A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisininbased combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nucleasebased 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 bisaminoquinoline 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

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens

A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine

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

Plasmodium falciparum LipB mutants display altered redox and carbon metabolism in asexual stages and cannot complete sporogony in Anopheles mosquitoes

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans. Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function

Global Spread of Mutant PfCRT and Its Pleiotropic Impact on Plasmodium falciparum Multidrug Resistance and Fitness

Our study defines the allelic distribution of pfcrt, an important mediator of multidrug resistance in Plasmodium falciparum, in Africa and Asia. We leveraged whole-genome sequence analysis and gene editing to demonstrate how current drug combinations can select different allelic variants of this gene and shape region-specific parasite population structures. We document the ability of PfCRT mutations to modulate parasite susceptibility to current antimalarials in dissimilar, pfcrt allele-specific ways. This study underscores the importance of actively monitoring pfcrt genotypes to identify emerging patterns of multidrug resistance and help guide region-specific treatment options.The global spread of Plasmodium falciparum chloroquine resistance transporter (PfCRT) variant haplotypes earlier caused the widespread loss of chloroquine (CQ) efficacy. In Asia, novel PfCRT mutations that emerged on the Dd2 allelic background have recently been implicated in high-level resistance to piperaquine, and N326S and I356T have been associated with genetic backgrounds in which resistance emerged to artemisinin derivatives. By analyzing large-scale genome sequencing data, we report that the predominant Asian CQ-resistant Dd2 haplotype is undetectable in Africa. Instead, the GB4 and previously unexplored Cam783 haplotypes predominate, along with wild-type, drug-sensitive PfCRT that has reemerged as the major haplotype. To interrogate how these alleles impact drug susceptibility, we generated pfcrt-modified isogenic parasite lines spanning the mutational interval between GB4 and Dd2, which includes Cam783 and involves amino acid substitutions at residues 326 and 356. Relative to Dd2, the GB4 and Cam783 alleles were observed to mediate lower degrees of resistance to CQ and the first-line drug amodiaquine, while resulting in higher growth rates. These findings suggest that differences in growth rates, a surrogate of parasite fitness, influence selection in the context of African infections that are frequently characterized by high transmission rates, mixed infections, increased immunity, and less recourse to treatment. We also observe that the Asian Dd2 allele affords partial protection against piperaquine yet does not directly impact artemisinin efficacy. Our results can help inform the regional recommendations of antimalarials, whose activity is influenced by and, in certain cases, enhanced against select PfCRT variant haplotypes

Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter

The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within-hos

Quinine Dimers Are Potent Inhibitors of the Plasmodium falciparum Chloroquine Resistance Transporter and Are Active against Quinoline-Resistant P. falciparum

Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparum is primarily conferred by mutations in the "chloroquine resistance transporter" (PfCRT). The resistance-conferring form of PfCRT (PfCRTCQR) mediates CQ resistance by effluxing the drug from the parasite's digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRTCQR can also decrease the parasite's susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRT CQR and a series of dimeric quinine molecules using a Xenopus laevis oocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H+ ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1 and 6 were also examined in vitro (against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo (against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRTCQR reported to date. Furthermore, the lead compounds (1 and 6) were not effluxed by PfCRTCQR from the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1 and 6 exhibited in vitro antiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1 and 6 in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRTCQR. This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum