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

Decreased susceptibilities of the human malaria parasite Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of PfKelch13, a homologue of the redox sensor Keap1 and other vertebrate BTB-Kelch proteins. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of PfKelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of PfKelch13 resulted in ring-stage survival rates around 40%. While the loss of the thiol group of C469 or of the potential disulfide bond between residues C580 and C532 had no effect on the artemisinin susceptibility, the thiol group of C473 could not be replaced. Furthermore, we detected two different forms of PfKelch13 with distinct electrophoretic mobilities around 85 and 95 kDa, suggesting an unidentified post-translational modification. We also established a protocol for the production of recombinant PfKelch13 and produced an antibody against the protein. Recombinant PfKelch13 adopted alternative oligomeric states and only two of its seven cysteine residues, C469 and C473, reacted with Ellman’s reagent. While common field mutations resulted in misfolded and completely insoluble recombinant PfKelch13, cysteine-to-serine replacements had no effect on the solubility except for residue C473. In summary, in contrast to residues C469, C532, and C580, the surface-exposed thiol group of residue C473 appears to be essential. However, not the redox properties but impaired folding of PfKelch13, resulting in a decreased PfKelch13 abundance, alters the artemisinin susceptibility and is the central parameter for mutant selection. We addressed the relevance of the redox and folding properties of the Keap1 homologue PfKelch13 for the artemisinin susceptibility of the malaria parasite Plasmodium falciparum. [Display omitted] •Establishment of a titratable glmS ribozyme-based knockdown system for PfKELCH13.•Knockdowns but not Cys-to-Ser mutations result in up to 40% ring-stage survival.•The thiol group of C473 but not of C469, C580 or C532 of PfKelch13 is essential.•Detection of a 10 kDa post-translational modification of PfKelch13.•Establishment of a production and purification protocol for recombinant PfKelch13.•Common field mutations destabilize PfKelch13 and affect the folding properties.