Differentiation and Signaling in Toxoplasmosis

弓形虫病的分化和信号转导

• 批准号: 6897808
• 负责人: Kami Kim
• 金额: $ 37.58万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6897808
• 关键词: Toxoplasma gondii; biological signal transduction; bioterrorism /chemical warfare; cell differentiation; cell growth regulation; cyclic AMP; enzyme linked immunosorbent assay; gene complementation; gene expression; green fluorescent proteins; intracellular parasitism; laboratory mouse; latent virus infection; mass spectrometry; matrix assisted laser desorption ionization; microarray technology; molecular cloning; pathologic process; polymerase chain reaction; protein kinase A; protein protein interaction; tissue /cell culture; toxoplasmosis; western blottings; yeast two hybrid system

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.

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疾病。弓形虫也是 B 类生物恐怖主义制剂。弓形虫感染导致通过速殖子形式在受感染宿主中传播。传播后，快速增殖的弓形虫速殖子分化成缓慢复制的缓殖子，在宿主的一生中一直潜伏在包囊内。艾滋病患者中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。缓殖子分化是应激介导的，我们的数据表明与其他真核病原体中 cAMP 调节的应激诱导分化相似。环核苷酸信号通路的操纵会影响缓殖子形成的频率。为了检查寄生虫信号传导途径的作用，我们克隆并表征了弓形虫 cAMP 依赖性激酶 PKA，它是 cAMP 信号传导的主要效应器。转染研究表明，cAMP 依赖性激酶 PKA 1 和 PKA2 的催化亚基的定位及其对寄生虫增殖的影响不同。我们假设 PKA 1 和 PKA2 在速殖子-缓殖子转变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缓殖子分化中的作用。在一系列独立的实验中，酵母 2 杂交筛选将鉴定与 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，用于检查全局基因表达模式，以响应已知诱导缓殖子形成的条件和影响 cAMP 信号传导的条件。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。