SSPA C-TERMINAL DOMAIN CONTRIBUTES TO STAPHYLOCOCCUS AUREUS PATHOGENESIS VIA ECM TETHERING - PROJECT SUMMARY/ABSTRACT STAPHYLOCOCCUS AUREUS, INCLUDING METHICILLIN-RESISTANT S. AUREUS (MRSA), IS A LEADING CAUSE OF SKIN AND SOFT- TISSUE INFECTIONS IN THE UNITED STATES AND IS KNOWN TO EXACERBATE ATOPIC DERMATITIS PATHOLOGY. A DEBILITATING SYMPTOM OF ATOPIC DERMATITIS IS A SEVERE, PERSISTENT ITCH THAT PERPETUATES A DESTRUCTIVE ITCH-SCRATCH CYCLE, EXACERBATING SKIN BARRIER DAMAGE AND WORSENING PATIENTS' QUALITY OF LIFE. RECENT WORK HAS IDENTIFIED THE S. AUREUS PROTEASE SSPA AS A DIRECT DRIVER OF ITCH BY ACTIVATING PROTEASE-ACTIVATED RECEPTOR 1 (PAR1) ON SENSORY NEURONS. HOWEVER, THE SPECIFIC MECHANISMS ENABLING THE POTENT AND EFFICIENT ACTIVITY OF SSPA ON THESE NEURONS REMAINS A CRITICAL KNOWLEDGE GAP. PRELIMINARY DATA SUGGESTS THAT SSPA HAS A UNIQUE AND CONSERVED C-TERMINAL DOMAIN (CTD) OF UNKNOWN FUNCTION THAT IS DISPENSABLE FOR CATALYSIS BUT IS REQUIRED FOR ITCH INDUCTION IN VIVO. ADDITIONALLY, THE CTD FACILITATES HIGH- AFFINITY BINDING OF SSPA TO TYPE I COLLAGEN, THE PRIMARY COMPONENT OF THE DERMAL EXTRACELLULAR MATRIX (ECM). THE CENTRAL HYPOTHESIS OF THIS PROJECT IS THAT THE SSPA CTD FUNCTIONS AS A NOVEL MOLECULAR TETHER, CONCENTRATING THE PROTEASE IN THE ECM NEAR PAR1-EXPRESSING SENSORY NEURONS, THEREBY ENABLING EFFICIENT NEURONAL ACTIVATION TO DRIVE ITCH. THE LONG-TERM OBJECTIVE IS TO DEFINE THIS NEW PARADIGM OF VIRULENCE FACTOR TARGETING AND ESTABLISH A FOUNDATION FOR NOVEL ANTI-VIRULENCE THERAPIES THAT DISARM THE PATHOGEN WITHOUT PROMOTING RESISTANCE. THIS PROJECT TESTS THE CENTRAL HYPOTHESIS THROUGH TWO COMPLEMENTARY AIMS. AIM 1 WILL DEFINE THE BIOCHEMICAL BASIS OF THE CTD-ECM INTERACTION USING SURFACE PLASMON RESONANCE TO QUANTIFY BINDING KINETICS AND WILL ASSESS THE ELECTROSTATIC NATURE OF THE INTERACTION WITH PHYSIOLOGICALLY RELEVANT COMPETITIVE INHIBITION ASSAYS. THIS AIM CULMINATES IN THE USE OF IMMUNOFLUORESCENCE MICROSCOPY TO CONFIRM THAT THE CTD MEDIATES SSPA LOCALIZATION TO THE ECM-RICH DERMAL-EPIDERMAL JUNCTION DURING MURINE SKIN INFECTION. AIM 2 WILL ELUCIDATE HOW THIS LOCALIZATION DRIVES NEUROGENIC ITCH BY USING LIVE-IMAGING OF SKIN EXPLANTS TO VISUALIZE SSPA ACCUMULATION ON NEURONS, QUANTIFYING PAR1 ACTIVATION IN PRIMARY SENSORY NEURONS VIA CALCIUM IMAGING, AND UTILIZING A CHIMERIC PROTEASE TO TEST THE SUFFICIENCY OF THE CTD TO CONFER ITCH-INDUCING ACTIVITY TO A HOMOLOGOUS PROTEASE IN VIVO. SUCCESSFUL COMPLETION OF THIS PROJECT WILL PROVIDE FUNDAMENTAL INSIGHTS INTO THIS CLINICALLY RELEVANT HOST-PATHOGEN INTERACTION AND WILL BE THE FIRST DESCRIBED FUNCTION OF THE SSPA CTD. BY IDENTIFYING THE CTD-ECM INTERACTION AS A CRITICAL AND DRUGGABLE POINT IN PATHOGENESIS, THIS WORK WILL PROVIDE A DIRECT TRANSLATIONAL PATH TOWARD DEVELOPING THERAPIES THAT CAN ALLEVIATE A DEBILITATING SYMPTOM OF S. AUREUS COLONIZATION AND INFECTION AND DISRUPT THE ITCH-SCRATCH CYCLE, DIRECTLY ADDRESSING THE MISSION OF NIAID.