title: Drug resistance-associated pfCRT mutations confer decreased Plasmodium falciparum digestive vacuolar pH.
abstract: Elucidating the altered physiology of various chloroquine resistant (CQR) strains of Plasmodium falciparum is essential for understanding the molecular basis of CQR. In this study, we have devised several new methods for analyzing digestive vacuolar (DV) pH for individual intraerythrocytic parasites under continuous perfusion. These use controlled illumination power and novel data acquisition software, and are based on either acridine orange (AO) emission spectra or ratiometric 5-(and 6-)carboxy-2',7'-dimethyl-3'-hydroxy-6'-N-ethylaminospiro [isobenzofuran-1(3H),9'-(9H)xanthen]-3-one (DM NERF) excitation. Results show that DV pH is more acidic for laboratory strains of CQR parasites relative to chloroquine sensitive (CQS). Using mutant pfcrt allelic exchange clones not previously exposed to chloroquine (CQ), we now show a direct association between acid DV pH, CQ resistance and mutation of pfcrt to either South American (7G8) or South East Asian (Dd2) CQR-associated alleles. Surprisingly, these alleles confer a similar degree of DV acidification. Verapamil (VPL) reversed acid DV pH for the Dd2 mutant C3(Dd2) clone, in a surprisingly rapid fashion, but did not reverse acid DV pH for the 7G8 mutant C6(7G8) clone. Thus, there is a direct link between expression of two major CQR-associated pfcrt alleles and altered parasite DV physiology. The data also support models that envision direct but allele-specific interaction between PfCRT and VPL.
authors.affiliation: Department of Chemistry, Biochemistry and Molecular Biology, and Program in Tumor Biology, Lombardi Cancer Center, Georgetown University, Washington, DC 20057, USA.
authors.forename: Tyler N
authors.initials: TN
authors.lastname: Bennett
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Kosar
authors.forename: Lyann M B
authors.initials: LM
authors.lastname: Ursos
authors.forename: Sergey
authors.initials: S
authors.lastname: Dzekunov
authors.forename: Amar Bir
authors.initials: AB
authors.lastname: Singh Sidhu
authors.forename: David A
authors.initials: DA
authors.lastname: Fidock
authors.forename: Paul D
authors.initials: PD
authors.lastname: Roepe
mesh_terms: D000165:Acridine Orange / Q000378:metabolism; D000483:Alleles; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D002738:Chloroquine / Q000494:pharmacology; D003592:Cytophotometry / Q000379:methods; D004351:Drug Resistance / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology*; D026901:Membrane Transport Proteins; D009154:Mutation; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000502:physiology* / Q000648:ultrastructure; D015800:Protozoan Proteins; D011995:Recombination, Genetic; D014617:Vacuoles / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D014700:Verapamil / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D002738:Chloroquine; D014700:Verapamil; D000165:Acridine Orange