The ART of Resistance.

Recent emergence and spread of artemisinin (ART) resistance in South-east Asia caused by mutations in Kelch13 in the background of other mutations including mutations in the macroautophagy/autophagy-related protein ATG18, has intensified studies towards understanding the molecular mechanisms of resistance. The autophagy pathway of the parasite has been hypothesized to engage in resistance-associated proteostasis involving enhanced phosphatidylinositol-3-phosphate vesiculation, oxidative stress, unfolded protein response and also reduced hemoglobin endocytosis resulting from nutrient-limiting conditions, albeit without any experimental evidence. We demonstrate that ART-induced ER stress leads to upregulation of parasite autophagy through the unfolded protein response pathway. In addition, we show elevated basal expression of autophagy proteins in the ART resistant Kelch13° isolate as compared to its isogenic counterpart WT Kelch13. When autophagy is induced through starvation, the expression levels of autophagy proteins increase further in the resistant parasites. The decreased IC50 of the autophagy-specific inhibitor MRT68921 in resistant parasites relative to its isogenic counterpart establishes that autophagy is the key parasite survival mechanism in ART resistance. Additionally, upon analyses of Kelch13 mutations from various field isolates, we observe a clear association between Kelch13 (C580Y, R539T and Y493H) and ATG18 (T38I) mutations. The copresence of ATG18 with Kelch13 on parasite cytostome-like and hemoglobin-containing vesicles provides further evidence that autophagy underpins various mechanisms of ART resistance. ART, artemisinin; DHA, dihydroartemisinin; eIF2A, eukaryotic translation initiation factor subunit eIF2A; ER, endoplasmic reticulum; PtdIns3P, phosphatidylinositol-3-phosphate; ATG18, autophagy-related protein 18; ATG8, autophagy-related protein 8; SEC62, translocation protein SEC62; PK4, eIF2A kinase; WT, wild type.