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.     

Structural and serological studies on Hafnia alvei O-specific polysaccharide of alpha-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223

On the basis of chemical and methylation analyses, one- and two-dimensional (1)H- and (13)C-NMR spectroscopy, including COSY, TOCSY, NOESY and (1)H, (13)C HSQC experiments, a neutral O-specific polysaccharide isolated from Hafnia alvei strain PCM 1223 lipopolysaccharide (LPS) was found to be an alpha-mannan composed of pentasaccharide repeating units having the following structure:-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->. Immunoblotting showed a strong cross-reactivity between anti-H. alvei PCM 1223 serum and LPSs of Escherichia coli O9 and Klebsiella pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions.     

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.     

Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1207 lipopolysaccharide.

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1207 lipopolysaccharide (LPS) has been investigated. Methylation analysis, partial acid hydrolysis, matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS, fast atom bombardment (FAB)-MS/MS and 1H- and 13C-NMR spectroscopy were the principal methods used. Glycerol phosphate was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: The polysaccharide is partially (approximately 10%) substituted with O-acetyl groups. The lipopolysaccharide was also subjected to high resolution magic angle spinning (HR-MAS) NMR analysis, which showed both the signals of the O-specific polysaccharide as well as several signals from unsubstituted core oligosaccharides. This confirmed the presence of the described structure in the native LPS.     

Structure of the O-specific polysaccharide isolated from the lipopolysaccharide of Citrobacter gillenii serotype O12a, 12b strain PCM 1544

A neutral O-specific polysaccharide was isolated from the lipopolysaccharide of Citrobacter gillenii strain PCM 1544, representing serotype O12a,12b. The polysaccharide was studied by sugar and methylation analyses and Smith degradation along with 1H and 13C NMR spectroscopy, including a ROESY experiment. The following structure of the tetrasaccharide repeating unit was established, in which substitution with terminal GlcNAc is approximately 60%. [structure: see text]     

Structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c.

The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: (struture: see text). The main D-mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.     

Structures of two O-polysaccharides of the lipopolysaccharide of Citrobacter youngae PCM 1538 (serogroup O9)

Mild acid degradation of the lipopolysaccharide of Citrobacter youngae O9, strain PCM 1538 released a homopolysaccharide of 4-acetamido-4,6-dideoxy-D-mannose (D-Rha4NAc, N-acetyl-D-perosamine). Studies by methylation analysis and (1)H and (13)C NMR spectroscopy, using two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and H-detected (1)H,(13)C HSQC experiments showed the presence of two structurally different polysaccharides consisting of the following units: -->)-alpha-D-Rhap4NAc-(1 --> and --> 3)-alpha-D-Rhap4NAc-(1 --> 3)-beta-D-Rhap4NAc-(1 -->.     

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.     

Structural analysis of the O-specific polysaccharide isolated from Plesiomonas shigelloides O51 lipopolysaccharide

Plesiomonasshigelloides strain CNCTC 110/92 (O51) was identified as a new example of plesiomonads synthesising lipopolysaccharides (LPSs) that show preference for a non-aqueous surrounding during phenol/water extraction. Chemical analyses combined with ¹H and ¹³C NMR spectroscopy, MALDI-TOF and ESI mass spectrometry showed that the repeating units of the O-specific polysaccharides isolated from phenol and water phase LPSs of P. shigelloides O51 have the same structure: →4)-β-d-GlcpNAc3NRA-(1→4)-α-l-FucpAm3OAc-(1→3)-α-d-QuipNAc-(1→, containing the rare sugar constituent 2,3-diamino-2,3-dideoxyglucuronic acid (GlcpNAc3NRA), and substituents such as d-3-hydroxybutyric acid (R) and acetamidino group (Am). The HR-MAS NMR spectra obtained for the isolated LPSs and directly on bacteria indicated that the O-acetylation pattern was consistent throughout the entire preparation. The ¹H chemical shift values of the structure reporter groups identified in the isolated O-antigens matched those present in bacteria. We have found that the O-antigens recovered from the phenol phase showed a higher degree of polymerisation than those isolated from the water phase.     

Structural and serological characterisation of an O-specific polysaccharide from Serratia plymuthica

Abstract The surface polysaccharides of a strain of Serratia plymuthica were characterised and shown to consist of a linear, acidic galactoglucomannan as well as a major and a minor neutral galactan. Immunoblotting results demonstrated cross-reactions between this strain and others with similar galactans ( S. marcescens O16 and O20, Klebsiella O1, and Pasteurella haemolytica T4 and T10).     

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.     

Structure of a phosphoethanolamine-containing O-polysaccharide of Citrobacter freundii strain PCM 1443 from serogroup O39 and its relatedness to the Klebsiella pneumoniae O1 polysaccharide

Lipopolysaccharide was extracted from cells of Citrobacter freundii PCM 1443 from serogroup O39 and degraded by mild acid hydrolysis to give an O-polysaccharide. Based on enzymatic and methylation analyses, along with 1H and 13C nuclear magnetic resonance spectroscopy, it was found that the lipopolysaccharide studied has two different linear polysaccharide chains of d-galactan type containing 3-substituted galactose residues. One of the galactans has the disaccharide repeating units of alpha-D-galactopyranose and beta-D-galactofuranose and the other is comprised of alpha-D-galactopyranose and beta-D-galactopyranose, the latter being substituted in 25% repeats with PEtN at O-6. An immunoblotting assay demonstrated that the lipopolysaccharide of C. freundii PCM 1443 is serologically related to that of Klebsiella pneumoniae O1, which contains the same galactan chains but is devoid of phosphoethanolamine.     

