Group A streptococcus (GAS, Streptococcus pyogenes) is a leading bacterial pathogen of the human pharynx and skin. In recent years, a striking resurgence in severe invasive GAS infections has been observed worldwide. GAS infections account for more than 650,000 cases of severe invasive disease annually. The invasive GAS infections, necrotizing fasciitis, cellulitis and erysipelas with concomitant scarlet fever and streptococcal toxic syndrome, are difficult to treat with antibiotics, and a GAS vaccine is urgently needed to combat this neglected disease. A major component of the GAS cell wall is the Group A Carbohydrate (GAC) covalently linked to peptidoglycan, consisting of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side-chains. GAC is an attractive vaccine candidate due to its conserved expression in all GAS serotypes and the absence of its constitutive component, rhamnose, in humans. Our genetic, biochemical and structural studies identified two novel modifications of GAC glycans: glycerol phosphate modification of GAC and de-N-acetylation of the GAC linkage unit. The goal of this proposal is to characterize the mechanisms of GAC biosynthesis and modification, and elucidate the roles of cell wall modifications in antimicrobial resistance mechanisms and GAS pathogenesis. To help answer these questions we will employ a variety of genetic, biochemical, analytical and structural approaches. The function of cell wall modifications in GAS pathogenesis will be studied in ex vivo and in vivo models of GAS infection. The proposed studies provide a platform for design of a safe and effective vaccine against this important human pathogen and should have broad application to other streptococci which express similar cell wall polysaccharides. Since the enzymes of the GAC biosynthesis pathway are attractive drug targets, the proposed studies will have important implications for drug design.