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

Abstract

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.

Identification of a Rabenosyn-5 like protein and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites

“…In contrast to the findings with the proteins studied in this work, the initial phase of hemoglobin endocytosis at the PPM seems to show strong differences to that in model organisms [3,15].…”

“…In contrast to the findings with the proteins studied in this work, the initial phase of hemoglobin endocytosis at the PPM seems to show strong differences to that in model organisms [3, 15]. A seemingly permanent structure termed the cytostome, an invagination of the PPM (in Plasmodium parasites also the PVM) containing a number of proteins in a ring structure around the neck of the invagination (the Kelch13 compartment) was implicated in this step in malaria and T. gondii parasites [3, 4, 8, 16–18].…”

“…Section of infected RBC showing ’initiation’ phase of endocytosis (with strongly parasite-specific characteristics [3, 15] feeding into an endosomal transport pathway that depends on a functional complex of VPS45, Rbsn5 and Rab5b (enlargement box) which indicates conserved elements playing a role in this phase of HCCU. FV, food vacuole; C, cytostome; E, endosome; PI3P, phosphatidylinositol-3-phosphate; RBC, red blood cell; PVM, parasitophorous vacuolar membrane; PPM (parasite plasma membrane); yellow arrow, direction of the pathway.…”

“…Currently, there are two categories of proteins affecting HCCU, those that act early when endocytic containers are generated and do not lead to HCC-filled vesicles when inactivated [3,15] and those that presumably act later and therefore lead to HCC-filled intermediates when inactivated ( [13]; this work). However, if these are indeed serial steps in the same pathway has not been tested.…”

“…Morphological studies implicated the cytostome, an invagination of the PPM and the surrounding PVM, as the site where HCCU is initiated at the PPM [9][10][11][12]. However, the mechanism of how endocytic structures are formed remains unclear [8], and only recently functional data directly implicated specific parasite proteins in this process [3,[13][14][15]. Most of these proteins are located at an electron dense collar surrounding the cytostomal neck and are involved in the early phase of HCCU (i.e.…”