Complementation and gain-of-function screens via inducible expression of a Trypanosoma brucei ORFeome library in Leishmania

通过利什曼原虫中布氏锥虫 ORFeome 文库的诱导表达进行互补和功能获得筛选

• 批准号: 10303810
• 负责人: PHILLIP A YATES
• 金额: $ 23.78万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-07 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303810
• 关键词: 3' Untranslated Regions; Ablation; African Trypanosomiasis; Binding; Binding Proteins; Biology; CRISPR/Cas technology; Cell Cycle; Cell Line; Cell surface; Cloning; Communities; Complement; Disease; Drug resistance; Excision; Exhibits; G1 Phase; Ganciclovir; Gene Expression; Gene Library; Genetic; Genetic Recombination; Genetic Screening; Glean; Goals; Growth; In Vitro; Incidence; Individual; Leishmania; Leishmania donovani; Libraries; Mediating; Melarsoprol; Messenger RNA; Modeling; Nucleotides; Nutrient; Open Reading Frames; Organism; Orthologous Gene; Parasites; Pathway interactions; Plasmids; Post-Transcriptional Regulation; Proteins; Proteome; Purines; RNA Interference; RNA library; RNA-Binding Proteins; Reaction; Regulation; Regulatory Element; Reporter; Repression; Reproducibility; Research; Resistance; Resources; Ribosomal RNA; Role; Simplexvirus; Specificity; Starvation; Stress; Structure; System; Temperature; Thymidine Kinase; Transfection; Trypanosoma brucei brucei; Trypanosoma cruzi; Visceral Leishmaniasis; biological adaptation to stress; design; detection of nutrient; environmental stressor; experience; expression vector; fitness; gain of function; genome-wide; human disease; inducible gene expression; innovation; loss of function; mutant; new therapeutic target; novel; novel therapeutics; overexpression; resistance gene; response; stem; tool; vector

Kinetoplastid parasites (Leishmania, Trypanosoma brucei, and Trypanosoma cruzi) cause devastating diseases that afflict ~8.5 million people worldwide. These parasites experience multiple environmental stressors throughout their lifecycles, including substantial fluctuations in temperature, pH, and nutrient availability. The ability to sense and respond to these stresses, while essential for parasite survival, is poorly understood. Genetic tools for understanding important questions in Leishmania biology are currently quite limited, especially compared to those available for T. brucei. The goals of this proposal are 1) to develop and validate a novel genome-scale Gain-of-Function (GoF) overexpression library as a resource for the Leishmania research community; and 2) to use the GoF library to identify RNA binding proteins (RBPs) that regulate L. donovani and T. brucei purine transporters in response to purine starvation, which is a model nutrient sensing and stress response pathway. Purines are essential nutrients obtained exclusively from the host. Upon removal of purines from the growth medium, L. donovani promastigotes arrest in the G1 phase of the cell cycle and enter a quiescent-like state in which they can persist for over 90 days in culture. As part of this robust stress response, both L. donovani and T. brucei increase expression of cell surface purine transporters such as LdNT3 and TbNT8.1, respectively. The 3'-untranslated regions (UTRs) of the LdNT3 and TbNT8.1 mRNAs encode a conserved regulatory element, predicted to form stem-loop structures, that represses expression of the transporters when purines are abundant; this repression is relieved when purines are scarce. Surprisingly, the TbNT8.1 stem-loop does not function in Leishmania, even though the loops differ by only three nucleotides. This intimates that the orthologous RBPs that bind this conserved stem-loop have evolved different binding specificities. We predict that expressing the cognate T. brucei RBP in trans will confer repressor activity to the TbNT8.1 stem-loop in L. donovani . We propose to exploit this possibility to identify the TbNT8.1 stem-loop binding protein via functional complementation in Leishmania. To achieve this, in Aim 1 we will take advantage of the fact that a plasmid library encoding the majority of T. brucei open reading frames (the TbORFeome) was recently constructed. Using an efficient in vitro recombination reaction (Gateway), we will transfer the TbORFeome to vectors we designed for inducible expression in L. donovani (the Ld_TbORFeome library). This new library will be transfected into an L. donovani reporter cell line that will allow us to identify TbORFs in the library that bind to the TbNT8.1 stem-loop and repress expression of the reporter (Aim 2). Completion of these Specific Aims will not only identify an RBP that regulates part of a highly conserved and essential stress response, but will also provide the community with a novel and versatile Gain-of-Function library for genetic screens.

动质体寄生虫（利什曼原虫、布氏锥虫和克氏锥虫）会引起毁灭性的疾病，困扰全世界约 850 万人。这些寄生虫在其整个生命周期中经历多种环境压力，包括温度、pH 值和营养物质可用性的大幅波动。感知和应对这些压力的能力虽然对于寄生虫的生存至关重要，但人们却知之甚少。目前，用于理解利什曼原虫生物学重要问题的遗传工具非常有限，特别是与可用于布氏利什曼原虫的工具相比。该提案的目标是 1) 开发和验证新型基因组规模的功能获得 (GoF) 过表达文库，作为利什曼原虫研究界的资源； 2) 使用 GoF 文库来识别 RNA 结合蛋白 (RBP)，该蛋白可调节杜氏乳杆菌和布氏锥虫嘌呤转运蛋白以应对嘌呤饥饿，这是一种营养传感和应激反应途径的模型。嘌呤是仅从宿主获得的必需营养素。从生长培养基中去除嘌呤后，杜氏乳杆菌前鞭毛体停滞在细胞周期的 G1 期，并进入类似静止的状态，在该状态下它们可以在培养物中持续存在 90 天以上。作为这种强烈应激反应的一部分，杜氏乳杆菌和布氏乳杆菌分别增加细胞表面嘌呤转运蛋白（例如 LdNT3 和 TbNT8.1）的表达。 LdNT3 和 TbNT8.1 mRNA 的 3'-非翻译区 (UTR) 编码保守的调节元件，预计会形成茎环结构，当嘌呤丰富时抑制转运蛋白的表达；当嘌呤缺乏时，这种抑制就会缓解。令人惊讶的是，TbNT8.1 茎环在利什曼原虫中不起作用，尽管环仅相差三个核苷酸。这表明结合该保守茎环的直系同源 RBP 已进化出不同的结合特异性。我们预测，以反式表达同源的 T. brucei RBP 将为杜氏乳杆菌中的 TbNT8.1 茎环赋予阻遏活性。我们建议利用这种可能性通过利什曼原虫中的功能互补来鉴定 TbNT8.1 茎环结合蛋白。为了实现这一目标，在目标 1 中，我们将利用最近构建的编码大多数 T. brucei 开放阅读框（TbORFeome）的质粒库这一事实。使用有效的体外重组反应（Gateway），我们将 TbORFeome 转移到我们设计的用于在杜氏乳杆菌中诱导表达的载体（Ld_TbORFeome 库）。这个新文库将被转染到杜氏乳杆菌报告细胞系中，这将使我们能够识别文库中与 TbNT8.1 茎环结合并抑制报告基因表达的 TbORF（目标 2）。完成这些具体目标不仅将鉴定出调节部分高度保守且重要的应激反应的 RBP，还将为社区提供用于遗传筛选的新颖且多功能的功能获得库。