Artemisinin and its derivatives (ARTs) are the most effective and recommended treatment for uncomplicated Plasmodium falciparum malaria. However, resistance has emerged to ARTs in South-East Asia and more recently in Africa. This study investigated specific biochemical changes associated with PfKelch13-mediated artemisinin resistance in P. falciparum parasites using an integrative multi-omics approach.
Metabolomics analysis revealed that ART resistant parasites have increased abundance of key antioxidant molecules such as glutathione (GSH) and its precursor gamma-glutamyl cysteine. Elevated GSH levels are thought to increase the parasite’s antioxidant capacity and its ability to manage ART-induced oxidative stress. We further analysed the role of GSH in ART resistance by showing that pre-incubation of ART-sensitive isolates with N-acetyl cysteine, a GSH precursor, reduced ART activity in a short-pulse assay (11.4% survival to 26.5%, p< 0.05) to a level comparable to the resistant isolates (25.3% survival), confirming thiol levels to play an essential role in ART resistance.
Peptidomics analysis of ART resistant parasites showed lower abundance of several haemoglobin-derived endogenous peptides compared to the sensitive isolate. Using activity based probes, we showed that ART resistant parasites have significantly lower activity of cysteine proteases involved in haemoglobin digestion, namely the falcipains. Haemoglobin digestion within the parasite releases free haem as a by-product, which is required for ART activation. Using drug stability experiments, we demonstrated ARTs to be two-fold more stable in early-trophozoite-stage resistant parasites compared to sensitive, suggesting reduced drug activation may contribute to resistance.
In conclusion, using unbiased metabolomics-based approaches we have identified glutathione metabolism and haemoglobin digestion as contributing factors to the mechanism of PfKelch13-mediated artemisinin resistance.