Translation initiation factors driving persistence of Toxoplasma gondii bradyzoites in neurons

项目摘要 原生动物寄生虫弓形虫由于其持续存在的能力， 作为患者体内的潜伏形式（缓殖子）。没有针对缓殖子囊肿的治疗方法， 进入免疫受损的破坏性增殖阶段（速殖子）。病人 重新激活的弓形虫病经常出现危及生命的神经系统问题， 脑内缓殖子囊肿的意义更好地理解推动 神经元中缓殖子的发育对于设计新的治疗方法来防止它们的形成是必要的， 持久性。为了满足这一需求，我们开发了一种神经元自发组织囊肿形成的新模型 使用隆德人中脑（LUHMES）细胞。我们将利用这一创新系统来确定 缓殖子形成所需的蛋白质合成变化的潜在机制。基于 我们以前的合作工作，我们假设翻译起始因子协调的变化， 关键mRNA的5 '前导序列，导致蛋白质合成的变化，诱导转化为 缓殖子翻译开始于eIF 4F复合物与5 '-帽的结合，然后招募eIF 2， 携带Met-tRNA我们以前曾发现TgIF 2在缓殖子转化过程中被磷酸化， 降低其丰度并可改变起始密码子选择。目标1将决定TgIF 2如何成为 磷酸化的，并且在自发的缓殖子转化过程中哪些mRNA优先翻译。 神经元我们的RiboSeq方法将揭示5 '-leader内调节翻译的区域，例如上游 开放阅读框（uORF）或二级结构，后者将通过产生第一个 Aim 2中弓形虫的RNA“结构组”。由于这些结构是通过eIF 4F的解旋酶活性解析的，因此目的 2还将描绘我们在寄生虫中发现的多种TgIF 4F复合物的功能。 总之，这些研究将确定细胞信号如何协调翻译起始因子进行重编程 在人类神经元中引发缓殖子自发形成的翻译体。完成本研究 将通过提供对复杂机制的重要新见解， 弓形虫用来在其宿主中持续存在，这将揭示治疗干预的新点。

PROJECT SUMMARY The protozoan parasite Toxoplasma gondii can cause recurring opportunistic infections due to its ability to persist as a latent form (bradyzoite) within patients. There is no treatment that targets bradyzoite cysts, which reconvert into the destructive proliferative stage (tachyzoites) in the immune compromised. Patients suffering from reactivated toxoplasmosis frequently present with life-threatening neurological problems, underscoring the significance of bradyzoite cysts in the brain. A better understanding of the mechanisms that drive the development of bradyzoites in neurons is necessary to devise new therapies that prevent their formation and persistence. To address this need, we developed a novel model of spontaneous tissue cyst formation in neurons using Lund human mesencephalic (LUHMES) cells. We will use this innovative system to determine the mechanisms underlying the changes in protein synthesis that are required for bradyzoite formation. Based on our previous collaborative work, we hypothesize that translation initiation factors coordinate changes in 5’-leader sequences of key mRNAs, resulting in changes in protein synthesis that induce conversion to bradyzoites. Translation begins with the binding of an eIF4F complex to the 5’-cap, which then recruits eIF2, which carries Met-tRNA. We previously showed TgIF2 is phosphorylated during bradyzoite conversion, which lowers its abundance and can alter start codon selection. Aim 1 will determine how TgIF2 becomes phosphorylated and which mRNAs are preferentially translated during spontaneous bradyzoite conversion in neurons. Our RiboSeq approach will reveal areas within 5’-leaders that regulate translation, such as upstream open reading frames (uORFs) or secondary structures, the latter of which will be identified by generating the first RNA “structurome” for Toxoplasma in Aim 2. As these structures are resolved by helicase activity of eIF4F, Aim 2 will also delineate the functions of the multiple TgIF4F complexes we have uncovered in the parasites. Together, these studies will determine how cellular signals coordinate translation initiation factors to reprogram the translatome to trigger the spontaneous formation of bradyzoites in human neurons. Completion of this study will have a sustained high impact on the field by providing significant new insights into the complex mechanisms that Toxoplasma uses to persists in its host, which will reveal novel points for therapeutic intervention.