title: CpsK of Streptococcus agalactiae exhibits alpha2,3-sialyltransferase activity in Haemophilus ducreyi.
abstract: Streptococcus agalactiae (GBS) is a major cause of serious newborn bacterial infections. Crucial to GBS evasion of host immunity is the production of a capsular polysaccharide (CPS) decorated with sialic acid, which inactivates the alternative complement pathway. The CPS operons of serotypes Ia and III GBS have been described, but the CPS sialyltransferase gene was not identified. We identified cpsK, an open reading frame in the CPS operon of most serotypes, which was homologous to the lipooligosaccharide (LOS) sialyltransferase gene, lst, of Haemophilus ducreyi. To determine if cpsK might encode a sialyltransferase, we complemented a H. ducreyi lst mutant with cpsK. CpsK was expressed in H. ducreyi and LOS was isolated and analysed for sialic acid content by SDS-PAGE and high-performance liquid chromatography (HPLC). Sialo-LOS was seen in the wild-type, cpsK- or lst-complemented mutant strains, but not in the mutant without cpsK. Addition of Neu5Ac to the LOS was confirmed by mass spectroscopy. Lectin binding studies detected terminal Neu5Ac(alpha 2-->3)Gal(beta 1- on LOS produced by the wild-type, cpsK or lst-complemented mutant strain LOS, compared with the mutant alone. Our data characterize the first sialyltransferase gene from a Gram- positive bacterium and provide compelling evidence that its product catalyses the alpha2,3 addition of Neu5Ac to H. ducreyi LOS and therefore the terminal side-chain of GBS CPS. Phylogenetic studies further indicated that lst and cpsK are related but distinct from sialyltransferases of most other bacteria and, along with their similar codon usage bias and G + C content, suggests acquisition by lateral transfer from an ancestral low G + C organism.
authors.affiliation: Department of Pediatrics, Division of Infectious Diseases, Children's Hospital and Regional Medical Center/University of Washington, Seattle, 98105, USA.
authors.forename: Donald O
authors.initials: DO
authors.lastname: Chaffin
authors.forename: Katherine
authors.initials: K
authors.lastname: McKinnon
authors.forename: Craig E
authors.initials: CE
authors.lastname: Rubens
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D006191:Haemophilus ducreyi / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013292:Streptococcus agalactiae / Q000201:enzymology* / Q000235:genetics; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; C117560:Lst protein, Haemophilus ducreyi; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase