[An unusual lipid A from a marine bacterium Chryseobacterium scophtalmum CIP 104199T].

The hydrolysis of defatted cells of the marine bacterium Chryseobacterium scophtalmum CIP 104199T with 10% acetic acid (3 h, 100 degrees C) led to an unusual lipid A (LA) (yield 0.6%), obtained for the first time. Using chemical analysis, FAB MS, and NMR spectroscopy, it was shown to be D-glucosamine 1-phosphate acylated with (R)-3-hydroxy-15-methylhexadecanoic and (R)-3-hydroxy-13-methyltetradecanoic acids at the C2 and C3 atoms, respectively. It is similar to the monosaccharide biosynthetic precursor of lipopolysaccharide (LPS), so-called lipid X (LX). Unlike LX, LA can be isolated by the treatment of bacteria with organic solvents only after the preliminary acidic hydrolysis of the cells, which suggests that LA might be strongly, probably chemically, linked to other components of the outer membrane. However, LPS cannot be such a component, because extraction with phenol-water or phenol-chloroform-petroleum ether mixtures in high yields (5.34% and 0.5%, respectively) leads to preparations that do not contain 3-deoxy-D-manno-oct-2-ulopyranosonic acid, 3-hydroxyalkanoic acids, or LA.

Mutual influence of plasmid profile and growth temperature on the lipid composition of Yersinia pseudotuberculosis bacteria.

The effect of a 82 MDa plasmid or its 25 MDa DNA fragment and growth temperature on the qualitative and the quantitative fatty acid and phospholipid composition of Yersinia pseudotuberculosis cells has been examined. In the cold, plasmid-containing and plasmid-free strains failed to differ appreciably in the contents of phospholipid and fatty acid. The exceptions were an elevated proportion of diphosphatidylglycerine and a decreased fatty acid unsaturation index in the plasmidless cells and those harbouring an incomplete 57 MDa plasmid in comparison with the strain containing the 82 MDa plasmid. At 37 degrees C, the lack of the 82 MDa plasmid or its 25 MDa fragment gave rise to a phospholipid of unknown structure, led to a sharp decrease in phospholipid content, in PE amount in particular, with a concurrent increase in the quantities of CL and LPE, and with a reduction in index of fatty acid unsaturation. The 82 MDa plasmid seems to be associated with a cancelling a temperature-dependent regulation of lipid synthesis and as a result, both the 'cold' and the 'warm' variants of the plasmid-containing strain possessed basically the close related lipid contents. Changes in composition of the polar head groups of the membrane phospholipids and in the extent of fatty acid unsaturation were suggested to be connected with an antibiotic hypersensitivity revealed earlier in plasmid-free Y. pseudotuberculosis.

[The structure of uncommon lipid A from the marine bacterium Marinomonas communis ATCC 27118T].

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118T with 1% AcOH. Using chemical analysis and ID and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be beta-(1',6)-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-[(R)-3-hydroxydecanoyloxy)]decanoic acid, and (R)-3-hydroxydecanoic acid at the C2, C2' and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M. communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.

The impact of abiotic factors (temperature and glucose) on physicochemical properties of lipids from Yersinia pseudotuberculosis.

