Inhibition by succinyl concanavalin A of strong adjuvant activity of lipopolysaccharides which possess mannans as the O-specific polysaccharide chains

It has been reported that lipopolysaccharides (LPS) from Klebsiella O3 and O5 and Escherichia coli O8 and O9 exhibit extraordinarily strong adjuvant activity in augmenting antibody responses against protein antigens in mice as compared with other kinds of LPS. These four kinds of LPS all possess homopolysaccharides consisting of mannose (mannans) as the O-specific side chains. When these kinds of LPS were mixed in vitro with succinyl concanavalin A (Con A) which is known to bind specifically to alpha-mannoside and alpha-glucoside, their strong adjuvant activity was inhibited. Degree of the inhibition of the adjuvant activity of Klebsiella O3 LPS by succinyl Con A was dependent upon the dose of succinyl Con A. However, phytohemagglutinin, which is known to bind specifically to N-acetyl-D-galactosamine, did not inhibit the adjuvant activity of Klebsiella O3 LPS and O5 LPS. When Klebsiella O3 LPS was mixed with succinyl Con A in the presence of excess amounts of alpha-methyl mannoside or the polysaccharide fraction isolated from Klebsiella O3 LPS, the inhibitory effect of succinyl Con A on the adjuvant activity of Klebsiella O3 LPS was blocked. By contrast, the activity of Klebsiella O3 LPS as a polyclonal B-cell activator was not affected by treatment with succinyl Con A. From these results it is concluded that the mannans, as the O-specific polysaccharide chains of the LPS, significantly contribute to expression of their strong adjuvant activity.     

Strong interaction of lipopolysaccharides possessing the mannose homopolysaccharides with complement and its relation to adjuvant action

LPS from Klebsiella pneumoniae O3 (KO3 LPS) exhibited an extremely high anticomplementary activity by the hemolysis assay using human sera. The free lipid A isolated from KO3 LPS by acid hydrolysis and R form LPS from a mutant lacking the O-specific polysaccharide portion possessed lower anticomplementary activity, and the O-specific polysaccharide fraction isolated from KO3 LPS alone did not activate the C system. It was suggested that the O-specific polysaccharide moiety enhanced the C activation by the lipid A portion. This was also supported by the finding that modification of the O-specific polysaccharide moiety with Con A or tyramine decreased anticomplementary activity of KO3 LPS, and that the other LPS preparations possessing the mannose homopolysaccharides as the O-specific polysaccharide portions such as KO3 LPS, such as LPS from Klebsiella O5, Escherichia coli O8 and O9, exhibited a high anticomplementary activity. KO3 LPS could activate the C system in either the classical or the alternative pathway, whereas the lipid A or R form LPS activated the classical pathway alone. The intensity of anticomplementary activity of LPS was parallel to that of their adjuvant action on antibody response to deaggregated BSA. The role of the anticomplementary activity in the expression of the adjuvant action of LPS is discussed.     

Adjuvant activity of Klebsiella O3 lipopolysaccharide: no contribution of proteins to the expression of adjuvant activity

Previously we found that Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant of strain Kasuya (O3: K1) or its decapsulated mutant strain LEN-1 (O3: K1-) exhibited very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice. The preparation of KO3 LPS after deproteinization by four cycles of treatment with chloroform-butanol (5: 1) usually contained a small percentage of proteins and a definite amount of another antigen which was destroyed by heating at 100 C for 1 hr. This antigen proved to be derived from type 1 fimbriae which are responsible for mannose-sensitive hemagglutination of guinea pig erythrocytes. The preparation of KO3 LPS isolated from culture supernatant of the strains which did not produce type 1 fimbriae exhibited strong adjuvant activity similar to that of the preparation from those which produced them. The preparation of KO3 LPS treated with hot phenol water which is known to remove lipid A-associated proteins exhibited a similar strong adjuvant activity. The preparation of KO3 LPS after extensive deproteinizing, two cycles of pronase treatment followed by ten cycles of treatment with chloroform-butanol, no longer contained detectable amounts of proteins and the fimbrial antigen, but this preparation also exhibited similar strong adjuvant activity. Moreover, there was no difference in strength of the adjuvant activity between the preparation of KO3 LPS isolated from culture supernatant and that isolated by the phenol method from bacterial cells. The present study demonstrates that the strong adjuvant activity of the preparation of KO3 LPS does not depend in any way on proteins contaminating the preparation.     

