The Shigella flexneri O-antigenic polysaccharide chain. Nature of the biological repeating unit

The sequence of monosaccharides in the biological repeating tetrasaccharide unit of Shigella flexneri variant Y O-antigenic polysaccharide chain was determined by subjecting three oligosaccharides of the polysaccharide, obtained by phage-Sf6-mediated enzymatic hydrolysis, to methylation analysis and proton nuclear magnetic resonance spectroscopy. The smallest saccharide was shown to be a tetrasaccharide with the structure alpha-L-Rhap-(1-2)-L-Rha. The next saccharide, an octasaccharide, was shown to be a dimer of the tetrasaccharide with the L-Rha residues linked alpha 1.3. The longest saccharide was shown to be a decasaccharide with the following structure: alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-beta-D-GlcpNAc-(1-2)-alpha-L-Rhap-(1-2)-alpha-L-Rhap++ + +-(1-3)-alpha-L-Rhap-(1-3)-beta-D-GlcpNAc-(1-2)-alpha-L-R hap-(1-2)-L-Rha. Thus the decasaccharide differed from the octasaccharide and tetrasaccharide by having the alpha-L-Rhap-(1-2)-L-Rhap disaccharide added in the terminal non-reducing end of the saccharide chain. This shows that the alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-D-GlcpNAc tetrasaccharide is the biological repeating unit of the O chain and that the repeating units are joined through a beta-D-GlcpNAc-(1-2)-L-Rhap linkage. Inhibition experiments utilizing the enzyme-linked immunosorbent assay (ELISA) with S. flexneri Y lipopolysaccharide/S. flexneri Y rabbit antiserum showed that the decasaccharide was the best inhibitor (threefold as active as the octasaccharide and sixtyfold as active as the tetrasaccharide); this supports the postulated structure of the biological repeating unit.     

Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella aerogenes type 54[A3(S1)]

Several strains of bacteriophage have been isolated that induce the formation of a polysaccharide hydrolase after infection of Klebsiella aerogenes type 54 [A3(S1)]. The action of this enzyme on polysaccharide solutions was to decrease their viscosity and increase their reducing value. These effects were associated with the release of two oligosaccharides (O1 and O2) from the polysaccharide. These two substances are not identical with any of the four oligosaccharides isolated from autohydrolysates. The two enzymically isolated fractions have been tentatively identified as tetrasaccharides, and oligosaccharide O2 is probably an acetylated version of oligosaccharide O1. This latter oligosaccharide differs in some way, still unknown, from the tetrasaccharide cellobiosylglucuronosylfucose found in acid hydrolysates of the slime polysaccharide. The enzyme is limited in its activity to the polysaccharide excreted by the A3 strain of K. aerogenes type 54 or by similar strains. It is also active on the polysaccharides altered by acid or alkaline treatment. The enzyme has optimum activity at pH6.5. A study of the products released by enzyme action has shown it to be a fucosidase splitting the fucosylglucose linkages found in the intact polysaccharide.     

The structural basis for the serospecificity of Actinobacillus suis serogroup O:2.

Actinobacillus suis is an important bacterial pathogen of healthly pigs. An O-antigen (lipopolysaccharide; LPS) serotyping system is being developed to study the prevalence and distribution of representative isolates from both healthy and diseased pigs. In a previous study, we reported that A. suis serogroup O:1 strains express LPS with a (1-->6)-beta-D-glucan O-antigen chain polysaccharide that is similar in structure to a key cell-wall component in yeasts, such as Saccharomyces cerevisiae and Candida albicans. This study describes the O-antigen polysaccharide chemical structure of an O:2 serogroup strain, A. suis H91-0380, which possesses a tetrasaccharide repeating block with the structure: -->3)-beta-D-Galp-(1-->4)-[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->6)-beta-D-GlcpNAc-(1-->. Studies have shown that A. suis serogroup O:2 strains are associated with severely diseased animals; therefore, work on the synthesis of a glycoconjugate vaccine employing O:2 O-antigen polysaccharide to vaccinate pigs against A. suis serogroup O:2 strains is currently underway.     

Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Structural studies of a neutral polymeric fraction from the lipopolysaccharide of Serratia marcescens C.D.C. 1783-57 (O14:H9)

A "neutral" polymer of glucose, galactose, and 2-acetamido-2-deoxyglucose (molar ratios 1:1:2) has been isolated from the lipopolysaccharide of Serratia marcescens strain C.D.C. 1783-57 (O14:H9). Degradative and spectroscopic studies established that the polysaccharide has a branched tetrasaccharide repeating-unit of the structure shown. The polymer was absent from other strains of serogroup O14 studied, but a polymer differing only in the configuration of the glucose residue has previously been isolated from a strain of S. marcescens O8. The polymer from strain C.D.C. 1783-57 also shares structural features with the Escherichia coli O18 antigen, which is known to be serologically related to the S. marcescens O8 antigen. (Formula: see text).     

