Dataset Information

Chloroquine transport in Plasmodium falciparum. 2. Analysis of PfCRT-mediated drug transport using proteoliposomes and a fluorescent chloroquine probe.

ABSTRACT: Mutations in the PfCRT protein cause chloroquine resistance (CQR), and earlier studies from our laboratory using plasma membrane inside-out vesicles (ISOV) prepared from yeast expressing recombinant PfCRT [Zhang, H., et al. (2004) Biochemistry 43, 8290-8296] suggested that the putative transporter mediates downhill facilitated diffusion of charged chloroquine (CQ). However, more recent experiments with a fluorescent CQ probe (NBD-CQ) presented in the accompanying paper (DOI 10.1021/bi901034r ) indicated that the CQR phenotype in live parasites is associated with a reduced rate of ATP-dependent CQ uptake into the digestive vacuole (DV). An altered rate constant for uptake has multiple interpretations. To further investigate this phenomenon, PfCRT proteins found in chloroquine-sensitive (CQS) and CQR strains of Plasmodium falciparum were purified from yeast engineered to express "yeast optimized" pfcrt genes, reconstituted into proteoliposomes (PL), and efflux of NBD-CQ was measured from these PL. A membrane-impermeant quencher was used to distinguish intra-PL NBD-CQ from extra-PL NBD-CQ vs time as well as resolve initial rates and rate constants for efflux. Efflux was investigated at a range of NBD-CQ concentrations, in the presence vs absence of pH gradients (DeltapH) and transmembrane potentials (DeltaPsi). Explicit turnover numbers for apparent PfCRT-mediated transport were then calculated under these conditions. Our data are consistent with a model wherein PfCRT catalyzes electrochemically downhill diffusion of NBD-CQ out of the DV, in response to DeltaPsi or DeltapH, at a rate that can partially compete with the ATP-dependent uptake of NBD-CQ by CQS parasites described in the previous paper. These data allow us to propose a refined model for altered CQ accumulation in CQR malarial parasites.

SUBMITTER: Paguio MF

PROVIDER: S-EPMC2778255 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Chloroquine transport in Plasmodium falciparum. 2. Analysis of PfCRT-mediated drug transport using proteoliposomes and a fluorescent chloroquine probe.

Paguio Michelle F MF Cabrera Mynthia M Roepe Paul D PD

Biochemistry 20091001 40

Mutations in the PfCRT protein cause chloroquine resistance (CQR), and earlier studies from our laboratory using plasma membrane inside-out vesicles (ISOV) prepared from yeast expressing recombinant PfCRT [Zhang, H., et al. (2004) Biochemistry 43, 8290-8296] suggested that the putative transporter mediates downhill facilitated diffusion of charged chloroquine (CQ). However, more recent experiments with a fluorescent CQ probe (NBD-CQ) presented in the accompanying paper (DOI 10.1021/bi901034r ) i ...[more]

PMID: 19725576

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Project description:BACKGROUND: In Plasmodium falciparum, resistance to chloroquine (CQ) is conferred by a K to T mutation at amino acid position 76 (K76T) in the P. falciparum CQ transporter (PfCRT). To date, at least 15 pfcrt genotypes, which are represented by combinations of five amino acids at positions 72-76, have been described in field isolates from various endemic regions. To identify novel mutant pfcrt genotypes and to reveal the genetic relatedness of pfcrt genotypes, a large-scale survey over a wide geographic area was performed. METHODS: Sequences for exon 2 in pfcrt, including known polymorphic sites at amino acid positions 72, 74, 75 and 76, were obtained from 256 P. falciparum isolates collected from eight endemic countries in Asia (Bangladesh, Cambodia, Lao P.D.R., the Philippines and Thailand), Melanesia (Papua New Guinea and Vanuatu) and Africa (Ghana). A haplotype network was constructed based on six microsatellite markers located -29 kb to 24 kb from pfcrt in order to examine the genetic relatedness among mutant pfcrt genotypes. RESULTS: In addition to wild type (CVMNK at positions 72-76), four mutant pfcrt were identified; CVIET, CVIDT, SVMNT and CVMNT (mutated amino acids underlined). Haplotype network revealed that there were only three mutant pfcrt lineages, originating in Indochina, Philippines and Melanesia. Importantly, the Indochina lineage contained two mutant pfcrt genotypes, CVIET (n = 95) and CVIDT (n = 14), indicating that CVIDT shares a common origin with CVIET. Similarly, one major haplotype in the Melanesian lineage contained two pfcrt genotypes; SVMNT (n = 71) and CVMNT (n = 3). In Africa, all mutant pfcrt genotypes were the CVIET of the Indochina lineage, probably resulting from the intercontinental migration of CQ resistance from Southeast Asia. CONCLUSIONS: The number of CQ-mutant lineages observed in this study was identical to that found in previous studies. This supports the hypothesis that the emergence of novel CQ resistance is rare. However, in the mutant pfcrt genotypes, amino acid changes at positions 72, 74 and 75 appear to have recently been generated at least several times, producing distinct pfcrt mutant genotypes. The occurrence of new mutations flanking K76T may yield stronger resistance to CQ and/or a higher fitness than the original pfcrt mutant.

Dataset Information

Chloroquine transport in Plasmodium falciparum. 2. Analysis of PfCRT-mediated drug transport using proteoliposomes and a fluorescent chloroquine probe.

Publications

Chloroquine transport in Plasmodium falciparum. 2. Analysis of PfCRT-mediated drug transport using proteoliposomes and a fluorescent chloroquine probe.

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