Streptococcus pyogenes is a ubiquitous and versatile human pathogen. Infections due to this organism range from the relatively mild and common pharyngitis and impetigo to the acute and life-threatening necrotizing fasciitis and toxic shock. The wide spectrum of streptococcal disease results in part from the production of the many and varied extracellular toxins and enzymes. The focus of this grant is a recently described streptococcal toxin we term Spy A, for S. pyogenes ADP-ribosylating toxin. SpyA transfers the ADP-ribose portion of NAD onto the cytoskeletal proteins vimentin, actin, and tropomyosin. While actin is modified by other ADP-ribosyltransferases, vimentin and tropomyosin are not common targets. Actin and vimentin filaments are needed for cellular functions requiring motility, such as cell migration during wound healing and phagocytosis. SpyA is a member of the C3 family of exotoxins, and similar to each member of this family, does not have an obvious receptor-binding domain, or associated binding domain as found with binary toxins. We proposed that SpyA gains entrance into the eukaryotic cell using a pore produced by the cholesterol dependent cytolysin SLO. The goals of this grant are (1) to determine the role of SpyA in streptococcal infections, (2) to characterize its novel entry mechanism and (3) to characterize the structure and biochemical activity of this toxin.