Structure of the O-specific polysaccharide of the Yersinia enterocolitica serovar O:4.32 lipopolysaccharide. Serologic relations of lipopolysaccharides of Y. enterocolitica O:4.32 and Y. intermedia O:4.33

O-Specific polysaccharide has been isolated on mild acid hydrolysis of the lipopolysaccharide from Yersinia enterocolitica O: 4.32 (strain 96) and shown to consist of yersiniose B (3,6-dideoxy-4-C-(1-hydroxyethyl)-D-xylo-hexose, YerB) acetylated at C1' and 2-acetamido-2-deoxy-D-galactose residues in a molar ratio 1:2. Acid hydrolysis, methylation and 13C NMR studies indicated the polysaccharide to be composed of trisaccharide repeating units of the following structure: (sequence; see text) The data obtained revealed structural and serological interrelation between O-antigens of Y. enterocolitica O:4.32 and Y. intermedia O:4.33.     

Structural and serological studies on a new 4-deoxy-d-arabino-hexose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter braakii PCM 1531 (serogroup O6).

The O-specific polysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) was isolated by mild acid hydrolysis of the lipopolysaccharide (LPS) and found to contain d-fucose, l-rhamnose, 4-deoxy-d-arabino-hexose and O-acetyl groups in molar ratios 2 : 1 : 1 : 1. On the basis of methylation analysis and 1H and 13C NMR spectroscopy data, the structure of the branched tetrasaccharide repeating unit of the O-specific polysaccharide was established. Using various serological assays, it was demonstrated that the LPS of strain PCM 1531 is not related serologically to other known 4-deoxy-d-arabino-hexose-containing LPS from Citrobacter PCM 1487 (serogroup O5) or C. youngae PCM 1488 (serogroup O36). Two other strains of Citrobacter, PCM 1504 and PCM 1505, which, together with strain PCM 1531, have been classified in serogroup O6, were shown to be serologically distinct from strain PCM 1531 and should be reclassified into another serogroup.     

Chemical structure of an acidic polysaccharide produced by serratia piscatorum

The acidic polysaccharide of Serratia piscatorum consists of L-rhamnopyranosyl, D-galactopyranosyl, and D-galactopyranosyluronic acid residues in the molar ratio of 2:1:1. Some of the D-galactopyranosyluronic acid residues are acetylated at O-2 or O-3, or both. Smith degradation and methylation analysis indicated that the L-rhamnopyranosyl, D-galactopyranosyl, and D-galactopyranosyluronic acid residues are substituted with glycosidic linkages at O-3, O-3, and O-4, respectively. Partial acid hydrolysis of the native polysaccharide gave four acidic oligosaccharides, each of which was isolated and characterized, suggesting the following tetrasaccharide repeating unit: →3)-L-Rhap-(1→4)-D-GalAp-(1→3)-L-Rhap-(1→3)-D-Galp-(1→.     

The lipopolysaccharide from Pseudomonas aeruginosa NCTC 8505. Structure of the O-specific polysaccharide

Structural studies have been carried out on the O-specific fraction from the lipopolysaccharide of Pseudomonas aeruginosa NCTC 8505, Habs serotype 03. The O-specific polysaccharide has a tetrasaccharide repeating-unit containing residues of L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and 2,4-diacetamido-2,4,6-trideoxy-D-glucose (BacNAc2). The following structure has been assigned to the repeating-unit: leads to 3)Rhap(beta 1 leads to 6)GlcpNAc(alpha 1 leads to 4)GalpNAcA(alpha 1 leads to 3)BacpNAc2(alpha 1 leads to. The parent lipopolysaccharide is a mixture of S, R, and SR species, and its high phosphorus content is partly due to the presence of triphosphate residues, as found for other lipopolysaccharides from P. aeruginosa. In addition to phosphorus, heptose, a 3-deoxyoctulosonic acid, and amide-bound alanine, the core oligosaccharide contains glucose, rhamnose, and galactosamine (molar proportions 3:1:1). The rhamnose and part of the glucose are present as unsubstituted pyranoside residues: other glucose residues are 6-substituted.     

