Translational control and latent Toxoplasma infection

描述(申请人提供)：弓形虫是一种胞内原生动物寄生虫，可导致先天性出生缺陷，以及免疫受损人群中危及生命的机会性疾病，如艾滋病患者或接受癌症化疗的患者。发病机制的核心是弓形虫从快速生长的速殖子转变为潜伏的包囊形式的能力，称为缓殖子。目前，我们对寄生虫如何发育成包囊的分子事件知之甚少，也没有药物可以清除体内的包囊。众所周知，由速殖子发育而来的缓殖子是由细胞压力启动的。我们发现，诱导包囊发育的同样的细胞应激也诱导真核细胞起始因子-2(TgIF2)的弓形虫版本的磷酸化。TgIF2的磷酸化与减速体标记物基因的整体翻译和诱导表达降低相关。TgIF2在缓殖子中仍然是磷酸化的，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，TgIF2的磷酸化既抑制了一般的蛋白质合成，又导致了关键转录因子的优先翻译，这些转录因子对缓殖体在应激反应中的发育至关重要。为了解决这一假设，我们提出了两个目标。目标1将阐述TgIF2磷酸化在缓殖子发育中的作用。我们将创造一个突变的弓形虫寄生虫，它将TgIF2中的磷酸化位点丝氨酸-71替换为丙氨酸，从而阻止应激过程中的磷酸化和翻译控制。我们认为，这样的突变体将对压力敏感，并且不能引发弓形虫从速殖子转化为缓殖子包囊所需的翻译控制机制。目的2将鉴定和鉴定优先翻译的参与包囊发育的mRNAs。我们将使用微阵列分析来自应激和非应激寄生虫的多聚体相关mRNA，以确定受优先翻译影响的关键调控因子。这项研究将解决TgIF2磷酸化和翻译控制对临床相关的缓殖子发育过程的要求。了解eIF2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。 与公共卫生相关：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以及接受器官移植的患者服用免疫抑制剂以减少器官排斥反应。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。我们正在采取一种创新的方法，重点研究控制蛋白质合成的机制，以阐明寄生虫包囊的发育过程。基因表达的调控在这一致病过程中起着关键作用；因此，我们的结果将对抗击弓形虫等机会性传染病的新疗法具有重要意义。

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.