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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

Hypothetical schematic model of the effects of PfCRT mutations.The FV is acidified by a vacuolar proton pump to create a suitable environment for hemoglobin digestion. The acidic nature of the FV also leads to near complete diprotonation of CQ, which diffuses across the FV membrane in an uncharged form (CQ) and accumulates as a charged form (either CQH+ or CQH22+, although predominantly CQH22+). CQH22+ interferes with the polymerization of toxic heme to non-toxic hemozoin, which leads to parasite death. In normal CQ-sensitive (CQS) parasites, PfCRT, which contains a positive charge in its pore (K76), exports its natural substrates but little, if any, CQH22+. Thus, CQH22+ accumulates in the FV and causes parasite death. In CQ-resistant (CQR) parasites, the positive change in the pore of PfCRT is lost (K76T) and both its natural substrates and CQH22+ are transported out of the FV. As CQH22+ cannot accumulate in the FV, the parasites become resistant to the drug. In 3D7L272F parasites (where the parent strain is already CQS), the mutation may reduce residual transport of CQH22+ out of the FV even further or completely, leading to a greater FV accumulation of CQH22+ and CQ-hypersensitivity or some other mechanism may be responsible for this phenomenon. The mutation also leads to a reduction in the export of natural substrates, resulting in a build-up of these substrates. This causes water to enter the FV by the process of osmosis, leading to swelling. In FCBC101F and Dd2Dd2 L272F parasites (where the parent strains are CQR), the mutations reduce the export of CQH22+ back towards levels measured in CQS lines and also reduce natural substrate export, leading to normal CQ sensitivity and FV swelling, respectively. Note mutations in PfCRT (orange graphic) are denoted by red transmembrane or loop regions, depending on the location of the amino acid change (see Fig. 1a).
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f6: Hypothetical schematic model of the effects of PfCRT mutations.The FV is acidified by a vacuolar proton pump to create a suitable environment for hemoglobin digestion. The acidic nature of the FV also leads to near complete diprotonation of CQ, which diffuses across the FV membrane in an uncharged form (CQ) and accumulates as a charged form (either CQH+ or CQH22+, although predominantly CQH22+). CQH22+ interferes with the polymerization of toxic heme to non-toxic hemozoin, which leads to parasite death. In normal CQ-sensitive (CQS) parasites, PfCRT, which contains a positive charge in its pore (K76), exports its natural substrates but little, if any, CQH22+. Thus, CQH22+ accumulates in the FV and causes parasite death. In CQ-resistant (CQR) parasites, the positive change in the pore of PfCRT is lost (K76T) and both its natural substrates and CQH22+ are transported out of the FV. As CQH22+ cannot accumulate in the FV, the parasites become resistant to the drug. In 3D7L272F parasites (where the parent strain is already CQS), the mutation may reduce residual transport of CQH22+ out of the FV even further or completely, leading to a greater FV accumulation of CQH22+ and CQ-hypersensitivity or some other mechanism may be responsible for this phenomenon. The mutation also leads to a reduction in the export of natural substrates, resulting in a build-up of these substrates. This causes water to enter the FV by the process of osmosis, leading to swelling. In FCBC101F and Dd2Dd2 L272F parasites (where the parent strains are CQR), the mutations reduce the export of CQH22+ back towards levels measured in CQS lines and also reduce natural substrate export, leading to normal CQ sensitivity and FV swelling, respectively. Note mutations in PfCRT (orange graphic) are denoted by red transmembrane or loop regions, depending on the location of the amino acid change (see Fig. 1a).

Mentions: An enlarged FV is also often observed in the presence of protease inhibitors, such as E64 or leupeptin35. Interference with the digestion of hemoglobin leads to a buildup of darkly staining FVs in electron micrographs and, eventually, to parasite death. The parasites described here have enlarged FVs but these are electron lucent (Figs 2 and 4), suggesting that the digestion of hemoglobin is relatively unaffected (further supported by the presence of visible hemozin within the FVs). The simplest explanation for these observations is that the C101F and L272F mutations interfere with the transport of the natural substrates of PfCRT out of the FV. The resulting increase in FV osmotic pressure would lead to water ingress and produce the unusual swelling observed in the FV of the FCBC101F, 3D7L272F, and Dd2Dd2 L272F parasites. Figure 6 presents a schematic model of this process. These morphological changes are associated with other phenotypic changes (which are discussed below). The natural substrate(s) of PfCRT are yet to be identified. Studies performed with other PfCRT expression systems have reported that the protein might function as a chloride channel, a proton pump, an activator of Na+/H+ exchangers and non-specific cation channels or, most recently, a transporter of cationic amino acids as well as a very broad range of other cations15. However, in many of these studies the insertion of PfCRT into the foreign membrane required its fusion to other proteins/polypeptides, and in the most recent study the additions to PfCRT were at both the N- and C- termini, almost doubled its size, and included a protein of undetermined function15. Moreover, in this and the previous studies, little or no interaction could be detected between PfCRTDd2 and known inhibitors of this protein (e.g. VP). Of significant note, the transport kinetics for the proposed natural substrates did not differ significantly between PfCRTDd2 and PfCRT3D7—despite multiple lines of evidence indicating that PfCRTDd2 imparts a substantial fitness cost13363738. Furthermore, the recent finding that much higher levels of acidic amino acids and/or short acidic peptides accumulate within CQ-resistant parasites than in CQ-sensitive strains71314 is not readily reconciled with PfCRT functioning as a chloride channel, a proton pump, or a non-specific cation channel/transporter. These, plus other inconsistencies in the data, suggest that PfCRT does not function correctly when fused to other proteins and that the natural function of PfCRT remains to be resolved.


