Deciphering the composite S-phase in Toxoplasma gondii

摘要 Apicomplexa寄生虫极大地增加了人类的疾病负担，包括约三分之一的人口 永久感染弓形虫。现有的治疗方法有限，对受影响最严重的人往往是有毒的。 免疫功能低下的患者群体。我们需要对寄生虫生物学有深刻的了解才能发展 有效的抗寄生虫药物。我们的小组专注于细胞周期机制的研究，这些机制是 寄生虫存活并提供了大量的可用药靶点。细胞周期程序协调细胞分裂和 确保遗传物质的遗传。Apicomplexan细胞周期与细胞明显不同 寄主的循环。尽管弓形虫速殖子按内生作用划分，类似于 传统的真核生物在细胞周期的组织和调控上存在着很大的差异。它包括 非典型的S期弓形虫内生性，这是我们研究的一个主要焦点。需要 适当的工具来研究复杂的顶端复合体细胞周期和非常规调控因子 严重阻碍了相关研究的开展。填补我们对人类基本生物学知识的一项重大空白 顶端复合体寄生虫和助推弓形虫细胞周期的研究，我们设计了一种新的荧光素 泛素化细胞周期指示剂(Fucci)探针。 在目标1的实验中，我们将检验弓形虫内生细胞周期包括一个 S/M/C复合阶段，占分区周期的近一半。使用我们的新ToxoFUCCI探测器，我们 将决定如何组织错综复杂的S/M/C阶段。在目标2中，我们将确定 S阶段的调控。设计的实验将检验这一假设，与常规的S阶段相反 细胞周期蛋白依赖性激酶(CDK)，CDK相关蛋白TgCrk5的控制释放调节DNA 速殖子中的复制。使用TgCrk5的条件表达式模型，我们将确定函数 隔离的TgCrk5和核TgCrk5，并鉴定TgCrk5底物。鉴于弓形虫缺乏 在传统的S-CDK底物上，我们有望发现一种新颖的TgCrk5网络。该项目将推进我们的 了解寄生虫生存的基本过程，并有很高的潜力发现未来的有效 毒品目标。

Abstract Apicomplexa parasites contribute significantly to human disease burden, including ~1/3 of human populations permanently infected with Toxoplasma gondii. Existing treatments are limited and often toxic to the most affected population of immunocompromised patients. We need a profound knowledge of parasite biology to develop efficient anti-parasitic drugs. Our group focuses on the studies of cell cycle mechanisms that are central to parasite survival and offer a wealth of druggable targets. The cell cycle program orchestrates cell division and ensures the inheritance of the genetic material. Apicomplexan cell cycles are strikingly different from the cell cycles of their hosts. Although T. gondii tachyzoite divides by endodyogeny that resembles a binary division of the conventional eukaryotes, there are substantial differences in cell cycle organization and regulation. It includes the atypical S-phase of Toxoplasma endodyogeny, which is a primary focus of our study. The need for appropriate tools to examine the intricacy of the apicomplexan cell cycle and unconventional regulators significantly impedes the related studies. To fill a major gap in our knowledge of the essential biology of apicomplexan parasites and boost the Toxoplasma cell cycle studies, we engineered a new Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probe. In the Aim 1 experiments, we will test the hypothesis that Toxoplasma endodyogenic cell cycle includes a composite S/M/C phase that runs for nearly half of the division cycle. Using our new ToxoFUCCI probes, we will determine how the intertwined S/M/C phase is organized. In Aim 2, we will determine the mechanism of the S-phase regulation. Designed experiments will test the hypothesis that, contrary to conventional S-phase cyclin dependent kinase (Cdk), controlled release of the sequestrated Cdk-related kinase TgCrk5 regulates DNA replication in the tachyzoites. Using the conditional expression model of TgCrk5, we will determine the functions of the sequestered and the nuclear TgCrk5 and identify the TgCrk5 substrates. Given that T. gondii lacks conventional S-Cdk substrates, we expect to discover a novel TgCrk5 network. The project will advance our knowledge of the fundamental process of parasite survival and have a high potential to discover future efficient drug targets.