Genetically-encoded Fluorescent Rna Sensors For Measuring Transport of Antibiotics Into The Cytoplasm of Gram-negative Pathogens and Development of Efflux Pump Inhibitors

Project SummaryComplications from infections caused by difficult to treat Gram-negative pathogens resistant tomultiple antibiotics (the MDR phenotype) has become a major public health threat. The basis ofresistance in the most important members of this group, including Pseudomonas aeruginosa, isthe poor penetration of the antibiotics through a relatively impermeable bacterial cell envelopeand their expulsion (efflux) by various pumps expressed by the microorganisms. Consequently,the intracellular concentrations of antibiotic are kept below bactericidal levels. This proposalaims at creating new classes of diagnostic tools and therapeutic agents to combat efflux-basedresistance by a project organized into two phases. First, we will develop genetically encodedfluorescent sensors activated by binding of antibiotics following their entry into the bacterialcytoplasm. This tool will enable us to measure antibiotic flux under different environmentalconditions and on a single cell basis. During the second phase of the project, working with ourindustrial partner, we will implement a screening program, using a library of several hundredbillion small molecules, to identify inhibitors targeting different components of efflux pumps.These inhibitors should activate the fluorescent detector by causing enhanced intracellularaccumulation of the antibiotics resulting from inhibition of efflux. Functional studies are proposedto assess the ability of the active compounds to promote killing of P. aeruginosa by antibioticsthat are normally expelled by the efflux pumps. Crystal structures of inhibitors bound to targetproteins will be determined and these will guide medicinal chemistry efforts with the goal ofcreating additional derivatives with increased potencies and expanded spectrum, capable ofinhibiting efflux pumps of other important Gram-negative pathogens.