Pneumococcal capsules are important in pneumococcal pathogenesis and vaccine development. Though conjugate vaccines have brought about a significant reduction in invasive pneumococcal disease (IPD) caused by vaccine serotypes, the relative serotype prevalence has shifted with dramatic emergence of serotype 24F in some countries. Herein, we describe fourteen isolates (thirteen IPD and one non-IPD) expressing a new capsule type, 24C, which resembles 24F but has a novel serological profile. We also describe the antigenic, biochemical, and genetic bases of 24F and 24C and the related serotypes 24A and 24B. Structural studies show that 24B, 24C, and 24F have identical polysaccharide backbones [β-Rib f- (1→4)-α-Rha p -(1→3)-β-Glc p NAc-(1→4)-β-Rha p -(1→4)-β-Glc p ] but with different side chains: 24F has arabinitol-phosphate, and 24B has ribitol-phosphate. 24C has a mixture of 24F and 24B repeating units, with ratio of ribitol to arabinitol being strain-dependent. In contrast, 24A capsule has a backbone without β-Rib f but with arabinitol-phosphate and phosphocholine side chains. These structures indicate that factor-sera 24d and 24e respectively recognize arabinitol and ribitol, which explains the serology of serogroup 24, including those of 24C. The structures can be genetically described by the bi-specificity of wcxG , capable of transferring arabinitol or ribitol when arabinitol is limiting. Arabinitol is likely not produced in 24B but is produced in reduced amounts in 24C due to various mutations in abpA or abpB genes. Our findings demonstrate how pneumococci modulate their capsule structure and immunologic properties with small genetic changes, thereby evading host immune responses. Our findings also suggest a potential for new capsule types within serogroup 24.
We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11A␣ and 11A. The result of our experiments is a revision to the old structure for S. pneumoniae 11A polysaccharide. The new structure differs from the old structure in both the primary connectivities and acetylation pattern. We also show that 11A contains an acetylglycerol-PO 4 moiety, a substitution that is heretofore unknown in the bacterial polysaccharide literature. The two clinical isolates were also structurally characterized. 11A␣ was determined to be identical to 11A. 11A is a new serotype, which differs from 11A in the absence of the acetylation of the glycerol-PO 4 moiety and a different acetylation pattern of the saccharides. Thus, we propose that the acetylglycerol is the structural basis for 11A␣ and 11A subtypes. The polysaccharide (PS)2 capsule of Streptococcus pneumoniae is recognized as the most important virulence factor of pneumococci. It is expressed by almost all pathogenic pneumococci and has been shown to increase the virulence of a pneumococcal strain by more than a million-fold in an animal model system (1). The capsule shields pneumococci from the host phagocytes, and its shielding capacity can be neutralized when the host produces antibodies to the capsule and to fix complement on pneumococci. Thus, the capsular PS is used as the antigen in all pneumococcal vaccines that are clinically used. Because of their impact on human health, pneumococcal capsules have been extensively investigated both serologically, genetically, and biochemically. Many years of serological studies have identified 91 serologically distinct capsule types (2, 3). Recently, the nucleotide sequences of the capsule gene loci of all 91 different capsule types have been determined (4, 5).With the use of monoclonal antibodies, serologic heterogeneity was noted recently among pneumococcal isolates that were typed as 11A using the quellung reaction (6). To avoid confusion, we have provisionally named the common variant 11A␣ and the less common variant 11A. Because 11A PS is a component of the 23-valent pneumococcal PS vaccine (7), it is important to understand the chemical basis of the serological heterogeneity among pneumococci that are typed as serotype 11A. However, there is only limited information on structure of pneumococcal serotype 11A PS, and the structure has not been examined with modern tools. Forty years ago, the structural model for S. pneumoniae serotype 11A PS was proposed to be a linear polymer containing D-glucose, D-galactose, glycerol, phosphate, and O-acetyl groups in the molar ratio of 2:2:1:1:2 based upon analysis of chemically modified PS (8). About 20 years ago, Richards et al. (9,10) proposed the model for 11A PS based on both NMR and chemical degradation studies. They proposed a linear repeating unit structure with four monosaccharides, a pendant glycerol phosphate moiety, and 2 mol of acetate as described in Structure ...
In follow-up work to this paper, it was found that the identification of an acetylglycerol in the polysaccharide repeating unit is incorrect. The proper placement of the O-acetate group is on -gal C6. The incorrect assignment occurred due to incorrect interpretation of NMR data, which was discovered while working on other polysaccharides that are structurally related to serotype 11A (Calix, J. J., Nahm, M. H., and Zartler, E. R. (2011) J. Bacteriol. 193,[5271][5272][5273][5274][5275][5276][5277][5278]. This correction does not impact the assignment of the other acetyl groups. Also, the correction does not change any genetic and serologic findings described in the paper. The first author, Edward R. Zartler, takes full responsibility for this matter and apologizes for any inconvenience caused. A detailed description of this correction is given in Calix, J. J., Nahm, M. H., and Zartler, E. R. (2011) ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.
The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.
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