Molecular Dissection of Early Toxoplasma gondii Bradyzoite Differentiation

简介（申请人提供）：刚地弓形虫是一种人畜共患寄生虫，分布在世界各地。在越来越多的免疫抑制患者中，疾病主要是由于休眠的慢殖子囊肿的重新激活。缓殖子形态的发育可以在体外研究，但这一重要过程背后的分子或遗传细节尚不清楚。利用一种新型多功能微阵列，下面概述的研究将提供一个全面的慢殖子形成的全基因组分析。根据迄今为止产生的初步数据，我们假设慢殖子分化的起始需要非常早期的基因产物，这将通过转基因和/或敲除寄生虫的靶向工程来研究。特异性目标1：鉴定慢殖子诱导的基因。1.。定义假定的慢殖子基因的表达模式。将采用多种诱导方法来确定独特的慢殖子特异性基因。1. b。聚焦下游基因靶向。通过合理的选择过程，结合生物信息学分析，基因将成为基因敲除实验的目标。特定目标2：通过遗传扰动进行关键阶段特异性转录本的分子解剖。2.。基因删除（敲除）感兴趣的选定基因。电转染和药物选择将用于产生等位基因敲除突变体。2. b。在体外评估假定的缓殖子诱导突变体。在体外慢殖子形成的多种方法中有缺陷的突变体将识别可能在分化过程中必不可少的基因。2.摄氏度。通过适应度测定和互补验证靶基因对慢殖子诱导的具体影响。为了确保表型与慢殖子形成直接相关，突变体将被补充并验证其生长和感染性适应性。该项目的长期目标是更全面地了解弓形虫普遍存在的致病慢殖子阶段。本研究中提出的实验有望导致鉴定一些参与弓形虫分化过程的基因。除了从一般生物学的角度来看，这些基因应该为慢殖子特异性治疗的发展提供新的见解。除了提高我的研究技能外，这可能会为开发针对慢殖子靶点的药物提供关键见解，或者为开发疫苗提供有用的疾病减毒生物体。

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.