Identification of a novel structural motif in the lipopolysaccharide of the galE/galK double mutant of Haemophilus influenzae strain Eagan |
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Authors: | Masoud Hussein Uhrin Dusan Moxon E Richard Richards James C |
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Institution: | a Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6 b Department of Biological Sciences, University of Jordan, 11942 Amman, Jordan c Molecular Infectious Diseases Group and Department of Paediatrics, Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford 0X3 3DU, UK |
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Abstract: | Defined mutants of the galactose biosynthetic (Leloir) pathway were employed to investigate lipopolysaccharide (LPS) oligosaccharide expression in Haemophilus influenzae type b strain Eagan. The structures of the low-molecular-mass LPS glycoforms from strains with mutations in the genes that encode galactose epimerase (galE) and galactose kinase (galK) were determined by NMR spectroscopy on O- and N-deacylated and dephosphorylated LPS-backbone, and O-deacylated oligosaccharide samples in conjunction with electrospray mass spectrometric, glycose and methylation analyses. The structural profile of LPS glycoforms from the galK mutant was found to be identical to that of the galactose and glucose-containing Hex5 glycoform previously identified in the parent strain Masoud, H.; Moxon, E. R.; Martin, A.; Krajcarski, D.; Richards, J. C. Biochemistry1997, 36, 2091-2103]. LPS from the H. influenzae strain bearing mutations in both galK and galE (galE/galK double mutant) was devoid of galactose. In the double mutant, Hex3 and Hex4 glycoforms containing di- and tri-glucan side chains from the central heptose of the triheptosyl inner-core unit were identified as the major glycoforms. The triglucoside chain extension, β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, identified in the Hex4 glycoform has not been previously reported as a structural element of H. influenzae LPS. In the parent strain, it is the galactose-containing trisaccharide, β-d-Galp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, and further extended analogues thereof, that substitute the central heptose. When grown in galactose deficient media, the galE single mutant was found to expresses the same population of LPS glycoforms as the double mutant. |
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Keywords: | DOC-PAGE deoxycholate-polyacrylamide gel electrophoresis GLC-MS gas liquid chromatography-mass spectrometry ESI-MS electrospray ionization-mass spectrometry COSY correlated spectroscopy TOCSY total correlated spectroscopy HMQC heteronuclear multiple quantum coherence NOE nuclear Overhauser effect NOESY two-dimensional NOE spectroscopy Kdo d-manno-octulsonic acid" target="_blank">3-deoxy-d-manno-octulsonic acid LPS lipopolysaccharide LPS-OH O-deacylated LPS Lipid A-OH O-deacylated lipid A OS oligosaccharide Etn ethanolamine PEtn phosphoethanolamine PPEtn pyrophosphoethanolamine PCho phosphocholine UDP uridine diphosphate EDTA ethylenediaminetetraacetic acid SDS sodium dodecyl sulfate |
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