Ewa Katzenellenbogen

Structural studies of the O-specific side-chains of the shigella sonnei phase I lipopolysaccharide

Abstract The structure of the O-specific side-chains of the Shigella sonnei phase I lipopolysaccharide has been investigated. The side chains are composed of disaccharide repeating-units containing two uncommon sugar components, one of witch, 2-amino-2-deoxy- L -altruronic acid, has been identified previously. The other has now been identified as 2-acetamido-4-amino-2,4,6-trideoxy- D -galactose. The uronic acid, as N-acetylated α-pyranosyl residues, is linked through O-4, and the diamino sugar, as β-pyranosyl residues, is linked through O-3. The pyranosyluronic acid residue assumes the 4C1 conformation in the polymer, with the carboxyl group in the axial position.

Structure, conformation and immunology of sialic acid-containing polysaccharides of human pathogenic bacteria

Capsular polysaccharides of types Ia, Ib, II and IIIGroup B Streptococcus and groups B and C Neisseria meni ngi ti di S contai n termi nal si al i c aci d i n di ffe rent molecular environments. Experimentation has identified sialic acid as an important factor in the virulence of these organisms and in the human antibody response to their capsular polysacchari de anti gens . Al though termi nal si al i c aci d i s not normally immunogenic it controls the determinants which are responsible for the production of protective antibodies. Using immunological and NMR spectroscopic techniques on the native and specifically modified polysaccharides, a number of these si al I c aci d-control 1 ed determi nants have been identified and located. These determinants are only formed in structures which can accommodate long—range interactions between sialic acid and other remote glycosyl residues. The carboxylate group of si ali c acid is essential for these interactions to occur.

The structure of glycerol teichoic acid-like O-specific polysaccharide of Hafnia alvei 1205

The O-specific polysaccharide of Hafnia alvei 1205 contained D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, 4-acetamido-4,6-dideoxy-D-glucose (Qui4NAc), glycerol, phosphate, and O-acetyl groups. On the basis of 1D and 2D shift-correlated homonuclear and 13C-1H heteronuclear NMR spectroscopy, methylation analysis, Smith degradation, and dephosphorylation with hydrofluoric acid, it was concluded that the O-antigen was a partially O-acetylated teichoic acid-like polysaccharide having the following structure: [formula: see text]

Structure of the O-specific polysaccharide of Hafnia alvei 1204 containing 3,6-dideoxy-3-formamido-D-glucose.

The O-specific polysaccharide of Hafnia alvei strain 1204 has a hexasaccharide repeating unit containing D-mannose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, and 3,6-dideoxy-3-formamido-D-glucose (Qui3NFo) in the ratios 2:1:1:1:1 as well as O-acetyl groups. On the basis of methylation analysis of the intact, carboxyl-reduced, and Smith-degraded polysaccharide as well as 1D and 2D NMR spectroscopy, including 1D total correlation spectroscopy, 1D NOE spectroscopy, 2D homonuclear shift-correlated spectroscopy (COSY), and 13C,1H heteronuclear COSY, the following structure of the O-deacetylated polysaccharide was established: -->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-beta-D-GlcpN Ac-(1--> -->2)-beta-D-Quip3NFo-(1-->3)-alpha-D-GalpNAc-(1-->4)-alpha-D-G lcpA-(1--> Location of the N-formyl group, occurring as two stereoisomers in the ratio approximately 3:1, was determined by an NOE on H-3 Qui3N arising on pre-irradiation of HCO of the minor (E) isomer. The O-acetyl groups are attached in nonstoichiometric amounts at position 3 of GlcA and position 6 of a mannose residue or GlcNAc.

Lipopolysaccharide core region of Hafnia alvei: structure elucidation using chemical methods, gas chromatography−mass spectrometry, and NMR spectroscopy

Sugar and methylation analysis with the use of gas chromatography-mass spectrometry and 1H NMR spectroscopy proved that the core oligosaccharides isolated from lipopolysaccharides of eight Hafnia alvei strains have the identical hexasaccharide skeleton. However, 1H, 31P heterocorrelated spectra showed that the phosphorylation pattern is not the same. The branched heptose for the ATCC 13337, 1187, 2, 1191, 1196, 1220, and 481L strains is phosphorylated as in the following formula, where P = -O-P(O)(O-)2 and P-PEtN = [-O-P(O)(O-)]2-O(CH2)2NH3+ [formula: see text] A different phosphorylation pattern was found for the 1211 strain, where the branched heptose residue is 6-substituted by a monophosphorylethanolamine group, ...-->3(-->7)(PEtN-->6)-alpha-LD-Hepp-(1-->3)..., where PEtN = -O-P(O)(O-)-O(CH2)2NH3+.

The structure of the O-specific polysaccharide from Hafnia alvei strain 38 lipopolysaccharide

The O-specific polysaccharide of the lipopolysaccharide from H. alvei strain 38 has been established by NMR spectroscopy (13C and 1H) and methylation analysis to have the repeating unit-->4)-beta-D-ManpNAc-(1-->4)-alpha-D-GlcpNAc(1-->.

The structure of the biological repeating unit of the O-antigen of Hafnia alvei O39

On mild acid-catalysed degradation of the lipopolysaccharide from Hafnia alvei O39 followed by gel filtration of Sephadex G-50, the O-specific polysaccharide and three oligosaccharides were obtained, which represent the core substituted with 0-2 O-antigen repeating-units. On the basis of sugar and methylation analyses, 13C-n.m.r. data, solvolysis of the polysaccharide with anhydrous hydrogen fluoride, and computer-assisted 13C-n.m.r. analysis of the Smith-degraded polysaccharide, it was concluded that the biological repeating unit of the O39 antigen was Formula; see text

The occurrence of glycine in bacterial lipopolysaccharides

The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [14C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100 degrees C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.

The structure of the O-specific polysaccharide of Hafnia alvei strain 1216

The O-specific polysaccharide of Hafnia alvei strain 1216 is composed of D-galactose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-D-glucose, and O-acetyl groups in the ratios 1:1:2:1:1. On the basis of sugar and methylation analyses of the intact and chemically degraded (O-deacetylated, carboxyl-reduced, Smith-degraded) polysaccharide and 1H and 13C NMR spectroscopy, including 2D shift-correlated (COSY, relayed COSY, 13C, 1H-COSY) and 1D NOE spectroscopy, it was concluded that the O-antigen is built up of linear pentasaccharide units having the following structure: [formula: see text]

Identification of the methyl phosphate substituent at the non-reducing terminal mannose residue of the O-specific polysaccharides of Klebsiella pneumoniae O3, Hafnia alvei PCM 1223 and Escherichia coli O9/O9a LPS.

O-specific polysaccharides of Gram-negative bacteria are synthesized by two different mechanisms: polymerization of the pre-formed O-repeating unit or sequential addition of the monosaccharides to the growing polysaccharide chain. In the second case, growth of the polymer can be further subdivided into two groups depending on the presence or absence of a special monosaccharide or non-sugar substituent that terminates the glycan. A family of polymannose O-polysaccharides provides prototypes for the chain terminating process. Polysaccharides of Klebsiella pneumoniae O3, Hafnia alvei PCM 1223, and Escherichia coli O9 have the same penta-mannose repeating unit. E. coli O9a has tetra-mannose repeat and this structure can be produced by mutants of E. coli O9. The mechanism of biosynthesis of H. alvei 1223 O-polysaccharide has not been reported. Here we show that all above polysaccharides contain the same modification at the non-reducing end; presence of a methyl phosphate group at O-3 of α-mannopyranose, that serves as the signal for termination of the chain elongation.