Acidic O-specific Polysaccharide Research Articles

Elucidation of the O-antigen structure of Escherichia coli O93 and characterization of its biosynthetic genes.

The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D 1H, 13C-HSQC, and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

ChemInform Abstract: Structure of a New Acidic O-Antigen of Proteus vulgaris O22 Containing O-Acetylated 3-Acetamido-3,6-dideoxy-D-glucose.

Abstract The acidic O-specific polysaccharide of Proteusvulgaris O22 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments, and the following structure for the branched pentasaccharide repeating unit was established: Download : Download full-size image where Qui3NAc is 3-acetamido-3,6-dideoxyglucose, O-acetylation of QuiNAc at position 4 is stoichiometric and at position 2 nonstoichiometric. Serological relationships of P. vulgaris O22 with some other Proteus strains were substantiated on the level of the O-antigen structures.

Structure of an acidic O-specific polysaccharide of the marine bacterium Pseudoalteromonas agarivorans KMM 232 (R-form)

An acidic O-specific polysaccharide containing L-rhamnose, 2-acetamido-2-deoxy-D-galactose, 2,6-dideoxy-2-(N-acetyl-L-threonine)amino-D-galactose, and 2-acetamido-2-deoxy-D-mannuronic acid was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Pseudoalteromonas agarivorans KMM 232 (R-form) followed by gel-permeation chromatography. The polysaccharide was subjected to Smith degradation to give a modified polysaccharide with trisaccharide repeating unit containing L-threonine. The initial and modified polysaccharides were studied by sugar analysis and 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, ROESY, and HSQC experiments, and the structure of the branched tetrasaccharide repeating unit of the polysaccharide was established.

Structure of acidic o-specific polysaccharide from the marine bacterium Cellulophaga baltica

The structure of an acidic O-specific polysaccharide from the marine bacterium Cellulophaga baltica was established by chemical methods of analysis and NMR spectroscopy. The polysaccharide was shown to consist of repeating tetrasaccharide units containing two mannose residues, one N-acetyl-D-glucosamine residue, and one D-glucuronic acid residue. An O-acetyl group was also found in the polysaccharide in nonstoichiometric amount. Thus, this polysaccharide had the following structure: [carbohydrate structure: in text].

Structure of the O-specific polysaccharide from Shewanella japonica type strain KMM 3299 T containing the rare amino sugar Fuc4NAc

An acidic O-specific polysaccharide (PS) of the agar-digesting bacterium Shewanella japonica with the type strain KMM 3299 T was obtained by mild acid hydrolysis of the lipopolysaccharide. The polysaccharide was studied by component analysis, methylation analysis, 1H and 13C NMR spectroscopy, including 2D NMR experiments. The PS was determined to have the following structure involving three unusual amino sugars: → 3 ) - α - d - Fuc p 4 NAc - ( 1 → 4 ) - α - d - Gal p A - ( 1 → 3 ) - α - l - Fuc p NAc - ( 1 → 3 ) - β - d - Qui p NAc 4 NAc - ( 1 →

Structure of the polysaccharide chain of the lipopolysaccharide from Flexibacter maritimus.

Flexibacter maritimus, a Gram-negative bacterium, is a fish pathogen responsible for disease in finfish species and a cause of cutaneous erosion disease in sea-caged salmonids. For the development of serology based diagnostics, protective vaccines, and a study of pathogenesis, the structural analysis of the lipopolysaccharide (LPS) produced by the bacterium has been undertaken. We now report that an acidic O-specific polysaccharide, obtained by mild acid degradation of the F. maritimus LPS was found to be composed of a disaccharide repeating unit built of 2-acetamido-3-O-acetyl-4-[(S)-2-hydroxyglutar-5-ylamido]-2,4,6-trideoxy-beta-glucose and 5-acetamido-7-[(S)-3-hydroxybutyramido]-8-amino-3,5,7,8,9-pentadeoxynonulopyranosonic acid (Sug) having the structure: The configuration of the C-2-C-7 fragment of the latter monosaccharide (B) was assigned beta-manno; however, the configuration at C-8 could not be established. NMR data indicate that the two monosaccharides have opposite absolute configurations. The repeating unit includes a linkage via a (S)-2-hydroxyglutaric acid residue, reported here for the first time as a component of a bacterial polysaccharide. The LPS was also found to contain a minor amount of a disaccharide beta-Sug-(2-3)-l-Rha, isolated from the products of the acidic methanolysis of the LPS.

