Deciphering the role of Plasmodium falciparum plasmepsin 2/3 amplifications in mutant pfcrt-driven piperaquine resistance

破译恶性疟原虫血浆蛋白酶 2/3 扩增在突变体 pfcrt 驱动的哌喹耐药中的作用

• 批准号: 10196128
• 负责人: Satish Kumar Dhingra
• 金额: $ 24.3万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-18 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10196128
• 关键词: Address; Adoption; Africa; Alleles; Amino Acids; Amodiaquine; Antimalarials; Artemisinins; Aspartic Endopeptidases; Biological Assay; Blood; CRISPR/Cas technology; Cambodia; Cambodian; Carrier Proteins; Caspase; Cells; Chloroquine; Chloroquine resistance; Cleaved cell; Combined Modality Therapy; Complex; Crystallization; Data; Defect; Dose; Drug Metabolic Detoxication; Drug Targeting; Drug usage; Early identification; Falciparum Malaria; Fractionation; Gene Amplification; Genes; Genetic; Heme; Hemoglobin; In Vitro; Investigation; Knowledge; Malaria; Mediating; Mediator of activation protein; Molecular; Monitor; Morbidity - disease rate; Morphology; Multiprotein Complexes; Mutate; Mutation; Parasites; Patients; Peptides; Pharmaceutical Preparations; Phenotype; Plasmodium falciparum; Policies; Predisposition; Process; Protein Biosynthesis; Proteins; Public Health; Refractory; Regulation; Reporting; Resistance; Role; South America; Southeastern Asia; Testing; Treatment Failure; Vacuole; Variant; artesunate; asexual; base; benflumetol; biomineralization; burden of illness; combat; defined contribution; fitness; hemozoin; insight; knock-down; member; mortality; mutant; new therapeutic target; novel; plasmepsin; programs; pyronaridine; response; sample fixation; tool

Project Summary Emerging resistance to artemisinin-based combination therapies, the first-line treatment against Plasmodium falciparum (Pf) malaria, poses a major public health problem. Resistance to piperaquine (PPQ) and dihydro- artemisinin has now swept across Southeast Asia, with treatment failures as high as 87%. The increasing use of this combination in Africa adds to the threat of PPQ resistance spreading across this continent, where malaria exerts its heaviest toll. There is thus an urgent need to understand the mechanistic basis of PPQ resistance, alongside other studies focused on mutant PfKelch13-mediated artemisinin resistance. Recent studies have associated PPQ resistance with the amplification of plasmepsins 2/3 (pfpm 2/3) that encode two Pf hemoglobinases, as well as recently emerged mutations in the Pf chloroquine resistance transporter (PfCRT). These PfCRT mutations, which enable the efflux of PPQ away from its heme target in the parasite’s digestive vacuole, always occur on an amplified pfpm 2/3 genetic background, indicating an important role for these amplifications. In Aim 1 we will test the hypothesis that pfpm 2/3 amplifications augment levels of PPQ resistance, utilizing isogenic clones that express a range of pfpm 2/3 copy numbers in two PPQ-resistant, mutant pfcrt Cambodian isolates. Leveraging CRISPR/Cas9-mediated gene editing, we will replace the endogenous mutant pfcrt allele in single and multicopy pfpm 2/3 clones with the parental PPQ-sensitive pfcrt Dd2 allele, and assess whether these amplifications alone can mediate a PPQ tolerance phenotype. We will also test the alternate, non-exclusive hypothesis that pfpm 2/3 amplifications compensate for a hemoglobin (Hb)-derived peptide accumulation defect caused by mutant PfCRT and thereby restore parasite fitness. Our studies will also determine the contribution of pfpm 2/3 copy number in regulating digestive vacuole morphology and Hb-derived peptide levels. For Aim 2, evidence suggests that PfPM 2/3 functions with the Pf heme detoxification protein (PfHDP) as part of the hemozoin (Hz) formation complex (HFC). We will explore the hypothesis that pfpm 2/3 amplifications increase the HFC-mediated conversion of Hb to Hz. We postulate that pfpm 2/3 amplifications increase Hb degradation and result in reduced PPQ inhibition of heme detoxification. Using heme fractionation assays, we will quantify Hb, free heme and Hz in single and multicopy pfpm 2/3 clones and assess whether their relative levels are altered by pfpm 2/3 amplifications. We will also generate translationally controlled TetR-DOZI- based conditional knockdowns of pfhdp in parasites with single or multicopy pfpm 2/3 and test whether these knockdowns modulate PPQ resistance and heme levels. These results will definitively assess the contribution of pfpm 2/3 amplifications to PPQ resistance. Mechanistic insights will also guide strategies to mitigate PPQ resistance by targeting the mediators of Hz formation, and inform the suitability of PfHDP as a novel drug target.

项目摘要 对基于青蒿素的联合疗法的新兴耐药性是针对疟原虫的一线治疗 恶性疟疾（PF）疟疾提出了一个重大的公共卫生问题。对载管（PPQ）和二氢的抗性 阿尔emisinin现在已经席卷了东南亚，治疗失败高达87％。越来越多的使用 在非洲的这种组合增加了PPQ抵抗在疟疾中蔓延的威胁 施加最重的损失。因此，迫切需要了解PPQ抗性的机理基础， 除其他研究以突变体PFKELCH13介导的青蒿素耐药性外。最近的研究 相关的PPQ抗性与血浆蛋白的扩增2/3（PFPM 2/3），该抗性编码两个PF 血红蛋白酶以及最近在PF氯喹转运蛋白（PFCRT）中出现的突变。 这些PFCRT突变，使PPQ的外流远离其血红素靶标的寄生虫消化 真空始终发生在扩增的PFPM 2/3遗传背景上，表明这些作用重要 放大。在AIM 1中，我们将检验以下假设：PFPM 2/3扩增增加了PPQ的水平 抗性，使用同基因克隆，在两个抗PPQ的突变体中表达一系列PFPM 2/3拷贝数 PFCRT柬埔寨分离株。利用CRISPR/CAS9介导的基因编辑，我们将替换内生 与父母PPQ敏感的PFCRT DD2等位基因的单一和多拷贝PFPM 2/3克隆中的突变PFCRT等位基因，以及 评估仅这些放大是否可以介导PPQ耐受性表型。我们还将测试 替代性的，非排他性的假设，即PFPM 2/3扩增补偿了血红蛋白（HB）的衍生 由突变体PFCRT引起的肽积累缺陷，从而恢复寄生虫适应性。我们的研究也将 确定PFPM 2/3拷贝数在确定消化真空形态和HB衍生的贡献 肽水平。对于AIM 2，有证据表明PFPM 2/3的功能具有PF血红素解毒蛋白 （PFHDP）作为血元（Hz）形成复合物（HFC）的一部分。我们将探讨PFPM 2/3的假设 扩增增加了HFC介导的HB转化为Hz。我们假设PFPM 2/3扩增 增加HB降解并导致PPQ抑制血红素排毒。使用血红素分馏 测定，我们将在单一和多拷贝PFPM 2/3克隆中量化HB，自由血红素和Hz，并评估其是否是否 相对水平通过PFPM 2/3扩增改变。我们还将生成翻译控制的Tetr-Dozi- 具有单一或多拷贝PFPM 2/3的寄生虫中PFHDP的有条件敲低 敲低调节PPQ电阻和血红素水平。这些结果将确定评估 PFPM 2/3对PPQ电阻的扩增。机械见解还将指导策略减轻PPQ 通过靶向Hz形成的介体来抗性，并为PFHDP作为新型药物靶标的适合性。