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.

动体寄生虫（利什曼原虫、布氏锥虫和克氏锥虫）引起的毁灭性疾病折磨着全世界约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。虽然对寄生虫的生存至关重要，但对这些压力的感知和反应能力却知之甚少。用于理解利什曼原虫生物学中重要问题的遗传工具目前相当有限，特别是与T。布鲁塞。该提案的目标是：1）开发和验证一种新的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社区的资源; 2）使用GoF文库鉴定调节利什曼原虫的RNA结合蛋白（RBP）。donovani和T.布鲁氏菌嘌呤转运蛋白响应嘌呤饥饿，这是一个模型营养传感和应激反应途径。嘌呤是人体必需的营养素，只能从宿主那里获得。在从生长培养基中除去嘌呤后，L. donovani前鞭毛体停滞在细胞周期的G1期并进入静止样状态，其中它们可以在培养物中持续超过90天。作为这种强大的压力反应的一部分，L。donovani和T.布氏杆菌分别增加细胞表面嘌呤转运蛋白如LdNT 3和TbNT 8.1的表达。LdNT 3和TbNT 8.1 mRNA的3 '-非翻译区（UTR）编码保守的调控元件，预测形成茎环结构，当嘌呤丰富时抑制转运蛋白的表达;当嘌呤缺乏时，这种抑制被解除。令人惊讶的是，TbNT8.1茎环在利什曼原虫中不起作用，即使环仅相差三个核苷酸。这暗示了结合该保守茎环的邻位RBPs已经进化出不同的结合特异性。我们预测，表达同源T。反式布氏杆菌RBP将赋予L. donovani。我们建议利用这种可能性，以确定TbNT8.1茎环结合蛋白通过功能互补利什曼原虫。为了实现这一点，在目的1中，我们将利用编码大部分T.最近构建了布鲁氏菌开放阅读框架（TbORFeome）。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L. Donovani（Ld_TbORFeome文库）。这个新的文库将被转染到L. donovani报告基因细胞系，这将允许我们鉴定文库中结合TbNT8.1茎环并抑制报告基因表达的TbORF（Aim 2）。这些特定目标的完成不仅将确定一个RBP，调节一个高度保守和必要的应激反应的一部分，但也将提供一个新的和通用的功能获得遗传筛选库的社会。