The structure of Proteus mirabilis O3 O-specific polysaccharide containing N-(2-hydroxyethyl)-D-alanine

O-Specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O3 lipopolysaccharide. The polysaccharide was dephosphorylated with 48% HF to give a linear polysaccharide and an amino acid, N-(2-hydroxyethyl)-D-alanine. The structure of the polysaccharide was determined by methylation, Smith degradation and computer-assisted analysis of the 13C-NMR spectra of original and dephosphorylated polymers and oligomers. The structure of the amino acid was investigated by using 1H and 13C-NMR spectroscopy and mass spectrometry (applied to the acetylated methyl ester derivative). Its absolute configuration was established by comparison of the optical rotation value and CD spectrum of the natural and synthetic product. On the basis of the data obtained, it was concluded that the repeating unit of P. mirabilis O3 O-specific polysaccharide has the following structure: (formula; see text) Removal of the amino acid phosphate substituent significantly decreased serological activity of the O-specific polysaccharide, thus showing the immunodominant role of this group. Serological cross-reactions between P. mirabilis O3 and O27 were demonstrated and tentatively substantiated.     

Structure and cross-reactivity of the O-antigen of Proteus vulgaris O8.

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O8 lipopolysaccharide followed by gel permeation chromatography. Studies of the polysaccharide by sugar and methylation analyses and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, demonstrated the presence of a tetrasaccharide repeating unit having the following structure: [sequence: see text] The role of an epitope associated with the alpha-L-FucpNAc-(1-->3)-D-GlcpNAc disaccharide in serological cross-reactivity of P. vulgaris O8 is discussed.     

Structural determination of the O-specific chain of the lipopolysaccharide from the mushrooms pathogenic bacterium Pseudomonas tolaasii

The complete structure of the O-specific polysaccharide of the lipopolysaccharide isolated from the cultivated mushrooms pathogen Pseudomonas tolaasii is described. The structural determination, achieved by chemical and spectroscopical analyses, indicates a novel tetrasaccharide repeating unit built up of two units of 2-acetamido-2,6-di-deoxy-glucopyranose (Quinovosamine, QuipNAc) and two units of 2-acetamido-2-deoxy-gulopyranuronamide (GulpNAcAN), one of which is acetylated at C-3 position:     

Occurrence of two extracellular acidic polysaccharides as products of Rhizobium meliloti 201

The extracellular polysaccharide of Rhizobium meliloti 201 consists of two acidic polysaccharides, APS-I and APS-II. APS-I is composed of D-glucose, D-mannose and D-glucuronic acid in a molar ratio of 3:3:2, whereas APS-II is composed of D-glucose, D-galactose, D-mannose and pyruvic acid in a molar ratio of 4:3:2:1.APS-II was separated from the extracellular polysaccharide preparation by hydrolysing APS-I to its octasaccharide repeating unit with a specific enzyme. APS-I and APS-II were also separated by treatment with cetylpyridinium chloride and by paper electrophoresis of the depyruvylated polysaccharide.     

Structural studies of the Vibrio cholerae O:3 O-antigen polysaccharide.

The structure of the Vibrio cholerae O:3 O-antigen polysaccharide has been investigated, mainly by n.m.r. spectroscopy, mass spectrometry, sugar and methylation analysis, and specific degradations, and is proposed to involve the following tetrasaccharide repeating-unit. [formula: see text]. In this structure, D-D-Hep is D-glycero-D-manno-heptose, Asc is 3,6-dideoxy-L-arabino-hexose (ascarylose), and Sug is 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine) in which N-2 is acetylated and N-4 is acylated with a 3,5-dihydroxyhexanoic acid. That the 2,4-diamino-2,4,6-trideoxy-D-glucose residue is linked through O-3 and not through one of the hydroxyl groups in the 3,5-dihydroxyhexanoyl group is indicated but not definitely proved. The configuration of the latter group has not been determined. The f.a.b.-mass spectrum of the methylated O-antigen indicates that the structure given above also represents the biological repeating-unit.     

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.     

Immunochemistry of Salmonella O-antigens: preparation of an octasaccharide-bovine serum albumin immunogen representative of Salmonella serogroup B O-antigen and characterization of the antibody response.

The O-antigenic polysaccharide of phenol-water extracted Salmonella typhimurium (O antigens 4, 12) lipopolysaccharide was enzymatically cleaved by phage P22 endorhamnosidase. An octasaccharide with the (formula: see text) structure Gal-Man-Rha-Gal-Man-Rha was isolated and shown to retain the O-antigen 4 specificity of the native polysaccharide. After oxidation of the terminal reducing rhamnose residue to the corresponding aldonic acid, the octasaccharide was covalently linked to bovine serum albumin (OLS-BSA) by use of a water-soluble carbodimide. The resulting conjugate showed O-antigen 4 specificity in enzyme-linked immunosorbent assay (ELISA) ans passive hemagglutination inhibition tests. Immunization of rabbits with the OLS-BSA conjugate gave rise to antibodies directed toward both the octasaccharide and the carrier protein. ELISA titration with synthetic disaccharide-protein conjugates as antigens revealed that the antibody titer against the mannose-rhamnose structure was higher than against the abequose-mannose structure. In rabbits immunized with heat-killed whole bacteria the titers against the two disaccharides were equal. The reason for this difference is not obvious. It is evident, however, that the OLS-BSA conjugate elicited in rabbits O-antibodies with the same specificity as whole bacteria.     

