Mechanism of a Male Killing Toxin in a Drosophila endosymbiont

项目摘要/摘要 细菌蛋白毒素及其靶标的共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，这些共生体在 感染。经母体传播的细胞内寄生虫普氏螺旋体编码一种这样的毒素。 在其自然宿主黑腹果蝇中导致雄性死亡的螺旋体雄杀素(Sprodium androsidin或SPEED)。这 拟议的项目旨在了解细菌毒素Sped是如何识别宿主细胞环境的 特别是杀死雄性。为此，我们使用了螺旋体-果蝇模型系统和 遗传学和分子生物学，以确定特定的宿主蛋白的目标和毒素如何结合 这些蛋白质。 先前的研究表明，螺旋体通过Spaid检测到男性特异性致死物的存在 在苍蝇身上很复杂。这种复合体负责雄性果蝇的剂量补偿，雄性果蝇缺乏第二个拷贝 在雄性的早期发育过程中形成，是果蝇性别决定的一部分。 路径。根据这一结果，我们假设果蝇对斯佩德存在天然抵抗力 SPEAD的靶点是剂量补偿复合体(DCC)的一部分，并且SPAID直接 绑定到DCC组件，改变它们的活动。我们强劲的初步数据表明，我们是正确的，因为我们 寻找果蝇的自然变种(在DGRP内)，在存在的情况下产生雄性 斯帕德和/或螺旋体。我们还发现，组织特异性的DCC组分的敲除部分抑制了 对男性组织有明显的毒性。 对于这一假设，在强劲的初步数据的指导下，我们建议追求两个具体目标 确定直接与Sped毒素相互作用的宿主蛋白，并表征宿主内的变异 蛋白质和Spay在分子和细胞水平上影响男性的死亡。我们将(1)使用以下工具确定付费目标 酵母双杂交筛选和免疫共沉淀法，(2)利用天然 果蝇的变异和遗传筛选。 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.