Project Summary/Abstract. Group A streptococci (GAS; Streptococcus pyogenes) are remarkable for the wide range of diseases they cause in humans, their sole biological host. Yet, most infections are mild and involve one of two tissues - the epithelial surface of the throat or skin - giving rise to pharyngitis or impetigo, respectively. A long-term goal is to better understand the distinct pathogenic mechanisms leading to pharyngitis and impetigo. A primary focus of the proposal is the regulation of pili expression in GAS. Pilus-associated proteins mediate adherence to epithelial cells and enhance superficial infection at the skin. Pili correspond to the T-antigens of GAS. All strains examined have pilus genes, however, many natural GAS isolates lack T-antigen. The hypothesis to be tested in Aim 1 states that organisms recovered from a carrier state and/or invasive disease are significantly more likely to have defects in pilus production, as compared to isolates derived from cases of pharyngitis or impetigo. Aim 1 seeks to define the relationship between defects in pilus expression and disease. The nra/rofA locus encodes a [unreadable]stand alone[unreadable] response regulator that affects the transcription of pilus genes; nra and rofA denote discrete lineages of alleles. Both Nra and RofA can have positive or negative regulatory effects on pilus gene transcription, depending on the GAS isolate or strain. The hypothesis to be tested in Aim 2 states that there are strain-specific differences among modulators of pilus gene expression that lie in a pathway upstream of Nra/RofA. Aim 2 seeks to identify regulators of pilus gene transcription having a differential presence among strains. The distribution of Nra and RofA among GAS is strongly correlated with subpopulations of strains having a tendency to cause infection at either the throat or skin. Nra and RofA are global regulators of GAS gene transcription. Two hypotheses will be addressed in Aim 3: (i), that co-regulated non-pilus genes act in concert with pili to cause disease; and (ii), that Nra and RofA confer differential transcription of downstream genes. Aim 3 seeks to identify genes of the Nra and RofA regulons, and to test their role in virulence. Through a better understanding of the molecular mechanisms used by GAS to persist in their primary ecological niches - the throat and skin of the human host - will come new knowledge on how best to interfere with these vital processes. Effective control and prevention measures that disrupt the chain of transmission of GAS will result in a decreased burden of the more severe GAS diseases (toxic shock syndrome, rheumatic heart disease) which have a high morbidity and mortality for many people throughout the world.