Essential Cell Cycle Mechanisms in Toxoplasma

描述(由申请人提供)：寄生虫负担增加是临床弓形虫病严重程度的关键因素，因此，弓形虫感染的致病机制主要是由寄生虫的生长引起的。最近的遗传分析表明，速殖子细胞周期缩短是弓形虫毒力的关键决定因素，但我们对这种寄生虫的复制是如何调控的分子细节知之甚少。弓形虫速殖子通过一个新的周期来划分，在这个周期中，一组复杂细胞器的复制与一个不寻常的双峰S期相协调，并且特定于门的萌发过程与有丝分裂的各个方面同步并可能调节。弓形虫速殖子进行内源性分裂为研究顶端复合体细胞周期提供了有利条件，尽管这些研究将广泛适用于该家族中其他病原体的生长，如疟原虫、艾美耳球虫和隐孢子虫，在这些地方我们对寄生虫细胞周期机制的了解同样不足。在这一应用中，我们提出了一项控制毒力I-RH速殖子复制的机制的全面研究。在目标1中，我们将通过分析我们通过化学诱变产生的大量温度敏感(Ts)生长突变(共165个)来检验这一假设，即G1、S早期和晚期以及有丝分裂至少有四个检查点调控RH速殖子的细胞周期。在目标2中，我们将检验这一假说，即通过基于粘粒的遗传互补，速殖子检查点控制需要已知和独特的顶复合体蛋白，这将确定特定ts突变所涉及的必要基因。最后，在目标3中，我们将定义子代/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的表型的方案，并且我们已经展示了在这种寄生虫中进行遗传互补的基于粘粒的新方法。这些研究将为调节寄生虫分裂的机制提供洞察力，并提供扰乱寄生虫增殖的新靶点。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。在这项建议中，我们将研究弓形虫细胞周期控制的遗传学基础。这些研究中确定的基本生长因子将与该家族中的其他病原体共享，如导致疟疾的疟疾，并可能代表导致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，在此基础上可能开发新的治疗方法。

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.