Structural, biosynthetic and serological interaction studies of the capsular polysaccharides from Streptococcus pneumoniae

Abstract

The rapid evolution of Streptococcus pneumoniae in response to clinical interventions, such as treatment with antibiotics and vaccination, has resulted in the change of prevalence in the carriage and invasive serotypes as well as frequent emergence of multidrug-resistant strains and novel serotypes. Thus, there are increasing needs in developing better diagnostic methods for the surveillance of Streptococcus pneumoniae and for gaining knowledge of non vaccine serotypes for the development of future vaccines. The capsular polysaccharides (CPS) are crucial virulence factors and the important antigen of Streptococcus pneumoniae. The structural elucidation of unknown pneumococcal CPS can provide insights into the function of CPS biosynthetic genes and enzymes, which is a significant addition to diagnosis based on gene sequencing methods. Moreover, the diagnostic antisera for pneumococcus are relying on serological reactivity of pneumococcal capsule, which also depends on CPS structures. Therefore, structure elucidation of pneumococcal CPS is fundamental for the development of better diagnostic methods and future pneumococcal vaccines. In this project, six previously unknown CPS structures of the pneumococcal serogroups 16, 24 and 28 (serotype 16F, 16A, 24A, 24B, 28F and 28A) and the newly discovered serotype 24X from serogroup 24 were thoroughly characterized by nuclear magnetic resonance (NMR) spectroscopy and other analytical methods. A further CPS biosynthetic analysis to correlate CPS structures with biosynthetic enzymes revealed the presumable function of glycosyltransferases (GTs), acetyltransferases, phosphotransferases and polymerases. The functions of GT WcxN and WcxT from serogroup 16 were proposed for the first time. The proposed function of GT WcxN is in agreement with determined CPS structures of serotype 28F and 28A, which also possess GT WcxT. Moreover, the GT WciU in serotypes 28F and 28A showed different sugar donor specificity toward α-D-Glcp and α-D-GlcNAcp, respectively. The critical residue position that could be responsible for the substrate specificity of WciU was predicted through bioinformatics tools and protein 3D structure homology modeling. In the serological study, the classic Quellung reaction and latex agglutination were applied to test the cross-reactions of related serotypes and the serological activities of partially degraded CPS. In addition, interactions between CPS and diagnostic antisera were studied by asymmetric flow field-flow fractionation coupled with multiangle light scattering, differential refractive index and ultraviolet detectors (AF4-MALS-dRI-UV), NMR titration and diffusion ordered spectroscopy (DOSY), which provides alternative methods with potential to be used for the evaluation of diagnostic antisera and for studying CPS-antibody interactions.