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Proteus mirabilis 2573 (ATCC 49565) produces an acidic capsular polysaccharide which was shown from glycose analysis, carboxyl reduction, methylation, periodate oxidation, and the application of one dimensional and two-dimensional high-resolution nuclear magnetic resonance techniques to be a high-molecular-weight polymer of branched trisaccharide units composed of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine), 2-acetamido-2,6-dideoxy-L-galactose (N-acetyl-L-fucosamine), and D-glucuronic acid, having the structure: [formula: see text] P. mirabilis 2573 also produces an O:6 serotype lipopolysaccharide in which the O-chain component has the same structure as the homologous capsular polysaccharide. This is the first report of a defined capsular polysaccharide in this bacterial genus.

The specific polysaccharide antigen of Streptococcus pneumoniae serotype 35A was shown, by a combination of one-and two-dimensional NMR methods and chemical analyses, to be a high-molecularmass polymer composed of D-galactose, D-glucose, mannitol, and phosphate (3 : 1 : 1 : 1). The pentasaccharide repeating unit is polymerized through phosphate diester linkages to give the structure, Ac AcAc0-Acetyl substituents are present at postions 5 and 6 of the 3)-P-~-Galfresidue and at position 2 of the 6)-P-~-Galf residue. The capsular polysaccharides of S. pneumoniae serotypes 20 and 35A both contain the disaccharide unit -3)-p-~-Galf-( l-?i)-p-~-Gkp-( 1-which is the probable structural determinant responsible for the serological cross reactivity of the two polysaccharides.Keywords: Streptococcus pneumoniae; polysaccharide ; structure ; antigenic determinant.Streptococcus pneumoniae is the most common etiological agent of bacterial pneumonia which until recently was easily treated with antibiotics. However, in the last 25 years the increase in antibiotic resistance of S. pneumoniae has presented major problems for patients and clinicians: there are as many as 40 000 deaths per year in the USA alone resulting from pneumococcal infections (Fedson et al., 1994). Since the capsular polysaccharides (CPS) of S. pneumoniae are responsible for stimulation of the host's immune system, vaccines have been developed from the CPS of the major pneumococcal serotypes responsible for infections in humans. Information on the structure (charge, hydrophobicity and conformational expression of epitopes) of the polysaccharide antigen is essential in the development of a new generation of vaccines and in explaining the serological cross-reactivity exhibited by S. pneumonine serogroups.Within the group 35 of S. pneumoniae four serotypes (35F, 35A, 35B, and 35C) have been recognized and, by the use of factor sera, seven factors (20b, 29b, 34b, 35a, 35b, 35c, and 42a) have been determined (Lund and Henrichsen, 1978 MATERIALS AND METHODSOrganisms and growth conditions. S. pneumoniae serotype 35A bacterial cells (Eddy 62, Statens Seruminstitut: NRCC 4757) were grown overnight on blood agar plates (Quelab) and then suspended in Bacto Todd-Hewitt broth (Difco, 15 g/l) and Bacto Columbia broth (Difco, 17.5 gll) in a 20-1 carboy, with the pH adjusted to between 7.50-7.80 with NaOH. The carboy was incubated in 5 % carbon dioxide, overnight, at 37°C. The cells were killed with 4% formalin (final concentration), left overnight at 4"C, prior to harvesting by centrifugation.Isolation and purification of capsular antigen. The cells were washed with 1 % phenol, the wash solution was dialysed, concentrated, and the CPS was recovered by precipitation with ethanol ( 5 vol.). It was further purified by precipitation with 1 % cetyltrimethylammonium bromide and, after keeping overnight at 4"C, the polysaccharide was recovered by centrifugation, redissolved in 10% sodium chloride, and precipitated with 5 vol. ethanol. The polysaccharide was dissolved in water, di...

. Can. J. Chem. 70, 2 18 (1992). The specific capsular polysaccharides of Streptococcus prleurnoniae types 19B and 19C (American types 58 and 59) were investigated by a combination of 'H, I3c, and 3 1~ nuclear magnetic resonance spectroscopy and analytical methods based on mass spectrometry. The two polysaccharides were found to be high molecular weight polymers composed of L-rhamnose, 2-amino-2-deoxy-D-mannose, D-ribose, D-glucose, and phosphate. Homo-and heteronuclear chemical shift correlation techniques and nuclear Overhauser enhancement experiments led to the unambiguous assignment of the 'H and I3c resonances from the glycose residues and established their sequence within repeating oligosaccharide units.The oligosaccharide units are polymerized through phosphate diester linkages. RBoth polysaccharides share a common hexasaccharide structural unit and they differ only in the degree of substitution at the branched 2-acetamido-2-deoxy-P-D-mannopyranosyl residue: the 19C polysaccharide is 0 -6 linked by P-D-glucopyranosyl end groups to form a heptasaccharide repeating unit, while the 19B polysaccharide is unsubstituted at that position. The serologic cross-reactivity between S. pneurnoniae serotypes 19B and 19C can now be related to the structural similarity of the antigenic capsular polysaccharides.

The antigenic lipopolysaccharide 0 polysaccharides of capsular serotypes 9 and 11 were examined by chemical, immunological, and nuclear magnetic resonance methods. Immunodiffusion tests carried out on these 0 antigens indicated that both contained common epitopes which were also shared by Actinobacilus peuropneumonae serotype 1. Chemical analysis and high-field nuclear magnetic resonance spectroscopy showed that the 0 antigens of serotypes 9 and 11 were high-molecular-weight polymers consisting of a backbone of repeating trisaccharide units composed of a-L-rhamnopyranosyl and a-D-glucopyranosyl residues (2:1). One of the c-L-rhamnose units forms a branch point and is stoichiometrically substituted with terminal 2-acetamido-2-deoxy-1-D-glucose residues in the serotype 11 0 polysaccharide, but only to the extent of 25% in the serotype 9 0 polysaccharide. Thus, the serotype 9 0 polysaccharide contains two different repeating units: a tetrasaccharide unit with the same structure as that of the serotype 11 0 polysaccharide and a trisaccharide unit:where R = j3-D-GlcpNAc for serotype 1 and 11 0 polysaccharides, and R = H (75%) and R = f-D-GlcPNAc (25%o) for serotype 9. The structure of the previously determined serotype 1 0 polysaccharide (E. Altman, J.-R Brisson, and M. B. Perry, Biochem. Cell. Biol. 64:17-25, 1986) is identical to that of the serotype 11 0 polysaccharide. We propose a more complete serotyping scheme for A. pleuropneumoniae which includes designation of both the capsular (K) and 0 antigens. Actinobacillus pleuropneumoniae is a gram-negative, pleomorphic rod which causes a highly contagious disease in pigs. The occurrence of A. pleuropneumoniae-associated pleuropneumonia has increased since 1976 (29) because of intensified pig production, and A. pleuropneumoniae lipopolysaccharide (LPS) is recognized as an important virulence factor (30). In attempts to combat the disease, serological methods of identification of infected animals based on the identification and characterization of serotype-specific antigens would greatly assist in solving the problem. Information on the structures of the LPS 0 antigens of A. pleuropneumoniae involved as virulence factors, in protection, and in serodiagnosis could provide data leading to the rational development of suitable, protective vaccines and the production of specific serotyping and serodiagnostic reagents.Currently, 12 serotypes ofA. pleuropneumoniae based on capsular polysaccharides (CPS) are recognized (26). Capsular serotypes 1, 5, and 7 are the predominant disease isolates from A. pleuropneumoniae outbreaks in the United States and Canada (28), whereas serotype 9 is predominant in The Netherlands (19). Serological cross-reactivity between A. pleuropneumoniae serotypes 1 and 9 has been detected by * Corresponding author. t NRCC no. 34260. complement fixation and gel diffusion methods (25). This cross-reactivity was attributed to common polysaccharide antigens by Mittal (24), who also concluded that these serotypes do not share CPS epitopes but that the cross...