Structure of the core oligosaccharide in the lipopolysaccharide isolated from Aeromonas salmonicida ssp. salmonicida.

The core oligosaccharide isolated from the lipopolysaccharide of Aeromonas salmonicida ssp. salmonicida has been investigated by methylation analysis, NMR spectroscopy (13C and 1H), oxidation with periodate and chromium trioxide, and Smith degradation. The following structure is proposed: [Formula: see text]

Large-scale fractionation of S-form lipopolysaccharide from Salmonella abortus equi. Chemical and serological characterization of the fractions.

The S-form lipopolysaccharide of Salmonella abortus equi was separated by a newly elaborated extraction method with organic solvents into three fractions of different chain length of the O-polysaccharide they contained. The three fractions were designated long-chain (20-50 repeating units), short-chain (0-6) and R-fraction (no repeating units) according to their migration pattern in polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. The nature of the fractions as long- and short-chain and as R-fraction was confirmed by chemical analysis.

Alterations in the chemical composition and antigenicity of Escherichia coli O89 lipopolysaccharide and lipid A after 60Co gamma irradiation.

The chemical composition and the antigenicity of Escherichia coli lipopolysaccharide (LPS) and lipid A were investigated after irradiation with 150 kGy 60Co-gamma ray. Compared to the original preparations, the irradiated LPS showed a significant reduction in glucosamine, glucose and galactose and a decrease in the phosphate content. The fatty acid components were reduced to a smaller degree.

Endotoxic properties of synthetic pentaacyl lipid A precursor Ib and a structural isomer.

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 Ib and the isomer exhibited similar or only slightly lower pyrogenic, lethal and Shwartzman-phenomenon-inducing activity than lipid A.

Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A.

A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B-cell mitogenicity, complement activation, Limulus amoebocyte lysate gelation) it expressed similar activity as Salmonella lipid A.

In vitro deacylation of lipopolysaccharide of Salmonella minnesota by Acanthamoeba castellanii enzymes.

Enzymatic deacylation of the lipopolysaccharide isolated from a Salmonella Rd mutant by a cell-free preparation from Acanthamoeba castellanii has been studied. The degradation was found to be dependent on the presence of a surface-active component (Triton X-100) in the reaction mixture. The lipid A part of the lipopolysaccharide was the primary target of the enzymes, which cleaved with high efficiency the ester-bound long-chain nonhydroxylated and 3-hydroxylated acyl residues, i.

Comparison of the capacity of two lipid A precursor molecules to express the local Shwartzman phenomenon.

It has been shown recently that a Salmonella lipid A precursor molecule (Ia) and its synthetic counterpart are inactive in expressing the local Shwartzman reaction in both homologous and heterologous systems in combination with lipid A. Precursor Ia contains a bisphosphoryl-beta-1,6-glucosamine disaccharide substituted by 4 mol of (D)-3-hydroxytetradecanoyl residues. Escherichia coli lipid A, on the other hand, which contains two additional non-hydroxylated acyl residues in the form of two 3-acyloxyacyl units, is highly active.

Free flow electrophoresis as a tool for enrichment of mutants with temperature-dependent lethal mutations in lipid A synthesis.

Free flow electrophoresis was shown to be a useful tool to enrich for mutants conditionally defective in lipid A synthesis. The method was based on the observation that electrophoretic mobility of bacterial cells is dependent on the structure of lipopolysaccharides and is influenced by lesions in the synthesis of the O-specific chains as well as by lesion in the synthesis of the complete 3-deoxy-D-manno-octulosonic acid (dOclA) lipid A region. Using this procedure a new mutant conditionally defective in dOclA-8-P synthesis was isolated (mutant Ts5).

Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities.

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.

Nature and location of amide-bound (R)-3-acyloxyacyl groups in lipid A of lipopolysaccharides from various gram-negative bacteria.

It has previously been demonstrated [Eur. J. Biochem.

Lipopolysaccharides: structural principles and biologic activities.

Structural principles of the three regions of the lipopolysaccharide molecule are briefly discussed. One of the recently available, chemically synthesized lipid A analogues has been tested for biologic activities. The preparation was found to be toxic, pyrogenic, and mitogenic.

Mitogenic activities of synthetic lipid A analogs and suppression of mitogenicity of lipid A.

The effect of synthetic lipid A analogs on murine spleen cells was studied. The preparations represented D-glucosamine and D-glucosaminyl-beta 1,6-D-glucosamine disaccharide derivatives substituted in different combinations by ester- and amide-bound fatty acids and by phosphate groups. Significant mitogenic activity was demonstrated with a number of synthetic disaccharide preparations; however, their potency was lower than that of lipid A.

Biological activities of synthetic lipid A analogs: pyrogenicity, lethal toxicity, anticomplement activity, and induction of gelation of Limulus amoebocyte lysate.

