC4 was indicated by demonstrating that peritoneal macrophages of endotoxin-low-responder mice or of mice rendered tolerant to endotoxin did not respond with the release of arachidonic acid metabolites on stimulation with Re-mutant lipopolysaccharide and that polymyxin B prevented the Re-lipopolysaccharide-induced LTC4 and PGE2 release. Physical measurements showed that the phase-transition temperatures of both free lipid A and S-form lipopolysaccharide were above 37 °C while those of R-mutant lipopolysaccharides were significantly lower (30-35 °C). Thus, with the materials investigated, an inverse relationship between the phase-transition temperature and the capacity to elicit LTC4 production was revealed. [ABSTRACT FROM AUTHOR]

The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests. [ABSTRACT FROM AUTHOR]

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12°C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetradecanoic acid at GlcN II is positionally replaced by Δ9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to Glecn II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to Glen I are free or only partially substituted. [ABSTRACT FROM AUTHOR]

The adsorption constant of the irreversible adsorption of the bacteriophage K3 to Escherichia coli K12 bacteria is strongly dependent on the incubation temperature. Two inflection points are observed in an Arrhenius plot. For cells grown at 37°C the inflection points are found at 20°C and 28°C whereas these inflection points shift to 10°C and 19°C for cells grown at 12°C. To study the lipid environment of the receptor the temperature dependence of the inactivation of bacteriophage K3 was measured in vitro in the presence of various lipids. The Arrhenius plots of the rate of inactivation of phage K3 by complexes of protein d and lipopolysaccharide are very similar to those observed for whole cells. With lipopolysaccharide isolated from cells grown at 37°C inflection points are observed at 20°C and 28°C. With lipopolysaccharide from cells grown at 12°C the inflection points are found at 10°C and 21°C. These results show that the environment of protein d in vivo can be mimicked perfectly in vitro by protein d/lipopolysaccharide complexes. The fatty acid composition of lipopolysaccharide isolated from cells grown at 37°C and at 12°C differs in that in the latter case the amounts of mono-unsaturated fatty acids (mainly palmitoleic acid) are increased at the expense of lauric acid. This difference in fatty acid composition probably explains the difference in the phase transition temperatures caused by the two lipopolysaccharide preparations. A transition at the inflection point of the highest temperature is also found for lipopolysaccharide using light-scattering measurements and appears to be a thermal transition, since it is also observed in differential scanning calorimetry. [ABSTRACT FROM AUTHOR]

A number of lipopolysaccharides derived from Salmonella and Escherichia coli S and R mutant strains were tested for toxicity and anticomplementary activity in the absence of added antiserum. Although all preparations were toxic, only a few exhibited high anticomplementary activity, while others proved to be of low or negligible activity. It was found that isolated lipid A from both active and reactive lipopolysaccharides was strongly anticomplementary as well as toxic, when made water-soluble with the aid of suitable carriers. Treatment of R form lipopolysaccharide with Mg2+ or Ca2+ led to complete precipitation of the lipopolysaccharide with consequent loss of toxicity and anticomplementary activity. This treatment had practically no effect on the anticomplementary activity and toxicity of S form lipopolysaccharides. When lipopolysaccharide, after reaction with complement, was reisolated and purified, the resulting preparation was found to be non-toxic and of negligible anticomplementary activity. No detectable alterations in either the sugar or the fatty acid composition could be detected. The only significant change was the loss of solubility in water. Treatment of the reisolated lipopolysaccharide with EDTA completely restored solubility m water, toxicity, and anti-complementary activity. [ABSTRACT FROM AUTHOR]

Lipid A preparations from the lipopolysaccharides of four Salmonella minnesota R mutants and one Escherichia coli 0100 R mutant were assayed as soluble complexes with bovine serum albumin for lethality in mice, pyrogenicity in rabbis and complement inactivation. Although generally less active than endotoxic lipopolysaceharides, these lipid · albumin complexes nevertheless exhibited strong biological activity. These results indicate that lipid A contains the endotoxic principle of bacterial lipopolysaccharides. [ABSTRACT FROM AUTHOR]

Abstract: The complex formation of lipopolysaccharide (LPS) with chitosan (Ch) was demonstrated using sedimentation velocity analysis in the analytical ultracentrifuge, centrifugation in glycerol gradient and isopicnic centrifugation in cesium chloride. An addition of Ch to the Escherichia coli and Yersinia pseudotuberculosis LPS solutions was found to result in formation of the stable LPS–Ch complexes. The interaction is a complicated process and depends on time and reaction temperature, as well as on the molecular weight of chitosan. A stable LPS–Ch complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 °C). It should be noted that process of LPS complexation with Ch is accompanied by additional dissociating of LPS. The complex formation was shown to be a result not only of ionic binding, but also of other types of interactions. The interaction of Ch with LPS was shown to modulate significantly the biological activity of LPS. The LPS–Ch complex (1:5 w/w) was shown to possess much lower toxicity in a comparison with the parent LPS at injection to mice in the similar concentration. The LPS–Ch complex was shown to maintain an ability to induce of IL-8 and TNF, but induction of IL-8 and TNF biosynthesis by the LPS–Ch complex was lower than that by the parent LPS. The complex LPS–Ch, similarly to the parent LPS, was found stimulated the formation of the IL-8 in the dose-dependent manner in the human embryonal kidney cells (HEK 293 cells) transfected with TLR4 in combination with MD2. [Copyright &y& Elsevier]