Potent adjuvant action of lipopolysaccharides possessing the O-specific polysaccharide moieties consisting of mannans in antibody response against protein antigen

It was previously reported that Klebsiella O3 lipopolysaccharide (LPS) exhibits extraordinarily strong adjuvant activity in augmenting antibody response against protein antigens in mice compared with other kinds of LPS, for example, LPS from Escherichia coli O55, O111, and O127 and Salmonella enteritidis. The present study was undertaken to clarify the relationship between the strong adjuvant activity in augmenting antibody response against deaggregated bovine gammaglobulin and the chemical structure of LPS. Among LPS from Klebsiella O1, O4, O5, and O7, only O5 LPS exhibited nearly the same degree of the strong adjuvant activity as did O3 LPS. The adjuvant activity of the other LPS was very weak in a degree similar to that of LPS from E. coli O55 and O127. Even when the natural forms of Klebsiella O3 LPS and O1 LPS were converted to various defined uniform salt forms, their adjuvant activity did not significantly differ from that of the respective natural forms. It is therefore unlikely that the difference in strength of the adjuvant activity between Klebsiella O3 LPS and O1 LPS is due to the difference in their salt forms. The common feature in the structures of Klebsiella O3 LPS and O5 LPS is their O-specific polysaccharide chains consisting of the mannose homopolysaccharides (mannans). LPS from E. coli O8 and O9, the O-specific polysaccharide chains of which consist of the mannans, also exhibited much stronger adjuvant activity than do LPS from E. coli O55 and O127, and the strength of the adjuvant activities of the former two was comparable to that of LPS from Klebsiella O3 and O5. On the other hand, LPS from Klebsiella O3 and O5 and E. coli O8 and O9 showed the ability to activate B lymphocytes polyclonally in vivo in a degree similar to that of the other kinds of LPS. From the present results it can be concluded that LPS possessing the O-specific polysaccharide moieties consisting of the mannans exhibit extraordinarily strong adjuvant activity in augmenting antibody response against protein antigen.     

A possible correlation between histological changes in regional subcutaneous tissue induced by bacterial lipopolysaccharides and their adjuvant activities

Previously it was demonstrated that Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) exhibited much stronger adjuvant action on antibody response to subcutaneously (s.c.) injected sheep red blood cells or deaggregated bovine serum albumin than did other kinds of LPS, the R-form LPS lacking the O-specific polysaccharide chain of KO3 LPS (R-LPS), and the lipid A fractionated from KO3 LPS. We compared histological changes in the regional subcutaneous tissues of mice injected subcutaneously (s.c.) with KO3 LPS, the lipid A, and R-LPS. At the early stage after injection, KO3 LPS induced the infiltration of a large number of inflammatory cells, mainly polymorphonuclear leukocytes (PMN), at the site of injection. Neither R-LPS nor the lipid A induced the accumulation of PMN so much as KO3 LPS did. When injected s.c. with LPS from Escherichia coli O111 (EO111 LPS) and O55 (EO55 LPS), and Salmonella enteritidis (Sent LPS), the appearance of PMN at the regional site was much less than KO3 LPS. KO3 LPS could accumulate more 51Cr-labeled leukocytes at the injection site than EO111 LPS and Sent LPS. Administration of acetylsalicylic acid, which can inhibit leukocyte migration in inflammatory lesions, suppressed its adjuvant action. It was therefore suggested that the strong adjuvant action of KO3 LPS in s.c. injection might be dependent on its potent capability of accumulating PMN at the regional subcutaneous tissue. Furthermore, at the late stage after injection, the formation of several lymphoid follicles at the regional site was seen only in mice injected with KO3 LPS. It might be also related to the strong adjuvant action of KO3 LPS.     

