Cyclin-mediated control of Toxoplasma development

项目摘要 弓形虫是一种重要的人类病原体，可引起免疫功能低下的严重疾病 个体，如接受化疗、器官移植和艾滋病患者。它也折磨着 在怀孕期间首次感染的妇女。健康的免疫系统或当前的药物 控制与临床弓形虫病相关的速殖子的复制。但有 没有有效的治疗方法来消除与包囊性缓殖子相关的慢性阶段，重要的是， 防止囊肿复发为了找到新的途径来对抗慢性和再活化的弓形虫病，我们 重点放在机制的速殖子和缓殖子相互转换，是知之甚少。的 速殖子和缓殖子的关键区别在于寄生虫的复制率和细胞周期 架构速殖子分裂快，G1期相对较短。相反，缓殖子很少分裂 并且在G1期花费的时间逐渐延长。寄生虫在G1期的时间是受调节的 在T中的限制检查点（R点）。弓形虫缺乏常规的调节剂。当前 该应用基于中心假设，即非典型TgCrk 2激酶和P型细胞周期蛋白定义了细胞周期蛋白。 新的G1检查点，控制急性和慢性弓形虫病之间的过渡。在我们的初步调查中 研究中，我们发现了三种P-细胞周期蛋白在快分裂（RH）和慢分裂（ME 49）T细胞中的差异表达。 弓形虫菌株，它们的分化能力也不同。我们还发现，所有三个P-细胞周期蛋白相互作用， 与G1激酶TgCrk 2的结合。我们认为，P-细胞周期蛋白差异调节TgCrk 2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，要么作为速殖子复制， 转化为静止的缓殖子为了证明我们的假设，我们将定义R点调节的机制 通过TgCrk 2激酶和细胞周期蛋白TgCycP 2、TgCycP 3在速殖子和缓殖子发育中的作用 体外（目的1）;在疾病的自然进展中使用小鼠模型（目的2）;并确定 新的R点组件TgCables 1和TgRch 1（目标3）。我们的建议是建立在一个坚实的基础上， 先进的遗传学，这是由我们在研究T. 弓形虫速殖子细胞周期使用一组具有新的生长素依赖性条件蛋白表达的菌株 我们将测试TgCrk 2/P-cyclin复合物调节的差异是否可以解释P-cyclin的动力学， 缓殖子分化和包囊复活。总的来说，我们的实验将开辟新的领域， 了解调节发育进程的发育开关的机制和调节， 疾病

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.