Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of lipopolysaccharide action on human line cells.
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The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed by TNF-alpha induction and NF-kappaB activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but all of the lipid As exerted strong activity on mouse cells and on Limulus gelation activity. Experiments using chemically synthesized E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A preparations strongly induced TNF-alpha release and activated NF-kappaB in mouse peritoneal macrophages and mouse macrophage-like cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-alpha and NF-kappaB activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS neither exhibited TNF-alpha induction activity on the cells when administered alone or together with lipid A nor inhibited the activity of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action that induced TNF-alpha release and NF-kappaB activation in THP-1 cells. (+info)
Biological activities of lipopolysaccharides are determined by the shape of their lipid A portion.
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Lipopolysaccharide (LPS) represents a major virulence factor of Gram-negative bacteria ('endotoxin') that can cause septic shock in mammals including man. The lipid anchor of LPS to the outer membrane, lipid A, has a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against LPS, a property that can be utilized in antisepsis treatment. We show here that these different biological activities are directly correlated with the molecular shape of lipid A. Only (hexaacyl) lipid A with a conical/concave shape, the cross-section of the hydrophobic region being larger than that of the hydrophilic region, exhibited strong interleukin-6 (IL-6)-inducing capacity. Most strikingly, a correlation between a cylindrical molecular shape of lipid A and antagonistic activity was established: IL-6 induction by enterobacterial LPS was inhibited by cylindrically shaped lipid A except for compounds with reduced headgroup charge. The antagonistic action is interpreted by assuming that lipid A molecules intercalate into the cytoplasmic membrane of mononuclear cells, and subsequently blocking of the putative signaling protein by the lipid A with cylindrical shape. (+info)
The role of lipopolysaccharide injected systemically in the reactivation of collagen-induced arthritis in mice.
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1. We investigated the role of bacterial lipopolysaccharide (LPS) in the reactivation of autoimmune disease by using collagen-induced arthritis (CIA) in mice in which autoimmunity to the joint cartilage component type II collagen (CII) was involved. 2. CIA was induced by immunization with CII emulsified with complete Freund's adjuvant at the base of the tail (day 0) followed by a booster injection on day 21. Varying doses of LPS from E. coli were i.p. injected on day 50. 3. Arthritis began to develop on day 25 after immunization with CII and reached a peak on day 35. Thereafter, arthritis subsided gradually but moderate joint inflammation was still observed on day 50. An i.p. injection of LPS on day 50 markedly reactivated arthritis on a dose-related fashion. Histologically, on day 55, there were marked oedema of synovium which had proliferated by the day of LPS injection, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells. The reactivation of CIA by LPS was associated with increases in anti-CII IgG and IgG2a antibodies as well as various cytokines including IL-12, IFN-gamma, IL-1beta, and TNF-alpha. LPS from S. enteritidis, S. typhimurium, and K. neumoniae and its component, lipid A from E. coli also reactivated the disease. Polymyxin B sulphate suppressed LPS- or lipid A-induced reactivation of CIA. 4. These results suggest that LPS may play an important role in the reactivation of autoimmune joint inflammatory diseases such as rheumatoid arthritis in humans. (+info)
Antibacterial agents that target lipid A biosynthesis in gram-negative bacteria. Inhibition of diverse UDP-3-O-(r-3-hydroxymyristoyl)-n-acetylglucosamine deacetylases by substrate analogs containing zinc binding motifs.
