Glutathione Transport: A New Role for PfCRT in Chloroquine Resistance

Abstract

Aims: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. Results: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. Innovation: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. Conclusion: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV. Antioxid. Redox Signal. 19,[683][684][685][686][687][688][689][690][691][692][693][694][695]

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

“…Given that these changes have been associated with impaired fitness in mutant PfCRT parasites expressing the CQ resistance-conferring Dd2 or 7G8 haplotypes [41], our results raise an obvious question: does Cam734 PfCRT impart a metabolic compensatory mechanism that allows these parasites to circumvent the normally deleterious effects of altered Hb digestion? Previous heterologous expression studies using Xenopus laevis oocytes have suggested that mutant PfCRT isoforms might selectively confer transport of the tripeptide glutathione [57], which was earlier proposed to facilitate the degradation of reactive heme and reduce heme-mediated toxicity [36,83]. However, we saw no significant differences in glutathione or any other redox-associated metabolites (see S4 Fig and S6 Table) between isogenic lines encoding Cam734 or Dd2 PfCRT, suggesting that major redox-related metabolic changes are unlikely to account for the improved fitness associated with the Cam734 pfcrt allele.…”

Characterization of the Chloroquine Resistance Transporter Homologue in Toxoplasma gondii

“…Knockdown of chloroquine-resistant PfCRT protein levels to approximately 65% of the wild-type levels lessened parasite resistance to chloroquine and led to an alkalinization of the DV (30), suggesting a possible role for CRT in acidification of this compartment. Plant chloroquine-like transporters function in transporting glutathione, and a recent study implicates chloroquine-resistant PfCRT (but not chloroquine-sensitive PfCRT) in glutathione transport into the DV (10,44).…”

“…Another possibility is that TgCRT, like plant chloroquine-like transporters and chloroquine resistance alleles of PfCRT (10,44), has a role in transporting glutathione. Dysregulation of glutathione functions in the VAC may then lead to the swelling of this organelle.…”

Inhibition of Glutathione Biosynthesis Sensitizes Plasmodium berghei to Antifolates

“…GSH has been implicated in modulating antimalarial sensitivity (17,(26)(27)(28). In order to evaluate this notion, the ␥-gcs-disrupted Pb-GFP (⌬gcs) strain was constructed by integrating linearized pL0017-⌬gcs (Fig.…”