Project Grant F31AI202940

INVESTIGATING THE REGULATION OF PEPTIDOGLYCAN AMIDASE ACTIVATOR NLPD IN ESCHERICHIA COLI - PROJECT SUMMARY AND ABSTRACT BACTERIAL CELL DIVISION IS AN ESSENTIAL PROCESS THAT REQUIRES THE CAREFUL REGULATION AND COORDINATION OF THE ACTIVITY OF MANY PROTEIN COMPLEXES. IN GRAM-NEGATIVE BACTERIA, LIKE ESCHERICHIA COLI, DIVISION REQUIRES THE MULTILAYERED CELL ENVELOPE, WHICH IS COMPRISED OF A THIN PEPTIDOGLYCAN (PG) CELL WALL HELD BETWEEN THE INNER AND OUTER MEMBRANE, TO BE COORDINATELY CONSTRICTED. DISRUPTED COORDINATION OR MISREGULATION AT ANY POINT IN CELL DIVISION HAS THE POTENTIAL TO CAUSE CELL LYSIS. THUS, IT IS NOT SURPRISING THAT THIS PATHWAY IS TARGETED BY MANY OF OUR MOST EFFECTIVE ANTIBIOTICS. WORK IN THE FIELD HAS DEMONSTRATED THAT AN ESSENTIAL MULTI-PROTEIN COMPLEX CALLED THE DIVISOME PROMOTES THE SYNTHESIS OF SEPTAL PG (SPG) AT MIDCELL TO BUILD THE NEW POLES OF THE DAUGHTER CELLS. A MAJOR GAP IN THE FIELD IS UNDERSTANDING HOW THE CELL REGULATES AND COORDINATES THE ACTIVITY OF THE PROTEINS THAT CLEAVE THE SHARED SPG TO ENABLE ENVELOPE CONSTRICTION AND SUCCESSFUL DAUGHTER CELL SEPARATION. IN E. COLI, THE CELL DIVISION AMIDASES SURGICALLY CLEAVE SPG WITHOUT DAMAGING THE DEVELOPING POLES. TO PREVENT ABERRANT CLEAVAGE, THE AMIDASES ARE AUTOINHIBITED AND REQUIRE DEDICATED ACTIVATORS, ENVC AND NLPD, TO ENABLE SPG HYDROLYSIS. THE REGULATORY MECHANISMS THAT CONTROL AMIDASE ACTIVATION BY ENVC AND THE PATHWAYS THAT COUPLE IT WITH CELL DIVISION HAVE BEEN WELL CHARACTERIZED. BY CONTRAST, THE NLPD REGULATORY PATHWAY REMAINS POORLY UNDERSTOOD. THIS PROPOSAL INVESTIGATES THE CONTROL MECHANISMS THAT FACILITATE PROPER NLPD ACTIVITY TO ENSURE THE COORDINATED SPG HYDROLYSIS REQUIRED FOR CELL DIVISION. IN MY PRELIMINARY STUDIES, I HAVE IDENTIFIED FTSN, AN ESSENTIAL CELL DIVISION PROTEIN REQUIRED FOR SPG SYNTHESIS, AS A POTENTIAL NEGATIVE REGULATOR OF NLPD. OVEREXPRESSION OF FTSN FULLY SUPPRESSES THE LETHAL PHENOTYPE OF MISREGULATED VARIANTS OF NLPD. ALPHAFOLD MODELING PREDICTS A HIGH CONFIDENCE INTERACTION BETWEEN THE NLPD AND FTSN THAT WOULD PREVENT AMIDASE ACTIVATION. IMPORTANTLY, ADDITION OF THE PG-BINDING SPOR DOMAIN OF FTSN INHIBITS AMIDASE ACTIVATION BY NLPD IN PG CLEAVAGE ASSAYS. IN AIM 1, I WILL TEST THE IMPORTANCE OF THIS PREDICTED INTERACTION IN CONTROLLING SPG HYDROLYSIS DURING CELL DIVISION. SEPARATELY, I WILL TAKE A MULTIPRONGED APPROACH IN AIM 2 TO INVESTIGATE AND IDENTIFY THE REGULATORY MECHANISMS THAT CONTROL NLPD ACTIVITY OUTSIDE OF THE DIVISION SITE. THE RESULTS FROM THIS PROPOSAL WILL ADDRESS IMPORTANT AND OUTSTANDING QUESTIONS IN THE REGULATION AND COORDINATION OF ACTIVITIES IMPORTANT FOR BACTERIAL CELL DIVISION. MY FINDINGS HAVE THE POTENTIAL TO IDENTIFY NEW VULNERABILITIES IN THIS ESSENTIAL PROCESS TO ENABLE THE DEVELOPMENT OF NOVEL ANTIBIOTICS. IN ADDITION TO THE POTENTIAL TO IMPACT PUBLIC HEALTH, THE PROPOSED RESEARCH AND TRAINING PLAN WILL FACILITATE MY PROFESSIONAL DEVELOPMENT AND SUPPORT MY CAREER GOAL OF BECOMING A SUCCESSFUL INDEPENDENT INVESTIGATOR. I WILL BE SUPPORTED BY DR. THOMAS BERNHARDT, A DEDICATED MENTOR, AND THE RESOURCES OF HARVARD MEDICAL SCHOOL. COMPLETION OF THIS TRAINING PLAN WILL EXPAND MY TECHNICAL SKILLS, CRITICAL THINKING, COMMUNICATION, AND ABILITY TO MENTOR OTHERS WHICH WILL ENABLE ME TO LEAD RIGOROUS BIOMEDICAL RESEARCH.