Investigation of the essential role of capsular polysaccharide in serotype 1 Streptococcus pneumoniae

Streptococcus pneumoniae remains a major global cause of morbidity and mortality, responsible for an estimated half million deaths annually despite widespread implementation of pneumococcal conjugate vaccines. Notably, invasive pneumococcal disease (IPD) caused by serotype 1 persists and disproportionately affects vulnerable populations in low-resource settings, despite inclusion of this serotype in available vaccines. Serotype 1 exhibits atypical biology characterized by short nasopharyngeal carriage and high invasive potential. This phenotype has been linked to capsule-mediated evasion of host immunity, including resistance to phagocytosis, complement deposition, opsonophagocytic killing, mucus entrapment, and neutrophil extracellular traps. The zwitterionic serotype 1 capsular polysaccharide is proposed to enhance traversal of the mucus layer and access to deeper tissues such as olfactory epithelium and brain. The genes required for capsule biosynthesis are located within the dexB-aliA cps locus; however, serotype 1 additionally requires genes outside this locus for AATGal biosynthesis and transfer to undecaprenyl phosphate (UndP). To elucidate the role of the serotype 1 capsule in pathogenicity, we performed genetic analysis and mutagenesis of cps-linked genes. Genes involved in capsule biosynthesis and assembly appear to be essential for viability because they could not be disrupted despite multiple attempts, unlike other genes on the capsule loci but not involved in capsule biosynthesis such as rlmA. To overcome the inability of constructing defined cps mutants, we used a serotype 2 capsule switch (D39::cps1) and an E. coli strain expressing the cps1 locus and generated defined capsule mutants (ΔwchB, ΔwchD, Δgla, Δugd). We used these mutants to quantify capsule thickness by FITC-dextran labeling and electron microscopy, measured complement deposition and antibody binding, and evaluated virulence in the Zebrafish infection model relative to the parental serotype 1 strains 519/43 and D39WT. Here, we report that although the D39::cps1 strain expresses a serotype 1 capsule and retains key associated properties-including increased capsule thickness and reduced antibody binding and complement deposition-its full virulence is not recapitulated. Instead, the capsule-switched strain exhibited significantly attenuated virulence compared with the parental serotype 1 strains suggesting that virulence is not entirely dependent on the capsule.