lipid-a and lauric-acid

Lipid A in lipopolysaccharide (LPS) of Escherichia coli mutant strains was modified by the introduction of myristoyltransferase gene cloned from Klebsiella pneumoniae. When the gene was introduced into the mutant having lipid A containing only 3-hydroxymyristic acids, it produced lipid A with two additional myristic acids (C

Topics: Escherichia coli; Fatty Acids; Interleukin-6; Klebsiella pneumoniae; Lauric Acids; Lipid A; Myristic Acids; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transformation, Genetic

Lauroyltransferase gene (lpxL), Myristoyltransferase gene (lpxM) and palmitoyltransferase gene (crcA) of Escherichia coli BL21 were independently disrupted by the insertional mutations. The knockout mutant of two transferase genes (lpxL and crcA) produced lipid A with no lauric or palmitic acids and only a little amount of myristic acid. The mutant was susceptible to polymyxin B, but showed comparable growth with the wild-type strain at 30°C. The palmitoyltransferase gene from E. coli (crcA) or Salmonella (pagP) was amplified by PCR, cloned in pUC119, and transferred into the double-knockout mutant by transformation. The transformant contained palmitic acid in the lipid A, and recovered resistance to polymyxin B. Mass spectrometric analysis revealed that palmitic acid was linked to the hydroxyl group of 3-hydroxymyristic acid at C-2 position of proximal (reducing-end) glucosamine. LPS from the double-knockout mutant showed reduced IL-6-inducing activity to macrophage-like line cells compared to that of the wild-type strain, and the activity was only slightly restored by the introduction of palmitic acid to the lipid A. These results suggested that the introduction of one palmitic acid was enough to recover the integrity of the outer membrane, but not enough for the stimulation of macrophages.

Topics: Acyltransferases; Animals; Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Gene Knockout Techniques; Humans; Interleukin-6; Lauric Acids; Lipid A; Macrophages; Mice; Microbial Sensitivity Tests; Mutation; Myristic Acid; Myristic Acids; Palmitic Acids; Polymyxin B; RAW 264.7 Cells; Salmonella; U937 Cells

Results from our previous studies demonstrated that activation of Toll-like receptor 4 (Tlr4), the lipopolysaccharide (LPS) receptor, is sufficient to induce nuclear factor kappaB activation and expression of inducible cyclooxygenase (COX-2) in macrophages. Saturated fatty acids (SFAs) acylated in lipid A moiety of LPS are essential for biological activities of LPS. Thus, we determined whether these fatty acids modulate LPS-induced signaling pathways and COX-2 expression in monocyte/macrophage cells (RAW 264.7). Results show that SFAs, but not unsaturated fatty acids (UFAs), induce nuclear factor kappaB activation and expression of COX-2 and other inflammatory markers. This induction is inhibited by a dominant-negative Tlr4. UFAs inhibit COX-2 expression induced by SFAs, constitutively active Tlr4, or LPS. However, UFAs fail to inhibit COX-2 expression induced by activation of signaling components downstream of Tlr4. Together, these results suggest that both SFA-induced COX-2 expression and its inhibition by UFAs are mediated through a common signaling pathway derived from Tlr4. These results represent a novel mechanism by which fatty acids modulate signaling pathways and target gene expression. Furthermore, these results suggest a possibility that propensity of monocyte/macrophage activation is modulated through Tlr4 by different types of free fatty acids, which in turn can be altered by kinds of dietary fat consumed.

Topics: Adenocarcinoma; Animals; Base Sequence; Cell Line; Colonic Neoplasms; Consensus Sequence; Cyclooxygenase 2; Docosahexaenoic Acids; Drosophila Proteins; Enzyme Induction; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Genes, Reporter; Humans; Isoenzymes; Lauric Acids; Lipid A; Lipopolysaccharides; Macrophages; Membrane Glycoproteins; Membrane Proteins; Mice; Monocytes; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Receptors, Cell Surface; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Cells, Cultured

