A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine.
A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine.
复制标题
DOI：
10.1128/mbio.00303-17
复制
发表时间：
2017-05-09
期刊：
影响因子：
6.4
通讯作者：
Fidock DA
中科院分区：
文献类型：
--
作者：
Dhingra SK;Redhi D;Combrinck JM;Yeo T;Okombo J;Henrich PP;Cowell AN;Gupta P;Stegman ML;Hoke JM;Cooper RA;Winzeler E;Mok S;Egan TJ;Fidock DA
目前减轻全球疟疾负担的努力受到了恶性疟原虫对青蒿素综合疗法的抗药性在整个亚洲迅速蔓延的威胁，其中包括柬埔寨使用双氢青蒿素加哌喹的临床失败率不断上升。使用基于锌指核酸酶的基因编辑，我们报告说，将C101 F突变添加到亚洲常见的氯喹（CQ）耐药PfCRT Dd 2亚型中，可以使培养的寄生虫具有PPQ耐药性。抗性表现为PPQ浓度显著升高，导致寄生虫生长抑制> 90%（IC 90）或寄生虫杀灭> 50%（50%致死剂量[LD 50]）。这种突变还逆转了Dd 2介导的CQ抗性，使寄生虫对阿莫地喹、奎宁和青蒿素敏感，并赋予金刚烷胺和杀稻瘟菌素抗性。使用血红素分馏测定，我们证明了PPQ导致活性游离血红素的积累，并抑制化学惰性的疟原虫色素晶体的形成。我们的数据唤起抑制血红素解毒寄生虫的酸性消化空泡作为主要模式的双-氨基喹啉PPQ和相关的4-氨基喹啉CQ。这两种药物在升高的浓度下也抑制血红蛋白蛋白水解，这表明了另一种作用模式。pfmdr 1拷贝数不同的等基因系表现出相同的PPQ敏感性。我们认为PfCRT的突变可能是导致田间分离株PPQ耐药的多因素基础。消除疟疾的全球议程取决于青蒿素类复方疗法的持续成功，这种疗法针对细胞内疟原虫的无性血液阶段。然而，对青蒿素的部分耐药性现已在东南亚建立，使伙伴药物面临更大的选择压力。恶性疟原虫对一线伙伴哌喹（PPQ）的抗药性目前在柬埔寨迅速蔓延，导致临床治疗失败。在这里，我们报告了一种变异形式的恶性疟原虫氯喹抗性转运蛋白，携带C101 F突变编辑成氯喹（CQ）抗性Dd 2亚型流行于亚洲，可以赋予PPQ抗性培养的寄生虫。这伴随着CQ电阻的损失。生化分析表明，PPQ，CQ一样，抑制反应性血红素的解毒，这是由寄生虫介导的catalysis宿主血红蛋白形成。我们提出，新的PfCRT变体出现在该领域可能有助于PPQ抗性的多基因基础。
Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates. The global agenda to eliminate malaria depends on the continued success of artemisinin-based combination therapies (ACTs), which target the asexual blood stages of the intracellular parasite Plasmodium. Partial resistance to artemisinin, however, is now established in Southeast Asia, exposing the partner drugs to increased selective pressure. Plasmodium falciparum resistance to the first-line partner piperaquine (PPQ) is now spreading rapidly in Cambodia, resulting in clinical treatment failures. Here, we report that a variant form of the Plasmodium falciparum chloroquine resistance transporter, harboring a C101F mutation edited into the chloroquine (CQ)-resistant Dd2 isoform prevalent in Asia, can confer PPQ resistance in cultured parasites. This was accompanied by a loss of CQ resistance. Biochemical assays showed that PPQ, like CQ, inhibits the detoxification of reactive heme that is formed by parasite-mediated catabolism of host hemoglobin. We propose that novel PfCRT variants emerging in the field could contribute to a multigenic basis of PPQ resistance.