Abstract
Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the re-emergence of the disease. Chloroquine (CQ) and artemisinin (ART) are thought to exert their anti-malarial activity inducing cytotoxicity in the parasite by blocking heme degradation (for CQ) and increasing oxidative stress. Besides the contribution of the CQ resistance transporter (PfCRT) and the multidrug resistant gene (pfmdr), CQ resistance has also been associated with increased parasite glutathione (GSH) levels. ART resistance was recently shown to be associated with mutations in the K13-propeller protein. To analyze the role of GSH levels in CQ and ART resistance, we generated transgenic Plasmodium berghei parasites either deficient in or overexpressing the gamma-glutamylcysteine synthetase gene (pbggcs) encoding the rate-limiting enzyme in GSH biosynthesis. These lines produce either lower (pbggcs-ko) or higher (pbggcs-oe) levels of GSH than wild type parasites. In addition, GSH levels were determined in P. berghei parasites resistant to CQ and mefloquine (MQ). Increased GSH levels were detected in both, CQ and MQ resistant parasites, when compared to the parental sensitive clone. Sensitivity to CQ and ART remained unaltered in both pgggcs-ko and pbggcs-oe parasites when tested in a 4 days drug suppressive assay. However, recrudescence assays after the parasites have been exposed to a sub-lethal dose of ART showed that parasites with low levels of GSH are more sensitive to ART treatment. These results suggest that GSH levels influence Plasmodium berghei response to ART treatment.
Highlights
The development of drug resistance by Plasmodium parasites has become one of the major obstacles in the efforts to control malaria
When an animal appeared to be in pain or disease the Veterinarian or Veterinary Technologist humanly euthanized the mouse by cervical dislocation or CO2 chamber following the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals
Integration of the construct into the genome of pbggcs-oe1 and pbggcs-oe2 parasites was confirmed by chromosome blots on field inverted gel electrophoresis (FIGE) separated chromosomes (Fig 1B) and by PCR analysis (Fig 1C)
Summary
The development of drug resistance by Plasmodium parasites has become one of the major obstacles in the efforts to control malaria. A marked increase in GSH levels and the activity and expression of GSH-related enzymes has been reported in P. berghei and P. falciparum lines resistant to CQ [7,8,9,10,11]. The increased levels of intracellular ROS, and the antimalarial activity of ART require the uptake and degradation of hemoglobin by Plasmodium parasites [15]. CQ sensitive P. falciparum parasites are more susceptible to BSO treatment than CQ resistant parasites [9, 20] These results support the association between increased GSH levels and CQ resistance in Plasmodium. To further investigate the potential contribution of GSH to Plasmodium drug resistance, the development of genetically engineered P. berghei parasites overexpressing the pbggcs gene and displaying high levels of GSH is reported . This study provides new insights into the GSH involvement in the mechanism(s) of action of ART
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