Haemophilus influenzae Type b Strain A2 Has Multiple Sialyltransferases Involved in Lipooligosaccharide Sialylation

• Published in: The Journal of biological chemistry 2002-04, Vol.277 (17), p.14598-14611
• Main Authors: Jones, Paul A., Samuels, Nicole M., Phillips, Nancy J., Munson, Robert S., Bozue, Joel A., Arseneau, Julie A., Nichols, Wade A., Zaleski, Anthony, Gibson, Bradford W., Apicella, Michael A.
• Format: Article
• Language: English
• Subjects: Base Sequence; Blotting, Southern; Blotting, Western; Cloning, Molecular; DNA Primers; Electrophoresis, Polyacrylamide Gel; Genetic Complementation Test; Haemophilus influenzae; Haemophilus influenzae - enzymology; lic3A gene; lic3A protein; lipooligosaccharides; Lipopolysaccharides - metabolism; lsgB gene; Mutagenesis, Site-Directed; N-Acetyllactosamine; N-Acetylneuraminic Acid - metabolism; siaA gene; sialylation; sialyltransferase; Sialyltransferases - chemistry; Sialyltransferases - genetics; Sialyltransferases - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110986200

The lipooligosaccharide (LOS) ofHaemophilus 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 lsgBaffected only the sialylated glycoforms containingN-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, andN-acetylneuraminic acid. Chromosomal complementation of thesiaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lackingN-acetylhexosamine and had no effect on sialylation of the terminal N-acetyllactosamine epitope. A double mutant insiaA 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 thesiaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction inN-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.