Project Summary Leishmania parasites cause a suite of devastating Neglected Tropical Diseases that afflict as many as one million people per year. At least 20,000 are killed each year by the deadly visceral form of the disease, which is caused by L. donovani. Genetic tools for understanding important questions in Leishmania biology are currently quite limited. Most Leishmania species lack the capacity for RNA interference (RNAi), which precludes the implementation of genome-scale RNAi library screens like those that have revolutionized molecular genetics in the related kinetoplastid T. brucei. As is the case for knocking down protein expression via RNAi, expressing proteins at higher levels than normal (i.e., overexpression) can confer resistance to drugs or otherwise enhance survival (Gain-of-Fitness = GnFt), or cause deleterious effects on cell fitness (Loss-of- Fitness = LsFt) that can reveal important pathways and protein functions. Genome-scale overexpression libraries have proven valuable for a wide range of genetic screens in multiple species, including T. brucei. The goal of this proposal is to generate and validate a novel, genome-scale, inducible overexpression library in Leishmania that will serve as a versatile and broadly applicable new genetic tool for the field. We propose to generate a library encoding the majority of L. donovani proteins (~7500 Open Reading Frames, or LdORFs). PCR amplified LdORFs will be directionally cloned into Gateway Entry vectors to facilitate transfer of the library to any Gateway compatible vector (Aim 1), resulting in a LdORFeome plasmid library. The LdORFeome will be transferred into novel inducible Leishmania expression vectors developed in our lab (Aim 1). The resulting library will be stably transfected into Leishmania to generate an inducible Leishmania donovani ORFeome overexpression library (the LdOX library). Because the overall goal is to make the LdOX library available as a resource for the community, we will validate the library by performing proof-of-principle overexpression screens to identify drug resistance genes (GnFt) and pathways required for normal growth (LsFt) (Aim2). The LdOX library will provide an innovative and much-needed new tool for high throughput genetic screens in Leishmania. We anticipate that screens with the LdOX library will have a sustained impact on the field by revealing mechanisms of drug action and resistance, and uncovering proteins and pathways required for surviving any growth condition or lifecycle stage.