Global spread of mutant PfCRT and its pleiotropic impact on plasmodium falciparum multidrug resistance and fitness

Abstract

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 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. IMPORTANCE 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.