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

阐明恶性疟原虫青蒿素耐药性的复杂遗传和分子基础。

• 批准号: 9406657
• 负责人: Leila Saxby Ross
• 金额: $ 0.06万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-10 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9406657
• 关键词: Admixture; Alleles; Anopheles Genus; Antioxidants; Artemisinins; Biological Assay; Blood; Cambodia; Cambodian; Cell Cycle Regulation; Cessation of life; Chloroquine resistance; Clinical; Coculture Techniques; Collaborations; Combined Modality Therapy; Complex; Complex Genetic Trait; Culicidae; Data; Disasters; Disease; Dissection; Drug resistance; Evaluation; Event; Excision; Falciparum Malaria; Ferredoxin; Genes; Genetic; Genetic Polymorphism; Genomic Segment; Genomics; Geographic Locations; Geography; Gold; Health; Human; Hypoxanthines; Immunoprecipitation; In Vitro; Individual; Infection; Investigation; Iron; Laboratories; Life Cycle Stages; Linkage Disequilibrium; Malaria; Mediating; Methods; Midgut; Molecular; Monitor; Morbidity - disease rate; Mosquito Control; Mutation; National Institute of Allergy and Infectious Disease; Oocysts; Oxidation-Reduction; Oxidative Stress; P-Glycoproteins; Parasite resistance; Parasites; Patient Isolators; Patients; Pennsylvania; Pharmaceutical Preparations; Pharmacotherapy; Plasmodium; Plasmodium falciparum; Population; Positioning Attribute; Prevalence; Research; Resistance; Risk; Role; Southeastern Asia; Testing; Time; Treatment Failure; Ubiquitin; United States National Institutes of Health; Universities; Vaccines; asexual; base; biological adaptation to stress; cost; drug sensitivity; efficacy study; feeding; fitness; gene function; genome wide association study; improved; innovation; insight; metabolome; metabolomics; molecular marker; mortality; mutant; novel strategies; novel therapeutics; permissiveness; prevent; protein degradation; public health relevance; pyrosequencing; resistance mechanism; resistance mutation; tool; transcriptomics; transmission process; vector control; vector mosquito; zinc finger nuclease

 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.

 描述(申请人提供)：疟疾是一种由感染疟原虫引起的疾病，其中恶性疟原虫是最致命的。世界上有一半的人口面临感染的风险。自2000年以来，更多地获得以青蒿素为基础的联合疗法和蚊媒控制举措，估计挽救了330万人的生命。尽管取得了这一重大进展，疟疾仍然是人类健康的主要负担，2012年约有627,000人死亡。目前还没有有效的疫苗。令人担忧的是，东南亚出现了青蒿素抗药性。我们必须了解抵抗的机制和程度，以帮助防止区域危机演变为全球灾难。最近，对青蒿素抗性寄生虫的基因组和转录组分析表明，K13基因发生了突变，与铁运输和泛素介导的蛋白质降解有关的基因起到了次要作用。我们使用了锌指核酸酶(ZFN)，一种变革性的基因编辑工具，表明从抗青蒿素的柬埔寨寄生虫中移除K13突变可以消除耐药性。在对青蒿素敏感的寄生虫品系中插入K13突变会产生不同程度的抗性，这表明了允许的遗传背景的影响。我们相信，我们正在对东南亚寄生虫进行的评估，以及从同基因、特征良好的寄生虫中快速添加或移除候选突变的能力相结合，将使我们能够全面剖析耐药性机制。我们将利用ZFN研究K13的功能以及遗传背景对青蒿素抗性产生和传播的影响。出现与抵抗机制有关，而传播与传播有关。我们将使用ZFN将潜在的抗药性突变单独或组合添加或移除到等基因寄生虫系中。为了研究突现，我们将使用环阶段存活试验(RSA0-3h)和改进的[~3H]-次黄嘌呤掺入试验来确定青蒿素的敏感性，并通过确定寄生虫氧化还原代谢体的变化、泛素介导的蛋白酶体降解、相互作用伙伴的定义以及K13的亚细胞定位来探索K13的功能。根据目前的数据，我们假设K13作为氧化应激反应的负调节因子，对铁介导的氧化还原损伤做出反应。我们假设K13的抗药性相关突变降低了功能，导致抗氧化能力增强。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的适合度比较。加强对青蒿素耐药性的了解将有助于指导在地理上有针对性地使用非青蒿素为基础的二线或新疗法，以减轻疟疾负担。