Defining P. falciparum resistance to artemisinin-based combination therapies

项目总结 以青蒿素(ART)为基础的联合疗法(ACTs)在全球范围内的采用有助于 自本世纪初以来将全球恶性疟疾负担减半；然而，取得了进展 自2015年以来一直停滞不前。疟疾的影响仍然巨大，2018年估计有40.5万人死亡。现在，PF抵抗运动 对抗逆转录病毒药物衍生品及其ACT合作伙伴的药物可能会压倒控制努力。具有抗药性的寄生虫 ART和哌喹(PPQ)分别由基因K13和pfcrt突变介导，具有 席卷了东南亚的低传输环境。非洲对ACT抗药性的发展， 全球疟疾负担的94%将是灾难性的。一个主要的警告信号是日益普遍的 卢旺达的抗逆转录病毒K13突变，以及邻近国家的其他K13突变。在此，我们 直面非洲出现的抗ACT药物的前景。在目标1中，我们将检验非洲PF的假设 菌株很少对导致抗逆转录病毒药物耐药性的K13突变构成生物学障碍。通过地理上的基因编辑 不同的非洲菌株，我们将确定最具耐药性的突变并评估寄生虫的影响 背景，这在很大程度上可以影响抵抗力和体能的水平。最近完成的 柬埔寨抗逆转录病毒K13突变体与对药物敏感的非洲寄生虫之间的两个PF基因杂交 NF54，我们将绘制并确认二级ART阻力调节器。我们还将测试抗药性是否可以 可归因于功能丧失突变，这种突变限制了环早期的血红蛋白内吞作用。在目标2中，我们 将检验这样的假设，即突变的K13将在非洲寄生虫中施加巨大的适应成本，这可能 阻碍其在高传输环境中的传播。一种方法是量化条形码K13的增长率 突变型和野生型等基因系。我们还假设，亚洲寄生虫中的ART抗性是通过一种 获得K13突变与补偿性适应突变相结合，并将使用我们的基因杂交后代 绘制并确认健身调节器。在目标3中，我们将探讨Pf抵抗ACT合作伙伴的决定因素 非洲寄生虫中的药物PPQ、鲁米芬(LMF)、咯萘啶(PND)和阿莫地喹(ADQ)。基因编辑 将被用来检验非洲PfCRT的单点突变可能导致PPQ耐药性的假设 单倍型。识别对LMF和PND的抗性决定因素的努力将采用具有高度... 变化无常的非洲路线。我们还将利用在我们的一个基因杂交中发现的两种成分 ADQ抗性的基础，包括pfcrt和12号染色体上的一个未知决定因素。鉴定 这些标记将为ADQ抗性的筛选提供有价值的工具。这项建议与 NIAID优先支持抗菌素耐药性的研究，旨在主动为 通过确定原因决定因素和突变，非洲出现抗逆转录病毒治疗和联合化疗耐药性的可能性极大 以及评估健身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.