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

着丝体寄生虫（利什曼原虫、布鲁氏锥虫和克氏锥虫）引起了毁灭性的疾病，全世界约有850万人受到影响。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，但人们对其了解甚少。用于理解利什曼原虫生物学重要问题的遗传工具目前相当有限，特别是与可用于布鲁氏体的遗传工具相比。本提案的目标是：1)开发和验证一个新的基因组尺度的功能获得（GoF）过表达文库，作为利什曼原虫研究界的资源；2)利用GoF文库鉴定多诺瓦氏杆菌和布鲁氏T.嘌呤饥饿时调节嘌呤转运体的RNA结合蛋白（rbp），这是一种典型的营养感知和应激反应途径。嘌呤是完全从宿主获得的必需营养素。从生长培养基中去除嘌呤后，L. donovani promastigotes停留在细胞周期的G1期，进入类似静止状态，在培养中可以持续90天以上。作为这种强大的应激反应的一部分，L. donovani和T. brucei分别增加了细胞表面嘌呤转运体如LdNT3和TbNT8.1的表达。LdNT3和TbNT8.1 mrna的3'-非翻译区（UTRs）编码一个保守的调控元件，预计形成茎环结构，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，尽管这两个环只有三个核苷酸的不同。这表明结合这种保守茎环的同源rbp进化出了不同的结合特异性。我们预测，通过反式表达同源的布氏T. brucei RBP将赋予L. donovani TbNT8.1茎环的抑制活性。我们建议利用这种可能性，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部分布鲁氏菌开放阅读框的质粒库（TbORFeome）。利用高效的体外重组反应（Gateway），我们将TbORFeome转移到我们设计的载体上，以便在L. donovani中诱导表达（Ld_TbORFeome文库）。这个新的文库将被转染到L. donovani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 （Aim 2）。这些特定目标的完成不仅将确定一个调控部分高度保守和必要的应激反应的RBP，而且还将为遗传筛选提供一个新颖和通用的功能获得文库。

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.