Mechanism of a Male Killing Toxin in a Drosophila endosymbiont

项目总结/摘要 细菌蛋白毒素与其靶蛋白的协同进化导致了许多新蛋白的发现 分子生物学中的修饰和应用的发展。节肢动物，最多样化的门 在地球上，是许多细菌共生体的宿主，这些细菌共生体分泌各种功能未知的毒素， 感染母体传播的胞内寄生虫螺原体编码一种这样的毒素 （螺原体androcidin或Spayed），在其自然宿主果蝇中导致雄性死亡。这 拟议的项目旨在了解细菌毒素Spaid如何识别宿主细胞环境， 专门杀死男性为此，我们使用螺原体-果蝇模型系统和以下组合： 遗传学和分子生物学，以确定Spaid靶向的特定宿主蛋白质以及毒素如何结合 这些蛋白质。 以前的工作表明，螺原体，通过Spaid，检测男性特定致命的存在， 复杂的苍蝇。这种复合物负责雄性果蝇的剂量补偿，而雄性果蝇缺乏第二个副本 的X.它在雄性果蝇的早期发育过程中形成，是果蝇性别决定的一部分 通路鉴于这一结果，我们假设果蝇对Spaid存在天然抗性 人群，Spaid的目标是剂量补偿复合物（dcc）的一部分，并且Spaid直接 与DCC成分结合，改变它们的活性。我们强大的初步数据表明，我们是正确的，因为我们 发现黑腹果蝇的自然变种（DGRP内），在存在 Spaid和/或Spiroplasma。我们还发现，组织特异性敲低dcc组分部分抑制了 在雄性组织中扩散毒性。 针对这一假设，并在强有力的初步数据的指导下，我们提出了两个具体目标， 鉴定与Spayed毒素直接相互作用的宿主蛋白质，并表征宿主内的变异 蛋白质和Spaid在分子和细胞水平上影响雄性杀伤。我们将（1）使用 酵母双杂交筛选和免疫共沉淀，（2）使用天然药物鉴定Spaid毒性的抑制剂 果蝇的基因变异和基因筛选。 这些实验的结果将说明细菌毒素如何操纵宿主细胞环境。

PROJECT SUMMARY/ABSTRACT The co-evolution of bacterial protein toxins and their targets has led to the discovery of many novel protein modifications and the development of applications in molecular biology. Arthropods, the most diverse phylum on Earth, are hosts to many bacterial symbionts that secrete diverse toxins of unknown function during infection. The maternally transmitted, intracellular parasite Spiroplasma poulsonii encodes one such toxin (Spiroplasma androcidin or Spaid) that causes male killing in its natural host Drosophila melanogaster. This proposed project aims to understand how the bacterial toxin Spaid recognizes the host cellular environment to specifically kill males. To that end, we use the Spiroplasma-Drosophila model system and a combination of genetics and molecular biology to identify specific host proteins targeted by Spaid and how the toxin binds these proteins. Previous work has suggested that Spiroplasma, via Spaid, detects the presence of the male specific lethal complex in flies. This complex is responsible for dosage compensation in male flies, which lack a second copy of the X. It is formed during early development in males and is part of the Drosophila sex determination pathway. Given this result, we hypothesized that natural resistance to Spaid would exist in Drosophila populations, that the target of Spaid is part of the dosage compensation complex (dcc), and that Spaid directly binds to dcc components, altering their activity. Our strong preliminary data suggest that we are correct, as we find natural variants of Drosophila melanogaster (within the DGRP), which produce males in the presence of Spaid and/or Spiroplasma. We also find that tissue specific knockdown of dcc components partially suppresses Spaid toxicity in male tissue. Towards this hypothesis, and guided by strong preliminary data, we propose to pursue two Specific Aims to identify host proteins that interact directly with the Spaid toxin and characterize how variation within host proteins and Spaid affect male killing at the molecular and cellular level. We will (1) Identify Spaid targets using a yeast 2-hybrid screen and co-immunoprecipitation, (2) identify suppressors of Spaid toxicity using natural variation in Drosophila and a genetic screen. Results from these experiments will illustrate how a bacterial toxin manipulates the host cellular environment.