Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdrl gene and cross-resistance to halofantrine and quinine ( drug reistance / malaria / P-glycoprotein / chloroquine )

Two chloroquine-resistant cloned isolates of Plasmodiumfalciparum were subjected to mefloquine selection to test if this resulted in alterations in chloroquine sensitivity and amplification of thepfmdrl gene. The mefloquine-resistant lines derived by this selection were shown to have amplified and overexpressed the pfmdrl gene and its protein product (Pghl). Macrorestriction maps of chromosome 5, where pfindrl is encoded, showed that this chromosome has increased in size in response to mefloquine selection, indicating the presence of a gene(s) in this area of the genome that confers a selective advantage in the presence of mefloquine. Concomitant with the increase in mefloquine resistance was a corresponding increase in the level ofresistance to halofantrine and quinine, suggesting a true multidrug-resistance phenotype. The mefloquineselected parasite lines also showed an inverse relationship between the level of chloroquine resistance and increased pfmdrl gene copy number. These results have important implications for the derivation of amplified copies of the pfmdrl gene in field isolates, as they suggest that quinine pressure may be involved. Plasmodium falciparum is the causative agent of the most severe form ofhuman malaria, and the ability of this parasite to develop resistance to antimalarial agents, such as chloroquine, makes it difficult to select appropriate drugs for both prophylaxis and treatment. Chloroquine resistance in P. falciparum involves a decrease in chloroquine concentration in the parasite, and the rate of chloroquine efflux has been shown to be 40to 50-fold more than in sensitive isolates (1). Other studies have suggested that decreased influx of chloroquine in resistant parasites is responsible for the phenotype (2). A P-glycoprotein homologue 1 (Pghl) encoded by the pfmdrl gene (3, 4) has been suggested to be involved in the chloroquine-resistance phenotype (5). The Pghl protein is localized on the membrane of the digestive vacuole of P. falciparum (6), and heterologous expression of this protein in Chinese hamster ovary (CHO) cells confers a chloroquinesensitive phenotype involving increased accumulation of the drug in the lysosomes (H. Van Es, S. Karcz, F. Chu, A.F.C., P. Gros, and E. Schurr, unpublished work). These results have suggested that Pghl is involved either directly or indirectly with the concentration of chloroquine in the P. falciparum food vacuole. The possible role of Pghl in concentrating chloroquine in the digestive vacuole is consistent with results of the selection for increased levels of chloroquine resistance obtained with three cloned lines of P. falciparum (7). The chloroquine pressure caused deamplification of the pfmdrl gene and decreased expression of the protein. It was also found that concomitant with the increase in chloroquine resistance was a decrease in the level of mefloquine resistance. In another study, selection for mefloquine resistance in the W2 isolate of P. falciparum showed a decrease in chloroquine resistance (8) and amplification of the pfmdrl gene (4). All these results suggested an inverse relationship between chloroquine and mefloquine resistance and a direct link with the level of Pghl expression. In this study we have selected two cloned lines of P. falciparum for increased mefloquine resistance and show that this has resulted in increased expression of the pfmdrl gene, a decrease in chloroquine resistance, and cross-resistance to halofantrine and quinine. MATERIALS AND METHODS Parasites. The P. falciparum isolate K1 (Thailand) was obtained from G. Knowles (Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea), and clone K1A2 from G. Brown (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) was used in all experiments described. Clone W2mef was obtained from D. Kyle (Walter Reed Army Institute of Medical Research, Washington) and clone 3D7 was from David Walliker (Department of Genetics, University of Edinburgh). Drug Assays. Parasites at an initial parasitemia of 1% were grown at 2% hematocrit in erythrocytes for 72 hr with dilutions of the appropriate drug. All assays were done in duplicate, and fresh drug was added with each daily medium change. The final parasitemia was assayed on the FACScan as described (9). The concentration of the drug that inhibits growth to 50%6 (IC50) was determined graphically. Parasites were grown in RPMI 1640/Hepes/5.8% NaHCO3/10%o human serum (10). Pulsed-Field Gel Electrophoresis (PFGE) and Chromosome Mapping. All PFGE experiments were done in a contoured clamp homogeneous electric field apparatus (11). Chromosome mapping was as described (12), and sizes were determined by comparison with chromosomes from Saccharomyces cerevisiae and bacteriophage A concatamers (Promega). DNA probes were labeled with [a-32P]dATP and hybridized as described (7). Quantitation of Pghl with Immunoblotting. Parasites were synchronized with 5% sorbitol and allowed to develop to trophozoite stage. We had reported (6) that between 2.5 X 105 and 5 x 106 trophozoites give a linear signal in immunoblotting experiments with affinity-purified anti-Pghl antibodies. Therefore, 5 x 105 trophozoites, purified free of uninfected erythrocytes by Percoll gradients, were separated by SDS/ PAGE, and immunoblots were probed with affinity-purified anti-Pghl and anti-Pfhsp70 antibodies followed by 1251labeled protein A. The intensity of the signal was determined Abbreviation: PFGE, pulsed-field gel electrophoresis. *To whom reprint requests should be addressed. 1143 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1144 Medical Sciences: Cowman et al. by using a Phosphorlmager (model 400; Molecular Dynamics) and Image Quant software.