Overexpression of the Escherichia coli msbB gene on high copy plasmids suppresses the temperature-sensitive growth associated with mutations in the htrB gene. htrB encodes the lauroyl transferase of lipid A biosynthesis that acylates the intermediate (Kdo)2-lipid IVA (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). Since msbB displays 27.5% identity and 42.2% similarity to htrB, we explored the possibility that msbB encodes a related acyltransferase. In contrast to htrB, extracts of strains with insertion mutations in msbB are not defective in transferring laurate from lauroyl acyl carrier protein to (Kdo)2-lipid IVA. However, extracts of msbB mutants do not efficiently acylate the product formed by HtrB, designated (Kdo)2-(lauroyl)-lipid IVA. Extracts of strains harboring msbB+ bearing plasmids acylate (Kdo)2-(lauroyl)-lipid IVA very rapidly compared with wild type. We solubilized and partially purified MsbB from an overproducing strain, lacking HtrB. MsbB transfers myristate or laurate, activated on ACP, to (Kdo)2-(lauroyl)-lipid IVA. Decanoyl, palmitoyl, palmitoleoyl, and (R)-3-hydroxymyristoyl-ACP are poor acyl donors. MsbB acylates (Kdo)2-(lauroyl)-lipid IVA about 100 times faster than (Kdo)2-lipid IVA. The slow, but measurable, rate whereby MsbB acts on (Kdo)2-lipid IVA may explain why overexpression of MsbB suppresses the temperature-sensitive phenotype of htrB mutations. Presumably, the acyloxyacyl group generated by excess MsbB substitutes for the one normally formed by HtrB.

Topics: Acylation; Acyltransferases; Bacterial Proteins; Carbohydrate Conformation; Carbohydrate Sequence; Cell-Free System; Chromatography, Ion Exchange; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Genes, Suppressor; Genotype; Lauric Acids; Lipid A; Molecular Sequence Data; Multigene Family; Substrate Specificity

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.

Topics: Acyltransferases; Bacteriophage P1; Bacteriophage P22; Crosses, Genetic; DNA, Bacterial; DNA, Recombinant; Escherichia coli; Flagella; Genes, Bacterial; Lauric Acids; Lipid A; Mutation; Myristic Acid; Myristic Acids; Palmitic Acid; Phenotype; Salmonella typhimurium; Transduction, Genetic; Virulence

By assaying lysates of Escherichia coli generated with the hybrid lambda bacteriophages of an ordered library (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508), we identified two clones (lambda232 and lambda233) capable of overexpressing the lauroyl transferase that functions after 3-deoxy-D-manno-octulosonic acid (Kdo) addition in lipid A biosynthesis (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). The E. coli DNA inserts in lambda232 and lambda233 suggested that a known gene (htrB) required for rapid growth above 33 degrees C might encode the lauroyl transferase. Using the intermediate (Kdo)2-lipid IVA as the laurate acceptor, extracts of strains with transposon insertions in htrB were found to contain no lauroyl transferase activity. Cells harboring hybrid htrB+ plasmids overproduced transferase activity 100-200-fold. The overproduced transferase was solubilized with a non-ionic detergent and purified further by DEAE-Sepharose chromatography. With lauroyl acyl carrier protein as the donor, the purified enzyme rapidly incorporated one laurate residue into (Kdo)2-lipid IVA. The rate of laurate incorporation was reduced by several orders of magnitude when either one or both Kdos were absent in the acceptor. With a matched set of acyl-acyl carrier proteins, the enzyme incorporated laurate 3-8 times faster than decanoate or myristate, respectively. Transfer of palmitate, palmitoleate, or R-3-hydroxymyristate was very slow. Taken together with previous studies, our findings indicate that htrB encodes a key, late functioning acyltransferase of lipid A biosynthesis.

Topics: Acylation; Acyltransferases; Bacterial Proteins; Bacteriophage lambda; Enzyme Stability; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Lauric Acids; Lipid A

Unlike Escherichia coli, living cells of Pseudomonas aeruginosa can complete the fatty acylation of lipid A when the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo) is inhibited (R. C. Goldman, C. C. Doran, S. K. Kadam, and J. O. Capobianco, J. Biol. Chem. 263:5217-5233, 1988). In this study, we demonstrate the presence of a novel enzyme in extracts of P. aeruginosa that can transfer lauroyl-acyl carrier protein (ACP) to a tetraacyl disaccharide-1,4'-bis-phosphate precursor of lipid A (termed lipid IVA) that accumulates in Kdo-deficient mutants of E. coli. Comparable E. coli extracts cannot transfer laurate from lauroyl-ACP to lipid IVA, only to (Kdo)2-lipid IVA (K. A. Brozek, and C. R. H. Raetz, J. Biol. Chem. 265:15410-15417, 1990). P. aeruginosa extracts do not utilize myristoyl- or R-3-hydroxymyristoyl-ACP instead of lauroyl-ACP to acylate lipid IVA. Laurate incorporation in P. aeruginosa extracts is dependent upon time, protein concentration, and the presence of Triton X-100 but is inhibited by lauroyl-coenzyme A. P. aeruginosa extracts transfer only one laurate to lipid IVA, whereas E. coli extracts can transfer two laurates to (Kdo)2-lipid IVA. These results demonstrate that incorporation of laurate into lipid A does not require prior attachment of Kdo in all gram-negative bacteria.