Structural determination of the O-specific polysaccharide from Aeromonas hydrophila strain A19 (serogroup O:14) with S-layer

Bacteria belonging to the genus Aeromonas are Gram-negative mesophilic and essentially ubiquitous in the microbial biosphere; moreover they are considered very important pathogens in fish and responsible for a great variety of human infections.The virulence of Gram-negative bacteria is often associated with the structure of lipopolysaccharides, which consist of three regions covalently linked: the glycolipid (lipid A), the oligosaccharide region (core region) and the O-specific polysaccharide (O-chain, O-antigen).The O-chain region seems to play an important role in host-pathogen interaction. In the case of Aeromonas hydrophila the majority of pathogenic strains belongs to serogroups O:11, O:16, O:18 and O:34. In this paper, we report the complete structure of the O-chain of A. hydrophila strain A19 (serogroup O:14), a pathogenic strain isolated from European eels, which showed high virulence when tested in trout or mice. Dried cells were extracted by the PCP (phenol/chloroform/petroleum ether) method obtaining the lipopolysaccharide. After mild acid hydrolysis the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by means of chemical analysis and one- and two-dimensional NMR spectroscopy. All the data collected are directed towards the following structure:     

Structural studies on the O-polysaccharide of the lipopolysaccharide produced by Citrobacter rodentium (ATCC 51459).

Citrobacter rodentium is the etiologic agent of transmissible murine colonic hyperplasia (TMCH) and is the only Citrobacter species known to possess virulence factors homologous to human enteropathogenic and enterohemorrhagic Escherichia coli. Members of this species are considered clonal and represent the only known attaching and effacing bacterial pathogen of mice and thus provides a useful animal model for studying the molecular basis of attaching and effacing pathology. The lipopolysaccharide (LPS) produced by C. rodentium has not been previously studied or its possible role as a virulence factor determined. The structure of the LPS has been undertaken as a first step in an investigation of its possible role in pathogenesis. The structure of C. rodentium (ATCC 51459, prototype TMCH isolate, original biotype 4280, previously designated DBS 100) LPS was determined from composition and methylation analyses, mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The antigenic O-polysaccharide was found to be a high molecular mass branched polymer of repeating pentasaccharide units composed of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), d-glucose (D-Glc), and L-rhamnose (L-Rha) in the molar ratio 2 : 2 : 1 linked through phosphate, and has the structure: [structure: see text]     

Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 8 from a new serogroup O67.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Proteus penneri 8 lipopolysaccharide and found to contain D-glucose, D-galacturonic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2,6-dideoxy-L-galactose (L-FucNAc) and 2-aminoethyl phosphate (PEtn) in the ratios 2 : 1 : 1 : 1 : 1 : 1. 1H and 13C NMR spectroscopy was applied to the intact and dephosphorylated polysaccharides, and the following structure of the hexasaccharide repeating unit was established: The O-specific polysaccharide has a unique structure, and, accordingly, we propose for P. penneri 8 a new Proteus O67 serogroup, in which this strain is at present the single representative. The nature of epitopes on LPS of P. penneri 34, P. mirabilis O16, P. mirabilis O23 and P. vulgaris O22, which cross-react with O-antiserum against P. penneri 8, is discussed.     

Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 63 from a new serogroup O68.

Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass O-specific polysaccharide that was isolated by gel-permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two-dimensional 1H, 1H COSY, TOCSY rotating-frame NOE spectroscopy, H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence (HMQC), and 1H, 13C HMQC-TOCSY experiments, demonstrated the following structure of the polysaccharide: where FucNAc is 2-acetamido-2,6-dideoxygalactose and PEtn is 2-aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of D-GlcNAc6PEtn and an alpha-L-FucNAc-(1-->3)-D-GlcNAc disaccharide, with other Proteus O-specific polysaccharides. A marked cross-reactivity of P. penneri 63 O-antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O-specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O-antigens, and therefore a new, separate serogroup, O68, is proposed for this strain.     

Structural studies of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide

The structure of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide has been investigated, different 1H- and 13C-n.m.r. techniques being the main methods used. It is concluded that they are composed of pentasaccharide repeating-units having the following structure, in which D-Fuc3NAc is 3-acetamido-3,6-dideoxy-D-galactose. ----4)-beta-D-GlcpNAc-(1----3)-alpha-L-Rhap-(1----2)-alpha-L-Rh ap-(1----3)-beta-L-Rhap-(1----2 increases 1 alpha-D-Fucp3NAc.     

Structure of the O-specific polysaccharide from the lipopolysaccharide of Serratia marcescens O8

Structural studies have been carried out on the O-specific polysaccharide from the lipopolysaccharide of the reference strain (CDC 1604-55) for serogroup O8 of Serratia marcescens. The polymer has a branched, tetrasaccharide repeating unit of D-galactose(Gal),D-glucose(Glc), and 2-acetamido-2-deoxy-D-glucose(GlcNAc) with the following structure: (Formula: see text). The anomeric configuration assigned to the glucose residue differs from that (beta) previously proposed [Tarcsay, L., Wang, C. S., Li, S.-C. and Alaupovic, P. (1973) Biochemistry 12, 1948-1955]. The structure of the O8 polymer is identical with that of one of two polymers present in the cell envelope of a strain (CDC 1783-57) of S. marcescens O14.     

Structural studies on the O-specific side-chains of the cell-wall lipopolysaccharide from Escherichia coli O 75

The structure of the O-specific side-chains of the cell-wall lipopolysaccharide of Escherichia coli O 75 has been investigated, using methylation analysis and Smith degradation as the principal methods. The O-specific side-chain was found to be composed of a tetrasaccharide repeating-unit of the following structure: