Regulation of cyst formation in the AIDS opportunistic pathogen Toxoplasma

项目总结 弓形虫是一种细胞内的原生动物，对患者构成重大威胁。 感染艾滋病毒/艾滋病。复制阶段(速殖子)发展为潜伏期(缓殖子) 以包囊的形式存在于宿主细胞内的大脑、心脏和骨骼肌组织中。组织囊肿 终生留在宿主中，因为它们不受免疫反应的影响，目前 经批准的药物，并引起反复再次激活的感染中的免疫损害 病人。尽管它在临床上很重要，但人们对它从 速殖子-缓殖子阶段。最近发现了一种名为BFD1的关键转录因子 这是在体外和小鼠体内形成包囊的必要条件和充分条件，但我们目前知道 没有关于这一因素的规定。我们将从我们的知识建设中填补这一空白 强有力的初步调查结果表明，BFD1在事后受到严格的监管- 转录水平。我们的研究表明，BFD1mRNA的翻译能力提高了30%以上。 折叠，以响应TgIF2α的磷酸化。与这一观察相一致的是， 在BFD1mRNA的5‘-前导序列中有大量的上游开放阅读框(UORF)。目标1将 测试我们的假设，即这些uORF在BFD1的优先翻译中发挥关键作用 发生在对缓殖子诱导的反应中。除了翻译控制，我们还有 更多的初步数据表明，BFD1通过赖氨酸在蛋白质水平上进行调节 乙酰化。我们对速殖子的乙酰化组分析发现，BFD1是在赖氨酸1720处乙酰化的 (K1720)，在用赖氨酸进行的酵母双杂交筛选中，BFD1是最热门的 乙酰转移酶TgGCN5a。目标2将验证我们的假设，即TgGCN5a调节BFD1 通过K1720乙酰化，采用免疫共沉淀法和突变分析。 这两个目标的完成将揭示对转录后的关键新见解 控制BFD1活性的机制，有望成为新的治疗点 对艾滋病毒/艾滋病患者的这种破坏性机会性感染进行干预。

PROJECT SUMMARY Toxoplasma gondii is an intracellular protozoan parasite that poses a major threat to patients with HIV/AIDS. The replicative stage (tachyzoite) develops into a latent stage (bradyzoite) that resides inside host cells as cysts in brain, heart, and skeletal muscle tissues. Tissue cysts remain in the host for life, as they are impervious to the immune response and currently approved drugs, and give rise to recurrent reactivation of infection in immune compromised patients. Despite its clinical importance, little is known about the complex transition from the tachyzoite to bradyzoite stage. A key transcription factor called BFD1 was recently discovered that is necessary and sufficient for cyst formation in vitro and in mice, but we currently know nothing about the regulation this factor. We will fill this gap in our knowledge building from our strong preliminary findings that BFD1 is subject to tight regulatory control at the post- transcriptional level. We have shown that translation of BFD1 mRNA is enhanced more than 30- fold in response to TgIF2α phosphorylation. Consistent with this observation is the presence of numerous upstream open reading frames (uORFs) in the 5’-leader of BFD1 mRNA. Aim 1 will test our hypothesis that these uORFs play a key role in the preferential translation of BFD1 that occurs in response to bradyzoite induction. In addition to translational control, we have additional preliminary data suggesting that BFD1 is regulated at the protein level through lysine acetylation. Our acetylome analysis of tachyzoites found that BFD1 is acetylated at lysine 1720 (K1720) and that BFD1 was the top hit in a yeast two-hybrid screen performed with the lysine acetyltransferase TgGCN5a. Aim 2 will test our hypothesis that TgGCN5a regulates BFD1 through K1720 acetylation using co-immunoprecipitation assays and mutational analysis. Completion of these two aims will reveal critical new insights into the post-transcriptional mechanisms that govern BFD1 activity, which promise to serve as novel points of therapeutic intervention to treat this devastating opportunistic infection in HIV/AIDS patients.