The linkage between O-specific caryan and core region in the lipopolysaccharide of Burkholderia caryophylli is furnished by a primer monosaccharide

From the lipopolysaccharide (LPS) fraction of the plant-pathogenic bacterium Burkholderia caryophylli, the linkage between O-specific caryan and core region was characterised. The LPS fraction was first treated with 48% aqueous HF at 4 degrees C and successively with 1% acetic acid at 100 degrees C. A main oligosaccharide representing the carbohydrate backbone of the core region and a portion of the caryan (three unit of caryose) was isolated by high-performance anion-exchange chromatography. Compositional and methylation analyses, matrix-assisted laser desorption/ionisation mass spectrometry and 2D NMR spectroscopy identified the structure: [carbohydrate structure: see text]. The above residues are alpha-linked pyranose rings, if not stated otherwise. Hep is L-glycero-D-manno-heptose, Car is 4,8-cyclo-3,9-dideoxy-L-erythro-D-ido-nonose and Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid. This finding indicates that QuiNAc residue is the primer monosaccharide, which connects the core oligosaccharide to caryan O-chain.     

Structural analysis of the core region of the lipopolysaccharides from eight serotypes of Klebsiella pneumoniae

The core regions of the lipopolysaccharides (LPS) from Klebsiella pneumoniae serotypes O1, O2a, O2a,c, O3, O4, O5, O8, and O12 were analysed using NMR spectroscopy, ESI-MS spectroscopy, and chemical methods. All the LPSs had similar core structures, as shown below, differing only in the number and position of beta-D-galacturonic acid substituents: [carbohydrate structure: see text] where P is H or alpha-Hep, J, K is H or beta-GalA. LPS from all serotypes contained varying proportions of structures having additional or missing phosphate substituents. The core from serotype O1 contained a minor amount of a previously described variant with alpha-DD-Hep-(1-->2)-alpha-DD-Hep-(1-->6)-alpha-GlcN-(1--> replacing the alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-alpha-GlcN-(1--> component.     

Isolation and characterisation of the lipopolysaccharide from Xanthomonas hortorum pv. vitians

Xanthomonas hortorum pv. vitians is a Gram-negative bacterium that acts as the causative agent of bacterial leaf spot and headrot in lettuce. The lipopolysaccharide (LPS) of this bacterium is suspected to be an important molecule for adhesion to the plants. We have isolated the LPS, prepared the lipid A and the polysaccharide moieties thereof, and characterised all preparations by compositional analysis. Main sugar components are rhamnose and 3-acetamido-3,6-dideoxy-galactose which presumably furnish the O-specific polysaccharide. Other sugars are mannose, glucose, 6-deoxygalactose (fucose), and galacturonic acid, which should be core region constituents, and glucosamine, which builds up the carbohydrate backbone of lipid A. The LPS contains several phosphate groups, most of which are present in the core region. The main fatty acids in the lipid A are C10:0, 3-OH-C10:0 and 3-OH-C12:0. The latter is the only amide-linked fatty acid. Two fatty acids present in small amounts were identified, C8:0 and C11:0.     

The structure of the linkage between the O-specific polysaccharide and the core region of the lipopolysaccharide from Salmonella enterica serovar Typhimurium revisited.

Salmonella enterica sv. Typhimurium strain 1135 possesses smooth(S)-form lipopolysaccharide (LPS). Although the structures of the core region and the O-specific polysaccharide were investigated intensively between the 1960s and the 1980s, the structure of the linkage region between the O-chain and the core was not elucidated unequivocally. By using modern MS and high-field NMR spectroscopy for analysis of the isolated carbohydrate backbone of the LPS, it has been shown that it is a beta-D-Galp residue that links the first repeating unit of the O-specific polysaccharide to O-4 of the last D-Glcp residue of the core region. Interestingly, this particular D-Galp residue is alpha-linked in all following repeating units. The data are discussed with regard to the ligation of O-specific polysaccharide and core region during LPS biosynthesis.     

