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

动体寄生虫(利什曼原虫、布氏锥虫和克氏锥虫)会造成毁灭性的疾病，全世界约有850万人受到这些疾病的折磨。这些寄生虫在其整个生命周期中经历了多种环境应激源，包括温度、PH值和养分供应的大幅波动。感知和应对这些压力的能力虽然对寄生虫的生存至关重要，但人们对此知之甚少。目前，用于理解利什曼原虫生物学中重要问题的遗传工具相当有限，特别是与布鲁氏毛滴虫可用的工具相比。这项建议的目标是：1)开发和验证一个新的基因组规模的功能增益(GOF)过表达文库作为利什曼原虫研究社区的资源；以及2)利用GOF文库识别调节杜氏利什曼原虫和布氏支原体嘌呤转运蛋白的RNA结合蛋白(RBPs)，以响应嘌呤饥饿，这是一个营养感知和应激反应的模型途径。嘌呤是从寄主那里获得的必需营养素。在去除生长介质中的嘌呤后，杜氏乳杆菌前鞭毛体停滞在细胞周期的G1期，并进入静止状态，在这种状态下，它们可以在培养中持续90天以上。作为这种强烈的胁迫反应的一部分，杜诺氏乳杆菌和布鲁氏锥虫都分别增加了细胞表面嘌呤转运蛋白的表达，如LdNT3和TbNT8.1。LdNT3和TbNT8.1mRNAs的3‘-非翻译区(UTRs)编码一个保守的调控元件，预计会形成茎环结构，当嘌呤丰富时抑制转运蛋白的表达；当嘌呤稀缺时，这种抑制作用被解除。令人惊讶的是，TbNT8.1茎环在利什曼原虫中不起作用，尽管这些环只有三个核苷酸的差异。这表明，与这个保守的茎环结合的同源限制性商业惯例已经进化出不同的结合特异性。我们预测，在反式系统中表达同源的布氏锥虫RBP将对杜诺瓦尼乳杆菌的TbNT8.1茎环具有抑制活性。我们建议利用这一可能性，通过利什曼原虫的功能互补来鉴定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布鲁氏锥虫开放阅读框架(TbORFeome)的质粒库(TbORFeome)。利用高效的体外重组反应(Gateway)，我们将TbORFeome转移到我们为在L.donovani中诱导表达而设计的载体(LD_TbORFeome文库)。这个新的文库将被转染到杜诺瓦尼乳杆菌的报告细胞系中，这将使我们能够在文库中鉴定与TbNT8.1茎环结合的TbORF，并抑制该报告基因的表达(目标2)。这些特定目标的完成不仅将识别调控部分高度保守和必要的应激反应的RBP，而且还将为该社区提供一个新的和多功能的基因筛选功能增益库。

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.