Characterization of the lipopolysaccharide O antigens of Actinobacillus pleuropneumoniae serotypes 9 and 11: antigenic relationships among serotypes 9, 11, and 1.

The antigenic lipopolysaccharide O polysaccharides of capsular serotypes 9 and 11 were examined by chemical, immunological, and nuclear magnetic resonance methods. Immunodiffusion tests carried out on these O antigens indicated that both contained common epitopes which were also shared by Actinobacillus pleuropneumoniae serotype 1. Chemical analysis and high-field nuclear magnetic resonance spectroscopy showed that the O antigens of serotypes 9 and 11 were high-molecular-weight polymers consisting of a backbone of repeating trisaccharide units composed of alpha-L-rhamnopyranosyl and alpha-D-glucopyranosyl residues (2:1). One of the alpha-L-rhamnose units forms a branch point and is stoichiometrically substituted with terminal 2-acetamido-2-deoxy-beta-D-glucose residues in the serotype 11 O polysaccharide, but only to the extent of 25% in the serotype 9 O polysaccharide. Thus, the serotype 9 O polysaccharide contains two different repeating units: a tetrasaccharide unit with the same structure as that of the serotype 11 O polysaccharide and a trisaccharide unit: [formula: see text] where R = beta-D-GlcpNAc for serotype 1 and 11 O polysaccharides, and R = H (75%) and R = beta-D-GlcpNAc (25%) for serotype 9. The structure of the previously determined serotype 1 O polysaccharide (E. Altman, J.-R. Brisson, and M. B. Perry, Biochem. Cell. Biol. 64:17-25, 1986) is identical to that of the serotype 11 O polysaccharide. We propose a more complete serotyping scheme for A. pleuropneumoniae which includes designation of both the capsular (K) and O antigens.     

Structure of a neutral O-specific polysaccharide of the bacterium Providencia alcalifaciens O5.

A neutral polysaccharide containing D-galactose, 2-acetamido-2-deoxy-D-glucose, and 3-acetamido-3,6-dideoxy-D-glucose (Qui3NAc) in the ratios 2:1:1 was obtained by mild acid degradation of lipopolysaccharide of the bacterium Providencia alcalifaciens O5 followed by gel chromatography and ion-exchange chromatography or treatment with anhydrous hydrogen fluoride. On the basis of full acid hydrolysis, methylation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), H-detected heteronuclear 1H,13C single-quantum coherence (HSQC), and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established:     

An Endo-(l → 6)-β-d-glucanase of Flavobacterium M64 Hydrolyzing the Octasaccharide Repeating Unit of Succinoglycan to Two Tetrasaccharides

An endo-(l → 6)-β-d-glucanase capable of hydrolyzing octasaccharide to two tetrasaccharides was isolated from cells of Flavobacterium M64. The octasaccharide represents the repeating unit of succinoglycan (SG-D). One tetrasaccharide was composed of d-glucose, succinic acid and pyruvic acid (4:1:1, molar ratio), and the other was composed of d-glucose and d-galactose (3:1, molar ratio). This enzyme hydrolyzed the (l → 6)-β-d-glucosidic linkage adjacent to the (1 → 6)-linked β-d-glucose residue in the octasaccharide repeating unit of succinoglycan and also hydrolyzed the octasaccharide repeating units of similar polysaccharides produced by many strains of Agrobacterium and Rhizobium species.     

Chemical and structural analysis of the capsular polysaccharide from Escherichia coli O9:K28(A):H- (K28 antigen)

The structure of the capsular polysaccharide from Escherichia coli O9:K28(A):H- (K28 antigen) has been determined by using the techniques of methylation, periodate oxidation, and partial hydrolysis. N.m.r. spectroscopy (1H and 13C) was used to establish the nature of the anomeric linkages. O-Acetyl groups were determined spectrophotometrically and were located using methyl vinyl ether as a protective reagent. The polysaccharide is comprised of repeating units of the tetrasaccharide shown (three-plus-one type) with 70% of the fucosyl residues carrying an O-acetyl substituent. (formula; see text) This structure resembles that of E. coli K27 and has the structural pattern of Klebsiella K54 polysaccharide.