Developing low-background inducible expression technology for Leishmania donovani

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 DESCRIPTION (provided by applicant): The goal of this proposal is to develop a low-background inducible expression system for Leishmania donovani that will serve as a valuable tool for molecular genetic dissection of basic metabolic, regulatory and virulence pathways in this important human pathogen. L. donovani is the causative agent of visceral leishmaniasis, a devastating disease that is estimated to infect 500,000 people annually, and killing approximately 50,000 people per year. Current anti-leishmanial therapies can be costly, are often poorly tolerated and the incidence of resistant parasites is increasing. Rational design of new chemotherapeutic strategies critically depends the development and implementation of new technologies to accelerate the rate at which knowledge of the basic biology of this parasite is acquired. Inducible expression systems have been invaluable tools in the related kinetoplastid parasite T. brucei and the absence a suitable system in Leishmania has been a widely acknowledged deficiency in the field. Several inducible promoter systems have been described for Leishmania, yet none has been widely adopted by the Leishmania research community, in part due to high background expression in the uninduced state, poor inducibilty, or non-physiological expression levels. This proposal posits several parameters critical for optimal performance of inducible expression systems based on T7 RNA polymerase (T7 pol) and Tet repressor (TetR) in Leishmania. The three most important of these include, 1) preventing non-specific background expression; 2) attaining the proper balance of T7 pol and TetR co-expression; and 3) avoiding position effects conferred by the site of inducible expression construct integration. These and other parameters are addressed in two specific aims. The first specific aim focuses on the construction the T7 pol and TetR expression vectors, inducible reporter constructs and other DNA reagents that will constitute the tetracycline inducible T7 promoter expression system. This aim includes novel strategies to prevent background expression, to optimize the relative amounts and ratios of co-expressed T7 pol and TetR, and for streamlined vector construction. The goal of the second specific aim is to generate L. donovani lines encoding a tetracycline inducible expression system with low background and optimal induction properties. Parasite cultures co-expressing T7 pol and TetR at several different ratios, and encoding a tetracycline inducible, T7 promoter-driven Renilla luciferase-green fluorescent protein (Rluc-GFP) reporter construct will be subjected to consecutive rounds of fluorescence activated cell sorting to select parasites with no background (GFP negative) in the uninduced state, and high expression upon induction (GFP positive). To avoid position effects in clones shown by luciferase assays to have ideal characteristics, the Rluc-GFP reporter construct will be replaced with a construct that tags the locus for the integration of future inducible expression constructs.