Lipopolysaccharide core structures and their correlation with genetic groupings of Shigella strains. A novel core variant in Shigella boydii type 16

Bacteria Shigella, the cause of shigellosis, evolved from the intestinal bacteria Escherichia coli. Based on structurally diverse O-specific polysaccharide chains of the lipopolysaccharides (LPSs; O-antigens), three from four Shigella species are subdivided into multiple serotypes. The central oligosaccharide of the LPS called core is usually conserved within genus but five core types called R1-R4 and K-12 have been recognized in E. coli. Structural data on the Shigella core are limited to S. sonnei, S. flexneri and one S. dysenteriae strain, which all share E. coli core types. In this work, we elucidated the core structure in 14 reference strains of S. dysenteriae and S. boydii. Core oligosaccharides were obtained by mild acid hydrolysis of the LPSs and studied using sugar analysis, high-resolution mass spectrometry and two-dimensional NMR spectroscopy. The R1, R3 and R4 E. coli core types were identified in 8, 3 and 2 Shigella strains, respectively. A novel core variant found in S. boydii type 16 differs from the R3 core in the lack of GlcNAc and the presence of a D-glycero-D-manno-heptose disaccharide extension. In addition, the structure of an oligosaccharide consisting of the core and one O-antigen repeat was determined in S. dysenteriae type 8. A clear correlation of the core type was observed with genetic grouping of Shigella strains but not with their traditional division to four species. This finding supports a notion on the existing Shigella species as invalid taxa and a suggestion of multiple independent origins of Shigella from E. coli clones.     

A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeri OX1 possessing two 4,6-O-(1-carboxy)-ethylidene residues in the outer core region.

Pseudomonas stutzeri OXI is a Gram-negative microorganism able to grow in media containing aromatic hydrocarbons. A novel lipo-oligosaccharide from P. stutzeri OX1 was isolated and characterized. For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration. The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-O-acylation) followed by de-N-acylation using hot KOH. The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups. The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy. These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A. All sugars are D-pyranoses and alpha-linked, if not stated otherwise. Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain. This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P. sutzeri OX1 to a hydrocarbon-containing environment.     

Biosynthesis of a novel 3-deoxy-D-manno-oct-2-ulosonic acid-containing outer core oligosaccharide in the lipopolysaccharide of Klebsiella pneumoniae

The core oligosaccharide region of Klebsiella pneumoniae lipopolysaccharide contains some novel features that distinguish it from the corresponding lipopolysaccharide region in other members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella. The conserved Klebsiella outer core contains the unusual trisaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-(2,6)-GlcN-(1,4)-GalUA. In general, Kdo residues are normally found in the inner core, but in K. pneumoniae, this Kdo residue provides the ligation site for O polysaccharide. The outer core Kdo residue can also be non-stoichiometrically substituted with an l-glycero-d-manno-heptopyranose (Hep) residue, another component more frequently found in the inner core. To understand the genetics and biosynthesis of core oligosaccharide synthesis in Klebsiella, the gene products involved in the addition of the outer core GlcN (WabH), Kdo (WabI), and Hep (WabJ) residues as well as the inner core HepIII residue (WaaQ) were identified. Non-polar mutations were created in each of the genes, and the resulting mutant lipopolysaccharide was analyzed by mass spectrometry. The in vitro glycosyltransferase activity of WabI and WabH was verified. WabI transferred a Kdo residue from CMP-Kdo onto the acceptor lipopolysaccharide. The activated precursor required for GlcN addition has not been identified. However, lysates overexpressing WabH were able to transfer a GlcNAc residue from UDP-GlcNAc onto the acceptor GalUA residue in the outer core.     

The structure of the core region of the lipopolysaccharide from Shewanella algae BrY, containing 8-amino-3,8-dideoxy-D-manno-oct-2-ulosonic acid

The structure of the carbohydrate backbone of the lipid A-core region of the LPS from Shewanella algae strain BrY was analysed. The LPS was N,O-deacylated to give three products, which were isolated and studied by chemical methods, NMR and mass spectrometry: [Carbohydrate structures: see text]. All monosaccharides except L-rhamnose had the D-configuration. This LPS presents a second example (after S. oneidensis) of the structure with a novel linking unit between the core and lipid A moieties, 8-amino-3,8-dideoxy-D-manno-oct-2-ulosonic acid (8-amino-Kdo).     

