Comparison of R- and S-form lipopolysaccharides fractionated from Escherichia coli UKT-B lipopolysaccharide in pyrogen and Limulus tests

Different LPS was shown to have a relatively different proportion of O-specific chain-less (R-form) LPS by polyacrylamide gel electrophoresis (PAGE) with sodium deoxycholate (DOC). By using DOC-PAGE, S-form LPS having O-chain with approximately 11 repeating units on average (S-Fr) and O-chain-less LPS (R-Fr) were separated from Escherichia coli UKT-B S-form LPS. Significantly stronger pyrogenicity was observed in R-Fr than in S-Fr when measured on the weight basis. Similar result was observed in Limulus test. Comparing biological activities of different S-form LPS, attention should be given to the amounts of co-existing R-form LPS.     

Modification of the chemical composition and structure of the U.S. Reference Standard Endotoxin (RSE) by 60Co radiation

A highly purified bacterial lipopolysaccharide (LPS) preparation was exposed in water to megadoses of ionizing radiation from a 60Co source. As evidenced by electrophoresis, the radiation treatment progressively degraded the lipopolysaccharide molecules by removing first the O-side chain units and then components of the R-core. Chemical analysis of the irradiated (LPS) preparations showed that, in accord with the structural changes, the most profound effects of ionizing radiation occurred in the hydrophilic oligo/polysaccharide moieties (R-core and O-side chain). Progressively higher doses of radiation degraded the simple sugars in decreasing order of galactose, galactosamine, glucosamine, glucose, and heptose. The R-core component 2-keto-3-deoxyoctonate was the most "resistant" sugar derivative to ionizing radiation. Due to its central position in the LPS aggregates in water, even at comparatively high doses of radiation the hydrophobic lipid A moiety of endotoxin was less affected than the sugar components. Of the fatty acids of lipid A, however, either partial conversion of beta-hydroxymyristic acid into myristic acid or selective loss of the former occurred. The observed structural and chemical changes of LPS are consistent with the effect of active oxygen radicals of radiolysis. In addition, the extensive physicochemical changes explain the altered biological reactivity of radiation-treated endotoxins.     

Conformation of Single and Interacting Lipopolysaccharide Surfaces Bearing O-Side Chains

The outer surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS) molecules exposing oligo- and polysaccharides to the aqueous environment. This unique, structurally complex biological interface is of great scientific interest as it mediates the interaction of bacteria with antimicrobial agents as well as with neighboring bacteria in colonies and biofilms. Structural studies on LPS surfaces, however, have so far dealt almost exclusively with rough mutant LPS of reduced molecular complexity and limited biological relevance. Here, by using neutron reflectometry, we structurally characterize planar monolayers of wild-type LPS from Escherichia coli O55:B5 featuring strain-specific O-side chains in the presence and absence of divalent cations and under controlled interaction conditions. The model used for the reflectivity analysis is self-consistent and based on the volume fraction profiles of all chemical components. The saccharide profiles are found to be bimodal, with dense inner oligosaccharides and more dilute, extended O-side chains. For interacting LPS monolayers, we establish the pressure-distance curve and determine the distance-dependent saccharide conformation.     

Characterization of biological activities of Brucella melitensis lipopolysaccharide

Biological activities of lipopolysaccharide (LPS) from Brucella melitensis 16M were characterized in comparison with LPS from Escherichia coli O55. LPS extracted from B. melitensis was smooth type by electrophoretic analysis with silver staining. The endotoxin-specific Limulus activity of B. melitensis LPS was lower than that of E. coli LPS. There was no significant production of tumor necrosis factor-alpha and nitric oxide in RAW 264.7 macrophage cells stimulated with B. melitensis LPS, although E. coli LPS definitely induced their production. On the other hand, B. melitensis LPS exhibited a higher anti-complement activity than E. coli LPS. B. melitensis LPS as well as E. coli LPS exhibited a strong adjuvant action on antibody response to bovine serum. The characteristic biological activities of B. melitensis are discussed.     

Relation of structure to function in bacterial endotoxins. VIII. Biological activities in a polysaccharide-rich fraction.

This is the first report describing in vivo biologic activities elicited by a non-toxic, polysaccharide-rich, water soluble fraction obtained by partial acidic hydrolysis from endotoxic lipopolysaccharide. The two activities present in this preparation were a) mouse bone marrow cell colony formation stimulation (CSF) and b) protection of mice against lethal irradiation. With polysaccharide-deficient rough mutants of salmonella minnesota, the CSF-inducing activity could be restricted to the "core" region of the LPS structure. Sixty-minute hydrolysis with 1 N HCl at 100 degrees C or 0.1 M sodium metaperiodate oxidation at cold room temperature completely abolished CSF-inducing activity of the preparation, whereas it showed considerable resistance to mild alkaline hydrolysis. These findings indicate that the active component in this preparation is carbohydrate in nature. Lipid preparations from smooth LPS or from Re rough mutants are either much less active or completely inactive in the above two assays. The fully active polysaccharide rich preparation was found to be inert in seven other characteristic endotoxicity parameters.     

