Combinatorial Genetic Modeling ofpfcrt-Mediated Drug Resistance Evolution inPlasmodium falciparum

Abstract

The emergence of drug resistance continuously threatens global control of infectious diseases, including malaria caused by the protozoan parasite Plasmodium falciparum. A critical parasite determinant is the P. falciparum chloroquine resistance transporter (PfCRT), the primary mediator of chloroquine (CQ) resistance (CQR), and a pleiotropic modulator of susceptibility to several first-line artemisinin-based combination therapy partner drugs. Aside from the validated CQR molecular marker K76T, P. falciparum parasites have acquired at least three additional pfcrt mutations, whose contributions to resistance and fitness have been heretofore unclear. Focusing on the quadruple-mutant Ecuadorian PfCRT haplotype Ecu1110 (K76T/A220S/N326D/I356L), we genetically modified the pfcrt locus of isogenic, asexual blood stage P. falciparum parasites using zinc-finger nucleases, producing all possible combinations of intermediate pfcrt alleles. Our analysis included the related quintuple-mutant PfCRT haplotype 7G8 (Ecu1110 + C72S) that is widespread throughout South America and the Western Pacific. Drug susceptibilities and in vitro growth profiles of our combinatorial pfcrt-modified parasites were used to simulate the mutational trajectories accessible to parasites as they evolved CQR. Our results uncover unique contributions to parasite drug resistance and growth for mutations beyond K76T and predict critical roles for the CQ metabolite monodesethyl-CQ and the related quinoline-type drug amodiaquine in driving mutant pfcrt evolution. Modeling outputs further highlight the influence of parasite proliferation rates alongside gains in drug resistance in dictating successful trajectories. Our findings suggest that P. falciparum parasites have navigated constrained pfcrt adaptive landscapes by means of probabilistically rare mutational bursts that led to the infrequent emergence of pfcrt alleles in the field.

Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic

“…In addition, several novel PfCRT mutations have been observed that can revert parasites to a CQ-sensitive status despite the presence of K76T, including C101F—which arose under pip-eraquine (PPQ) or amantadine pressure in vitro 42,166 —or C350R, which has recently spread throughout French Guiana 167 . Some PfCRT variants also mediate resistance to the active metabolite of AQ (monodesethyl-AQ), which might have been an important driving force for selection 168,169 . Importantly, all mutant CQ-resistant pfcrt alleles known so far increase P. falciparum susceptibility to lumefantrine, both in vitro and in field settings, and often increase sensitivity to ART derivatives, benefiting the clinical efficacy of the first-line ACT AL 170–172 .…”

“…Most mutant pfcrt alleles come with a fitness cost, indicated in the field by a decrease in their allelic prevalence in the absence of CQ pressure 177 , and in vitro as reduced growth rates when compared to recombinant isogenic parasites expressing the wild-type allele 35,169,178 . Reduced fitness might involve less efficient Hb catabolism and peptide transport in PfCRT mutants, as well as other digestive-vacuole-related physiological processes 178,179 .…”

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

“…Among CQ-resistant field isolates, PfCRT isoforms are comprised of geographically distinct clusters of single-nucleotide polymorphisms (SNPs), namely K76T and 3 to 8 additional point mutations. PfCRT K76T is a critical, albeit insufficient, determinant of parasite in vitro CQR [16]. This mutation also predicts in vivo CQ treatment failure with high sensitivity but lower specificity [17].…”

“…At the cellular level, PfCRT is a multi-pass transporter embedded in the intra-erythrocytic parasite’s digestive vacuole (DV) membrane, with enigmatic functions that may include transport of ions and/or peptides [18–21]. In the absence of PfCRT structural information, mutational approaches have guided studies into the effect of specific PfCRT mutations on drug transport and parasite growth [16,22–24]. …”

“…falciparum suggests that pfcrt evolution occurred via punctuated periods of mutation that were too brief to allow fixation of partially mutated alleles (i.e. bearing 1 to 3 SNPs), shedding light on physiologic constraints that explain the rarity of mutant pfcrt emergence in the field [16]. Intriguingly, studies from Cambodia, an epicenter of multidrug resistance in P .…”