The Mechanism of Cytoplasmic Incompatibility

项目总结： 沃尔巴克氏菌是一种内共生细菌，它是一种有希望的、经济有效的工具来遏制寨卡病毒和 登革热虫媒病毒的传播基于两个关键方面。首先，沃尔巴克氏菌通过以下方式阻断致病RNA病毒 抑制它们在节肢动物体内的复制。其次，沃尔巴克氏杆菌自私地改变精子和卵子的过程被称为 细胞质不亲和性(CI)可驱使细菌进入宿主群体。CI以胚胎的形式表达 受感染的雄性和未受感染的雌性杂交的致命性，但这种致命性在杂交中得到挽救 在感染的雄性和感染的雌性之间，这是沃尔巴克氏菌的传播性。因此，CI是 在现场试验中部署，以抑制蚊子种群数量或用 对虫媒病毒感染具有抵抗力的感染者。我们最近发现了一种脑梗塞的遗传模型， 两个基因(cifA和cifB)在睾丸表达时会导致胚胎死亡，而cifA和cifB基因的表达 其中一个相同的基因(CIFA)在卵巢中表达时可以拯救致命性。在胚胎致死之前，有几个 受精后会出现缺陷，包括父亲核膜的延迟破裂，有丝分裂停止，以及 染色质桥接。由于沃尔巴克氏杆菌在精子发生过程中被剥离，CIFA造成的缺陷 而cifB可能是由于受精前对精子完整性的不明特征的损害。尽管四十年来 密集的研究和当前应用于病媒控制的努力，围绕这些受精前的细节 损伤仍然是一个中心谜团。建议研究的首要假设是CIFA和CIFB。 编码的蛋白质会改变精子的完整性，从而导致CI。在目标1中，我们将使用细胞化学、酶和 转基因检测以确定精子改变的类型、强度和遗传基础 世界蚊子计划在野外试验中释放了来自wMel Wolbachia的CIF蛋白。在目标2中，我们将 研究Cif蛋白在精子发生和储存过程中的定位模式。我们还将确定 Cif蛋白与宿主蛋白或Wolbachia蛋白之间的重要相互作用。基因敲除和转基因 实验将询问蛋白质-蛋白质相互作用对CI表达的必要性。终于在AIM 3，我们将评估天然CI蛋白质或蛋白质复合体能否通过实验分离并成功 注射到未感染的宿主中，总结CI和抢救。在这些实验中，我们将评估一部小说 沃尔巴克氏菌Cif蛋白与WO噬菌体颗粒的相关性研究研究还没有得出结论 对困扰配子的天然CI缺陷的机械性突破，以及对部署的兴趣的上升 为了遏制虫媒病毒的传播，迫切需要了解支撑沃尔巴克氏菌的事件 CI驱动系统。如果成功，这项研究将从根本上推进CI修改的研究，并为 沃尔巴克氏杆菌作为一种控制全球节肢动物的天然工具，其持续的效力和交付。

Project Summary: Wolbachia are a genus of endosymbiotic bacteria that comprise a promising, cost-effective tool to curb Zika and dengue arboviral transmission based on two key facets. First, Wolbachia block pathogenic RNA viruses by inhibiting their replication in arthropods. Second, Wolbachia selfishly alter sperm and egg via a process termed cytoplasmic incompatibility (CI) that can drive the bacteria into host populations. CI is expressed as embryonic lethality in crosses between infected males and uninfected females, but this lethality is rescued in crosses between infected males and infected females, which are the transmitting sex of Wolbachia. Consequently, CI is deployed in field trials to either suppress mosquito population sizes or replace uninfected populations with infected individuals resistant to arboviral infection. We recently exposed a genetic model of CI wherein expression of two genes (cifA and cifB) causes embryonic lethality when expressed in testes, and expression of one of the same genes (cifA) rescues lethality when expressed in ovaries. Prior to embryonic lethality, several post-fertilization defects arise including delayed breakdown of the paternal nuclear envelope, mitotic arrest, and chromatin bridging. As Wolbachia are stripped from sperm during spermatogenesis, the defects caused by cifA and cifB may be due to uncharacterized pre-fertilization impairments to sperm integrity. Despite four decades of intense research and current applications to vector control efforts, the details surrounding these pre-fertilization impairments remain a central enigma. The overarching hypothesis of the proposed research is cifA and cifB encode proteins that alter sperm integrity to cause CI. In Aim 1, we will use cytochemical, enzymatic, and transgenic assays to determine the types, strength, and genetic bases of sperm modifications imposed by the Cif proteins from wMel Wolbachia released in field trials by the World Mosquito Program. In Aim 2, we will investigate localization patterns of the Cif proteins during spermatogenesis and storage. We will also identify important interactions between the Cif proteins and either host or Wolbachia proteins. Knockout and transgenic experiments will interrogate the necessity of the protein-protein interactions for expression of CI. Finally in Aim 3, we will evaluate if the natural CI proteins or protein complex can be experimentally isolated and successfully injected into uninfected hosts to recapitulate CI and rescue. In these experiments, we will evaluate a novel association between the Cif proteins and bacteriophage WO particles from Wolbachia. Studies have yet to yield a mechanistic breakthrough for the natural CI defects afflicting gametes, and the rising interest in deploying Wolbachia to curb arbovirus transmission necessitates an urgent understanding of the events underpinning the CI drive system. If successful, this research will fundamentally advance studies of CI modifications and inform Wolbachia's ongoing efficacy and delivery as a natural tool to control arthropods worldwide.