Glutathione metabolism of Plasmodium falciparum

[摘要] Apicomplexan parasites of the genus Plasmodium are the causative agent of malaria, one of the most prevalent infectious diseases worldwide. Five different Plasmodium species can cause malaria in humans, leading to a total of approximately 500 million cases each year and of these, P. falciparum causes the most deadly form of the disease and is responsible for more than 1 million deaths annually. A major problem in the global fight against malaria is the widespread resistance of the parasites against the currently available drugs. It is of great importance to identify new drug target as well as to understand the mechanisms that lead to drug resistance in the first instance in order to potentially reverse the resistant phenotypes and to avoid the development of resistance in the future.The tripeptide glutathione (GSH) or γ-glutamylcysteinyl-glycine is the most abundant low molecular weight thiol in most eukaryotic organisms and serves a number of important functions as sulfhydryl-buffer, cofactor for enzymes and for the detoxification of xenobiotics and drugs. GSH is an important component of the antioxidant machinery and because malaria parasites live in an environment rich in iron and oxygen and thus increased oxidative stress, they depend on functional antioxidant systems. The biosynthesis pathway for GSH, consisting of γ-glutamylcysteine synthetase (γGCS) and glutathione synthetase (GS) is present in malaria parasites as well as in their host cells. Previous studies have shown that depletion of GSH has an antimalarial effect, but it remained unclear whether parasites were killed directly or died because their host cell could not survive the depletion of GSH. To address this question, the knockout of both genes encoding the enzymes of the GSH biosynthesis pathway in P. falciparum was attempted. While both gene loci were targeted by control constructs, the knockout of either pfγgcs or pfgs was impossible, indicating both genes are essential for parasite survival in the erythrocytic stages. To analyse the localization of γGCS and GS, GFP-tagged recombinant fusion proteins were expressed in the parasites and showed that GSH biosynthesis is cytosolic.Apart form its other functions GSH has previously been suggested to be involved in resistance to the antimalarial drug chloroquine (CQ). CQ was for a long time the first line antimalarial drug due to its high efficiency, low cost and low toxicity, but is now widely inefficient in the treatment of the disease. CQ resistance is associated with mutations in the CQ resistance transporter (PfCRT), a membrane protein of the digestive vacuole that allows the efflux of the drug form its site of action. However, PfCRT mutations alone cannot explain the full array of phenotypes found in resistant parasites. GSH is able to degrade heme, the target of CQ, in vitro and it has been suggested that elevated GSH levels contribute to CQ resistance. However, analyses of isogenic parasite lines bearing different forms of PfCRT in this study revealed lower GSH levels and higher susceptibility to inhibition of GSH biosynthesis in the CQ resistant lines. These changes did not correlate with changes in the expression of enzymes involved in the de novo biosynthesis or consumption of GSH. However, the cellular accumulation ratio for CQ indicated a decrease of free heme in the resistant parasites. Mutant forms of PfCRT expressed in oocytes of Xenopus laevis were able to transport GSH, while the sensitive wild-type form did not transport the tripeptide. The findings of this study suggest that in parasites bearing mutant PfCRT, GSH is transported into the digestive vacuole where it is able to contribute to resistance by degrading heme, before the tripeptide itself is degraded by peptidases inside the vacuole, consistent with the overall reduction of GSH levels in CQ resistant parasites.

[发布机构] University:University of Glasgow;Department:Institute of Infection Immunity and Inflammation

[关键词] Plasmodium falciparum, glutathione, gamma-glutamylcysteine synthetase, glutathione synthetase, chloroquine resistance, chloroquine resistance transporter