Cyclin-mediated control of Toxoplasma development

细胞周期蛋白介导的弓形虫发育控制

• 批准号: 10161722
• 负责人: Elena Suvorova
• 金额: $ 37.38万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-08 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10161722
• 关键词: Acquired Immunodeficiency Syndrome; Acute; Affect; Animal Model; Animals; Architecture; Auxins; Biochemical; Biological; Biological Assay; Biology; Cell Cycle; Cell division; Cells; Chronic; Chronic Disease; Clinical; Complex; Cyclins; Cyst; Development; Differentiation and Growth; Disease; Disease Outbreaks; Disease Progression; Drug Targeting; Ensure; Equilibrium; Eukaryota; Evaluation; Foundations; Frequencies; G1 Phase; Gene Expression; Genetic; Genetic Models; Genome; Goals; Human; Immune; Immune response; Immune system; Immunity; Immunocompromised Host; In Vitro; Individual; Infection; Kentucky; Life; Link; Measures; Mechanics; Mediating; Modeling; Monitor; Mothers; Mus; Organ Transplantation; Parasites; Parasitic infection; Patients; Pharmaceutical Preparations; Phosphotransferases; Population; Post-Translational Protein Processing; Pregnancy; Proteomics; Readiness; Regulation; Rest; Role; Site; Specialist; Testing; Time; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Universities; Woman; base; chemotherapy; differential expression; effective therapy; experience; experimental study; human pathogen; immune function; in vitro activity; in vivo; mouse model; mutant; novel; prenatal; prevent; protein expression; success; transmission process

Project Abstract Toxoplasma gondii is an important human pathogen that causes severe disease in immunocompromised individuals, such as those undergoing chemotherapy, organ transplantation, and AIDS patients. It also afflicts women who become infected for the first time during pregnancy. A healthy immune system or current drug regiment controls the replication of the tachyzoites associated with clinical toxoplasmosis. However, there are no effective therapies to eliminate the chronic stages associated with encysted bradyzoites and, importantly, to prevent the cyst reactivation. To find new avenues for combating the chronic and reactivated toxoplasmosis, we focus on the mechanisms of the tachyzoite and bradyzoite interconversions that are poorly understood. The critical difference between a tachyzoite and a bradyzoite is the rate of parasite replication and the cell cycle architecture. A tachyzoite divides fast and has a relatively short G1 period. In contrast, a bradyzoite rarely divides and spends a progressively longer time in the G1 phase. The time parasite spends in the G1 period is regulated by the RESTRICTION checkpoint (R-point) that in T. gondii lacks conventional regulators. The current application is based on the central hypothesis that the atypical TgCrk2 kinase and P-type cyclins define the novel G1 checkpoint that governs transitions between acute and chronic toxoplasmosis. In our preliminary studies, we showed a differential expression of three P-cyclins in the fast- (RH) and slow-dividing (ME49) T. gondii strains, which also differ in their ability to differentiate. We also showed that all three P-cyclins interact with G1 kinase TgCrk2 in vivo. We believe that P-cyclins differentially regulate levels of TgCrk2 activity, therefore, facilitate or block R-point passage. This dictates the parasite’s choice to either replicate as a tachyzoite or to convert into a resting bradyzoite. To prove our hypothesis, we will define the mechanism of the R-point regulation by TgCrk2 kinase and cyclins TgCycP2, and TgCycP3 in the tachyzoite and bradyzoite development in vitro (Aim 1); in the natural progression of the disease using mouse model (Aim 2); and determine function of the novel R-point components TgCables1 and TgRch1 (Aim 3). Our proposal is built on a strong foundation of advanced genetics, which is bolstered by our extensive experience in studying cyclin/Crk regulators of the T. gondii tachyzoite cell cycle. Using a panel of strains with novel auxin-dependent conditional protein expression of P-cyclins, we will test whether differences in regulation of TgCrk2/P-cyclin complexes explain the dynamics of the bradyzoite differentiation and cyst reactivation. Altogether, our experiments will break new ground in understanding the mechanics and regulation of the developmental switch that regulates the progression of the disease.

项目摘要 弓形虫是一种重要的人类病原体，可导致免疫功能低下者患严重疾病 个体，例如接受化疗、器官移植的人和艾滋病患者也会受到影响。 怀孕期间首次感染的女性 健康的免疫系统或当前的药物。 然而，存在与临床弓形虫病相关的速殖子的复制。 没有有效的疗法可以消除与包囊缓殖子相关的慢性阶段，并且重要的是， 防止囊肿重新激活 为了寻找对抗慢性和重新激活的弓形虫病的新途径，我们 重点关注人们知之甚少的速殖子和缓殖子相互转化的机制。 速殖子和缓殖子之间的关键区别在于寄生虫复制速率和细胞周期 速殖子分裂速度快，G1 期相对较短，而缓殖子分裂很少。 并且在 G1 阶段花费的时间逐渐延长 寄生虫在 G1 时期花费的时间受到调节。 弓形虫中缺乏常规调节器的限制检查点（R点）。 应用基于非典型 TgCrk2 激酶和 P 型细胞周期蛋白定义的中心假设 在我们的初步研究中，控制急性和慢性弓形虫病之间转变的新型 G1 检查点。 研究中，我们发现快速分裂 (RH) 和慢速分裂 (ME49) T 中三种 P-细胞周期蛋白的差异表达。 弓形虫菌株，它们的分化能力也不同。我们还表明所有三种 P-细胞周期蛋白相互作用。 体内 G1 激酶 TgCrk2 我们认为 P-cyclins 差异性调节 TgCrk2 活性水平，因此， 促进或阻止 R 点通过，这决定了寄生虫的选择是作为速殖子复制还是以速殖子形式复制。 为了证明我们的假设，我们将定义 R 点调节的机制。 TgCrk2 激酶和细胞周期蛋白 TgCycP2 和 TgCycP3 在速殖子和缓殖子发育中的作用 体外（目标 1）；使用小鼠模型研究疾病的自然进展（目标 2）； 新颖的 R 点组件 TgCables1 和 TgRch1（目标 3）建立在坚实的基础之上。 先进的遗传学，得到了我们在研究 T 细胞周期蛋白/Crk 调节因子方面的丰富经验的支持。 使用一组具有新型生长素依赖性条件蛋白表达的菌株。 P-细胞周期蛋白，我们将测试 TgCrk2/P-细胞周期蛋白复合物的调节差异是否可以解释 P-细胞周期蛋白的动态 总而言之，我们的实验将在缓殖子分化和包囊再激活方面取得新突破。 了解调节发育进程的发育开关的机制和调节 疾病。