A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019 |
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Authors: | Mikael KR Engskog Jianjun Li Mary Deadman Elke KH Schweda |
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Institution: | a Clinical Research Centre, Karolinska Institutet and Södertörn University, NOVUM, S-141 86 Huddinge, Sweden b Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6 c Molecular Infectious Diseases Group, University of Oxford, Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford 0X3 9DS, UK |
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Abstract: | Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-l-glycero-d-manno-heptosyl inner-core moiety (l-α-d-Hepp-(1→2)-PEtn→6]-l-α-d-Hepp-(1→3)-β-d-GlcIp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdop) to which addition of β-d-Glcp to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a β-d-Galp is linked to O-4 of GlcI. In order to test the hypothesis that the lex2 locus is involved in the expression of β-d-Galp-(1→4-β-d-Glcp-(1→ from HepI, lex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESIMSn on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only β-d-Glcp extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a β-d-Galp to O-4 of GlcI in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product. |
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Keywords: | AnKdo-ol reduced anhydro Kdo CE capillary electrophoresis Gly glycine Hep l-glycero-d-manno-heptose" target="_blank">l-glycero-d-manno-heptose Hex hexose HexNAc N-acetyl-hexosamine Kdo d-manno-oct-2-ulosonic acid" target="_blank">3-deoxy-d-manno-oct-2-ulosonic acid Lipid A-OH O-deacylated lipid A LPS lipopolysaccharide LPS-OH O-deacylated lipopolysaccharide MSn multiple-step tandem mass spectrometry Neu5Ac N-acetylneuraminic acid NTHi non-typeable Haemophilusinfluenzae OS oligosaccharide P monophosphate PCho phosphocholine PEtn phosphoethanolamine PPEtn pyrophosphoethanolamine tHep terminal heptose |
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