Group A Streptococcus (S. pyogenes or GAS) is responsible for millions of cases of pharyngitis and skin infections each year in the United States with attendant morbidity and absenteeism from school and work. The organism also causes approximately 10,000 cases annually of invasive infections in the U.S., including bacteremia, necrotizing fasciitis, and streptococcal toxic shock. Globally, GAS infections and complications of the postinfectious syndrome of acute rheumatic fever account for an estimated 500,000 deaths per year. No vaccine is yet available, and treatment failures, relapses, and recurrences of infection remain common. With rare exceptions, clinical isolates of GAS produce the pore-forming hemolytic toxin streptolysin O (SLO). SLO is a member of the cholesterol-dependent cytolysins, a family of secreted proteins produced by many Gram- positive bacteria that share the property of binding to cholesterol-containing membranes where they oligomerize and insert to form large pores. Experimental infection studies have demonstrated a modest contribution of SLO to GAS virulence; however, animal models are not necessarily sensitive to factors that influence colonization in the human host. Colonization of the human oropharynx is central to GAS infection, and our recent results implicate SLO in this process through its ability to enhance GAS intracellular survival in oropharyngeal keratinocytes. SLO-mediated pore-formation and the capacity of SLO to deliver the co-toxin NAD-glycohydrolase to the cytosol of infected cells result in prolonged GAS survival. If these toxins are critical contributors to GAS adaptation for survival in the human host, they are important potential targets for pharmacologic intervention and vaccine development. Accordingly, Aim 1 of the project is to investigate how SLO and NADase promote resistance to killing by oropharyngeal epithelial cells as a potential mechanism for GAS persistence in the pharynx. We will determine how NADase increases expression or stability of SLO at both the transcriptional level and through protein-protein interactions. We will use anthrax toxin-based delivery of NADase as a novel means to dissociate the cellular effects of NADase from those of SLO, which is normally required for NADase translocation into host cells. This model system will permit direct study of how NADase subverts endosomal and autophagic maturation and promotes GAS survival in infected keratinocytes. Aim 2 will pursue the association of NADase production with the emergence of invasive GAS disease over the past 3 decades. If NADase-producing GAS can survive in macrophages, these cells may serve as a Trojan horse to initiate or extend invasive infection. To investigate this hypothesis, we will determine whether SLO and NADase mediate GAS resistance to killing by human macrophages, how NADase is delivered to these cells, and how SLO and NADase modify intracellular trafficking of GAS in macrophages. Understanding how SLO and NADase enhance GAS colonization of the human host and promote invasive infection will inform novel strategies to treat and prevent GAS infection.