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

大多数昆虫携带的细菌生活在它们的细胞内；这些细菌是从它们的母亲那里遗传的。这种细菌可能通过改善携带这种细菌的雌性宿主的健康或数量来操纵它们的昆虫宿主的繁殖。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”或“CI”）。目前，已知有两种常见的、不相关的细菌会引起昆虫的CI：沃尔巴克氏菌和本研究的重点——赫蒂吉红基数菌。该项目的主要目标是发现红雀胺引起昆虫CI的分子机制。这项工作的好处至少是双重的。像Cardinium这样的细菌以动物细胞分裂为目标，在研究Cardinium如何干扰细胞分裂时，可以更好地理解这一基本过程。其次，引起ci的细菌可用于有害生物或病媒管理。沃尔巴克氏体病原体降低了携带它的昆虫对病毒的易感性，目前正在世界各地的蚊子种群中引入，对减少病媒传播疾病和全球生物经济具有重大影响。该项目还将通过面向小学（亚利桑那大学昆虫发现和昆虫节）、高中（北卡罗来纳州立大学制作《动物操纵寄生虫》一书）和爱荷华州立大学乔治·华盛顿·卡弗实习计划)和本科生（通过在这三所机构的研究机会）的外展项目，为代表性不足的群体提供有吸引力的科学教育。对于感染Encarsia spp.（白蝇的寄生黄蜂）的Cardinium菌株，基因组学和转录组学研究已经确定了可能在CI和/或共生中重要的候选基因。主要的假设是：(1)在精子发生过程中参与修饰宿主DNA的Cardinium效应蛋白与睾丸有关，其他参与修饰精子的Cardinium效应蛋白存在于受感染黄蜂的卵巢中，(2)这些蛋白的作用可以通过鉴定它们相互作用的宿主蛋白来推断，(3)维生素B生物素在CI和/或共生中起关键作用。该项目提供了一个独特的机会，用不同的方法来测试这些假设，有四个目标：(1)比较雄性蛹Encarsia（“修饰”阶段）中Cardinium定位和CI候选基因表达与成虫中观察到的模式。(2)用差异蛋白质组学方法鉴定与CI相关的Cardinium和Encarsia蛋白。(3)利用异源表达鉴定CI候选蛋白的相互作用宿主蛋白。(4)研究Cardinium合成生物素对宿主蛋白的修饰作用。这个项目将阐明由Cardinium进行的复杂的CI生殖操作。Cardinium受到的关注相对较少，但它产生的CI表型与沃尔巴克氏体几乎相同，具有完全独立的真核相互作用基因，质粒而不是噬菌体，以及不寻常的6型分泌系统。对cardinum候选基因功能、表达和宿主靶点的了解将阐明这两个系统中的CI和共生关系。该奖项反映了美国国家科学基金会的法定使命，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.