Elucidating the complex genetic and molecular basis of Plasmodium falciparum artemisinin resistance.

 描述（由申请人提供）：疟疾是一种由疟原虫感染引起的疾病，其中恶性疟原虫是最致命的。世界上一半的人口面临感染的风险。自2000年以来，青蒿素类复方疗法和蚊媒控制举措的普及估计挽救了330万人的生命。尽管取得了这一重大进展，但疟疾仍然是人类健康的一个重大负担，2012年约有627 000人死亡。没有有效的疫苗。令人担忧的是，青蒿素耐药性已在东南亚出现。我们必须了解抵抗的机制和程度，以帮助防止区域危机成为全球灾难。青蒿素耐药寄生虫的基因组和转录组学分析最近涉及K13基因的突变，与参与铁转运和泛素介导的蛋白质降解的基因的次要贡献。我们使用锌指核酸酶（ZFN），一种变革性的基因编辑工具，证明从青蒿素抗性柬埔寨寄生虫中去除K13突变可以消除抗性。将K13突变插入青蒿素敏感的寄生虫品系中，可产生不同程度的抗性，表明受容许遗传背景的影响。我们相信，我们对东南亚寄生虫的持续评估以及从等基因的、充分表征的寄生虫中快速添加或删除候选突变的能力的结合，将允许全面剖析耐药机制。我们将使用ZFN来研究K13的功能以及遗传背景对青蒿素抗性的出现和传播的影响。出现与抵抗机制有关，而传播与传播有关。我们将使用ZFN单独或组合添加或去除潜在的抗性突变到等基因寄生虫系中。为了研究羽化，我们将使用环阶段存活测定（RSA 0 - 3 h）和改良的[3 H]-次黄嘌呤掺入测定来确定青蒿素敏感性，并通过鉴定寄生虫氧化还原代谢组的变化，泛素介导的蛋白酶体降解，相互作用伴侣的定义和K13亚细胞定位来探索K13功能。鉴于目前的数据，我们假设K13作为氧化应激反应的负调节剂，对铁介导的氧化还原损伤作出反应。我们认为K13中的抗性相关突变降低了功能，导致抗氧化能力增加。为了研究传播，我们将对无性血液阶段和有性蚊子阶段的寄生虫进行成对适合度比较。更好地了解青蒿素耐药性将有助于指导有针对性地使用非青蒿素二线或新疗法，以减轻疟疾负担。

 DESCRIPTION (provided by applicant): Malaria is a disease caused by infection with Plasmodium parasites, of which P. falciparum is the deadliest. Half of the world's population is at risk of infection. Increased access to artemisinin-based combination therapies (ACTs) and mosquito vector control initiatives have saved an estimated 3.3 million lives since the year 2000. Despite this substantial progress, malaria remains a major burden to human health with approximately 627,000 deaths in 2012. There is no effective vaccine. Alarmingly, artemisinin resistance has emerged in Southeast Asia. It is imperative that we understand the mechanism and extent of resistance to help prevent a regional crisis from becoming a global disaster. Genomic and transcriptomic analyses of artemisinin-resistant parasites have recently implicated mutations in the K13 gene, with secondary contributions from genes involved in iron transport and ubiquitin-mediated protein degradation. We used zinc-finger nucleases (ZFNs), a transformative genetic editing tool, to show that removing K13 mutations from artemisinin-resistant Cambodian parasites ablates resistance. Inserting K13 mutations into artemisinin-sensitive parasite lines conferred variable degrees of resistance, indicating the influence of a permissive genetic background. We believe that our combination of ongoing evaluation of parasites from Southeast Asia and the ability to swiftly add or remove candidate mutations from isogenic, well-characterized parasites will allow a comprehensive dissection of the resistance mechanism. We will use ZFNs to study the function of K13 and the influence of genetic background on the emergence and spread of artemisinin resistance. Emergence is tied to the mechanism of resistance, whereas spread is tied to transmission. We will use ZFNs to add or remove potential resistance mutations into isogenic parasite lines alone or in combination. To study emergence, we will use both the ring-stage survival assay (RSA0-3h) and a modified [3H]-hypoxanthine incorporation assay to determine artemisinin sensitivity, and explore K13 function through identifying changes in the parasite redox metabolome, ubiquitin-mediated proteasomal degradation, the definition of interacting partners, and K13 subcellular localization. Given current data, we hypothesize that K13 functions as a negative regulator of oxidative stress responses that respond to iron-mediated redox damage. We posit that resistance-associated mutations in K13 reduce function, leading to increased antioxidant capability. To study transmission, we will perform pairwise fitness comparisons of parasites in both asexual blood stages and sexual mosquito stages. An improved understanding of artemisinin resistance will help guide the targeted geographical use of non-artemisinin-based second-line or novel therapies to reduce the burden of malaria.