Reduced susceptibility of Plasmodium falciparum to artemisinin in vivo is associated with increased parasitaemia clearance times in treated patients. This correlates with increased survival of ring-stage trophozoites, but not later parasite stages, treated with short pulses of 700nM artemisinin in vitro, and with particular variants of the K13 kelch domain protein. These variants have markedly increased in prevalence in certain areas of SE Asia in the last decade. However, slow clearance or persistence of parasites in vivo can also be associated with variants of AP2μ and UBP1 in the absence of variant K13.
The P. falciparum orthologues of variant AP2μ and UBP1, first identified in drug-selected P. chabaudi, were generated by CRISPR-Cas9 transgenesis. Ring-stage susceptibility to brief pulses of 700nM dihydroartemisinin was assessed by flow cytometry. HA-tagged AP2μ was visualised by fluorescent microscopy throughout the intra-erythrocytic cycle in order to determine its cellular location.
Ring-stage survival following pulsed DHA treatment will be presented for each transgenic parasite clone, and compared to survival of parental lines, and of Cambodian slow clearing parasite isolates. Initial localisation experiments strongly suggest a non-canonical role for the AP2 adaptin complex in Plasmodium, as no evidence of association with clathrin, nor with endocytic vesicles, has thus far been found.
The AP2μ-associated phenotypes described will be discussed in light of similarites to K13-generated reduced susceptibility, and the possibility that K13 and the AP2 adaptin complex may be linked by trafficking events early in the intra-erythrocytic cell cycle. Implications for rational use of current anti-malarial combinations will be considered.