PROJECT SUMMARY The host range, or tropism, of pathogen virulence factors is a key determinant of infection. A detailed understanding of host and pathogen mutations that control species tropism is required in order to assess the risk of future zoonotic disease outbreaks, improve animal infection models, and design new therapeutics that take advantage of host specificity. The objective of this proposal is to define barriers to cross-species activity in bacterial virulence factors at high resolution, leveraging staphylococcal exfoliative toxins as a study system. Exfoliative toxins encoded by pathogenic bacteria in the genus Staphylococcus cause life- threatening skin diseases including staphylococcal scalded skin syndrome and bullous impetigo characterized by widespread blistering and damage to the epidermis. Exfoliative toxins are proteases that act by selectively cleaving the skin desmosomal cadherin desmoglein-1, leading to loss of epidermal barrier function and blister formation. Our central hypothesis is that virulence factor activity is dependent on genetic compatibilities between hosts and pathogens, and that interrogating these compatibilities will uncover specific barriers to host tropism. In preliminary work, we found that genes encoding desmoglein-1 have undergone rapid evolution and repeated natural selection across non-human primates within a small protein surface sufficient to restrict toxin activity. We have also developed tractable in vitro assays to measure toxin cleavage of recombinant desmglein-1 from various host species. The Specific Aims of this proposal are to 1) generate a high-resolution map of mutations in desmoglein-1 that restrict toxin tropism, and 2) test how toxin mutations at the desmoglein-1 binding interface contribute to host tropism. In Aim 1 we will perform combinatorial mutagenesis of the toxin recognition surface in desmoglein-1 to produce a genetic map defining the barriers of host recognition. In Aim 2 we will apply structural and biochemical approaches to resolve the desmoglein- exfoliative toxin binding interface combined with genetics to assess how toxin mutations at this surface control host tropism. Collectively this proposal will establish a framework to define genetic barriers to bacterial infections at high-resolution, applied towards the goal of anticipating and preventing future disease outbreaks.