The Plasmodium falciparum artemisinin resistance-associated protein Kelch 13 is required for formation of normal cytostomes

Reviewer #1 (Public Review):

In this paper, the authors investigated the localization and function of the protein Kelch 13 (K13) in Plasmodium falciparum. Mutations of K13 confer parasite resistance to artemisinin derivatives, the first-line treatment for malaria. Previous studies have shown that K13 is located at the cytostome - a structure that the parasite uses to take up hemoglobin - and that K13 mutations confer artemisinin resistance by dampening hemoglobin endocytosis. Digestion of host hemoglobin is thought to be essential for artemisinin activation through the production of haem. However, the exact function of K13 is currently unknown, and direct evidence for a role of K13 in the production of haem (and artemisinin activation) is missing.

The authors used fluorescent dextran to visualize endocytosis, and show an accumulation of dextran-positive structures colocalizing with GFP-tagged K13. They confirm the localization of K13 to cytostomes by immune-electron microscopy, showing that the protein is localized to the cytostomal collar. Using a genetic knock-sideways strategy, the authors show that mislocalization of K13 results in defects in cytostome formation and morphology, with the disappearance of the electron-dense cytostomal collar, as evidenced by serial block face scanning electron microscopy and transmission electron tomography. Finally, they provide direct evidence that K13 mislocalization leads to a decrease in haemoglobin digestion products, haem and hemozoin.

The paper is very well written and the work is very well performed, relying on a validated genetic approach and high-quality imaging. While conceptually the study does not bring many novel insights, the confirmation of K13 localization and, most importantly, the demonstration that K13 is required for cytostome formation and function constitute important pieces that consolidate the current model of artemisinin resistance. However, the exact role of K13 at the cytostomal collar remains undefined. Whether other proteins of the K13 compartments also play a role in cytostome formation remains to be determined. In addition, the study does not address whether the formation of abnormal cytostomes is also seen in artemisinin-resistant K13 mutant field isolates and is a general mechanism underlying resistance to artemisinin.

Reviewer #2 (Public Review):

Summary of major findings:
The manuscript "The Plasmodium falciparum artemisinin resistance-associated protein Kelch 13 is required for formation of normal cytostomes" authored by Tutor et al. provides evidence that Kelch13 is necessary for the formation and maintenance of cytostomes. The group provides compelling evidence using multiple state-of-the-art microscopy imaging techniques to demonstrate that when Kelch13 is mislocalized to the nucleus, cytostomes are decreased, cytostome morphology is aberrant, and there are decreased levels of heme within the parasite.

Impact of the study:
Mutations in Kelch13 have been associated with artemisinin resistance. The biological function of Kelch13 has been a question of great interest. Kelch13 was shown to associate with proteins in the endocytic machinery although not with clathrin. It was previously shown that Kelch13 mutants have decreased levels of hemoglobin digestion-derived peptides, decreased Kelch13 protein (although levels are not decreased at all asexual stages), and decreased heme. Here, the authors show that when Kelch13 is mislocalized, there are decreased numbers of properly-formed cytostomes that lead to decreased heme within parasites. Although not formally demonstrated, it is thus possible that there is decreased subsequent heme-mediated activation of artemisinin, which would explain the connection between Kelch13 and artemisinin resistance.

**Reviewer #3 (Public Review):
**
Tutor et al. present their work on Kelch13/K13 from Plasmodium falciparum, the causative agent of malaria. This protein is involved in resistance against artemisinin (ART), one of the most commonly used drugs to treat malaria. Despite having identified the mutation in K13 that leads to resistance to ART, the exact molecular mechanism, function of K13, and impact of the K13 mutations still need to be elucidated. This is where the authors step in to investigate the relationship between endocytosis and K13, as well as the impact of depleting the protein using knock-sideway (KS). Using light microscopy, the authors demonstrate how K13-YFP forms a pore associated with fluorescently labeled dextran, which is taken up into tubules that move toward the digestive vacuole. This tubule formation is not sensitive to jasplakinolide (JAS) treatment. Using electron microscopy, they show that K13 is localized at the dark contrast border of the cytostome, and knocking down K13 leads to the disruption of the cytostome structure. Upon removal of K13, the structure changes, and the opening enlarges. The impact of KS induction on the cytostome was quantified using TEM and tomography. The authors also provide reconstructions of the cytostome in both induced and non-induced parasites. Finally, they measure the impact of KS on haem degradation. These data provide clear information on the function of K13 in cytostome formation and the implication of this structure in endocytosis for Plasmodium falciparum.

The conclusions of this paper are well supported by the data, but some data analysis should be clarified and extended, and some complementary experiments would further strengthen the authors' claims.