Abstract
Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.
Highlights
AND RESULTSP. aeruginosa Strains-The neutral polysaccharide was isolated from a clinical strain of P . aeruginosa determined to be an immunotype strain by agglutination with antiseraraised
Previous work hadsescribed small molecular weight they directly substitute the LPS molecule [2,3,4]
Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 “C!, 2 h) LPS from the seven Fisher immunotype strains of P . aeruginosa bound well to the neutral polysaccharide.Antibodies affinitypurified lated from a Fisher immunotype 4 strain of P. aeruginosa
Summary
P. aeruginosa Strains-The neutral polysaccharide was isolated from a clinical strain of P . aeruginosa determined to be an immunotype strain by agglutination with antiseraraised. The neutral polysaccharide was elutedwith 0.01 M buffer.Lipopolysaccharide antigens from the seven Fisherimmunotypestrains of P. aeruginosa were prepared asdescribed elsewhere [5,6,7,8]. Monosaccharides were identified using a Techniconsugaranalyzerandinthe form of the alditol acetates using gas-liquid chromatography/mass spectrometry as described [3].Acid hydrolysis of the polysaccharide yielded glucose, ribose, and rhamnose in a ratio of -1:1:2. Rhamnose was isolated from the hydrolysate of the polysaccharide and proved to be L on the basis of its specific optical rotation, [a]L3+ 7.7" (c 0.2, water) (cf Ref. 12, +8.9" (water)).Methylationanalysis of this polysaccharide yielded 2,4-di-O-methylrhamnose an3d,5di-0-methylribose in a ratio of 4 1 Both residues of rhamnose are substituteadt position 3, and thepolysaccharide resulting from Smith degradation has thfeollowing structure (I),where Rha is rhamnose.
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