Ultrastructure of Klebsiella O3 Lipopolysaccharide Isolated from Culture Supernatant: Comparison with Other Lipopolysaccharides

Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from the culture supernatant, which was found to exhibit a very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice, was examined by electron microscopy. When negatively stained with uranyl acetate or ammonium molybdate, the KO3 LPS was found to consist principally of flat ribbon-like structures branching freely (average width 16 nm and average thickness 7 nm) and to contain a small proportion of spheres (diameter 20–50 nm), both structures covered with fine hairy structures (average length approximately 10 nm). When the polysaccharide of KO3 LPS was stained by the periodic acid-thiosemicarbazide-silver proteinate procedure, silver granules were deposited on the ribbon-like structures and around the spheres, suggesting that the polysaccharide moiety is located on their surface and that the fine hairy structures consist of the polysaccharide moiety. Comparison by means of preparations stained with uranyl acetate or ammonium molybdate showed that KO3 LPS isolated from the culture supernatant has structural features in common with KO3 LPS isolated from bacterial cells, Escherichia coli O9 LPS isolated from the culture supernatant, and E. coli O127 LPS isolated from bacterial cells. On the basis of the present results, schematic representations of the common physical structure of LPS were drawn; the fine hairy structures attach to the wide surface of the flat ribbon-like structures along their lateral margin.     

The role of the structural parts of bacterial lipopolysaccharide in its direct immunosuppressive activity]

The direct immunosuppressive activity of lipopolysaccharides (LPS) and their structural parts (O-chains, R-core, lipid A), obtained from Salmonella, Pseudomonas and Burkholderia, was studied. LPS preparations were extracted by the phenol-water method. Structural parts of LPS were obtained by acetic acid hydrolysis and gel filtration. The study demonstrated that all these preparations, when injected intraperitoneally into mice, did not affect the level of delayed-type hypersensitivity (DTH) to the test antigen in the animals. After redox treatment all LPS preparations became capable of suppressing DTH. After redox treatment such immunosuppressive activity could be observed in lipid A, while O-specific chains and R-core remained inactive. After phenol treatment immunosuppressive activity disappeared. Chemical groups capable of activation were likely to be located in lipid A or in lipid A-associated protein.     

Structural studies of the side chain of outer membrane lipopolysaccharide from Pseudomonas syringae pv. coriandricola W-43.

The lipopolysaccharide (LPS) was isolated from Pseudomonas syringae pv. coriandricola W-43 by hot phenol-water extraction. Rhamnose and 3-N-acetyl-3-deoxyfucose were found to be the major sugar constituents of the LPS together with N-acetylglucosamine, N-acetylgalactosamine, heptose, and 3-deoxy-D-manno-octulosonic acid (Kdo). The main fatty acids of lipid A of the LPS were 3-OH-C:10, C12:0, 2-OH-C12:0, and 3-OH-C12:0. The O-specific polysaccharide liberated from the LPS by mild-acid hydrolysis was purified by gel permeation chromatography. The compositional analysis of the O-specific polysaccharide revealed the presence of L-rhamnose and 3-N-acetyl-3-deoxy-D-fucose in a molar ratio of 4:1. The primary structure of the O-specific polysaccharide was established by methylation analysis together with 1H and 13C nuclear magnetic resonance spectroscopy, including two-dimensional shift-correlated and one-dimensional nuclear Overhauser effect spectroscopy. The polysaccharide moiety was found to consist of a tetrasaccharide rhamnan backbone, and 3-N-acetyl-3-deoxy-D-fucose constitutes the side chain of the branched pentasaccharide repeating unit of the polysaccharide.     

Chemical characteristics and endotoxic activity of the lipopolysaccharide of Rahnella aquatilis 2-95

The lipopolysaccharide (LPS) from a new Enterobacteriaceae species, Rahnella aquatilis 2-95, was isolated and investigated. The structural components of the LPS molecule, namely, lipid A, core oligosaccharide, and O-specific polysaccharide, were obtained by mild acid hydrolysis. In lipid A, 3-oxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. The major monosaccharides of the core oligosaccharide were galactose, arabinose, fucose, rhamnose, and an unidentified component. The O-specific polysaccharide was found to be assembled of a repeated trisaccharide unit of the following structure: [structure: see text]. The R. aquatilis 2-95 LPS is less toxic and more pyrogenic as compared to the one from the R. aquatilis 1-95 strain studied earlier. Both acyl and phosphate groups are essential for toxic and pyrogenic activity of R. aquatilis 2-95 LPS.     

