A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H. influenzae strains.
In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by 1H NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS/5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.
Bauer
S H
SH
Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, NOVUM, S-141 86 Huddinge, Sweden.
Månsson
M
M
Hood
D W
DW
Richards
J C
JC
Moxon
E R
ER
Schweda
E K
EK
D002852:Chromatography, Ion Exchange / Q000379:methods / Q000592:standards; D006193:Haemophilus influenzae / Q000737:chemistry* / Q000145:classification; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D009439:Neuraminidase / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization
D008070:Lipopolysaccharides; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid