Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain. A unique teichoic acid-like polysaccharide and the group O antigen which is a C-polysaccharide in common with pneumococci.

The cell wall of Streptococcus mitis biovar 1 strain SK137 contains the C-polysaccharide known as the common antigen of a closely related species Streptococcus pneumoniae, and a teichoic acid-like polysaccharide with a unique structure. The two polysaccharides are different entities and could be partially separated by gel chromatography. The structures of the two polysaccharides were determined by chemical methods and by NMR spectroscopy. The teichoic acid-like polymer has a heptasaccharide phosphate repeating unit with the following structure: The structure neither contains ribitol nor glycerol phosphate as classical teichoic acids do, thus we have used the expression teichoic acid-like for this polysaccharide. The following structure of the C-polysaccharide repeating unit was established: where AAT is 2-acetamido-4-amino-2,4, 6-trideoxy-D-galactose. It has a carbohydrate backbone identical to that of one of the two structures of C-polysaccharide previously identified in S. pneumoniae. C-polysaccharide of S. mitis is characterized by the presence, in each repeating unit, of two residues of phosphocholine and both galactosamine residues in the N-acetylated form. Immunochemical analysis showed that C-polysaccharide constitutes the Lancefield group O antigen. Studies using mAbs directed against the backbone and against the phosphocholine moiety of the C-polysaccharide revealed several different patterns of these epitopes among 95 S. mitis and Streptococcus oralis strains tested and the exclusive presence of the group O antigen in the majority of S. mitis biovar 1 strains.

The pneumococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine.

The structure of the pneumococcal common antigen, C-polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C-polysaccharide is composed of a backbone of tetrasaccharide-ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a D-ribitol residue and position 6 of a beta-D-glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N-acetylated. O)-P-Cho | 6 6)-beta-D-Glcp-(1-->3)-alpha-AATp-(1-->4)-alpha-D-GalpNAc-(1-->3)- bet a-D-GalpNAc-(1-->1)-D-ribitol-5-P-(O--> where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N-acetylation, from those reported previously for pneumococcal C-polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry 19, 4712-4719; Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem. 71, 644-648]. The structures of the C-polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C-polysaccharides was identical and in all cases both galactosamine residues appeared to be N-acetylated.

Relationship between cell surface carbohydrates and intrastrain variation on opsonophagocytosis of Streptococcus pneumoniae.

Streptococcus pneumoniae undergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci.

The chemical structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A.

The structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A have been determined by means of NMR spectroscopy as the principal method. It is concluded that both polysaccharides are composed of tetrasaccharide repeating units with a phosphorylcholine (PCho) group linked to the 3-position of the 4-substituted beta-L-rhamnose (Rha) residue. Both polysaccharides are substituted with one O-acetyl group at the 2-position of the same beta-L-rhamnose residue. In addition, the type-32A polysaccharide is substituted with another O-acetyl group at the 4-position of the 2,3-disubstituted alpha-D-glucose residue, i.e. the branch-point residue. An unusual detail in the structure is that the side chain is composed of a rhamnosyl phosphate. [chemical structure: see text] In the type-32F polysaccharide R=H, and in the type-32A polysaccharide R=Ac. The structure of C-polysaccharide found in our preparations of type-32F and type-32A capsular polysaccharides is in agreement with that published previously for the pneumococcal common antigen C-polysaccharide [Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D. W., Young, N. M. & Jennings, H. J. (1993) Can. J. Chem. 71, 644-648].

Structural elucidation of the capsular polysaccharide from Streptococcus pneumoniae type 18B.

The structure of the capsular polysaccharide from Streptococcus pneumoniae type 18B has been determined using NMR spectroscopy and methylation analysis as the principal methods. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with a glycerol phosphate substituting the 3-position of the branch point residue. The carbohydrate backbone in type 18B is identical to that in S. pneumoniae type 18F but without the O-acetyl groups present in that type. [formula: see text] In this structure, the absolute configuration of the glycerol phosphate moiety has not been determined but should be D, in analogy with that determined for the capsular polysaccharide from S. pneumoniae type 18A [T. Rundlöf, G. Widmalm, Anal. Biochem., 243 (1996) 228-233].

A modified enzyme-linked immunosorbent assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies.

