Essential Cell Cycle Mechanisms in Toxoplasma

弓形虫的基本细胞周期机制

• 批准号: 7780037
• 负责人: Michael W White
• 金额: $ 36.42万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-05 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7780037
• 关键词: Acute; Affect; Apicomplexa; Biological; Categories; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cells; Chemicals; Chromosome Mapping; Chromosome Segregation; Chromosomes; Clinical; Code; Collection; Complement; Complex; Cosmids; Cryptosporidium; Cytoskeleton; DNA biosynthesis; Daughter; Defect; Disease; Drug Delivery Systems; Eimeria; Electrons; Essential Genes; Experimental Models; Family; Genes; Genetic; Genome; Genomic Library; Genomics; Growth; Growth Factor; High temperature of physical object; In Situ; Infection; Investigation; Knowledge; Light; Link; Location; Malaria; Measures; Messenger RNA; Methods; Microscopic; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Monitor; Mutagenesis; Nuclear; Organelles; Parasite Control; Parasites; Pathogenesis; Phase; Phenotype; Plasmodium; Population; Process; Proteins; Protocols documentation; Regulation; Role; Set protein; Severities; Staging; Techniques; Temperature; Testing; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Validation; Virulence; Virulent; Yeasts; asexual; base; burden of illness; chromosome replication; genetic analysis; insight; mutant; novel; nuclear division; pathogen; protein expression; public health relevance; research study; scaffold; segregation; spatial relationship; temperature sensitive mutant

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.

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，弓形虫弓形虫感染的发病机理主要由寄生虫的生长引起。从最近的遗传分析中可以清楚地看出，缩短的tachyzoite细胞周期是弓形虫中的关键毒力决定因素，但是关于该寄生虫如何调节复制的分子细节很少。弓形虫速氮族族除以一个新的循环，其中一组复杂的细胞器的重复与不寻常的双峰相协调，并且可能调节有符合有丝利分子的方面，并且可能调节特异性的发芽过程。弓形虫速二鼠的二元分裂正在接受内部发育，这为研究Apicomplexan细胞周期提供了优势，尽管这些研究将广泛地适用于该家族中其他病原体的增长，例如疟原虫，eimeriia和cryptosporidium，parasite细胞周期机械的知识是公平的。在此应用中，我们提出了一项全面的研究，对控制有毒的I型RH速二氮族复制的机制。在AIM 1中，我们将测试以下假设：G1，早期和晚期中至少有四个检查点，以及有丝分裂通过分析大量温度敏感（TS）生长突变体（165个总计），从而调节RH tachyzoite细胞周期，这是我们由化学诱变产生的。在AIM 2中，我们将研究以下假设，即tachyzoite检查点通过基于宇宙的遗传互补进行控制所需的以及独特的Apicomplexan蛋白，这将确定与特定TS-突变剂有关的基本基因。最后，在AIM 3中，我们将定义女儿/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，我们已经建立了用于产生和分析细胞周期突变体表型的高吞吐量方案，并且我们证明了在该寄生虫中基于强大的基于新的cosmid的基于新的cosmid方法。这些研究将提供有关调节寄生虫分裂的机制，并提供破坏寄生虫增殖的新靶标。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，但是很明显，通过寄生虫细胞周期的进展速率对于宿主中的寄生虫数量至关重要。在此提案中，我们将研究弓形虫弓形虫中细胞周期控制的遗传基础。这些研究中确定的基本生长因子将由该家族中的其他病原体（例如引起疟疾的疟原虫）共享，并可能代表负责寄生虫生长的新型蛋白质。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标可以在哪些新疗法上进行。