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.     

Structure of the extracellular gelling polysaccharide produced by Enterobacter (NCIB 11870) species

The gelling polysaccharide produced by a species of Enterobacter (NCIB 11870) contains L-fucose, D-glucose, and D-glucuronic acid in the ratios 1:2:1. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide revealed terminal non-reducing glucose, (1----3)-linked fucose, (1----3,1----4)-linked glucose, and (1----4)-linked glucuronic acid in the ratios 1:1:1.2:0.8. From the results of Smith degradation of the polysaccharide and spectroscopic studies of the acidic tetra- and octa-saccharides produced by bacteriophage-induced enzymic depolymerization of the polysaccharide, the following tetrasaccharide repeating-unit is proposed. (Formula: see text). This repeating-unit is identical to that of the capsular polysaccharide produced by Klebsiella aerogenes serotype K54 except for the absence of O-acetyl groups. The effects of the O-acetyl groups on the secondary structure and rheological properties of these polysaccharides are discussed.     

Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica (serotype T4) organisms

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype T4 organisms was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, has the backbone structure ----(2-glycerol-l)----(phosphate)----(6-alpha-D-galactose-1)---- and is partially O-acetylated on the C2 and C3 galactose residues. Chemical removal of O-acetyl groups from the polysaccharide destroyed both its ability to precipitate with antiserum raised against killed whole serotype T4 organisms and its ability to adhere to sheep erythrocytes in passive haemagglutination experiments. Attempts to elicit antisera using the purified polymer were unsuccessful but a partially purified material was immunogenic.     

Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H

Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.     

Structural elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis using high-resolution NMR spectroscopy.

The capsule of Bacteroides fragilis is unusual in that it consists of two distinct capsular polysaccharides. Using a combination of high-resolution NMR spectroscopy, theoretical calculations, and as few chemical procedures as required, the structure of both polysaccharide antigens (polysaccharides A and B) was elucidated. Using the above procedures, it was possible to obtain the complete structures using minimal quantities of polysaccharides A and B (8 and 5 mg, respectively). Only small amounts of each subjected to chemical analysis were not recoverable. Polysaccharide A is composed of the following repeating unit: [----3)alpha-D-AATp(1----4)[beta-D-Galf(1----3)]alpha-D- GalpNAc(1----3)beta-D-Galp(1----], where AAT is 2-acetamido-4-amino-2,4,6-trideoxygalactose. A pyruvate substituent having the R configuration spans O-4 and O-6 of the beta-D-galactopyranosyl residue. Polysaccharide B is composed of the following repeating unit: [----4)alpha-L-QuipNAc(1----3)beta-D-QuipNAc(1----4)[alpha-L - Fucp(1----2)beta-D-GalpA(1----3)beta-D-GlcpNAc(1----3)]alpha -D-Galp(1----]. A 2-aminoethylphosphonate substituent is situated on O-4 of the N-acetyl-beta-D-glucopyranosyl residue.     

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 acidic polysaccharide from cell wall of Propionibacterium acnes strain C7

The structure of polysaccharide prepared by lysozyme digestion from the cell wall of Propionibacterium acnes strain C7 was examined. The polysaccharide fraction was composed of glucose, galactose, mannose, galactosamine, and diaminomannuronic acid in a molar ratio of 1:1:0.3:1:2. By Smith degradation of the polysaccharide, diaminouronic acid-containing fractions were obtained, and the configuration of diaminouronic acid was identified as 2,3-diacetamido-2,3-dideoxymannuronic acid [Man(NAc)2A] by means of 1H-NMR and 13C-NMR spectroscopic analyses. The results of analyses involving methylation and partial acid hydrolysis led to the conclusion that the polysaccharide has the repeating unit----6)Gal(alpha 1----4)Man(NAc)2A(beta 1----6)Glc(alpha 1----4)Man(NAc)2A (beta 1----3)GalNAc(beta 1--. In addition, a portion of the galactose residues were substituted at C-4 by alpha 1----2 linked mannotriose.     

Structure of the O-antigen of Francisella tularensis strain 15.

The O-specific polysaccharide, obtained by mild acid degradation of the lipopolysaccharide of Francisella tularensis strain 15, contained 2-acetamido-2,6-dideoxy-D-glucose (D-QuiNAc), 4,6-dideoxy-4-formamido-D-glucose (D-Qui4NFm), and 2-acetamido-2-deoxy-D-galacturonamide (D-GalNAcAN) in the ratios 1:1:2. Tri- and tetra-saccharide fragments were obtained on treatment of the polysaccharide with anhydrous hydrogen fluoride and partial hydrolysis with 0.1 M hydrochloric acid, respectively. On the basis of 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and the saccharides, it was concluded that the O-antigen had the structure: ----4)-alpha-D-GalpNAcAN-(1----4)-alpha-D-GalpNAcAN-(1----3) -beta-D-QuipNAc-(1----2)-beta-D-Quip4NFm-(1----. This O-antigen is related in structure to those of Pseudomonas aeruginosa O6, immunotype 1, and IID 1008, and Shigella dysenteriae type 7.     