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain l-rhamnose, 2-acetamido-4-[ d-3-hydroxybutyramido)]-2,4,6-trideoxy- d-glucose ( d-BacNAc4NHbu), and 2-amino-2,6-dideoxy- l-galactose, N-acylated by the 4-carboxyl group of l-malic acid ( l-malyl-(4→2)-α- l-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established: -3)- α- d - BacNAc4NHbu-(1 →3)- α- l - Rha-(1 →2)- α- l - Rha-(1 →2)- l - malyl-(4 →2)- α- l - FucN-(1- The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.

Structure of the O-specific polysaccharide of Proteus vulgaris O15 containing a novel regioisomer of N-acetylmuramic acid, 2-acetamido-4- O-[( R)-1-carboxyethyl]-2-deoxy- d-glucose

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O15 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, and H-detected 1H, 13C HMQC experiments. The polysaccharide was found to contain an ether of GlcNAc with lactic acid, and the following structure of the repeating unit was established: →3)- α- d - Glcp NAc4(R- Lac)6 Ac-(1 →2)- β- d - Glcp A-(1 →3)- α- l -6 dTalp2 Ac-(1 →3)- β- d - Glcp NAc-(1 → where l-6dTal and d-GlcNAc4( R-Lac) are 6-deoxy- l-talose and 2-acetamido-4- O-[( R)-1-carboxyethyl]-2-deoxy- d-glucose, respectively. The latter sugar, which to our knowledge has not been hitherto found in nature, was isolated from the polysaccharide by solvolysis with anhydrous triflic acid and identified by comparison with the authentic synthetic compound. Serological studies with the Smith-degraded polysaccharide showed an importance of 2-substituted GlcA for manifesting of the immunospecificity of P. vulgaris O15.

Structure of the O-specific polysaccharide of Proteus mirabilis D52 and typing of this strain to Proteus serogroup O33.

The acidic O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Proteus mirabilis strain D52 was studied using chemical analyses along with 1H-NMR and 13C-NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C and 1H,31P HMQC experiments. The polysaccharide was found to contain D-ribitol 5-phosphate (D-Rib-ol-5-P) and ethanolamine phosphate (Etn-P) and has the following structure: D-Rib-ol-5-P (3) approximately 75% EtnP(6)-->2)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-GlcpNAc-(1-->). This structure is identical with that of the O-polysaccharide of P. mirabilis O33 strain 59/57, and, hence, P. mirabilis D52 belongs to the same Proteus serogroup O33. Serological studies with O-antiserum against P. mirabilis D52 confirmed this but showed that the LPS species of P. mirabilis 59/57 and D52 are not identical, having different epitopes in the core region. A serological cross-reactivity of P. mirabilis D52 O-antiserum was observed with LPS of two other Proteus strains, P. mirabilis O16 and P. penneri 103, which have structurally different O-polysaccharides. The role of charged groups, Rib-ol-5-P and Etn-P in the immunospecificity is discussed.

Structure of the O-specific polysaccharide of Hafniaalvei PCM 1196

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established: →4)- α- d- Galp A-(1 →3)- β- d- Glcp NAc-(1 →2)- β- d- Galp-(1 →6)- α- d- Glcp-(1 →6)- α- d- Glcp NAc-(1 →

Structure of an O-acetylated acidic O-specific polysaccharide of Proteus vulgaris O46

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O46 and studied by chemical methods (O-deacetylation, sugar and methylation analyses, partial solvolysis) and 1H and 13C NMR spectroscopy. Solvolysis of the O-deacetylated polysaccharide with trifluoromethanesulfonic acid resulted in a α- d-Glc pNAc-(1→3)- d-GlcA disaccharide that demonstrated the usefulness of this reagent for selective cleavage of heteropolysaccharides. The following structure for the polysaccharide was established: →4)- α- d - Glcp6 Ac(1 →3)- β- d - Glcp A4Ac-(1 →3)- α- d - Glcp NAc-(1 →3)- β- d - Glcp A4Ac-(1 → where the degree of O-acetylation is ∼65% at position 6 of Glc and 80–95% at position 4 of GlcA residues.

