The synthesis of two repeating units of Haemophilus influenzae type a capsular antigen

The repeating units 2-O-beta-D-glucopyranosyl-L-ribitol 4'- and 1-phosphate of Haemophilus influenzae type a capsular antigen have been synthesised by condensation of an alpha-D-glucopyranosyl bromide derivative with 5-O-allyl-1,2,3-tri-O-benzyl-D-ribitol followed by selective deprotection of HO-4' or HO-1, phosphorylation, and removal of the blocking groups.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 87/D2 and international type strains from E. coli O128

The O-antigen of the lipopolysaccharide (LPS) from the enteroaggregative Escherichia coli strain 87/D2 has been determined by component analysis together with NMR spectroscopy. The polysaccharide has pentasaccharide repeating units in which all the residues have the galacto-configuration. The repeating unit of the O-antigen, elucidated using the O-deacylated LPS, is branched with the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately 0.7 per repeating unit) distributed over two positions. Subsequent analysis showed that the galactose residue carries acetyl groups at either O-3 or O-4 in a ratio of approximately 2:1. The international reference strain from E. coli O128ab was investigated and the repeating unit of the O-antigens has the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately one per repeating unit) distributed over two positions. The integrals of the resonances for the O-acetyl groups indicated similarities between the O-antigen from E. coli O128ab and that of E. coli strain 87/D2, whereas the O-acetyl substitution pattern in the E. coli O128ac O-antigen differed slightly. Enzyme immunoassay using specific anti-E. coli O128ab and anti-E. coli O128ac rabbit sera confirmed the results.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 1. Structural analysis of the highly hydrophobic lipopolysaccharide, including the O-antigen, its biological repeating unit, the core oligosaccharide, and the linkage between them

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [-->2)-beta-d-Quip3NAcyl-(1-->3)-alpha-l-Rhap2OAc-(1-->3)-alpha-d-FucpNAc-(1-->] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--> residue and the -->3)-beta-d-FucpNAc-(1-->4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The -->3)-beta-d-FucpNAc-(1--> residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.     

Structure of a new 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii PCM 1542

The O-specific polysaccharide of Citrobacter gillenii PCM 1542 from serotype O-12a,12 b is composed of one residue each of D-glucose, D-GlcNAc, 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose (D-GlcNAcyl) and two GalNAc residues. On the basis of sugar and methylation analyses of the intact and Smith degraded polysaccharides, along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched pentasaccharide repeating unit of the O-specific polysaccharide was established:This structure differs significantly from that of the O-specific polysaccharide of C. gillenii PCM 1544 from the same serotype O-12a,12 b, which has been established earlier (Kübler-Kielz.shtsls;b, J. et al. Carbohydr. Res. 2001, 331, 331-336). Serological studies confirmed that the two O-antigens are not related and suggested that strains PCM 1542 and 1544 should be classified into different O-serogroups.     

Determination of the structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with the capsule from type b Haemophilus influenzae

The structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with that from type b Haemophilus influenzae, was determined by using a combination of chemical and spectroscopic techniques. The structure of the K100 repeating unit was found to be----3)-beta-D-Ribf-(1----2)-D-ribitol-5-(PO4----. The K100 polysaccharide is thus identical in composition to, but different in linkage from, the H. influenzae type b capsular polysaccharide, which has beta-D-Ribf-(1----1)-D-ribitol linkages.     

The nucleotide sequence of oocyte 5S DNA in Xenopus laevis. II. The GC-rich region

The primary sequence of the GC-rich half of the repeating unit in X. laevis 5S DNA has been determined in both a single plasmid-cloned repeating unit and in the total population of repeatig units. The GC-rich half of the repeating unit contains a single long duplication of 174 nucleotides. The duplicated segment commences 73 nucleotides preceding the 5' end of the gene and terminates at nucleotide 101 of the gene. The duplicated portion of the gene, termed the pseudogene, differs by 10 nucleotides from the corresponding portion of the gene, and the remaining duplicated sequence of 73 nucleotides differs by 13 nucleotides. The plasmid-cloned repeating unit differs from the dominant sequence in the total population repeating units by 6 nucleotides in the GC-rich region. Evidence is provided that most of the CpG dinucleotides in 5S DNA are at least partially methylated.     

The type-specific substance from Pneumococcus type 10A(34). The phosphodiester linkages

1. The phosphate groups in the type-specific substance S. 10A from Pneumococcus type 10A (34) were shown to join the hydroxyl group at position 1 or 5 of ribitol and the hydroxyl group at position 5 or 6 of a d-galactofuranosyl residue in the next repeating unit. 2. A partial formula of the type-specific substance was derived.     