We have developed an ELISA for antibody determination, superior to others hitherto described, in which optimal coating is achieved using phenylated pneumococcal capsular polysaccharides as coating antigen. The specificity of the assay is ensured by complete inhibition of antibodies against the species-specific pneumococcal antigen, C-polysaccharide (C-Ps). The method is sensitive, specific, reproducible, fast and easy to work with and can be used for both immunoglobulin class and subclass antibody determinations.

Typing of pneumococci by using 12 pooled antisera.

A new simplified chessboard system for typing of Streptococcus pneumoniae is described. It is intended for typing or grouping of 90 to 95% of the pneumococcal strains most commonly isolated from blood or cerebrospinal fluid and is based on 12 pooled diagnostic antisera, each reacting with 7 to 11 single types, together covering the 23 different vaccine-related types as well as 25 other cross-reacting types. Worldwide surveillance of the type distribution is important in order to ensure an optimal formulation of pneumococcal polysaccharide vaccines and, in the future, of polysaccharide-protein conjugate vaccines. The simplified typing system described in this paper makes it easier to carry out surveillance in other than specialized reference laboratories. Finally, it takes advantage of the fact that some types cause disease more often in children--as opposed to adults--than do others.

Antibodies against pneumococcal C-polysaccharide are not protective.

The ability of antibodies against C-polysaccharide (C-Ps) to protect against experimental pneumococcal infection was examined in a mouse model. No protection against types 6A and 14 pneumococcal infection could be demonstrated neither with mouse monoclonal antibodies against C-Ps, specific for phosphorylcholine (PC) or for C-Ps backbone, nor for polyclonal rabbit immunsera against C-Ps. The monoclonal antibody with PC-specificity was protective against infection with type 27 pneumococcus, that has PC as part of its capsular polysaccharide. Type-specific mono- and polyclonal antibodies were highly protective against infection with the homologous type. We conclude that no species-specific protection can be achieved against intraperitoneal Streptococcus pneumoniae infection with optimally capsulated bacteria in outbred mice by passive immunization with antibodies to C-Ps.

Capsular polysaccharide is linked to the outer surface of type 6A pneumococcal cell walls.

The in situ attachment of capsular polysaccharide of type 6A pneumococci was examined by immunoelectron microscopy using anti-type 6A monoclonal antibody. The result discloses an asymmetrical cross-section of pneumococcal cell walls because capsular polysaccharides are located on the outer surface of the walls only, in contrast to the cell wall polysaccharide, which has been shown to be located on both surfaces.

Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods.

The attachment of capsular polysaccharide to Streptococcus pneumoniae was examined using monoclonal and polyclonal antibodies. Among the strains examined, the capsular polysaccharide of types 2, 4, 6A, 6B, 7F, 8, 14, 19F and 23F was bound to the pneumococci whereas that of a type 3 strain was not. Sequential treatment with 2% SDS at 100 degrees C, pronase, and EDTA did not dissociate the capsular polysaccharide from the pneumococci. Treatment of the cells with mutanolysin, a muramidase that degrades the cell wall peptidoglycan of pneumococci and other streptococci, released both the capsular and the cell wall C-polysaccharide (C-Ps). Type 6A capsular polysaccharide released from cell walls by mutanolysin treatment, was fractionated by high performance liquid chromatography and examined by immunoelectrophoresis. It was found to be bound to both the C-Ps and the peptidoglycan. The bond between the capsular polysaccharide and the peptidoglycan has not yet been identified but is probably covalent, as the two components could not be dissociated after boiling in SDS. Based on our studies with type 6A, we propose that capsular polysaccharide and C-Ps of the pneumococcus are linked to the peptidoglycan at different sites and, thereby, indirectly to each other. Studies in mice showed that the peptidoglycan enhanced the serum antibody response to C-Ps but not to type 6A polysaccharide.

Local production of IgG to pneumococcal C-polysaccharide in upper airway secretions from children with recurrent acute otitis media.

