Targeting isoprenoid biosynthesis in Plasmodium falciparum

描述(由申请人提供)：与NIAID任务相关：本申请描述了一项为期5年的培训计划，旨在发展儿科传染病的学术生涯，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。研究设计和方法：由于恶性疟原虫耐药性的传播，迫切需要新的抗疟疾药物。了解恶性疟原虫的基本生物学是这些药物开发努力的关键。在所有生物体中一个重要的代谢途径是类异戊二烯分子的生物合成，类异戊二烯分子是对细胞呼吸、膜结构和信号转导至关重要的各种细胞化合物的基本构件。我们推测这一途径对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氧戊酸(DXP)途径产生的。寄生虫的DXP途径在生化上不同于人类的甲氧戊酸途径，证据表明这一途径是寄生虫生存所必需的。研究将集中在这一途径的两种酶，脱氧果糖磷酸还原异构酶(DXR)和甲基赤藓糖醇环二磷酸合成酶(ISPF)。为了研究DXR和ISPF的生物学和生化特性，我们提出了生化和遗传学的双管齐下的方法。具体目标包括：(1)DXR和ISPF的异源表达，适合高通量筛选的体外检测方法的建立，以及这两种酶的生化特性；(2)通过开发表达DXR和ISPF GFP融合基因的恶性疟原虫转基因菌株，定位DXR和ISPF在寄生虫内的位置；(3)如果可能，建立恶性疟原虫DXR和ISPF干扰株，并详细分析DXR破坏株和DXR小分子抑制剂福米霉素处理的株抑制异戊二烯生物合成的表型效应。与公众健康相关：这些实验探索恶性疟原虫基本代谢途径--类异戊二烯生物合成的基本生物学。异戊二烯类化合物，包括苯二酚、光合色素和甾醇，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提供见解，并最终提供治疗方法。

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.