Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae: incorporation of disialic acid.
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., 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 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.
Fox
Kate L
KL
Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, United Kingdom. k.fox@uq.edu.au
Cox
Andrew D
AD
Gilbert
Michel
M
Wakarchuk
Warren W
WW
Li
Jianjun
J
Makepeace
Katherine
K
Richards
James C
JC
Moxon
E Richard
ER
Hood
Derek W
DW
D002240:Carbohydrate Sequence; D005819:Genetic Markers; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D006801:Humans; D008070:Lipopolysaccharides / Q000097:blood / Q000737:chemistry*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D012091:Repetitive Sequences, Nucleic Acid; D015610:Serum Bactericidal Test; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism*; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D005819:Genetic Markers; D008070:Lipopolysaccharides; D009097:Multienzyme Complexes; D012794:Sialic Acids; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase