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Isolation, purification and partial analysis of the lipopolysaccharide antigenic determinant recognized by a monoclonal antibody to Legionella pneumophila serogroup 1.

Monoclonal antibody II-6-18 recognizes a serogroup-1-specific Legionella pneumophila antigenic determinant which has been shown to be virulence-associated. We previously reported the physicochemical characterization by means of a quantitative fluorometric assay of monoclonal antibody II-6-18 binding to L. pneumophila, and its implications concerning the nature of the antigen. We describe here the isolation and the purification of the antigen by chemical and immunological methods, followed by its partial chemical analysis. The results demonstrate that the epitope--an immunodominant carbohydrate which includes a fucosamine-like residue--is part of the cell wall lipopolysaccharide (LPS). It is localized in the polysaccharide moiety of the LPS which contains KDO, rhamnose, mannose, glucosamine and an unidentified aminodideoxyhexose X1, but no heptose. The aminodideoxyhexose X1 could be fucosamine and is probably the immunodominant residue in the epitope, localized, at least partially, at the end of the polysaccharide chain.

Antibodies, Monoclonal↗

PubMed2005

GC/MS identification and quantification of constituents of bacterial lipids and glycoconjugates obtained after methanolysis as heptafluorobutyrate derivatives.

In previous articles [Anal. Biochem. 284 (2000) 201; J. Lipid Res. 43 (2002) 794], we reported that the GC/MS identification and quantification of nearly all constituents of glycolipids could be obtained on the same sample in a single GC/MS analysis as heptafluorobutyrate derivatives of the products liberated using acid-catalyzed methanolysis. The same type of data could be obtained on glycoproteins and proteoglycans [Biochemistry 42 (2003) 8342]. These experiments were performed on material from higher organisms, and there was no evidence that bacteria-specific constituents could also be identified and quantified. The current article reports that the GC/MS analysis of compounds liberated by acid-catalyzed methanolysis as heptafluorobutyrate derivatives allows the simultaneous qualitative and quantitative determinations of pentoses, deoxyhexoses, hexoses, hexosamines, uronic acids, Kdo, Mur, heptose, Kdn, and neuraminic acid as well as of most fatty acids (including hydroxylated fatty acids). This approach provides a way of obtaining fingerprints of bacterial constituents and quantification of the overall effect of gene inactivation or of culture conditions.

Bacteria↗

PubMed2003

Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient.

Vibrio parahaemolyticus strain KX-V212 of a novel serotype, which does not belong to any of the known 13 O-serotypes of this vibrio, was isolated from a patient. Its O-antigen harbors a unique strain-specific O-antigenic factor(s), in addition to that shared by the O-antigen of V. parahaemolyticus serotype O2. A carbohydrate backbone nonasaccharide was isolated from the lipopolysaccharide (LPS) of strain KX-V212 by dephosphorylation, reduction and deacylation and found to consist of one residue each of D-glucose, D-galactose, D-GlcN, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5-acetamido-7-(N-acetyl-D-alanyl)amino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (Non5Ac7Ala), and two residues each of D-GlcA and L-glycero-D-manno-heptose (LD-Hep). Analysis of the isolated and deacylated lipid A showed that this oligosaccharide was an artifact resulting from a loss of one GlcN residue from the lipid A backbone. Therefore, the carbohydrate backbone of the LPS is a decasaccharide having the structure shown below. The initial LPS contains also D-GalA and phosphoethanolamine at unknown positions. Both similarity and differences are observed between the LPS of V. parahaemolyticus serotype O2 and strain KX-V212. [carbohydrate structure: see text]

Carbohydrate Sequence↗

PubMed2003

Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3).

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.

Carbohydrate Conformation↗

PubMed2003

Structure of the core-oligosaccharide with a characteristic D-glycero-alpha-D-talo-oct-2-ulosylonate-(2-->4)-3-deoxy-D-manno-oct-2-ulosonate [alpha-Ko-(2-->4)-Kdo] disaccharide in the lipopolysaccharide from Burkholderia cepacia.

The core oligosaccharide in the lipopolysaccharide (LPS) of Burkholderia cepacia GIFU 645(T) was investigated. After mild acid hydrolysis of the LPS, a heptasaccharide was isolated and identified by chemical analyses, GLC-MS, FABMS, and NMR spectroscopy as follows: [carbohydrate structure: see text] where L-alpha-D-Hep stands for L-glycero-alpha-D-manno-heptose, Ko for D-glycero-D-talo-oct-2-ulosonic acid, and Kdo for 3-deoxy-D-manno-oct-2-ulosonic acid.

