GCN5-mediated transcription in AIDS pathogen Toxoplasma

描述（由申请人提供）：刚地弓形虫是一种对艾滋病患者构成重大风险的原生动物寄生虫，被NIAID列为生物防御的B类优先病原体。致病过程的基础是寄生虫发展成潜伏的组织囊肿的能力，这种组织囊肿可以在免疫力受损时以快速生长的形式重新出现。加深对这一发育过程的了解将为治疗干预提供新的机会。我们在第一个奖项期间的工作重点是GCN5组蛋白乙酰转移酶（HATs），并导致发现组蛋白乙酰化与弓形虫发育相关的基因表达相关。我们采用遗传学方法来确定我们在弓形虫中发现的两个不同的gcn5家族hat （TgGCN5-A和-B）的作用。我们发现TgGCN5缺失会损害弓形虫上调关键发育表达基因的能力。我们还确定，在酵母双杂交筛选中，与TgGCN5相互作用的一些蛋白质具有典型的转录调节结构域；这一点很重要，因为在弓形虫基因组中检测不到dna结合转录因子，这阻碍了了解基因表达是如何被调节的努力。弓形虫如何在急性期调节转录有效生长，并在应激期发展成潜伏的囊肿，这是我们知识的主要空白，阻碍了我们对抗机会性感染的能力。第一个奖项产生的数据使我们能够将重点放在定义寄生虫发育背景下GCN5介导的基因调控机制上，这是寄生虫发病机制的基础。我们假设TgGCN5 hat通过与调节发育基因表达的新蛋白形成不同的复合物，在弓形虫的发病机制中发挥关键作用。我们的具体目标包括：(1)确定TgGCN5 hat在发病机制中的作用；(2)阐明弓形虫发育过程中TgGCN5复合物的差异；(3)明确TgGCN5被募集到靶基因启动子以协调发育性基因表达的机制。拟议的研究利用了我们已经开发的新试剂、技术和转基因寄生虫。所产生的数据将阐明弓形虫的发育转变背后的机制，这些转变负责疾病进展，从而揭示新的治疗干预点。公共卫生相关性：刚地弓形虫是一种原生动物寄生虫，作为艾滋病患者的机会性感染导致重大疾病。慢性弓形虫病目前是无法治愈的，因为寄生虫能够发展成囊肿，一直潜伏到免疫抑制。驱动这些囊肿发展的机制是我们研究的重点，因为了解弓形虫如何发展将确定新的治疗干预点。我们正在采用一种创新的方法，利用转录调节因子作为阐明寄生虫发育机制的手段。基因表达调控在这一致病过程中起关键作用；因此，我们的研究结果很有可能转化为对抗弓形虫等机会性传染病的有用新疗法。

DESCRIPTION (provided by applicant): Toxoplasma gondii is a protozoan parasite that poses a significant risk to AIDS patients and is listed by NIAID as a Category B Priority Pathogen for biodefense. Fundamental to the pathogenic process is the ability of the parasite to develop into a latent tissue cyst that can re-emerge as a rapidly growing form upon impairment of immunity. Increased understanding of this developmental process will provide new opportunities for therapeutic intervention. The work in our first award period focused on GCN5 histone acetyltransferases (HATs) and led to the discovery that histone acetylation correlates with gene expression pertinent to Toxoplasma development. We have taken genetic approaches to define the roles of two distinct GCN5-family HATs we have characterized in Toxoplasma (TgGCN5-A and -B). We have found that the loss of TgGCN5 impairs the ability of Toxoplasma to up-regulate key developmentally expressed genes. We have also determined that some of the proteins interacting with TgGCN5 in a yeast two-hybrid screen have domains typical of transcriptional regulators; this is significant because the lack of DNA-binding transcription factors detectable in the Toxoplasma genome has hampered efforts to understand how gene expression is regulated. How Toxoplasma regulates transcription to grow effectively during the acute stage, and develop into a latent cyst during stress, represent major gaps in our knowledge that hinder our ability to fight the opportunistic infection. The data generated by the first award allow us to focus this renewal on defining mechanisms of GCN5- mediated gene regulation in the context of parasite development, which underlies pathogenesis. We hypothesize that the TgGCN5 HATs play key roles in Toxoplasma pathogenesis by forming distinct complexes with novel proteins that regulate developmental gene expression. Our specific aims include (1) Determine the roles of TgGCN5 HATs in pathogenesis; (2) Elucidate differences in TgGCN5 complexes during Toxoplasma development; (3) Define the mechanism by which each TgGCN5 is recruited to target gene promoters to coordinate developmental gene expression. The proposed research capitalizes on the novel reagents, techniques, and transgenic parasites that we have developed. The data generated will illuminate the mechanism behind developmental transitions in Toxoplasma that are responsible for disease progression, thus exposing novel points of therapeutic intervention. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is a protozoan parasite that causes significant disease as an opportunistic infection of AIDS patients. Chronic toxoplasmosis is currently incurable because the parasite is able to develop into cysts that remain latent until immunosuppression. The mechanisms driving the development of these cysts are the focus of our studies, as learning how Toxoplasma develops will identify novel points of therapeutic intervention. We are taking an innovative approach to use transcriptional regulators as a means to elucidate the mechanisms of parasite development. The regulation of gene expression plays a key role in this pathogenic process; therefore, our results stand a high probability of translating into useful new therapies to combat opportunistic infectious diseases like Toxoplasma.