The AP2 factors required for Toxoplasma replication

描述(申请人提供)：Apicomplexan寄生虫是人类的重要病原体，可引起疾病，对全球健康造成广泛影响。这些专性细胞内寄生虫的生命周期是复杂的，涉及确保寄生虫成功传播的多个增殖期和非增殖期。病原学、致病力和疾病严重性受到无性阶段生长速度的严重影响，无性阶段生长速度可能导致寄生虫生物量增加和显著的组织破坏和炎症。因此，控制寄生虫生长速度的机制对毒力很重要。建立新的子寄生虫需要严格控制蛋白质产品的传递，在疟原虫和弓形虫中，2800个mRNA的级联在每个分裂周期中连续展开。顶端复合体病原菌的转录机制目前还知之甚少，然而，最近在顶端复合体中发现的类植物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地表达，其时间分布在整个分裂周期。我们假设，这些因素是制造新寄生虫所需的基因产品“及时”交付的原因。TgAP2因子通过与特定启动子DNA序列结合，调节细胞周期转录，从而影响弓形虫复制的效率和保真度。在这个建议中，我们将通过确定关键细胞周期TgAP2蛋白的准确表达顺序，通过全基因组蛋白结合研究发现它们调节的启动子，并通过产生丢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。这些研究将提供一套产生寄生虫复制所需的新机制的基础知识，这些机制与弓形虫在免疫受损的人类宿主中的生长引起的急性疾病有关。

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.