Topics: Acyl Carrier Protein; Acylation; ADP Ribose Transferases; Bacterial Toxins; Cell-Free System; Cytoplasm; Exotoxins; Glycolipids; Glycoproteins; Lauric Acids; Lipid A; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Spectrometry, Mass, Fast Atom Bombardment; Substrate Specificity; Sugar Acids; Transferases; Virulence Factors

In previous studies we described enzyme(s) from Escherichia coli that transfer two 3-deoxy-D-manno-octulosonate (KDO) residues from two CMP-KDO molecules to a tetraacyldisaccharide-1,4'-bis-phosphate precursor of lipid A, termed lipid IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem. 264, 6956-6966). The product, designated (KDO)2-IVA, can be prepared in milligram quantities and/or radiolabeled with 32P at position 4' of the IVA moiety. We now demonstrate the presence of enzymes in E. coli extracts that transfer laurate and/or myristate residues from lauroyl or myristoyl-acyl carrier protein (ACP) to (KDO)2-IVA. Thioesters of coenzyme A are not substrates. The cytosolic fraction catalyzes rapid acylation with lauroyl-ACP, but not with myristoyl, R-3-hydroxymyristoyl, palmitoyl, or palmitoleoyl-ACP. The membrane fraction transfers both laurate and myristate to (KDO)2-IVA. Evidence for the enzymatic acylation of (KDO)2-IVA is provided by (a) conversion of [4'-32P](KDO)2-IVA to more rapidly migrating products in the presence of the appropriate acyl-ACP, (b) incorporation of [1-14C]laurate or [1-14C]myristate into these metabolites in the presence of (KDO)2-IVA, (c) fast atom bombardment-mass spectrometry, and (d) 1H NMR spectroscopy. At protein concentrations less than 0.5 mg/ml, the acylation of (KDO)2-IVA by the cytoplasmic fraction is absolutely dependent upon the addition of exogenous acyl-ACP. These acyltransferases cannot utilize lipid IVA as a substrate, demonstrating that they possess novel KDO recognition domains. The unusual substrate specificity of these enzymes provides compelling evidence for their involvement in lipid A biosynthesis. Depending on the conditions it is possible to acylate (KDO)2-IVA with 1 or 2 lauroyl residues, with 1 or 2 myristoyl residues, or with 1 of each.

Topics: Acyl Carrier Protein; Acylation; Escherichia coli; Kinetics; Lauric Acids; Lipid A; Myristic Acid; Myristic Acids; Sugar Acids

The present paper describes the isolation and characterization of a mutant (mutant Ts7) of Salmonella typhimurium that is conditionally defective in the incorporation of dodecanoic and tetradecanoic acid into lipopolysaccharide precursor structures. Enrichment of mutant Ts7 was achieved by free-flow electrophoresis and was based on a previous observation that at least some Salmonella mutants conditionally blocked in the synthesis of the 3-deoxy-D-manno-octulosonic acid (dOc1A)-lipid-A region exhibit higher electrophoretic mobilities than cells with intact dOc1A-lipid-A regions. Under nonpermissive conditions (42 degrees C) mutant Ts7 accumulates at least two incomplete dOc1A-lipid-A structures. One is made up of glucosamine, phosphate, dOc1A, and 3-hydroxytetradecanoic acid in a molar ratio 1.0:1.3:1.0:2.2 and is devoid of dodecanoic and tetradecanoic acid. The other structure has the same basic structure but contains hexadecanoic acid.

Topics: Acetylation; Chromatography, DEAE-Cellulose; Chromatography, Paper; Electrophoresis; Glycolipids; Lauric Acids; Lipid A; Lipopolysaccharides; Mutation; Myristic Acid; Myristic Acids; Salmonella typhimurium