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

项目摘要 利什曼原虫导致一系列毁灭性的被忽视的热带疾病，这些疾病折磨着多达 每年有一百万人。每年至少有2万人死于这种致命的内脏形式的疾病， 它是由L.donovani引起的。理解利什曼原虫生物学中重要问题的遗传工具有 目前相当有限。大多数利什曼原虫缺乏RNA干扰(RNAi)能力， 排除了基因组规模的RNAi文库屏幕的实施，这些屏幕已经带来了革命性的变化 与之相关的布氏锥虫的分子遗传学研究。就像下调蛋白质表达的情况一样 通过RNAi，表达比正常水平更高的蛋白质(即过度表达)可以产生抗药性 或以其他方式提高存活率(健身增益=GnFt)，或对细胞健康造成有害影响(丧失 适合度=LsFt)，可以揭示重要的途径和蛋白质功能。基因组规模的过度表达 文库已被证明对包括布氏毛滴虫在内的多个物种的广泛遗传筛选具有价值。这个 该方案的目标是构建和验证一个新的、基因组规模的、可诱导的过表达文库 利什曼原虫将成为该领域一种多功能和广泛适用的新遗传工具。 我们建议建立一个编码多诺瓦氏杆菌大部分蛋白的文库(~7500个开放阅读 帧，或LdORF)。PCR扩增的LdORF将被定向克隆到Gateway Entry载体中 促进文库转移到任何Gateway兼容载体(Aim 1)，产生LdORFeome质粒 图书馆。LdORFeome将被转移到新的可诱导利什曼原虫表达载体中 我们的实验室(目标1)。得到的文库将被稳定地导入利什曼原虫以产生可诱导的 杜氏利什曼原虫ORFeome高表达文库(LdOX文库)。因为总体目标是 将LdOX库作为社区资源提供，我们将通过执行以下操作来验证库 原理证明过表达筛查以确定耐药基因(GnFt)和所需的途径 正常生长(LsFt)(AIM2)。 LdOX文库将为高通量基因研究提供一个创新和急需的新工具 利什马尼亚的银幕。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的机制，以及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都能存活。

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.