The structure of the core region of the lipopolysaccharide from Geobacter sulfurreducens

The structure of the core part of the LPS from Geobacter sulfurreducens was analysed. The LPS contained no O-specific polysaccharide (O-side chain) and upon mild hydrolysis gave a core oligosaccharide, which was isolated by gel chromatography. It was studied by chemical methods, NMR and mass spectrometry, and the following structure was proposed. [carbohydrate structure: see text] where Q = 3-O-Me-alpha-L-QuiNAc-(1-->or H (approximately 3:2).     

MD-2 binds to bacterial lipopolysaccharide

The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex. MD-2 binds to highly purified rough LPS derived from Salmonella minnesota and Escherichia coli in five different assays; one assay yielded an apparent KD of 65 nm. MD-2 binding to LPS did not require LPS-binding proteins LBP and CD14; in fact LBP competed with MD-2 for LPS. MD-2 enhanced the biological activity of LPS in toll-like receptor 4-transfected Chinese hamster ovary cells but inhibited LPS activation of U373 astrocytoma cells and of monocytes in human whole blood. These data indicate that MD-2 is a genuine LPS-binding protein and strongly suggest that MD-2 could play a role in regulation of cellular activation by LPS depending on its local availability.     

Biological activities of lipopolysaccharide-like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton

The biological activities of lipopolysaccharide-like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton by the hot phenol-water method were studied in mice. The addition of 12.5 micrograms/ml or more of LLS fraction increased the incorporation of [3H]thymidine into in vitro cultured spleen cells of C57BL/6 mice, while the activity of the LLS fraction was about 20 times weaker than that of Salmonella typhimurium lipopolysaccharide (LPS). Pretreatment of murine spleen cells with rabbit anti-mouse thymocyte antiserum did not diminish the mitogenic activity of leptospiral LLS, and the LLS could not increase the incorporation of [3H]thymidine into thymocytes, suggesting that LLS acts on a B-lymphocyte population of lymphocytes. When sheep erythrocytes and LLS fraction were injected intraperitoneally into BALB/c mice, LLS exhibited an enhancing effect on antibody response in vivo. However, lethal toxicity of the LLS fraction was about 500 times lower than that of LPS in C57BL/6 mice loaded with galactosamine. No antitumor activity of leptospiral LLS (250-1,000 micrograms/mouse) against the ascites form of Ehrlich carcinoma in ddY mice was observed. The biological activities of the LLS fraction from the organism were weaker than those of gram-negative bacterial LPS, suggesting that Leptospira possesses no typical LPS.     

Characterization of lipopolysaccharide fractions and their interactions with cells and model membranes.

The role of the length of the O-antigen polysaccharide side chain of bacterial lipopolysaccharide (LPS) in biological and model membrane systems was investigated. LPS from Salmonella typhimurium ATCC 14028 was chromatographed on a Sephadex G-200 column in the presence of sodium deoxycholate and separated into three fractions on the basis of molecular size. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot (immunoblot), and chemical analyses indicated that these fractions differed from each other primarily in the number of repeating units in the O-antigen polysaccharide side chain. In a biological system fractions 2 and 3 had the same effects to induce mitogenesis in murine lymphocytes, but fraction 1 was less effective than the other two fractions. In a model membrane system, LPS induced changes in small unilamellar vesicles (SUVs) which were measured by changes in the behavior of a fluorescent probe, 1,6-diphenylhexa-1,3,5-triene (DPH), and interaction of increasing amounts of all LPS fractions with SUVs gradually increased DPH anisotropy. Fractions 2 and 3 had similar effects on the SUVs as detected by changes in DPH anisotropy, while fraction 1 had almost twice as much activity as the other two fractions. These results suggest that the polysaccharide side chain of LPS may modulate the ability of biologically active lipid A to interact with cells and model membranes. In addition, factors other than changes in membrane fluidity may play a role in mediating LPS-induced cell activation.     

Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.     

