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

项目摘要 对以青蒿素为基础的联合疗法产生的耐药性，这是治疗疟疾的一线药物 恶性疟疾是一个重大的公共卫生问题。对哌喹(PPQ)和二氢呋喃的抗性 青蒿素现已席卷东南亚，治疗失败率高达87%。不断增加的使用量 在非洲，这种组合增加了PPQ耐药性在整个非洲大陆蔓延的威胁，那里的疟疾 付出了最沉重的代价。因此迫切需要了解PPQ抗性的机制基础， 与其他研究一起，重点是突变体PfKelch13介导的青蒿素耐药性。最近的研究表明 PPQ抗性与编码两个pF的血浆蛋白2/3(pfpm 2/3)的扩增有关 血红蛋白酶，以及最近出现的PF氯喹抗性转运蛋白(PfCRT)突变。 这些PfCRT突变使PPQ在寄生虫消化中的外流远离其血红素目标 液泡，总是发生在扩增的pfpm 2/3的遗传背景上，这表明这些 放大。在目标1中，我们将测试pfpm 2/3扩增提高ppq水平的假设。 抗性，利用两个PPQ抗性突变体中表达一系列pfpm 2/3拷贝数的等基因克隆 柬埔寨分离株。利用CRISPR/Cas9介导的基因编辑，我们将取代内源性 单拷贝和多拷贝pfpm 2/3克隆中突变的pfcrt等位基因与亲本ppq敏感的pfcrt DD2等位基因，以及 评估这些扩增是否可以单独调节PPQ耐受表型。我们还将测试 另一种非排他性假设，即pfpm 2/3扩增补偿血红蛋白(Hb)来源的 突变PfCRT引起的多肽蓄积缺陷，从而恢复寄生虫的适合性。我们的研究也将 确定pfpm 2/3拷贝数在调节消化液泡形态和Hb来源中的作用 多肽水平。对于目标2，证据表明PfPM 2/3与PF血红素解毒蛋白起作用 (PfHDP)作为血球蛋白(Hz)形成复合体(HFC)的一部分。我们将探索假设pfpm 2/3 放大增加了HFC介导的Hb到Hz的转换。我们假设pfpm 2/3扩增 增加Hb降解率，减少PPQ对血红素解毒的抑制作用。使用血红素分级法 我们将对单个和多拷贝pfpm 2/3克隆中的Hb、游离血红素和赫兹进行量化，并评估它们的 相对水平被pfpm 2/3扩增改变。我们还将生成翻译控制的TetR-dozi- 基于单拷贝或多拷贝pfpm 2/3寄生虫中pfhdp的条件性敲除，并测试这些 基因敲除调节PPQ抗性和血红素水平。这些结果将最终评估 PPQ抗性的Pfpm 2/3扩增。机械性的见解也将指导缓解PPQ的战略 通过靶向HZ形成的介体来产生耐药性，并告知PfHDP作为新的药物靶点的适宜性。

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.