Maria Bogulska

Structure of an abequose-containing O-polysaccharide from Citrobacter freundii O22 strain PCM 1555

The lipopolysaccharide of Citrobacter freundii O22 (strain PCM 1555) was degraded under mild acidic conditions and the O-polysaccharide released was isolated by gel chromatography. Sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H ROESY and (1)H,(13)C HMBC experiments, showed that the repeating unit of the O-polysaccharide has the following structure: alpha-Abep 1 -->3 --> 2)-alpha-D-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Galp-(1--> where Abe is abequose (3,6-dideoxy-D-xylo-hexose). SDS-PAGE and immunoblotting revealed that the O-antigen of C. freundii O22 is serologically indistinguishable from those of Salmonella group B serovars (Typhimurium, Brandenburg, Sandiego, Paratyphi B) but not related to other abequose-containing O-antigens tested (Citrobacter werkmanii O38 and Salmonella Kentucky) or colitose (l enantiomer of abequose)-containing O-antigen of Escherichia coli O111.

Structure of the O-Specific Polysaccharide of Hafnia Alvei 23 Having an Oligosaccharide-Phosphate Repeating Unit

ABSTRACT Lipopolysaccharide (LPS) of Hafnia alvei 23 has an acid-labile O-specific polysaccharide (OPS) with a pentasaccharide-phosphate repeating unit containing D-Glc1P, D-GlcNAc, L-Fuc, 6-deoxy-D-talose (D-6dTal), 4-acetamido-4,6-dideoxy-D-glucose (D-Qui4NAc), and an O-acetyl group. A partially degraded OPS was obtained by hydrolysis of LPS with 0.25 M sodium acetate in aqueous 0.5% acetic acid. Fractionation of LPS on Sephadex G-200 in DOC buffer allowed isolation of long-chain LPS species which, together with OPS, were studied by methylation analysis, chemical degradations (O-deacetylation, dephosphorylation with 48% hydrofluoric acid, Smith degradation), and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H,13C heteronuclear single-quantum coherence (HSQC) experiments. The following structure of the repeating unit of OPS was established:

Structure of the O-specific polysaccharide, containing a glycerol phosphate substituent, of Hafnia alvei strain 1220 lipopolysaccharide

The O-specific polysaccharide of the lipopolysaccharide produced by Hafnia alvei strain 1220 contained D-glucose, D-galactose, N-acetyl-D-glucosamine, N-acetyl-L-fucosamine (2-acetamido-2,6-dideoxy-L-galactose), glycerol, and phosphate. It was proved by composition and methylation analyses, Smith degradation, dephosphorylation, and one- and two-dimensional 1H NMR spectroscopy to be a teichoic acid-like polymer with a branched hexasaccharide repeating unit of the following structure. [sequence: see text]

Structures of a unique O-polysaccharide of Edwardsiella tarda PCM 1153 containing an amide of galacturonic acid with 2-aminopropane-1,3-diol and an abequose-containing O-polysaccharide shared by E. tarda PCM 1145, PCM 1151 and PCM 1158

Lipopolysaccharides of four strains of Edwardsiella tarda were degraded by mild acid hydrolysis, and the released O-polysaccharides were isolated by GPC and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H, (1)H COSY, TOCSY, ROESY, (1)H, (13)C HMBC, HSQC and HSQC-TOCSY experiments. The O-polysaccharide from E. tarda PCM 1153 was found to contain D-GalA, D-GlcNAc, D-Gal and 2-amino-1,3-propanediol (GroN). In the tetrasaccharide repeating unit, GroN is amide-linked to one of the GalA residues, and Gal is non-stoichiometrically 2- or 3-O-acetylated (~45% at each position): [structure: see text]. Three other E. tarda strains examined (PCM 1145, PCM 1151 and PCM 1158) share the following O-polysaccharide structure: [structure: see text] where Abe indicates 3,6-dideoxy-D-xylo-hexose (abequose). This structure resembles those of Citrobacter freundii O22 (PCM 1555) and Salmonella enterica O4. In accordance with the structural data, SDS-PAGE and immunoblotting of the lipopolysaccharides with anti-C. freundii O22 serum demonstrated that the O-antigens of the three E. tarda strains are serologically identical to each other and to the O-antigens of C. freundii O22 and S. enterica O4.

