title: Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae.
abstract: We have identified a gene for the addition of N-acetylneuraminic acid (Neu5Ac) in an alpha-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase-variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N-acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-alpha-(2-3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an alpha-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene.
authors.affiliation: Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK. dhood@molbiol.ox.ac.uk
authors.forename: D W
authors.initials: DW
authors.lastname: Hood
authors.forename: A D
authors.initials: AD
authors.lastname: Cox
authors.forename: M
authors.initials: M
authors.lastname: Gilbert
authors.forename: K
authors.initials: K
authors.lastname: Makepeace
authors.forename: S
authors.initials: S
authors.lastname: Walsh
authors.forename: M E
authors.initials: ME
authors.lastname: Deadman
authors.forename: A
authors.initials: A
authors.lastname: Cody
authors.forename: A
authors.initials: A
authors.lastname: Martin
authors.forename: M
authors.initials: M
authors.lastname: Månsson
authors.forename: E K
authors.initials: EK
authors.lastname: Schweda
authors.forename: J R
authors.initials: JR
authors.lastname: Brisson
authors.forename: J C
authors.initials: JC
authors.lastname: Richards
authors.forename: E R
authors.initials: ER
authors.lastname: Moxon
authors.forename: W W
authors.initials: WW
authors.lastname: Wakarchuk
mesh_terms: D001770:Blood Bactericidal Activity; D002240:Carbohydrate Sequence; D019075:Electrophoresis, Capillary; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D012799:Sialyltransferases