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

 描述（由申请方提供）：本提案阐述了弓形虫潜伏期形成和再激活的核心分子机制，弓形虫是一种原生动物寄生虫，可导致AIDS/HIV患者发生危及生命的机会性感染。弓形虫从增殖期（速殖子）转化为潜伏组织包囊（缓殖子）的能力 引起了折磨艾滋病患者的危及生命的慢性机会性疾病。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再活化的机制认识不足，这一努力受到阻碍。我们发现弓形虫真核起始因子2 α亚基（TgIF 2 α）的磷酸化有助于缓殖子的发育。使用我们用于弓形虫的多核糖体分析，我们发现TgIF 2 α磷酸化导致与缓殖子发育相关的一部分mRNA优先翻译。翻译控制在复制和潜伏阶段都具有关键功能的进一步支持来自我们使用药物salubrinal（SAL）和guanabenz（GA）的新研究，这些药物抑制TgIF 2 α去磷酸化并阻断缓殖子的再激活。在这里，我们表明，GA也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，我们的遗传学、生物化学和药理学实验支持我们的假设，即由TgIF 2 α磷酸化介导的翻译控制对于寄生虫阶段互变至关重要。我们提出的实验将解决这一假设，并1）建立TgIF 2 α激酶如何协调转化为缓殖子，2）鉴定启动缓殖子发育和再转化为复制速殖子的受控制的mRNA，3）确定TgIF 2 α去磷酸化和感染再激活的潜在机制。这些目标的完成将有助于确定弓形虫阶段转换的机制，着眼于开发艾滋病弓形虫病急需的新疗法。

 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.