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.

动力质体寄生虫（Leishmania，Brucei锥虫和Cruzi锥虫）引起毁灭性疾病，全世界约有850万人。这些寄生虫在整个生命周期中都经历了多种环境压力源，包括温度，pH和养分的可用性大幅波动。对这些压力的感觉和反应的能力，尽管对寄生虫的生存至关重要，但对这些压力的理解很少。目前，与T. Brucei可用的遗传工具相比，在利什曼尼亚生物学中了解重要问题的遗传工具是非常有限的。该提案的目标是1）开发和验证一种新型的基因组规模的功能获得（GOF）过表达图书馆，作为利什曼尼亚研究社区的资源； 2）使用GOF文库识别RNA结合蛋白（RBP），该蛋白（RBP）调节了Donovani和T. brucei嘌呤转运蛋白对嘌呤饥饿的响应，这是一种模型的营养感应和应力反应途径。嘌呤是仅从宿主那里获得的必需营养素。在从生长培养基中取出嘌呤后，多诺瓦尼乳杆菌前抗物在细胞周期的G1阶段停滞，并进入静态状态，在这种状态下，它们可以在培养物中持续90天以上。作为这种强大的应激反应的一部分，牛乳杆菌和t。brucei分别增加了细胞表面嘌呤转运蛋白的表达，例如ldnt3和tbnt8.1。 LDNT3和TBNT8.1 mRNA的3'-非翻译区域（UTRS）编码一个保守的调节元件，预测形成了干循环结构，当嘌呤是附加的时，会抑制转运蛋白的表达；当嘌呤稀缺时，这种压抑就会缓解。令人惊讶的是，即使环路仅通过三个核苷酸不同，TBNT8.1茎环在利什曼尼亚不起作用。这暗示着结合这种保守的茎环的直系同源RBP已进化出不同的结合特异性。我们预测，在反式中表达同源的t. brucei rbp将赋予抑制剂活性，以donovani中的tbnt8.1茎环。我们建议利用这种可能性，以通过利什曼尼亚的功能补充来鉴定TBNT8.1茎环结合蛋白。为了实现这一目标，在AIM 1中，我们将利用以下事实：最近构建了编码大多数T. Brucei开放式阅读框（Tborfeome）的质粒库。使用有效的体外重组反应（网关），我们将将tborfeome转移到我们为Donovani（LD_Tborfeome库）中诱导表达的矢量转移到我们设计的载体。这个新的库将被转染到多诺瓦尼记者细胞系中，该细胞系将使我们能够在库中识别与tbnt8.1 stem-loop结合的tborfs，并抑制了记者的表达（AIM 2）。这些特定目标的完成不仅将确定调节高度保守和基本压力反应的一部分的RBP，而且还将为社区提供用于基因筛查的新颖而多功能的功能奖励库。