Essential Cell Cycle Mechanisms in Toxoplasma

描述（由申请方提供）：寄生虫负荷增加是临床弓形虫病严重程度的关键因素，因此，弓形虫感染的发病机制主要由寄生虫生长引起。从最近的遗传分析，它已经变得很清楚，一个缩短的速殖子细胞周期是一个关键的毒力决定因素，在弓形虫，但我们有一些分子的细节如何复制在这种寄生虫的调节。弓形虫速殖子划分一个新的周期，其中一组复杂的细胞器的复制是协调与一个不寻常的双峰S期和门特定的出芽过程是同步的，并可能调节，有丝分裂的方面。弓形虫速殖子进行endodyrification的二元分裂提供了优势的apicomplexan细胞周期的调查，虽然这些研究将广泛适用于其他病原体在这个家庭，如疟原虫，艾美球虫，隐孢子虫，我们的知识寄生虫细胞周期机制的增长同样不足。在这个应用中，我们提出了一个全面的研究机制控制复制的毒性I-RH型速殖子。在目的1中，我们将测试的假设，至少有四个检查点在G1，早期和晚期S，和有丝分裂调节RH速殖子细胞周期通过分析大量收集的温度敏感（TS）的生长突变体（165个），我们已经产生了化学诱变。在目标2中，我们将研究的假设，即已知的，以及独特的顶复体蛋白所需的速殖子检查点控制通过粘粒为基础的遗传互补，这将确定参与特定的TS突变体的必需基因。最后，在目标3中，我们将定义子细胞/有丝分裂细胞骨架在调节控制染色体复制的检查点中的作用。在初步研究中，我们已经建立了高通量的生产和分析细胞周期突变体的表型协议，我们已经证明了强大的新的基于粘粒的方法，在这种寄生虫的遗传互补。这些研究将提供深入了解调节寄生虫分裂的机制，并提供新的靶点来破坏寄生虫增殖。公共卫生相关性：最近对寄生虫毒力的遗传分析证实，寄生虫负担增加与弓形虫引起的疾病之间存在重要联系。控制寄生虫分裂周期的因素尚不清楚，但很明显，寄生虫细胞周期的进展速度对宿主中的寄生虫数量至关重要。在本研究中，我们将探讨弓形虫细胞周期调控的遗传基础。这些研究中确定的基本生长因子将被该家族中的其他病原体共享，例如导致疟疾的疟原虫，并且可能代表负责寄生虫生长的新蛋白质。因此，通过对寄生虫细胞周期的分子基础的研究，将确定新的潜在药物靶点，从而可以开发新的治疗方法。

DESCRIPTION (provided by applicant): Increased parasite burden is a key factor in the severity of clinical toxoplasmosis and thus, pathogenesis in Toxoplasma gondii infections is caused primarily by the growth of the parasite. From recent genetic analysis it has become clear that a shortened tachyzoite cell cycle is a key virulence determinant in Toxoplasma, yet we have few molecular details about how replication is regulated in this parasite. Toxoplasma tachyzoites divide by a novel cycle where the duplication of a complex set of organelles is coordinated with an unusual bimodal S phase and a phylum-specific budding process is synchronized with, and may regulate, aspects of mitosis. The binary division of Toxoplasma tachyzoites undergoing endodyogeny offers advantages for the investigation of the apicomplexan cell cycle, although these studies will apply broadly to the growth of other pathogens in this family, such as Plasmodium, Eimeria, and Cryptosporidium, where our knowledge of parasite cell cycle mechanisms is equally deficient. In this application, we propose a comprehensive study of the mechanisms controlling the replication of virulent Type I-RH tachyzoites. In Aim 1, we will test the hypothesis that at least four checkpoints in G1, early and late S, and mitosis regulate the RH tachyzoite cell cycle through the analysis of a large collection of temperature sensitive (ts) growth mutants (165 total), which we have produced by chemical mutagenesis. In Aim 2, we will examine the hypothesis that known as well as unique apicomplexan proteins are required for tachyzoite checkpoint control through cosmid-based genetic complementation, which will identify the essential genes involved in specific ts-mutants. Finally, in Aim 3, we will define the role of the daughter/mitotic cytoskeletons in regulating checkpoints that control chromosome replication. In preliminary studies, we have established high throughput protocols for producing and analyzing the phenotype of cell cycle mutants and we have demonstrated robust new cosmid-based methods for genetic complementation in this parasite. These studies will provide insight into the mechanisms regulating parasite division and provide new targets upon which to disrupt parasite proliferation. PUBLIC HEALTH RELEVANCE: Recent genetic analysis of parasite virulence confirms that there is an important link between increased parasite burden and disease caused by Toxoplasma gondii. The factors that control the parasite division cycle are not understood, but it is clear that the rate of progression through the parasite cell cycle is critical to parasite numbers in the host. In this proposal, we will investigate the genetic basis for cell cycle control in Toxoplasma gondii. The essential growth factors identified in these studies will be shared by other pathogens in this family, such as Plasmodium, which causes malaria, and will likely represent novel proteins responsible for parasite growth. Therefore, through this investigation of the molecular basis of the parasite cell cycle, new potential drug targets will be identified upon which novel therapies may be developed.