Resistance to the frontline antimalarial, artemisinin, poses a critical threat to global malaria control efforts. Artemisinin resistance is predominantly driven by mutations in Kelch 13 (K13). K13 plays a crucial role in the regulation of a specialised double-membraned invagination termed the cytostome; the apparatus that facilitates haemoglobin uptake from the host red blood cell into the Plasmodium parasite. Haemoglobin digestion supplies essential amino acids and creates space within the red blood cell to facilitate parasite growth, while releasing haem-iron required for artemisinin activation. Parasites harbouring K13 mutations exhibit slower feeding rates and reduced haem levels, leading to decreased artemisinin activation and reduced parasite death. However, the precise mechanism by which K13 mutations impair parasite feeding remains unclear, limiting our ability to effectively combat this emerging threat.
We propose that K13 mutations reduce protein stability and abundance, impacting cytostome formation and maintenance, thus impacting parasite feeding. Using ultrastructure expansion microscopy coupled with super-resolution microscopy, we resolved K13 as distinctive ring structures (~160 nm diameter) localising to the parasite periphery surrounding cytostome necks. We also performed live-cell lattice light-sheet microscopy, enabling systematic analysis of cytostome dynamics across the asexual life cycle in mutant and wild-type parasites using an automated machine learning pipeline. Critically, K13 mutant parasites formed new cytostomes at significantly slower rates than wild-type controls and exhibited a 4-hour developmental delay. Quantitative analysis revealed that mutants formed 18% fewer K13 rings despite producing similar numbers of progeny merozoites. Additionally, mutants displayed aberrant cytostome morphologies, directly correlating with decreased haemoglobin uptake efficiency.
These findings fundamentally advance our understanding of artemisinin resistance by providing the first mechanistic explanation for K13-mediated feeding defects. This mechanistic insight enhances our ability to predict resistance emergence and could inform molecular surveillance strategies and partner drug selection in artemisinin combination therapies, ensuring therapeutic efficacy against resistant parasites.