Translational control and latent Toxoplasma infection

描述（由申请人提供）：弓形虫是一种细胞内原生动物寄生虫，可导致先天性出生缺陷，以及免疫功能低下患者（如艾滋病患者或接受癌症化疗的患者）的危及生命的机会性疾病。发病机制的核心是弓形虫从快速生长的形式（称为速殖子）转化为潜伏的包囊形式（称为缓殖子）的能力。目前，我们对寄生虫如何发展成囊肿的分子事件知之甚少，也没有药物可以清除身体的囊肿形式。已经确定，速殖子发育为缓殖子是由细胞应激启动的。我们发现，诱导包囊发育的相同细胞应激也诱导弓形虫真核起始因子-2（TgIF 2）的磷酸化。TgIF 2的磷酸化与缓殖子标记基因的整体翻译降低和诱导表达相关。TgIF 2在缓殖子中保持磷酸化，这表明翻译控制对于维持缓殖子包囊的静止状态也很重要。我们的假设是，TgIF 2磷酸化的结果在一般蛋白质合成的抑制和优先翻译的关键转录因子，这是关键的缓殖子的发展，以应对压力。为了解决这个问题，我们提出了两个目标。目的1将阐明TgIF 2磷酸化在缓殖子发育中的作用。我们将创建一个突变的弓形虫寄生虫，取代磷酸化位点，丝氨酸-71在TgIF 2丙氨酸，因此阻断磷酸化和翻译控制在压力。我们建议，这样的突变体将敏感的压力，不能引起翻译控制机制所需的弓形虫从速殖子转换为缓殖子包囊。目的2将识别和表征参与囊肿发育的优先翻译的mRNA。我们将使用微阵列分析应激和非应激寄生虫的多核糖体相关mRNA，以确定优先翻译的关键调控因子。本研究将解决TgIF 2磷酸化和翻译控制的缓殖子发育的临床相关过程的要求。了解eIF 2激酶介导的通路将为弓形虫感染和相关寄生虫病的治疗提供新的治疗靶点。 公共卫生相关性：弓形虫是一种原生动物寄生虫，可导致艾滋病患者、接受化疗的癌症患者和接受器官移植的患者发生严重疾病，这些患者服用免疫抑制剂以尽量减少器官排斥反应。慢性弓形虫病目前是不可治愈的，因为寄生虫能够发展成囊肿，保持潜伏，直到免疫抑制。我们正在采取一种创新的方法，专注于控制蛋白质合成的机制，以阐明寄生虫包囊发育的过程。基因表达的调控在这一致病过程中起着关键作用;因此，我们的研究结果对于对抗机会性感染性疾病（如弓形虫）的新疗法将是重要的。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an intracellular protozoan parasite that causes congenital birth defects, as well as life-threatening opportunistic disease in the immunocompromised, such as AIDS patients or patients undergoing cancer chemotherapy. Central to pathogenesis is the ability of Toxoplasma to convert from a rapidly growing form, referred to as the tachyzoite, to a latent cyst form, called the bradyzoite. Currently, we know little about the molecular events underlying how the parasite develops into cysts, and no drugs exist that can rid the body of the cyst form. It is well established that the development of bradyzoites from tachyzoites is initiated by cellular stress. We discovered that the same cellular stresses that induce cyst development also induce the phosphorylation of the Toxoplasma version of eukaryotic initiation factor -2 (TgIF2). Phosphorylation of TgIF2 correlates with lowered global translation and induced expression of bradyzoite marker genes. TgIF2 remains phosphorylated in bradyzoites, suggesting that translational control is also important for maintenance of the quiescent state of bradyzoite cysts. Our hypothesis is that TgIF2 phosphorylation results in both repression of general protein synthesis and preferential translation of key transcription factors, which are critical for bradyzoite development in response to stress. To address this hypothesis, we propose two Aims. Aim 1 will address the role of TgIF2 phosphorylation in bradyzoite development. We will create a mutant Toxoplasma parasite that substitutes the phosphorylated site, serine-71 in TgIF2 to alanine, therefore blocking phosphorylation and translational control during stress. We propose that such a mutant will be sensitized to stress and be unable to elicit translational control mechanisms required for Toxoplasma to convert from the tachyzoite to the bradyzoite cyst. Aim 2 will identify and characterize preferentially translated mRNAs involved in cyst development. We will use microarray analyses of polysome-associated mRNA from stressed and unstressed parasites to identify key regulatory factors subject to preferential translation. This study will address the requirement of TgIF2 phosphorylation and translational control for the clinically relevant process of bradyzoite development. Knowledge of eIF2 kinase-directed pathways will provide new therapeutic targets for the treatment of Toxoplasma infections and related parasitic diseases. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is a protozoan parasite that causes significant disease in AIDS patients, cancer patients undergoing chemotherapy, and organ-transplant recipients taking immunosuppressants to minimize organ rejection. Chronic toxoplasmosis is currently incurable because the parasite is able to develop into cysts that remain latent until immunosuppression. We are taking an innovative approach focused on mechanisms controlling protein synthesis to elucidate the process of parasite cyst development. The regulation of gene expression plays a key role in this pathogenic process; therefore, our results will be important for new therapies to combat opportunistic infectious diseases, such as Toxoplasma.