Project Grant F32AI191529

DEFINING THE UNIQUE DIVISOME OF CLOSTRIDIOIDES DIFFICILE - PROJECT SUMMARY/ABSTRACT THE ESSENTIAL PROCESS OF BACTERIAL CELL DIVISION IS MEDIATED BY A MULTIPROTEIN COMPLEX CALLED THE "DIVISOME." THE DIVISOME DIRECTS PEPTIDOGLYCAN SYNTHASES TO MAKE THE SEPTAL PEPTIDOGLYCAN THAT BISECTS A BACTERIAL CELL INTO TWO DAUGHTER CELLS. RECENT PHYLOGENETIC ANALYSES INDICATE THAT THE MECHANISMS CONTROLLING CELL DIVISION - IDENTIFIED THROUGH DECADES OF WORK IN A HANDFUL OF MODEL ORGANISMS - ARE WIDELY CONSERVED AMONG BACTERIA. DESPITE THESE FINDINGS, OUR LAB RECENTLY DISCOVERED THAT THE MAJOR NOSOCOMIAL PATHOGEN CLOSTRIDIOIDES DIFFICILE MEDIATES VEGETATIVE CELL DIVISION USING A MECHANISM THAT FUNDAMENTALLY DIFFERS FROM PREVIOUSLY STUDIED BACTERIA. IN PARTICULAR, THE MAJORITY OF CORE DIVISOME PROTEINS CHARACTERIZED IN OTHER BACTERIA ARE EITHER MISSING OR DISPENSABLE FOR VEGETATIVE CELL DIVISION IN C. DIFFICILE. INDEED, C. DIFFICILE LACKS ORTHOLOGS OF THE FTSW-FTSI SEPTAL PEPTIDOGLYCAN SYNTHASE COMPLEX THAT WAS PREVIOUSLY THOUGHT TO BE UNIVERSALLY REQUIRED FOR CELL DIVISION IN BACTERIA. INSTEAD, C. DIFFICILE USES THE ENZYME PBP1, WHICH BELONGS TO A DISTINCT CLASS OF ENZYMES CALLED CLASS A PBPS, TO DRIVE SEPTAL PEPTIDOGLYCAN SYNTHESIS. GIVEN THAT C. DIFFICILE DIVISION OCCURS IN THE ABSENCE OF THE MAJORITY OF CANONICAL DIVISOME PROTEINS, THE PROTEINS THAT COMPRISE C. DIFFICILE'S UNIQUE DIVISOME AND THUS GOVERN PBP1 RECRUITMENT TO THE SITE OF DIVISION AND LICENSE ITS SEPTAL PEPTIDOGLYCAN SYNTHASE ACTIVITY REMAIN UNKNOWN. TO ADDRESS THIS CRITICAL GAP IN KNOWLEDGE, THIS PROPOSAL SEEKS TO IDENTIFY AND CHARACTERIZE C. DIFFICILE'S UNIQUE CELL DIVISION MACHINERY AND TO DETERMINE THE ORDER OF ASSEMBLY OF THIS NOVEL COMPLEX. I WILL EMPLOY A FORWARD GENETIC SELECTION, AS WELL AS A COMPLEMENTARY CANDIDATE-BASED SCREENING APPROACH, TO IDENTIFY NEW ESSENTIAL CELL DIVISION GENES. IN PARALLEL, I WILL BIOCHEMICALLY DEFINE THE DIVISOME COMPLEX BY IDENTIFYING PROTEINS THAT INTERACT WITH ONE OF THREE KNOWN CORE DIVISOME COMPLEX PROTEINS IN C. DIFFICILE. TO DELINEATE THE ORDER OF ASSEMBLY, I WILL LEVERAGE FLUORESCENT PROTEIN FUSIONS AND GENETIC KNOCK-DOWNS TO DETERMINE THE LOCALIZATION DEPENDENCIES OF DIVISOME PROTEIN CANDIDATES. THESE ANALYSES WILL THUS UNCOVER CRITICAL COMPONENTS OF THE C. DIFFICILE DIVISION MACHINERY AND DELINEATE HOW THEY ASSEMBLE INTO A FUNCTIONAL DIVISOME. BY PROVIDING NOVEL INSIGHT INTO C. DIFFICILE'S DIVISION MECHANISM, THE PROPOSED ANALYSES WILL ALSO HIGHLIGHT THAT THERE IS GREATER DIVERSITY IN BACTERIAL CELL DIVISION MECHANISMS THAN PREVIOUSLY THOUGHT BASED ON MODEL SYSTEMS. BY IDENTIFYING UNIQUE FACTORS THAT ARE ESSENTIAL FOR C. DIFFICILE CELL DIVISION, THIS WORK WILL ULTIMATELY FACILITATE THE DEVELOPMENT OF C. DIFFICILE-SPECIFIC THERAPIES THAT SPARE THE COMMENSAL GUT FLORA. SUCH C. DIFFICILE-SPECIFIC THERAPIES ARE CRITICALLY NEEDED GIVEN THAT THE MOST EFFECTIVE THERAPIES FOR TREATING C. DIFFICILE INFECTIONS ARE THOSE THAT PRESERVE THE NATIVE GUT MICROBIOTA.