Structure of the glycerol phosphate‐containing O‐specific polysaccharide and serological studies on the lipopolysaccharides of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14)

The O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14) and found to contain D-glucose, D-glucosamine and glycerol-1-phosphate in molar ratios 2 : 2 : 1. Based on methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy data, it was established that the O-specific polysaccharides from both strains have the identical branched tetrasaccharide repeating unit with 3,6-disubstituted GlcNAc, followed by 2,4-disubstituted Glc residues carrying at the branching points lateral residues of Glc and GlcNAc at positions 6 and 2, respectively. Glycerol-1-phosphate is linked to position 6 of the chain Glc. All sugars have a beta configuration, except for the side-chain Glc, which is alpha. Serological studies revealed a close relatedness of the lipopolysaccharides of C. werkmanii PCM 1548 and PCM 1549, both belonging to serogroup O14. In immunoblotting, anti-C. werkmanii PCM 1548 serum showed no cross-reactivity with the O-polysaccharide bands of the lipopolysaccharides of Citrobacter youngae PCM 1550 (serogroup O16) and Hafnia alvei PCM 1207, also containing a lateral glycerol phosphate residue.

Structure of the O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii O11, strain PCM 1540

The O-specific polysaccharide of the lipopolysaccharide of Citrobacter gillenii PCM 1540 (serogroup O11) consists of D-Glc, D-Man, D-GalNAc, D-GlcNAc, 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) and O-acetyl groups in the ratios 2:1:1:1:1:1. On the basis of sugar and methylation analyses and Smith-degradation along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched hexasaccharide repeating unit was established: [structure: see text]. Citrobacter werkmanii PCM 1541 belonging to the same serogroup O11 was found to have an R-form lipopolysaccharide devoid of the O-specific polysaccharide.

Structure of the lipopolysaccharide core region of Hafnia alvei strains 1185 and 1204.

Sugar and methylation analyses using gas chromatography/mass spectrometry and NMR spectroscopy proved that the core oligosaccharides of Hafnia alvei strains 1185 and 1204 have the following formula: carbohydrate sequence [see text] where Kdo = 3-deoxy-oct-2-ulosonic acid and P-PEtN = diphosphorylethanolamine. The structure shown above is a slight modification of the typical core region of H. alvei lipopolysaccharides. The difference refers to one sugar only: terminal galactose is present in the core of strains of 1185 and 1204, while terminal glucose in the typical core.

Structure of the O-polysaccharide of Edwardsiella tarda PCM 1150 containing an amide of D-glucuronic acid with L-alanine.

Mild acid degradation of the lipopolysaccharide of Edwardsiella tarda PCM 1150 afforded an O-polysaccharide, which was isolated by GPC on Sephadex G-50 and studied by sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopies, including experiments performed in a 9:1 H2O/D2O mixture to detect NH protons and their correlations with CH protons. The O-polysaccharide was found to contain an amide of d-glucuronic acid with l-alanine (d-GlcA6Ala) and the following structure of the branched hexasaccharide repeating unit was established: -->4)-β-D-GlepA6Ala-(1-->4)-α-L-Fucp-(1-->4)-α-D-Glcp-(1-->4)-α-D-Quip-(1-->3)-β-D-GlcpNAc-(1-->3<--1α-D-GalpNAc.

Immunochemical studies of the lipopolysaccharides of Hafnia alvei PCM 1219 and other strains with the O-antigens containing D-glucose 1-phosphate and 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose

Introduction:Hafnia alveiis the only species of the genus Hafnia, which belongs to the family of Enterobacteriaceae. These Gram-negative bacteria are commonly distributed in the natural environment and are often the cause of human opportunistic infections. Their lipopolysaccharides (LPSs) are important surface antigens which are responsible for the serological specificity and numerous cross-reactions with other enterobacterial genera. So far, 29 different O-polysaccharide (OPS, O-antigen) structures in Hafnias LPSs have been established and for some of them the molecular basis of the serological activity has been elucidated.Materials and Methods:OPS from H. alvei strain PCM 1219 was obtained by mild acid hydrolysis of the LPS followed by gel permeation chromatography of carbohydrate material on Sephadex G-50 column. The polysaccharide structure was determined using chemical methods as well as 13C NMR and 1H NMR spectroscopy. For serological studies, SDS-PAGE, immunoblotting, and passive hemagglutination tests were used.Results:The serological studies revealed a cross-reactivity of the LPSs of H. alvei PCM 1219 and a group of H. alvei strains with an O-antigen containing D-glucose 1-phosphate and [(R)-3-hydroxybutyramido]-D-glucose. The following structure of the OPS was established: where Acyl stands for (R)-3-hydroxybutyryl and the degree of O-acetylation is ~70%. The structure of the core oligosaccharide was found to be typical of the genus Hafnia.Conclusions:Based on the OPS structure and serological results it was concluded that H. alvei strain PCM 1219 should be classified in the same serogroup as the H. alvei type strain ATCC 13337 and five other strains containing D-glucose 1-phosphate and 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose in their O-antigens.

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.