Antibodies against C-polysaccharide (C-Ps), a common cell wall component of all pneumococci, may be of importance for the elimination of decaying pneumococci. By means of ELISA with phenylated C-Ps, anti-C-Ps IgG was measured in samples of plasma and upper airway secretions from otitis-prone children (OP), children with fewer episodes of recurrent acute otitis media (rAOM), and children with no previous history of AOM, but suffering from secretory otitis media (SOM). All children were free from acute illness at the time of sampling. No statistically significant differences of anti-C-Ps IgG in plasma or in nasopharyngeal secretions (NPS) were found between any of the groups. Based on calculations of the correlation between levels of anti-C-Ps IgG in plasma and NPS, and of the transudation ratios of anti-C-Ps IgG, total IgG, and albumin from plasma to NPS, we suggest that a significant amount of the anti-C-Ps IgG in NPS must be locally produced. The additional finding that OP children had significantly higher levels of anti-C-Ps IgG in their middle ear effusions (MEE) than SOM children points in the same direction. Anti-C-Ps IgA and IgM were detected in very low concentrations in plasma and secretion samples.

Ultrastructural localization of capsules, cell wall polysaccharide, cell wall proteins, and F antigen in pneumococci.

The localization of pneumococcal capsular and cell wall antigens was examined by immunoelectron microscopy. C polysaccharide (C-Ps), a common component of all pneumococci, was uniformly distributed on both the inside and outside of the cell walls. The thickness of the C-Ps varied with the strain. Encapsulated strains were covered by varied amounts of capsular polysaccharide concealing the C-Ps of the bacteria so as to render it inaccessible to anti-C-Ps antibodies. In addition to C-Ps, protein antigens were demonstrable on the surface of nonencapsulated pneumococci. The proteins were not masked by the C-Ps layer. An extra layer on the cell walls was conspicuous on electron micrographs of both rough and encapsulated pneumococci. The nature of this extra layer has not been disclosed. F antigen, another common antigen of pneumococci, was uniformly distributed on the surface of the plasma membranes. During the course of the experimental work a reproducible method of gold labeling immunoglobulins was developed.

Cross-reactions between pneumococci and other streptococci due to C polysaccharide and F antigen.

By serological methods, all 83 known types of Streptococcus pneumoniae could be shown to possess C polysaccharide and F antigen. Cross-reactions due to these two antigens between pneumococci and a broad range of most other commonly encountered streptococci were examined. The presence of an antigen closely similar or identical to pneumococcal C polysaccharide was demonstrated in some strains of Streptococcus mitior. Therefore, we conclude that pneumococci cannot be identified serologically from mixed samples without culture. Streptococcal group C antiserum was found to cross-react with pneumococcal F antigen.

Monoclonal phosphorylcholine antibody binds to beta-lipoprotein from different animal species.

Previously published observations have demonstrated that murine antibody with specificity for phosphorylcholine (PC) binds to a variety of PC-containing polysaccharides and to human beta-lipoprotein. In this investigation it was found that monoclonal anti-PC antibody binds not only to human beta-lipoprotein, but also to beta-lipoprotein from the serum of all vertebrate species examined. beta-Lipoprotein from mouse reacted with autologous antibody; therefore, this antibody must be an autoantibody. This raises the question whether PC antibodies may exert unwanted effects after having been passively transferred or induced by PC-containing antigens, e.g., C-polysaccharide from pneumococci.

Serotyping of Vibrio anguillarum.

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.

C-polysaccharide in a pneumococcal vaccine.

The 14-valent pneumococcal vaccine, Pneumovax, was found to contain approximately 25o micrograms per ml of the cell-wall antigen, C-polysaccharide (C-Ps), in addition to 100 micrograms per ml of each of the 14 capsular polysaccharides. Rabbit anti-C-Ps antiserum recognized four components of this C-Ps with different antigenic determinants, corresponding to four fragments with different molecular sizes. By lectin affinity chromatography it was demonstrated that some of the C-Ps in the vaccine contained cell-wall residues. The methods used in this study can also be used for the characterization of other polysaccharide preparations.

Phosphorylcholine determinants in six pneumococcal capsular polysaccharides detected by monoclonal antibody.

The presence of phosphorylcholine in pneumococcal capsular polysaccharides was examined by using monoclonal antiphosphorylcholine antibody. Of the 83 known capsular types of Streptococcus pneumoniae, 6 types, viz., 24A, 27, 28F, 28A, 32F, and 32A, gave a positive capsular reaction (quellung) which could be inhibited by phosphorylcholine. The capsular polysaccharides of these six types, therefore, contain phosphorylcholine.