Chemical structure, conjugation, and cross-reactivity of Bacillus pumilus Sh18 cell wall polysaccharide.
Bacillus pumilus strain Sh18 cell wall polysaccharide (CWP), cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b, was purified and its chemical structure was elucidated using fast atom bombardment mass spectrometry, nuclear magnetic resonance techniques, and sugar-specific degradation procedures. Two major structures, 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), with the latter partially substituted by 2-acetamido-2-deoxy-alpha-galactopyranose (13%) and 2-acetamido-2-deoxy-alpha-glucopyranose (6%) on position O-2, were found. A minor component was established to be a polymer of -->3-O-(2-acetamido-2-deoxy-beta-glucopyranosyl)-1-->4-ribitol-1-OPO3-->. The ratios of the three components were 56, 34, and 10 mol%, respectively. The Sh18 CWP was covalently bound to carrier proteins, and the immunogenicity of the resulting conjugates was evaluated in mice. Two methods of conjugation were compared: (i) binding of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-activated hydroxyl groups of the CWP to adipic acid dihydrazide (ADH)-derivatized protein, and (ii) binding of the carbodiimide-activated terminal phosphate group of the CWP to ADH-derivatized protein. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with the homologous polysaccharide and with a number of other bacterial polysaccharides containing ribitol and glycerol phosphates, including H. influenzae types a and b and strains of Staphylococcus aureus and Staphylococcus epidermidis.
Laboratory of Developmental and Molecular Immunity, NIH/NICHD, 9000 Rockville Pike, Bldg. 6, Rm. 1A05, Bethesda, MD 20892, USA. kielbj@mail.nih.gov.
Joanna
J
Kubler-Kielb
Bruce
B
Coxon
Rachel
R
Schneerson
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001407:Bacillus / Q000737:chemistry* / Q000276:immunology; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology; D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000737:chemistry*; D003429:Cross Reactions; D005260:Female; D007114:Immunization; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D018074:Vaccines, Conjugate / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D011135:Polysaccharides, Bacterial; D018074:Vaccines, Conjugate