Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

Abstract

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the -subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to <2% for the 3D7 parental line (P = 0.0011 for each clone). In immunoprecipitation and localisation studies of HA-tagged AP-2 we identified interacting partners including AP-2 AP-1/2 AP-2 and a kelch-domain protein encoded on chromosome 10 of P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

No Evidence of Plasmodium falciparum k13 Artemisinin Resistance-Conferring Mutations over a 24-Year Analysis in Coastal Kenya but a Near Complete Reversion to Chloroquine-Sensitive Parasites

“…Therefore, it appears to be restricted to Africa, as it has also been observed in Ghana (55). Recent evidence points to a different mutation, I592T (not detected in this population), that showed increased ring-stage survival following a dihydroartemisinin pulse (56). Further work is required to evaluate the potential role of ap2-mu mutations on the ACT response.…”

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

“…Trophozoites, which have high levels of free heme from degrading hemoglobin, are more sensitive to artemisinins than ring or liver stages, which are thought to rely predominantly on their endogenous heme biosynthetic pathways 38,76,77 . The implication of endocytic machinery in artemisinin sensitivity 18,20,21 could also be related to reduced hemoglobin uptake and digestion, which would limit or delay the availability of free heme.…”

“…A further 65 genes were significantly enriched in individual screens ( Supplementary Table 4). Because K13 resistance alleles retain function and have not been recovered by selection in culture, the absence of K13 from our loss-of-function screens was expected 12,13,17,18,20,21 .…”

“…The mechanism underlying this effect remains under scrutiny, but it is thought to involve changes in the unfolded protein response, autophagy, and PI3K activity [14][15][16][17] . Decreased ART sensitivity has also been attributed to mutations unrelated to K13, such as coronin 18 , clathrin adaptors [19][20][21] , and cysteine proteases 22 . P. falciparum strains without identified causative mutations have displayed decreased clearance as well 23,24 .…”