The emergence of artemisinin resistance in the malaria parasite, Plasmodium falciparum poses a major threat to the control and elimination of malaria. The underlying mechanism associated with artemisinin resistance is poorly understood, but point mutations in the PfKelch13 protein strongly correlate with resistance. However, using PfKelch13 genomic sequence as a molecular marker is resource-intensive and has limited sensitivity and specificity, as mutations do not always lead to resistance to artemisinin. Therefore this study aimed to identify novel quantitative biomarkers for artemisinin resistance using an integrative multi-omics approach, which combines proteomics, peptidomics and metabolomics to analyse global differences between drug-resistant and drug-sensitive parasite strains.
When applied to P. falciparum infected red blood cells, our multi-omics platform facilitated the identification of approximately ~2300 proteins, 800 putative metabolites and 971 naturally abundant peptides. Proteomics analysis from three independent artemisinin resistant lines found artemisinin resistance to be associated with decreased expression of PfKelch13 protein by approximately 2 fold. As the PfKelch13 sequence is already a genetic biomarker for artemisinin resistance, we show for the first time that the abundance of this protein is also associated with the resistance phenotype.
Metabolomics analysis from two independent artemisinin resistant lines showed significant accumulation of glutathione and a reduction in the abundance of nine metabolites, primarily involved in amino acid metabolism. Peptidomics analysis revealed lower abundance of several endogenous peptides derived from haemoglobin (HBα and HBβ) in the resistant strains. This dysregulation of specific endogenous metabolites, peptides and the PfKelch13 protein itself, provides additional insight into the mechanism of artemisinin resistance, and novel candidates which could potentially be used as quantitative biomarkers for artemisinin resistance.