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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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Localization of PfMRP in sexual stages. Row 1, green, anti-PfMRP; row 2, red, anti-PfMDV-1; row 3, blue, 4′,6′-diamino-2-phenylindole dye; row 4, differential interference contrast; row 5, merged rows 1–4. A, stage I gametocyte identified by anti-PfMDV-1 staining. B, stage II gametocyte showing two broad straight lines stained strongly with anti-PfMRP. C, stage III–V gametocyte showing surface membrane staining and some internal membrane networks. D and E, iso-surface model of the stage II gametocyte reconstructed from serial Z-sections of confocal images. D, side view of a stage II gametocyte showing two heavily stained structures (green) parallel to the straight side of the D-shaped parasite. Internal vesicles with PfMRP (white arrowhead) could also be seen inside the parasite. E, bottom view of the same stage II gametocyte. The red shown in this transparent iso-surface model indicates the distribution of PfMDV-1 that is a gametocyte-specific protein located on gametocyte outside membrane and internal vesicles (33).
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fig7: Localization of PfMRP in sexual stages. Row 1, green, anti-PfMRP; row 2, red, anti-PfMDV-1; row 3, blue, 4′,6′-diamino-2-phenylindole dye; row 4, differential interference contrast; row 5, merged rows 1–4. A, stage I gametocyte identified by anti-PfMDV-1 staining. B, stage II gametocyte showing two broad straight lines stained strongly with anti-PfMRP. C, stage III–V gametocyte showing surface membrane staining and some internal membrane networks. D and E, iso-surface model of the stage II gametocyte reconstructed from serial Z-sections of confocal images. D, side view of a stage II gametocyte showing two heavily stained structures (green) parallel to the straight side of the D-shaped parasite. Internal vesicles with PfMRP (white arrowhead) could also be seen inside the parasite. E, bottom view of the same stage II gametocyte. The red shown in this transparent iso-surface model indicates the distribution of PfMDV-1 that is a gametocyte-specific protein located on gametocyte outside membrane and internal vesicles (33).

Mentions: Expression and Localization of PfMRP in Asexual and Sexual Stages—PfMRP was predicted to be a membrane protein with 11 transmembrane domains and was transcribed in both asexual and sexual stages (28–30). A previous study also suggested that PfMRP was expressed on the plasma membrane of trophozoite and schizont stages but was not detectable at ring stage or on the membrane of food vacuole (27). To further characterize the protein expression patterns and localization of PfMRP in different developmental stages, we performed immunofluorescent assay using antibodies against PfMRP, PfMSP-1 (a protein on merozoite plasma membrane), PfCRT (on food vacuole membrane), and PfMDV-1 (gametocyte-specific). We found that PfMRP was expressed in all of the asexual and sexual stages that we investigated, from merozoite to mature schizont, and from young gametocyte to mature gametocyte (Figs. 6 and 7; supplemental Fig. 2).


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)

Localization of PfMRP in sexual stages. Row 1, green, anti-PfMRP; row 2, red, anti-PfMDV-1; row 3, blue, 4′,6′-diamino-2-phenylindole dye; row 4, differential interference contrast; row 5, merged rows 1–4. A, stage I gametocyte identified by anti-PfMDV-1 staining. B, stage II gametocyte showing two broad straight lines stained strongly with anti-PfMRP. C, stage III–V gametocyte showing surface membrane staining and some internal membrane networks. D and E, iso-surface model of the stage II gametocyte reconstructed from serial Z-sections of confocal images. D, side view of a stage II gametocyte showing two heavily stained structures (green) parallel to the straight side of the D-shaped parasite. Internal vesicles with PfMRP (white arrowhead) could also be seen inside the parasite. E, bottom view of the same stage II gametocyte. The red shown in this transparent iso-surface model indicates the distribution of PfMDV-1 that is a gametocyte-specific protein located on gametocyte outside membrane and internal vesicles (33).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Localization of PfMRP in sexual stages. Row 1, green, anti-PfMRP; row 2, red, anti-PfMDV-1; row 3, blue, 4′,6′-diamino-2-phenylindole dye; row 4, differential interference contrast; row 5, merged rows 1–4. A, stage I gametocyte identified by anti-PfMDV-1 staining. B, stage II gametocyte showing two broad straight lines stained strongly with anti-PfMRP. C, stage III–V gametocyte showing surface membrane staining and some internal membrane networks. D and E, iso-surface model of the stage II gametocyte reconstructed from serial Z-sections of confocal images. D, side view of a stage II gametocyte showing two heavily stained structures (green) parallel to the straight side of the D-shaped parasite. Internal vesicles with PfMRP (white arrowhead) could also be seen inside the parasite. E, bottom view of the same stage II gametocyte. The red shown in this transparent iso-surface model indicates the distribution of PfMDV-1 that is a gametocyte-specific protein located on gametocyte outside membrane and internal vesicles (33).
Mentions: Expression and Localization of PfMRP in Asexual and Sexual Stages—PfMRP was predicted to be a membrane protein with 11 transmembrane domains and was transcribed in both asexual and sexual stages (28–30). A previous study also suggested that PfMRP was expressed on the plasma membrane of trophozoite and schizont stages but was not detectable at ring stage or on the membrane of food vacuole (27). To further characterize the protein expression patterns and localization of PfMRP in different developmental stages, we performed immunofluorescent assay using antibodies against PfMRP, PfMSP-1 (a protein on merozoite plasma membrane), PfCRT (on food vacuole membrane), and PfMDV-1 (gametocyte-specific). We found that PfMRP was expressed in all of the asexual and sexual stages that we investigated, from merozoite to mature schizont, and from young gametocyte to mature gametocyte (Figs. 6 and 7; supplemental Fig. 2).

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