Chemically synthesized lipid A analogs were investigated for several endotoxic activities, including pyrogenicity, lethal toxicity, anticomplement activity, and the capacity to gelate Limulus amoebocyte lysate in comparison to natural lipid A. The synthetic preparations contained D-glucosamine or D-glucosamine-beta-1,6-D-glucosamine disaccharide substituted by ester- and amide-bound hydroxylated or non-hydroxylated fatty acids and by phosphate groups in different combinations. Some preparations which were insoluble in water were succinylated and thus rendered more soluble.

Endotoxic properties of chemically synthesized lipid A part structures. Comparison of synthetic lipid A precursor and synthetic analogues with biosynthetic lipid A precursor and free lipid A.

Synthetic lipid A part structures corresponding structurally to a biosynthetic lipid A disaccharide precursor have been analyzed for endotoxic activity in several systems in vivo and in vitro. It was found that a synthetic beta-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxytetradecanoyl residues in positions 2, 3, 2' and 3' and phosphoryl groups in positions 1 and 4' (preparation 406), exhibited lethal toxicity, B lymphocyte mitogenicity, the capacity to engender prostaglandin formation in macrophages and to induce endotoxic tolerance, as well as serological lipid A antigenicity. On a weight basis, preparation 406 was of comparable activity to lipid A precursor and bacterial free lipid A.

Structural studies on the O-antigen of Aeromonas salmonicida.

Lipopolysaccharide from a strain of Aeromonas salmonicida salmonicida was isolated from cells by the aqueous phenol method in 2.3% yield (based on dry weight of bacteria). Hydrolysis of the lipopolysaccharide in 1% acetic acid afforded O-polysaccharide (19% by weight), core-oligosaccharide (12.

Isolation of a 3-deoxy-D-mannooctulosonic acid disaccharide from Salmonella minnesota rough-form lipopolysaccharides.

Lipopolysaccharides of Salmonella minnesota rough mutants were treated with 20 mM acetate buffer pH 4.4 at 70 degrees C/3 h. After dialysis of the hydrolysates about one third of the total 3-deoxy-D-mannooctulosonic acid (dOclA) content but no neutral sugars were found in the dialysate.

Fatty acid in lipopolysaccharides of Salmonella species grown at low temperature. Identification and position.

Salmonella minnesota R 595 (Re) and other Salmonella strains incorporate cis-delta 9-16:1 (palmitoleic acid) at the expense of mainly dodecanoic acid into the lipid-A portion of lipopolysaccharides, when the cells are grown at low temperature (12 degrees C). It has recently been shown, that in S. minnesota R 595 grown at 37 degrees C dodecanoic acid is linked to the 3-hydroxyl group of an amide-bound 3-hydroxytetradecanoic acid.

Lipid A, the lipid component of bacterial lipopolysaccharides: relation of chemical structure to biological activity.

Lipopolysaccharides are integral components of the outer membrane of Gram-negative bacteria and they participate in various membrane functions essential for bacterial growth and survival. Lipopolysaccharides also represent the endotoxins of Gram-negative bacteria and possibly play a role for the pathogenesis and manifestations of bacterial infections. These biological activities are mediated mainly by the lipid component of lipopolysaccharides, termed lipid A.

Distribution and antigenic properties of the O-determinants of Salmonella zuerich (1, 9, 27 46).

Fractionation of the O-polysaccharide derived from Salmonella zuerich (1, 9, 27, 46) on a concanavalin A polymer permitted immunological and chemical analysis on the different fractions. The S. zuerich O-polysaccharide preparation is composed of two distinct populations of molecules: one, ZB1-, devoid of O-antigenic determinant 1, and the other, ZB1+, carrying the determinant 1.

The chemical structure of lipid A. Demonstration of amide-linked 3-acyloxyacyl residues in Salmonella minnesota Re lipopolysaccharide.

In Salmonella minnesota lipopolysaccharide the lipid A backbone, a substituted diphosphorylated beta 1,6-linked D-glucosamine disaccharide molecule, carries approximately seven residues of fatty acids: one each of dodecanoic, hexadecanoic, D-3-hydroxytetradecanoic and D-3-O-(tetradecanoyl)-tetradecanoic acid in ester linkage and two of D-3-hydroxytetradecanoic acid in amide linkage. In the present study it is shown that treatment of the lipopolysaccharide with alkali at elevated temperature leads, through a beta-elimination reaction, to the generation of amide-bound delta 2-tetradecanoic acid. This suggested that the 3-hydroxyl group of amide-bound hydroxy fatty acids carried a substituent.

Effect of ionizing radiation on chemical and biological properties of Salmonella minnesota R595 lipopolysaccharide.

Lipopolysaccharide of the Salmonella minnesota Re mutant R595 was irradiated with 60Co gamma doses of 50, 100, 150 and 200 kGy. The irradiated preparations were less toxic, less active in the Shwartzman reaction and as activators of the complement system, but they had retained the protection activity against the lethal action of endotoxin. The irradiation resulted in a dose-dependent decrease in the amounts of constituents (glucosamine, KDO, fatty acids) of the original lipopolysaccharide.