Structure of an acidic O-specific polysaccharide of Pseudoalteromonashaloplanktis type strain ATCC 14393 containing 2-acetamido-2-deoxy- d- and - l-galacturonic acids and 3-( N-acetyl- d-alanyl)amino-3,6-dideoxy- d-glucose

An acidic O-specific polysaccharide was obtained from the lipopolysaccharide of Pseudoalteromonas haloplanktis ATCC 14393 and found to contain d-galactose, 3-( N-acetyl- d-alanyl)amino-3,6-dideoxy- d-glucose ( d-Qui3N dAlaAc), 2,4-diacetamido-2,4,6-trideoxy- d-glucose ( d-QuiNAc4NAc), 2-acetamido-2-deoxy- d- and - l-galacturonic acids ( d- and l-GalNAcA), and O-acetyl groups. On the basis of Smith degradation and 1H and 13C NMR spectroscopic studies, including 2D COSY, TOCSY, NOESY, 1H, 13C HMQC, and HMBC experiments, the following structure of the pentasaccharide repeating unit of the polysaccharide was established: →4)- α- l - Galp NAcA-(1 →3)- β- d - Quip NAc4NAc-(1 →2)- β- d - Quip3 N d AlaAc-(1 →4)- α- d - Galp NAcA-(1 →4)- α- d - Galp2,6 Ac 2-(1 → where O-acetylation of the galactose residue at each position is partial (50–70%).

Structure of a new acidic O-antigen of Proteusvulgaris O22 containing O-acetylated 3-acetamido-3,6-dideoxy- d-glucose

The acidic O-specific polysaccharide of Proteus vulgaris O22 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, and the following structure for the branched pentasaccharide repeating unit was established: where Qui3NAc is 3-acetamido-3,6-dideoxyglucose, O-acetylation of QuiNAc at position 4 is stoichiometric and at position 2 nonstoichiometric. Serological relationships of P. vulgaris O22 with some other Proteus strains were substantiated on the level of the O-antigen structures.

Structural and serological studies on a new acidic O-specific polysaccharide of Proteus vulgaris O32.

The following structure of the O-specific polysaccharide chain (O-antigen) of the Proteus vulgaris 032 lipopolysaccharide (LPS) was established by 1H-NMR and 13C-NMR spectroscopy, including two-dimensional NOESY and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments: -->2)-alpha-L-RhapI-(1-->2)-alpha-L-RhapII-(1-->4)-beta-D-++ +GalpA(I)-(1-->3)-beta-D-GlcpNAc-(1-->4)-alpha-D-GalpA(II)-(1-- >. In addition, an O-acetyl group was detected, which, most probably, is located at position 3 of a part of RhapI residues. Serological studies, using rabbit polyclonal anti-(P. vulgaris 032) serum, homologous and heterologous Proteus O-antigens and related artificial antigens, revealed the importance of an a-D-GalA-associated epitope in manifesting the immunospecificity of P. vulgaris 032 and substantiated serological relationships between the O-antigen studied and those of some other Proteus strains.

Structure of a highly acidic O-specific polysaccharide of lipopolysaccharide of Pseudoalteromonas haloplanktis KMM 223 (44-1) containing l-iduronic acid and d-QuiNHb4NHb

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated by phenol–water extraction of Pseudoalteromonas haloplanktis strain KMM 223 (44-1). l-Iduronic acid (IdoA) was found to be a component of the polysaccharide and identified by NMR spectroscopy and after carboxyl-reduction followed by acid hydrolysis and acetylation, by GLC-MS as 2,3,4-tri- O-acetyl-1,6-anhydroidose. On the basis of 1H and 13C NMR spectroscopic studies, including 1D NOE, 2D NOESY, HSQC and HMBC experiments, the following structure of the branched pentasaccharide repeating unit of the polysaccharide was established: where QuiNAc4NAc and QuiNHb4NHb are 2,4-diacetamido-2,4,6-trideoxyglucose and 2,4,6-trideoxy-2,4-di[( S)-3-hydroxybutyramido]glucose, respectively. This is the first report of l-iduronic acid in a lipopolysaccharide and of d-QuiNHb4NHb in nature.

Structural and serological studies of the O-specific polysaccharide of the bacterium Proteus mirabilis O10 containing L-altruronic acid, a new component of O-antigens

An acidic O-specific polysaccharide from the lipopolysaccharide of Proteus mirabilis O10 contains 2-acetamido-2deoxy- d-glucose, 2-acetamido-2-deoxy- d-galactose, d-galacturonic acid, and l-altruronic acid, the last-named sugar having not been found hitherto in O-antigens. Structure of a branched tetrasaccharide repeating unit of the polysaccharide was established by 1H and 13C NMR spectroscopy, including two-dimensional COSY and rotating-frame NOE spectroscopy. The lateral l-altruronic acid residue plays the immunodominant role in manifestation of the O10 specificity of Proteus, whereas a disaccharide fragment of the main chain in common with the O-specific polysaccharide of P. mirabilis O43 provides the one-way serological cross-reactivity between anti-O10 serum and O43-antigen.

