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

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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.