Translational control during stage conversion of Toxoplasma, an opportunistic infection of HIV/AIDS

 描述(由申请人提供)：这项提案涉及弓形虫潜伏期形成和重新激活的核心分子机制，弓形虫是一种原生动物寄生虫，会在艾滋病/艾滋病毒患者中造成威胁生命的机会性感染。弓形虫从增殖期(速殖子)向潜伏组织囊(缓殖子)转化的能力 引发了威胁生命的慢性机会性疾病，这种疾病折磨着艾滋病患者。那里 目前迫切需要新的方法来治疗艾滋病--弓形虫病，但由于对潜伏期发展和重新活跃的机制了解不足，这一努力受到了阻碍。我们发现真核细胞起始因子-2的弓形虫α亚基(TgIF2α)的磷酸化参与了缓殖子的发育。利用我们在弓形虫中使用的多聚体图谱，我们证明了TgIF2mRNA的磷酸化导致了与缓殖子发育相关的α子集的优先翻译。进一步支持翻译控制在复制和潜伏期都具有关键功能的新研究来自我们使用药物salubrinal(SAL)和Ganabenz(GA)的研究，这两种药物抑制TgIF2α去磷酸化并阻止缓殖子的重新激活。在这里，我们显示GA还保护急性感染弓形虫的小鼠，并减少慢性感染小鼠的包囊数量。总之，我们的遗传、生化和药理学实验支持了我们的假设，即TgIF2α磷酸化介导的翻译控制对寄生虫阶段的相互转换至关重要。我们提出的实验将解决这一假设，并1)建立TgIF2mRNAs如何协调转换为缓体，2)识别翻译控制的α启动缓体发育和重新转换为复制型速殖子，以及3)确定TgIF2α去磷酸化和感染重新激活的机制。完成这些目标将有助于确定弓形虫阶段转换的机制，着眼于开发急需的治疗艾滋病-弓形虫病的新疗法。

 DESCRIPTION (provided by applicant): This proposal addresses molecular mechanisms that are central for formation and reactivation of latent stages of Toxoplasma gondii, a protozoan parasite that causes life-threatening opportunistic infection in AIDS/HIV patients. The ability of Toxoplasma to convert from its proliferative stage (tachyzoite) to latent tissue cysts (bradyzoite) gives rise to the life-threatening chronic opportunistic disease that afflicts AIDS patients. There is a vital need for new approaches for treatment of AIDS-toxoplasmosis, but this effort has been hindered by an insufficient understanding of the mechanisms by which the latent stage develops and reactivates. We discovered that phosphorylation of the Toxoplasma alpha subunit of eukaryotic initiation factor-2 (TgIF2α) contributes to bradyzoite development. Using polysome profiling, which we adapted for use in Toxoplasma, we showed that TgIF2α phosphorylation leads to preferential translation of a subset of mRNAs that are linked to bradyzoite development. Further support that translational control has critical functions during both replicative and lateny stages comes from our new studies using the drugs salubrinal (SAL) and guanabenz (GA), which inhibit TgIF2α dephosphorylation and block the reactivation of bradyzoites. Here we show that GA also protects mice acutely infected with Toxoplasma and decreases the number of cysts in chronically infected mice. Together, our genetic, biochemical, and pharmacological experiments support our hypothesis that translational control mediated by TgIF2α phosphorylation is critical for parasite stage interconversion. Our proposed experiments will address this hypothesis and 1) establish how TgIF2α kinases coordinate conversion to bradyzoites, 2) identify translationally controlled mRNAs initiating bradyzoite development and reconversion into replicating tachyzoites, and 3) determine the mechanisms underlying TgIF2α dephosphorylation and reactivation of infection. Completion of these aims will help define the mechanics of Toxoplasma stage conversion, with an eye towards developing critically needed novel therapies for AIDS-toxoplasmosis.