Identification of the Common Antigenic Determinant Shared by Streptococcus pneumoniae Serotypes 35A and 20 Capsular Polysaccharides — Structural Analysis of the Streptococcus pneumoniae Serotype 35A Capsular Polysaccharide

Abstract

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...

WciG O -Acetyltransferase Functionality Differentiates Pneumococcal Serotypes 35C and 42

“…The chemical structures suggest that WcjE acetylates the 5-and 6-positions of 3-␤-Galf in the polysaccharides of both serotypes. This is consistent with other reports of O-acetylation in serotypes 20, 31, 33A, 35A, and 47A (2), which also carry wcjE (11,16). Thus, WciG acetylates the 2-position of the 6-␤-Galf in the serotype 35C repeat unit, and 2-OAc␤Galf ("OAc" represents O-acetylation) is essential for factor serum 35a recognition.…”

Serological and Genetic Diversity of Capsular Polysaccharides in Enterococcus faecalis

“…However, one of the serum types (i.e., the type 2-specific serum) showed a high degree of cross-reactivity with a different prototype strain (i.e., E. faecalis type 5). This may be explained by chemical and/or structural similarities of the different polysaccharides, a situation not uncommon for polysaccharide antigens from other bacteria (5,25). Alternatively, a given strain may be able to produce two different variant forms of carbohydrate antigen, depending on the conditions used.…”

Biochemical, Genetic, and Serological Characterization of Two Capsule Subtypes among Streptococcus pneumoniae Serotype 20 Strains

“…These genes are also present in the serotype 35A cps locus (11), whose PS repeat unit also contains 2-OAc-␤Galf and 5,6-OAc-␤Galf (10). The PS repeat unit of serotype 33F also contains two ␤Galf residues, but only ␤Galf-2-O-acetylation (24).…”

“…That 33F cps locus contains an intact wciG gene and a frameshift mutation in wcjE (11) suggests that the wciG gene product mediates 2-O-acetylation. Factor serum 20b was proposed to bind a 5,6-OAc-␤Galf-mediated epitope in serotypes 35A and 20 (10). Factor serum 20b does not bind serotype 33F but does bind serotype 33A, whose cps locus contains an intact wcjE gene and is otherwise identical to the 33F cps locus (11).…”

“…The capsular PS of the reference serotype 20 strain included in PPV-23 is composed of a branched hexasaccharide repeat unit (see Fig. 1A) that contains ϳ2.2 molar equivalents of O-acetate (OAc) per repeat unit (9), including OAc substitutions on carbons 5 and 6 of the nonbranching ␤-galactofuranose (10). Remaining OAc groups were not assigned to specific positions because of limitations of the techniques employed to analyze this PS.…”