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Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

Pulcini S, Staines HM, Lee AH, Shafik SH, Bouyer G, Moore CM, Daley DA, Hoke MJ, Altenhofen LM, Painter HJ, Mu J, Ferguson DJ, Llinás M, Martin RE, Fidock DA, Cooper RA, Krishna S - Sci Rep (2015)

Bottom Line: These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains.Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes.Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Infection and Immunity, St. George's, University of London, London SW17 0RE, UK.

ABSTRACT
Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

No MeSH data available.


Related in: MedlinePlus

Morphological comparisons of parasites with mutations in pfcrt compared with their parental controls.(a) Parasites were synchronized by sorbitol lysis and areas of FVs and parasites (approximately 38 h post-invasion) were measured and expressed as ratios (AFV/AParasite). 3D7, FCB and Dd2Dd2 (open bars; n = 72, 31 and 42, respectively) and 3D7L272F, FCBC101F and Dd2Dd2 L272F (closed bars; n = 84, 58 and 88, respectively) parasites were analyzed and significant enlargement of the FV was confirmed in 3D7L272F, FCBC101F and Dd2Dd2 L272F parasites relative to 3D7, FCB and Dd2Dd2, respectively (*p < 0.0001: two-tailed, unpaired, Student’s t-test). (b,c) Transmission electron micrographs of 3D7 and 3D7L272F parasites, respectively, showing the food vacuole (FV) and nucleus (N). Note the enlarged electron lucent FV in 3D7L272F (suggesting changes in the process of hemoglobin degradation and formation of hemozoin crystals). RBCs infected with 3D7L272F displayed approximately 7.5-fold more knobs (arrowheads) on the host surface than 3D7-infected RBCs, although this is likely due to sub-population selection rather than a direct link to the mutation in pfcrt. (insert) Detail of a knob. Bars represent 1 μm (b,c) and 100 nm (insert).
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f3: Morphological comparisons of parasites with mutations in pfcrt compared with their parental controls.(a) Parasites were synchronized by sorbitol lysis and areas of FVs and parasites (approximately 38 h post-invasion) were measured and expressed as ratios (AFV/AParasite). 3D7, FCB and Dd2Dd2 (open bars; n = 72, 31 and 42, respectively) and 3D7L272F, FCBC101F and Dd2Dd2 L272F (closed bars; n = 84, 58 and 88, respectively) parasites were analyzed and significant enlargement of the FV was confirmed in 3D7L272F, FCBC101F and Dd2Dd2 L272F parasites relative to 3D7, FCB and Dd2Dd2, respectively (*p < 0.0001: two-tailed, unpaired, Student’s t-test). (b,c) Transmission electron micrographs of 3D7 and 3D7L272F parasites, respectively, showing the food vacuole (FV) and nucleus (N). Note the enlarged electron lucent FV in 3D7L272F (suggesting changes in the process of hemoglobin degradation and formation of hemozoin crystals). RBCs infected with 3D7L272F displayed approximately 7.5-fold more knobs (arrowheads) on the host surface than 3D7-infected RBCs, although this is likely due to sub-population selection rather than a direct link to the mutation in pfcrt. (insert) Detail of a knob. Bars represent 1 μm (b,c) and 100 nm (insert).

Mentions: A monstrously swollen FV was observed at all stages that ordinarily display a vacuole in the asexual cycle of both parasite lines FCBC101F and 3D7L272F (Fig. 2). This phenotype was stably maintained in the parasites following repeated rounds of parasite culture and cryopreservation. The enlarged FVs were already apparent in the early to mid trophozoite stages of the FCBC101F line, when compared with FVs from FCB parental controls (Fig. 2a left and right panels). In more mature FCBC101F parasites, the FVs were strikingly clear in appearance, with hemozoin crystals apparently marginalized to the FV periphery and opposite the developing nuclei, although live imaging suggests that the hemozoin is distributed normally (Fig. 2b). The immature ring stages of development were indistinguishable from those of the parental strain. Similar findings were evident in the parasite line 3D7L272F when compared with 3D7 (Fig. 2c left and right panels). Measurement of the area of the FV was also undertaken and expressed as a ratio of the parasite’s area to correct for parasite age (Fig. 3a). This confirmed that FCBC101F and 3D7L272F parasites have a relative FV/parasite area that is approximately twice that of FCB and 3D7, respectively (p < 0.0001). Neither FCBC101F nor 3D7L272F parasites appeared to be enlarged within their host red blood cells (RBCs).


Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

Pulcini S, Staines HM, Lee AH, Shafik SH, Bouyer G, Moore CM, Daley DA, Hoke MJ, Altenhofen LM, Painter HJ, Mu J, Ferguson DJ, Llinás M, Martin RE, Fidock DA, Cooper RA, Krishna S - Sci Rep (2015)

Morphological comparisons of parasites with mutations in pfcrt compared with their parental controls.(a) Parasites were synchronized by sorbitol lysis and areas of FVs and parasites (approximately 38 h post-invasion) were measured and expressed as ratios (AFV/AParasite). 3D7, FCB and Dd2Dd2 (open bars; n = 72, 31 and 42, respectively) and 3D7L272F, FCBC101F and Dd2Dd2 L272F (closed bars; n = 84, 58 and 88, respectively) parasites were analyzed and significant enlargement of the FV was confirmed in 3D7L272F, FCBC101F and Dd2Dd2 L272F parasites relative to 3D7, FCB and Dd2Dd2, respectively (*p < 0.0001: two-tailed, unpaired, Student’s t-test). (b,c) Transmission electron micrographs of 3D7 and 3D7L272F parasites, respectively, showing the food vacuole (FV) and nucleus (N). Note the enlarged electron lucent FV in 3D7L272F (suggesting changes in the process of hemoglobin degradation and formation of hemozoin crystals). RBCs infected with 3D7L272F displayed approximately 7.5-fold more knobs (arrowheads) on the host surface than 3D7-infected RBCs, although this is likely due to sub-population selection rather than a direct link to the mutation in pfcrt. (insert) Detail of a knob. Bars represent 1 μm (b,c) and 100 nm (insert).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588581&req=5

f3: Morphological comparisons of parasites with mutations in pfcrt compared with their parental controls.(a) Parasites were synchronized by sorbitol lysis and areas of FVs and parasites (approximately 38 h post-invasion) were measured and expressed as ratios (AFV/AParasite). 3D7, FCB and Dd2Dd2 (open bars; n = 72, 31 and 42, respectively) and 3D7L272F, FCBC101F and Dd2Dd2 L272F (closed bars; n = 84, 58 and 88, respectively) parasites were analyzed and significant enlargement of the FV was confirmed in 3D7L272F, FCBC101F and Dd2Dd2 L272F parasites relative to 3D7, FCB and Dd2Dd2, respectively (*p < 0.0001: two-tailed, unpaired, Student’s t-test). (b,c) Transmission electron micrographs of 3D7 and 3D7L272F parasites, respectively, showing the food vacuole (FV) and nucleus (N). Note the enlarged electron lucent FV in 3D7L272F (suggesting changes in the process of hemoglobin degradation and formation of hemozoin crystals). RBCs infected with 3D7L272F displayed approximately 7.5-fold more knobs (arrowheads) on the host surface than 3D7-infected RBCs, although this is likely due to sub-population selection rather than a direct link to the mutation in pfcrt. (insert) Detail of a knob. Bars represent 1 μm (b,c) and 100 nm (insert).
Mentions: A monstrously swollen FV was observed at all stages that ordinarily display a vacuole in the asexual cycle of both parasite lines FCBC101F and 3D7L272F (Fig. 2). This phenotype was stably maintained in the parasites following repeated rounds of parasite culture and cryopreservation. The enlarged FVs were already apparent in the early to mid trophozoite stages of the FCBC101F line, when compared with FVs from FCB parental controls (Fig. 2a left and right panels). In more mature FCBC101F parasites, the FVs were strikingly clear in appearance, with hemozoin crystals apparently marginalized to the FV periphery and opposite the developing nuclei, although live imaging suggests that the hemozoin is distributed normally (Fig. 2b). The immature ring stages of development were indistinguishable from those of the parental strain. Similar findings were evident in the parasite line 3D7L272F when compared with 3D7 (Fig. 2c left and right panels). Measurement of the area of the FV was also undertaken and expressed as a ratio of the parasite’s area to correct for parasite age (Fig. 3a). This confirmed that FCBC101F and 3D7L272F parasites have a relative FV/parasite area that is approximately twice that of FCB and 3D7, respectively (p < 0.0001). Neither FCBC101F nor 3D7L272F parasites appeared to be enlarged within their host red blood cells (RBCs).

Bottom Line: These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains.Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes.Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Infection and Immunity, St. George's, University of London, London SW17 0RE, UK.

ABSTRACT
Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

No MeSH data available.


Related in: MedlinePlus