Molecular Dissection of Early Toxoplasma gondii Bradyzoite Differentiation

描述(申请人提供)：弓形虫是一种人畜共患寄生虫，分布于世界各地。在越来越多的免疫抑制患者中，疾病主要是由于休眠的缓体包囊重新激活所致。缓殖子形式的发育可以在体外进行研究，但人们对这一重要过程背后的分子或遗传学细节知之甚少。利用一种新的多功能微阵列，下面概述的研究将提供对缓殖子形成的全面的全基因组分析。根据到目前为止产生的初步数据，我们假设非常早期的基因产物是启动缓殖子分化所必需的，这将通过转基因和/或敲打寄生的靶向工程来研究。具体目标1：确定定义缓殖子诱导的基因。1.a.确定可能的缓殖子基因的表达模式。将使用多种诱导方法来确定独特的缓殖子特异基因。1.B.关注下游的基因靶向。利用合理的选择过程和生物信息学分析，基因将成为基因敲除实验的目标。具体目标2：通过遗传扰动对关键阶段特定转录本的分子解剖。2.a.从基因上删除(敲除)选定的感兴趣基因。电转染和药物筛选将被用来产生等位基因敲除突变。2.B.体外评估可能的缓殖子诱导突变体。在体外形成缓殖子的多种方法中存在缺陷的突变株将识别可能在分化过程中至关重要的基因。2.C.通过适合度分析和互补性验证目的基因对缓殖子诱导的特异性影响。为了确保表型与缓殖体的形成直接相关，将对突变株进行补充，并验证其生长和传染性适合性。该项目的长期目标是建立对弓形虫普遍存在的致病缓殖子阶段的更全面的了解。这项研究中提出的实验有望导致识别出一些参与弓形虫分化过程的基因。除了从一般的生物学观点来看是有意义的，这些基因还应该为开发针对缓殖子的治疗方法提供新的见解。除了提高我的研究技能外，这可能会为开发针对缓殖体靶标的药物提供关键见解，也就是开发疫苗所用的减病生物。

DESCRIPTION (provided by applicant): Toxoplasma gondii is a zoonotic human parasite with worldwide distribution. Disease in the growing ranks of immunosuppressed patients is primarily due to reactivation of dormant bradyzoite cysts. Development of the bradyzoite form can be studied in vitro, but few molecular or genetic details underlying this important process are known. Taking advantage of a novel multifunctional microarray, the research outlined below will provide a comprehensive genome-wide analysis of bradyzoite formation. Based on preliminary data generated to date, we hypothesize that very early gene products are required for the initiation of bradyzoite differentiation, which will be studied through targeted engineering of transgenic and/or knock-put parasites. Specific Aim 1: Identification of genes that define bradyzoite induction. 1.a. Define expression patterns for putative bradyzoite genes. Multiple induction methods will be employed to ascertain unique bradyzoite-specific genes. 1.b. Focus downstream gene targeting. Using a rational selection process coupled with bioinformatic analysis, genes will be targeted for genetic knock-out experiments. Specific Aim 2: Molecular dissection of critical stage-specific transcripts through genetic perturbation. 2.a. Genetically delete (knock-out) selected genes of interest. Electro-transfection and drug selection will be used to generate allelic knock-out mutants. 2.b. Assess putative bradyzoite induction mutants in vitro. Mutants defective in multiple methods of in vitro bradyzoite formation will identify genes likely to be essential in the differentiation process. 2.c. Verify specific influence of the target gene on bradyzoite induction through fitness assays and complementation. To ensure that phenotypes are directly related to bradyzoite formation, mutants will be complemented and verified for growth and infectivity fitness. The long-term goal of this project is to establish a more complete understanding of the ubiquitous and pathogenic bradyzoite stage of T. gondii. The experiments proposed in this study are expected to lead to the identification of a number of genes involved in the differentiation process in T. gondii. Besides being of interest from a general biological standpoint, such genes should provide novel insights into the development of bradyzoite-specific therapeutics. In addition to increasing my research skills, this may provide key insights leading to the development of drugs designed against bradyzoite targets, or disease-attenuated organisms useful in developing vaccines.