UC San Diego

Group A Streptococcus (GAS, Streptococcus pyogenes) is one of the most important human bacterial pathogens, causing hundreds of millions of infections around the globe annual in a wide spectrum of disease presentations. In this dissertation work, I will examine 2 different molecules of GAS: the group A carbohydrate (GAC) and Cas9 nuclease. Both molecules were initially discovered a century ago, but regained popularity and interest in recent years with re-examination of potential roles in pathogenesis and virulence.GAC has since been explored beyond its structural function in the streptococcal cell, with putative roles in evasion of host immune factors and development of lasting host immunity. The highly conserved and essential nature of the GAC posits the molecule as a candidate as an universal GAS vaccine antigen. However, due to the N-acetylglucosamine (GlcNAc) sidechain as the hypothesized driver of post-streptococcal autoimmune sequelae, modification of the GAC to remove the autoreactive epitope is necessary to make a safe antigen (GACpr). To develop our safe but immunogenic GAC-conjugate, we collaborated with Vaxcyte Inc. to use cell-free protein synthesis technology to incorporate non-native amino acids (nnAA) as site-specific attachment sites for the polysaccharide. This technology allows for the generation of precise, clean glyco-conjugates with GACpr covalently linked to streptococcal virulence factor SpyAD. Finally, we test our SpyAD-GACpr conjugate for efficacy in a multi-valent vaccine in primary neutrophil opsonophagocytic assay and murine infection challenges. Cas9, now famous for the precise gene-editing technology, was originally discovered in the GAS type IIA CRISPR system. Notably, this CRISPR system was frequently found in bacterial pathogens and evaluated for potential roles in virulence regulation and control. I examine the role of Cas9 with regards to GAS pathogenesis. Using proteomics, we identify key protein regulators of GAS virulence networks significantly reduced with the absence of endogenous Cas9. Follow-up studies confirm Cas9-deficient GAS has reduction of specific virulence factors and significantly reduced virulence in whole human blood and mouse skin infection model, providing strong evidence that Cas9 plays a regulatory role in virulence, though the precise molecular mechanism is unknown.