GCN5-mediated transcription in AIDS pathogen Toxoplasma

描述(申请人提供)：弓形虫是一种原生动物寄生虫，对艾滋病患者构成重大风险，并被NIAID列为B类优先生物防御病原体。致病过程的基础是寄生虫有能力发展成潜伏的组织包囊，一旦免疫受损，包囊就会以快速生长的形式重新出现。对这一发育过程的进一步了解将为治疗干预提供新的机会。我们第一个获奖期的工作重点是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有关。我们采用遗传学方法来确定我们在弓形虫中描述的两个不同的GCN5家族帽子(TgGCN5-A和-B)的作用。我们发现TgGCN5的缺失削弱了弓形虫上调关键发育表达基因的能力。我们还确定了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控域；这一点很重要，因为在弓形虫基因组中缺乏可检测到的DNA结合转录因子，阻碍了了解基因表达是如何调控的努力。弓形虫如何调节转录在急性期有效生长，并在应激状态下发育成潜伏的包囊，代表了我们知识中的主要空白，阻碍了我们抗击机会性感染的能力。第一个奖项产生的数据使我们能够集中精力在寄生虫发育的背景下定义GCN5介导的基因调控机制，这是发病的基础。我们推测，TgGCN5 HATS通过与调节发育基因表达的新蛋白形成不同的复合体，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 HATS在发病机制中的作用；(2)阐明TgGCN5复合体在弓形虫发育过程中的差异；(3)确定每个TgGCN5被招募到靶基因启动子以协调发育基因表达的机制。这项拟议的研究利用了我们开发的新试剂、技术和转基因寄生虫。产生的数据将阐明弓形虫发育转变背后的机制，这些转变是疾病进展的原因，从而揭示了治疗干预的新观点。公共卫生相关性：弓形虫是一种原虫寄生虫，作为艾滋病患者的一种机会性感染，会导致重大疾病。慢性弓形虫病目前是无法治愈的，因为这种寄生虫能够发育成包囊，在免疫抑制之前保持潜伏状态。推动这些包囊发展的机制是我们研究的重点，因为了解弓形虫是如何发展的将确定新的治疗干预点。我们正在采取一种创新的方法，利用转录调节因子作为一种手段来阐明寄生虫的发育机制。基因表达的调节在这一致病过程中起着关键作用；因此，我们的结果很有可能转化为有用的新疗法，以对抗弓形虫等机会性传染病。

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.