Structural elucidation of a novel core oligosaccharide backbone of the lipopolysaccharide from the new bacterial species Agrobacterium larrymoorei

Agrobacterium larrymoorei is a Gram-negative phytopathogenic bacterium, which produces tumours on Ficus benjamina plants and differs from other Agrobacteria both genetically and biochemically. The lipopolysaccharide (LPS) plays an important role in the pathogenesis of Agrobacteria. The present paper is the first report on the molecular primary structure of the core region of an Agrobacterium LPS. The following structure of the core and lipid A carbohydrate backbone of an R-form LPS of A. larrymoorei was determined by chemical degradations and 1D and 2D NMR spectroscopy: [carbohydrate structure: see text] All sugars are alpha-D-pyranoses if not stated otherwise, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Qui3NAcyl is 3,6-dideoxy-3-(3-hydroxy-2,3-dimethyl-5-oxoprolylamino)glucose, GlcAN and GalAN are amides of GlcA and GalA.     

A novel type of carbohydrate-protein linkage region in the tyrosine-bound S-layer glycan of Thermoanaerobacterium thermosaccharolyticum D120-70.

The surface-layer (S-layer) protein of Thermoanaerobacterium thermosaccharolyticum D120-70 contains glycosidically linked glycan chains with the repeating unit structure -->4)[alpha-D-Galp-(1-->2)]-alpha-L-Rhap-(1-->3)[beta-D-Glcp-(1--> 6)] -beta-D-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Glcp-(1--> . After proteolytic degradation of the S-layer glycoprotein, three glycopeptide pools were isolated, which were analyzed for their carbohydrate and amino-acid compositions. In all three pools, tyrosine was identified as the amino-acid constituent, and the carbohydrate compositions corresponded to the above structure. Native polysaccharide PAGE showed the specific heterogeneity of each pool. For examination of the carbohydrate-protein linkage region, the S-layer glycan chain was partially hydrolyzed with trifluoroacetic acid. 1D and 2D NMR spectroscopy, including a novel diffusion-edited difference experiment, showed the O-glycosidic linkage region beta-D-glucopyranose-->O-tyrosine. No evidence was found of additional sugars originating from a putative core region between the glycan repeating units and the S-layer polypeptide. For the determination of chain-length variability in the S-layer glycan, the different glycopeptide pools were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry, revealing that the degree of polymerization of the S-layer glycan repeats varied between three and 10. All masses were assigned to multiples of the repeating units plus the peptide portion. This result implies that no core structure is present and thus supports the data from the NMR spectroscopy analyses. This is the first observation of a bacterial S-layer glycan without a core region connecting the carbohydrate moiety with the polypeptide portion.     

Structural analysis of the heptose/hexose region of the lipopolysaccharide from Escherichia coli K-12 strain W3100.

The disaccharide L-glycero-D-manno-heptosyl-D-glucose was isolated from the lipopolysaccharide (LPS) of Escherichia coli K-12 strain W3100 after partial hydrolysis with acid, and the structure was determined by methylation analysis, n.m.r. spectroscopy, and comparison with a synthetic standard. In addition, the oligosaccharides L,D-Hep-D-Glc-D-Glc and L,D-Hep-D-Glc-D-Glc-D-Glc were isolated, and their structures were established by g.l.c.-m.s. and methylation analysis. The results indicated that L-glycero-D-manno-heptose, a characteristic constituent of the inner core region, may also occur in the outer core region which, in E. coli, is generally composed of hexoses. A revised structure of the carbohydrate backbone of the hexose/heptose region of the LPS is given.     

Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of Lipid A fatty acid distribution

Lipid A is the lipophilic moiety of lipopolysaccharides (LPSs), the major components of the external membrane of almost all gram-negative bacteria. It is responsible for the toxicity of LPS and has a heterogeneous structure composed of a bis-phosphorylated glucosamine disaccharide backbone that is acylated at the positions 2, 3 of the GlcN I (proximal) and GlcN II (distal) residue with O- and N-linked 3-hydroxy fatty acids (primary substitution). These fatty acids are further acylated by means of their 3-hydroxy groups (secondary substitution). The toxicity of Lipid A is dependent on its primary structure; the number, the length, and the distribution of the fatty acids on the disaccharide backbone strongly influence the endotoxic activity. In this paper a general and easy methodology to obtain secondary fatty acid distribution, which is one of the most difficult issues in the structural determination of Lipid A, is proposed. The method combines ammonium hydroxide hydrolysis and matrix assisted laser desorption ionization (MALDI)-mass spectrometry analysis and has been successfully proven with five different Lipid A species. The procedure exploits the lower stability under mild alkaline conditions of acyl and acyloxyacyl esters with respect to that of the acyl and acyloxyacyl amides. The partially degraded Lipid A species obtained are analyzed by MALDI-MS. The generality of this approach was tested on five Lipid As, namely those arising from Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas reactans, and Burkholderia caryophylli.     

Structure of the lipid A-inner core region and biological activity of Plesiomonas shigelloides O54 (strain CNCTC 113/92) lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative rod associated with episodes of intestinal infections and outbreaks of diarrhea in humans. The extraintestinal infections caused by this bacterium, for example, endopthalmitis, meningitidis, bacteremia, and septicemia, usually have gastrointestinal origin and serious course. The lipopolysaccharide (LPS, endotoxin) as virulence factor is important in enteropathogenicity of this bacterium. LPSs of P. shigelloides and especially their lipid A part, that is, the immunomodulatory center of LPS, have not been extensively investigated. The structure of P. shigelloides O54 lipid A was determined by chemical analysis combined with MALDI-TOF mass spectrometry, and the intact Kdo-containing core region was investigated by NMR spectroscopy on deacylated LPS. Products from alkaline deacylation of LPS, containing 4-substituted uronic acids, are usually very complex and difficult to separate. Since Kdo residues, like sialic acids, form complexes with serotonin, we used immobilized serotonin for one-step isolation of oligosaccharide containing the intact Kdo region from the reaction mixture by affinity chromatography. The major form of lipid A was built of beta-d-GlcpN4PPEtn-(1-->6)-alpha-d-GlcpN1P disaccharide substituted with 14:0(3-OH), 12:0(3-OH), 14:0(3-O-14:0), and 12:0(3-O-12:0) acyl groups at N-2, O-3, N-2', and O-3', respectively. This is a novel structure among known lipid A molecules. Analysis of intact Kdo-lipid A region, lipid A and its linkage with the core oligosaccharide completes the structural investigation of P. shigelloides O54 LPS, resolving the entire molecule. Biological activities and observed discrepancy between in vitro and in vivo activity of P. shigelloides and Escherichia coli LPS are discussed.     

Structural analyses of the lipopolysaccharides from Chlamydophila psittaci strain 6BC and Chlamydophila pneumoniae strain Kajaani 6.

Lipopolysaccharides (LPSs) of Chlamydophila psittaci 6BC and Chlamydophila pneumoniae Kajaani 6 contain 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), GlcN, organic bound phosphate, and fatty acids in the molar ratios of approximately 3:2:2.2:4.8 and approximately 2.9:2:2.1:4.9, respectively. The LPSs were immunoreactive with a monoclonal antibody against a family-specific epitope of chlamydial LPS. This finding, together with methylation analyses of both LPSs and MALDI-TOF MS experiments on de-O-, and de-O,N-acylated LPSs, indicate the presence of a Kdo trisaccharide proximal to lipid A having a structure alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo, which appears to be the main component of the core region in the native chlamydial LPSs. In the de-O-acylated LPSs from Chl. psittaci 6BC and Chl. pneumoniae Kajaani 6, two major molecular species are present that differ in distribution of amide-bound hydroxy fatty acids over both GlcN. It appears that either two (R)-3-hydroxy-18-methylicosanoic acids or one (R)-3-hydroxy-18-methylicosanoic acid and one (R)-3-hydroxyicosanoic acid are attached to the GlcN residues. In contrast, the de-O-acylated LPS of Chl. psittaci PK 5082 contains one major molecular species that has two (R)-3-hydroxyicosanoic acid residues attached to two GlcN residues.     

