Mutations in the Plasmodium falciparum chloroquine resistance transporter: Balancing drug resistance and fitness

Mutations in the Plasmodium falciparum chioroquine resistance transporter (PfCRT) render malaria parasites resistant to chloroquine. High rates of resistance to chloroquine have prompted many malarious countries to switch to alternative therapies. In Malawi, the replacement of chloroquine has led to the reemergence of wild type chioroquine-sensitive parasites, a finding that was attributed to a decreased fitness of parasites with mutant pfcrt. However, in Southeast Asia mutant parasites were maintained in the population after the replacement of chioroquine with various other antimalarial drugs. I reasoned that local Southeast Asian pfcrt alleles might either be selected by the regionally used replacement drugs or do not confer a fitness cost as observed with chloroquine-resistant alleles in Malawi.;To examine the properties of Southeast Asian PfCRT haplotypes, I used an allelic exchange strategy to create isogenic parasites that expressed the Cam734 pfcrt allele from Cambodia and the Phi and Ph2 alleles from the Philippines, respectively. To test the influence of PfCRT haplotype on drug susceptibility, I assayed these recombinant lines with various antimalarial drugs. I found that not only the established K76T mutation but also the C725 polymorphism influences chloroquine resistance in the Philippine haplotypes. The only amino acid difference between the two Philippine PfCRT haplotypes occurs in residue 72 and leads to chloroquine sensitivity in Phi (C72) and chloroquine resistance in Ph2 (72S). Additionally, different levels of amodiaquine resistance were found to depend on the geographic origin of the mutant pfcrt allele, with high resistance in the Philippines haplotypes, intermediate levels in Cambodian and Brazilian haplotypes and low levels in the Asian/African haplotype. I also showed that lumefantrine susceptibilities are influenced by pfcrt alleles, with the wild type allele conferring resistance and mutant alleles leading to susceptibility to this drug. Examination of recombinant parasites, which expressed either PfCRT K76 or 76T, revealed additionally that this polymorphic residue was the main determinant of pfcrt-dependent lumefantrine susceptibility and established the validity of K76T as a marker for lumefantrine resistance. These in vitro data has been confirmed by a clinical trial in Tanzania, which investigated the association of lumefantrine treatment with the recrudescence of parasites expressing wild type pfcrt alleles. The results obtained from these drug susceptibility experiments provide important information for the treatment of falciparum malaria with the appropriate drug in Cambodia and the Philippines depending on the predominant pfcrt allele on a regional level.;I also utilized the recombinant lines I generated to test whether PfCRT haplotype influences parasite fitness, as has been suggested from observations in Malawi. For this, a fitness assay was designed that utilized pyrosequencing to quantify changes in the abundance of two co-cultured isogenic parasite lines over time. Indeed, expression of different pfcrt alleles led to distinct relative fitness values. Interestingly, the recombinant line expressing Ph1pfcrt was more fit but less resistant to chloroquine than the Ph2 expressing recombinant and vice versa, which could be attributed to the exchange of only one amino acid at position 72. Also, fitness results from the Cam734 allele from Cambodia were intriguing because parasites expressing Cam734 pfcrt displayed an even higher relative fitness value than parasites expressing the wild type allele despite increased chloroquine resistance. This has important implications for the future geographical distribution of this mutant Cambodian PfCRT haplotype, possibly expanding at the expense of the wild type allele and other less fit mutant parasites due to its high fitness.;Finally, I created a parasite line that stably expresses PfCRT fused to a tandem affinity purification tag, which is delivered to the digestive vacuole (DV) surface. Besides the essential hemoglobin degradation pathway, few other metabolic processes have been localized to the DV. The expression of tagged PfCRT on the DV surface allows the immuno-precipitation of highly purified DV preparations as well as PfCRT binding partners, which then can be subjected to mass spectrometry analysis. This tool might lead to the identification of new drug targets and assign new metabolic pathways to the DV.