Structural studies of the Escherichia coli O78 O-antigen polysaccharide

The structure of the O-antigen polysaccharide from Escherichia coli O78 has been investigated; methylation analysis, partial solvolysis with liquid hydrogen fluoride, and 2D-n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the following structure.----3)-beta-D-GlcpNAc-(1----4)-beta-D-GlcpNAc- (1----4)-beta-D-Manp-(1----4)-alpha-D-Manp-(1----     

Isolation and structural studies of phosphate-containing oligosaccharides from alkaline and acid hydrolysates of Streptococcus pneumoniae type 6B capsular polysaccharide

The capsular polysaccharide of Streptococcus pneumoniae serotype 6B [----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap-( 1----4)- D-RibOH-(5-P----]n was depolymerised under alkaline (NaOH) and acidic (HF) conditions. The former treatment yielded, as the major component, alpha-2-P-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-5- P-RibOH. The latter treatment at -16 degrees gave alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH-(5-P----2)- alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH and at 4 degrees gave alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH. These oligosaccharides were characterised by sugar analysis, f.a.b.-m.s., and 1H- and 13C-n.m.r. spectroscopy.     

Antigenic polysaccharides of bacteria. 34. Structure of O-specific polysaccharide chains of lipopolysaccharides from Pseudomonas cepacia strains IMV 4207 (Serotype A) and IMV 598/2

On the basis of non-destructive analysis by means of 1H and 13C NMR spectroscopy and calculation of specific optical rotation, it was concluded that O-specific polysaccharide of Pseudomonas cepacia strain IMV 4207 (serotype A) has the structure (I): (formula; see text) Two structurally different polysaccharides were found in the ratio of approximately 2.5:1 in P. cepacia strain IMV 598/2 which is serologically related to serotype A in Nakamura classification and serotype 2 in Heidt classification. The minor polysaccharide has the structure (I) whereas the major one possesses the structure (II) which is characteristic of the formerly studied O-specific polysaccharide of P. cepacia strain IMV 4137 belonging to serotype 2: ----4)-beta-D-Galp-(1----2)-alpha-L-Rhap-(1----.     

Structural studies of the capsular polysaccharide of Acinetobacter calcoaceticus BD4

Compositional analysis of the intact and carboxyl-reduced capsular polysaccharide of Acinetobacter calcoaceticus BD4 (PS-4) showed it to consist of L-rhamnose, D-glucose, D-glucuronic and D-mannose in molar ratios of 4:1:1:1. 13C-nuclear magnetic resonance spectroscopy, methylation analysis, oligosaccharide analysis and base-catalyzed beta-elimination were used to elucidate the primary structure. Oligosaccharides were obtained by enzymatic depolymerization with a specific bacteriophage-induced depolymerase and by partial acid hydrolysis. Form the results it is concluded that PS-4 consists of repeating units of the heptasaccharide (Formula: see text). The bacteriophage-induced depolymerase was found to be an endo-beta-D-glucosidase that hydrolyzed the bond beta-D-Glc-(1----3)-L-Rha to generate a heptasaccharide in 40% yield.     

Structure of the K95 antigen from Escherichia coli O75:K95:H5, a capsular polysaccharide containing furanosidic KDO-residues

The structure of the K95 antigenic capsular polysaccharide (K95 antigen) of Escherichia coli O75:K95:H5 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and methylation analysis. The K95 polysaccharide, which contains furanosidic 3-deoxy-D-manno-2-octulosonic acid (KDOf) residues, consists of----3)-beta-D-Rib-(1----8)-KDOf-(2----repeating units, has a molecular weight of approximately 25,000 (approximately 65 repeating units), and is randomly O-acetylated (1 acetyl group per repeating unit at unknown positions).     

Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica (serotype A1) organisms

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A1 organisms was purified and characterized by chemical analysis and NMR spectroscopy. The polymer has the structure----3)-O-(2-acetamido-2-deoxy-4-O-acetyl-beta-D-mannopyranos yluronic acid)-(1----4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranose)-(1----. The polysaccharide was immunogenic (able to evoke production of antibodies) for sheep but not for rabbits. Immuno electron-microscopy studies using the Protein A-gold technique showed the polysaccharide to be peripherally located on the bacterial surface. Reduction, oxidation and de-O-acetylation of the polymer did not appear to alter its immunological precipitability with specific antiserum, but all three treatments destroyed its ability to adhere to sheep erythrocytes at neutral pH. De-N-acetylation of the polymer destroyed both immunological precipitability and erythrocyte adherence.     