Burkholderia cepacia↗

PubMed2004

Structural studies on the R-type lipopolysaccharide of Aeromonas hydrophila.

A rough strain of Aeromonas hydrophila, AH-901, has an R-type lipopolysaccharide with the complete core. The following core structure was established by chemical degradations followed by sugar and methylation analyses along with ESIMS and NMR spectroscopy: [formula: see text] where D-alpha-D-Hep and l-alpha-D-Hep stand for D-glycero- and l-glycero-alpha-D-manno-heptose, respectively; Kdo stands for 3-deoxy-D-manno-oct-2-ulosonic acid; all monosaccharides are in the pyranose form; the degree of substitution with beta-D-Gal is approximately 50%. Lipid A of the lipopolysaccharide has a 1,4(')-bisphosphorylated beta-D-GlcN-(1-->6)-alpha-D-GlcN disaccharide backbone with both phosphate groups substituted with 4-amino-4-deoxyarabinose residues.

Aeromonas hydrophila↗

PubMed2004

The structure of the carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-79104.

The structure of the carbohydrate backbone of the lipopolysaccharide from Pectinatus frisingensis strain VTT E-79104 was analyzed using chemical degradations, NMR spectroscopy, mass spectrometry, and chemical methods. The LPS contains two major structural variants, differing in the presence or absence of an octasaccharide fragment. The largest structure of the carbohydrate backbone of the LPS, that could be deduced from experimental results, consists of 20 monosaccharides arranged in a nonrepetitive sequence: [carbohydrate structure: see text] where R is H or 4-O-Me-alpha-L-Fuc-(1-2)-4-O-Me-beta-Hep-(1-3)-alpha-GlcNAc-(1-2)-beta-Man-(1-3)-beta-ManNAc-(1-4)-alpha-Gal-(1-4)-beta-Hep-(1-3)-beta-GalNAc-(1- where Hep is a residue of D-glycero-D-galacto-heptose; all monosaccharides have the D-configuration except for 4-O-Me-L-Fuc and L-Ara4N. This structure is architecturally similar to the oligosaccharide system reported previously in P. frisingensis VTT E-82164 LPS, but differs from the latter in composition and also in the size of the outer region.

Acetylation↗

PubMed2004

The complete structure of the lipooligosaccharide from the halophilic bacterium Pseudoalteromonas issachenkonii KMM 3549T.

Novel lipooligosaccharide components were isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Pseudoalteromonas issachenkonii type strain KMM 3549T. The complete structure was achieved by chemical analysis, 2D NMR spectroscopy and MALDI mass spectrometry as the following: [carbohydrate formula see text] All sugars are d-pyranoses. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked. In addition, by MALDI mass spectrometry of the intact LOS, the lipid A moiety was also identified as a mixture of penta-, tetra- and triacylated species.

Bacillaceae↗

PubMed2004

The structure of the phosphorylated carbohydrate backbone of the lipopolysaccharide of the phytopathogen bacterium Pseudomonas tolaasii.

A novel core-lipid A backbone oligosaccharide was isolated and identified from the lipopolysaccharide fraction of the mushrooms pathogen bacterium Pseudomonas tolaasii. The oligosaccharide was obtained by alkaline treatment of the lipopolysaccharide fraction. Since the repeating unit of the O-antigen contained one residue of -->4)-alpha-l-GulpNAcAN, the hydrolysis was accompanied by beta-elimination on this residue and following depolymerization, producing a mixture of oligosaccharides. The complete structural elucidation showed the presence of a single core glycoform and was achieved by chemical analysis and by (1)H, (31)P, and (13)C NMR spectroscopy applying various 1D and 2D experiments. [structure: see text]. All sugars are alpha-d-pyranoses, if not stated otherwise. Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid, P is phosphate. QuiN and DeltaGulNA are present in nonstoichiometric amount.

Carbohydrate Conformation↗

PubMed2004

Structure of minor oligosaccharides from the lipopolysaccharide fraction from Pseudomonas stutzeri OX1.

A minor oligosaccharide fraction was isolated after complete de-acylation of the lipooligosaccharide extracted from Pseudomonas stutzeri OX1. The full structure of this oligosaccharide was obtained by chemical degradation, NMR spectroscopy and MALDI-TOF MS spectrometry. These experiments showed the presence of two novel oligosaccharides (OS1 and OS2): [structure: see text] where R=(S)-Pyr(-->4,6) in OS1 and alpha-Rha-(1-->3) in OS2. All sugars are D-pyranoses, except Rha, which is L-pyranose. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Pyr is pyruvic acid, P is phosphate.

