How to starve a parasite: Manipulating CoA biosynthesis to control Plasmodium development in the mosquito

项目摘要 疟疾寄生虫需要来自昆虫和哺乳动物宿主的泛酸(PAN)来合成 辅酶A(CoA)和乙酰辅酶A(AC)。此外，蚊子阶段的寄生虫不能摄取预制的CoA 因此，它们完全依赖于蚊盘的可获得性。因此，我们假设 通过增加PAK活性减少蚊子体内的PAN储存，进而促进CoA的生物合成 将限制寄生虫在蚊子中的存活，而不会影响蚊子本身对CoA/AC的可用性。 Pank是CoA生物合成途径中的限速酶，也是我们方法的合理靶点。在这 研究我们将专注于增加蚊子中PANK的活性，将PANK转化为CoA，从而使疟疾挨饿 这种重要前体的寄生虫。为了实现这一点，我们将利用PANK靶向的小分子或 潘他津与潘克在本研究中寄主斯氏按蚊的遗传操作。我们会放映 泛津来自我们在圣犹大儿童医院的合作者开发的化合物图书馆。在……里面 目标1，我们将使用去不去的策略进行潘他嗪筛查，最终测试 选择潘他津以减少斯氏疟原虫中恶性疟原虫和约氏疟原虫感染。目标2将 验证目标1中筛选出的候选泛嗪的生物活性和特异性。特异性 将通过RNAi或CRISPR/Cas9基因敲除来评估用于激活Pank的候选泛津 Pank，然后描述了对PAN、CoA、AC和寄生虫感染成功的影响。 在目标1和目标2的同时，我们将产生具有更高Pank活性和 确定对PAN水平和AIM中寄生虫存活率的影响3.转基因蚊子的产生 随着中肠Pank表达的增加，将有助于我们对Pank依赖的PAN耗竭的评估 储存寄生虫感染以及与媒介能力有关的蚊子生物学的其他方面。 具体地说，我们将通过潘他津治疗和分子治疗来定义蚊虫Pank激活的影响 操纵对斯氏沼虾寿命、应激反应、新陈代谢和繁殖的影响。这些研究将 揭示推动寄生虫感染的营养驱动的蚊子-寄生虫相互作用的重要新见解 成功，他们将支持未来的努力，以优化潘他津和新的转基因品系作为不同的 以蚊子为目标的疟疾控制战略。

Project Summary Malaria parasites require pantothenate (Pan) from both the insect and mammalian hosts to synthesize coenzyme A (CoA) and acetyl-CoA (AC). Further, mosquito-stage parasites cannot take up preformed CoA from the insect host, so they are entirely dependent on mosquito Pan availability. Thus, we hypothesize that reducing Pan stores in the mosquito by increasing Pan kinase (PanK) activity and, in turn, CoA biosynthesis will limit parasite survival in the mosquito, without impacting the availability of CoA/AC to the mosquito itself. PanK is the rate-limiting enzyme in the CoA biosynthesis pathway and a logical target for our approach. In this study we will focus on increasing PanK activity in the mosquito to convert Pan into CoA and starve the malaria parasite of this essential precursor. To accomplish this we will utilize PanK-targeted small molecules or pantazines and genetic manipulation of PanK in our study host Anopheles stephensi. We will screen pantazines from a library of compounds developed by our collaborators at St. Jude Children’s Hospital. In Aim 1, we will use a Go-No Go strategy for pantazine screening that culminates in testing the capacity of selected pantazines to reduce P. falciparum and Plasmodium yoelii infections in A. stephensi. Aim 2 will validate the bioactivity and specificity of candidate pantazines identified in the screen in Aim 1. The specificity of candidate pantazines to activate PanK will be assessed through RNAi or CRISPR/Cas9 knockdown of PanK, followed by a characterization of the impact on Pan, CoA, AC and parasite infection success. Concurrent with Aims 1 and 2, we will generate transgenic A. stephensi with increased PanK activity and determine the impact on Pan levels and parasite survival in Aim 3. The generation of transgenic mosquitoes with increased midgut PanK expression will contribute to our assessment of PanK-dependent depletion of Pan stores on parasite infection as well as other aspects of mosquito biology related to vectorial capacity. Specifically, we will define the effects of mosquito PanK activation, via both pantazine treatment and molecular manipulations, on A. stephensi lifespan, stress responses, metabolism and reproduction. These studies will reveal important new insights into nutrient-driven mosquito-parasite interactions that drive parasite infection success and they will support future efforts to optimize pantazines and novel transgenic lines as distinct strategies for mosquito-targeted malaria control.