Modulation of Retrograde Transport by a Novel Parasite-Derived GTPase

描述(由申请人提供)：尽管决定寄生虫成败的分子机制提供了对免疫的关键见解，但到目前为止，很少有自然宿主-寄生虫相互作用的高度特征。我们开发了一个基于共同进化的宿主-寄生虫对的模型系统来分析黑腹果蝇及其寄生蜂(Leptopilina spp.)攻击武器库中潜在的相互作用。这个系统提供了复杂和微妙的剖析先天免疫激活、沉默和颠覆。D.黑猩猩的遗传基因是易处理的、快速的、低影响的。从苍蝇到人类，先天免疫功能被广泛保守，我们的长期目标是提供翻译结果。细毛藻Leptopilina spp.是免疫抑制的微结构，称为VLP。细胞特异性药物传递和治疗性免疫调节是我们对这些独特的寄生虫衍生颗粒功能的研究的两种可能结果。VLP的蛋白质组成是这些果蝇寄生虫攻击成功的关键。这项工作标志着对单一VLP蛋白的第一次功能研究。我们选择了一个仅在毒力最强的细毛虫黄蜂中表达的基因产物，该基因产物在VLP蛋白质组中高度丰富，并具有推测的GTP结合和水解位点。当在全基因组遗传相互作用筛查中进行分析时，这种GTP酶会导致与逆行运输受损相关的合成生长抑制。在目标1中，我们将确认这些遗传相互作用的结果，然后在GTP/GDP锁定的突变以及对正常囊泡运输产生负面影响的突变的背景下对它们进行测试。在目标2中，我们将在果蝇血细胞亚群中表达该蛋白，以检测其亚细胞定位和蛋白质-蛋白质相互作用。我们将测试它对苍蝇幼虫中依赖于NF-κB的信号的影响。Toll-NF-κB途径是果蝇先天免疫系统细胞臂和体液臂激活的基础，我们认为这种基于GTP酶的逆行运输调节可能会抑制正常的免疫信号。这项工作中使用的策略取决于配对模型系统方法。我们已经通过一个强大的酵母筛选获得了一种新蛋白的细胞功能和靶向的初步信息，该筛选提供了数千种遗传相互作用的信息。这种方法使我们能够在果蝇身上设计适当和有效的实验计划。我们的目标将为PI提供酵母和果蝇遗传学、转基因、高通量全基因组遗传筛选、免疫组织化学和荧光成像方面的深入技能。这项工作将得到一个包括跨学科环境领域专家的小组的大力支持，该小组将在#年为PI提供高级培训。 免疫学、多模式系统、遗传学和先进的分子生物学技术。

DESCRIPTION (provided by applicant): Few natural host-parasite interactions have been highly characterized to date, even though the molecular mechanisms that determine parasite success or failure offer critical insights into immunity. We have developed a model system based on a coevolved host-parasite pair to analyze the reciprocal interactions underlying immunity in Drosophila melanogaster and the attack arsenals of its parasitic wasps (Leptopilina spp.). This system offers the complexity and subtlety to dissect innate immune activation, silencing, and subversion. D. melanogaster genetics are tractable, fast, and low-impact. Innate immune functions are widely conserved from fly to human and our long-term goal is to provide translational results. The most unique venom products of Leptopilina spp. are immunosuppressive microstructures, referred to as VLPs. Cell-specific drug delivery and therapeutic immune modulation represent two possible outcomes our investigations of the function of these unique parasite-derived particles. The protein composition of VLPs is central to the attack success of these parasites of Drosophila. This work marks the first functional investigation into a singular VLP protein. We have selected a gene product that is expressed only by the most virulent of Leptopilina wasps, is highly abundant in the VLP proteome, and possesses a putative site for GTP binding and hydrolysis. When analyzed in a genome-wide genetic interaction screen, this GTPase causes synthetic growth repression associated with impaired retrograde transport. In Aim 1, we will confirm these genetic interaction results and then test them in the context of GTP/GDP-locked mutants, as well as mutations that negatively impact normal vesicular trafficking. In Aim 2, we will express this protein in sub-populations of Drosophila blood cells to examine its subcellular localization and protein-protein interactions. We will test its impact on NF-κB-dependent signaling in fly larvae. The Toll- NF-κB pathway underlies activation of both the cellular and humoral arms of Drosophila's innate immune system and we believe that this GTPase-based modulation of retrograde transport may suppress normal immune signaling. The strategies utilized in this work depend on a paired model system approach. We have obtained preliminary information on the cellular function and targeting of a novel protein via a powerful yeast screen that provides information on thousands of genetic interactions. This approach has allowed us to design appropriate and efficient experimental plans in Drosophila. Our aims will provide the PI with in-depth skills in yeast and Drosophila genetics, transgenics, high-throughput genome-wide genetic screens, immunohistochemistry, and fluorescence imaging. This work will be strongly supported by a team that includes experts in the field in an interdisciplinary environment that will provide high-level training to the PI in immunity, multiple model system, genetics, and advanced molecular biology techniques.