Structures of Capsular Polysaccharide Serotypes 35F and 35C of Streptococcus pneumoniae Determined by Nuclear Magnetic Resonance and Their Relation to Other Cross-Reactive Serotypes

Abstract

The structures of Streptococcus pneumoniae capsular polysaccharides (CPSs) are essential for defining the antigenic as well as genetic relationships between CPS serotypes. The four serotypes that comprise CPS serogroup 35 (i.e., types 35F, 35A, 35B, and 35C) are known to cross-react with genetically related type 20, 29, 34, 42, or 47F. While the structures of CPS serotype 35A (CPS35A) and CPS35B are known, those of CPS35F and CPS35C are not. In the present study, the serotypes of CPS35F and CPS35C were characterized by high-resolution heteronuclear magnetic resonance (NMR) spectroscopy and glycosyl composition analyses to reveal the following repeat unit structures:Glc␣1 where OAc indicates O-acetylated. Importantly, CPS35F, the immunizing serotype for the production of group 35 serum, more closely resembles CPS34 and CPS47F than other members of serogroup 35. Moreover, CPS35C is distinct from either CPS35F or CPS35B but closely related to CPS35A and identical to de-O-acetylated CPS42. The findings provide a comprehensive view of the structural and genetic relations that exist between the members of CPS serogroup 35 and other cross-reactive serotypes. T he virulence of Streptococcus pneumoniae depends in part on the production of capsular polysaccharides (CPSs) that protect invading bacteria from phagocytosis and subsequent killing by host neutrophils (1). When administered in vaccines, these polysaccharides elicit the formation of opsonic antibodies that confer serotype-specific protective immunity. Currently available CPS-based vaccines include the 23-valent polysaccharide vaccine and the 13-valent conjugate vaccine. The widespread use of these vaccines, while highly effective in preventing invasive pneumococcal disease, may also favor the emergence of nonvaccine, replacement serotypes (2) or possible new serotypes by recombination of existing strains (3). Surveillance of both vaccine and nonvaccine pneumococcal serotypes is thus essential for monitoring and maintaining vaccine efficacy. IMPORTANCE Cross-reactions of diagnostic rabbit antisera withTwenty-four of the 92 presently recognized CPS serotypes (4-6) are distinct in the Danish system of nomenclature, while the remaining 68 are distributed among 21 serogroups that contain from two to five members each. For the most part, cross-reactions between serotypes are limited to members of the same CPS serogroup. Exceptions do, however, occur, most notably among a group of nonvaccine serotypes (7) that includes the members of CPS serogroup 35 (i.e., 35F, 35A, 35B, and 35C) and other individual types (i.e., 20, 29, 34, 42, and 47F). Examples of crossreactions between these types include group 35 serum with types 42 and 47F (8), factor 34b serum with type 35F, factor 20b serum

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

“…The two O-acetyl peaks at 2.156/21.18 and 2.096/21.03 in 1 H/ 13 C represented di-O-acetyl groups at 5-and 6-positions of residue A (3-␤-Galf). The relative heights of these peaks in the HSQC spectra suggested 100% di-O-acetylation forms in both; however, in our previously reported structure of commercial serotype 42 polysaccharide, there was one form with 54% O-acetylation at both A5 and A6, a second form with 30% acetylation at A6, and a third form without acetylation (13).…”

“…5) clearly demonstrated the distinct difference between the two polysaccharides. CPS from SP147 had two O-acetyl peaks at the same chemical shifts as those of the SP155 CPS but contained an additional peak at 2.140 ppm in 1 H/21.12 ppm in 13 C absent in SP155 (Fig. 5A).…”

“…4); thus, the unique feature of serotype 35C must arise from O-acetylation of residue C. In all resonances of this residue, the peak at 5.192 ppm in 1 H/106.70 ppm in 13 C was characteristic of an anomeric center. HSQCtotal correlation spectroscopy (TOCSY) showed cross peaks between 5.192 ppm (C1-H) and 84.40 ppm (C2-C) and 76.41 ppm (C3-C).…”

“…Pool G typing antiserum, which recognizes serotypes 35C and 42 (15), strongly stained all putative 35C and 42 isolates. Each isolate was positive for factor sera 35c and 42a, whereas binding by factor sera 35b and 29b was at or below the levels of the irrelevant serotype (12,13). The wciG genes of all isolates serologically classified as serotype 35C encoded an intact WciG (see Table 2 for accession numbers).…”

“…2A]) (11). However, biochemical studies showed that serotype 42 capsular polysaccharide (CPS) has two O-acetylations on its 3-linked ␤-galactofuranose (Galf) (12,13), while a commercial serotype 35C capsule preparation contained no O-acetylation (13). In addition, both serotypes are known to bind ficolin-2, which targets O-acetyl groups in some pneumococcal serotypes (14).…”