Decreased K13 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance

Abstract

Highlights d K13 artemisinin resistance-conferring mutations lead to reduced K13 abundance d K13 is localized to cytostome-like structures at the parasite periphery d Reduced K13 abundance impairs hemoglobin catabolism and lessens artemisinin activation d Reduced artemisinin activation limits cell damage, permitting parasite survival

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

“…These data confirm that mutant K13 does not confer resistance in a dominant-negative manner and suggest that overexpression of the mutant protein mostly reverts K13 mutant parasites to DHA sensitivity. Our results agree with recently published evidence that the RSA 0-3h phenotype inversely correlates with K13 abundance and that K13 levels are reduced in mutant parasites relative to WT [27,28].…”

“…We also obtained evidence of K13 localizing near cytostomes, sites where the plasma membrane invaginates to deliver endocytosed hemoglobin to the parasite DV. These results extend prior observations of K13 associating with the ER or vesicular structures as well as sites of cytostomal formation, as defined using GFP-tagged endogenous K13 or polyclonal K13-specific antibodies [25,27,28,35]. Evidence of K13 localizing to cytostomes was also obtained recently using correlative light and electron microscopy [27], a highly-specialized technique that was unavailable for our study.…”

“…Increased overall expression of mutant K13 via co-expression of the endogenous protein and second mutant copy in trans thus presumably ablates resistance by compensating for a loss of function in the endogenous locus (Fig 1F). In broad agreement with these results, a recent study using K13 conditional knock-sideways parasites showed that mislocalization of K13 can lead to resistance [28]. Further studies will be required to assess whether K13 mutations can differ in their impact on protein stability and activity, how this varies between strains, and to what extent these effects correlate with ART resistance.…”

“…Mechanistic studies have led to several proposals for how mutant K13 might counter ARTmediated cellular toxicity. These proposals include lowering the levels of the heme activator of ART including via reduced hemoglobin endocytosis in rings [27][28][29][30], upregulating endoplasmic reticulum (ER) stress-response pathways [31], reducing the levels of ubiquitinated proteins [32], promoting translational arrest via differential phosphorylation of the translation initiation factor eIF2α [33], or increasing levels of the phospholipid phosphatidylinositol-3phosphate (PI3P) [34,35]. To gain additional insight into the biology of this protein, we raised K13-specific monoclonal antibodies (mAbs) and used these to interrogate this protein's subcellular localization in DHA-exposed or vehicle-treated asexual blood-stage parasites.…”

Exposure to artemisinin at the trophozoite stage increases sexual conversion rates in the malaria parasite Plasmodium falciparum

“…ART resistance is associated with mutations in the PfKelch13 protein (Ariey et al, 2014) that prevent 105 haemoglobin degradation in early ring-stage parasites. This in turn prevents ART activation, resulting in resistance of early rings to the drug (Birnbaum et al, 2020;Yang et al, 2019). Nowadays, ART resistance is frequently accompanied by simultaneous resistance to partner drugs such as mefloquine, piperaquine or amodiaquine, resulting in high rates of treatment failure and limiting treatment 110 options (Mairet-Khedim et al, 2020;Phyo et al, 2016;van der Pluijm et al, 2019).…”