Differentiation and Signaling in Toxoplasmosis

描述(申请人提供)：弓形虫是一种机会性原虫寄生虫，可导致艾滋病患者的中枢神经系统疾病。弓形虫也是B类生物恐怖分子。感染弓形虫会通过速殖子在受感染的宿主中传播。在传播后，快速增殖的弓形虫速殖子分化为缓慢复制的缓体，这些缓体在宿主的一生中潜伏在包囊内。临床上，艾滋病患者的明显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所致。缓殖子的分化是由胁迫介导的，我们的数据表明，在其他真核病原体中，cAMP调控的胁迫诱导的分化与之相似。环核苷酸信号通路的操作影响缓殖子形成的频率。为了研究寄生虫信号通路的作用，我们克隆并鉴定了弓形虫cAMP依赖的激酶PKA，它是cAMP信号的主要效应者。转染法研究表明，cAMP依赖的激酶催化亚基PKA 1和PKA2在定位和对寄生虫增殖的影响上存在差异。我们推测，在速殖子-缓殖子转变过程中，PKA-1和PKA2对新陈代谢和细胞周期的调节具有相反的调节功能。我们计划破坏PKA基因，以进一步确定它们在寄生虫增殖和缓殖体分化中的作用。在一系列独立的实验中，酵母2杂交筛选将识别与PKA 1和PKA2相互作用的蛋白质。最后，我们将使用7-。弓形虫cDNA微阵列检测全球基因表达模式，以响应已知的诱导缓殖子形成的条件和影响cAMP信号的条件。基因表达模式将与带有PKA亚单位突变或缺失的寄生虫进行比较。这些研究将进一步阐明弓形虫中的cAMP信号通路，并确定cAMP信号通路在弓形虫分化为潜伏缓殖子过程中的作用。

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.