Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

Abstract

Decreased susceptibilities of Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of Kelch13, which is the homologue of the redox sensor Keap1 in vertebrates. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of Kelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of Kelch13 resulted in ring-stage survival rates around 40%. While the loss of a potential disulfide bond between residues C580 and C532 had no effect on the artemisinin suceptibility, the thiol group of C473 could not be replaced. We also established a protocol for the production of recombinant Kelch13. In contrast to cysteine-to-serine replacements, common field mutations resulted in misfolded and insoluble protein. In summary, not the redox properties but impaired folding of Kelch13, resulting in a decreased Kelch13 abundance, is the central parameter for mutant selection.

Impact of different mutations on Kelch13 protein levels, ART resistance and fitness cost inPlasmodium falciparumparasites

“…ART-resistance, measured by RSA, decreased as the rapalog concentration and hence K13 knock sideways decreased, confirming that RSA-survival is a function of K13 levels in the parasites (Figure 3C). These results confirm observations made on parasites in which the K13 abundance was titrated on mRNA level through the glmS system (19). To confirm that this was due to a reduction in endocytosis, we performed a second titration using KIC7, a K13-compartment protein, which was also knocked aside using the same decreased rapalog concentrations as for K13 (Figure 3D).…”

“…We found that the amount of K13 and KIC7 at the K13 compartment in the cell determine the level of in vitro resistance, confirming that the endocytic process is important for ART resistance and suggesting that the amount of protein directly influences the rate of endocytosis and level of ART susceptibility. The same effect on resistance was observed in a k13 mRNA titration recently (19) and a role of K13 protein abundance rather than a specific functional change in resistance is also supported by the finding that increasing levels of K13 C580Y reverts resistant parasite back to sensitive (10). The finding that KIC7 titration had a similar effect strengthens this conclusion and further supports that resistance is due to altered endocytosis levels.…”

“…R539T and k13 C580Y contain reduced amounts of K13 (10,(16)(17)(18). Artificially modulating K13 levels indicated that the reduced K13 levels lead to resistance (10,16,19). It is currently unclear whether other k13 mutations, like R561H, confer resistance through the same mechanism or affect the functionality of K13.…”