Regulation of cyst formation in the AIDS opportunistic pathogen Toxoplasma

项目摘要 刚地弓形虫是一种细胞内的原生动物寄生虫，对患者构成重大威胁 艾滋病毒/艾滋病。复制阶段（速殖子）发育成潜伏阶段（缓殖子）， 存在于宿主细胞内，如脑、心脏和骨骼肌组织中的包囊。组织囊肿 在宿主体内终生存在，因为它们不受免疫反应的影响， 批准的药物，并引起免疫受损的感染复发性再激活 患者尽管它的临床重要性，很少有人知道从复杂的过渡， 速殖子到缓殖子阶段。最近发现了一种名为BFD 1的关键转录因子 这对于体外和小鼠中的囊肿形成是必要的和足够的，但我们目前知道， 没有任何关于这个因素的规定。我们将填补我们的知识建设从我们的差距， 强有力的初步调查结果表明，BFD 1在后 转录水平。我们已经证明，BFD 1 mRNA的翻译增强超过30- 40倍。 对TgIF 2 α磷酸化的响应增加一倍。与这一观察相一致的是， 在BFD 1 mRNA的5 '-前导序列中存在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BFD 1的优先翻译中起关键作用， 是由缓殖子诱导引起的除了翻译控制，我们还有 额外的初步数据表明，BFD 1是通过赖氨酸在蛋白质水平上调节的， 乙酰化我们对速殖子的乙酰基组分析发现，BFD 1在赖氨酸1720处被乙酰化 （K1720），并且BFD 1是用赖氨酸进行的酵母双杂交筛选中的最高命中。 乙酰转移酶TgGCN 5a。目的2将验证我们的假设，即TgGCN 5a调节BFD 1 通过使用免疫共沉淀测定和突变分析的K1720乙酰化。 这两个目标的完成将揭示关键的新见解转录后 控制BFD 1活性的机制，这有望成为新的治疗点， 艾滋病毒/艾滋病患者的这种毁灭性的机会性感染。

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.