lipid-a and 2-keto-3-deoxyoctonate

Methods for rapid and simple analysis of lipopolysaccharide (LPS) from bacterial whole-cell lysates or membrane preparations have contributed to advancing our knowledge of the genetics of the LPS biogenesis. LPS, a major constituent of the outer membranes in Gram-negative bacteria, has a complex mechanism of synthesis and assembly that requires the coordinated participation of many genes and gene products. This chapter describes a collection of methods routinely used in our laboratory for the characterization of LPS in Escherichia coli and other bacteria.

Topics: Bacteriological Techniques; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Glycine; Lipid A; Lipopolysaccharides; Molecular Probe Techniques; O Antigens; Silver Staining; Sugar Acids; Sulfhydryl Compounds

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.

Topics: Carbohydrate Sequence; Endotoxins; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glycosylation; Heptoses; Lipid A; Lipopolysaccharides; Molecular Sequence Data; O Antigens; Polysaccharides, Bacterial; Sugar Acids

Topics: Animals; Antigens, Bacterial; Endotoxins; Gram-Negative Bacteria; Lipid A; Lipopolysaccharides; Molecular Structure; O Antigens; Sugar Acids

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen

Topics: Animals; Bacterial Proteins; Blood Proteins; Chickens; Computational Biology; Drug Resistance, Bacterial; Ethanolaminephosphotransferase; Ethanolamines; Factor For Inversion Stimulation Protein; Galactose; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Heptoses; Isoenzymes; Lipid A; Mutation; Nuclear Proteins; Pasteurella Infections; Pasteurella multocida; Phylogeny; Protein Precursors; Sugar Acids; Transcriptome

Most Gram-negative organisms produce lipopolysaccharide (LPS), a complex macromolecule anchored to the bacterial membrane by the lipid A moiety. Lipid A is synthesized via the Raetz pathway, a conserved nine-step enzymatic process first characterized in Escherichia coli. The Epsilonproteobacterium Helicobacter pylori uses the Raetz pathway to synthesize lipid A; however, only eight of nine enzymes in the pathway have been identified in this organism. Here, we identify the missing acyltransferase, Jhp0255, which transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A, an activity similar to that of E. coli LpxM. This enzyme, reannotated as LpxJ due to limited sequence similarity with LpxM, catalyses addition of a C12:0 or C14:0 acyl chain to the 3'-linked primary acyl chain of lipid A, complementing an E. coli LpxM mutant. Enzymatic assays demonstrate that LpxJ and homologues in Campylobacter jejuni and Wolinella succinogenes can act before the 2' secondary acyltransferase, LpxL, as well as the 3-deoxy-d-manno-octulosonic acid (Kdo) transferase, KdtA. Ultimately, LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homologue, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homologue.

Topics: Acylation; Acyltransferases; Bacteria; Bacterial Proteins; Epsilonproteobacteria; Genetic Complementation Test; Lipid A; Multigene Family; Mutation; Phenotype; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Sugar Acids

Lipopolysaccharide (LPS) is an important virulence factor of Burkholderia cepacia, an opportunistic bacterial pathogen that causes life-threatening disease in cystic fibrosis patients and immunocompromised individuals. B. cepacia LPS comprises an O-specific polysaccharide covalently linked to a core oligosaccharide (OS) which in turn is attached to a lipid A moiety. The complete structure of the LPS core oligosaccharide from B. cepacia serotype O4 was investigated by detailed NMR and mass spectrometry (MS) methods. High- (HMW) and low-molecular-weight (LMW) OSs were obtained by deacylation, dephosphorylation, and reducing-end reduction of the LPS. Glycan and NMR analyses established that both OSs contain a common inner-core structure consisting of D-glucose, L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, 3-deoxy-D-manno-octulsonic acid, and D-glycero-D-talo-2-octulosonic acid. The structure of the LMW OS differed from that of the HMW OS in that it lacks a tetra-rhamnosyl GlcNAc OS extension. These structural conclusions were confirmed by tandem MS analyses of the two OS fractions as well as an OS fraction obtained by alkaline deacylation of the LPS. The location of a phosphoethanolamine substituent in the core region was determined by ESI-MS and methylation analysis of O-deacylated LPS and core OS samples. A polyclonal antibody to B. cepacia serotype O4 core OS was cross-reactive with several other serotypes indicating common structural features.

Topics: Burkholderia cepacia; Ethanolamines; Glucose; Heptoses; Lipid A; Lipopolysaccharides; Methylation; Models, Chemical; Nuclear Magnetic Resonance, Biomolecular; O Antigens; Polysaccharides, Bacterial; Serotyping; Sugar Acids; Tandem Mass Spectrometry

The lipopolysaccharide of Vibrio cholerae has been reported to contain a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue that is phosphorylated. The phosphorylated Kdo sugar further links the hexa-acylated V. cholerae lipid A domain to the core oliogosaccharide and O-antigen. In this report, we confirm that V. cholerae possesses the enzymatic machinery to synthesize a phosphorylated Kdo residue. Further, we have determined that the presence of the phosphate group on the Kdo residue is necessary for secondary acylation in V. cholerae. The requirement for a secondary substituent on the Kdo residue (either an additional Kdo sugar or a phosphate group) was also found to be critical for secondary acylation catalyzed by LpxL proteins from Bordetella pertussis, Escherichia coli, and Haemophilus influenzae. Although three putative late acyltransferase orthologs have been identified in the V. cholerae genome (Vc0212, Vc0213, and Vc1577), only Vc0213 appears to be functional. Vc0213 functions as a myristoyl transferase acylating lipid A at the 2'-position of the glucosamine disaccharide. Generally acyl-ACPs serve as fatty acyl donors for the acyltransferases required for lipopolysaccharide biosynthesis; however, in vitro assays indicate that Vc0213 preferentially utilizes myristoyl-CoA as an acyl donor. This is the first report to biochemically characterize enzymes involved in the biosynthesis of the V. cholerae Kdo-lipid A domain.

Topics: Acyl Coenzyme A; Acylation; Acyltransferases; Bacterial Proteins; Genome, Bacterial; Lipid A; O Antigens; Phosphorylation; Sugar Acids; Vibrio cholerae

In Escherichia coli, FtsH (HflB) is a membrane-bound, ATP-dependent metalloendoprotease belonging to the AAA family (ATPases associated with diverse cellular activities). FtsH has a limited spectrum of known substrates, including the transcriptional activator sigma32. FtsH is the only known E. coli protease that is essential, as it regulates the concentration of LpxC, which carries out the first committed step in the synthesis of lipid A. Here we identify a new FtsH substrate--3-deoxy-D-manno-octulosonate (KDO) transferase--which carries out the attachment of two KDO residues to the lipid A precursor (lipid IVA) to form the minimal essential structure of the lipopolysaccharide (LPS) (KDO2-lipid A). Thus, FtsH regulates the concentration of the lipid moiety of LPS (lipid A) as well as the sugar moiety (KDO-based core oligosaccharides), ensuring a balanced synthesis of LPS.

Topics: ATP-Dependent Proteases; Escherichia coli; Escherichia coli Proteins; Lipid A; Lipopolysaccharides; Oligosaccharides; Sugar Acids; Transferases

Differences in the pattern and chemical nature of fatty acids of lipid A of Neisseria meningitides lipooligosaccharides (LOS) and Escherichia coli lipopolysaccharides (LPS) may account for differences in inflammatory properties. Furthermore, there are indications that dimeric 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) moieties of LOS and LPS enhance biological activities. Heterogeneity in the structure of lipid A and possible contaminations with other inflammatory components have made it difficult to confirm these observations. To address these problems, a highly convergent approach for the synthesis of a lipid A derivative containing KDO has been developed, which relies on the ability to selectively remove or unmask in a sequential manner an isopropylidene acetal, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonate (Alloc), azide, and thexyldimethylsilyl (TDS) ether. The strategy was employed for the synthesis of N. meningitidis lipid A containing KDO (3). Mouse macrophages were exposed to the synthetic compound and its parent LOS, E. coli lipid A (2), and a hybrid derivative (4) that has the asymmetrical acylation pattern of E. coli lipid A, but the shorter lipids of meningococcal lipid A. The resulting supernatants were examined for tumor necrosis factor alpha (TNF-alpha) and interferon beta (IFN-beta) production. The lipid A derivative containing KDO was much more active than lipid A alone and just slightly less active than its parent LOS, indicating that one KDO moiety is sufficient for full activity of TNF-alpha and IFN-beta induction. The lipid A of N. meningitidis was a significantly more potent inducer of TNF-alpha and IFN-beta than E. coli lipid A, which is due to a number of shorter fatty acids. The compounds did not demonstrate a bias towards a MyD88- or TRIF-dependent response.