Carbohydrate Conformation↗

PubMed2005

Structural analysis of the lipopolysaccharide of Pasteurella multocida strain VP161: identification of both Kdo-P and Kdo-Kdo species in the lipopolysaccharide.

The structure of the lipopolysaccharide from the Pasteurella multocida strain VP161 was elucidated. The lipopolysaccharide was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the lipopolysaccharides were determined on the basis of the combined data from these experiments. [structure: see text]. Based on the NMR data, all sugars were found in pyranose ring forms, and Kdo is 2-keto-3-deoxy-octulosonic acid, L-alpha-D-Hep is L-glycero-D-manno-heptose, PPEtn is pyrophosphoethanolamine and PCho is phosphocholine. Intriguingly, when the O- and fully deacylated LPS was examined, it was evident that there was variability in the arrangement of the Kdo region of the molecule. Glycoforms were found with a Kdo-P moiety, as well as glycoforms elaborating a Kdo-Kdo group. Furthermore the Glc II residue was not attached to Hep I when two Kdo residues were present, but it was attached when the Kdo-P arrangement was elaborated, suggesting a biosynthetic incompatibility due to either steric hindrance or an inappropriate acceptor conformation. This variation in the Kdo region of the LPS was also observed in several other Pasteurella multocida strains investigated including the genome strain Pm70.

Carbohydrate Sequence↗

PubMed2005

Structural analysis of the core oligosaccharide from Pasteurella multocida strain X73.

The structure of the core oligosaccharide region of the lipopolysaccharide from the Pasteurella multocida strain X73 was elucidated. The lipopolysaccharide was subjected to a variety of degradative procedures. The structure of the purified oligosaccharide was established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure illustrates a similar structure to the recently identified oligosaccharide from another P. multocida strain VP161, but with additional symmetrical substitution of the terminal galactose residues with phosphoethanolamine moieties, where based on the NMR data all sugars were found in pyranose ring forms and Kdo is 3-deoxy-alpha-D-manno-2-oct-2-ulosonic acid, l,D-alpha-Hep is l-glycero-D-manno-heptose, PEtn is phosphoethanolamine and PCho is phosphocholine.

Acylation↗

PubMed2005

The complete structure of the core carbohydrate backbone from the LPS of marine halophilic bacterium Pseudoalteromonas carrageenovora type strain IAM 12662T.

The complete novel structure of the components of the core oligosaccharide fraction from the LOS of the halophilic marine bacterium Pseudoalteromonas carrageenovora was characterized. The fully de-acylated lipooligosaccharide was studied by means of compositional analysis, matrix-assisted laser desorption/ionization mass spectrometry and complete (1)H and (13)C and (31)P NMR spectroscopy. The core oligosaccharide is composed by a mixture of species differing for the length of the sugar chain and the phosphorylation pattern: [carbohydrate structure]; see text. All sugars are D-pyranoses. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked.

Acylation↗

PubMed2005

Cold temperature-induced modifications to the composition and structure of the lipopolysaccharide of Yersinia pestis.

Following a report of variations in the lipopolysaccharide (LPS) structure of Yersinia pestis at mammalian (37 degrees C) and flea (25 degrees C) temperatures, a number of changes to the LPS structure were observed when the bacterium was cultivated at a temperature of winter-hibernating rodents (6 degrees C). In addition to one of the known Y. pestis LPS types, LPS of a new type was isolated from Y. pestis KM218 grown at 6 degrees C. The core of the latter differs in: (i) replacement of terminal galactose with terminal d-glycero-d-manno-heptose; (ii) phosphorylation of terminal oct-2-ulosonic acid with phosphoethanolamine; (iii) a lower content of GlcNAc, and; (iv) the absence of glycine; lipid A differs in the lack of any 4-amino-4-deoxyarabinose and presumably partial (di)oxygenation of a fatty acid(s). The data obtained suggest that cold temperature switches on an alternative mechanism of control of the synthesis of Y. pestis LPS.

Carbohydrate Sequence↗

PubMed2005

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.

Acetic Acid↗

PubMed2005

Isolation and characterisation of the lipopolysaccharide from Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian copper mine.

