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. [ 3 H]hypoxanthine incorporation assays demonstrated up to a 40% decrease in chloroquine with or without verapamil IC 50 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.

The pH of the digestive vacuole ofPlasmodium falciparumis not associated with chloroquine resistance

“…The concentration of dextran in each case was ~28 M. There were no significant differences in IC 50 for either CQ or mefloquine, or in CQ uptake (P>0.4). Nor was there any significant difference in Waller et al, 2003) have been interpreted as inferring that CQR parasites have a lower pH DV than CQS parasites, and that verapamil restores the pH DV of CQR parasites to levels similar to those observed in CQS parasites Ursos et al, 2000). In attempting to reconcile these results with the CQ accumulation data it was proposed that because CQ does not bind as efficiently to insoluble haematin , and because haematin becomes less soluble as pH DV is reduced , a reduction in pH DV in CQR parasites produces a concomitant reduction in the availability of the drug target.…”

Antimalarial drugs disrupt ion homeostasis in malarial parasites

“…The antimalarial drug chloroquine is thought to accumulate in acidic compartments, although its mechanisms of action are controversial (Slater & Cerami 1992, Dorn et al 1995, Waller et al 2003. The resistance to chloroquine observed in P. falciparum is dependent to mutations in specific transporter (PfCRT), by using heterologous expression Reeves et al (2006) observed a increase of lysosomes acidification in mammalian cells expressing PfCRT, providing new information about acidification process and chloroquine resistance.…”

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

“…This conjugate further enhanced sensitivity of this strain to chloroquine. It is noteworthy that modulating PfCRT levels in 7G8, another chloroquine-resistant strain, also resulted in a similar increase in chloroquine sensitivity (41).…”