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-␣-galactopyranose (13%) and 2-acetamido-2-deoxy-␣-glucopyranose (6%) on position O-2, were found. A minor component was established to be a polymer of 33-O-(2-acetamido-2-deoxy--glucopyranosyl)-134-ribitol-1-OPO 3 3. 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 ( Cell wall polysaccharides (CWP) of gram-positive bacteria, also referred to as teichoic acids, are structurally diverse linear polymers of polyols or carbohydrates linked through phosphodiester bonds, often substituted with different glycosyl or amino acid residues and terminally linked to the muramic acid of peptidoglycan (5). The functions and structures of different types of teichoic acids have been reviewed (23). These compounds comprise 20 to 50% of the weight of the cell wall and are major surface antigens in noncapsulated gram-positive bacteria. In capsulated bacteria, the capsule is the surface antigen covering other structures such as CWP in gram-positive bacteria and lipopolysaccharides in gram-negative bacteria. Antibodies to the capsular polysaccharides (CP) of pathogenic bacteria are protective, and the CP are used as vaccines, either alone or as protein conjugates (1).Bacillus pumilus strain Sh18, a nonpathogenic, enteric grampositive bacterium, produces a CWP, hitherto of unknown structure, reported to cross-react with the H. influenzae type b (Hib) CP. This cross-reactivity has been attributed to poly (ribitol phosphate), a known component of teichoic acids of bacilli. Ribitol phosphate, but not ribitol alone, inhibited precipitation of anti-Hib serum with B. pumilus Sh18 CWP (3, 9). No cross-reactivity, however, was observed with H. influenzae type a (Hia) CP, also containing ribitol phosphate in its subunit (Fig. 1). The induction of antibodies against Hia would be of interest, since this type has been reported in several countries to cause up to 10% of systemic infections due to H. influenzae (26,35,36). Here, we describe the purification of B. pumilus Sh18 CWP, the determination of its chemical structure, and the preparation and characterization of CWP-protein conjugates.
MATERIALS AND METHODSBacteria and cultivation. B. pumilus Sh18 and Sh17, Hib strains Eagan and Rab, Hia strain Harding, Staphylococcus aureus type 5 strain Lowenstein, and Sta...