Topics: Animals; Carbohydrate Conformation; Escherichia coli; Immunity, Innate; Immunologic Tests; Interferon-beta; Lipid A; Lipopolysaccharides; Macrophages; Mice; Molecular Structure; Neisseria meningitidis; Stereoisomerism; Sugar Acids; Tumor Necrosis Factor-alpha

Lipopolysaccharide (LPS) is a major surface component of Chlamydia trachomatis, as with all Gram-negative bacteria. The effect of C. trachomatis LPS on C. trachomatis infectivity of human epithelial cells was investigated. C. trachomatis LPS and C. trachomatis LPS antibody significantly reduced infectivity, mostly in a dose-dependent manner. As the structure of LPS in C. trachomatis is simple and consists only of lipid A and 3-deoxy-D-manno-octulosonic acid (Kdo), we investigated whether lipid A or Kdo was inhibitory to chlamydial infectivity. Polymyxin B, as a lipid A inhibitor, and Kdo considerably reduced C. trachomatis infectivity. With all the LPS inhibitors used, there was greater inhibition against serovar E than serovar LGV. These results suggest a role for LPS in chlamydial infectivity. Elucidation of how LPS acts in infectivity and identification of host-cell receptors would help in understanding pathogenicity.

Topics: Cell Line, Tumor; Chlamydia Infections; Chlamydia trachomatis; Epithelial Cells; Humans; Immunohistochemistry; Lipid A; Lipopolysaccharides; Sugar Acids

Gram-negative bacteria possess an asymmetric lipid bilayer surrounding the cell wall, the outer membrane (OM). The OM inner leaflet is primarily composed of various glycerophospholipids, whereas the outer leaflet predominantly contains the unique amphiphilic macromolecule, lipopolysaccharide (LPS or endotoxin). The majority of all gram-negative bacteria elaborate LPS containing at least one 2-keto 3-deoxy-D-manno-octulosonate (Kdo) molecule. The minimal LPS structure required for growth of Escherichia coli has long been recognized as two Kdo residues attached to lipid A, inextricably linking viability to toxicity. Here we report the construction and characterization of the nonconditional E. coli K-12 suppressor strain KPM22 that lacks Kdo and is viable despite predominantly elaborating the endotoxically inactive LPS precursor lipid IV(A). Our results challenge the established E. coli Kdo2-lipid A dogma, indicating that the previously observed and well-documented dependence of cell viability on the synthesis of Kdo stems from a lethal pleiotropy precipitated after the depletion of the carbohydrate, rather than an inherent need for the Kdo molecule itself as an indispensable structural component of the OM LPS layer. Inclusion of the inner membrane LPS transporter MsbA on a multicopy plasmid partially suppresses the lethal deltaKdo phenotype directly in the auxotrophic parent strain, suggesting increased rates of nonglycosylated lipid A transport can, in part, compensate for Kdo depletion. The unprecedented nature of a lipid IV(A) OM redefines the requisite LPS structure for viability in E. coli.

Topics: Carbohydrate Sequence; Cell Membrane; Endotoxins; Escherichia coli; Gram-Negative Bacteria; Lipid A; Lipid Bilayers; Lipopolysaccharides; Molecular Sequence Data; Phospholipases; Sugar Acids; Thermodynamics

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

Topics: Helicobacter pylori; Hydrolases; Lipid A; Lipopolysaccharides; Phosphates; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sugar Acids; Temperature; Transferases

Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants of Neisseria meningitidis that contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS alpha or beta chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an approximately 10-fold (P < 0.0001) reduction in biologic activity compared to KDO2-containing meningococcal LOS. Removal of KDO2 from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO2 linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.

Topics: Animals; Antigens, Surface; Cell Line; Humans; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Lymphocyte Antigen 96; Macrophage Activation; Macrophages; Membrane Glycoproteins; Mice; Neisseria meningitidis; Receptors, Cell Surface; Respiratory Burst; Structure-Activity Relationship; Sugar Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; U937 Cells

Lipopolysaccharide, lipooligosaccharide (LOS), or endotoxin is important in bacterial survival and the pathogenesis of gram-negative bacteria. A necessary step in endotoxin biosynthesis is 3-deoxy-D-manno-octulosonic acid (Kdo) glycosylation of lipid A, catalyzed by the Kdo transferase KdtA (WaaA). In enteric gram-negative bacteria, this step is essential for survival. A nonpolar kdtA::aphA-3 mutation was created in Neisseria meningitidis via allelic exchange, and the mutant was viable. Detailed structural analysis demonstrated that the endotoxin of the kdtA::aphA-3 mutant was composed of fully acylated lipid A with variable phosphorylation but without Kdo glycosylation. In contrast to what happens in other gram-negative bacteria, tetra-acylated lipid IV(A) did not accumulate. The LOS structure of the kdtA::aphA-3 mutant was restored to the wild-type structure by complementation with kdtA from N. meningitidis or Escherichia coli. The expression of a fully acylated, unglycosylated lipid A indicates that lipid A biosynthesis in N. meningitidis can proceed without the addition of Kdo and that KdtA is not essential for survival of the meningococcus.

Topics: Alleles; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Genetic Complementation Test; Genetic Vectors; Genotype; Lipid A; Lipopolysaccharides; Molecular Structure; Mutation; Neisseria meningitidis; Phosphorylation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sugar Acids; Transferases

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Chlamydophila psittaci; Chloroform; Chromatography, Ion Exchange; Chromatography, Thin Layer; Hydrazines; Hydroxides; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Mice; Molecular Sequence Data; Phenol; Potassium Compounds; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sugar Acids; Time Factors; Water

Lipooligosaccharide (LOS) glycoforms from Haemophilus influenzae 2019 were profiled using the high-resolution and accurate mass capabilities of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Sequence and linkage for two previously unknown LOS glycoforms were subsequently obtained through MSn analyses on FT-ICR and quadrupole ion trap (qIT) instruments. MSn analysis of negative ion precursors confirmed structural details within the lipid moiety, while CID spectra of sodiated precursor ions provided monosaccharide sequence and linkage for the oligosaccharide portion of the molecule. Results obtained in this study indicate that extensive heterogeneity exists within the oligosaccharide moieties in LOS from H. influenzae 2019. More importantly, the data suggest that additional hexose moieties, which are added onto the LOS, are not simple extensions of one particular core structure but rather that structural isomers with different connectivities are present within the heterogeneous mixture.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Cyclotrons; Disaccharides; Fourier Analysis; Haemophilus influenzae; Heptoses; Hexoses; Lipid A; Lipopolysaccharides; Mass Spectrometry; Molecular Sequence Data; Phosphorylation; Polysaccharides; Sugar Acids; Trisaccharides

The lipopolysaccharides LPS I and LPS II, isolated from the hypovirulent EV40 strain of Yersinia pestis, are composed only of type R lipopolysaccharides. This type consists of two forms a and b, depending on their solubility pattern in a solvent mixture containing varying proportions of chloroform, methanol, hexane, and hydrochloric acid. LPS I consists of one subtype, RIb, while LPS II consists of two subtypes, RIIa and RIIb. Analysis by gel electrophoresis shows that the mass of these lipopolysaccharide forms are in the vicinity of 2000-3000 Da. The RIb and RIIb subtypes, which are found in the majority of lipopolysaccharide I and II fractions, are composed of ketoses and amines that are similar to those occurring in LPS I and LPS II. In contrast, the two subtypes RIIa and RIIb are different both in terms of the composition of lipid A and the extent of its substitution. Certain fractions of RIIa contain only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), while other fractions of RIIb possess a lipid A, which is not substituted by arabinose. The whole set of these R-type lipopolysaccharide forms are excellent models for the study of the role of the primary structure of the polysaccharide region, and for the effect of lipid A substitution on the biological activity of bacterial lipopolysaccharides.

Topics: Electrophoresis, Polyacrylamide Gel; Lipid A; Lipopolysaccharides; Molecular Weight; Solubility; Sugar Acids; Virulence; Yersinia pestis

Lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue, linked to the 6' position of lipid A. In Escherichia coli and related organisms, a Kdo disaccharide is attached to lipid A. In previous studies, we cloned the gene (kdtA) encoding the E. coli Kdo transferase and demonstrated that homogeneous preparations of KdtA polypeptide catalyzed the attachment of both Kdo groups to the precursor, lipid IVA. E. coli KdtA produced only traces of mono-glycosylated product. We now show that a single Kdo is transferred to lipid IVA in extracts of H. influenzae. The mono-functional Kdo transferase of H. influenzae is membrane-bound, and the reaction is dependent upon a CMP-Kdo-generating system, as in E. coli. The specific activity of Kdo transfer to lipid IVA is 0.5-1 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, milligram quantities of Kdo-lipid IVA were prepared for analysis. Matrix-assisted laser desorption/ionization mass spectrometry revealed a parent ion (M - H)- at m/z 1626.0, consistent with the addition of a single Kdo moiety. Like lipid IVA, Kdo-lipid IVA was an excellent substrate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IVA was further phosphorylated in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid IVA with a parent ion (M - H)- at m/z 1703.9. The identification of the mono-functional H. influenzae Kdo transferase, which is encoded by a KdtA homologue that displays 50% identity to its E. coli counterpart, should facilitate the mechanistic dissection of more complex multi-functional Kdo transferases, like those of E. coli and Chlamydia trachomatis.

Topics: Escherichia coli; Haemophilus influenzae; Lipid A; Mass Spectrometry; Phosphorylation; Phosphotransferases; Sugar Acids; Transferases

Various elicitors were examined by Northern blot analysis to investigate the simultaneous induction of gene expression of antibacterial proteins such as cecropin B, attacin and lebocin from the silkworm, Bombyx mori. Lipopolysaccharide (LPS), lipid A, 2-keto-3-deoxyoctonate (KDO) and peptidoglycan (PG) triggered efficiently and simultaneously the gene expression of antibacterial proteins. Effects of inhibitors for signal transduction on the gene expression of Bombyx mori (Bm) cecropin B triggered by lipid A were observed using isolated adherent hemocytes consisting of granular cells and plasma cells. H-7, H-89 but not W-7 inhibited gene expression, suggesting that protein kinase C and A but not myosine light chain kinase may participate in signal transduction.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Anti-Infective Agents; Bombyx; Cell Adhesion; Enzyme Inhibitors; Gene Expression; Genes, Insect; Hemocytes; Insect Hormones; Insect Proteins; Isoquinolines; Larva; Lipid A; Lipopolysaccharides; Peptidoglycan; Protein Biosynthesis; Signal Transduction; Sugar Acids; Sulfonamides

A lipopolysaccharide isolated from Coxiella burnetti strain Nine Mile in avirulent phase II contains in the lipid A proximal region D-mannose, D-glycero-D-manno-heptose, and 3-deoxy-D- manno-oct-2-ulosonic acid (Kdo) in the molar ratio 2:2:3. The primary structure 1 of the heptasaccharide was determined by glycose analysis, methylation analysis, ESI-MS, and FABMS. [sequence: see text]

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Coxiella burnetii; Electrophoresis, Polyacrylamide Gel; Heptoses; Lipid A; Lipopolysaccharides; Mannose; Methylation; Molecular Sequence Data; Oligosaccharides; Q Fever; Spectrometry, Mass, Fast Atom Bombardment; Sugar Acids; Virulence

The lipopolysaccharide of Rhizobium leguminosarum differs from that of other Gram-negative organisms. R. leguminosarum lipid A lacks phosphate groups, but it contains a galacturonic acid residue at the 4'-position and an aminogluconate moiety in place of the usual glucosamine 1-phosphate unit. R. leguminosarum lipid A is esterified with a peculiar long chain fatty acid, 27-hydroxyoctacosanoate, not found in enteric Gram-negative bacteria, and the inner core of R. leguminosarum contains mannose and galactose in place of heptose. Despite these differences, the biosynthesis of R. leguminosarum lipid A is initiated by the same seven enzyme pathway as in Escherichia coli (Raetz, C. R. H. (1993) J. Bacteriol. 175, 5745-5753) to form the phosphorylated precursor, (Kdo)2-lipid IVA, which is then processed differently. We now describe several novel Rhizobium-specific enzymes that recognize and modify (Kdo)2-lipid IVA. The 1- and 4'-phosphatases were detected using (Kdo)2-[1-32P]-lipid IVA and (Kdo)2-[4'-32P]-lipid IVA, respectively, as shown by release of 32Pi. In the presence of GDP-mannose and/or UDP-galactose, membranes of R. leguminosarum first transferred mannose and then galactose to (Kdo)2-[4'-32P]-lipid IVA. In addition, at least two hydrophobic metabolites were generated from (Kdo)2-[4'-32P]-lipid IVA in a manner that was dependent upon both membranes and a cytosolic factor from R. leguminosarum. These compounds are attributed to novel acylations of (Kdo)2-[4'-32P]-lipid IVA. E. coli membranes and cytosol did not catalyze any of the unique reactions detected in R. leguminosarum extracts. Our findings establish the conservation and versatility of (Kdo)2-lipid IVA as a lipid A precursor in bacteria.

Topics: Acyltransferases; Adenosine Triphosphate; Carbohydrate Sequence; Cytosol; Escherichia coli; Galactosyltransferases; Glycolipids; Guanosine Diphosphate Mannose; Lipid A; Lipopolysaccharides; Mannosyltransferases; Models, Biological; Molecular Sequence Data; Phosphoprotein Phosphatases; Rhizobium leguminosarum; Sugar Acids; Uridine Diphosphate Galactose

The lipopolysaccharide structure of the nitrogen-fixing bacterium Rhizobium leguminosarum differs from that of Escherichia coli in several ways, one of which is the sugar composition of the core. The E. coli inner core consists of 3-deoxy-D-manno-octulosonic acid (Kdo) and L-glycero-D-manno-heptose (heptose), while the inner core of R. leguminosarum contains 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo), mannose, galactose, and galacturonic acid. The two Kdo residues and their linkages appear to be identical in both species. The linkages of heptose in E. coli and of mannose in R. leguminosarum to Kdo are both alpha1-5. We now characterize a membrane-associated glycosyl transferase in R. leguminosarum extracts that incorporates mannose into nascent lipopolysaccharide, using Kdo2-lipid IVA as the acceptor and GDP-mannose (or synthetic ADP-mannose) as the donor. The mannosyl transferase is associated with the inner membrane. The apparent Km values for GDP-mannose and Kdo2-lipid IVA are 4.3 microM and 7.1 microM, respectively, in the presence of excess co-substrate. Extracts of E. coli do not catalyze GDP-mannose-dependent glycosylation of Kdo2-lipid IVA, but they are active when ADP-mannose is substituted for GDP-mannose. Given the structural similarity of ADP-mannose to ADP-heptose, we examined the possibility that heptosyl transferase I of E. coli (the product of the rfaC gene) catalyzes mannose transfer from ADP-mannose to Kdo2-lipid IVA. Extracts of E. coli mutants defective in the rfaC gene are unable carry out ADP-mannose-dependent glycosylation of Kdo2-lipid IVA. Plasmids bearing rfaC+ not only restore the missing activity but also direct its overexpression. Our assay using ADP-mannose as a substitute for ADP-heptose (which is not readily available) should facilitate the purification and characterization of heptosyl transferase I of E. coli. The GDP-mannose-dependent enzyme of R. leguminosarum may represent a functional equivalent of E. coli RfaC.

Topics: Escherichia coli; Glucosyltransferases; Glycolipids; Glycosylation; Guanosine Diphosphate Mannose; Kinetics; Lipid A; Lipopolysaccharides; Mannose; Mannosyltransferases; Rhizobium leguminosarum; Subcellular Fractions; Sugar Acids

Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a beta1'-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4'. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4'-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo)2-[4'-32P]-lipid IVA as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf* contains the consensus sequence, DSLD, for attachment of 4'-phosphopantetheine. When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids. Orf* is a new kind of acyl carrier protein because it is only approximately 25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf* confirmed the presence of 4'-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24, and 26 carbon fatty acids were also observed. Given the specialized function of Orf* in lipid A acylation, we suggest the new designation AcpXL.

Topics: Acyl Carrier Protein; Amino Acid Sequence; Bacterial Proteins; Base Sequence; Carbohydrate Sequence; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Fatty Acids, Unsaturated; Glycolipids; Hydroxylamine; Hydroxylamines; Hydroxylation; Lipid A; Molecular Sequence Data; Open Reading Frames; Rhizobium leguminosarum; Sugar Acids

Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane, and for Rhizobium spp. has been shown to play a critical role in the establishment of an effective nitrogen-fixing symbiosis with a legume host. Many genes required for O-chain polysaccharide synthesis are in the lps alpha region of the CE3 genome; this region may also carry lps genes required for core oligosaccharide synthesis. The LPSs from several strains mutated in the alpha region were isolated, and their mild acid released oligosaccharides, purified by high performance anion-exchange chromatography, were characterized by electrospray- and fast atom bombardment-mass spectrometry, NMR, and methylation analysis. The LPSs from several mutants contained truncated O-chains, and the core region consisted of a (3-deoxy-D-manno-2-octulosomic acid) (Kdo)-(2-->6)-alpha-Galp-(1-->6)-[alpha-GalpA-(1-->4)]-alpha-Ma np-(1-->5)- Kdop (3-deoxy-D-manno-2-octulosomic acid) (Kdo)pentasaccharide and a alpha-GalpA-(1-->4)-[alpha-GalpA-(1-->5)]-Kdop trisaccharide. The pentasaccharide was altered in two mutants in that it was missing either the terminal Kdo or the GalA residue. These results indicate that the lps alpha region, in addition to having the genes for O-chain synthesis, contains genes required for the transfer of these 2 residues to the core region. Also, the results show that an LPS with a complete core but lacking an O-chain polysaccharide is not sufficient for an effective symbiosis.

Topics: Carbohydrate Sequence; Lipid A; Lipopolysaccharides; Molecular Sequence Data; O Antigens; Oligosaccharides; Polysaccharides, Bacterial; Rhizobium; Sugar Acids

Lipids A are the hydrophobic domains of bacterial endotoxic lipopolysaccharides. Since they are responsible for most of the biological activities (both pathogenic and beneficial) of endotoxins, the characterization of their structure is crucial to the understanding of their mode of action. However, the inadequacy of existing methods for preparing certain lipids A has prompted us to devise a new, mild procedure which gives intact products. Use was made of the special features of 252Cf-plasma desorption mass spectrometry for forming molecular ions from these species and giving qualitative and quantitative information from the primary mass spectrum.

Topics: Bordetella pertussis; Californium; Chromatography, Thin Layer; Escherichia coli; Lipid A; Mass Spectrometry; Moraxella catarrhalis; Shigella flexneri; Sugar Acids

Structural analysis of the 3-deoxy-D-manno-2-octulosonic acid (Kdo) region in a lipopolysaccharide (LPS) II isolated from Coxiella burnetii strain Nine Mile in avirulent phase II revealed the presence of three variously linked Kdo residues. The lipid A proximal Kdo is substituted at C-4 by a Kdo-(2-->4)-Kdo disaccharide and this structural arrangement of the Kdo residues is similar to that of enterobacterial LPSs.

Topics: Coxiella burnetii; Gas Chromatography-Mass Spectrometry; Lipid A; Lipopolysaccharides; Molecular Structure; Sugar Acids; Virulence

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

Lipopolysaccharides (LPSs) are prominent structural components of the outer membranes of gram-negative bacteria. In Rhizobium spp. LPS functions as a determinant of the nitrogen-fixing symbiosis with legumes. LPS is anchored to the outer surface of the outer membrane by the lipid A moiety, the principal lipid component of the outer bacterial surface. Several notable structural differences exist between the lipid A of Escherichia coli and that of Rhizobium leguminosarum, suggesting that diverse biosynthetic pathways may also exist. These differences include the lack of phosphate groups and the presence of a 4'-linked GalA residue in the latter. However, we now show that UDP-GlcNAc plays a key role in the biosynthesis of lipid A in R. leguminosarum, as it does in E. coli. 32P-labeled monosaccharide and disaccharide lipid A intermediates from E. coli were isolated and tested as substrates in cell extracts of R. leguminosarum biovars phaseoli and viciae. Six enzymes that catalyze the early steps of E. coli lipid A biosynthesis were also present in extracts of R. leguminosarum. Our results show that all the enzymes of the pathway leading to the formation of the intermediate 3-deoxy-D-manno-2-octulosonic acid (Kdo2)-lipid IVA are functional in both R. leguminosarum biovars. These enzymes include (i) UDP-GlcNAc 3-O-acyltransferase; (ii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase; (iii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcN N-acyltransferase; (iv) disaccharide synthase; (v) 4'-kinase; and (vi) Kdo transferase. Our data suggest that the early steps in lipid A biosynthesis are conserved and that the divergence leading to rhizobial lipid A may occur at a later stage in the pathway, presumably after the attachment of the Kdo residues.

Topics: Acetates; Acyltransferases; Amidohydrolases; Carbohydrate Sequence; Glycolipids; Lipid A; Models, Biological; Molecular Sequence Data; N-Acetylglucosaminyltransferases; Phosphates; Phosphotransferases (Alcohol Group Acceptor); Rhizobium leguminosarum; Sugar Acids; Transferases

The content of 4-amino-4-deoxy-L-arabinopyranose (L-Arap4N) and the phosphate substitution pattern of the LPS of various strains from Salmonella minnesota, Yersinia enterocolitica and Proteus mirabilis was determined by GC/MS, HPLC and 31P-NMR. These data allowed us to examine the possible role of these components for the polymyxin B-binding capacity of LPS and for the minimal inhibiting concentration (MIC) and the minimal bactericidal concentration (MBC) of polymyxins B and E towards the respective R-mutants. Contrary to other investigated Re-, Rd- and Rc-mutants of S. minnesota, strain R595 (Re-mutant) showed about a 90% substitution of the ester-linked phosphate-group with L-Arap4N, whereas the L-Arap4N content of the other S. minnesota strains amounted to 17-25%. Neither the binding capacity of LPS to polymyxin B, determined by a bioassay, nor the MIC- and MBC-values of the R-mutants were significantly affected by this alteration. Similar results were obtained after using the temperature-dependent changes in the L-Arap4N-content and phosphate substitution pattern of Y. enterocolitica 75R. In order to explore the relevant polymyxin B binding site, lipid A samples with or without substitution of their ester-linked phosphate group were prepared and subjected to the polymyxin-binding assay. The results obtained so far indicated that the inner core bound L-Arap4N, detected in all resistant strains investigated, may play a decisive role in the decreased binding of polymyxin B, responsible for the bacterial resistance towards polymyxin(s).

Topics: Amino Sugars; Enterobacteriaceae; Lipid A; Lipopolysaccharides; Microbial Sensitivity Tests; Mutation; Polymyxins; Sugar Acids

Lactoferrin (LF), a cationic 80-kDa protein present in polymorphonuclear leukocytes and in mucosal secretions, is known to have antibacterial effects on gram-negative bacteria, with a concomitant release of lipopolysaccharides (LPS, endotoxin). In addition, LF is known to decrease LPS-induced cytokine release by monocytes and LPS priming of polymorphonuclear leukocytes. Its mechanism of action is incompletely understood. We have now demonstrated by in vitro-binding studies that LF binds directly to isolated lipid A and intact LPS of clinically relevant serotypes of the species which most frequently cause bacteremia (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), as well as to lipid A and LPS of mucosal pathogens (among others, Neisseria meningitides and Haemophilus influenzae). Binding to LPS was inhibitable by lipid A and polymyxin B but not by KDO (3-deoxy-D-manno-octulosonate), a glycoside residue present in the inner core of LPS. Binding of LF to lipid A was saturable, and an affinity constant of 2 x 10(9) M-1 was calculated for the LF-lipid A interaction. Our data may explain, in part, the mechanism whereby LF exerts its antibacterial and anti-endotoxic effects. Further studies on the ability of LF to block the detrimental effects of LPS, both in vitro and in vivo, are warranted.

Topics: Carrier Proteins; Humans; Lactoferrin; Lipid A; Lipopolysaccharides; Sugar Acids

The synthesis of the lipopolysaccharide fragment O-(4,5,7,8-tetra-O-acetyl-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide)-(2-->6)-O-(2-deoxy-2-[(3R)-3- dodecanoyloxytetradecanamido]-4-O- phosphono-3-O-tetradecanoyl-beta-D-glucopyranosyl)-(1-->6)-1-O-acetyl-2- deoxy-2 - [(3R)-3-dodecanoyloxytetradecanamido]-3-O-tetradecanoyl-alpha-D- glucopyranose (35 alpha) is performed via anomeric O-alkylation. With this objective, the 2-azido-3-O-benzyl-2-deoxy-6-O-trifluoromethanesulfonyl-beta-D-glu copyranosides 5, 7, and 19 alpha, beta were synthesized from D-glucal and employed as alkylating agents. Reaction of 5 with the O-cyclohexylidene-protected Kdo-derivative 10 afforded the desired alpha-linked disaccharide, tert-butyldimethylsilyl 4-O-allyl-2-azido-3-O-benzyl-2-deoxy-6- O-(4,5:7,8-di-O-cyclohexylidene-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide)-beta-D-glucopyranoside (11); even better yields of the structurally related disaccharide 12 were obtained with the 4-O-unprotected 7 as alkylating agent. 1-O-Desilylation of 12 furnished the lactol 20, which could be alkylated at the anomeric position with 1-O-allyl protected alkylating agents 19 alpha and 19 beta, both of which furnished exclusively the desired beta-(1-->6)-linked trisaccharides allyl O-(4,5:7,8-di-O-cyclohexylidene-3- deoxy-N-methyl-alpha-D-manno-2-octulopyranosylonamide)-(2-->6)-O-( 2-azido-3- O-benzyl-2-deoxy-beta-D-glucopyranosyl)-(1-->6)-2-azido-3, 4-di-O-benzyl-2-deoxy-alpha- (21 alpha) and -beta-D-glucopyranoside (21 beta), respectively. Phosphorylation with diphenyl phosphorochloridate, replacement of the O-cyclohexylidene protective group by O-triethylsilyl (TES) protective groups, removal of the 1-O-allyl group, azido group reduction, subsequent N-acylation, and then O-acetylation provided the key 1-O-acetyl protected intermediate 30 alpha. Removal of the O-TES groups, subsequent O-acetylation, and hydrogenolytic O-debenzylation furnished O-[4,5:7,8-tetra-O-acetyl-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide]-(2-->6)-O-(2-deoxy-4-O-diphenoxyphospho ryl-2-[(3R)- 3-dodecanoyloxytetradecanamido]-beta-D-glucopyranosyl)-(1-->6)-1-O -acetyl-2- deoxy-2[(3R)-dodecanoyloxytetradecanamido]-alpha-D-glucopyranose (33 alpha), which underwent the required selective O-tetradecanoylation at the 3-O- and 3'-O-position, thus furnishing, after hydrogenolytic O-dephenylation of the diphenoxyphosphoryl group, the target molecule 35 alpha.