The Klebsiella O3 lipopolysaccharide isolated from culture fluid: structure of the polysaccharide moiety

In a series of our earlier studies, the O3 antigen isolated from culture supernatant of Klebsiella pneumoniae strain Kasuya (O3:K1) (KO3) was shown to exhibit very strong adjuvant activity in mice. KO3 obtained was homogeneous in analyses by either gel filtration or ultracentrifugation. Its molecular weight determined by ultracentrifugal analysis was greater than 2 X 10(6). It contained 37.9% C, 6.20% H, 0.24% N, and less than 0.1% P. KO3 was degraded into the polysaccharide moiety and lipid moiety (about 20%) by hydrolysis with 1% acetic acid at 100 C for 1 hr. The molecular weight of the polysaccharide moiety obtained by the hydrolysis was 16,200 as determined by the Somogyi-Nelson method. Chemical analyses using methylation analysis and Smith degradation as the principal methods indicated that the polysaccharide moiety consisted of a mannan which has a pentasaccharide repeating unit of alpha-mannosyl-1,3-alpha-mannosyl-1,2-alpha-mannosyl-1,2-alpha-mannosyl-1, 2-alpha-mannose joined through alpha-1,3-mannosyl linkages. The number of repetitions was less than 20. The fact that minor components such as 2-keto-3-deoxyoctonate and glucose were detected suggests the presence of a core oligosaccharide, but its precise structure is unknown.     

Adjuvant activity of Klebsiella O3 lipopolysaccharide: comparative study using defined uniform salt forms

Previously we showed that Klebsiella O3 lipopolysaccharide (KO3 LPS) is much more potent than other kinds of LPS including Escherichia coli O127 LPS (EO127 LPS) in adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens and in the ability to enlarge the regional lymph node. Various defined uniform salt forms, the triethylamine, sodium, potassium, ammonium, tris(hydroxymethyl)aminomethane, and calcium salt forms, of KO3 LPS and EO127 LPS were prepared by removing basic materials present in LPS preparations by electrodialysis and neutralizing the electrodialyzed LPS preparations with various kinds of alkali. The triethylamine salt form showed the best solubility and consisted of the smallest granules and, on the other hand, the calcium salt form showed the lowest solubility, compared with the natural form and the other uniform salt forms. Even if the natural forms of KO3 LPS and EO127 LPS were converted to the defined uniform salt forms, adjuvanticity of KO3 LPS and EO127 LPS in augmenting delayed-type hypersensitivity to ovalbumin and the ability to enlarge the regional lymph node did not significantly differ from those of the respective natural forms. From these results it is concluded that the difference in strength of the adjuvanticity between KO3 LPS and EO127 LPS is not due to the difference in their salt forms, solubility or physical state. Moreover, there were no significant differences in lethal toxicity for mice by the intraperitoneal route among the natural form and all the uniform salt forms of KO3 LPS tested.     

Structure and properties of the lipopolysaccharide of Pseudomonas fluorescens IMV 2366 (biovar III)

The lipopolysaccharide (LPS) preparation isolated from the bacterial mass of Pseudomonas fluorescens IMV 2366 (biovar III) by Westphal's method and purified by repeated ultracentrifugation was characterized by the presence of the S- and R-forms of molecules. The following structural portions of the LPS molecule were obtained in the individual state and characterized: lipid A, core oligosaccharide, and O-specific polysaccharide. The main components of the lipid A hydrophobic moiety were 3-hydoxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, and hexadecanoic fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic moiety. Rhamnose, glucose, galactose, glucosamine, galactosamine, alanine, phosphoethanolamine, phosphorus, 2-keto-3-desoxyoctulosonic acid (KDO), as well as 2-amino-2,6-didesoxygalactose (FucN) and 3-amino-3,6-didesoxyglucose (Qui3N), were revealed in the composition of the core oligosaccharide fractions. O-specific polysaccharide chains were established to be composed of repeating trisaccharide units consisting of residues of L-rhamnose (L-Rha), 2-acetamido-2,6-didesoxy-D-galactose (D-FucNAc), and 3-acylamido-3,6-didesoxy-D-glucose (D-Qui3NAcyl), where Acyl = 3-hydroxy-2,3-dimethyl-5-hydroxyprolyl. Neither double immunodiffusion in agar not the immunoenzyme assay revealed serological relations between the strain studied and the P. fluorescens strains studied earlier.     