The type-specific substance from Pneumococcus type 13

1. The type-specific substance, S.13, from Pneumococcus type 13 was subjected to hydrolysis with alkali, followed by enzymic dephosphorylation, to yield a pentasaccharide. 2. The pentasaccharide, corresponding to the dephosphorylated repeating unit of S.13, was shown to be O-beta-d-galactopyranosyl-(1-->4)-O-beta- d-glucopyranosyl-(1-->3)-O-beta-d- galactofuranosyl-(1-->4)-O-2-acetamido-2-deoxy-beta-d- glucopyranosyl-(1-->2)-ribitol. 3. The phosphodiester linkages in S.13 join the hydroxyl group at position 1 of ribitol and the hydroxyl group at position 4 of a galactopyranosyl residue in the next repeating unit. 4. Ester groups, presumably O-acetyl, are located on positions 2 or 3 of most glucopyranosyl residues in S.13. 5. A partial structure for S.13 is proposed.     

The conformations of the O-specific polysaccharides of Shigella dysenteriae type 4 and Escherichia coli O159 studied with molecular mechanics (MM3) filtered systematic search

The branched O-antigens of Escherichia coli O159 and Shigella dysenteriae type 4 are structurally related and are known to show cross-reactivity with antibodies. In the present study, conformational analyses were performed on these two O-antigens using molecular mechanics MM3(96) with filtered systematic search. The results show very strong steric restrictions for the trisaccharide at the branch point of the E. coli O159 antigen, especially for the β-d-GlcNAc-(1 → 3)-β-d-GlcNAc linkage of the main chain. For the type 4 O-antigen the calculations show essentially a single conformation with respect to the α-d-GlcNAc-(1 → 3)-α-d-GlcNAc linkage of the main chain and three different favoured conformations for the fucose branch. Consecutive repeating units of the S. dysenteriae type 4 and E. coli O159 O-antigens form linear extended chains with significant flexibility between the branches. Comparative calculations carried out with the SWEET server indicate that our method of filtered systematic search is a superior method in the case of branched, constrained oligosaccharides. Based on the results of the MM3 calculations, we propose that the common epitope explaining the cross-reactivity comprises the fucose branch, the downstream GlcNAc and part of the uronic acid.     

The specific substance from Pneumococcus type 34 (41). The phosphodiester linkages

1. The phosphate groups in the type-specific substance S.34 from Pneumococcus type 34 (U.S. type 41) were shown to join the hydroxyl group at position 1 or 5 of ribitol and the hydroxyl group at position 3 of a d-galactofuranosyl residue in the next repeating unit. 2. A partial structure of the type-specific substance was derived. 3. New syntheses of d-galactose 2-phosphate and d-galactose 3-phosphate are described.     

Acceptor specificity of mannosyl transferases from Salmonella of serotypes C2 and C3

Synthetic mono- and disaccharide derivatives of moraprenyl pyrophosphate were studied as mannose acceptors during the assembly of the repeating unit Rha-Man-Man-Gal of the Salmonella newport (serogroup C2) and S. kentucky (serogroup C3) O-antigens. Mannosyl transferases revealed strict specificity towards the configuration of terminal monosaccharide residue at C1 as well as to the type of linkage between monosaccharide residues in the disaccharide acceptor. The specificity of mannosyl transferases towards the structure of subterminal monosaccharide was not absolute. Alpha-D-Glucose and alpha-D-mannose derivatives were found not to serve as mannosyl residue acceptors, whereas those of alpha-D-talose, alpha-D-fucose, 4-deoxy-D-xylo-hexose and Man (alpha 1-3) glucose were substrates in enzymatic mannosylation with formation of polyprenyl pyrophosphate trisaccharides. These derivatives could serve as substrates for two subsequent enzymatic reactions: rhamnosylation and polymerization of the repeating units, yielding 40-60% of the polysaccharides.     

