The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation

Abstract

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13. Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent. IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum. In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

“…Of note, K13 colocalized with AP-2μ, although this protein was not identified by affinity purification of K13 in either the Birnbaum study or our own. A separate study also did not identify K13 upon affinity purification of HA-tagged AP-2μ protein [60]. We cannot yet explain the discrepancy between the data we obtained by co-IP with our K13 mAb and that generated using the DiQ-BioID method, although we note that DiQ-BioID will preferentially identify proteins in the same compartment as K13 rather than proteins physically bound to K13.…”

Artemisinin susceptibility in the malaria parasitePlasmodium falciparum: propellers, adaptor proteins and the need for cellular healing

“…Just as post-artemisinin slow clearance in vivo does not impart full resistance to typical treatment regimens, the reported estimates of fractional in vitro survival of K13 mutant parasites in the RSA vary dramatically between 2% and 45%, with laboratory protocols varying in the duration of drug exposure (Straimer et al . 2015 ; Henrici, van Schalkwyk and Sutherland 2019a , 2020 ). Adequate standardisation to allow robust inter-laboratory comparisons is needed and requires widely available standard parasite lines that can be shared among teams (see the section 'Detailed studies of K13 mutants in vitro ').…”

“…While AP-2 function has only recently been studied in apicomplexans (Henrici et al . 2020 ), the clathrin heavy chain and AP-1 have been localised to post-Golgi secretory structures in P. falciparum and the related Toxoplasma gondii (Ngô et al . 2003 ; Pieperhoff et al .…”

“… 2020 ; Henrici et al . 2020 ). AP-3 and AP-4 have not yet been studied or implicated in drug responses in apicomplexans, though are likely to participate in post-Golgi transport as in other eukaryotes.…”

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility

“…Two recent publications attributed this decreased susceptibility to a hemoglobin import defect that presumably renders parasites less able to activate ART 14,15 . This hypothesis may help explain how mutants in other components of the endocytic machinery, such as coronin 16 and clathrin adaptors [17][18][19] , and in hemoglobin digestion 20 also confer decreased susceptibility to ART. Previous studies have also postulated that the altered ART susceptibility of K13 C580Y parasites involves changes in the unfolded protein response, autophagy, and PI3K activity, which may contribute to cellular survival following protein alkylation [21][22][23][24] .…”