Transcriptional mechanisms in Toxoplasma gondii

描述（由申请人提供）：刚地弓形虫感染是一种公共卫生危害，每年有数百万人接触这种病原体，特别是那些免疫系统因衰老、化疗或艾滋病而减弱的人；以及在怀孕期间感染的女性。弓形虫的暴露是通过接触环境源和受污染的食物发生的，仅次于细菌病原体李斯特菌和沙门氏菌，是食源性死亡的第三大原因。目前没有一种治疗方法能够预防由囊状慢殖子期引起的终身感染，药物毒性限制了高危个体的长期治疗。因此，迫切需要开发能够预防慢性弓形虫感染的新方法，尽管由于缺乏对组织囊肿形成的分子基础的了解，这一目标的实现受到阻碍。最近，我们完成了弓形虫中间生命周期转录组的全面SAGE分析（由R21奖AI53815资助）。这些研究表明，导致组织囊肿形成的主要发育转变伴随着分散在所有寄生虫染色体上的基因的一组暂时有序的转录事件，因此，由反式作用蛋白因子共同调节。这项探索性资助旨在通过描述核苷酸序列元件和鉴定参与已知和新型慢殖子特异性基因表达的蛋白质因子，更好地了解与寄生虫发育相关的核心转录机制。我们开发了基于高通量克隆方法的双荧光素酶测定，从功能上证明了控制速殖子和慢殖子特异性基因表达需要位于弓形虫基因组编码区上游的基因近端序列。在本研究中，我们将采用相同的方法来确定5个慢殖子启动子的最小序列要求，并利用这些信息通过生化和遗传策略来识别合适的DNA结合蛋白。这里提出的研究将解决我们理解弓形虫寄生虫（和其他顶复虫）如何调节基因表达的根本空白，并最终导致更好地理解这些寄生虫如何控制发展成临床重要的组织囊肿。

DESCRIPTION (provided by applicant): Toxoplasma gondii infections are a public health hazard for millions of individuals that contact this pathogen annually, particularly those with immune systems weakened by aging, chemotherapy or AIDS; and women that acquire the infection during pregnancy. Exposure to this pathogen occurs through contact with environmental sources and contaminated food ranking T. gondii only behind bacterial pathogens, Listeria and Salmonella, as the third-leading cause of food-borne death. No current therapy is able to prevent life- time infections caused by the encysted bradyzoite stage and drug toxicity limits long-term treatment for at risk individuals. Thus, there is a critical need to develop new approaches capable of preventing chronic Toxoplasma infection, although achieving this goal is hampered by a lack of understanding of the molecular basis for tissue cyst formation. Recently, we have completed a comprehensive SAGE analysis (funded by R21 award AI53815) of the transcriptome of the Toxoplasma intermediate life cycle. These studies demonstrate primary developmental transitions leading to tissue cyst formation are accompanied by a temporally ordered set of transcriptional events from genes that are dispersed across all parasite chromosomes, and are therefore, co-regulated by trans-acting protein factors. This exploratory grant seeks to gain a greater understanding of the core transcriptional mechanisms associated with parasite development by characterizing the nucleotide sequence elements and identifying the protein factors involved in the expression of known and novel bradyzoite-specific genes. We have developed dual- luciferase assays based on high-throughput cloning methods to functionally demonstrate that the control of tachyzoite- and bradyzoite-specific gene expression requires gene proximal sequences lying upstream of coding regions in the Toxoplasma genome. In this proposal, we will employ these same methods to define the minimal sequence requirements for five bradyzoite promoters, and in turn, use this information to identify the appropriate DNA binding proteins through biochemical and genetic strategies. The studies proposed here will address a fundamental gap in our understanding how Toxoplasma parasites (and other Apicomplexa) regulate gene expression, and will ultimately, lead to a better understanding of how these parasites control development into the clinically important tissue cyst.