Differentiation and Signaling in Toxoplasmosis

描述（申请人提供）：刚地弓形虫是一种机会性原生动物寄生虫，可引起艾滋病患者中枢神经疾病。弓形虫也是B类生物恐怖主义制剂。弓形虫的感染通过速殖子形式在被感染的宿主中传播。传播后，快速繁殖的弓形虫速殖子分化为缓慢复制的慢殖子，这些慢殖子在宿主的一生中都潜伏在包囊内。临床上明显的艾滋病患者弓形虫病被认为是由于免疫功能减弱时潜伏的慢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调节的应激诱导分化与缓殖子相似。操纵环核苷酸信号通路影响慢殖子形成的频率。为了研究寄生虫信号通路的作用，我们克隆并鉴定了弓形虫cAMP依赖性激酶，PKA， cAMP信号通路的主要效应体。转染研究表明，cAMP依赖性激酶的催化亚基pka1和PKA2在定位和对寄生虫增殖的影响上存在差异。我们假设pka1和PKA2在速殖子-慢殖子过渡过程中对代谢和细胞周期调节具有相反的调节功能。我们计划破坏PKA基因，以进一步表征它们在寄生虫增殖和慢殖子分化中的作用。在一系列独立的实验中，酵母2杂交筛选将识别与pka1和PKA2相互作用的蛋白质。最后，我们用7-。弓形虫cDNA微阵列检测全球基因表达模式，以响应已知诱导慢殖子形成的条件和影响cAMP信号的条件。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cAMP信号通路在刚地弓形虫中的作用，并确定cAMP信号通路在刚地弓形虫向潜伏慢殖虫分化过程中的作用。

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.