The structure of the O-specific polysaccharide of Proteus penneri 52.

An acidic O-specific polysaccharide isolated from Proteus penneri 52 contains D-galacturonic acid, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose in the ratio 1:2:2. On the basis of sugar analysis and NMR spectroscopy, which included one-dimensional TOCSY, two-dimensional COSY, heteronuclear 13C, 1H-COSY, and rotating-frame NOE spectroscopy, the following structure of the pentasaccharide repeating unit of the O-specific polysaccharide was established: [sequence: see text] Cross-reactivity of the anti-P-penneri 52 O-serum with other strains of P. penneri isolated in Germany. Poland, USA, and Canada is discussed and a new Proteus serogroup is proposed.

The structure and serological specificity of Proteus mirabilis O43 O antigen.

On the basis of sugar analysis and NMR spectroscopy, including selective spin-decoupling, one-dimensional NOE, two-dimensional homonuclear and 13C,1H-heteronuclear correlation spectroscopy, the following structure of the acidic O-specific polysaccharide of Proteus mirabilis O43 was established: -->4)-alpha-D-GalpA-(1-->3)-alpha-D-GalpA-(1-->3)-alpha-D-Gl cpNAc-(1-->4)-alpha - D-Glcp-(1-->, where GalA is galacturonic acid and Galp is galactopyranose. No serological cross-reactivity was observed between lipopolysaccharides of P. mirabilis O43 and other studied Proteus strains, except for P. mirabilis O10. The O-specific polysaccharide of P. mirabilis O43 was serologically active in precipitation and inhibition tests but the activity was lost after periodate oxidation. These data suggest that the O43 specificity is determined by a wide epitope with the immunodominant role of 4-substituted D-Glc or/and D-GalA, which are destroyed by periodate oxidation.

The Structure and Serological Specificity of Proteus mirabilis O43 O Antigen

On the basis of sugar analysis and NMR spectroscopy, including selective spin-decoupling, one-dimensional NOE, two-dimensional homonuclear and 13C,1H-heteronuclear correlation spectroscopy, the following structure of the acidic O-specific polysaccharide of Proteus mirabilis O43 was established: -->4)-alpha-D-GalpA-(1-->3)-alpha-D-GalpA-(1-->3)-alpha-D-Gl cpNAc-(1-->4)-alpha - D-Glcp-(1-->, where GalA is galacturonic acid and Galp is galactopyranose. No serological cross-reactivity was observed between lipopolysaccharides of P. mirabilis O43 and other studied Proteus strains, except for P. mirabilis O10. The O-specific polysaccharide of P. mirabilis O43 was serologically active in precipitation and inhibition tests but the activity was lost after periodate oxidation. These data suggest that the O43 specificity is determined by a wide epitope with the immunodominant role of 4-substituted D-Glc or/and D-GalA, which are destroyed by periodate oxidation.

Somatic Antigens of Shigella

The specific polysaccharide was obtained from the lipopolysaccharide of Shigella newcastle by mild acid hydrolysis and further purified by permeation chromatography on Sephadex G-50. It was found to consist of L-rhamnose, 2-acetamido-2-deoxy-D-galactose, D-galacturonic acid residues and O-acetyl groups in the molar ratios of 2:1:1:1. On the basis of 1H and 13C nuclear magnetic resonance spectroscopy, methylation analysis, partial acid hydrolysis, Smith degradation, and chromium trioxide oxidation, the following structure can be assigned to the repeating oligosaccharide unit of the polysaccharide:-4)DGalA(beta 1-3)DGalNAc-(beta 1-2)LAc3Rha(alpha 1-2)LRha(alpha 1-, where GalA = galacturonic acid. GalNAc = N-acetylgalactosamine, Ac3Rha = 3-O-acetylrhamnose. The structural and immunochemical data presented prove that Sh. newcastle lipopolysaccharide belongs to a 'non-classical' type of somatic antigens with acidic O-specific polysaccharide chains.