The impact of the availability of glucose in nutrition medium and growth temperature on the composition and thermotropic behavior of lipids from Yersinia pseudotuberculosis (Enterobacteriaceae) was studied. Y. pseudotuberculosis was grown in nutrition broth (NB) with/without glucose at 8 and 37 degrees C, corresponding to the temperatures of saprophytic and parasitic phases of this bacterium life. The decrease of phosphatidylethanolamine, phosphatidylglycerol and unsaturated fatty acids and the parallel increase of lysophosphatidylethanolamine and diphosphatidylglycerol and saturated and cyclopropane acids were the most significant changes with temperature in bacterial phospholipid (PL) classes and fatty acids, respectively. Glucose did not effect the direction of temperature-induced changes in the contents of PLs, fatty acids, however it enhanced (for PLs) or diminished (for fatty acids) intensity of these changes. The thermally induced transitions of lipids were studied by differential scanning calorimetry (DSC). It was revealed that the addition of glucose to NB induced a sharp shift of DSC thermograms to lower temperatures in the "warm" variants of bacteria. The peak maximum temperature (Tmax) of thermal transitions dropped from 50 to 26 degrees C that is the optimal growth temperature of Y. pseudotuberculosis. Tmax of total lipids of the cells grown at 8 degrees C without glucose in NB was equal to growth temperature that corresponded to the classical mechanism of homeoviscous adaptation of bacteria. An addition of glucose to NB at this growth temperature caused the subsequent reduction of Tmax to -8 degrees C, while the temperature ranges of thermograms were not substantially changed. So, not only the temperature growth of bacteria, but also the presence of glucose in NB can modify the physical state of lipids from Y. pseudotuberculosis. In this case, both factors affect additively. It is suggested that glucose influences some membrane-associated proteins and then the fluidity of lipid matrix through temperature-inducible genes.

New glycolipids (chitooligosaccharide derivatives) possessing immunostimulating and antitumor activities.

New glycolipids, derived from chitooligosaccharides of dp 2-4 and containing both free and acylated amino groups, were synthesized. The structure of the key compounds (di-, tri-, and tetra-saccharides acylated with different fatty acids) were elucidated by 13C NMR spectroscopy. Only the amino group of the reducing end of the chitooligosaccharides was found to be acylated when equimolecular amounts of reagents were used. The compounds obtained were shown to possess a low toxicity and certain immunostimulatory and antitumor activities. An induction of interleukin-1 and tumor necrosis factor by the immunocompetent cells and an augmentation by 140-180% of the mean life of mice with the Erlich carcinoma were observed.

[Rapid method of isolation of lipid A from Yersinia pseudotuberculosis]. / Bystryi sposob polucheniia lipida A iz bakterii Yersinia pseudotuberculosis.

The possibility of preparing the lipid A (LA) from Yersinia pseudotuberculosis Serovar IB by the hydrolysis of whole cells instead of the preliminary isolation of lipopolysaccharide (LPS) was demonstrated. Direct extraction with an organic solvent of the bacterial mass preliminary treated with 10% acetic acid or 1 M HCl was shown to result in a di- (LAAcOH) or monophosphoryl derivative (LAHCl), respectively. These were completely extractable only after treatment with strong hydrolyzing agents. We concluded that two forms of LA (and LPS) exist in the pseudotuberculosis bacterium which differ in the stability of their bonding to the bacterial outer membrane.

[New method of isolation of O-specific polysaccharide from gram-negative bacteria Yersinia pseudotuberculosis]. / Novyi sposob vydeleniia O-spetsificheskogo polisakharida iz gramotritsatel'noi bakterii Yersinia pseudotuberculosis.

Using bacteria Yersinia pseudotuberculosis as an example, two procedures for obtaining the O-specific polysaccharide of endotoxins of gram-negative bacteria were compared: the direct acidic hydrolysis of whole cells and the traditional procedure based on preliminary isolation of the corresponding lipopolysaccharide. Analysis of the resulting polysaccharides showed that the isolation from the bacterial biomass gave a high yield of the polysaccharide; the polysaccharide is not degraded; and judging from the 13C NMR data, its structure was completely identical to that of the corresponding product of lipopolysaccharide hydrolysis. Therefore, this procedure is useful for obtaining O-specific polysaccharides for structural studies.

[Effect of a culturing method and growth phase on composition of lipopolysaccharides in Yersinia pseudotuberculosis]. / Vliianie sposoba kul'tivirovaniia i fazy rosta na lipopolisakharidnyi sostav Yersinia pseudotuberculosis.

The effects of the culturing method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lypopolysaccharide (LPS) composition of Yersinia pseudotuberculosis (O:Ib serovar, strain KS 3058) grown in cold (5 degrees C) were studied. The amount the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and achieved maximum in the stationary growth phase for both media. The bacteria culturing on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultured in the liquid medium. It was proposed that the culturing of Yersinia pseudotuberculosis in cold as colonies on the agar surface causes an increase in the bacterial virulence.

