Role of novel mutations in food vacuole transporters beyond K13-mediated artemisinin resistance in Plasmodium falciparum.

Malaria remains one of the leading causes of morbidity and mortality worldwide, mainly because of the emergence of drug resistance against current antimalarials. The Plasmodium falciparum food vacuole (FV) proteins, P. falciparum chloroquine (CQ) resistance transporter (PfCRT), PfMDR1 and the cytosolic protein PfKelch13 have been linked to CQ and artemisinin resistance, respectively. Here, we aimed to identify the associations of these resistance markers with mutations in other FV transporters in several field isolates. In this study, we isolated intact P. falciparum FVs and carried out detailed proteome analysis to identify new FV transporters. Furthermore, we carried out co-existing mutational analysis for these transport proteins identified in the FV-enriched fraction with known PfKelch13 and PfCRT polymorphisms via single-nucleotide polymorphism (SNP) data from the Pf3K and MalariaGEN databases. Proteome analysis identified 16 transporter proteins in Plasmodium FVs. Comparative amino acid analysis of these transporter proteins revealed a coassociation of mutations in several transport proteins identified in the FV-enriched fraction with mutations in the PfKelch13, PfCRT, and PfMDR1 proteins. SNP data analysis of the Pf3K and MalariaGEN databases for 2,517 samples revealed the coassociation of six mutations in four transporter genes, PfCRT, PfNT1, PfCTR2, and PfMDR2, with the PfKelch13 polymorphisms (P < 0.0001), suggesting the contribution of additional parasite transporters to the evolution of CQ and artemisinin resistance. Furthermore, functional complementation with the wild-type PfNT1 and PfMFR5 proteins and their mutant forms (PfNT1-F394L, PfMFR5-S278T, and PfMFR5-Y570F) in Saccharomyces cerevisiae resulted in resistance to mutant phenotypes in the presence of dihydroartemisinin, suggesting a possible role of these mutations in the acquisition of drug resistance. Together, the genome sequence data from field isolates and yeast complementation analysis of the mutant phenotypes identified novel loci related to PfKelch13-mediated antimalarial resistance and revealed unexplored contributions of transporters to artemisinin resistance.