Defining P. falciparum resistance to artemisinin-based combination therapies

项目摘要 在世界范围内采用青蒿素（ART）为基础的联合疗法（ACT）有助于 自21世纪初以来，全球恶性疟原虫（Pf）疟疾负担减半;然而， 自2015年以来停滞不前。疟疾的影响仍然巨大，2018年估计有405，000人死亡。Pf电阻 抗逆转录病毒药物衍生物及其抗逆转录病毒药物伙伴药物的威胁压倒了控制努力。抗寄生虫 ART和哌喹（PPQ）主要分别由基因k13和pfcrt突变介导， 席卷了东南亚的低传播环境。非洲ACT耐药性的发展， 占全球疟疾负担的94%，将是灾难性的。一个主要的警告信号是， 卢旺达的ART耐药K13突变体，以及附近国家的其他K13突变体。在此我们 非洲出现ACT耐药性的前景。在目标1中，我们将检验非洲Pf 菌株很少对驱动ART抗性的K13突变造成生物学障碍。通过基因编辑 我们将确定最具抗药性的突变，并评估寄生虫的影响 背景，这可以大大影响阻力和健身水平。最近完成了 柬埔寨ART耐药K13突变体和药物敏感的非洲寄生虫之间的两个Pf遗传杂交 NF 54，我们将绘制并确认二级ART抗性调节剂。我们还将测试抵抗力是否能 这可能归因于在早期环阶段限制血红蛋白内吞作用的功能丧失突变。在目标2中， 将测试突变K13将在非洲寄生虫中产生大量适应性成本的假设，这可能 阻止其在高传播环境中传播。一种方法将是量化条形码K13的生长速率， 突变体和野生型等基因系。我们还假设亚洲寄生虫的抗逆转录病毒疗法耐药性是通过一种 获得K13突变与补偿适应性突变相结合，并将使用我们的遗传杂交后代， 映射并确认适应性调节器。在目标3中，我们将探讨Pf对ACT伴侣耐药的决定因素 药物PPQ、苯芴醇（LMF）、咯萘啶（PND）和阿莫地喹（ADQ）在非洲寄生虫中的作用。基因编辑 将用于检验PPQ耐药性可通过非洲PfCRT中的单点突变产生的假设 单倍型。确定LMF和PND抗性的决定因素的努力将采用具有超 多变的非洲血统我们还将利用我们的一个基因杂交的发现， ADQ抗性的基础包括pfcrt和12号染色体上的未知决定簇。识别 这些标记物将为ADQ抗性筛选提供有价值的工具。该提案符合 NIAID优先支持抗菌药物耐药性研究，旨在积极准备 通过确定因果决定因素和突变，非洲出现ART和ACT耐药性的可怕可能性 以及评估适应性成本是否可以阻止耐药性在非洲传播环境中的传播。

PROJECT SUMMARY The worldwide adoption of artemisinin (ART)-based combination therapies (ACTs) has been instrumental in halving the global burden of Plasmodium falciparum (Pf) malaria since the early 2000s; however, progress has stalled since 2015. Malaria’s impact remains vast, with an estimated 405,000 deaths in 2018. Now, Pf resistance to ART derivatives and their ACT partner drugs threatens to overwhelm control efforts. Parasites resistant to ART and piperaquine (PPQ), mediated primarily by mutations in the genes k13 and pfcrt respectively, have swept through the low-transmission setting of Southeast Asia. The development of ACT resistance in Africa, with 94% of the global malaria burden, would be calamitous. A major warning sign is the increasing prevalence of an ART resistance-conferring K13 mutant in Rwanda, and of other K13 mutations in nearby countries. Herein we confront the prospect of ACT resistance emerging in Africa. In Aim 1, we will test the hypothesis that African Pf strains rarely pose a biological obstacle to K13 mutations driving ART resistance. By gene editing geographically diverse African strains, we will identify the most resistant mutations and assess the impact of the parasite background, which can substantially influence the levels of resistance and fitness. Having recently completed two Pf genetic crosses between Cambodian ART-resistant K13 mutants and the drug-sensitive African parasite NF54, we will map and confirm secondary ART resistance modulators. We will also test whether resistance can be attributed to loss-of-function mutations that restrict hemoglobin endocytosis in early ring stages. In Aim 2, we will test the hypothesis that mutant K13 will exert a substantial fitness cost in African parasites, which could impede its spread in high-transmission settings. One approach will be to quantify growth rates of barcoded K13 mutant and wild-type isogenic lines. We also hypothesize that ART resistance in Asian parasites evolved via a gain of K13 mutations combined with compensatory fitness mutations, and will use our genetic cross progeny to map and confirm fitness modulators. In Aim 3, we will pursue determinants of Pf resistance to the ACT partner drugs PPQ, lumefantrine (LMF), pyronaridine (PND) and amodiaquine (ADQ) in African parasites. Gene editing will be used to test the hypothesis that PPQ resistance can arise through single point mutations in African PfCRT haplotypes. Efforts to identify determinants of resistance to LMF and PND will employ selections with hyper- mutable African lines. We will also leverage the discovery in one of our genetic crosses of a two-component basis of ADQ resistance that includes pfcrt and an unknown determinant on chromosome 12. The identification of these markers will provide a valuable tool to screen for ADQ resistance. This proposal, which aligns with the NIAID priority of supporting research on antimicrobial drug resistance, is designed to proactively prepare for the dire possibility of ART and ACT resistance emerging in Africa by identifying causal determinants and mutations and assessing whether fitness costs can impede the spread of resistance in African transmission settings.