Developing low-background inducible expression technology for Leishmania donovani

 描述（由申请人提供）：本提案的目标是开发杜氏利什曼原虫的低背景诱导表达系统，该系统将作为对该重要人类病原体的基本代谢、调节和毒力途径进行分子遗传学解剖的有价值的工具。L. donovani是内脏利什曼病的病原体，内脏利什曼病是一种毁灭性的疾病，估计每年感染500，000人，每年造成约50，000人死亡。目前的抗利什曼原虫治疗可能是昂贵的，往往耐受性差，耐药寄生虫的发病率正在增加。新的化疗策略的合理设计关键取决于新技术的开发和实施，以加快获得这种寄生虫的基本生物学知识的速度。诱导表达系统在相关动质体寄生虫T.布氏杆菌的感染和利什曼原虫中缺乏合适的系统是该领域广泛承认的缺陷。已经描述了几种用于利什曼原虫的诱导型启动子系统，但没有一种被利什曼原虫研究界广泛采用，部分原因是在未诱导状态下的高背景表达、差的诱导性或非生理表达水平。该建议提出了几个关键参数的诱导表达系统的最佳性能的基础上T7 RNA聚合酶（T7 pol）和泰特阻遏物（TetR）在利什曼原虫。其中三个最重要的包括，1）防止非特异性背景表达; 2）获得T7 pol和TetR共表达的适当平衡;和3）避免由诱导型表达构建体整合位点赋予的位置效应。这些和其他参数在两个具体目标中得到处理。第一个具体目标集中于构建T7 pol和TetR表达载体、诱导型报告基因构建体和其他DNA试剂，其将构成四环素诱导型T7启动子表达系统。该目的包括防止背景表达、优化共表达的T7 pol和TetR的相对量和比率以及用于流线型载体构建的新策略。第二个具体目标的目标是生成L。donovani系编码四环素诱导表达系统，具有低背景和最佳诱导特性。以几种不同比例共表达T7 pol和TetR，并编码四环素诱导型、T7启动子驱动的海肾胰蛋白酶-绿色荧光蛋白（Rluc-GFP）报告基因构建体的寄生虫培养物将进行连续轮的荧光激活细胞分选，以选择在未诱导状态下无背景（GFP阴性），诱导后高表达（GFP阳性）的寄生虫。为了避免在通过荧光素酶测定显示具有理想特征的克隆中的位置效应，Rluc-GFP报告基因构建体将被标记基因座的构建体替换，用于整合未来的诱导型表达构建体。

 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.