title: Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT.
abstract: Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.
authors.affiliation: Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-8132, USA.
authors.forename: Bronwen
authors.initials: B
authors.lastname: Naudé
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Brzostowski
authors.forename: Alan R
authors.initials: AR
authors.lastname: Kimmel
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wellems
mesh_terms: D000143:Acids / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000962:Antimalarials / Q000737:chemistry / Q000493:pharmacokinetics*; D002738:Chloroquine / Q000737:chemistry / Q000493:pharmacokinetics*; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004023:Dictyostelium / Q000187:drug effects* / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D015870:Gene Expression; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D010641:Phenotype; D010802:Phylogeny; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics*; D017354:Point Mutation; D015800:Protozoan Proteins; D011802:Quinidine / Q000493:pharmacokinetics; D011803:Quinine / Q000493:pharmacokinetics; D011804:Quinolines / Q000737:chemistry / Q000493:pharmacokinetics; D014162:Transfection; D010879:Piperazines
chemical_list: D000143:Acids; D000962:Antimalarials; D002738:Chloroquine; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D015800:Protozoan Proteins; D011802:Quinidine; D011803:Quinine; D011804:Quinolines; C416369:PfCRT protein, Plasmodium falciparum; C034759:piperaquine; D010879:Piperazines