Studies of polysaccharide fractions from the lipopolysaccharide of Pseudomonas aeruginosa N.C.T.C. 1999

Abstract

Two polymeric water-soluble fractions were isolated by gel filtration after mild acid hydrolysis of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 1999. The fraction of higher molecular weight retained the O-antigenic specificity of the lipopolysaccharide and may be 'side-chain' material. This fraction was rich in N (about 10%) and gave several basic amino compounds on acid hydrolysis; fucosamine (at least 2.8% w/w) was the only specifc component identified. The fraction of lower molecular weight was a phosphorylated polysaccharide apparently corresponding to 'core' material. The major components of this fraction and their approximate molar proportions were: glucose (3-4); rhamnose (1); heptose (2); 3-deoxy-2-octulonic acid (1); galactosamine (1); alanine (1-1.5); phosphorus (6-7). In the intact lipopolysaccharide this fraction was probably linked to lipid A via a second residue of 3-deoxy-2-octulonic acid, and probably also contained additional phosphate residues and ethanolamine. The residues of 3-deoxy-2-octulonic acid were apparently substituted in the C-4 or C-5 position, and the phosphorylated heptose residues in the C-3 position. The rhamnose was mainly 2-substituted, though a little 3-substitution was detected. The glucose residues were either unsubstituted or 6-substituted. Four neutral oligosaccharides were produced by partial acid hydrolysis and were characterized by chemical, enzymic, chromatographic and mass-spectrometric methods of analysis. The structures assigned were: Glcpalpha1-6Glc; Glcpbeta1-2Rha; Rhapalpha1-6Glc; Glcpbeta1-2Rhapalpha1-6Glc. The galactosamine was substituted in the C-3 or C-4 position, the attachment of alanine was indicated, and evidence that the amino sugar linked the glucose-rhamnose region to the 'inner core' was obtained.

Structure of the Core Oligosaccharide from the Lipopolysaccharide of Pseudomonas aeruginosa PAC1R and Its Defective Mutants

“…It was not possible to assign signals to C5 of dOclA and not always to C4, but the values quoted in the literature are tentative [25] and no values for substituted C4 and C5 of dOclA are available. Our data would support the view of Wilkinson and his colleagues [12] that dOclA may be linked to heptose through either C4 or (2.5, the other of these two carbons being substituted possibly by acetylation. Evidence for this substitution comes from the very low values obtained for dOclA using the thiobarbiturate assay compared with the semicarbazide method [12].…”

“…Our data would support the view of Wilkinson and his colleagues [12] that dOclA may be linked to heptose through either C4 or (2.5, the other of these two carbons being substituted possibly by acetylation. Evidence for this substitution comes from the very low values obtained for dOclA using the thiobarbiturate assay compared with the semicarbazide method [12]. The presence of additional resonances at 174.4 ppm and 173.9 ppm in our PACIR fraction could perhaps be attributed to carbonyl groups of acetyl residues.…”

“…All three spectra showed a clear signal at 96.9 ppm attributable to the free anomeric carbon 2 of dOclA. This would be expected since the acetic acid hydrolysis which split the oligosaccharide from the lipid A is thought to cleave the lipopolysaccharide at dOclA and to release a second dOclA residue [12]. All the spectra contained resonances attributable to unsubstituted C8, C7, C6 and C3 atoms of dOclA.…”

“…Xylose (100 -200 pg) was added as an internal standard. The free neutral sugars were reduced with NaBH4 [I61 and acetylated by treating for 70 min using the method described by Drewry et al [12]. Amino sugars were analysed either by converting them to their 2,5-anhydro derivatives after hydrolysis with 2 M HCI (105 T, 1 h) [I71 or by washing the 1 M HCI hydrolysates repeatedly with n-propanol [7] before reduction and acetylation.…”

“…The repeating units of some of these 0-antigenic side chains have recently been elucidated t9-111. A partial structure for the core oligosaccharide of P. aeruginosa NCTC 1999 was proposed by Wilkinson and his colleagues [12], but it is not known whether this would apply to other strains. Indirect evidence for the sequence and role of the core structure came from our work with mutants of P. aeruginosa PAClR which lacked the 0-antigenic side chains and parts of the core itself [13] and from studies of the pyocin and bacteriophage-binding sites in these mutants [2].…”