Abstract
Plasmodium falciparum multidrug resistance constitutes a major obstacle to the global malaria elimination campaign. Specific mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediate resistance to the 4-aminoquinoline drug chloroquine and impact parasite susceptibility to several partner agents used in current artemisinin-based combination therapies, including amodiaquine. By examining gene-edited parasites, we report that the ability of the wide-spread Dd2 PfCRT isoform to mediate chloroquine and amodiaquine resistance is substantially reduced by the addition of the PfCRT L272F mutation, which arose under blasticidin selection. We also provide evidence that L272F confers a significant fitness cost to asexual blood stage parasites. Studies with amino acid-restricted media identify this mutant as a methionine auxotroph. Metabolomic analysis also reveals an accumulation of short, hemoglobin-derived peptides in the Dd2 + L272F and Dd2 isoforms, compared with parasites expressing wild-type PfCRT. Physiologic studies with the ionophores monensin and nigericin support an impact of PfCRT isoforms on Ca2+ release, with substantially reduced Ca2+ levels observed in Dd2 + L272F parasites. Our data reveal a central role for PfCRT in regulating hemoglobin catabolism, amino acid availability, and ionic balance in P. falciparum, in addition to its role in determining parasite susceptibility to heme-binding 4-aminoquinoline drugs.
Highlights
Higher concentrations in the acidic DV wherein its diprotonated form binds to reactive heme and inhibits its detoxification[5,6,7,8]
To define the impact of the L272F mutation on both CQ susceptibility and drug potentiation by VP, we examined four P. falciparum lines: Dd2, Dd2Dd2, Dd2Dd2 L272F, and GC03
In the presence of VP, the Dd2Dd2 L272F line showed a CQ IC50 value that was close to that of the CQS GC03 line (Fig. 1; Supplementary Table S1). md-CQ assays yielded IC50 values in Dd2Dd2 L272F that were 4.8–fold lower as compared with Dd2Dd2, yet remained 5.5–fold above those of GC03. These data with the md-CQ metabolite show a much higher level of resistance imparted by the Dd2 allele as compared with its effect on the parent compound CQ, consistent with the idea that md-CQ might be the more important selective agent31. md-CQ results showed some degree of VP reversibility with the L272F mutant
Summary
Higher concentrations in the acidic DV wherein its diprotonated form binds to reactive heme and inhibits its detoxification[5,6,7,8]. Transport studies suggest that PfCRT mutations can enable this electrochemical potential-driven transporter to mediate the facilitated diffusion of protonated CQ, which itself might bind to and inhibit PfCRT10,12,15,21,29. Studies with Xenopus oocytes have suggested that VP is a partial mixed-type inhibitor of CQR PfCRT-mediated CQ transport[29]. In this model, PfCRT harbors a substrate-recognition cavity with various overlapping substrate binding sites. PfCRT mutations can decrease or increase parasite susceptibility to several other drugs These include various partner agents employed in globally adopted artemisinin-based combination therapies, notably piperaquine, amodiaquine (ADQ), lumefantrine, and mefloquine[5,11,32,33,34,35,36]. Consistent with its complex patterns of evolution in multiple geographic regions subject to varied drug selection pressures, there are currently 57 known PfCRT isoforms worldwide, including four observed recently in piperaquine-resistant parasites from Cambodia that have recently been shown to confer piperaquine resistance[17,34,36,37,38]
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