Project Grant F31AI203017

IDENTIFICATION OF A POTENTIAL TERMINATOR OF CELL WALL SYNTHESIS IN ACINETOBACTER BAUMANNII - PROJECT SUMMARY/ABSTRACT BACTERIAL CELLS SYNTHESIZE AN ESSENTIAL STRUCTURE CALLED THE CELL WALL THAT PROVIDES THE CELL WITH ITS SHAPE AS WELL AS RESISTANCE TO INTERNAL TURGOR PRESSURE. THE WALL IS COMPOSED OF A POLYMER CALLED PEPTIDOGLYCAN (PG), WHICH IS ASSEMBLED AT THE OUTER SURFACE OF THE CYTOPLASMIC MEMBRANE BY PG SYNTHESIS MACHINERY THAT POLYMERIZES AND CROSS-LINKS A LIPID-LINKED MOLECULE CALLED LIPID II. MANY OF OUR MOST EFFECTIVE ANTIBIOTICS TARGET CELL WALL SYNTHESIS. THEREFORE, STUDYING THE REGULATORY MECHANISMS GOVERNING CELL WALL SYNTHESIS HAS THE POTENTIAL TO IDENTIFY NEW TARGETS FOR DEVELOPING ANTIBIOTICS AGAINST MULTIDRUG-RESISTANT PATHOGENS. ALTHOUGH MUCH IS KNOWN ABOUT THE MECHANISMS THAT DRIVE CELL WALL SYNTHESIS, MUCH LESS IS UNDERSTOOD ABOUT HOW THIS PROCESS IS TERMINATED. TERMINATION IS CRUCIAL BECAUSE IT ALLOWS NEWLY MADE PG TO BE FULLY INCORPORATED INTO THE MATURE WALL. IT ALSO SUPPORTS THE RECYCLING OF THE LIPID CARRIER MOIETY OF LIPID II. OUR PRELIMINARY DATA SUGGEST THAT PROTEINS CONTAINING A DOMAIN OF UNKNOWN FUNCTION (DUF218), WHICH IS PREDICTED TO HAVE ENZYMATIC ACTIVITY, ARE INVOLVED IN TERMINATING CELL WALL SYNTHESIS. ELDM, A MEMBER OF THIS FAMILY IN THE GRAM- NEGATIVE, MULTIDRUG-RESISTANT PATHOGEN ACINETOBACTER BAUMANNII, INFLUENCES CELL SHAPE AND BETA-LACTAM RESISTANCE, FURTHER IMPLICATING THESE PROTEINS IN THE REGULATION OF CELL WALL SYNTHESIS. BASED ON OUR INITIAL FINDINGS, WE HYPOTHESIZE THAT ELDM INTERACTS WITH THE PG SYNTHESIS MACHINERY TO CLEAVE PG POLYMERS AND THEREBY TERMINATE CELL WALL SYNTHESIS. WE WILL TEST THIS HYPOTHESIS THROUGH TWO SPECIFIC AIMS. IN AIM 1, WE WILL ASSESS WHETHER ELDM AND A PREDICTED FUNCTIONAL ORTHOLOG IN ESCHERICHIA COLI, SANA, CAN CLEAVE LIPID II IN VITRO, TO DETERMINE IF DUF218-CONTAINING PROTEINS HAVE ENZYMATIC ACTIVITY THAT COULD TERMINATE CELL WALL SYNTHESIS. ADDITIONALLY, WE WILL EXAMINE WHETHER THE LOSS OF ELDM AND SANA RESULTS IN AN INCREASE IN PG LINKED TO THE INNER MEMBRANE, INDICATING A DEFECT IN RELEASING THE PG POLYMER FROM ITS MEMBRANE-ANCHOR. AS PRELIMINARY DATA LINKS ELDM TO THE ACTIVITY OF A PG SYNTHESIS ENZYME CALLED PBP1A, IN AIM 2, WE WILL EXPLORE THE NATURE OF THE ELDM-PBP1A INTERACTION. WE WILL TEST IF ELDM AND PBP1A INTERACT INSIDE CELLS USING A PULL-DOWN EXPERIMENT AND EXAMINE THE LOCALIZATION OF ELDM AND PBP1A IN CELLS WITH FLUORESCENCE MICROSCOPY. FINALLY, WE WILL CONDUCT GENETIC EXPERIMENTS TO DETERMINE HOW THE INTERACTION BETWEEN ELDM AND PBP1A IMPACTS ANTIBIOTIC RESISTANCE. OVERALL, THE INSIGHTS GAINED FROM THIS PROJECT WILL SHED LIGHT ON HOW CELL WALL SYNTHESIS IS REGULATED AND REVEAL A NEW MECHANISM OF ANTIBIOTIC RESISTANCE IN A SIGNIFICANT ANTIBIOTIC- RESISTANT PATHOGEN.