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

Stanislaw J Gabryszewski,Timothy J Egan,Paul D Roepe,David A Fidock,Satish K Dhingra,Paul S Callaghan,Lise Musset,Matthew R Hassett,Nina F Gnädig,Jill M Combrinck,Ian A Lewis,Philipp P Henrich,Amila Siriwardana,Andrew H Lee,Manuel Llinás

doi:10.1371/journal.ppat.1005976

Abstract

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.

Highlights

Human malaria remains a leading global health scourge in part due to multidrug resistance mechanisms evolved by Plasmodium falciparum, the protozoan species responsible for the most severe forms of disease [1]
We reported that the highly mutated Cambodian Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoform Cam734 is fitness-neutral in terms of parasite growth, unlike other less fit isoforms such as Dd2 that are outcompeted by wild-type parasites in the absence of CQ pressure
Using pfcrt-specific zinc-finger nucleases to genetically dissect the Cam734 allele, we report that its unique constituent mutations directly contribute to CQ resistance and collectively offset fitness costs associated with intermediate mutational steps

Summary

Introduction

Human malaria remains a leading global health scourge in part due to multidrug resistance mechanisms evolved by Plasmodium falciparum, the protozoan species responsible for the most severe forms of disease [1]. The multi-focal emergence and spread of CQ resistance (CQR) contributed to stalled control measures and substantial increases in malaria-associated hospitalizations and deaths [6]. Studies of infections with CQ-resistant P. falciparum strains in Guinea-Bissau recently revealed a ~5–fold increase in CQ efficacy upon doubling the standard dose in children aged

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