Chemical characteristics and endotoxic activity of the lipopolysaccharide of Rahnella aquatilis 2-95

The lipopolysaccharide (LPS) from a new Enterobacteriaceae species, Rahnella aquatilis 2-95, was isolated and investigated. The structural components of the LPS molecule, namely, lipid A, core oligosaccharide, and O-specific polysaccharide, were obtained by mild acid hydrolysis. In lipid A, 3-oxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. The major monosaccharides of the core oligosaccharide were galactose, arabinose, fucose, rhamnose, and an unidentified component. The O-specific polysaccharide was found to be assembled of a repeated trisaccharide unit of the following structure: The R. aquatilis 2-95 LPS is less toxic and more pyrogenic than the LPS from the R. aquatilis 1-95 strain studied earlier. Both acyl and phosphate groups are essential for toxic and pyrogenic activity of R. aquatilis 2-95 LPS.     

The immunochemical characteristics of the lipopolysaccharides of Pseudomonas syringae (pathovars atrofaciens and phaseolicola) and P. holci (serogroup VI)]

Lipopolysaccharides (LPS) of the representatives of strains of serogroup VI Pseudomonas syringae (P. syringae pv. atrofaciens 2399, pv. phaseolicola 120a, 7842 and P. holci 8299) possessing virulence and confinement to the host-plant are characterized by high serological activity in direct and cross reactions of the binary diffusion in agar, immunoelectrophoresis, passive hemagglutination and inhibition of passive hemagglutination. A supernatant and a sediment obtained after ultracentrifugation of LPS preparations possessed O-antigenic activity. O-specific polysaccharide (PS) is serologically less active than the LPS preparations. Problems of the intergroup and intragroup serological affinity in connection with the structure of O-specific PS. It is proved that the basic chain of O-specific polysaccharide (D-rhamnane) plays definite (but not a single) part in displaying antigenic properties of the whole LPS macromolecule.     

Correlation between strong adjuvanticity of Klebsiella O3 lipopolysaccharide and its ability to induce lnterleukin-1 secretion

Adjuvant activity of Klebsiella O3 lipopolysaccharide (KO3 LPS) in augmenting antibody response and delayed-type hypersensitivity to protein antigens in SMA mice was much stronger than that of LPS from Escherichia coli O55 and O127 (EO55 LPS and EO127 LPS). Relationship between strength of the adjuvant activity and that of the ability to induce interleukin-1 (IL-1) secretion by peritoneal macrophages from C3H/HeN or SMA mice was investigated using these three kinds of LPS. When supernatant samples of macrophages cultured at 37 °C for 24 hr in the presence of 5 μg/ml LPS were assayed by their mitogenic effect on thymocytes from C3H/HeJ mice, KO3 LPS induced the secretion of about four to six times greater amounts of IL-1 activity than did EO127 LPS. When concentration of LPS used for stimulation of macrophages was varied from 0.1 to 50 μg/ml, KO3 LPS induced the secretion of definitely greater amounts of IL-1 activity than did EO55 LPS and EO127 LPS throughout the LPS concentrations tested. Nearly the same amount of IL-1 activity as that produced by 10 μg/ml EO55 LPS or 50 μg/ml EO127 LPS could be produced by 1.0 μg/ml or lower concentrations of KO3 LPS.     

Structure and Properties of the Lipopolysaccharide of Pseudomonas fluorescens IMV 2366 (Biovar III)

The lipopolysaccharide (LPS) preparation isolated from the bacterial mass of Pseudomonas fluorescens IMV 2366 (biovar III) by Westphal's method and purified by repeated ultracentrifugation contained S- and R-forms of molecules. The structural components of the LPS molecule—lipid A, core oligosaccharide, and O-specific polysaccharide—were obtained in the individual state and characterized. The main components of the lipid A hydrophobic moiety were 3-hydoxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, and hexadecanoic fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic moiety. Rhamnose, glucose, galactose, glucosamine, galactosamine, alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as 2-amino-2,6-dideoxygalactose (FucN) and 3-amino-3,6-dideoxyglucose (Qui3N), were revealed in the composition of the core oligosaccharide fractions. O-specific polysaccharide chains were composed of repeating trisaccharide units consisting of residues of L-rhamnose (L-Rha), 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc), and 3-acylamido-3,6-dideoxy-D-glucose (D-Qui3NAcyl), where Acyl = 3-hydroxy-2,3-dimethyl-5-hydroxyprolyl. Neither double immunodiffusion in agar not the immunoenzymatic assay revealed serological relations between the strain studied and the P. fluorescens strains studied earlier.     

