Mapping Epistatic Interactions in Molecular Evolution of Antibiotic Resistance

PROJECT SUMMARY Evolution of antibiotic resistance is a global public health problem. How evolution renders antibioticmolecules ineffective by altering antibiotic targets is an interesting phenomenon from both clinical and basicscience perspectives. In pathogenic bacteria, there is only a handful of drug target enzymes such as DNAgyrases, RNA polymerases, fatty acid synthetases, and enzymes involved in folic acid synthesis. Therefore, amechanistic understanding of resistance-conferring mutations in these enzymes is clinically critical fordesigning new drugs or drug variants that can inhibit resistant bacteria. In this project, we propose to study evolution of the Escherichia coli dihydrofolate reductase (DHFR)enzyme and map epistatic interactions between DHFR mutations. DHFR is a ubiquitous enzyme with anessential role in the folic acid synthesis pathway and is used as a drug target in antibacterial, anticancer, andantimalarial therapies. In bacteria, an antibiotic named trimethoprim competitively binds to DHFR and blocks itscatalytic activity. Therefore, DHFR mutations that either confer resistance or compensate for reduced catalyticactivity of resistant DHFR mutants are selected for bacterial survival. We will use laboratory evolution experiments to identify functional DHFR mutations and reproduciblegenetic trajectories leading to elevated trimethoprim resistance. We will characterize these mutations by usingin vitro biochemical assays and deep-sequencing based fitness measurements for calculating epistaticinteractions between DHFR mutations. We will use molecular dynamics along with other computational toolsand nuclear magnetic resonance (NMR) spectroscopy to reveal structural changes responsible for resistanceand epistatic interactions. The combination of these approaches presents a unique opportunity to quantitativelyevaluate evolutionary paths leading to trimethoprim resistance and create a discovery pipeline for studyingprotein evolution. By creating a deeper understanding for the evolutionary dynamics of an important drug targetenzyme, our proposal will develop experimental and computational tools for studying protein evolution with theultimate goal of improving human health. Indeed, our preliminary analyses suggest that we will be able todesign and test novel trimethoprim derivatives that can selectively inhibit DHFR mutants that carry the L28Rreplacement, a common and synergistic DHFR mutation. We propose to synthesize trimethoprim-Dihydrofolatehybrid molecules that will possess the salient structural features of both DHF and trimethoprim moleculesselectively inhibit DHFR mutants with the L28R replacement. We will evolve pan sensitive E. coli strains in themorbidostat in order to quantify the efficacy of the mutant specific trimethoprim derivatives in impedingresistance evolution and accordingly develop new strategies for better use of it.