Antigenicity of a viral peptide displayed on β-galactosidase fusion proteins is influenced by the presence of the homologous partner protein

Abstract In an earlier study of the distribution of O-serotypes among clinical isolates of Serratia marcescens , two apparently new serotypes were identified, represented by strains S1254 and S3255. Studies using ELISA, immunoblotting and the Quellung reaction have shown that they qualify for inclusion in the O-antigenic typing scheme on three counts: (1) they possess chemically distinct O-antigenic repeating units, (2) the O-antigens are serologically distinguishable from all others, and (3) they are found in a significant proportion of clinical S. marcescens strains (13% and 6% respectively). S1254, the type strain for serotype O27, is an acapsular strain which expressed a glucorhamnan with a disaccharide repeating unit as its lipopolysaccharide side chain. It cross-reacts with serotype O4, the O antigen of which is an O-acetylated form of the O27 glucorhamnan, but this cross-reaction can be eliminated by reciprocal cross-absorption. S3255, the type strain for serotype O28, has a mannose homopolymer as its O-antigen and is the only S. marcescens serotype with a trimeric repeating-unit structure. However, it cross-reacts with the O5 serotype strain due to similarities in their acidic capsular polysaccharides. Cross-absorption and the production of serum to an acapsular variant of serotype strain O28 produced typing reagents which could differentiate serotypes O5 and O28.     

Structure of the type 5 capsular polysaccharide of Staphylococcus aureus

The Staphylococcus aureus type 5 capsular polysaccharide is composed of 2-acetamido-2-deoxy-L-fucose (1 part), 2-acetamido-2-deoxy-D-fucose (1 part), and 2-acetamido-2-deoxy-D-mannuronic acid (1 part). On the basis of methylation analysis, optical rotation, high-field one- and two-dimensional 1H- and 13C-n.m.r. experiments, and selective cleavage with 70% aqueous hydrogen fluoride, the polysaccharide was found to be a partially O-acetylated (50%) polymer of the repeating trisaccharide unit, [----4)-3-O-Ac-beta-D-ManpNAcA-(1----4)-a-L-FucpNAc-(1----3) -beta-D-FucpNAc-(1----]n.     

Structure of the capsular polysaccharide of Haemophilus pleuropneumoniae serotype 5

The capsular polysaccharide of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 (ATCC 33377) was found to be a linear type polysaccharide of a repeating disaccharide unit composed of 2-acetamido-2-deoxy-D-glucose and 3-deoxy-D-manno-2-octulosonic acid (dOclA). By composition analysis, methylation, partial hydrolysis and 1H and 13C nuclear magnetic resonance studies, it was concluded that the capsular polysaccharide is a high-molecular-mass unbranched polymer having the structure: [6)-alpha-D-GlcNAcp-(1-5)-beta-dOclAp-(2]n.     

The type-specific substance from Pneumococcus type 29

1. A pentasaccharide, corresponding to the dephosphorylated repeating unit of the specific substance, S.29, from Pneumococcus type 29, was obtained by hydrolysis with alkali followed by enzymic dephosphorylation. 2. The pentasaccharide was shown to be O-2-acetamido-2-deoxy-beta-d-galactopyranosyl-(1-->6)-O-beta-d-galactofuranosyl-(1-->3)-O-beta-d-galactopyranosyl-(1-->6)-O-beta-d-galactofuranosyl-(1-->1)-ribitol. 3. The phosphodiester linkages in S.29 join the hydroxyl group at position 5 of ribitol and the hydroxyl group at position 3 or 4 of a 2-acetamido-2-deoxy-d-galactose residue in the next repeating unit. 4. A partial structure for S.29 was deduced from these experiments.     

Partial composition of the O-antigens of O-group Escherichia

The partial composition of O-antigens in Escherichia known as the causative agents of escherichioses localized in the intestine and other organs has been studied. Escherichia of groups 01, 02 and 03 are characterized by different partial composition of their O-antigens. This composition can be expressed by the formulae: 01a, 1b; 01a, 1b, 1c; 01a, 1b, 1c, 1d; 01a, 1e; 02a, 2b, 2c; 02a, 2b, 2c, 2d, 2e; 02a, 2b, 2d, 2e, 2g; 02a, 2c, 2e; 06a, 6b; 06a, 6c. No pronounced correlation between the partial composition of O-antigens within the O-group and the presence of different K- or H-antigens in the strain has been established. The scheme of adsorption has been developed and anti-Escherichia factor O-sera have been obtained. The use of these sera allows one to differentiate Escherichia strains, isolated from patients, within the O-groups according to the partial composition of their antigens, which is of both diagnostic and epidemiological importance.     

Structural heterogeneity in the lipopolysaccharides of Pseudomonas syringae with O-polysaccharide chains having different repeating units.

