Essential Cell Cycle Mechanisms in Toxoplasma

描述（由申请人提供）：增加的寄生虫负担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是由寄生虫的生长引起的。从最近的遗传分析中可以清楚地看出，缩短的速殖子细胞周期是弓形虫毒力的关键决定因素，但我们对这种寄生虫如何调节复制的分子细节知之甚少。弓形虫速殖子通过一个新的周期进行分裂，在这个周期中，一组复杂的细胞器的复制与一个不寻常的双峰S期相协调，一个门特异性的出芽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生发育的二元分裂为研究顶复合体细胞周期提供了优势，尽管这些研究将广泛应用于该家族中其他病原体的生长，如疟原虫、艾美耳虫和隐孢子虫，在这些领域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，我们建议对控制毒性I-RH型速殖子复制的机制进行全面研究。在Aim 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）生长突变体（共165个），验证G1、S早期和S晚期以及有丝分裂中至少有四个检查点调节RH速殖子细胞周期的假设。在Aim 2中，我们将通过基于cosmid的遗传互补来研究速殖子检查点控制所需的已知以及独特的顶复合体蛋白的假设，这将确定特定ts突变体中涉及的必要基因。最后，在Aim 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方案来产生和分析细胞周期突变体的表型，我们已经证明了在这种寄生虫中基于cosmid的遗传互补的强大的新方法。这些研究将为寄生虫分裂的调控机制提供新的见解，并为破坏寄生虫增殖提供新的靶点。公共卫生相关性：最近对寄生虫毒力的遗传分析证实，寄生虫负担增加与刚地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体内的寄生虫数量至关重要。在本研究中，我们将研究刚地弓形虫细胞周期控制的遗传基础。在这些研究中确定的基本生长因子将与该家族的其他病原体共享，例如引起疟疾的疟原虫，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分子基础的研究，可以发现新的潜在药物靶点，从而开发出新的治疗方法。

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.