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UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A, a unique amphiphilic molecule found in the outer membranes of virtually all Gram-negative bacteria. Since lipid A biosynthesis is required for bacterial growth, inhibitors of LpxC have potential utility as antibiotics. The enzymes of lipid A biosynthesis, including LpxC, are encoded by single copy genes in all sequenced Gram-negative genomes. We have now cloned, overexpressed, and purified LpxC from the hyperthermophile Aquifex aeolicus. This heat-stable LpxC variant (the most divergent of all known LpxCs) displays 32% identity and 51% similarity over 277 amino acid residues out of the 305 in Escherichia coli LpxC. Although A. aeolicus LpxC deacetylates the substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine at a rate comparable with E. coli LpxC, a phenyloxazoline-based hydroxamate that inhibits E. coli LpxC with K(i) of approximately 50 nM (Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Galloway, S. M., Hyland, S. A., Anderson, M. S., and Raetz, C. R. H. (1996) Science 274, 980-982) does not inhibit A. aeolicus LpxC. To determine whether or not broad-spectrum deacetylase inhibitors can be found, we have designed a new class of hydroxamate-containing inhibitors of LpxC, starting with the structure of the physiological substrate. Several of these compounds inhibit both E. coli and A. aeolicus LpxC at similar concentrations. We have also identified a phosphinate-containing substrate analog that inhibits both E. coli and A. aeolicus LpxC, suggesting that the LpxC reaction proceeds by a mechanism similar to that described for other zinc metalloamidases, like carboxypeptidase A and thermolysin. The differences between the phenyloxazoline and the substrate-based LpxC inhibitors might be exploited for developing novel antibiotics targeted either against some or all Gram-negative strains. We suggest that LpxC inhibitors with antibacterial activity be termed "deacetylins." (+info)
Characterization of a novel lipid A containing D-galacturonic acid that replaces phosphate residues. The structure of the lipid a of the lipopolysaccharide from the hyperthermophilic bacterium Aquifex pyrophilus.
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According to the 16 S rRNA phylogenetic tree, the hyperthermophilic bacterium Aquifex pyrophilus represents the deepest and shortest branching species of the kingdom Bacteria. We show for the first time that an organism, which is phylogenetically ancient on the basis of its 16 S rRNA and that exists at extreme conditions, may contain lipopolysaccharide (LPS). The LPS was extracted from dried bacteria using a modified phenol/water method. SDS-polyacrylamide gel electrophoresis and silver stain displayed a ladder-like pattern, which is typical for smooth-form LPS (possessing an O-specific polysaccharide). The molecular masses of the LPS populations were determined by matrix-assisted laser-desorption ionization mass spectrometry. Lipid A was precipitated after mild acid hydrolysis of LPS. Its complete structure was determined by chemical analyses, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser-desorption ionization mass spectrometry, and one- and two-dimensional NMR spectroscopy. The lipid A consists of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-D-glucopyranose (DAG) disaccharide carrying two residues each of (R)-3-hydroxytetradecanoic acid and (R)-3-hydroxyhexadecanoic acid in amide linkage and one residue of octadecanoic acid in ester linkage. Each DAG moiety carries one residue of each 3-hydroxytetradecanoic and 3-hydroxyhexadecanoic acid. In the nonreducing DAG, the octadecanoic acid is attached to the 3-hydroxy group of 3-hydroxytetradecanoic acid. Each DAG is substituted by one D-galacturonic acid residue, which is linked to O-1 of the reducing and to O-4 of the nonreducing end. This structure represents a novel type of lipid A. (+info)
High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium.
