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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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GSH accumulation assays. Parasitized RBCs or uninfected RBCs were incubated with radioactive GSH (the curves shown are from 3 nm GSH), and the radioactivities in the RBCs were measured at different time points. Unincorporated radioactive materials were removed by spinning the RBC through silicon oil. The data were from three independent repeats. Similar accumulation curve patterns were obtained using 1 and 6 nm radioactive labeled GSH (data not shown).
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fig3: GSH accumulation assays. Parasitized RBCs or uninfected RBCs were incubated with radioactive GSH (the curves shown are from 3 nm GSH), and the radioactivities in the RBCs were measured at different time points. Unincorporated radioactive materials were removed by spinning the RBC through silicon oil. The data were from three independent repeats. Similar accumulation curve patterns were obtained using 1 and 6 nm radioactive labeled GSH (data not shown).

Mentions: Increased GSH Accumulation in the W2/MRPΔ Parasite— MRP has been shown to transport GSH and its conjugates such as GSSG, GSH drug adducts, and other metabolites outside mammalian cells (47). The ability to transport GSH conjugates outside the parasite cell may be related to how parasites respond to stresses caused by accumulation of metabolic toxins in in vitro culture, which may explain why the W2/MRPΔ could not grow to a parasitemia higher than 5% when culture medium was changed once a day. We therefore investigated GSH accumulation in 3D7, W2/MRPΔ, and W2 parasites. 3D7 is a CQS parasite that has wild type PfCRT and PfMRP and was included as a control for PfCRT effects on drug accumulation. For all the parasites, accumulation of GSH peaked at ∼20 min after addition of radioactive GSH, and the GSH concentrations stayed at similar levels until the experiments were terminated at 60 min (Fig. 3). Although W2 and 3D7 accumulated approximately the same amount of radioactive GSH, the W2/MRPΔ parasite accumulated approximately twice as much radiolabeled GSH as the WT W2 did (Fig. 3), suggesting that PfMRP may play a role in pumping GSH or GSH conjugates outside the cell and that disruption of PfMRP affects the transport process. RBC accumulated very minimum radioactive GSH, possibly because of active transport of GSH by human MRP and other transporters present on the RBC membrane.


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)

GSH accumulation assays. Parasitized RBCs or uninfected RBCs were incubated with radioactive GSH (the curves shown are from 3 nm GSH), and the radioactivities in the RBCs were measured at different time points. Unincorporated radioactive materials were removed by spinning the RBC through silicon oil. The data were from three independent repeats. Similar accumulation curve patterns were obtained using 1 and 6 nm radioactive labeled GSH (data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: GSH accumulation assays. Parasitized RBCs or uninfected RBCs were incubated with radioactive GSH (the curves shown are from 3 nm GSH), and the radioactivities in the RBCs were measured at different time points. Unincorporated radioactive materials were removed by spinning the RBC through silicon oil. The data were from three independent repeats. Similar accumulation curve patterns were obtained using 1 and 6 nm radioactive labeled GSH (data not shown).
Mentions: Increased GSH Accumulation in the W2/MRPΔ Parasite— MRP has been shown to transport GSH and its conjugates such as GSSG, GSH drug adducts, and other metabolites outside mammalian cells (47). The ability to transport GSH conjugates outside the parasite cell may be related to how parasites respond to stresses caused by accumulation of metabolic toxins in in vitro culture, which may explain why the W2/MRPΔ could not grow to a parasitemia higher than 5% when culture medium was changed once a day. We therefore investigated GSH accumulation in 3D7, W2/MRPΔ, and W2 parasites. 3D7 is a CQS parasite that has wild type PfCRT and PfMRP and was included as a control for PfCRT effects on drug accumulation. For all the parasites, accumulation of GSH peaked at ∼20 min after addition of radioactive GSH, and the GSH concentrations stayed at similar levels until the experiments were terminated at 60 min (Fig. 3). Although W2 and 3D7 accumulated approximately the same amount of radioactive GSH, the W2/MRPΔ parasite accumulated approximately twice as much radiolabeled GSH as the WT W2 did (Fig. 3), suggesting that PfMRP may play a role in pumping GSH or GSH conjugates outside the cell and that disruption of PfMRP affects the transport process. RBC accumulated very minimum radioactive GSH, possibly because of active transport of GSH by human MRP and other transporters present on the RBC membrane.

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