Role of the pfmdr1 Gene in Plasmodium falciparum Resistance to Antimalarial Treatment

Plasmodium falciparum is an intracellular protozoan that causes the most severe form of malaria. A major hindrance to disease control efforts is the spread of drug resistant parasite populations, which has been linked to the Plasmodium falciparum multidrug resistance (pfmdr1) gene. Pfmdr1 encodes a transporter protein that pumps solutes, including antimalarials, into the parasite food vacuole. Elevated pfmdr1 gene copy number (CN) is thought to decrease parasite sensitivity to numerous antimalarial compounds, including quinine (QN) and mefloquine (MFQ). Therefore, our central hypothesis was that the efficacy of antimalarials is dependent upon the CN and expression of the pfmdr1 gene in P. falciparum. This hypothesis was addressed in the following objectives: (1) to determine pfmdr1 expression after MFQ treatment, (2) to determine the frequency of parasite subpopulations with elevated pfmdr1 CN in patients that fail antimalarial treatment, and (3) to determine the subcellular localization of QN. We found that MFQ exposure resulted in a clear upregulation of pfmdr1. Concomitant morphology analyses revealed that MFQ treatment delayed maturation through the normal intraerythrocytic cycle at the ring stage. Our data show that MFQ-induced increases in pfmdr1 expression are the direct result of the maturation delay and indicate that pfmdr1 is primarily expressed during the ring stage of development. Next, to understand the role of elevated pfmdr1 CN in clinical treatment outcome, we developed a limiting dilution assay to evaluate pfmdr1 CN in parasite subpopulations within an individual patient. We found that even small proportions of parasites with multiple pfmdr1 copies could affect the treatment outcome, which suggests that elevated CN has a stronger influence over treatment response than previously reported. Lastly, we explored the effect of multiple pfmdr1 copies on the subcellular localization of the fluorescent antimalarial compound, QN. We found that, regardless of pfmdr1 copy number, QN overlapped with hemozoin but did not colocalize with the acidotropic dye. These results suggest that QN localizes to a non-acidic compartment within the food vacuole and that pfmdr1 CN does not affect QN subcellular localization. Our results reveal novel data on pfmdr1 gene expression and the degree to which CN can affect clinical treatment outcome