Topics: Glycosides; Lipid A; Lipopolysaccharides; Sugar Acids

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophilus and Neisseria species is evident from the multiplicity of components observed with electrophoretic analyses. Knowledge of the precise structures that make up these diverse LOS molecules is clearly the key to reaching an understanding of pathogenic processes such as phase variation and molecular mimicry. Except for a few cases, little is known about the specific structural features of LOS that underlie phase variation and molecular mimicry, partly because of the inherent difficulties in the structural elucidation of these complex glycolipids. In the lipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia coli, rough, or R-type, mutants have been isolated that have provided insight into the biosynthetic pathways and associated genetics that control LPS expression. Nonetheless, recent work has shown that these R-type LPS are more complex than originally thought, and significant heterogeneity is still observed, primarily in their phosphorylation states. In order to investigate the structures of LPS and LOS in a more rapid fashion, we have determined the precise molecular weights of LOS (and LPS) preparations from various Haemophilus, Neisseria, and Salmonella species by electrospray ionization-mass spectrometry. The LOS (or LPS) were first O-deacylated under mild hydrazine conditions to remove O-linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M-nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Mass spectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heterogeneity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the variable phosphorylation of the core of S. typhimurium LPS have afforded insights into the biosynthetic pathways used by these organisms. Information of this type is important for understanding the underlying genetic and environmental factors controlling LOS and LPS expression.

Topics: Antigens, Bacterial; Carbohydrate Sequence; Esters; Gas Chromatography-Mass Spectrometry; Genetic Variation; Haemophilus; Haemophilus ducreyi; Haemophilus influenzae; Ions; Lipid A; Lipopolysaccharides; Molecular Sequence Data; N-Acetylneuraminic Acid; Neisseria; Neisseria meningitidis; Phosphorylation; Protons; Salmonella typhimurium; Sialic Acids; Sugar Acids; Virulence

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.

Topics: Antigens, Bacterial; Caulobacter crescentus; Cell Membrane; Galactose; Genetic Variation; Glucose; Heptoses; Lauric Acids; Lipid A; Lipids; Lipopolysaccharides; Mannose; Oligosaccharides; Sugar Acids

The interaction of the polycationic decapeptide polymyxin B with asymmetric planar bilayers from lipopolysaccharide and phospholipid monolayers, which resemble the lipid matrix of the outer membrane of Gram-negative bacteria, was investigated. The addition of polymyxin B in micromolar amounts to the lipopolysaccharide side of the asymmetric bilayers resulted, under voltage-clamp conditions, in a fast macroscopic increase of their ionic conductance, whereas the polymyxin B nonapeptide induced no significant conductance changes. The polymyxin B induced macroscopic conductance exhibited large fluctuations and was strongly dependent on the amplitude and polarity of the transmembrane potential. The temporal pattern and amplitudes of the fluctuations were characterized by power spectra of the membrane currents and their variances, respectively. In the initial phase following peptide addition, the conductance changes appeared to be channellike discrete fluctuations. The lifetimes of the fluctuations were exponentially distributed, and the mean lifetimes were strongly voltage-dependent, ranging from approximately 30 ms at +80 mV (positive at the side opposite to peptide addition) to less than 5 ms at reverse polarity. The conductance amplitudes of the single fluctuations exhibited a broad distribution with a mean of 2 nS. A comparison of the features of the macroscopic conductance and of the discrete fluctuations showed that the former can basically be understood as a superposition of a large number of the latter. From the amplitudes of the fluctuations, the diameter of the polymyxin-induced lesions was estimated to about 3 nm. The experimental findings can be understood by assuming a detergent-like action of polymyxin B.

Topics: Cardiolipins; Electric Conductivity; Kinetics; Lipid A; Lipid Bilayers; Lipopolysaccharides; Membrane Potentials; Models, Biological; Molecular Conformation; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polymyxin B; Salmonella; Sugar Acids

The chemical structure of the saccharide portion of Vibrio parahaemolyticus serotype 012 lipopolysaccharide was studied. Using chemical degradation and modification, as well as methylation analysis in combination with GLC-MS, laser-desorption mass spectrometry and 1H-NMR and 13C-NMR spectroscopy, the carbohydrate backbone of the lipopolysaccharide was characterized as a branched decasaccharide with the following structure: (formula; see text) In the native lipopolysaccharide two additional phosphate groups are present and 3-deoxy-D-threo-hexulosonic acid and D-galacturonic acid are bound via acid-labile linkages.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Chromium; Chromium Compounds; Gas Chromatography-Mass Spectrometry; Hexuronic Acids; Lasers; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methylation; Molecular Sequence Data; Molecular Structure; Periodic Acid; Phosphates; Sugar Acids; Vibrio parahaemolyticus

The titer of IgG and IgM antibodies to two fragments of endotoxin derived from Salmonella minnesota R 595, lipid A width a 2-keto-3-deoxyoctanate oliosaccharide (KDO lipid A), and monophosphoryl lipid A (mono-P lipid), were measured in normal children, children with cystic fibrosis, and children with SLE and all forms of chronic juvenile arthritis. Elevated titers of IgM anti mono-P lipid A were found in all of the rheumatic diseases, but not in cystic fibrosis. The elevated IgM titers were not correlated with concentration of activation fragments of C3 or C4, but IgG anti mono-P lipid A titers, even though not usually elevated, did correlate with C3a and C3d concentrations. The elevated IgM titers to mono-P lipid A may represent a genetically determined hyper-reactivity to normal gastrointestinal antigens, an increased antigenic stimulus from the intestinal tract, or polyspecificity of an antibody of undetermined primary reactivity.

Topics: Antibodies, Bacterial; Child; Complement Activation; Humans; Immunoglobulin M; Lipid A; Rheumatic Diseases; Sugar Acids

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021-7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR-type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A. actinomycetemcomitans to consist of short sugar chains. Chemical analysis revealed the presence of thiobarbituric-acid-positive material (3-deoxy-D-manno-octulosonic acid equivalents) and four neutral sugars: glucose, galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose. Phosphate, glucosamine, glycine, and the fatty acids, 3-hydroxymyristic acid, myristic acid and palmitic acid, comprised the remainder of the molecule. The structure of the free lipid A revealed it to consist of a 1,6-glucosamine disaccharide esterified at C4' by a phosphomonoester. The hydroxyl group at C3 and the amide group of the non-reducing glucosamine were both acylated by 3-myristoylmyristic acid; analogous sites on the reducing glucosamine were acylated by 3-hydroxymyristic acid. Hydroxyl groups at C4 and C6' in the free lipid A were unsubstituted, with C6 being the proposed attachment site of the polysaccharide moiety. Chemical analysis revealed the presence of glycine in the intact LPS; its exact location in the A. actinomycetemcomitans LPS is still to be determined. Both intact LPS and free lipid A were highly lethal to galactosamine-sensitized mice, comparable to that of Salmonella.

Topics: Actinobacillus; Animals; Electrophoresis, Polyacrylamide Gel; Fatty Acids; Female; Galactose; Glucose; Heptoses; Lipid A; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methylation; Mice; Mice, Inbred C57BL; Molecular Structure; Sugar Acids; Thiobarbiturates

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

Coupling of methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosyl) onate-(2----4)-methyl (5,7,8-tri-O-acetyl-3-deoxy-alpha-D-manno-octulopyranosyl bromide)onate with benzyl O-(methyl [3-deoxy-7,8,O-(tetraisopropyldisiloxan 1,3-diyl)-alpha-D-manno-octulopyranosylonate)-2-----6)-O-([( R)-3-benzyloxytetradecanoylamino]-2-deoxy-3,4-O-(tetraisopropyl disiloxan-1,3-diyl)-beta-D-glucopyranosyl)-(1----6)-3-O-benz yl- 2-[(R)-3-benzyloxytetradecanoylamino]-2-deoxy-alpha-D-glucopyranos ide gave stereo- and regio-selectively a pentasaccharide that was deprotected into alpha-Kdo-(2----4)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-D-GlcNh m-(1----6)-D-GlcNhm [Nhm = 2-deoxy-(R)-3-hydroxytetradecanoylamino].

