Defining the Role of PfCRT and PfMDR1 as Pleiotropic Mediators of Plasmodium falciparum Multidrug Resistance

项目摘要 全世界采用青蒿素类复方疗法有助于将艾滋病毒/艾滋病发病率减少一半， 自21世纪初以来，恶性疟原虫（Pf）疟疾的全球负担。尽管如此， 仍然很大，仅在非洲每年就有超过40万人死亡。现在，Pf对ACT的耐药性威胁到 压倒了疟疾防治工作。ACT双氢青蒿素+哌喹（PPQ）治疗失败率 目前，在柬埔寨超过50%，在亚洲其他地方也观察到对其他青蒿素综合疗法的耐药性。的 在非洲出现和传播抗青蒿素综合疗法耐药性将是灾难性的。研究确定了以下方面的核心作用： Pf氯喹耐药转运蛋白PfCRT和多药耐药转运蛋白PfMDR 1在 调节Pf对ACT伴侣药物PPQ、阿莫地喹（ADQ）、咯萘啶（PND）的敏感性， 本芴醇（LMF）和甲氟喹（MFQ）。这些研究包括我们最近基于基因编辑的发现， 柬埔寨存在的新型PfCRT突变可赋予PPQ抗性。通过分析> 2，500 Pf的数据集， 从亚洲和非洲的基因组样本中，我们现在发现了这两种基因的大量新的序列变异。在 目的1，我们将应用基因编辑技术结合综合药物敏感性分析来测试 这些新的PfCRT突变被选择介导ACT伴侣耐药性的假设。 在亚洲，我们预测这些变异主要影响PPQ、LMF或MFQ的易感性。在非洲，我们 观察到以前未表征的单倍型的优势，并将表征这些单倍型是否 变体介导对伴侣药物的易感性降低和/或使适应性成本最小化。在目标2中，我们将测试 假设目前的ACT正在亚洲和非洲选择PfMDR 1的新突变。基于 我们最近发现PfMDR 1作为配子母细胞对抗疟药耐药的介导者，我们将 还评估突变PfMDR 1亚型是否可以增强耐药Pf寄生虫的传播。在 目标3，我们建议确定对PfCRT施加相反选择压力的抗疟组合， PfMDR 1，从而排除获得多药耐药性。使用突变pfcrt的选择方法 和pfmdr 1系，我们的研究将实验性地解决两个正在进行的三重ACT临床试验的前提， 柬埔寨，正在测试双伙伴药物组合LMF+ADQ和PPQ+MFQ， 有效治疗抗药性疟疾并防止其进一步演变和传播。在目标4中，我们将使用细胞- 基于分析来测试假设，即血红蛋白内吞作用和随后的加工在Pf 消化空泡是大多数ACT药物作用方式的关键方面，其效力可能受到影响 PfMDR 1或PfCRT的突变亚型。该提案符合NIAID的优先事项， 抗菌药物耐药性的研究，将改变我们对ACT合作伙伴耐药性的理解， 研究的目的是提供新的生物标志物，并确定有效治疗抗药性Pf疟疾的策略。

PROJECT SUMMARY The worldwide adoption of artemisinin-based combination therapies (ACTs) has been instrumental in halving the global burden of Plasmodium falciparum (Pf) malaria since the early 2000s. Malaria’s impact nonetheless remains vast, with over 400,000 yearly deaths in Africa alone. Now, Pf resistance to ACTs threatens to overwhelm malaria control efforts. Treatment failure rates with the ACT dihydroartemisinin+piperaquine (PPQ) currently exceed 50% in Cambodia, and resistance to other ACTs has been observed elsewhere in Asia. The emergence and spread of ACT resistance in Africa would be calamitous. Studies have identified core roles for the Pf chloroquine resistance transporter PfCRT and the multidrug resistance transporter PfMDR1 in modulating Pf susceptibility to the ACT partner drugs PPQ, amodiaquine (ADQ), pyronaridine (PND), lumefantrine (LMF) and mefloquine (MFQ). These studies include our recent gene editing-based finding that novel PfCRT mutations present in Cambodia can confer PPQ resistance. By analyzing a dataset of >2,500 Pf genomes sampled across Asia and Africa, we now find a plethora of new sequence variants of both genes. In Aim 1, we will apply gene editing techniques coupled with comprehensive drug susceptibility profiling to test the hypothesis that these novel PfCRT mutations have been selected to mediate ACT partner drug resistance. In Asia, we predict that these variants primarily affect susceptibility to PPQ, LMF, or MFQ. In Africa, we observe a predominance of previously uncharacterized haplotypes, and will characterize whether these variants mediate reduced susceptibility to partner drugs and/or minimize fitness costs. In Aim 2, we will test the hypothesis that current ACTs are selecting for novel mutations in PfMDR1 throughout Asia and Africa. Based on our recent discovery of a role for PfMDR1 as a mediator of gametocyte resistance to antimalarials, we will also assess whether mutant PfMDR1 isoforms can enhance the transmission of drug-resistant Pf parasites. In Aim 3, we propose to identify antimalarial combinations that exert opposing selective pressures on PfCRT and PfMDR1 and thus preclude the acquisition of multidrug resistance. Using selection methods with mutant pfcrt and pfmdr1 lines, our studies will experimentally address the premise of two ongoing triple ACT clinical trials in Cambodia, which are testing the dual partner drug combinations LMF+ADQ and PPQ+MFQ as approaches to effectively treat drug-resistant malaria and prevent its further evolution and spread. In Aim 4, we will use cell- based assays to test the hypothesis that hemoglobin endocytosis and subsequent processing in the Pf digestive vacuole are key aspects of the modes of action of most ACT drugs, whose potency can be impacted by mutant isoforms of PfMDR1 or PfCRT. This proposal, which aligns with the NIAID priority of supporting research on antimicrobial drug resistance, will transform our understanding of ACT partner drug resistance and modes of action, provide new biomarkers, and identify strategies to effectively treat drug-resistant Pf malaria.