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When Escherichia coli are grown on LB broth containing 25 mm NH(4)VO(3), complex modifications to the lipid A anchor of lipopolysaccharide are induced. Six modified lipid As (EV1-EV6) have been purified. Many of these variants possess 4-amino-4-deoxy-l-arabinose (l-Ara4N) and/or phosphoethanolamine (pEtN) substituents. Here we use NMR spectroscopy to investigate the attachment sites of the l-Ara4N and pEtN moieties on underivatized, intact EV3 and EV6 and on precursors II(A) and III(A) from kdsA mutants of Salmonella. CDCl(3)/CD(3)OD/D(2)O (2:3:1, v/v) is shown to be a superior solvent for homo- and heteronuclear one- and two-dimensional NMR experiments. The latter were not feasible previously because available solvents caused sample decomposition. Selective inverse decoupling difference spectroscopy is used to determine the attachment sites of substituents on EV3, EV6, II(A), and III(A). l-Ara4N is attached via a phosphodiester linkage to the 4'-phosphates of EV3 and EV6 and has the beta anomeric configuration. pEtN is attached by a pyrophosphate linkage to the 1-phosphate of EV6. The l-Ara4N and pEtN substituents of lipids II(A) and III(A) are attached in the opposite manner, with l-Ara4N on the 1-phosphate of II(A) and pEtN on the 4'-phosphate of III(A). Determination of the proper attachment sites of these substituents is necessary for elucidating the enzymology of lipid A biosynthesis and for characterizing polymyxin-resistant mutants, in which l-Ara4N and pEtN substituents are greatly increased. (+info)
Monophosphoryl lipid A and QS21 increase CD8 T lymphocyte cytotoxicity to herpes simplex virus-2 infected cell proteins 4 and 27 through IFN-gamma and IL-12 production.
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We have shown previously that IFN-gamma pretreatment of human epidermal cells (ECs) cultured in vitro partially reverses down-regulation of surface MHC class I by HSV infection, allowing recognition by CD8 CTLs, and that HSV immediate early (IE)/early (E) proteins are the predominant targets for CD8 CTLs. In this study of 25 subjects, CD8 CTLs recognized the HSV-2 IE infected cell protein 27 (ICP27) (expressed in autologous IFN-gamma-pretreated, Vaccinia virus recombinant-infected ECs) in all subjects studied, ICP4 in 89%, and ICP0 in 11%. The main hierarchy of recognition was ICP27 > ICP4. ICP27 was the dominant target in 89% of subjects but showed great individual variability in the degree of cytotoxicity. CD8 cytotoxicity specific for HSV-2 IE proteins was enhanced by 48-67% when CD8 CTLs were coincubated with the combination of monophosphoryl lipid A and QS21 adjuvants at the time of Ag presentation. These adjuvants also significantly enhanced IL-12 and IFN-gamma production from nonadherent mononuclear cells stimulated by HSV-2-infected ECs. Addition of IL-12 and IFN-gamma at the time of initial Ag presentation enhanced CD8 cytotoxicity to levels comparable with those stimulated by the adjuvants. Addition of neutralizing Abs to IL-12 or IFN-gamma inhibited CD8 T cell cytotoxicity up to 95% when a combination of the Abs were added at the time of initial Ag presentation. Therefore, the mechanism for the enhancement of CD8 T cell cytotoxicity by adjuvants in this system appears to be via increased levels of IL-12 and IFN-gamma. (+info)
Intrinsic conformation of lipid A is responsible for agonistic and antagonistic activity.
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Lipopolysaccharides (LPS, endotoxin) represent a major virulence factor of Gram-negative bacteria, which can cause septic shock in mammals, including man. The lipid anchor of LPS to the bacterial outer membrane, lipid A, exhibits a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is also responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against endotoxically active LPS. By applying orientation measurements with attenuated total reflectance (ATR) infrared spectroscopy on hydrated lipid A samples, we show here that these different biological activities are directly correlated to the intrinsic conformation of lipid A. Bisphosphoryl-hexaacyl lipid A molecules with an asymmetric (4/2) distribution of the acyl chains linked to the diglucosamine backbone have a large tilt angle (> 45 degrees ) of the diglucosamine backbone with respect to the membrane surface, a conical molecular shape (larger cross-section of the hydrophobic than the hydrophilic moiety), and are endotoxically highly active. Monophosphoryl hexaacyl lipid A has a smaller tilt angle, and the conical shape is less expressed in favour of a more cylindrical shape. This correlates with decreasing endotoxic activity. Penta- and tetraacyl lipid A or hexaacyl lipid A with a symmetric acyl chain distribution (3/3) have a small tilt angle (< 25 degrees ) and a cylindrical shape and are endotoxically inactive, but may be antagonistic. (+info)