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)

Hypothetical schematic model of the effects of PfCRT mutations.The FV is acidified by a vacuolar proton pump to create a suitable environment for hemoglobin digestion. The acidic nature of the FV also leads to near complete diprotonation of CQ, which diffuses across the FV membrane in an uncharged form (CQ) and accumulates as a charged form (either CQH+ or CQH22+, although predominantly CQH22+). CQH22+ interferes with the polymerization of toxic heme to non-toxic hemozoin, which leads to parasite death. In normal CQ-sensitive (CQS) parasites, PfCRT, which contains a positive charge in its pore (K76), exports its natural substrates but little, if any, CQH22+. Thus, CQH22+ accumulates in the FV and causes parasite death. In CQ-resistant (CQR) parasites, the positive change in the pore of PfCRT is lost (K76T) and both its natural substrates and CQH22+ are transported out of the FV. As CQH22+ cannot accumulate in the FV, the parasites become resistant to the drug. In 3D7L272F parasites (where the parent strain is already CQS), the mutation may reduce residual transport of CQH22+ out of the FV even further or completely, leading to a greater FV accumulation of CQH22+ and CQ-hypersensitivity or some other mechanism may be responsible for this phenomenon. The mutation also leads to a reduction in the export of natural substrates, resulting in a build-up of these substrates. This causes water to enter the FV by the process of osmosis, leading to swelling. In FCBC101F and Dd2Dd2 L272F parasites (where the parent strains are CQR), the mutations reduce the export of CQH22+ back towards levels measured in CQS lines and also reduce natural substrate export, leading to normal CQ sensitivity and FV swelling, respectively. Note mutations in PfCRT (orange graphic) are denoted by red transmembrane or loop regions, depending on the location of the amino acid change (see Fig. 1a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Hypothetical schematic model of the effects of PfCRT mutations.The FV is acidified by a vacuolar proton pump to create a suitable environment for hemoglobin digestion. The acidic nature of the FV also leads to near complete diprotonation of CQ, which diffuses across the FV membrane in an uncharged form (CQ) and accumulates as a charged form (either CQH+ or CQH22+, although predominantly CQH22+). CQH22+ interferes with the polymerization of toxic heme to non-toxic hemozoin, which leads to parasite death. In normal CQ-sensitive (CQS) parasites, PfCRT, which contains a positive charge in its pore (K76), exports its natural substrates but little, if any, CQH22+. Thus, CQH22+ accumulates in the FV and causes parasite death. In CQ-resistant (CQR) parasites, the positive change in the pore of PfCRT is lost (K76T) and both its natural substrates and CQH22+ are transported out of the FV. As CQH22+ cannot accumulate in the FV, the parasites become resistant to the drug. In 3D7L272F parasites (where the parent strain is already CQS), the mutation may reduce residual transport of CQH22+ out of the FV even further or completely, leading to a greater FV accumulation of CQH22+ and CQ-hypersensitivity or some other mechanism may be responsible for this phenomenon. The mutation also leads to a reduction in the export of natural substrates, resulting in a build-up of these substrates. This causes water to enter the FV by the process of osmosis, leading to swelling. In FCBC101F and Dd2Dd2 L272F parasites (where the parent strains are CQR), the mutations reduce the export of CQH22+ back towards levels measured in CQS lines and also reduce natural substrate export, leading to normal CQ sensitivity and FV swelling, respectively. Note mutations in PfCRT (orange graphic) are denoted by red transmembrane or loop regions, depending on the location of the amino acid change (see Fig. 1a).
Mentions: An enlarged FV is also often observed in the presence of protease inhibitors, such as E64 or leupeptin35. Interference with the digestion of hemoglobin leads to a buildup of darkly staining FVs in electron micrographs and, eventually, to parasite death. The parasites described here have enlarged FVs but these are electron lucent (Figs 2 and 4), suggesting that the digestion of hemoglobin is relatively unaffected (further supported by the presence of visible hemozin within the FVs). The simplest explanation for these observations is that the C101F and L272F mutations interfere with the transport of the natural substrates of PfCRT out of the FV. The resulting increase in FV osmotic pressure would lead to water ingress and produce the unusual swelling observed in the FV of the FCBC101F, 3D7L272F, and Dd2Dd2 L272F parasites. Figure 6 presents a schematic model of this process. These morphological changes are associated with other phenotypic changes (which are discussed below). The natural substrate(s) of PfCRT are yet to be identified. Studies performed with other PfCRT expression systems have reported that the protein might function as a chloride channel, a proton pump, an activator of Na+/H+ exchangers and non-specific cation channels or, most recently, a transporter of cationic amino acids as well as a very broad range of other cations15. However, in many of these studies the insertion of PfCRT into the foreign membrane required its fusion to other proteins/polypeptides, and in the most recent study the additions to PfCRT were at both the N- and C- termini, almost doubled its size, and included a protein of undetermined function15. Moreover, in this and the previous studies, little or no interaction could be detected between PfCRTDd2 and known inhibitors of this protein (e.g. VP). Of significant note, the transport kinetics for the proposed natural substrates did not differ significantly between PfCRTDd2 and PfCRT3D7—despite multiple lines of evidence indicating that PfCRTDd2 imparts a substantial fitness cost13363738. Furthermore, the recent finding that much higher levels of acidic amino acids and/or short acidic peptides accumulate within CQ-resistant parasites than in CQ-sensitive strains71314 is not readily reconciled with PfCRT functioning as a chloride channel, a proton pump, or a non-specific cation channel/transporter. These, plus other inconsistencies in the data, suggest that PfCRT does not function correctly when fused to other proteins and that the natural function of PfCRT remains to be resolved.

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