lipid-a and alpha-hydroxymyristic-acid

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella PhoP/PhoQ-activated gene pagP is required for resistance to cationic antimicrobial peptides and for biosynthesis of hepta-acylated lipid A species containing palmitate. Interestingly, pagP encodes an unusual enzyme of lipid A biosynthesis localized in the outer membrane, whereas all previously characterized lipid A enzymes are cytoplasmic or associated with the inner membrane. PagP is not unique, however, as pagL encodes another outer membrane enzyme in Salmonella that deacylates the 3 position of lipid A.S. typhimurium also synthesizes S-2-hydroxymyristate modified lipid A in a PhoP/PhoQ-dependent manner. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Using well-characterized dioxygenase sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A. The S. typhimurium aspartyl beta-hydroxylase homologue (lpxO) was cloned and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression induces O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO may be an inner membrane enzyme that catalyzes Fe(2+)/ascorbate/alpha-ketoglutarate dependent hydroxylation of lipid A. We propose that 2-hydroxymyristate released from LPS inside infected animal cells might be converted to 2-hydroxymyristoyl coenzyme A, a potent inhibitor of protein N-myristoyl transferase.

Topics: Bacterial Proteins; Carboxylic Ester Hydrolases; Catalysis; Cations, Divalent; Escherichia coli; Hydrogen-Ion Concentration; Lipid A; Magnesium; Mixed Function Oxygenases; Models, Chemical; Molecular Structure; Myristic Acids; Salmonella typhimurium; Transcription Factors

Lipid A of Salmonella typhimurium can be resolved into multiple molecular species. Many of these substances are more polar than the predominant hexa-acylated lipid A 1,4'-bisphosphate of Escherichia coli K-12. By using new isolation methods, we have purified six lipid A subtypes (St1 to St6) from wild type S. typhimurium. We demonstrate that these lipid A variants are covalently modified with one or two 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties. Each lipid A species with a defined set of polar modifications can be further derivatized with a palmitoyl moiety and/or a 2-hydroxymyristoyl residue in place of the secondary myristoyl chain at position 3'. The unexpected finding that St5 and St6 contain two l-Ara4N residues accounts for the anomalous structures of lipid A precursors seen in S. typhimurium mutants defective in 3-deoxy-d-manno-octulosonic acid biosynthesis in which only the 1-phosphate group is modified with the l-Ara4N moiety (Strain, S. M., Armitage, I. M., Anderson, L., Takayama, K., Quershi, N., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 16089-16098). Phosphoethanolamine (pEtN)-modified lipid A species are much less abundant than l-Ara4N containing forms in wild type S. typhimurium grown in broth but accumulate to high levels when l-Ara4N synthesis is blocked in pmrA(C)pmrE(-) and pmrA(C)pmrF(-) mutants. Purification and analysis of selected compounds demonstrate that one or two pEtN moieties may be present. Our findings show that S. typhimurium contains versatile enzymes capable of modifying both the 1- and 4'-phosphates of lipid A with l-Ara4N and/or pEtN groups. PmrA null mutants of S. typhimurium produce lipid A species without any pEtN or l-Ara4N substituents. However, PmrA is not needed for the incorporation of 2-hydroxymyristate or palmitate.

Topics: Amino Sugars; Bacterial Proteins; Carbohydrate Sequence; Chromatography; Escherichia coli; Ethanolamines; Hydrolysis; Lipid A; Models, Chemical; Molecular Sequence Data; Mutation; Myristic Acids; Palmitic Acid; Protein Binding; Protein Conformation; Salmonella typhimurium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on the S. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132-43144), ArnT utilizes the novel lipid undecaprenyl phosphate-alpha-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

Topics: Amino Sugars; Bacterial Proteins; Carbohydrate Sequence; Cell Membrane; Chromatography; Escherichia coli; Ethanolamines; Hexosyltransferases; Hydrolysis; Intracellular Membranes; Lipid A; Magnetic Resonance Spectroscopy; Models, Biological; Models, Chemical; Molecular Sequence Data; Mutation; Myristic Acids; Palmitic Acid; Polymyxins; Protein Binding; Protein Conformation; Salmonella typhimurium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

Topics: Amino Sugars; Anti-Bacterial Agents; Bacterial Proteins; Carbohydrate Sequence; Carbohydrates; Cell Nucleus; Cell-Free System; Chromatography; DEAE-Cellulose; Escherichia coli; Ethanolamines; Hydrolysis; Lipid A; Lipids; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Sequence Data; Mutation; Myristic Acids; Palmitic Acid; Periplasm; Phosphorus; Polymyxins; Protein Binding; Protein Conformation; Protein Prenylation; Salmonella typhimurium; Silicic Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The PhoP-PhoQ two-component system is necessary for the virulence of Salmonella spp. and is responsible for regulating several modifications of the lipopolysaccharide (LPS). Mutagenesis of the transcriptional regulator phoP resulted in the identification of a mutant able to activate transcription of regulated genes approximately 100-fold in the absence of PhoQ. Sequence analysis showed two single-base alterations resulting in amino acid changes at positions 93 (S93N) and 203 (Q203R). These mutations were individually created, and although each resulted in a constitutive phenotype, the double mutant displayed a synergistic effect both in the induction of PhoP-activated gene expression and in resistance to antimicrobial peptides. The constitutive phoP gene was placed under the control of an arabinose-inducible promoter to examine the kinetics of PhoP-activated gene induction and the resultant modifications of LPS. Gene induction and 2-hydroxymyristate modification of the lipid A were shown to occur within minutes of the addition of arabinose and to peak at 4 h. As the first constitutive mutant of phoP identified, this allele will be invaluable to future genetic and biochemical studies of this and likely other regulatory systems.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Microbial; Gene Expression Regulation, Bacterial; Lipid A; Lipopolysaccharides; Membrane Proteins; Molecular Sequence Data; Myristic Acids; Peptides; Phenotype; Point Mutation; Salmonella typhimurium; Sequence Homology, Amino Acid; Serotyping; Trans-Activators; Transcription Factors; Transcriptional Activation

Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacyl chains. S. typhimurium has recently been shown to synthesize its S-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O(2). Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of (18)O(2) confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/asparaginyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhimurium aspartyl/asparaginyl beta-hydroxylase homologue (designated lpxO) was cloned into pBluescriptSK and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression is sufficient to induce O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO very likely is a novel Fe(2+)/alpha-ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends. We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase.

Topics: Aerobiosis; Amino Acid Sequence; Anaerobiosis; Cloning, Molecular; Consensus Sequence; Lipid A; Mixed Function Oxygenases; Molecular Sequence Data; Myristic Acids; Oxygen Consumption; Recombinant Proteins; Salmonella typhimurium; Sequence Alignment