The AP2 factors required for Toxoplasma replication

描述（由申请人提供）：顶复合体寄生虫是人类的重要病原体，引起对全球健康有广泛影响的疾病。这些专性细胞内寄生虫的生命周期是复杂的，包括多个增殖和非生长阶段，确保寄生虫成功传播。发病机制、毒力和疾病严重程度受到无性期生长速度的严重影响，无性期生长速度可导致寄生虫生物量增加和显著的组织破坏和炎症。因此，控制寄生虫生长速率的机制对毒力很重要。构建新的子体寄生虫需要严格控制蛋白质产物的传递，而在疟原虫和弓形虫中，在每个分裂周期中，会有1万8千个mrna级联序列展开。人们对顶复合体病原体的转录知之甚少，然而，最近在顶复合体中发现的植物样转录因子揭示了一组可能控制这些寄生虫细胞周期转录的重要因子。近三分之一的弓形虫AP2基因在速殖子复制过程中周期性表达，其时间分布在分裂周期中。我们假设这些因素负责“及时”传递构建新寄生虫所需的基因产物。TgAP2因子通过与特定启动子DNA序列结合，调控细胞周期转录，从而影响弓形虫复制的效率和保真度。在本研究中，我们将通过确定关键细胞周期TgAP2蛋白的精确表达顺序，通过全基因组蛋白结合研究发现它们调节的启动子，以及通过产生缺失和错误表达突变体来验证这些蛋白在寄生虫复制中的体内功能，来表征TgAP2因子在寄生虫细胞周期中的作用。这些研究将为产生与弓形虫在免疫功能低下的人类宿主中生长引起的急性疾病相关的寄生虫复制所需的一套新机制提供基础知识。

DESCRIPTION (provided by applicant): Apicomplexan parasites are important pathogens of humans that cause diseases with widespread impacts on global health. The life cycles of these obligate intracellular parasites are complex, involving multiple proliferative and non-growing stages that ensure successful parasite transmission. Pathogenesis, virulence, and disease severity are critically influenced by asexual stage growth rates that can lead to increased parasite biomass and significant tissue destruction and inflammation. Consequently, mechanisms controlling parasite growth rate are important to virulence. Building new daughter parasites requires tight control over the delivery of protein products, and in Plasmodium and Toxoplasma a cascade of >2800 mRNAs serially unfolds during each division cycle. Transcription in apicomplexa pathogens is poorly understood, however, the recent discovery of plant-like transcription factors in the Apicomplexa has uncovered an important set of factors that may control cell cycle transcription in these parasites. Nearly a third of all Toxoplasma AP2 genes are periodically expressed during tachyzoite replication with a timing distributed across the division cycle. We hypothesize that these factors are responsible for the "just-in-time" delivery of gene products required to build new parasites. Through their binding to specific promoter DNA sequence, TgAP2 factors regulate cell cycle transcription, and thereby influence the efficiency and fidelity of Toxoplasma replication. In this proposal, we will characterize the role of TgAP2 factors in the parasite cell cycle by determining the precise order of expression for key cell cycle TgAP2 proteins, by discovering the promoters they regulate through whole-genome protein binding studies, and by validating the in vivo function of these proteins in parasite replication through the generation of loss and mis- expression mutants. These studies will provide fundamental knowledge of a new set of mechanisms required to produce parasite replication that is relevant to acute disease caused by the growth of Toxoplasma in the immunocompromised human host. 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 parasite replication are not understood, but it is clear that the factors regulating progression through the parasite cell cycle are critical to parasite burden in the host. In this proposal, we will investigate the genetic program responsible for the delivery of proteins at the right time and in the correct amount to produce viable progeny. The factors orchestrating this complex process are not found in the human host, and thus, new potential drug targets will be identified upon which novel therapies may be developed.