Targeting isoprenoid biosynthesis in Plasmodium falciparum

描述（由申请人提供）：与NIAID使命的相关性：本申请描述了一个为期5年的儿科传染病学术生涯发展培训计划，目标是独立指导寄生虫生物学，发病机制和治疗方面的研究。研究设计与方法：恶性疟原虫耐药蔓延，迫切需要新的抗疟药物。了解恶性疟原虫的基本生物学是这些药物开发工作的关键。类异戊二烯分子的生物合成是所有生物体中重要的代谢途径。类异戊二烯分子是各种细胞化合物的基本组成部分，对细胞呼吸、膜结构和信号传导至关重要。我们推测这一途径对于恶性疟原虫的正常发育和繁殖也是必不可少的。在疟疾物种中，类异戊二烯是通过非甲羟戊酸（DXP）途径产生的。寄生虫的DXP途径在生物化学上不同于人类的甲羟戊酸途径，有证据表明该途径是寄生虫生存所必需的。研究将集中在该途径的两种酶，脱氧纤维素磷酸还原异构酶（DXR）和甲基赤四醇环二磷酸合成酶（IspF）。为了研究DXR和IspF的生物学和生化特性，我们提出了生物化学和遗传学双管齐下的方法。具体目标包括：(1)DXR和IspF的异源表达，开发适合高通量筛选的体外检测方法，并对这两种酶进行生化表征；(2)通过培养表达DXR和IspF gfp融合物的恶性疟原虫转基因菌株，在疟原虫内定位DXR和IspF；(3)如有可能，生成恶性疟原虫DXR和IspF破坏菌株，并详细分析疟原虫破坏菌株和DXR小分子抑制剂fosmidomycin处理的菌株抑制类异戊二烯生物合成的表型效应。与公共卫生的相关性：这些实验探索了恶性疟原虫的基本代谢途径，类异戊二烯生物合成的基本生物学。类异戊二烯化合物，包括醌类、光合色素和固醇，对细胞功能至关重要。这一领域的研究有望为寄生虫的发病机制和最终的治疗方法提供见解。

DESCRIPTION (provided by applicant): Relevance to NIAID mission: This application describes a 5-year training program for the development of an academic career in Pediatric Infectious Diseases, with a goal of independently directing research into parasite biology, pathogenesis, and therapy. Research design and methods: New antimalarial agents are urgently needed due to the spread of drug resistance in the pathogen Plasmodium falciparum. Understanding the fundamental biology of P. falciparum is key to these drug development efforts. An important metabolic pathway in all organisms is the biosynthesis of isoprenoid molecules, fundamental building blocks for diverse cellular compounds vital for cellular respiration, membrane structure, and signaling. We hypothesize that this pathway is also essential for the normal development and reproduction of Plasmodium falciparum. In malaria species, isoprenoids are made via the non-mevalonate (DXP) pathway. The parasite DXP pathway is biochemically distinct from the mevalonate pathway in humans, and evidence suggests this pathway is required for parasite survival. Research will focus on two enzymes of this pathway, deoxyxylulose phosphate reductoisomerase (DXR) and methylerythritol cyclodiphosphate synthase (IspF). To study the biological and biochemical characteristics of DXR and IspF, we propose a dual-pronged biochemical and genetic approach. The specific aims include the following: (1) Heterologous expression of DXR and IspF, development of in vitro assays suitable for high-throughput screening, and biochemical characterization of both enzymes; (2) Localization of DXR and IspF within the parasite by development of transgenic strains of P. falciparum that express GFP-fusions of DXR and IspF; (3) Generation of DXR and IspF disruption strains of P. falciparum, if possible, and detailed analysis of the phenotypic effects of inhibition of isoprenoid biosynthesis in both parasite disruption strains and strains treated with a small-molecule inhibitor of DXR, fosmidomycin. Relevance to public health: These experiments explore the basic biology of a fundamental metabolic pathway, isoprenoid biosynthesis, of Plasmodium falciparum. Isoprenoid compounds, which include quinones, photosynthetic pigments, and sterols, are vital to cellular function. This area of research is expected to provide insights into parasite pathogenesis, and ultimately therapeutics.