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（由适用提供）：疟疾是由疟原虫寄生虫感染引起的疾病，其中恶性疟原虫是最致命的。世界一半人口有感染的风险。自2000年以来，基于青蒿素的联合疗法（使徒行为）和蚊子矢量控制计划的使用量增加了估计的330万生命。尽管取得了重大进展，但疟疾仍然是人类健康的重大烧伤，2012年的死亡人数约为627,000。令人震惊的是，东南亚已经出现了青蒿素抵抗。我们必须了解抵抗的机制和程度，以防止区域危机成为全球灾难。抗毒素抗性寄生虫的基因组和转录组分析最近在K13基因中实现了突变，其中涉及铁转运和泛素蛋白介导的蛋白质降解的次要贡献。我们使用了一种变换的遗传编辑工具锌指核（ZFNS）来表明从抗贫青其他柬埔寨寄生虫中去除K13突变，消除了耐药性。将K13突变插入甲萎缩蛋白敏感的寄生虫线中，赋予了可变程度的抗性，表明了允许的遗传背景的影响。我们认为，我们对来自东南亚的寄生虫的持续评估以及从等源性，良好的寄生虫中迅速添加或去除候选突变的能力将允许对抗性机制进行全面的解剖。我们将使用ZFN研究K13的功能以及遗传背景对青蒿素耐药性的出现和传播的影响。出现与电阻机理有关，而扩散与传播相关。我们将使用ZFN单独或组合将潜在的抗性突变添加或去除潜在的抗性突变。为了研究出现，我们将同时使用环阶段的生存测定法（RSA0-3H）和经过改进的[3H] - 黄嘌呤结合结合测定法来确定青蒿素的敏感性，并通过确定寄生虫氧化还原氧化氧化物氧化代谢，泛素介导蛋白介导的蛋白酶体质量质量的定义和K13的定义分子和k13的变化，并探索K13。鉴于当前的数据，我们假设K13是氧化应激反应的负调节剂，对铁介导的氧化还原损伤做出了反应。我们阳性K13中与抗性相关的突变降低了功能，从而增加了抗氧化能力。为了研究传播，我们将在无性血液阶段和性蚊子阶段进行寄生虫的成对适应性比较。对青蒿素耐药性的改进理解将有助于指导非基于非甲虫素的二线或新型疗法的靶向地理使用，以减少疟疾的燃烧。