Differentiation and Signaling in Toxoplasmosis

描述（由申请人提供）：弓形虫是一种机会性原生动物寄生虫，可引起艾滋病患者的中枢神经系统疾病。T.弓形虫也是一种B类生物恐怖剂。感染猫弓首蛔虫弓形虫通过速殖子形式在整个感染宿主中传播。传播后迅速增殖的T.弓形虫速殖子分化成缓慢复制的缓殖子，其在宿主的一生中保持潜伏在包囊内。艾滋病患者临床上明显的弓形虫病被认为是由于免疫功能减弱时潜伏的缓殖子重新激活而发生的。缓殖子的分化是应激介导的，我们的数据表明在其他真核病原体中cAMP调节应激诱导的分化的相似性。环核苷酸信号通路的操纵影响缓殖子形成的频率。为了研究寄生虫信号通路的作用，我们克隆了T。弓形虫cAMP依赖性激酶，PKA，cAMP信号传导的主要效应子。转染研究已经揭示了cAMP依赖性激酶PKA 1和PKA 2的催化亚基在定位和它们对寄生虫增殖的影响方面不同。我们推测PKA 1和PKA 2在速殖子-缓殖子转换过程中对代谢和细胞周期的调控具有相反的功能。我们计划破坏PKA基因，以进一步表征其在寄生虫增殖和缓殖子分化中的作用。在一个独立的实验中，酵母2杂交筛选将鉴定与PKA 1和PKA 2相互作用的蛋白质。最后，我们将使用7-。用弓形虫cDNA微阵列检测在已知诱导缓殖子形成的条件和影响cAMP信号传导的条件下的整体基因表达模式。基因表达模式将与PKA亚基突变或缺失的寄生虫进行比较。这些研究将进一步阐明T.并确定cAMP信号通路在T.弓形虫分化为潜伏的缓殖子。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an opportunistic protozoan parasite that causes central nervous disease in AIDS patients. T. gondii is also a Category B bioterrorism agent. Infection with T. gondii results in dissemination throughout the infected host via the tachyzoite form. After dissemination, the rapidly proliferating T. gondii tachyzoites differentiate into slowly replicating bradyzoites that remain latent within cysts for the life of the host. Clinically apparent toxoplasmosis in AIDS patients is thought to occur due to reactivation of latent bradyzoites as immune function wanes. Bradyzoite differentiation is stress mediated, and our data suggest similarities to cAMP regulated stress-induced differentiation in other eukaryotic pathogens. Manipulation of cyclic nucleotide signaling pathways affects the frequency of bradyzoite formation. To examine the role of parasite signaling pathways, we have cloned and characterized the T. gondii cAMP dependent kinase, PKA, the major effector of cAMP signaling. Transfection studies have revealed that the catalytic subunits of the cAMP dependent kinase, PKA 1 and PKA2, differ in localization and in their effect on parasite proliferation. We hypothesize that PKA 1 and PKA2 have opposing regulatory functions on metabolism and cell cycle regulation during tachyzoite-bradyzoite transition. We plan to disrupt PKA genes to further characterize their roles in parasite proliferation and bradyzoite differentiation. In an independent line of experiments, yeast 2 hybrid screens will identify proteins that interact with PKA 1 and PKA2. Finally, we will use 7-. gondii cDNA microarrays to examine global gene expression patterns in response to conditions known to induce bradyzoite formation and conditions that affect cAMP signaling. Gene expression patterns will be compared to parasites with mutations or deletions of PKA subunits. These studies will further elucidate cAMP signaling pathways in T. gondii and determine the role of cAMP signaling pathways during T. gondii differentiation into latent bradyzoites.