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Oligosaccharides; Sugar Acids

A new class of antibacterial agents for Gram-negative bacteria, rationally designed to inhibit the incorporation of 3-deoxy-D-manno-octulosonate into lipopolysaccharide (LPS), was recently reported. In Salmonella typhimurium, where the lipid A species are well characterised, it was previously demonstrated that the addition of a compound which inhibits the enzyme 3-deoxy-manno-octulosonate cytidylytransferase (CMP-KDO synthetase; EC 2.7.7.38) leads to rapid accumulation of lipid A derivatives. The major lipid A species, IVA (O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1-6)-2-amino-2-deoxy-alpha-D - glucose, acylated at positions 2, 3, 2', 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'), was shown to be converted mainly to LPS by pulse-chase experiments in the absence of inhibitor. Labelled precursor (IVA) was also chased to other more polar lipid A derivatives. During chase in the presence of inhibitor, there was no conversion to LPS, while the major lipid A species was converted to the same polar lipid A derivatives as in chase without inhibitor. Our data indicate that despite the accumulation of several species of lipid A derivatives during inhibition of LPS synthesis, only IVA is destined for synthesis of mature LPS when LPS synthesis resumes. The more polar lipid A derivatives would thus represent aberrant side reaction products which occur when the pathway is inhibited.

Topics: Amino Sugars; Anti-Bacterial Agents; Bacterial Proteins; Gram-Negative Bacteria; Lipid A; Lipopolysaccharides; Nucleotidyltransferases; Prodrugs; Salmonella typhimurium; Sugar Acids

Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs.

Topics: Cytidine Triphosphate; Cytoplasm; Escherichia coli; Gas Chromatography-Mass Spectrometry; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Substrate Specificity; Sugar Acids; Time Factors; Transferases

The immunopharmacological activities of 2-keto-3-deoxyoctonic acid (KDO)-(alpha 2----6)-linked lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents, were compared with those of the corresponding analogs without KDO, GLA-27, GLA-47, and GLA-60. Among the analogs tested, GLA-60, a 4-O-phosphono-D-glucosamine carrying N-3-hydroxytetradecanoyl and 3-O-3-tetradecanoyloxytetradecanoyl groups, exhibited the most intensive activities in terms of mitogenicity, adjuvanticity, and mediator (tumor necrosis factor and colony-stimulating factor) induction. Binding (alpha 2----6) of KDO to GLA-60 failed to enhance the activities. Similarly, the activities of GLA-27 and GLA-47 were also decreased by introduction of KDO to the O-6 of the analogs. This indicates that the strengths of the activities of the subunit analogs depend on the kinds of N- and 3-O-linked acyl substituents and not on the presence of the KDO linked to the O-6.

Topics: Adjuvants, Immunologic; Animals; Colony-Stimulating Factors; Female; Lipid A; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred ICR; Mitogens; Structure-Activity Relationship; Sugar Acids; Tumor Necrosis Factor-alpha

Inhibition of lipopolysaccharide (LPS) synthesis in Pseudomonas aeruginosa at the stage of incorporation of 3-deoxy-D-manno-octulosonate (KDO) caused accumulation of a lipid A precursor which contained all of the fatty acids present on the lipid A of mature LPS. The enzyme CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from P. aeruginosa is inhibited by the KDO analog alpha-C-[1,5-anhydro-8-amino-2,7,8-trideoxy-D-manno-octopyranosyl] carboxylate (I), and I is effectively delivered to P. aeruginosa following attachment by amide linkage to the carboxyl terminus of alanylalanine. Intracellular hydrolysis releases the free inhibitor (I) which then inhibits activation of KDO by CMP-KDO synthetase causing accumulation of lipid A precursor and subsequent growth stasis. The major lipid A precursor species accumulated was purified and found to contain glucosamine, phosphate, C12:O, 2OH-C12:O and 3OH-C10:0 (in ester linkage), and 3OH-C12:0 (in amide linkage) in molar ratios of 1:1:0.5:0.5:1:1. Analysis of precursor by fast atom bombardment mass spectroscopy yielded a major ion (M - H)- of mass 1616 and fragments which were consistent with the structure of lipid A from P. aeruginosa. In contrast, Salmonella typhimurium, Escherichia coli, Citrobacter sp., Serratia marcescens, Enterobacter aerogenes, and Enterobacter cloacae all accumulated underacylated lipid A precursors which only contained 3-OH-C14:0, glucosamine, and phosphate. This difference and species-specific patterns of major and minor precursor species show that early steps in the assembly of lipid A are similar, but not identical in enteric and nonenteric Gram-negative bacteria.

Topics: Acetylglucosamine; Acylation; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Glycolipids; Gram-Negative Bacteria; Lipid A; Lipopolysaccharides; Nucleotidyltransferases; Pseudomonas aeruginosa; Species Specificity; Sugar Acids

Antibacterial agents which specifically inhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity were used to block the incorporation of 3-deoxy-D-manno-octulosonate (KDO) into lipopolysaccharide. Lipopolysaccharide synthesis ceased, molecules similar in structure to lipid A accumulated, and bacterial growth ceased following addition of such agents to cultures of Salmonella typhimurium and Escherichia coli. Although four major species of lipid A accumulated in S. typhimurium, their kinetics of accumulation were different. The least polar of the major species was IVA [O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-a lph a- D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'], a molecule previously isolated from bacteria containing a kdsA mutation (C. R. H. Raetz, S. Purcell, M. V. Meyer, N. Qureshi, and K. Takayama, J. Biol. Chem. 260:16080-16088, 1985). Species IVA accumulated first and to the greatest extent following addition of the inhibitor, with other more polar derivatives appearing only after IVA attained half its maximal level. In contrast, only two major species of precursor accumulated in E. coli following addition of the inhibitor. One of these species was identical to IVA from S. typhimurium on the basis of chemical composition, fast atom bombardment mass spectroscopy, and comigration on Silica Gel H, and it also accumulated prior to a more polar species of related structure. We conclude that the addition of KDO to precursor species IVA is the major pathway of lipid A-KDO formation in both S. typhimurium LT2 and E. coli and that accumulation of the more polar species lacking KDO only occurs in response to accumulation of species IVA following inhibition of the normal pathway.

Topics: Anti-Bacterial Agents; Chemical Phenomena; Chemistry; Chromatography, DEAE-Cellulose; Escherichia coli; Kinetics; Lipid A; Lipopolysaccharides; Mass Spectrometry; Nucleotidyltransferases; Prodrugs; Salmonella typhimurium; Sugar Acids

Due to the formation of micelles, severance of the hydrophilic (poly- or oligosaccharide) and hydrophobic ("Lipid A") domains of bacterial lipopolysaccharides at pH 3.4 or 4.5 and 100 degrees is slow and sometimes does not proceed at all; partially degraded fragments are usually formed. At pH 3.4 (100 degrees) in aqueous 1% sodium dodecylsulphate (SDS), both lipopolysaccharides of the Bordetella pertussis endotoxin are cleaved within 20-30 min, but 80% of the glycosidically bound phosphate present in the hydrophobic domain is lost. Other endotoxins behave similarly. At pH 4.5 (100 degrees) and in the absence of detergent, hydrolysis of the glycosidic bonds of 3-deoxy-D-manno-2-octulosonic acid residues of the B. pertussis endotoxin is negligible but, in aqueous 1% SDS, severance of the two regions of LPS 1 is complete within 1 h (that of LPS-2 requires 3-4 h), and the glycosidically bound phosphate of the isolated hydrophobic region is preserved. Comparison of the rate of acid-catalysed hydrolysis of the glycosidically bound phosphate present in this "isolated Lipid A" preparation with that of 2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-alpha- and -beta-D-glucopyranose 1-phosphates established that the former 1-phosphate was the alpha anomer.

Topics: Bordetella pertussis; Endotoxins; Fatty Acids; Hexosamines; Hydrolysis; Isomerism; Kinetics; Lipid A; Lipopolysaccharides; Phosphates; Sodium Dodecyl Sulfate; Sugar Acids

Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42 degrees C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grow at 42 degrees C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10(-6), required exogenous KDO for growth at 42 degrees C. The growth rate at 42 degrees C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent Km for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of KDO per min per 10(8) cells. Bacteria incorporated exogenous [3H]KDO exclusively into LPS, with less than 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (3H, 13C, and 14C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.

Topics: DNA Transposable Elements; Genotype; Kinetics; Lipid A; Lipopolysaccharides; Mutation; Phenotype; Salmonella typhimurium; Sugar Acids

Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42 degrees C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on [3H]KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-KDO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent Km for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of KDO incorporation in toluenized cells with 50% inhibitory concentrations comparable to those obtained in purified CMP-KDO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying Kis of 4.2 microM for A-60478 and 2.5 microM for A-60821. These data indicated that the inactivity of the KDO analogs on intact bacteria was the result of poor permeation into cells rather than intracellular inactivation.

