Haemophilus influenzae type b strain A2 has multiple sialyltransferases involved in lipooligosaccharide sialylation.
The lipooligosaccharide (LOS) of Haemophilus influenzae contains sialylated glycoforms, and a sialyltransferase, Lic3A, has been previously identified. We report evidence for two additional sialyltransferases, SiaA, and LsgB, that affect N-acetyllactosamine containing glycoforms. Mutations in genes we have designated siaA and lsgB affected only the sialylated glycoforms containing N-acetylhexosamine. A mutation in siaA resulted in the loss of glycoforms terminating in sialyl-N-acetylhexosamine and the appearance of higher molecular weight glycoforms, containing the addition of phosphoethanolamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Chromosomal complementation of the siaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lacking N-acetylhexosamine and had no effect on sialylation of the terminal N-acetyllactosamine epitope. A double mutant in siaA and lic3A resulted in the complete loss of sialylation of the terminal N-acetyllactosamine epitope and expression of the higher molecular weight sialylated glycoforms seen in the siaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction in N-acetyllactosamine containing glycoforms. A triple mutant of siaA, lic3A, and lsgB contained no sialylated glycoforms. These results demonstrate that the sialylation of the LOS of H. influenzae is a complex process involving multiple sialyltransferases.
Jones
Paul A
PA
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.
Samuels
Nicole M
NM
Phillips
Nancy J
NJ
Munson
Robert S
RS
Bozue
Joel A
JA
Arseneau
Julie A
JA
Nichols
Wade A
WA
Zaleski
Anthony
A
Gibson
Bradford W
BW
Apicella
Michael A
MA
D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D006193:Haemophilus influenzae / Q000201:enzymology*; D008070:Lipopolysaccharides / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid