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Disruption of a Plasmodium falciparum multidrug resistance-associated protein (PfMRP) alters its fitness and transport of antimalarial drugs and glutathione.

Raj DK, Mu J, Jiang H, Kabat J, Singh S, Sullivan M, Fay MP, McCutchan TF, Su XZ - J. Biol. Chem. (2008)

Bottom Line: More recently, many transporters were associated with higher IC(50) levels in responses to chloroquine (CQ) and quinine (QN) in field isolates.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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Malaria and Vector Research, Research Technologies Branch, and Biostatistical Research Branch, NIAID, National Institutes of Health, Bethesda, Maryland 20892-8132, USA.

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 IC(50) 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/MRPDelta) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPDelta 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.

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CQ and QN accumulation assays with or without GSH. A, CQ accumulation. B, CQ radioactivity at 30 min. C, QN accumulation. D, QN radioactivity at 30 min. Parasites and treatments with or without GSH are as indicated. The standard deviations were from three independent experiments.
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fig4: CQ and QN accumulation assays with or without GSH. A, CQ accumulation. B, CQ radioactivity at 30 min. C, QN accumulation. D, QN radioactivity at 30 min. Parasites and treatments with or without GSH are as indicated. The standard deviations were from three independent experiments.

Mentions: Increased CQ and QN Accumulation in the W2/MRPΔ Parasite—One of the mechanisms for many ABC transporters to confer drug resistances is to rapidly pump drugs outside the cells (4, 10, 11). To investigate the possibility of PfMRP playing a role in drug efflux, we performed drug accumulation assays comparing W2, W2/MRPΔ, and 3D7. The parasites were incubated with radioactively labeled CQ and QN, and the radioactivity within the parasites was measured at various time points. Compared with the W2 parasite, the W2/MRPΔ 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. The CQS 3D7 parasite accumulated approximately two to three times more CQ or QN than both W2 and W2/MRPΔ did, whereas RBC accumulated minimum radioactive CQ and QN. Accumulation of more radioactive CQ and QN in 3D7 could be due to retention of more CQ and QN in the parasite food vacuole, because 3D7 has a WT PfCRT, making the drug less accessible to PfMRP and other transporters.


Disruption of a Plasmodium falciparum multidrug resistance-associated protein (PfMRP) alters its fitness and transport of antimalarial drugs and glutathione.

Raj DK, Mu J, Jiang H, Kabat J, Singh S, Sullivan M, Fay MP, McCutchan TF, Su XZ - J. Biol. Chem. (2008)

CQ and QN accumulation assays with or without GSH. A, CQ accumulation. B, CQ radioactivity at 30 min. C, QN accumulation. D, QN radioactivity at 30 min. Parasites and treatments with or without GSH are as indicated. The standard deviations were from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2658063&req=5

fig4: CQ and QN accumulation assays with or without GSH. A, CQ accumulation. B, CQ radioactivity at 30 min. C, QN accumulation. D, QN radioactivity at 30 min. Parasites and treatments with or without GSH are as indicated. The standard deviations were from three independent experiments.
Mentions: Increased CQ and QN Accumulation in the W2/MRPΔ Parasite—One of the mechanisms for many ABC transporters to confer drug resistances is to rapidly pump drugs outside the cells (4, 10, 11). To investigate the possibility of PfMRP playing a role in drug efflux, we performed drug accumulation assays comparing W2, W2/MRPΔ, and 3D7. The parasites were incubated with radioactively labeled CQ and QN, and the radioactivity within the parasites was measured at various time points. Compared with the W2 parasite, the W2/MRPΔ 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. The CQS 3D7 parasite accumulated approximately two to three times more CQ or QN than both W2 and W2/MRPΔ did, whereas RBC accumulated minimum radioactive CQ and QN. Accumulation of more radioactive CQ and QN in 3D7 could be due to retention of more CQ and QN in the parasite food vacuole, because 3D7 has a WT PfCRT, making the drug less accessible to PfMRP and other transporters.

Bottom Line: More recently, many transporters were associated with higher IC(50) levels in responses to chloroquine (CQ) and quinine (QN) in field isolates.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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Malaria and Vector Research, Research Technologies Branch, and Biostatistical Research Branch, NIAID, National Institutes of Health, Bethesda, Maryland 20892-8132, USA.

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 IC(50) 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/MRPDelta) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPDelta 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.

Show MeSH
Related in: MedlinePlus