Interaction of Porin from Yersinia pseudotuberculosis with Different Structural Forms of Endogenous Lipopolysaccharide

The interaction of Yersinia pseudotuberculosis porin solubilized in deoxycholate with the S- and R-forms of endogenous lipopolysaccharide (LPS) was studied by the quenching of intrinsic protein fluorescence. The samples of S-LPS differed both in the length of O-specific polysaccharide (n = 1 and 4) and in the acylation degree of the 3-hydroxytetradecanoic acid residues of the lipid A moiety (12-66%). R-LPS (12%) binding to porin was found to occur with positive cooperativity on two integrated structural regions of the R-LPS macromolecule, namely, core oligosaccharide and lipid A. The mode of porin interaction with low-acylated S-LPSs (15 or 20%) coincided with a model involving three types of binding sites. The shape of Scatchard curves of binding indicates that a complex formation between porin and low-acylated S-LPS is cooperative at low and moderate ligand concentration, whereas at near-saturating LPS concentrations porin binds to LPS independently on two types of binding sites. The O-specific polysaccharide chain in the S-LPS macromolecule increases the affinity of its interaction with porin in comparison with R-LPS–porin binding. A significant increase (to 66%) in the degree of S-LPS acylation substantially changed its porin-binding character: the process becomes anti-cooperative with lowered affinity. Thus, the features of LPS–porin interaction significantly depend on the conformational changes in the LPS molecule due to expanding of its hydrophobic region.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

High state of order of isolated bacterial lipopolysaccharide and its possible contribution to the permeation barrier property of the outer membrane.

The conformational properties of the isolated S form of Salmonella sp. lipopolysaccharide (LPS), of Re mutant LPS, and of free lipid A were investigated by using X-ray diffraction and conformational energy calculations. The data obtained showed that LPS in a dried, in a hydrated, and probably also in an aqueous dispersion state is capable of forming bilayered lamellar arrangements similar to phospholipids. From the bilayer packing periodicities, a geometrical model of the extensions of the LPS regions lipid A, 2-keto-3-deoxyoctulosonic acid, and O-specific chain along the membrane normal could be calculated. Furthermore, the lipid A component was found to assume a remarkably high ordered conformation: its fatty acid chains were tightly packed in a dense hexagonal lattice with a center-to-center distance of 0.49 nm. The hydrophilic backbone of lipid A showed a strong tendency to form domains in the membrane, resulting in a more or less parallel arrangement of lipid A units. According to model calculations, the hydrophilic backbone of lipid A appears to be oriented approximately 45 degrees to the membrane surface, which would lead to a shed roof-like appearance of the surface structure in the indentations of which the 2-keto-3-deoxyoctulosonic acid moiety would fit. In contrast, the O-specific chains assume a low ordered, heavily coiled conformation. Comparison of these structural properties with those known for natural phospholipids in biological membranes indicates that the high state of order of the lipid A portion of LPS might be an important factor in the structural role and permeation barrier functions of LPS in the outer membrane of gram-negative bacteria.     

The distinctive features of the structure of the Pseudomonas fluorescens IMV 247 (biovar II) lipopolysaccharide

The results of the study of the Pseudomonas fluorescens IMV 247 (biovar II) lipopolysaccharide (LPS) isolated from the dry bacterial mass by Westphal's method and purified by repeated ultracentrifugation are presented. The macromolecular organization of the LPS is characterized by the presence of S and R forms of LPS molecules in a 1:1 ratio. The structural components of the LPS molecule--lipid A, the core oligosaccharide, and the O-specific polysaccharide--were isolated and characterized. 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, and dodecanoic acids proved to be the main lipid A fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic portion. Glucose, galactose, arabinose, rhamnose, glucosamine, alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulonate (KDO) were revealed in the heterogeneous fraction of the core oligosaccharide. The O-specific polysaccharide chain was composed of repeating tetrasaccharide units consisting of L-rhamnose (L-Rha), 3,6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido-2,4,6-trideoxy-4[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb), and 2-acetamido-2-deoxy-D-galacturonic acid (D-GalNAcA) residues. A peculiarity of the O-specific polysaccharide was that it released, upon partial acid hydrolysis, the nonreducing disaccharide GalNAcA-->QuiNAc4NHb with a 3-hydroxybutyryl group glycosylated intramolecularly with a QuiN4N residue. Double immunodiffusion in agar and lipopolysaccharide precipitation reactions revealed no serological interrelationship between the strain studied and the P. fluorescens strains studied earlier.