High throughput approach to identify new therapeutics in the treatment of Lyme disease

Goals and ObjectivesGoal 1: Develop population level and single-cell analysis platforms to screen compound libraries to identify new, more effective therapeutics that inhibit Borrelia burgdorferi growth.Objectives 1.1: Live microscopy validation of targets. We will develop an imaging strategy that couples high-throughput culture methods with live-cell, epifluorescent microscopy. We aim to make our workflow fully automated such that we will be able to screen tens of thousands of cells per experimental drug treatment condition, and quantitatively analyze single-cell information, but at the population level. We will utilize this technology to screen novel inhibitors that interfere with the incorporation of fluorescent PG precursor HADA.Objectives 1.2: In vivo kinetics of peptidoglycan synthesis. Fluorescent polarization (FP) uses stochastic, but predictable, fluorescent particle motion to determine its state (i.e. free vs bound). In tandem with our high-throughput microscopy screen we will utilize FP to quantify the rate of B. burgdorferi PG synthesis and the effects of drug intervention.Goal 2: Identification and biochemical characterization of B. burgdorferi PBPs.Objectives 2.1:In vitroprobing to identify and screenB. burgdorferiPBPs.Beta-lactam libraries utilized in live cell analysis studies above will be screened for efficacy and specificityin vitro. Using functional probes, we will determine the actual protein target(s) of therapies utilized to treat Lyme disease. This method will determine the mechanistic basis for penicillin/cephalosporin sensitivity and possible specificity, and also will be used to identify new compounds that interfere withB. burgdorferiPBPs.Objectives 2.2: Biochemical activity of specific PG synthesis inhibitors. We will produce recombinant versions of each protein and test the biological activity, specificity, and kinetics of select PG synthesis inhibitors.