Studies by sugar and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy revealed a structural heterogeneity in the O-polysaccharides of Pseudomonas syringae pvs. coronafaciens IMV 9030 and atrofaciens IMV 8281 owing to the presence of different types of repeating units. In strain IMV 9030, the major repeating units are a linear alpha-L-rhamnose trisaccharide and a tetrasaccharide (A, n=0 or 1). A minor repeating unit is a branched pentasaccharide with an alpha-L-rhamnose main chain and a lateral 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) residue (B, X=2, n=1). In strain IMV 8281, all repeating units are branched and differ in size and position of substitution of one of the alpha-L-rhamnose residues (tetrasaccharide, B, X=3, n=0; pentasaccharides, B, X=2 or 3, n=1). [structure--see text] Reinvestigation of the structure of the branched O-polysaccharide of P. syringae pv. tomato IPGR 140 showed that, together with the major tetrasaccharide repeating unit (B, X=3, n=0) [Knirel, Y. A., et al. Carbohydr. Res. 1993, 243, 199-204], it has a minor pentasaccharide repeating unit (B, X=3, n=1).     

Somatic antigens of Shigella. The strucuture of the specific polysaccharide chain of Shigella dysenteriae type 5 lipopolysaccharide.

The specific polysaccharide was released from Shigella dysenteriae type 5 lipopolysaccharide by mild acidic hydrolysis and then purified by gel chromatography on Sephadex G-50. The polysaccharide was built up of residues of D-mannose, 2-acetamido-2-deoxy-D-glucose, 3-0-(D-1-carboxyethyl)-L-rhamnose (rhamnolactylic acid) and 0-acetyl groups in a ratio 2:1:1:1. On the basis of radiospectroscopy, methylation analysis, Smith degradation, and chromium trioxide oxidation, the repeating oligosaccharide unit of the polysaccharide can be assigned the following structure: (formula: see text) where GlcNAc is 2-acetamido-2-deoxy-D-glucopyranose, Manp is mannopyranose, RhaLcA is rhammolacytic acid and Ac is an acetyl group. The serological properties of Sh. dysenteriae somatic antigens are discussed in relation to the chemical structures of their specific polysaccharides.     

C3b binding, but not its breakdown, is affected by the structure of the O-antigen polysaccharide in lipopolysaccharide from Salmonellae

Bacteria whose lipopolysaccharide contains O-antigen side chains activate complement via the alternative pathway. We have shown previously that three strains of Salmonella, differing in the chemical structure of their O-antigens, consumed C3 to different extents when incubated in C4-deficient guinea pig serum. Moreover, sheep erythrocytes coated with lipopolysaccharide purified from these strains mimicked whole cells in C3 consumption, proving that lipopolysaccharide alone could account for these results. We have now measured the deposition of 125I-C3 in this system, and found that C3 deposition parallels C3 consumption in rate and extent, and differs for surfaces bearing different O-antigens, whether tested with bacteria or with erythrocytes coated with purified lipopolysaccharide. We have also examined the fate of C3 on these Salmonellae by measuring the size and quantity of 125I-C3 breakdown fragments by SDS-PAGE, and have determined the kinetics of conversion of C3b to iC3b by using conglutinin, a molecule that binds specifically to iC3b. There is no difference in breakdown of C3b deposited on cells with different O-antigens: all show partial conversion to iC3b and C3dg as indicated by 68,000, 44,000, and 41,000 m.w. bands on reduced SDS gels. Furthermore, for all strains, the Ka of conglutinin binding to iC3b is similar (0.49 to 0.69 X 10(8) M-1), as is the rate of generation of iC3b and the final ratio of iC3b:C3b + iC3b (0.62 to 0.72). We therefore postulate that the fine structure of the O-antigen in lipopolysaccharide determines the magnitude of alternative pathway activation on the bacterial surface by affecting the rate and extent of C3b deposition, but not the rate and extent of breakdown of C3b.     

Structural determination and immunochemical characterization of the type V group B Streptococcus capsular polysaccharide

The type V capsular polysaccharide of group B Streptococcus has been isolated and purified, and its repeating unit structure determined. The native type V polysaccharide contains D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and sialic acid in a molar ratio of 3:2:1:1. Methylation analysis and 1H NMR and 13C NMR analysis of the native type V polysaccharide and of its specifically degraded products permitted the determination of the repeating unit structure of the type V polysaccharide: [formula: see text] The type V polysaccharide has certain structural features in common with other group B streptococcal capsular polysaccharides but is antigenically distinct: no immunologic cross-reactivity was observed between type V and types Ia, Ib, II, III, or IV polysaccharides. Studies of antibody binding to the partially degraded forms of the type V polysaccharide indicated that the native epitope is complex, involving most if not all of the sugar residues of the repeating unit.