Adjuvant activity of Klebsiella O3 lipopolysaccharide: inhibition of the adjuvant activity by concanavalin A

Klebsiella O3 lipopolysaccharide (KO3 LPS) was found to exhibit extraordinarily strong adjuvant activity in augmenting antibody responses and delayed-type hypersensitivity (DTH) to protein antigens in mice. The O-specific polysaccharide chain of KO3 LPS consists of alpha-mannoside. We investigated the effect of concanavalin A (Con A) or succinyl Con A, which is known to bind to alpha-mannoside, on the adjuvant activity of KO3 LPS in augmenting DTH to ovalbumin. When KO3 LPS was mixed with Con A prior to injection, the strong adjuvant activity of KO3 LPS in augmenting DTH was inhibited and the degree of inhibition depended upon the dose of Con A. An equal amount of Con A elicited nearly complete inhibition of the adjuvant activity of KO3 LPS, Con A at 1/10 the amount of LPS elicited partial inhibition, and Con A at 1/100 the amount of LPS showed no inhibition. An equal amount of succinyl Con A, which induced less marked aggregation of KO3 LPS than Con A, elicited inhibition of the adjuvant activity of KO3 LPS to an extent similar to that by Con A. On the other hand, Con A or succinyl Con A bound to KO3 LPS did not impair in any way the lethal toxicity of KO3 LPS for mice which is known to be due to the lipid A moiety. From these findings it is concluded that the strong adjuvant activity of KO3 LPS does not solely depend upon the lipid A moiety but the O-specific polysaccharide moiety plays an important role in expression of the adjuvant activity.     

The Effect of galE Gene Inactivation on Lipopolysaccharide Profile of Helicobacter pylori

The galE gene product, UDP-galactose 4-epimerase, mediates the incorporation of galactose in extracellular polysaccharide materials such as the O-side chain of lipopolysaccharide (LPS). The O-side chain in H. pylori LPS has been shown to cross-react with Lewis x and/or y blood group antigens, suggesting its potential involvement in H. pylori-linked autoimmune disease. To study its role in H. pylori LPS biosynthesis, the galE gene was cloned, sequenced, and a galE-knockout H. pylori strain was constructed. The H. pylori galE gene encoded a protein of 344 amino acids with a molecular weight of 39K. The LPS profile from the galE-knockout H. pylori strain showed a lower molecular weight than that of the parental strain, indicating the involvement of the galE gene in LPS biosynthesis of H. pylori. Received: 15 December 1997 / Accepted: 10 March 1998     

The immunogenic and serological properties of the O-specific polysaccharide (L-hapten) in Shigella]

O-specific polysaccharide (L-hapten) was isolated earlier (Zh. mikrobiol. epidemiol. immunobiol., 1989, No. 11, pp. 8-11). In this paper L-hapten was shown to be unable, even at high concentrations (up to 2,000 micrograms/ml), to sensitize sheep red blood cells for passive hemagglutination by O-antibodies. At the same time classical LPS and heat-activated LPS were active at concentrations ot 32 and 8 micrograms/ml respectively. The O-antibody-neutralizing activity of L-hapten was lower than that of LPS 10(3)-10(4) times in the passive hemagglutination test and 25-50 times in competitive ELISA. The immunogenicity of isolated L-hapten was very weak: primary response in mice to the i.v. injection of 1-10 micrograms of L-hapten was similar to the effect produced by 10(-3)-10(-4) micrograms of LPS. No protective activity of L-hapten was noted in mice when the challenge dose of virulent shigellae was 16 LD50 or more, and only a weak protective effect was observed with a low challenge dose (8 LD50). The molecular basis of low serological and biological activity of L-hapten is discussed. The most probable explanation of the results obtained in this study is that L-hapten contains some nonspecific carbohydrates, inserted in or complexed with the O-side chain. Despite its low immunogenicity, L-hapten can be an important component of effective bacterial vaccines provided it is included into a suitable delivery system as is the case with Shigella ribosomal vaccine.     

Release of membrane vesicles containing endotoxic lipopolysaccharide in Escherichia coli O157:H7 clinical isolates

Membrane vesicles released by E. coli O157:H7 strains were investigated by immuno-electron microscopy using anti-O157 antibody. Anti-O157 antibody enhanced the negative-staining of vesicles and we found numerous small vesicles clearly formed around bacterial cells. An immunogold-electron microscopic examination confirmed that lipopolysaccharide (LPS) including the O-side chain is present on the surface of the vesicles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified vesicles showed that the vesicles contained LPS consisting of a lipid-A and an O polysaccharide. In addition, the endotoxic activity of the vesicle was confirmed by a limulus test. These results suggest that the vesicles may play an important role in the pathogenesis of Escherichia coli O157:H7.     