Topics: Cell Membrane; Cell Membrane Permeability; Chromatography, DEAE-Cellulose; Cytidine Triphosphate; Gas Chromatography-Mass Spectrometry; Glycolipids; Hydrogen-Ion Concentration; Kinetics; Lipid A; Mutation; Nucleotidyltransferases; Salmonella typhimurium; Sugar Acids; Toluene

Lipid A (LA), ketodeoxyoctonate (KDO) and lipoteichoic acids (LTA) were used to produce homologous polyclonal antibodies. These haptens were administered to rabbits in differing immunogenic forms, using multiple intradermal and intraperitoneal injections with complete Freund adjuvant. Booster injections were either made intradermally with incomplete Freund adjuvant or intravenously in saline. The immune-response was monitored regularly with an enzyme-immunoassay. Lipid A and KDO covalently linked to bovine serum albumin (BSA), with hapten densities per BSA molecule of 17 and 9, respectively, produced nondetectable immune-response. Acid-hydrolysed and intact cells of Salmonella minnesota Re 595 used as LA and KDO immunogens, respectively, produced significant immune-response when administered intradermally or intraperitoneally. Good immune-response was obtained with LTA covalently linked to BAS. However, a better result was obtained with crude LTA, containing 21.5% proteins. Generally, the lengthy immunization schedules used produced IgG antibodies to the antigens and the highest reciprocal titres attained were 75,000, 55,000 and 150,000 for LA, KDO and LTA, respectively. Meaningful expression of antisera titres by enzyme-immunoassay is discussed. We defined titre as the reciprocal antiserum dilution of the intercept of the mid-point on the linear section ending at 0.2 absorbance on the antiserum dilution curve.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Cell Membrane; Female; Immunization; Immunization, Secondary; Immunoenzyme Techniques; Injections, Intradermal; Injections, Intraperitoneal; Lacticaseibacillus casei; Lipid A; Lipopolysaccharides; Rabbits; Salmonella; Sugar Acids; Teichoic Acids

The relationship between lipopolysaccharide (LPS) composition and virulence of Haemophilus ducreyi strains was investigated. Glycoses identified in LPS by gas-liquid chromatography were glucose, galactose, and their amino derivatives glucosamine and galactosamine. Fucose was found in trace amounts but mannose and rhamnose, characteristic of the O-side chain of LPS in many species, were not detected. Qualitatively, the LPS composition of the eight strains examined was similar and differences were mainly quantitative. The total glycose:KDO ratio of the LPS of virulent strains exceeded that of avirulent strains. All strains had similar fatty-acid composition but lacked lauric acid. SDS-polyacrylamide gel electrophoresis of the LPS of virulent and avirulent strains also revealed differences in their electrophoretic mobilities. The LPS profiles of avirulent strains were similar, but differed from those of virulent strains. These profiles lacked high mol. wt bands representing O-side chain repeating units. Thus, differences in the electrophoretic mobilities of the LPS of virulent and avirulent strains may reflect differences in the amount of carbohydrates associated with the core polysaccharide.

Topics: Electrophoresis, Polyacrylamide Gel; Fatty Acids; Haemophilus ducreyi; Hexoses; Lipid A; Lipopolysaccharides; Phosphates; Polysaccharides, Bacterial; Sialic Acids; Spectrophotometry, Infrared; Structure-Activity Relationship; Sugar Acids

The mitogenicity, lethal toxicity, and Shwartzman reaction of three derivatives of chemically synthesized 2-keto-3-deoxyoctonic acid-linked 2,3-diacyloxyacylglucosamine-4-phosphate (KDO-GlcN-4-P) were determined. The compounds, A-301 (with di-3-hexadecanoyloxytetradecanoyl at the C-2 and C-3 positions), A-303 (di-3-tetradecanoyloxytetradecanoyl), and A-305 (3-dodecanoyloxytetradecanoyl and 3-tetradecanoyloxytetradecanoyl), induced a significant incorporation of [3H]thymidine into splenocytes of C57BL/6 mice. The compounds A-301 and A-303 showed lethality at a high dose of 50 micrograms per mouse in C57BL/6 mice sensitized with D-galactosamine, whereas A-305 caused toxicity even at a dose of 10 micrograms per mouse. However, the three compounds did not elicit the local Shwartzman reaction in rabbits. These findings indicate that the addition of 2-keto-3-deoxyoctonic acid enhances the mitogenic activity of 2,3-diacyloxyacylglucosamine-4-phosphate but does not affect the lethal toxicity and the induction of Shwartzman reaction.

Topics: Animals; Glucosamine; Glucosephosphates; Lipid A; Lymphocyte Activation; Male; Mice; Rabbits; Shwartzman Phenomenon; Structure-Activity Relationship; Sugar Acids

Lipid A that contains mainly 2,3-diamino-2,3-dideoxy-D-glucose, phosphate and fatty acids in the molar ratio 2:1:5-6 was found in Pseudomonas diminuta lipopolysaccharide. The lipid A was considered to have a diamino-sugar disaccharide structure that carries a nonglycosidic phosphomonoester group and amide-bound acyloxyacyl and 3-hydroxy fatty acyl groups. The lipopolysaccharide exhibited endotoxic activities including lethal toxicity, pyrogenicity, local Shwartzman activity, body weight-decreasing toxicity and Limulus activity. The free lipid A was also endotoxic.

Topics: Animals; Chromatography, Thin Layer; Fatty Acids; Glucosamine; Glucose; Heptoses; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Male; Mass Spectrometry; Mice; Pseudomonas; Sugar Acids

Topics: Glucosamine; Lipid A; Mitogens; Sugar Acids

Topics: Animals; Cells, Cultured; Glucosamine; Lipid A; Mice; Mice, Inbred C57BL; Mitogens; Sugar Acids

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-D-manno-octulosonic acid region of lipopolysaccharides. Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. The precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-D-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue. The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-D-manno-octulosonic acid and/or other polar substituents.

Topics: Chromatography, DEAE-Cellulose; Chromatography, Paper; Chromatography, Thin Layer; Glycolipids; Lipid A; Mass Spectrometry; Mutation; Palmitic Acids; Salmonella typhimurium; Sugar Acids

Temperature-sensitive mutants of Salmonella typhimurium that are defective in the biosynthesis of 3-deoxy-D-manno-octulosonate are known to accumulate disaccharide precursor(s) of lipid A at 42 degrees C (Rick, P. D., Fung, L. W.-M., Ho, C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4904-4912). We have devised new methods for purifying this material by chromatography on DEAE-cellulose and silicic acid columns and have fractionated it into eight related anionic components that fall into four sets, as judged by their charge. Substances IA and IB have an apparent net charge of -1, IIA and IIB of -2, IIIA and IIIB of -3, and IVA and IVB of -4. Negative ion fast atom bombardment mass spectrometry reveals that the simplest component is IVA [( M - H]- at m/z 1404). Compound IVA is also the most abundant, representing 30-50% of the accumulated lipids after 3 h at 42 degrees C. Structural studies of IVA, including NMR spectroscopy described in the accompanying paper, reveal that it consists of O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-alpha - D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl moieties and bearing phosphate groups at positions 1 and 4'. Compound IIIA ([M - H]- at m/z 1527) contains an additional phosphoethanolamine residue, while IIA ([M - H]- m/z 1535) bears an aminodeoxypentose substituent, presumably 4-amino-4-deoxy-L-arabinose. Compound IA ([M - H]- at m/z 1658) bears both a phosphoethanolamine and an aminodeoxypentose. The compounds of the less abundant B series are further derivatized with an ester-linked palmitoyl moiety. Our results demonstrate that these precursors are far more heterogeneous than previously suspected.

Topics: Glycolipids; Lipid A; Lipids; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mutation; Salmonella typhimurium; Species Specificity; Sugar Acids; Temperature

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.

Topics: Acylation; Carbohydrate Sequence; Disaccharides; Lipid A; Magnetic Resonance Spectroscopy; Mutation; Salmonella typhimurium; Sugar Acids

A lipopolysaccharide (LPS) was isolated from the Lyme disease spirochete by a modification of the hot phenol-water method. The material was composed of 45% carbohydrate, 8% protein, 44% lipid A, and 1% 3-deoxy-D-mannooctulosonic acid and accounted for approximately 1.5% of the cellular dry weight. The isolated LPS possessed several biologic activities characteristic of endotoxins. The LPS was pyrogenic for rabbits, mitogenic for human mononuclear cells and murine splenocytes, capable of clotting limulus lysate, and cytotoxic for murine macrophages. LPS extracted from Borrelia burgdorferi by the petroleum-ether:chloroform:liquid-phenol procedure was also characterized. The results show that the Lyme disease spirochete contains a hitherto unknown LPS that is biologically active in vitro, and the expression of such activities in vivo may play an important role in the pathogenesis of Lyme disease. Some of the clinical manifestations of other spirochetal disease may be explained by similar endotoxins in those organisms. To our knowledge this is the first report of an LPS extracted from a spirochete that is known to be a human pathogen.