The lipopolysaccharide (LPS) of the Gram-negative Acidiphilium strain GS18h/ATCC55963, a new soil isolate, exhibited very low endotoxic activity as determined by Limulus gelation activity, lethal toxicity in galactosamine (GalN) sensitised mice, and level of tumor necrosis factor alpha (TNFalpha) in the blood serum of BALB/c mice. Analysis of the LPS, specially of lipid A which usually accounts for the toxicity, revealed the latter to contain glucosamine and phosphate besides fatty acids, of which 14:0(3-OH), 18:0(3-OH), 18:1 and 19:0(cyclo) are the major components, while 12:0, 16:0, 19:1, 20:0(3-OH) and 20:1(3-OH) are present in small amounts. The 14:0(3-OH) and 18:0(3-OH) fatty acids are amide-linked, whereas the rest are ester bound. Glucose, galactose, mannose, rhamnose, heptose, galacturonic acid and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) were present in the polysaccharide part of this LPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the LPS showed a macromolecular heterogeneity distinctly different from those of Escherichia coli or Salmonella. The toxicity of this LPS being extremely low attributed to fatty acid composition of its lipid A, promises potential therapeutic application.

Acidiphilium↗

PubMed2005

Candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: developmental chemistry and investigation of immunological responses following immunization of mice and rabbits.

Glycoconjugates were prepared by covalently linking the immunogenic protein carrier CRM(197) to O-deacylated lipopolysaccharide (LPS) derived from Neisseria meningitidis (strain H44/76), immunotype L3 galE LPS. This mutant strain elaborates a truncated LPS structure that displays immunological epitopes characteristic of 76% of Group B meningococcal (NmB) strains. CRM(197) was covalently linked either to the reducing glucosamine residue of the lipid A region of the O-deacylated LPS or to a 2-keto-3-deoxy-octulosonic acid (Kdo) residue in the inner core region of the O-deacylated LPS. In both rabbits and mice a much stronger IgG response to the immunising antigen was generated in those animals that received conjugates linked via the lipid A region. Sera from mice that were immunized with these conjugates were assayed for their reactivity with LPS, both mutant and wild-type, of several homologous and heterologous NmB strains. Sera obtained from mice immunized with conjugates in which the carrier protein was linked via the Kdo moiety were only able to react with O-deacylated, but not fully acylated (native), LPS from the homologous strain. However, sera obtained from mice that were immunized with conjugates, in which the carrier protein was coupled to the lipid A region, reacted predominately with inner core epitopes that contained phosphoethanolamine at the same 3-position of the distal heptose residue (HepII) of the inner core LPS as was present on the immunising antigen. Additionally it was observed that sera from rabbits immunised with lipid A linked conjugates, unlike the mice responses, were generally not as specific for LPS antigens that contained phosphoethanolamine at the same 3-position as was present on the immunising antigen, but showed a broader inner core recognition, whereas those rabbits that received the Kdo-linked conjugates gave only a very weak non-specific response to all immunotypes. Finally, the sera from two out of six mice that had received lipid A linked conjugates had bactericidal activity against L3 wild-type NmB strain 8047 and one of these was able to passively protect against meningococcal infection in an infant rat model. This study demonstrates evidence towards the proof-in-principle that by using Nm inner core LPS conjugates coupled via the lipid A region with an intact phosphoethanolamine at the O-3 position of the HepII of the inner core LPS, it is possible to elicit functional and protective antibodies against meningococcal infection.

Animals↗

PubMed2006

Production of a D-glycero-D-manno-heptosyltransferase mutant of Mannheimia haemolytica displaying a veterinary pathogen specific conserved LPS structure; development and functionality of antibodies to this LPS structure.

Previous structural studies of the lipopolysaccharides from the veterinary pathogens Mannheimia haemolytica (Mh), Actinobacillus pleuropneumoniae (Ap) and Pasteurella multocida (Pm) had identified a conserved inner core oligosaccharide structure that was present in all strains investigated. In order to examine the potential of this inner core structure as a vaccine, a mutagenesis strategy was adopted to interrupt a D-glycero-D-manno-heptosyltransferase gene (losB) of Mh. This gene encodes the enzyme responsible for the addition of a D-glycero-D-manno-heptose residue, the first residue beyond the conserved inner core, and its inactivation exposed the conserved inner core structure as a terminal unit on the mutant LPS molecule. Subsequent analyses confirmed the targeted structure of the mutant LPS had been obtained, and complementation with losB in trans confirmed that the losB gene encodes an alpha-1,6-D-glycero-D-manno-heptosyltransferase. Monoclonal antibodies raised in mice to this LPS structure were found to recognise LPS and whole-cells of the truncated mutant and wild-type Mh. The antibodies were bactericidal against a wild-type Mh strain and were able to passively protect mice in a model of Mh disease. This illustrates that it is possible to raise functional antibodies against the conserved inner core LPS structure.

Animals↗