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

用于利什曼原虫基因组规模遗传筛选的新型诱导过表达文库的生成和验证

• 批准号: 10818854
• 负责人: PHILLIP A YATES
• 金额: $ 3.97万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-13 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10818854
• 关键词: Biology; Cells; Communities; Disease; Drug resistance; Expression Library; Generations; Genetic; Genetic Screening; Genomic Library; Glean; Goals; Growth; Leishmania; Leishmania donovani; Libraries; Life Cycle Stages; Molecular Genetics; Open Reading Frames; Parasites; Pathway interactions; Persons; Pharmacotherapy; Plasmids; Proteins; RNA Interference; Resistance; Resources; Transfection; Trypanosoma brucei brucei; Validation; Visceral; Visceral Leishmaniasis; drug action; expression vector; fitness; genome-wide; human disease; innovation; knock-down; neglected tropical diseases; novel; novel therapeutics; overexpression; protein expression; protein function; resistance gene; tool; vector

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.

项目概要 利什曼原虫寄生虫会引起一系列毁灭性的、被忽视的热带疾病，影响多达 每年一百万人。每年至少有 20,000 人死于这种致命的内脏疾病， 这是由杜诺瓦尼乳杆菌引起的。用于理解利什曼原虫生物学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，这 阻碍了基因组规模RNAi文库筛选的实施，就像那些已经彻底改变的 相关动质体 T. brucei 的分子遗传学。就像敲低蛋白质表达的情况一样 通过 RNAi，以高于正常水平的蛋白质表达（即过度表达）可以赋予药物耐药性 或以其他方式提高生存率（适应度增益= GnFt），或对细胞适应度造成有害影响（丧失- Fitness = 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 事实证明，文库对于包括布氏锥虫在内的多个物种的广泛遗传筛选很有价值。这 该提案的目标是生成并验证一个新颖的、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多功能且广泛适用的新遗传工具。 我们建议生成一个编码大多数杜氏乳杆菌蛋白的文库（~7500 Open Reading 框架，或 LdORF）。 PCR 扩增的 LdORF 将定向克隆到 Gateway Entry 载体中，以 促进文库转移至任何 Gateway 兼容载体（目标 1），从而产生 LdORFeome 质粒 图书馆。 LdORFeome 将被转移到 2017 年开发的新型诱导型利什曼原虫表达载体中。 我们的实验室（目标 1）。所得文库将稳定转染至利什曼原虫中，以产生诱导型 杜氏利什曼原虫 ORFeome 过表达文库（LdOX 文库）。因为总体目标是 使 LdOX 库可作为社区的资源，我们将通过执行来验证该库 原理验证过表达筛选，以确定耐药基因 (GnFt) 和耐药所需的途径 正常生长 (LsFt)（目标 2）。 LdOX 文库将为高通量遗传提供创新且急需的新工具 利什曼原虫屏幕。我们预计带有 LdOX 库的筛选将对 通过揭示药物作用和耐药机制，并揭示所需的蛋白质和途径 用于在任何生长条件或生命周期阶段生存。