Enhancement of endotoxicity and reactivity with carbocyanine dye by sonication of lipopolysaccharide

The specificity of endotoxin (lipopolysaccharide, LPS) in the carbocyanine dye reaction was investigated, and then a stoichiometric study of the dye-LPS interaction was conducted with attention to the relationship of biological activities of LPS to the reactivity with the dye. Absorption maxima of some bacterial components in the dye reaction were as follows; LPS from both Escherichia coli and Pseudomonas aeruginosa and lipid A from E. coli LPS, 465 nm; Shigella flexneri LPS, 460 nm; Salmonella minnesota R595 glycolipid, 470 nm; polysaccharide from E. coli LPS, 650 nm; yeast RNA, 620 nm; streptococcal M protein and pyrogenic exotoxin, 610 nm; and free fatty acids, 445-450 nm. The absorbance at 465 nm was increased approximately threefold by sonicating LPS for 1-3 min, which roughly paralleled the decrease in turbidity of the LPS aqueous solution. The Limulus amoebocyte lysate (LAL) gelation activity of LPS increased 10-fold when LPS was sonicated for 0.5-5 min, but it decreased to the control level after further treatment. This decrease, however, was overcome by sonication in the presence of 5 mmol of L-ascorbic acid used as an antioxidant. The LAL gelation activity of LPS was inactivated in parallel with an increase in the ratio (w/w) of dye to LPS from 1.73 to 6.90 in the dye-LPS mixture. Pyrogenicity of LPS was also clearly inactivated when the ratio was over 1.73. The ratios of the height of the beta band at 465 nm (dye-LPS complex) to that of the alpha band at 510 nm (free dye) were increased by sonicating LPS, indicating that the binding character, or stacking tendency, was increased by sonicating LPS.     

DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth.

The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was interferon (IFN)-gamma and that to inhibit viral growth was IFN-alpha/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS-insensitive C3H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including 3 of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G-C motif(s) are required for induction of IFNs and activation of NK cells with synthetic 30-mer oligonucleotides.     

Production of pectinases by Polyporus squamosus in aqueous two-phase system

The ability of Polyporus squamosus to grow and produce pectinases in an aqueous two-phase medium composed of polyethylene glycol and crude dextran is reported. Fungal growth was restricted to the bottom phase leaving the top phase cell free. Amounts of produced biomass and endo and exo-pectinase activities were superior or equal to those obtained in homogeneous medium. The partition coefficient for the endo-pectinase was 1.52 followed by a top phase yield of 70.86%. Although the phase system composition favours partition of a greater part of exo-pectinase activity to the bottom phase (K(exo) was 0.6 and yield in top phase 48.56%) the partitioned activity in the top phase was equal to that produced in homogeneous cultivation.     

Isolation and biological activities of limulus anticoagulant (anti-LPS factor) which interacts with lipopolysaccharide (LPS)

Exposure of limulus hemocytes to bacterial endotoxins (lipopolysaccharide, LPS) results in the activation of the intracellular clotting system, consisting of several protein components. During the separation of these components, a potent anticoagulant, named anti-LPS factor, which inhibits the endotoxin-mediated activation of the coagulation cascade, was found in hemocytes from both Tachypleus tridentatus and Limulus polyphemus (Tanaka, S., et al. (1982) Biochem. Biophys. Res. Commun. 105, 717-723). The principle isolated from the Tachypleus hemocyte lysate by column chromatographies on dextran sulfate-Sepharose CL-6B and Sephadex G-50 under sterile conditions was a simple basic protein with an apparent molecular weight of 15,000. It consisted of a single chain polypeptide containing a total of 128 amino acids. The COOH-terminal end was presumed to be histidine, but no NH2-terminal end reactive to phenylisothiocyanate was detected. The isolated anti-LPS factor specifically inhibited the endotoxin-mediated activation of factor C, which has recently been identified as an LPS-sensitive serine protease zymogen in the hemocytes. This inhibition appeared to be due to the binding of anti-LPS factor with LPS. Moreover, anti-LPS factor had an antibacterial effect on the growth of Gram-negative bacteria (Salmonella minnesota R595 and 1114W) but not on that of Gram-positive bacteria (Staphylococcus aureus 209P). These biological activities of the isolated anti-LPS factor suggest an important role in cellular defence of limulus against microbial invasion.     

Characterization of the lipopolysaccharides and capsules of Shewanella spp

Electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining and (1)H, (13)C, and (31)P-nuclear magnetic resonance (NMR) were used to detect and characterize the lipopolysaccharides (LPSs) of several Shewanella species. Many expressed only rough LPS; however, approximately one-half produced smooth LPS (and/or capsular polysaccharides). Some LPSs were affected by growth temperature with increased chain length observed below 25 degrees C. Maximum LPS heterogeneity was found at 15 to 20 degrees C. Thin sections of freeze-substituted cells revealed that Shewanella oneidensis, S. algae, S. frigidimarina, and Shewanella sp. strain MR-4 possessed either O-side chains or capsular fringes ranging from 20 to 130 nm in thickness depending on the species. NMR detected unusual sugars in S. putrefaciens CN32 and S. algae BrY(DL). It is possible that the ability of Shewanella to adhere to solid mineral phases (such as iron oxides) could be affected by the composition and length of surface polysaccharide polymers. These same polymers in S. algae may also contribute to this opportunistic pathogen's ability to promote infection.