[Elucidation of structure of lipid A from the marine Gram-negative bacterium Pseudoalteromonas haloplanktis ATCC 14393T]. / Ustanovlenie struktury lipida A iz morskoi gramotritsatel'noi bakterii Pseudoalteromonas haloplanktis ATSS 14393T.

The chemical structure of lipid A from the marine gamma-proteobacterium Pseudoalteromonas haloplanktis ATCC 14393T, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be beta-1,6-glucosaminobiose 1,4'-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3' and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2', respectively.

[The use of 13C-NMR spectra of lipopolysaccharide-protein complexes to determine the structure of O-specific polysaccharides]. / Ispol'zovanie spektrov 13C-IaMR lipopolisakharidov-belkovykh kompleksov dlia ustanovleniia stroeniia O-spetsificheskikh polisakharidov.

Lipopolysaccharide-protein complexes are shown to be useful for elucidation of O-specific polysaccharide structures. This approach may be especially useful in studying polysaccharides with labile linkages in the molecule.

[Cooperative interaction between protein-porin and lipopolysaccharide]. / Kooperativnoe vzaimodeistvie mezhdu belkom-porinom i lipopolisakharidom.

The interaction of the pore-forming protein (porin) from the outer membrane of Yersinia pseudotuberculosis with the S- and R-forms of lipopolysaccharide (LPS) from this bacterium was studied. Analysis of the equilibrium binding of 125I-labeled S- and R-LPS, as well as the competitive inhibition of this reaction by lipid A, core oligosaccharide, and O-specific polysaccharide, suggests that there are binding sites on the porin molecule specific to these fragments of the LPS molecule. The binding of R-LPS occurs at independent sites of two classes with Ka 1.7 x 10(5) and 1.1 x 10(5) M-1. S-LPS interacts with porin with positive cooperation (h 1.6) and Ka 0.8 x 10(5) M-1. The number of binding sites was found to be nine and four R- and S-LPS, respectively. Molecular mechanisms of the interaction are discussed.

[Synthesis of lipid A analogs. Preparation of serologically active glycolipids on the basis of beta-1,4-glucosaminobiose (chitobiose)]. / Sintez analogov lipida A. Poluchenie serologicheski aktivnykh glikolipidov na osnove beta-1,4-gliukozaminobiozy (khitobiozy).

Synthesis of beta-1,4-glucosaminobiose (chitobiose) 4'-phosphates N,N'-diacylated with (R)-3-hydroxymyristic acid is described, the structure being corroborated by 13C-NMR spectra. It was shown that activity of the components in various reactions with antibodies to lipid A from Yersinia pseudotuberculosis is similar to the lipid A activity.

Influence of lipopolysaccharides and lipids A from some marine bacteria on spontaneous and Escherichia coli LPS-induced TNF-alpha release from peripheral human blood cells.

Some endotoxic properties of lipopolysaccharides (LPS) and lipids A (LA) from the marine bacteria Marinomonas communis ATCC 27118(T), Marinomonas mediterranea ATCC 700492(T), and Chryseobacterium indoltheticum CIP 103168(T) were studied. The preparations tested were shown to have high 50% lethal doses (4 microg per mouse for LPS from M. mediterranea and more than 12 microg per mouse for two other LPS and LA from C. indoltheticum) and were moderate (371 +/- 37 pg/ml at 10 microg/ml of C. indoltheticum LPS), weak (148 +/- 5 pg/ml at 1 microg/ml of M. mediterranea LPS), and zero (LA and LPS from M. communis and LA from C. indoltheticum) inducers of tumor necrosis factor alpha (TNF-alpha) release from peripheral human blood cells. The capacity of the LA and LPS samples from marine bacteria to inhibit TNF-alpha release induced by LPS from Escherichia coli O55 : B5 (10 ng/ml) was also studied.

Synthesis of lipopolysaccharides in the bacterium Yersinia pseudotuberculosis: effect of the pVM82 plasmid and growth temperature.

