Analysis of the cellular and molecular mechanisms of chloroquine resistance in Plasmodium falciparum

Chloroquine resistance (CQR) is caused by mutations in the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) and is characterized by decreased chloroquine susceptibility as well as reduced chloroquine (CQ) accumulation in CQR parasites. The cellular and molecular mechanisms that characterize CQR are still not completely understood and the objective of this work is to distinguish between the different models proposed for CQR based on analysis of drug transport and susceptibility, and the contribution of PfCRT to both.;Mechanisms that have been proposed for altered CQ flux in CQR include reduced energy dependent influx, and facilitated outward diffusion or active efflux of CQ out of the digestive vacuole (DV) of the parasite. Using a novel fluorescent CQ analogue, single cell photometry, and spinning disk confocal microscopy techniques with live, synchronized intraerythrocytic parasites, this study was able to obtain very high kinetic resolution drug transport data. These data elucidate energy dependent rate constants for CQ influx in CQ sensitive (CQS) parasites and energy independent, decreased rate constants for CQ influx in CQR parasites. The data also show that changes in influx for CQR parasites are due to mutations in PfCRT. Analysis of drug efflux data for CQS and CQR parasites suggests distinguishing between free versus bound CQ in the DV is essential for proper kinetic analysis of efflux.;The CQR phenotype has traditionally been defined at low nanomolar cytostatic CQ concentrations but these do not correspond to medically relevant cytotoxic doseages. Thus, CQ accumulation was also measured using 3H-CQ and infected red blood cells at external CQ that corresponds to cytotoxic vs cytostatic concentrations. It was discovered that reduced CQ accumulation is not the underlying cause of cytotoxic CQR.;Also addressed in this work are molecular studies of PfCRT biochemistry. Crosslinkers were used to successfully covalently crosslink PfCRT in yeast membranes or purified reconstituted protein, however, the effects of CQ on crosslinking oligomeric PfCRT were equivocal. Uptake of basic amino acids in yeast cells or vesicles expressing various isoforms of PfCRT was measured and it was discovered that PfCRT may transport the basic amino acid arginine.