Sweet Immunization: Conserved glycan motifs for vaccines against streptococcal pathogens

Abstract

The emergence and spread of antimicrobial resistance warrant novel strategies to prevent and treat bacterial infections. Bacterial surface glycans, which serve as a protective layer around the bacteria and function as an important physical interface between host and pathogen, have proven to be successful vaccine targets. Capsular polysaccharide (CPS) vaccines have been very effective in reducing mortality and morbidity globally for a number of pathogens, including Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis. However, CPS is diverse in structure, even within species, and vaccination with CPS often elicits limited cross-reactive immunity. In addition to CPS, many streptococci produce rhamnose-rich polysaccharide (RPS), which is covalently anchored to the peptidoglycan mesh, abundantly expressed, and often exhibits less structural variation, making it an attractive candidate for broad-spectrum vaccines. This thesis explores the genetic, structural and immunogenic characteristics of conserved glycan motifs in RPS of streptococcal pathogens, with the aim of informing the development of broad-spectrum vaccines. Particular focus is placed on Streptococcus suis, a zoonotic pathogen that expresses over 29 CPS types and has over 2,000 multilocus sequence types, and Streptococcus uberis, a genetically diverse bovine mastitis pathogen. In Chapter 2, the biosynthesis gene cluster and glycosyl linkage patterns of S. suis RPS were characterized. S. suis from pathogenic lineages contains a 14-gene cluster contributing to RPS biosynthesis. Despite allelic variation in a glycosyltransferase leading to side-chain diversity, a conserved glycan ‘core’ structure was identified. Functional studies revealed that deletion of the intact or partial side-chain affected S. suis morphology and lysozyme resistance. Importantly, an immunization study in pigs demonstrated that antibodies raised against the conserved glycan core cross-reacted with S. suis strains from different CPS types and sequence types. These findings underscore the potential of the RPS core structure as a target for the development of broadly protective vaccine against S. suis. In contrast, a comparative genomics study of global S. uberis genomes (Chapter 4) revealed diversity in the RPS biosynthesis gene cluster, but the majority of strains possessed genes required for the rhamnan backbone biosynthesis. Importantly, the rhamnan motif was shown to be immunogenic and accessible to bovine IgG, supporting its candidacy as a universal vaccine target. RPS of Streptococcus pyogenes and Streptococcus mutans are anionic glycopolymers due to glycerol phosphate (GroP) modifications on the side-chain. This thesis confirmed the presence and established the location of the GroP modification on S. suis and S. uberis RPS using nuclear magnetic resonance analysis. In Chapter 3, SrpH was identified as the GroP transferase for S. suis RPS. Using a combination of genetic manipulation and biochemical quantification, SrpH was shown to catalyze GroP modification of both side-chain variants, further clarifying the molecular basis of RPS biosynthesis and charge modulation. Together, these findings provide bioinformatic and biochemical insights into S. suis and S. uberis RPS, demonstrating that conserved RPS motifs in these pathogens are structurally and immunologically significant. This thesis provides new insights into the development of next-generation glycoconjugate vaccines, whereby shared-glycan motifs could overcome antigenic diversity and protect against genetically diverse streptococcal pathogens.