DETERMINING THE ROLE OF S. AUREUS YJBH CLINICAL MUTATIONS IN QUORUM SENSING AND INFECTION - PROJECT SUMMARY/ABSTRACT STAPHYLOCOCCUS AUREUS IS A GRAM-POSITIVE BACTERIUM THAT CAUSES A RANGE OF DISEASES FROM MILD TO SEVERE, AND IT HAS BEEN ESTIMATED THAT OVER 1 MILLION DEATHS WORLDWIDE IN 2019 CAN BE ATTRIBUTED TO THIS BACTERIUM. ITS ABILITY TO CAUSE THESE INFECTIONS RELIES ON NUMEROUS VIRULENCE FACTORS, INCLUDING TOXINS, PROTEASES, ADHESINS, IMMUNE- EVASIVE PROTEINS, AND MORE THAT HELP IN INFECTION. THESE VIRULENCE FACTORS ARE TIGHTLY CONTROLLED, ENABLING THE BACTERIA TO ADJUST THEIR RESPONSE FOR SUCCESSFUL COLONIZATION AND PERSISTENCE. WHILE STRIDES ARE BEING MADE TO UNDERSTAND HOW VIRULENCE FACTOR REGULATION AFFECTS PATHOGENESIS AND ANTIBIOTIC RESISTANCE, THE INDIVIDUAL CONTRIBUTION OF EACH OF THESE FACTORS IS LARGELY UNKNOWN. ONE REGULATORY NETWORK IN GRAM-POSITIVE BACTERIA THAT CONTRIBUTES TO GENE EXPRESSION IS YJBH AND SPX, WHICH HAVE BEEN LINKED TO STRESS RESPONSES AND VIRULENCE IN DIVERSE BACTERIAL SPECIES. HOWEVER, FEW STUDIES HAVE IDENTIFIED HOW THIS SYSTEM IMPACTS VIRULENCE IN S. AUREUS. IN ADDITION, CLINICAL ISOLATES HAVE BEEN FOUND WITH MUTATIONS IN YJBH, AND HOW THIS IMPACTS FUNCTION AND DISEASE OUTCOMES IS UNKNOWN. THIS APPLICATION USES TWO INDEPENDENT, YET COMPLEMENTARY, SPECIFIC AIMS TO ADVANCE OUR UNDERSTANDING OF HOW YJBH IMPACTS THE PHYSIOLOGY OF S. AUREUS AND ITS CLINICAL IMPACT ON PATHOGENESIS AND ANTIBIOTIC RESISTANCE. FURTHERMORE, IT WILL CLARIFY THE ROLE OF YJBH IN THE CELL SINCE OUR PRELIMINARY DATA AND SEVERAL STUDIES IN OTHER BACTERIA POINT TO SPX-INDEPENDENT ROLES FOR YJBH, THOUGH THIS HAS NOT BEEN CAREFULLY DEFINED TO DATE. AIM 1 WILL UTILIZE A COMBINATION OF GENETIC MANIPULATION, RT-QPCR, QUANTITATIVE HEMOLYSIS, AND PROTEOMICS TO DETERMINE HOW YJBH AFFECTS AGR ACTIVITY. AIM 2 WILL BE A COMBINATION OF BOTH IN VITRO AND IN VIVO EXPERIMENTS ASSESSING THE MUTATIONS IN YJBH FOUND CLINICALLY AND THEIR RELEVANCE TO PATHOGENESIS AND VIRULENCE FACTOR REGULATIONS. THIS WILL BE ACCOMPLISHED WITH GENETIC MANIPULATION, QUANTITATIVE HEMOLYSIS, AND MURINE MODELS OF CHRONIC LUNG INFECTION AND ACUTE BACTEREMIA. TO ACCOMPLISH THESE GOALS, A SPONSOR AND CO-SPONSOR WITH COMPLEMENTARY EXPERTISE IN BACTERIAL GENETICS AND INFECTION MODELS WILL LEAD TO ROBUST TRAINING FOR THE TRAINEE IN A COLLABORATIVE RESEARCH ENVIRONMENT.