Resistance against the frontline antimalarial, artemisinin, is predominantly driven by mutations in the Kelch 13 (K13) protein. K13 plays a crucial role in the formation of a double-membraned invagination termed the cytostome; the apparatus which facilitates the uptake of haemoglobin from the host red blood cell into the Plasmodium parasite. As the parasite progresses through its asexual life cycle, it forms new cytostomes to increase the rate of haemoglobin uptake. Haemoglobin digestion supplies essential amino acids and creates space within the red blood cell to facilitate parasite growth, while also releasing haem-iron as a waste byproduct which is required for the activation of artemisinin. Parasites harbouring K13 mutations exhibit a slower feeding rate, characterised by a reduction in haem biosynthesis and delayed growth. Consequently, this reduced level of haem leads to less artemisinin activation, resulting in less parasite death. However, the exact mechanism by which K13 mutations result in impaired parasite feeding remains unclear.
We propose that mutation of K13 reduces its stability and abundance, impacting the formation and maintenance of new cytostomes and thus parasite feeding. By employing immunofluorescence assays alongside ultrastructure expansion microscopy (u‑ExM) combined with super-resolution microscopy techniques, we resolved K13 as ring-shaped structures approximately 160 nm in diameter, which localise to the periphery of the parasite surrounding the neck of the cytostome. We performed live cell and u-ExM at various stages throughout the asexual life cycle and the morphology and number of K13 rings were compared between mutant and wild-type (WT) parasites. Our findings suggest that K13 mutant parasites form new K13 rings at a slower rate than WT parasites, and in some cases, the cytostomes in K13 mutants displayed altered morphologies. These findings provide new insights into how K13 mutations could reduce haemoglobin uptake, potentially linking this defect to artemisinin resistance.