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1. (Article ID: 3145)
Fox KL, Cox AD, Gilbert M, Wakarchuk WW, Li J, Makepeace K, Richards JC, Moxon ER, Hood DW
Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae: Incorporation of disialic acid
Journal of Biological Chemistry 281(52) (2006)
40024-40032
The lipopolysaccharide (LPS) of non-typeable H. influenzae (NTHi) can be substituted at various positions by N-acetylneuraminic acid (Neu5Ac). LPS sialylation plays an important role in pathogenesis. The only LPS sialyltransferase characterised biochemically to date in H. influenzae is Lic3A, an α-2,3- sialyltransferase responsible for the addition of Neu5Ac to a lactose acceptor (1). Here we describe a second sialyltransferase, Lic3B, which is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in E. coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both α-2,3- and α-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterise the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared to mono-sialylated or non-sialylated species, indicating that this modification has biological significance.
Lipopolysaccharide, Haemophilus influenzae, sialyltransferase, sialylation
NCBI PubMed ID: 17071616Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: k.fox
uq.edu.au
Institutions: Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, United Kingdom and Institute for Biological Sciences, National Research Council, Ottawa, Ontario, K1A OR6, Canada
Methods: ESI-MS, electrophoresis, genetic methods, biochemical methods
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