Project Grant F32AI203130

EVALUATION OF TRISCATECHOLATE SIDEOPHORE ANTIBIOTIC CONJUGATES TOWARDS ENTERIC BIOFILM-FORMING PATHOGENS - PROJECT SUMMARY ALTHOUGH SIDEROPHORE-MEDIATED ANTIBIOTIC DELIVERY HAS BEEN EXPLORED FOR DECADES, ONLY A SINGLE SIDEROPHORE- BASED ANTIBIOTIC HAS BEEN APPROVED FOR THE TREATMENT OF COMPLEX GRAM-NEGATIVE BACTERIAL INFECTIONS. THE LONG- TERM GOAL OF THIS WORK IS TO DEVELOP SIDEROPHORE-ANTIBIOTIC CONJUGATES (SACS) BASED ON THE NATIVE TRISCATECHOLATE SIDEROPHORES, ENTEROBACTIN AND DIGLUCOSYLATED ENTEROBACTIN (ENT & DGE), AS NARROW-SPECTRUM ANTIBIOTICS TOWARDS ENTERIC PATHOGENS AND PATHOBIONTS. THE OVERALL OBJECTIVES OF THIS PROPOSAL ARE TO (I) EVALUATE ENDOGENOUS SIDEROPHORE BIOSYNTHESIS AND TRANSPORT WITHIN NATIVE BACTERIAL BIOFILMS, AND (II) INVESTIGATE THE TRANSPORT, INTERNALIZATION, AND ANTIBACTERIAL ACTIVITY OF EXOGENOUS ENT & DGE-BASED SACS TO ENTERIC GRAM-NEGATIVE PATHOGENS AND PATHOBIONTS WITHIN THE BIOFILM STATE. THE CENTRAL HYPOTHESIS IS THAT ENT & DGE-BASED SACS WILL DISPLAY ENHANCED DELIVERY AND ANTIBIOTIC ACTIVITY TOWARDS NON-TYPHOIDAL SALMONELLA (NTS) AND ESCHERICHIA COLI (EC) BACTERIAL BIOFILMS, RELATIVE TO THE UNCONJUGATED ANTIBIOTIC. THE RATIONALE FOR THIS PROPOSAL IS THAT BIOFILM FORMATION PROMOTES ANTIBACTERIAL RESISTANCE IN MOST CHRONIC BACTERIAL INFECTIONS, AND IS REQUIRED FOR PROLONGED COLONIZATION IN THE HUMAN GUT; THEREFORE, EXAMINING THE TRANSPORT AND ANTIBIOTIC ACTIVITY OF ENT & DGE-BASED SACS IS PHYSIOLOGICALLY RELEVANT. THE CENTRAL HYPOTHESIS WILL BE TESTED BY PURSUING TWO SPECIFIC AIMS: (I) STUDY AND MODEL ENDOGENOUS ENT & DGE PRODUCTION AND TRANSPORT WITHIN NTS & EC BIOFILMS; AND (II) EVALUATE EXOGENOUS SIDEROPHORE TRANSPORT AND SAC ANTIBACTERIAL ACTIVITY WITHIN NTS & EC BIOFILMS. IN BOTH AIMS, WE WILL CONSTRUCT A MICROFLUIDIC CULTURING SYSTEM TO PROMOTE GROWTH OF MATURE NTS & EC BIOFILMS THAT WILL BE MONITORED BY CONFOCAL MICROSCOPY. IN THE FIRST AIM, FLUORESCENT REPORTERS FOR ENT & DGE BIOSYNTHESIS AND TRANSPORT WILL BE CONSTRUCTED TO VISUALIZE THE USE OF ENT & DGE WITHIN THE NTS & EC BIOFILMS, PROVIDING FUNDAMENTAL INSIGHT INTO IRON TRAFFICKING AND DISTRIBUTION WITHIN THE BIOFILM. ADDITIONALLY, MUTANTS LACKING ONE OR MULTIPLE ENT & DGE OUTER MEMBRANE RECEPTORS WILL BE UTILIZED TO CHARACTERIZE HOW LOSS OF SIDEROPHORE UPTAKE MACHINERY ALTERS THE ARCHITECTURE OF MATURE NTS & EC BIOFILMS. IN THE SECOND AIM, WE WILL SYNTHESIZE ENT & DGE-BASED SIDEROPHORE FLUOROPHORE CONJUGATES TO VISUALIZE THE DIFFUSION AND TRANSPORT OF EXOGENOUS SIDEROPHORES THROUGHOUT THE NTS & EC BIOFILMS. WE WILL ALSO SYNTHESIZE ENT & DGE-BASED SACS TO EVALUATE EFFICACY TOWARDS NTS & EC BIOFILMS BY MONITORING PERTURBATION OF THE BIOFILM STRUCTURE, SAC INTERNALIZATION, AND ANTIBACTERIAL ACTIVITY. THE RESEARCH PROPOSED IS INNOVATIVE BECAUSE IT WILL FOCUS ON AN UNEXPLORED AND PHYSIOLOGICALLY RELEVANT REALM OF SIDEROPHORE-MEDIATED ANTIBIOTIC DELIVERY, THE BACTERIAL BIOFILM. THE PROPOSED RESEARCH IS SIGNIFICANT BECAUSE IT IS EXPECTED TO PROVIDE STRONG SCIENTIFIC JUSTIFICATION FOR THE CONTINUED DEVELOPMENT OF ENT & DGE-DERIVED SACS, WHERE SUCH KNOWLEDGE HAS THE POTENTIAL TO ADVANCE ENT & DGE-DERIVED SACS AS NARROW-SPECTRUM ANTIBIOTICS. THE MULTIDISCIPLINARY ENVIRONMENT AND THE COLLABORATIVE COMMUNITY OF SCHOLARS AT MIT WILL ENABLE SUCCESS OF THE PROPOSED RESEARCH, AND FACILITATE THE PROFESSIONAL DEVELOPMENT OF THE APPLICANT FOR AN INDEPENDENT CAREER IN ACADEMIC RESEARCH.