The structure of the core part of Proteus penneri strain 16 lipopolysaccharide

The structure of the carbohydrate backbone of the lipid A-core region of the lipopolysaccharide (LPS) from Proteus penneri strain 16 was determined using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of the LPS, and of the products of alkaline deacylation of the LPS: formula [see text]. Incomplete substitution is indicated by bold italics. All sugars are in the pyranose form, alpha-Hep is the residue of L-glycero-alpha-D-manno-Hep, alpha-DD-Hep is the residue of D-glycero-alpha-D-manno-Hep, Bu is the (R)-3-hydroxybutyryl residue.     

Intrachain disulfide bond in the core hinge region of human IgG4.

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgG1 and thus may permit formation of a stable intrachain disulfide bond more readily.     

Structural features involved in the mitogenic activity of bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice

Abstract Spleen cells from the C3H/HeJ mouse strain cannot be stimulated by many smooth-type lipopolysaccharides (LPSs), and by the main biologically-active region (lipid A) of these molecules. The genetic origin of this defect (expression of the mutant allele Lps^d at the chromosome 4 locus) was established over 20 years ago, but its biochemical nature has remained undefined. Several investigators have noted, however, that some particular LPSs can bypass this defect, and stimulate the proliferation of C3H/HeJ B lymphocytes. In this study we compare the mitogenic activities of the LPSs isolated from a wild strain (1414) and from a mutant ‘rough’ strain (A100) of Bordetella pertussis . Both LPS-1414 and LPS-A100 were mitogenic for C3H/HeJ spleen cells, but their lipid A fragments were not. This indicates that a carbohydrate structure proximal to lipid A is involved in the mitogenic activity. However, the isolated polysaccharides were not mitogenic. Four sugars are common to both LPS-1414 and LPS-A100: an heptose, and three sugars bearing free amino groups. After removal of these four sugars from the LPSs by nitrous acid treatment, the recovered lipooligosaccharides were not mitogenic in Lps^d spleen cells. The results suggest that substructures present in lipid A and in this group of four sugars are both required for induction of a mitogenic effect in Lps^d splenocytes, whereas lipid A alone can stimulate Lpsⁿ spleen cells.     

Structural studies of the core region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: [carbohydrate: see text] where R=alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1--> or alpha-D-Galp-(1--> (approx. ratio 4:3). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida.     

Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme).

The lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype 06 rough-type mutant A28 was isolated by a modified phenol-chloroform-petroleum ether extraction method. Deoxycholate-polyacrylamide gel electrophoresis indicated a single band with mobility similar to that of the complete core region of the wild-type parent serotype 06 (International Antigenic Typing Scheme) strain. Compositional analysis of the LPS indicated that the core oligosaccharide was composed of D-glucose (three units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (two units), 3-deoxy-D-manno-octulosonic acid (two units), L-alanine (one unit), and phosphate (two units). Under the mild conditions of hydrolysis with methanolic hydrogen chloride, a 7-O-carbamoyl substituent was observed on the second heptose residue. The glycan structure of the LPS was determined by employing one- and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry-based methods with a backbone oligosaccharide that was obtained from the LPS by deacylation, dephosphorylation, and reduction of the terminal glucosamine. On the basis of the results of the present study and our earlier work with the P. aeruginosa 06-derived core-defective mutant R5 (H. Masoud, E. Altman, J. C. Richards, and J. S. Lam, Biochemistry, 33:10568-10578, 1994), a structural model for the complete core oligosaccharide is proposed.