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 proteins PfCRT and PfMDR1 and the cytosolic protein PfKelch13 have been linked to chloroquine (CQ) and artemisinin resistance, respectively. Here, we aimed to identify the associations of these resistance markers with mutations in other food vacuole transporters in several field isolates. In this study, we isolated intact P. falciparum food vacuoles (FVs) and carried out detailed proteome analysis to identify new food vacuole transporters. Furthermore, we carried out coexisting mutational analysis for these newly identified transporters with known PfKelch13 and PfCRT polymorphisms via 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 newly identified FV transporters with mutations in the PfKelch13, PfCRT and PfMDR1 proteins. SNP data analysis of the Pf3K and MalariaGEN databases for 2517 samples revealed the coassociation of 6 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 (F394L_PfNT1, S278T_PfMFR5 and Y570F_PfMFR5) in S. cerevisiae resulted in resistance to mutant phenotypes in the presence of dihydroartemisinin (DHA), 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 as well as food vacuole dynamics.