Generation and validation of a novel inducible overexpression library for genome-scale genetic screens in Leishmania

项目摘要 利什曼原虫引起一系列毁灭性的被忽视的热带病， 每年一百万人。每年至少有2万人死于这种致命的内脏疾病， 这是由L. donovani。用于理解利什曼原虫生物学中重要问题的遗传工具是 目前相当有限。大多数利什曼原虫物种缺乏RNA干扰（RNAi）的能力， 排除了基因组规模的RNAi文库筛选的实施，就像那些革命性的 相关动质体T.布鲁塞。就像敲低蛋白质表达一样 通过RNAi，以高于正常水平的水平表达蛋白质（即，过度表达）可赋予对药物的抗性 或以其他方式增强存活（适应性增加= GnFt），或对细胞适应性造成有害影响（适应性丧失= GnFt）。 适合度= LsFt），可以揭示重要的途径和蛋白质功能。基因组规模过表达 文库已被证明对多个物种的广泛遗传筛选有价值，包括T.布鲁塞。的 该建议的目标是产生和验证一种新的，基因组规模的，诱导型过表达文库， 利什曼原虫，将作为一个多功能和广泛适用的新的遗传工具的领域。 我们建议生成编码大部分L的库。donovani蛋白（~7500开放阅读 框架或LdORF）。将PCR扩增的LdORF定向克隆到Gateway Entry载体中， 促进文库转移至任何Gateway相容载体（Aim 1），产生LdORFeome质粒 图书馆LdORFeome将被转移到新的诱导型利什曼原虫表达载体中， 我们的实验室（目标1）。将所得文库稳定转染到利什曼原虫中，以产生诱导型表达载体。 杜氏利什曼原虫ORFeome过表达文库（LdOX文库）。因为总的目标是 为了使LdOX库作为社区的资源可用，我们将通过执行以下操作来验证库 原理验证过表达筛选，以鉴定耐药基因（GnFt）和 正常生长（LsFt）（Aim 2）。 LdOX文库将为高通量遗传学研究提供创新的和急需的新工具。 利什曼原虫的屏幕我们预计，带有LdOX库的屏幕将对 通过揭示药物作用和耐药性机制，并揭示所需的蛋白质和途径， 在任何生长条件或生命周期阶段存活。

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.