Collaborative Research: The saboteur's tools: mechanisms for host reproductive manipulation by the bacterial arthropod endosymbiont Cardinium hertigii

大多数昆虫携带生活在细胞内的细菌;这些细菌是从它们的母亲那里遗传来的。这些细菌可以操纵昆虫宿主的繁殖，从而改善携带细菌的雌性宿主的健康或数量。与这项研究相关的是，这些细菌“共生体”可以破坏宿主精子，使得没有细菌的雌性产下的受精卵在生命早期死亡（“细胞质不相容性”或“CI”）。目前，已知两种常见的、不相关的细菌在昆虫中引起CI：沃尔巴克氏体和本研究的重点--赫氏Cardinium hertigium。该项目的主要目标是发现Cardinium引起昆虫CI的分子机制。这项工作的好处至少是双重的。如Cardinium的细菌靶向动物细胞分裂，当检查Cardinium如何干扰它时，可以更好地理解这一基本过程。其次，CI引起的细菌可用于害虫或媒介管理。CI剂Wolbachia降低了携带它的昆虫对病毒的易感性，目前正在世界各地引入蚊子种群，对减少病媒传播疾病和全球生物经济具有重大意义。该项目还将通过小学（亚利桑那大学昆虫发现和昆虫节）、高中（在北卡罗来纳州和爱荷华州乔治华盛顿卡弗实习计划中制作一本书“动物操纵寄生虫”）和本科生（通过所有三个机构的研究机会）的外展计划，为代表性不足的群体提供参与性科学教育。对于感染恩蚜小蜂属的Cardinium菌株，从粉虱的寄生蜂中，基因组学和转录组学研究已经鉴定了可能在CI和/或共生中重要的候选基因。主要假设是：（1）在精子发生过程中参与修饰宿主DNA的Cardinium效应蛋白与睾丸相关，而其他参与拯救修饰精子的Cardinium效应蛋白存在于受感染黄蜂的卵巢中，（2）这些蛋白质的作用可以通过鉴定它们与之相互作用的宿主蛋白质来推断，以及（3）B族维生素生物素在CI和/或CI/B中具有关键作用。或共生关系。该项目提供了一个独特的机会，以测试这些假设与各种各样的方法，在四个目标：（1）比较Cardinium本地化和CI候选基因表达的雄性蛹Encarsia，“修改”阶段，在成年人中观察到的模式。(2)用差异蛋白质组学鉴定CI中涉及的Cardinium和Encarsia蛋白。(3)使用异源表达来鉴定Cl候选蛋白的相互作用宿主蛋白。(4)研究Cardinium合成的生物素在宿主蛋白质修饰中的作用。该项目将揭示Cardinium复杂的CI生殖操纵。Cardinium受到的关注相对较少，但产生了与Wolbachia几乎相同的CI表型，似乎是完全独立的真核生物相互作用基因，质粒而不是噬菌体，以及不寻常的6型分泌系统。关于Cardinium候选基因功能、表达和宿主靶点的了解将阐明两个系统中的CI和共生关系。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Most insects carry bacteria that live within their cells; these bacteria are inherited from their mothers. The bacteria may manipulate their insect hosts’ reproduction in ways that improves the health or number of female hosts carrying the bacterium. Relevant to this study, these bacterial “symbionts” can sabotage host sperm such that fertilized eggs laid by females without the bacterium die early in life (“cytoplasmic incompatibility,” or “CI”). Currently, two common, unrelated bacteria are known to cause CI in insects: Wolbachia, and Cardinium hertigii, the focus of this study. The main goal of this project is to discover the molecular mechanism by which Cardinium causes CI in insects. The benefits of this work are at least two-fold. Bacteria such as Cardinium target animal cell division, a fundamental process that can be understood better when examining how Cardinium interferes with it. Secondly, CI-causing bacteria may be used for pest or vector management. The CI agent Wolbachia reduces the susceptibility to viruses of insects that carry it, and is currently being introduced around the world to mosquito populations, with major implications for reduction of vector-borne disease and the global bioeconomy. The project will also provide engaging science education targeted for under-represented groups through outreach programs for elementary school (U Arizona Insect Discovery and Insect Festival), high school (production of a book “Animal Manipulating Parasites at NC State, and Iowa State George Washington Carver Internship program) and undergraduate students (via research opportunities at all three institutions). For Cardinium strains that infect Encarsia spp., parasitic wasps of whiteflies, genomic and transcriptomic studies have identified candidate genes that are likely to be important in CI and/or symbiosis. The central hypotheses are that (1) Cardinium effector proteins involved in modifying host DNA during spermatogenesis are associated with the testes, and other Cardinium effectors involved with rescue of modified sperm are present in the ovaries of infected wasps, (2) the role of these proteins can be inferred by identifying the host proteins they interact with and (3) that the B vitamin biotin has a critical role in CI and/or the symbiosis. The project presents a unique opportunity to test these hypotheses with a diverse array of approaches, in four objectives: (1) Compare Cardinium localization and CI candidate gene expression in male pupal Encarsia, the “modification” stage, with patterns observed in adults.(2) Identify Cardinium and Encarsia proteins involved in CI with differential proteomics. (3) Use heterologous expression to identify interacting host proteins of CI candidate proteins. (4) Investigate the role of Cardinium synthesized biotin in modification of host proteins. This project will shed light on the sophisticated CI reproductive manipulation by Cardinium. Cardinium has received comparatively little attention, but produces a virtually identical CI phenotype to Wolbachia with what appears to be completely independent eukaryote-interacting genes, a plasmid and not a phage, and an unusual type 6 secretion system. What is learned about Cardinium candidate gene function, expression and host targets will illuminate CI and symbiosis in both systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.