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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

PfCRT mutations.(a) Schematic representation of PfCRT and positions of previously identified polymorphisms871 from field isolates (green circles) and from drug-pressured laboratory lines (purple circles). The critical CQ resistance mutation site (K76) is shaded red, and the two residues at which mutations are described in this study are shaded in orange (C101) and blue (L272). (b) PfCRT haplotypes included this study.
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f1: PfCRT mutations.(a) Schematic representation of PfCRT and positions of previously identified polymorphisms871 from field isolates (green circles) and from drug-pressured laboratory lines (purple circles). The critical CQ resistance mutation site (K76) is shaded red, and the two residues at which mutations are described in this study are shaded in orange (C101) and blue (L272). (b) PfCRT haplotypes included this study.

Mentions: SNPs were identified in pfcrt in two different P. falciparum lines (Fig. 1a,b). The first was discovered after isolating amantadine (AMT)-resistant mutants of the CQ-resistant parasite strain FCB, following selection with 80 μM of this antiviral agent. Viable parasites were observed in one of four drug-pressured flasks at 42 days, whereas none had emerged within the remaining flasks by 60 days. PCR amplification and sequencing of pfcrt in four clonal lines derived from the AMT-resistant culture detected a single non-synonymous SNP, g302t. This encoded the amino acid mutation C101F. These lines were therefore designated FCBC101F. Position 101 is predicted to lie within the second TMD of PfCRT (Fig. 1a). This mutation was earlier observed in a CQ-resistant Dd2 parasite line derived by continuous piperaquine (PPQ) pressure22, although that study did not describe any changes in parasite morphology.


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)

PfCRT mutations.(a) Schematic representation of PfCRT and positions of previously identified polymorphisms871 from field isolates (green circles) and from drug-pressured laboratory lines (purple circles). The critical CQ resistance mutation site (K76) is shaded red, and the two residues at which mutations are described in this study are shaded in orange (C101) and blue (L272). (b) PfCRT haplotypes included this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4588581&req=5

f1: PfCRT mutations.(a) Schematic representation of PfCRT and positions of previously identified polymorphisms871 from field isolates (green circles) and from drug-pressured laboratory lines (purple circles). The critical CQ resistance mutation site (K76) is shaded red, and the two residues at which mutations are described in this study are shaded in orange (C101) and blue (L272). (b) PfCRT haplotypes included this study.
Mentions: SNPs were identified in pfcrt in two different P. falciparum lines (Fig. 1a,b). The first was discovered after isolating amantadine (AMT)-resistant mutants of the CQ-resistant parasite strain FCB, following selection with 80 μM of this antiviral agent. Viable parasites were observed in one of four drug-pressured flasks at 42 days, whereas none had emerged within the remaining flasks by 60 days. PCR amplification and sequencing of pfcrt in four clonal lines derived from the AMT-resistant culture detected a single non-synonymous SNP, g302t. This encoded the amino acid mutation C101F. These lines were therefore designated FCBC101F. Position 101 is predicted to lie within the second TMD of PfCRT (Fig. 1a). This mutation was earlier observed in a CQ-resistant Dd2 parasite line derived by continuous piperaquine (PPQ) pressure22, although that study did not describe any changes in parasite morphology.

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