Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O5 (Lanyi) and immunotype 6 (Fisher)

Abstract

Lipolysaccharides were isolated from dry bacterial cells of Pseudomonas aeruginosa OSa,b,c, OSa,b,d, 05a,d (Lanyi classification) and immunotype 6 (Fisher classification) by the Westphal procedure. Their polysaccharide chains were built up of trisaccharide repeating units containing ~-xylose, 2-acetamido-2,6-dideoxy-~-galactose and a new sialic acid-like sugar, the di-N-acyl derivative of 5,7-diarnino-3,5,7,9-tetradeoxy-~-glycero-~-mannononulosonic (pseudaminic) acid. Formyl, acetyl and (R)-3-hydroxybutyryl groups were identified as the N-acyl substituents of the last monosaccharide; O5a,b,c and O5a,b,d lipopolysaccharides also contained 0-acetyl groups.The glycosidic linkage of pseudaminic acid was extremely labile towards acids, and mild acid degradation of the lipopolysaccharides produced, instead of the 0-specific polysaccharides, their trisaccharide fragments with pseudaminic acid at the reducing terminus. Similar degradation of immunotype 6 lipopolysaccharides, followed by oxidation with sodium metaperiodate, resulted in a disaccharide fragment due to destruction of xylose. In contrast the glycosidic linkage of pseudaminic acid proved to be more stable towards treatment with hydrogen fluoride than those of xylose and N-acetylfucosamine. As a result, solvolysis of immunotype 6 lipopolysaccharide with hydrogen fluoride in methanol gave methyl glycosides of a disaccharide and a trisaccharide with pseudaminic acid at the non-reducing terminus. Mild acid hydrolysis of these oligosides afforded free 5-N-acetyl-7-Nformylpseudaminic acid, which was identified by the 'H and 13C nuclear magnetic resonance data, as well as by the mass spectrum of the corresponding fully methylated aldonic acid.As a result of the identification of all oligosaccharides obtained and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and lipopolysaccharides the following structures were estab- +~) -D -X~~~-(~~+~) -D -F~C N A C -(~~+~) -P S~~N A C~N F~-(~where D-xyl = ~-xylose, D-FUCNAC = 2-acetamido-2,6-dideoxy-~-galactose, Pse5N7NFm = 5-amino-3,5,7,9-tetradeoxy-7-formamido-~-glycero-~-manno-nonulosonic acid (7-N-formylpseudaminic acid). All the polysaccharides have an identical carbohydrate skeleton and differ from each other by the acyl substituent at N-5 of pseudaminic acid [acetyl or (R)-3-hydroxybutyryl group] or by the presence or absence of the 0-acetyl group at position 4 of N-acetylfucosamine. The data obtained account properly for the 0 specificity of the studied P. aeruginosa strains.The data on composition and structure of lipopolysaccharides, determining the 0 specificity of gram-negative bacteria, are employed to work out intraspecies chemotype schemes. Investigation of the lipopolysaccharides of an opportunistic bacterial pathogen P. aeruginosa showed many [2, 81, acetamidino [5, 6, 91, (R)- [7] and (S)-3-hydroxybutyryl [3] groups.In the present communication we report the establishing of the structures of the 0-specific polysaccharide chains of P. aeruginosa 0 5 (Lanyi classifi...

Functional Characterization of the Flagellar Glycosylation Locus in Campylobacter jejuni 81–176 Using a Focused Metabolomics Approach

“…The relatively small difference in proton chemical shifts for H3ax and H3eq (⌬␦ H 0.59 ppm) indicated that I had the ␣ configuration with the C1 carboxyl group occupying the equatorial position. In ␤-Pse, the difference for H3ax and H3eq tends to be larger and ranges from ⌬␦ H 0.77 to 1.10 ppm (29,32,34). The chemical shift for C6 at ␦ C 71.9 ppm also supported that C1 occupies the equatorial position since when C1 is axial, C6 typically resonates downfield at ␦ C 74.2 ppm (34).…”

“…By comparing chemical shifts and coupling constants to those reported for Pse in the literature, the absolute configuration of CMPPse5NAc7Am (I) was determined to be L-glycero-L-manno (14,29,30,(32)(33)(34). The relatively small difference in proton chemical shifts for H3ax and H3eq (⌬␦ H 0.59 ppm) indicated that I had the ␣ configuration with the C1 carboxyl group occupying the equatorial position.…”

The structure of the O‐specific chain of Legionella pneumophila serogroup 1 lipopolysaccharide

“…The difference between chemical shifts of the signals for H3,, and H3,, in the 'H-NMR spectrum of PS, , , and PSNaOAc, Bt3N amounts to 0.84-1 .OO ppm, being characteristic [7,8,10, 111 for the axial orientation of carboxyl group in glycosides of neuraminic acid and related sugars (a difference less than 0.6ppm was reported [12] for glycosides of 5,7-diamino-3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic (pseudaminic) acid with the equatorial carboxyl…”

“…The threo configuration of the fragment C7-8 followed from the "C chemical shift 19.4 ppm of the signal for C9 in the 13C-NMR spectrum of PSNaOAc, Et3N (according to the published data [7,11,12,14,151 this resonance lies at 19.6-20.3 ppm in the isomer with the threo configuration and at 16.2-17.7 pprn in that with the erythro configuration). The configuration of the side chain of the higher sugar was confirmed independently by the NOE observed on H6 at 4.25 ppm as a result of pre-irradiation of H9 at 1.27 ppm.…”

Structural determination of a 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-L-glycero-L-manno-nonulosonic acid-containing homopolysaccharide isolated from Sinorhizobium fredii HH103

“…The sugar residues are attached via a glycosidic linkage to the OH group of the 3-hydroxybutyramido substituent ; monomers are thus linked via both glycosidic and amidic linkages. Different pseudaminic acid derivatives have been described previously as components of the polysaccharides isolated from Pseudomonas aeruginosa, Shigella boydii and Vibrio cholerae [6][7][8][9][10][11] but this is the first report of the completely determined structure of a pseudaminic acid-containing homopolysaccharide in a rhizobial bacterium. Although different homopolysaccharides composed of a higher sugar of this class have been reported [12][13][14][15], and a polysaccharide with this class of glycosidic linkage has been described [9], this is the first report of a homopolymer of pseudaminic acid derivative with this type of glycosidic linkage.…”