The composition and structure of lipopolysaccharides (LPS) of three isogenic strains of Yersinia pseudotuberculosis serovar O:1b (without plasmids (82-) and with plasmids pVM82 (82+) or p57 (57+)) grown at 8 or 37 degrees C were studied by chemical and immunochemical methods, SDS-polyacrylamide gel electrophoresis, and 13C-NMR spectroscopy. At the lower temperature, the (82-) and (82+) strains synthesized S-form of LPS with similar structure characterized by high acylation and immunochemical activity. On the other hand, LPS of the (82+) strain had shorter carbohydrate chains than LPS of the (82-) strain. The contents of LPS were decreased in cells of the plasmid-free strain grown at the higher temperature. LPS isolated from these cells were of the R-form and had low acylation and immunochemical activity. Total LPS content in cells of the (82+) strain did not significantly depend on the growth temperature. LPS of the warm variant of these bacteria contained a polysaccharide fragment and had moderate immunochemical activity. The cells of the (57+) strain at both growth temperatures had low LPS contents and produced LPS of low acylation without O-specific chains (cold variant) or containing O-polysaccharide with low polymerization degree (bacteria grown at 37 degrees C). The data indicate that in the absence of the plasmids, LPS synthesis is encoded by the chromosomal genes in pseudotuberculosis bacteria. Expression of the genes involved in LPS synthesis is regulated by the temperature of bacterial growth. Genes responsible for temperature-dependent regulation of LPS biosynthesis are located on chromosomal DNA. The pVM82 plasmid includes two gene groups; one group is localized in a 57-mD fragment of DNA and inhibits LPS synthesis, suppressing temperature-dependent regulation of the synthesis. The genes located in a 25-mD fragment of the pVM82 plasmid are de-repressors of the 57-mD fragment, and they restore the ability of pseudotuberculosis bacteria to synthesize relatively long LPS at both growth temperatures.

Effects of growth temperature and pVM82 plasmid on fatty acids of lipid A from Yersinia pseudotuberculosis.

Effects of cultivation temperature (8 or 37 degrees C) and plasmid profile on the lipid A fatty acids of three isogenic Yersinia pseudotuberculosis strains (plasmidless (82-) and strains containing pVM82 (82+) or p57 (57+) plasmids) obtained by alkaline hydrolysis of the whole bacterial cells and differentiated from fatty acids of other membrane lipids were investigated. On the basis of the analysis, it is concluded that lipids A of all studied samples contain 3-hydroxytetradecanoic and dodecanoic acids, a part of which exists as the 3-dodecanoyloxytetradecanoic derivative. The effect of temperature appears in the higher contents of ester- and amide-linked 3-acyloxyalkanoic residues in lipid A from the "cold" variants of the bacteria and is determined by chromosomal genes. The plasmid effect is seen as various responses of the isogenic derivatives to change of growth temperature: in cells of strains 82+ and 82- grown in the cold, the share of lipid A fatty acids in the total population of cellular fatty acids is reduced, while in strains with plasmid p57 it is increased. The temperature variants of the 57+ strain differ by the low contents of amide-linked 3-acyloxyalkanoic acids. Finally, lack of plasmid pVM82 in the "warm" variants of the bacteria results in accumulation of glycolipid molecules deprived of dodecanoic acid. Correlation between growth temperature and plasmid profiles, on one hand, and lipid A fatty acid composition and potential pathogenic properties of the Y. pseudotuberculosis, on the other hand, and also possible mechanisms of thermal adaptation of this organism are discussed.

D-3-Dodecanoyltetradecanoic acid as a constituent of lipid A from the lipopolysaccharide of Yersinia pseudotuberculosis.

D-3-Dodecanoyltetradecanoic acid has been separated from the lipid A of Yersinia pseudotuberculosis and its structure has been established by chromato-mass-spectrometry and 13C NMR spectroscopy, by comparison with authentic samples.

Heterogeneity of lipopolysaccharides from Yersinia pseudotuberculosis: chemical characterization of various molecular types.

Prior studies have shown some unusual changes in the lipopolysaccharides (LPSs) from Yersinia pseudotuberculosis that occur when the microbe is grown at low temperature; the specific features of these LPSs in comparison with the LPSs from other enteropathogens may be due to unusual thermal adaptation mechanisms. To gain insight into this question, the chemical composition of Y. pseudotuberculosis LPS has been determined. The data indicate that two different S-form LPS species are produced in "cold"-grown bacteria. These have an identical set of bands after SDS-PAGE, similar elution profiles during gel-filtration on a Sephadex G-200 column in the presence of sodium deoxycholate, identical monosaccharide and fatty acid compositions, and similar polymerization degrees, but they have different acylation degree. On the whole, the macromolecularly different LPS populations, varying not only in their smooth or rough nature and hydrophobicity, but also in their localization in the outer membrane and, probably, their interactions with other cell components, are synthesized in "cold"-grown Y. pseudotuberculosis. The biological sense of the heterogeneity and its connection with psychrophilic and pathogenic properties of pseudotuberculosis organisms are discussed.

Glucose as a growth medium factor regulating lipid composition of Yersinia pseudotuberculosis.

Effects of glucose and growth temperature on Yersinia pseudotuberculosis O:1b serovar lipid composition have been studied. These growth parameters were shown to have drastic effects on biosynthetic processes in the pseudotuberculosis bacteria. The temperature effect is the most universal, extending to cell growth and to free lipid and lipopolysaccharide content and composition; it is most conspicuous in the bacteria cultivated on glucose-containing nutrient broth. The effect of glucose is selective, affecting only free lipids and depending on temperature (glucose favors phospholipid (PL) synthesis in the cold and inhibits it at 37 degrees C); the effect of glucose is more evident in the cold. Determination of the contents of individual PL in percent dry bacterial weight indicates that the most obvious effect of glucose and/or growth temperature is on phosphatidylethanolamine (PE) content: on both media and at both temperatures an overall decrease in PL content stems from the inhibition of PE synthesis and is attended by decreasing ratio of neutral to acidic lipids.

Studies on lipid A from Yersinia pseudotuberculosis lipopolysaccharide. Isolation and general characterization.

Lipid A was isolated from lipopolysaccharide of Yersinia pseudotuberculosis S form (strain 341, subtype IB) using mild hydrolysis with acetic acid. The purified material (yield about 25%, molecular weight about 2900) contained D-glucosamine (11%), fatty acids (54%), protein concomitant (9.7%) and phosphorus (approximately 2%). Dodecanoic and 3-hydroxy-tetradecanoic acids in a molar ratio of 1 : 3.6 were detected as major fatty acid constituents. The hydroxyl groups of D-glucosamine were acylated with the residues of both fatty acids, while the amino groups were substituted with the residue of 3-hydroxy-tetradecanoic acid. Such a simple fatty acid composition is reminiscent of that found in lipid A in Y. pestis.

Interaction of bacterial endotoxins with chitosan. Effect of endotoxin structure, chitosan molecular mass, and ionic strength of the solution on the formation of the complex.

The interaction of endotoxins of different structure (lipopolysaccharides (LPS) and lipopolysaccharide-protein complexes (LPPC)) with chitosan has been studied. It was shown that the mechanism of interaction is rather complicated and depends on the macromolecular organization of endotoxin as well as on the degree of polymerization of the chitosan. Chitosan with molecular mass of 20 kD reveals higher affinity to LPS than chitosan with molecular mass of 140 kD. Endotoxins with long O-specific chains can bind completely with chitosan with the formation of LPS-chitosan and LPPC-chitosan complexes with weight ratios between the original components of 1:1 and 1:5. When endotoxins with higher degree of hydrophobicity and short O-specific chains were mixed with chitosan, a part of the LPS remained unbound. The stability of the complexes formed depends on ionic strength. It was shown that, in addition to electrostatic forces, other types of forces take part in the formation of the complexes. A decrease in acute toxicity of various LPSs is observed on their binding with chitosans.