title: Chloroquine resistance modulated in vitro by expression levels of the Plasmodium falciparum chloroquine resistance transporter.
abstract: Plasmodium falciparum malaria is increasingly difficult to treat and control due to the emergence of parasite resistance to the major antimalarials, notably chloroquine. Recent work has shown that the chloroquine resistance phenotype can be conferred by multiple amino acid mutations in the parasite digestive vacuole transmembrane protein PfCRT. Here, we have addressed whether chloroquine resistance can also be affected by changes in expression levels of this protein. Transient transfection reporter assays revealed that truncation of the pfcrt 3'-untranslated region just prior to putative polyadenylation sites resulted in a 10-fold decrease in luciferase expression levels. Using allelic exchange on a chloroquine-resistant line (7G8 from Brazil), this truncated 3'-untranslated region was inserted downstream of the pfcrt coding sequence, in the place of the endogenous 3'-untranslated region. The resulting pfcrt-modified "knockdown" clones displayed a marked decrease in pfcrt transcription and an estimated 30-40% decrease in PfCRT protein expression levels. [3H]hypoxanthine incorporation assays demonstrated up to a 40% decrease in chloroquine with or without verapamil IC50 levels of pfcrt knockdown clones, relative to the 7G8 parent. Single-cell photometric analyses were consistent with an altered intracellular pH in the knockdown clones, providing further evidence for a relationship between PfCRT, pH regulation, and chloroquine resistance. Genetic truncation of 3'-untranslated regions provides a useful approach for assessing the impact of candidate genes on drug resistance or other quantifiable phenotypes in P. falciparum.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Karena L
authors.initials: KL
authors.lastname: Waller
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Muhle
authors.forename: Lyann M
authors.initials: LM
authors.lastname: Ursos
authors.forename: Paul
authors.initials: P
authors.lastname: Horrocks
authors.forename: Dominik
authors.initials: D
authors.lastname: Verdier-Pinard
authors.forename: Amar Bir Singh
authors.initials: AB
authors.lastname: Sidhu
authors.forename: Hisashi
authors.initials: H
authors.lastname: Fujioka
authors.forename: Paul D
authors.initials: PD
authors.lastname: Roepe
authors.forename: David A
authors.initials: DA
authors.lastname: Fidock
mesh_terms: D020413:3' Untranslated Regions; D000483:Alleles; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002738:Chloroquine / Q000494:pharmacology*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D026901:Membrane Transport Proteins; D016253:Microscopy, Immunoelectron; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D010963:Plasmodium falciparum / Q000187:drug effects / Q000378:metabolism*; D015800:Protozoan Proteins; D013997:Time Factors; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D000962:Antimalarials; D018076:DNA, Complementary; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D002738:Chloroquine; D008156:Luciferases