Synthesis of a tetrasaccharide unit of the capsular polysaccharide of Streptococcus pneumoniae type 9V

In the synthesis of 8-methoxycarbonyloctyl O-(alpha-D-galactopyranosyl)-(1----3)-O-(2-acetamido-2-deoxy-beta-D- mannopyranosyl)-(1----4)-O-(beta-D-glucopyranosyl)-(1----4)-alpha-D- glucopyranoside, which represents a component of the capsular polysaccharide of Streptococcus pneumoniae type 9V, the key step was the coupling of alpha-D-Galp-(1----3)-beta-D-ManpNAc-(1----4)-D-Glc as glycosyl donor with 8-ethoxy-carbonyloctyl 6-O-acetyl-2,3-di-O-benzyl-alpha-D-glucopyranoside as glycosyl acceptor by use of the imidate method. Only the beta-imidate of the trisaccharide could be employed in this glycosidation reaction to give stereoselectively the tetrasaccharide in high yield. The alpha-imidate of the trisaccharide led to hydrolysis of the imidate group.     

Structure of the O-specific polysaccharide of Vibrio fluvialis serovar 3]

O-Specific polysaccharide composed of L-rhamnose and 2-acetamido-2-deoxy-D-mannose was obtained on mild acid degradation of the V. fluvialis lipopolysaccharide. On the basis of the 13C-NMR data and methylation studies, the following structure was suggested for the polysaccharide repeating unit: ----4)-alpha-L-Rhap-(1----3)-beta-D-ManpNAc-(1---- This structure was confirmed by calculations using known glycosidation effects on 13C chemical shifts.     

Antigenic polysaccharides of bacteria. 25. Structure of the O-specific polysaccharide chain of Pseudomonas cepacia 673/2 lipopolysaccharide containing L-glycero-D-manno-heptose

O-Specific polysaccharide, consisting of D-rhamnose and L-glycero-D-manno-heptose (LD-Hep) in a 2 : 1 ratio, was obtained on the mild acid degradation of the Pseudomonas cepacia IMV 673/2 lipopolysaccharide; monosaccharide LD-Hep has not previously been found in O-specific chains of lipopolysaccharides. On the basis of methylation and 13C-NMR data, it was concluded that the polysaccharide is composed of trisaccharide repeating units having the following structure: ----3)-alpha-D-Rha-(1----3)-alpha-D-Rha-(1----2)-alpha-LD-Hep-(1----     

Antigenic bacterial polysaccharides. 13. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from Pseudomonas cepacia strain IMV 4137

On mild acid degradation of a lipopolysaccharide from Pseudomonas cepacia strain IMV 4137, a serologically active O-specific polysaccharide was obtained and shown to contain L-rhamnose and D-galactose. According to 1H- and 13C-NMR data as well as methylation analysis, the polysaccharide is made up of disaccharide repeating units of the following structure:----2)-alpha-L-Rhap-(1----4)-alpha-D-Galp-(1----.     

Antigenic polysaccharides of bacteria of the genus Shigella. Determination of the structure of polysaccharide chains of Shigella boydii type 9 lipopolysaccharide and detection of unusually high molecular weight glycolipid

Specific acidic polysaccharide has been isolated from the Shigella boydii type 9 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and L-rhamnose. From the results of methylation analysis, partial acid hydrolysis and 13C NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [----4)DGlcp(alpha 1----4)DGlcAp(beta 1----3)DGlcNAcp(alpha 1----3)LRhap(alpha 1----]n. The lipopolysaccharide from Sh. boydii 9 was fractionated by gel chromatography on the Sephadex G-200 column in a buffer containing sodium deoxycholate into three fractions. PAGE-SDS of the fractions obtained, 13C NMR- and chromato-mass-spectrometry data indicated that the three fractions contained the O-specific polysaccharide as the only carbohydrate component. The substance from the most high-molecular weight fraction contained unusually long O-specific chains (60,000 dalton). In the fat acid composition this fraction differed from other lipopolysaccharides by absence of beta-hydroxymyristic acid.     

Structural studies on the fucosamine-containing O-specific polysaccharide of Proteus vulgaris O19

The polysaccharide chain of Proteus vulgaris O19 lipopolysaccharide contains D-galactose, N-acetyl-D-glucosamine N-acetyl-D-galactosamine and N-acetyl-L-fucosamine in the ratio 1:1:1:1. The structure of the polysaccharide was established by full acid hydrolysis and methylation analysis, as well as by non-destructive methods, i.e. the computer-assisted evaluation of the 13C-NMR spectrum and computer-assisted evaluation of the specific optical rotation by Klyne's rule. The polysaccharide is regular and built up of tetrasaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp-(1----3)-beta-D-GlcNAcp-(1----3)-alph a-D-Galp- (1----4)-alpha-D-GalNAcp-(1---- The O19-antiserum cross-reacts with lipopolysaccharide from P. vulgaris O42, the structure of which is still unknown. No cross-reactions were observed with O-polysaccharides Pseudomonas aeruginosa O7 and Salmonella arizonae O59 in spite of some structural similarities.