Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae - Incorporation of disialic acid

Fox, Kate L., Cox, Andrew D., Gilbert, Michel, Wakarchuk, Warren W., Li, Jianjun, Makepeace, Katherine, Richards, James C., Moxon, E. Richard and Hood, Derek W. (2006) Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae - Incorporation of disialic acid. Journal of Biological Chemistry, 281 52: 40024-40032. doi:10.1074/jbc.M602314200

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Author Fox, Kate L.
Cox, Andrew D.
Gilbert, Michel
Wakarchuk, Warren W.
Li, Jianjun
Makepeace, Katherine
Richards, James C.
Moxon, E. Richard
Hood, Derek W.
Title Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae - Incorporation of disialic acid
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
Publication date 2006-12
Sub-type Article (original research)
DOI 10.1074/jbc.M602314200
Open Access Status File (Publisher version)
Volume 281
Issue 52
Start page 40024
End page 40032
Total pages 9
Place of publication Bethesda, M.D, U.S.A.
Publisher Amer Soc Biochemistry Molecular Biology Inc
Language eng
Subject 0601 Biochemistry and Cell Biology
Abstract 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 alpha-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., Mansson, 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 alpha-2,3- and alpha-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.
Keyword Biochemistry & Molecular Biology
Sialic-acid
Biofilm Formation
Otitis-media
Structural-characterization
Phase Variation
Middle-ear
Biosynthesis
Genes
Electrophoresis
Sialylation
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Chemistry and Molecular Biosciences
 
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Created: Fri, 25 Jan 2008, 17:01:06 EST