Topics: Animals; Bacterial Proteins; Borrelia; Borrelia burgdorferi; Carbohydrates; Cell Survival; Fever; Humans; Limulus Test; Lipid A; Lipopolysaccharides; Lyme Disease; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C3H; Rabbits; Sugar Acids

Lipopolysaccharides (LPS) from the non-nodulating Rhizobium trifolii 24SM 15 and from the nodulating R. trifolii 24SM 13 were isolated and examined by means of gas-liquid chromatography and mass spectrometry. Analysis of LPS showed these preparations from both strains examined contained Lipid A, 2-keto-3-deoxyoctonate, neutral sugars, amino sugars, and trace amounts of amino acids. In 24SM 13 LPS prevailed glucose and rhamnose whereas LPS from the non-nodulating strain SM 15 contained mainly mannose, galactose and heptose. Quinovosamine and mannosamine were detected only in the nodulating strain. The ratio of glucosamine phosphate to glucosamine was higher in the LPS of the non-nodulating strain SM 15 than in the corresponding material of the nodulating one. An unknown component producing a peak at the position of glyceryl-S-cysteine on amino acid analysis profiles was detected in SM 15 LPS. The differences in LPS composition were associated with the alterations in the sensitivity to phage 3H, and nodulation ability.

Topics: Amino Acids; Genes, Bacterial; Glucosamine; Lipid A; Lipopolysaccharides; Mutation; Rhizobium; Sugar Acids

An incomplete precursor of lipid A produced by a mutant of Salmonella typhimurium conditionally defective in synthesis of 3-deoxy-D-mannooctulosonate (KDO) (Rick, P.D., and Osborn, M.J. (1977) J. Biol. Chem. 252, 4895-4903) is poorly translocated to the outer membranes. Approximately 40% of the lipid A precursor synthesized under nonpermissive conditions was recovered in the isolated inner (cytoplasmic) membrane fraction, and the rate of translocation to outer membrane in pulse-chase experiments was only 20% that of lipopolysaccharide. However, integration of the incomplete molecule into the outer membrane appeared to be similar to that of lipopolysaccharide in stability and irreversibility. The ultimate extent of translocation pulse-labeled precursor was comparable to that of lipopolysaccharide and the process was functionally unidirectional. Little or no reverse translocation from outer to inner membrane was detected during conversion of preformed lipid A precursor to lipopolysaccharide following return to permissive conditions.

Topics: Cell Membrane; Kinetics; Lipid A; Lipopolysaccharides; Membrane Lipids; Mutation; Salmonella typhimurium; Sugar Acids

The incomplete lipid A precursor produced by a mutant conditionally defective in synthesis of 3-deoxy-D-mannoctulosonate (KDO) is rapidly converted to lipopolysaccharide when the mutant culture is shifted from nonpermissive to permissive conditions (Osborn, M.J., Rick, P.D., and Rasmussen, N.S. (1980) J. Biol. Chem. 255, 4246-4251). An intermediate product which accumulates transiently during this conversion has been isolated and identified as a derivative of the lipid A precursor containing 2 residues of KDO. The intermediate lacks the saturated O-fatty acyl chains of the completed lipid A and is indistinguishable in composition and chromatographic properties from the previously described product obtained by enzymatic addition of KDO to isolated lipid A precursor (Munson, R.S., Jr., Rasmussen, N.S., and Osborn, M.J. (1978) J. Biol. Chem. 253, 1503-1511). The intermediate produced in vivo is rapidly converted to lipopolysaccharide under conditions in which its continued formation is interrupted by return of the culture to nonpermissive temperature. The results provide direct evidence that transfer of KDO to lipid A precedes addition of the saturated fatty acid residues.

Topics: Acetates; Acetylglucosamine; Kinetics; Lipid A; Lipopolysaccharides; Mutation; Salmonella typhimurium; Sugar Acids

The effect of cerulenin on conversion of an acyl-deficient precursor of lipid A to lipopolysaccharide was investigated in a mutant of Salmonella typhimurium (PRX22H9) conditionally defective in synthesis of 3-deoxy-D-mannooctulosonate (KDO). The precursor lacks both KDO and the saturated O-fatty acyl chains of lipopolysaccharide and contains beta-hydroxymyristate as sole fatty acid. Concentrations of cerulenin which inhibited de novo synthesis of fatty acids and lipopolysaccharide more than 95% had no effect on the rate or extent of conversion of preformed lipid A precursor to a lipopolysaccharide product. The product was identified as a polymer containing the Rc type core polysaccharide of PRX22H9 linked to the acyl-deficient lipid A unit of the precursor. The acyl-deficient lipopolysaccharide was translocated to the outer membrane at a normal rate. Lipopolysaccharide deficient in saturated fatty acids was also produced by a fabD mutant of Escherichia coli under conditions of limited endogenous fatty acid synthesis. The results indicate that prior incorporation of the saturated O-acyl chains of lipid A is not necessary for extension of the core polysaccharide chain and that synthesis of underacylated lipopolysaccharides occurs under conditions of restricted fatty acid synthesis which permit formation of the beta-hydroxymyristate-containing lipid A precursor.

Topics: Escherichia coli; Kinetics; Lipid A; Lipopolysaccharides; Membrane Lipids; Salmonella typhimurium; Species Specificity; Sugar Acids; Temperature

The cell envelope fraction of Salmonella typhimurium contains an enzyme system which catalyzes transfer of 3-deoxyoctulosonate (KDO) from CMP-KDO to an incomplete, KDO-deficient precursor of lipid A. The enzyme system is firmly membrane-bound, but has been solubilized by treatment with nonionic detergent at alkaline pH and partially purified. Both the particulate and partially purified fractions catalyzed formation of a single reaction product containing 2 residues of KDO. Periodate oxidation of the purified product permitted tentative identification of the KDO disaccharide structure as KDO2-4KDO.

Topics: Cell Membrane; Ketoses; Kinetics; Lipid A; Lipopolysaccharides; Salmonella typhimurium; Sugar Acids; Transferases

We describe here experiments which determine at which stage in the lipid A biosynthesis the polar head groups 4-aminoarabinose, phosphorylethanolamine and 3-deoxy-D-manno-octulosonic acid are transferred to the diphosphorylated glucosamine backbone of the lipid A structure. Use was made of a conditional lethal mutant of Salmonella typhimurium (Ts1) which is defective in the synthesis of 3-deoxy-D-manno-octulosonic acid 8-phosphate and accumulates under nonpermissive conditions an underacylated lipid A intermediate [Lehmann, Rupprecht and Osborn (1977) Eur. J. Biochem. 76, 41-49]. Pulse-chase experiments, including a detailed analysis of radioactive pulse and chase products, demonstrated that this underacylated compound is a key intermediate in the lipid A synthesis. It can serve as direct acceptor for the incorporation of the polar head groups 4-aminoarabinose, phosphorylethanolamine and 3-deoxy-D-manno-octulosonic acid. On the basis of these findings some steps in the sequence of reactions involved in the lipid A biosynthesis are proposed.

Topics: Acetylglucosamine; Amino Sugars; Arabinose; Ethanolamines; Glucosamine; Ketoses; Lipid A; Lipopolysaccharides; Models, Biological; Molecular Weight; Mutation; Organophosphorus Compounds; Phosphates; Salmonella typhimurium; Sugar Acids

A procedure is described for the selection of conditional 3-deoxy-D-manno-octulosonic-acid--Lipid A mutants which depends on temperature sensitivity for both synthesis of complete lipopolysaccharide and for growth. Using this procedure new types of mutants were isolated which cease growth and accumulate lipid A precursors following a shift to nonpermissive temperatures. All precursor molecules differ in their charge as judged by DEAE-cellulose chromatography. While they all contain glucosamine, phosphate and 3-hydroxymyristic acid, they lack detectable 3-deoxy-D-manno-octulosonic acid (dOclA) as well as the nonhydroxylated fatty acids of the complete lipid A structure. Three mutants proved to be conditionally defective in dOclA metabolism, whereas one seems to be blocked at a relatively early step in lipid A synthesis. The phenotypes of all these mutants appear to be due to single mutations by reversion analysis and by characterization of the temperature-resistant revertants. Studies of these mutants may shed light on the essential role of the complete dOclA--lipid A part of lipopolysaccharides in membrane function.

Topics: Aldehyde-Lyases; Animals; Cell Survival; Galactose; Ketoses; Lipid A; Lipopolysaccharides; Mutation; Pentosephosphates; Salmonella Phages; Salmonella typhimurium; Species Specificity; Sugar Acids; Temperature