Drug resistance-associated pfCRT mutations confer decreased Plasmodium falciparum digestive vacuolar pH

• Published in: Molecular and biochemical parasitology Vol. 133; no. 1; pp. 99 - 114
• Main Authors: Bennett, Tyler N, Kosar, Andrew D, Ursos, Lyann M.B, Dzekunov, Sergey, Singh Sidhu, Amar Bir, Fidock, David A, Roepe, Paul D
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 2004
• Subjects: Acridine orange; Acridine Orange - metabolism; Alleles; Animals; Antimalarials - pharmacology; Chloroquine; Chloroquine - pharmacology; Cytophotometry - methods; DM NERF; Drug Resistance - genetics; Hydrogen-Ion Concentration; Malaria; Membrane Proteins - genetics; Membrane Proteins - physiology; Membrane Transport Proteins; Mutation; pfcrt; Plasmodium falciparum; Plasmodium falciparum - drug effects; Plasmodium falciparum - genetics; Plasmodium falciparum - physiology; Plasmodium falciparum - ultrastructure; Protozoan Proteins; Recombination, Genetic; Vacuolar pH; Vacuoles - chemistry; Vacuoles - physiology; Vacuoles - ultrastructure; Verapamil - metabolism; Verapamil - pharmacology; SCP; CQS; Malaria; CQR; BCECF; Chloroquine; FCCP; ROI; AO; Acridine orange; DV; RBC; VPL; QD; pfcrt; PMT; DM NERF; QN; Vacuolar pH
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2003.09.008

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(3
H),9′-(9
H)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.