Developing low-background inducible expression technology for Leishmania donovani

 描述(申请人提供)：本提案的目标是为杜氏利什曼原虫开发一种低背景可诱导表达系统，该系统将作为一种有价值的工具，用于对这种重要的人类病原体的基本代谢、调节和毒力途径进行分子遗传剖析。多诺瓦尼乳杆菌是内脏利什曼病的病原体，这是一种毁灭性的疾病，估计每年感染50万人，每年导致约5万人死亡。目前的抗利什曼疗法可能昂贵，往往耐受性差，耐药寄生虫的发生率正在增加。合理设计新的化疗策略至关重要地依赖于新技术的开发和实施，以加快获得这种寄生虫基本生物学知识的速度。诱导表达系统在相关的动质体寄生虫T.brucei中一直是非常有价值的工具，而在利什曼原虫中缺乏合适的表达系统一直是该领域公认的缺陷。利什曼原虫的诱导启动子系统已经被描述，但没有一个被利什曼原虫研究界广泛采用，部分原因是在非诱导状态下背景表达高，诱导性差，或非生理性表达水平。这项建议提出了几个参数，对利什曼原虫基于T7RNA聚合酶(T7Poll)和Tet Repressor(TetR)的诱导表达系统的最佳性能至关重要。其中最重要的三点包括：1)防止非特异性背景表达；2)达到T7ol和TetR共表达的适当平衡；3)避免可诱导表达结构整合位点所带来的位置效应。这些参数和其他参数在两个具体目标中得到解决。第一个具体目的是构建T7 POL和TetR表达载体、可诱导的报告载体和其他DNA试剂，以构成四环素诱导的T7启动子表达系统。这一目标包括防止背景表达的新策略，优化共表达的T7ol和TetR的相对量和比例，以及简化载体构建。第二个特定目标的目标是获得编码四环素可诱导表达系统的杜诺瓦尼乳杆菌株系，该系统具有低背景和最佳诱导特性。寄生虫培养物以几种不同的比例共表达T7pol1和TetR，并编码四环素可诱导的、T7启动子驱动的Renilla荧光素酶-绿色荧光蛋白(Rluc-GFP)报告结构，将进行连续几轮荧光激活细胞分选，以筛选在未诱导状态下无背景(GFP阴性)和诱导后高表达(GFP阳性)的寄生虫。为了避免荧光素酶分析显示具有理想特性的克隆中的位置效应，Rluc-GFP报告结构将被标记用于未来诱导表达构建物整合的位置的结构所取代。

 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.