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J. Biol. Chem., Vol. 281, Issue 52, 40024-40032, December 29, 2006

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Identification of a Bifunctional Lipopolysaccharide Sialyltransferase in Haemophilus influenzae

INCORPORATION OF DISIALIC ACID^*

Kate L. Fox, Recipient of a studentship¹, Andrew D. Cox, Michel Gilbert, Warren W. Wakarchuk, Jianjun Li, Katherine Makepeace, James C. Richards, E. Richard Moxon², and Derek W. Hood²

From the 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

The lipopolysaccharide (LPS) of non-typeable Haemophilus 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 characterized biochemically to date in H. influenzae is Lic3A, an -2,3-sialyltransferase responsible for the addition of Neu5Ac to a lactose acceptor (Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh, S., Deadman, M. E., Cody, A., Martin, A., Månsson, M., Schweda, E. K., Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W. (2001) Mol. Microbiol. 39, 341-350). Here we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both -2,3- and -2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that 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 characterize 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 with mono-sialylated or non-sialylated species, indicating that this modification has biological significance.

Received for publication, March 13, 2006 , and in revised form, October 26, 2006.

^* This work was supported in part by the Medical Research Council, United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2.

² Funded by a Program Grant.

¹ To whom correspondence should be addressed: Dept. of Microbiology and Parasitology, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, QLD 4072, Australia. Tel.: 61-73365-1892; Fax: 61-73365-4620; E-mail: k.fox{at}uq.edu.au.

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