Ribosome structure determination from Apicomplexan parasites

项目摘要/摘要 Apicomplexa门含有许多寄生虫，这些寄生虫会感染人类和动物并导致疾病， 包括导致疟疾、弓形虫病和巴贝斯虫病的病原体。巴贝斯虫是一种由壁虱传播的细胞内病毒。 引起严重到致命疾病的寄生虫，由于气候变化，地理分布日益扩大 以及增加林区的郊区开发。目前还没有针对人类巴贝斯虫病的疫苗，而且 目前的治疗方法疗效有限。考虑到巴贝斯虫物种复杂的生活史，它们的生存 依赖于对它们基因表达的精确控制，这主要是在转录后调节的(例如， 在蛋白质合成期间)。巴贝斯虫物种有一种独特的非光合作用细胞器，称为质外体， 源自次要的内生共生事件。除了在细胞质中有核糖体和 线粒体，质外体含有自己的核糖体。人们对这些核糖体的结构知之甚少， 尽管它们被认为是高度简化的，含有类似原核生物的成分。它们被抑制在 抗寄生虫活性有限的抗菌药物。任何一种分子结构的缺失 顶体核糖体限制了抗生素的合理设计，使其具有更好的抗寄生虫性能。在第一个 本研究的目的是，我们将建立一株表达血凝素(HA)的巴贝斯虫分离株。 质外体外膜上标记的磷酸三糖转运体。这一策略将允许亲和力 纯化高质量的细胞器制剂，然后纯化质外体核糖体。在 第二个目标，我们将定义质外体核糖体的成分，并使用低温冷冻技术确定它们的结构。 电子显微镜(冷冻-EM)。因此，我们的目标是阐明 顶端复合体寄生虫用来在高度专门化的质外体细胞器中合成蛋白质。我们的工作将 为研究这些寄生虫的基因调控机制奠定了基础，并有助于启发 设计更好的抗寄生虫剂。

PROJECT SUMMARY/ABSTRACT The Apicomplexa phylum contains many parasites that infect and cause disease in humans and animals, including the agents that cause malaria, toxoplasmosis, and babesiosis. Babesia is a tick-borne intracellular parasite that causes severe to fatal diseases, with broadening geographic distribution because of climate change and increasing suburban developments in forested areas. There is no vaccine against human babesiosis, and the current therapies have limited efficacy. Considering the complex life cycle of Babesia species, their survival depends on the precise control of their gene expression, which is mostly regulated post-transcriptionally (e.g., during protein synthesis). Babesia species have a unique, non-photosynthetic organelle named apicoplast, derived from a secondary endosymbiotic event. In addition to having ribosomes in its cytoplasm and mitochondria, the apicoplast contains its own ribosomes. Little is known about the structure of these ribosomes, although they are thought to be highly simplified, with prokaryotic-like components. They are inhibited by antibacterial drugs that have some limited antiparasitic activity. The absence of a molecular structure of any apicoplast ribosomes limits the rational design of antibiotics with improved antiparasitic properties. In the first aim of this research proposal, we will establish a Babesia divergens strain expressing an hemagglutinin (HA)- tagged triose phosphate transporter on the outer membrane of the apicoplast. This strategy will allow the affinity purification of a high-quality organelle preparation, followed by the purification of apicoplast ribosomes. In the second aim, we will define the components of apicoplast ribosomes and determine their structure using cryo- electron microscopy (cryo-EM). Our goal is thus to elucidate the fundamental molecular machinery that apicomplexan parasites use to synthesize proteins in the highly specialized apicoplast organelle. Our work will lay the foundation for studies that interrogate these parasites' gene regulation mechanisms and help inspire the design of better antiparasitic agents.