PROJECT SUMMARY Itch is an unpleasant sensation that evokes a desire to scratch. While itch accompanies many skin infections, a causative role for microbes in itch has not been previously investigated. Itch-induced scratching can contribute to significant skin damage and bacterial pathogenesis. Here, we investigate the role of the human bacterial pathogen Staphylococcus aureus and its secreted proteases in driving itch and inflammation. S. aureus colonizes 90% of skin lesions caused by Atopic Dermatitis, a disease characterized by chronic itch. S. aureus also causes impetigo, a contagious skin disease characterized by itchy lesions. We hypothesize that S. aureus secretes proteases that can directly act on host sensory neurons to drive itch, scratch-induced skin damage, and neuroimmune crosstalk. Our preliminary data shows that S. aureus epicutaneous infection of mice induces robust itch behaviors (alloknesis, spontaneous itch) and resulting skin pathology. Using isogenic mutant strains, we find that secreted S. aureus proteases, and in particular the serine protease V8 (SspA) is required for itch production during infection. In Specific Aim 1, we will determine the role of S. aureus V8 protease in driving itch, neuronal activation, and skin inflammation. We will utilize isogenic S. aureus mutant and complemented strains for V8, as well as recombinant V8 to elucidate the necessity and sufficiency of this protease in inducing itch, scratch induced damage, and inflammation caused by S. aureus. Itch is mediated by dorsal root ganglia (DRG) sensory neurons. DRG neuron calcium imaging will be performed to investigate specific neuronal responses to V8 protease. Cheek intradermal injections and neurobehavioral analysis will be performed to distinguish itch vs. pain behaviors in mice. In Specific Aim 2, we will determine whether neurons and host cells detect S. aureus V8 protease through specific protease-activated receptors (PARs). Preliminary data indicates that PAR1 may be an important host receptor that is activated by V8. We will utilize biochemical and luminescence-based approaches to determine the V8 cleavage site on PAR1. We will treat mice with pharmacological antagonists against PAR1 and utilize PAR1-/- mice to determine effects on V8-protease and S. aureus induced itch. In Specific Aim 3, we will utilize genetic approaches to ablate specific skin-innervating neurons (Nav1.8+, Mrgprd+, and Trpv1+) to assay their roles in S. aureus induced inflammation. We hypothesize that neurons will drive both itch/scratch-induced damage, and the release of neural mediators that directly signal to immune cells. We will use targeted approaches and proteomics to assess neuronal release of proinflammatory mediators. Our work could elucidate novel molecular crosstalk between S. aureus, host neurons and immune cells in itch. The three aims of this study leverage the complementary skills of Dr. Chiu and Dr. Horswill, combining neurobiological, immunological, and mic...