Abstract
BackgroundThe Plasmodium falciparum multidrug resistance 1 transporter, PfMDR1, contains five amino acid polymorphisms that are suggested to be involved in altered drug transport from the parasite’s cytosol into the digestive vacuole (DV). Transport of a substrate into another intracellular compartment influences drug availability at its site of action, therefore making the parasite more susceptible or resistant to a drug. Fluo-4 is a known fluorescent substrate that can be used as a molecular tool to investigate transport dynamics of PfMDR1 in many parasite strains.MethodsSix P. falciparum strains with varying PfMDR1 mutations were loaded with Fluo-4 AM. Accumulation of the fluorophore in the DV was measured using confocal microscopy. The role of a key amino acid mutation was verified using selected parasite clones with point mutations at PfMDR1 amino acid position 1042. Equal expression of PfMDR1 was confirmed by Western blot.ResultsFluo-4 was transported by PfMDR1 into the DV of most drug-sensitive and -resistant parasites. Asparagine at PfMDR1 amino acid position 1042 was crucial for Fluo-4 transport, while the N1042D substitution abolished Fluo-4 transport. Competition studies of Fluo-4 with chloroquine, quinine and mefloquine were performed on parasites harbouring asparagine at position 1042. A distinct Fluo-4 transport inhibition pattern for each tested anti-malarial drug was observed in parasite strains of different genetic background.ConclusionThis study demonstrates that Fluo-4 can be used to investigate PfMDR1 transport dynamics in both drug-sensitive and -resistant parasites. Furthermore, direct evidence of altered Fluo-4 transport in PfMDR1 is linked to a single amino acid mutation in the substrate binding pocket. This system offers a great tool to investigate the role of substrate transport by PfMDR1 and the mutations necessary to support transport, which would lead to new insights for the development of novel anti-malarial drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0791-3) contains supplementary material, which is available to authorized users.
Highlights
The Plasmodium falciparum multidrug resistance 1 transporter, PfMDR1, contains five amino acid polymorphisms that are suggested to be involved in altered drug transport from the parasite’s cytosol into the digestive vacuole (DV)
To identify PfMDR1 mutation(s) crucial for Fluo-4 transport, several drug-sensitive and -resistant P. falciparum strains of different genetic backgrounds were tested for accumulation of Fluo-4 in the DV
While PfMDR1 has been suggested to play a role in CQ resistance, the key genetic indicator for CQS versus CQR parasites is attributed to the amino acid mutation K76T in the P. falciparum chloroquine resistance transporter (PfCRT) [21]
Summary
The Plasmodium falciparum multidrug resistance 1 transporter, PfMDR1, contains five amino acid polymorphisms that are suggested to be involved in altered drug transport from the parasite’s cytosol into the digestive vacuole (DV). Fluo-4 is a known fluorescent substrate that can be used as a molecular tool to investigate transport dynamics of PfMDR1 in many parasite strains. Researchers found a correlation between antimalarial resistance and the Plasmodium falciparum multidrug resistance 1 transporter (PfMDR1) [2, 3]. PfMDR1 is a P-glycoprotein homologue (Pgh1) and belongs to the ATP binding cassette (ABC) transporter superfamily. It is a 162 kDa protein with two nucleotide binding domains (NBD) and twelve transmembrane domains (TMDs), with a putative substrate binding pocket in TMD11 [4]. The transporter is located in the membrane of the digestive vacuole (DV) [5], transporting substrates from the parasite’s cytoplasm into the DV [6]
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