Determination of the structural features of A-band lipopolysaccharide from a rough mutant of Pseudomonas aeruginosa

Abstract

<p>"A-band" lipopolysaccharide (LPS) is believed to be widely distributed among serotypes of the bacterium Pseudomonas aeruginosa. The present work is a study of the structural features of the polysaccharide portion of A-band LPS ("A-PS") from strain AK1401. The A-PS was found to consist principally of D-rhamnose, with lesser amounts of 3-O-methylrhamnose, ribose, mannose, and glucose. These monosaccharides were found to exist as 1,3-linked rhamnopyranose, 1,2-linked rhamnopyranose (2:1 ratio), 1,4-linked 3-O-methylrhamnopyranose, and 1,4-linked manno- and gluco-pyranoses; the ring form and linkage positions of ribose could not be determined directly, but could be inferred to be 1,3-linked ribopyranose. ¹H- and ¹³C-NMR of the intact A-PS showed the presence of three principal monosaccharides, all of which were α-D-rhamnosides, suggesting a trisaccharide repeating unit structure composed of two α-(1,3)-linked rhamnopyranosides and one α-(1,2)-linked rhamnopyranoside. The ¹H-NMR spectrum of this repeating unit was assigned by use of hononuclear correlation spectroscopy (COSY) and relayed coherence transfer (RELAY) experiments. Nuclear Overhauser enhancement experiments allowed the assignment of the sequence of monosaccharides. The deduced structure of the repeating unit of AK1401 A-PS is: (→2)-α-D-Rhap-(1→3)-α-D-Rhap-(1→3)-α-D-Rhap-(1→)m. This work represents the first assignment of the ¹H-NMR spectrum of this repeating unit structure. The A-PS was subjected to sodium periodate oxidation, reduction, and mild acid hydrolysis (Smith degradation), which yielded an oligosaccharide of predictable structure as the major product. The peracetate derivatives of the minor oligosaccharide products were studied in an attempt to learn more about the role of the monosaccharides that are not included in the repeating unit. Gas chromatography-mass spectrometry (GC-MS) revealed the presence of at least five oligosaccharides in addition to the major product. The structures of two of these could be inferred from the mass spectra as: [equation omitted] A fraction containing oligosaccharides which were too involatile to be analyzed by GC-MS was isolated by liquid chromatography and analyzed by direct chemical ionization (DCI) mass spectrometry. This revealed the presence of at least two components, one of which was inferred to be the peracetate of: (3-O-MeRha)₄-(3-O-MeHex)₂-Gly-ol. These components provide some insight into the structure of the non-repeating portion of the A-PS.</p>