Disruption of a Plasmodium falciparum Multidrug Resistance-associated Protein (PfMRP) Alters Its Fitness and Transport of Antimalarial Drugs and Glutathione

Dipak Kumar Raj,Jianbing Mu,Hongying Jiang,Juraj Kabat,Subash Singh,Margery Sullivan,Michael P Fay,Thomas F Mccutchan,Xin-Zhuan Su

doi:10.1074/jbc.m806944200

Abstract

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC50 levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPΔ) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPΔ parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

Highlights

Genes encoding ATP-binding cassette (ABC)2 transporters belong to a supergene family present in organisms from pro
One example is that mutations in the gene encoding a putative P. falciparum chloroquine resistance transporter (PfCRT) can convert a clinical CQ-sensitive (CQS) P. falciparum parasite into a CQ-resistant (CQR) one [23]; parasites with the same mutant PfCRT allele can display very different levels of resistance to CQ, suggesting contribution from proteins such as PfPgh-1 and other molecules [24, 25]
We showed that when the gene encoding the PfMRP in a CQR parasite (W2) was disrupted, the parasite growth was affected and became more sensitive to multiple antimalarial drugs

Summary

EXPERIMENTAL PROCEDURES

Parasite and Parasite Culture—P. falciparum parasites used in this study have been described [24, 33]. Compared with the W2 parasite, the W2/MRP⌬ parasite accumulated more radioactive labeled CQ (59.0%) and QN (55.2%) than W2 did (Fig. 4), suggesting that PfMRP plays a role in pumping CQ and QN outside the parasite cell. Partial reduction (37.4%) of QN accumulation was observed in the W2/MRP⌬ parasite (Fig. 4, C and D) These results indicated that GSH could enhance the transport of drugs outside the parasite cell at least in some parasites, possibly through pumping of GSH drug adducts by PfMRP and other transporters. Compared with its WT W2, the W2/MRP⌬ parasite became significantly more sensitive to five of the eight drugs tested (CQ, QN, ART, PQ, and PRQ; Table 1) These results showed that PfMRP played a role in parasite response to multiple antimalarial drugs, possibly by transporting the drugs outside the parasite cell; PfMRP might not be a key molecule that can convert a clinically sensitive parasite into a resistant parasite, because the five sig-. Note that CQ, QN, ART, PQ, and PRQ are significant even after correcting for multiple tests using Bonferroni’s adjustment (p Ͻ 0.05)

No repeats

Findings

DISCUSSION