The Kelch13 compartment contains highly divergent vesicle trafficking proteins in malaria parasites

Single amino acid changes in the parasite protein Kelch13 (K13) result in reduced susceptibility of P. falciparum parasites to artemisinin and its derivatives (ART). Recent work indicated that K13 and other proteins co-localising with K13 (K13 compartment proteins) are involved in the endocytic uptake of host cell cytosol (HCCU) and that a reduction in HCCU results in reduced susceptibility to ART. HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins among the few proteins so far functionally linked to this process. Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site. Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these and previously identified proteins showed that vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance or endocytosis (or both), comprising one group of K13 compartment proteins. While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process. Another group of K13 compartment proteins did not influence endocytosis or ART susceptibility and lacked detectable vesicle trafficking domains. We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion. Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of endocytosis in malaria parasites. Malaria parasites growing in red blood cells take up host cell cytosol by a poorly understood endocytic mechanism. Kelch13, the protein mutated in parasites with lowered susceptibility to Artemisinin-drugs (ART), is involved in this process and the rate of endocytosis was linked to ART susceptibility. To better understand the endocytosis process we here screened further candidates identified in previous searches for proteins in proximity to Kelch13 (the K13 compartment) and found 4 additional K13 compartment proteins. Two of these had a function in endocytosis but not in early stage parasites, the stage associated with lowered ART susceptibility. One of these proteins was a Myosin, indicating involvement of actin in endocytosis in later stage parasites. Analysing the AlphaFold structures of the here and previously confirmed K13 compartment proteins revealed many with vesicle trafficking domains in unusual configurations. This indicates that the K13 compartment is involved in a highly parasite-specific vesicle trafficking process, suggesting an unusual endocytosis mechanism. We also found one K13 compartment protein with a likely function in invasion, indicating K13 compartment proteins can have functions outside endocytosis, although for this particular protein this function may also be attributable to an additional cellular location we observed.

Automated summary

This study identified novel proteins involved in endocytosis and at the K13 compartment in malaria parasites. It also provided insights into the highly adaptive nature of endocytosis in these parasites.