Cytosine DNA Methylation and Transcriptional Repression in African Trypanosomes

说明(申请人提供)：4.4项目6：布鲁氏锥虫是引起人类非洲锥虫病(HAT)的原虫寄生虫，又称非洲睡眠病。由于没有有效的疫苗，迫切需要对这种疾病采取新的控制策略。许多用于治疗HAT的药物有毒且无效。对寄生虫生命周期的全面了解可能会揭示治疗干预的新靶点。寄生虫的生命周期需要在采采蝇和哺乳动物宿主中生长和分化。寄生虫在每个独特环境中的适应和复制能力取决于在整个生命周期中调节特定蛋白质存在的能力。我们的生物信息学研究表明，非洲锥虫含有一种名为5-甲基胞嘧啶的修饰碱基，通过抑制基因转录，这种修饰碱基是依赖生命周期的蛋白质表达所必需的。我们将使用分子、生物化学和遗传策略的组合来检验这一假设。首先，5-甲基胞嘧啶的亚细胞定位和发育调节将通过使用针对5-甲基胞嘧啶的抗体探测纯化的布氏毛滴虫的核和线粒体DNA来确定。接下来，我们将确定在布氏毛滴虫中负责5-甲基胞嘧啶产生的酶机制。布鲁氏毛滴虫胞嘧啶-5DNA甲基转移酶将从大肠杆菌中生产和纯化。在体外，DNA甲基化反应将被用来确定该蛋白质是否真的具有合成5-甲基胞嘧啶的能力，并确定该反应的生化要求。最后，我们将确定5-甲基胞嘧啶与转录抑制的关系。利用RNA干扰技术构建缺乏5-甲基胞嘧啶的寄生虫，并利用Northern blotts分析其对靶基因转录沉默的影响。我们的研究可能会改善对HAT的控制。我们预测，5-甲基胞嘧啶途径是寄生虫调节表面抗原表达和逃避人类免疫系统所必需的。因此，针对这种DNA修饰的药物可能会损害寄生虫的传播或致病性。4.4项目说明迫切需要新的疗法来预防和治疗人类非洲锥虫病，因为目前没有有效的疫苗，药物治疗是有毒和不可靠的。我们对布鲁氏锥虫胞嘧啶DNA甲基化的研究可能揭示这种寄生虫调节表面抗原表达和逃避人类免疫系统的机制。针对胞嘧啶DNA甲基化的药物可能会消除寄生虫免疫系统的逃避或传播。

DESCRIPTION (provided by applicant): 4.4 Item 6: Project Summary Trypanosoma brucei is the protozoan parasite responsible for Human African Trypanosomiasis (HAT), otherwise known as African Sleeping Sickness. There is an urgent need for new control strategies for this disease, as there is no effective vaccine. Many of the drugs used to treat HAT are toxic and ineffective. A complete understanding of the parasite's life cycle will potentially reveal novel targets for therapeutic intervention. The life cycle of the parasite requires growth and differentiation in both the tsetsfe fly and mammalian host. The ability of the parasites to adapt and replicate in each unique environment is dependent on the ability to regulate the presence of specific proteins throughout the life cycle. Our bioinformatic studies suggest that African trypanosomes contain a modified base called 5-methylcytosine, and this modified base is required for life cycle-dependent protein expression by repressing gene transcription. We will test this hypothesis using a combination of molecular, biochemical and genetic strategies. First, the subcellular localization and developmental regulation of 5-methylcytosine will be determined by probing purified nuclear and mitochondrial DNA from T. brucei parasites with an antibody specific to 5-methylcytosine. Next, we will identify the enzymatic machinery responsible for 5-methycytosine generation in T. brucei. The putative T. brucei cytosine-5 DNA methyltransferase will be produced and purified from E. coli. In vitro DNA methylation reactions will be used to determine if this protein truly has the ability to synthesize 5-methylcytosine, and determine the biochemical requirements for this reaction. Last, we will determine the relationship between 5-methlycytosine and transcriptional repression. Parasites lacking 5- methylcytosine will be constructed using RNA interference technology, and the effect on transcriptional silencing of target genes will be analyzed using northern blots. Our studies will potentially improve control of HAT. We predict that the 5-methylcytosine pathway is required for the parasite's ability to regulate surface antigen expression and evade the human immune system. Thus, drugs targeting this DNA modification will likely compromise parasite transmission or pathogenicity. 4.4 Item 7. Project Narrative There is an urgent need for new therapeutics to prevent and treat Human African Trypanosomiasis, as there is currently no effective vaccine and drug treatments are toxic and unreliable. Our studies of cytosine DNA methylation in Trypanosoma brucei may reveal the mechanism by which the parasites regulate expression of surface antigens and evade the human immune system. Drugs that target cytosine DNA methylation could possibly eliminate parasite immune system evasion or transmission.