MCA2 is part of the K13 compartment and its truncation reduces ART susceptibility.

(A) Schematic representation and nomenclature of MCA2 fusion proteins (not to scale) expressed in the parasite by modification of the endogenous gene. GFP in green, 3xHA in red and metacaspase domain in grey. Amino acid positions are indicated by numbers. (B) Localisation of MCA2Y1344STOP-GFP by live-cell microscopy across the intra-erythrocytic development cycle. Nuclei were stained with DAPI. Scale bar, 5 μm. (C) Live cell microscopy images of parasites expressing the truncated MCA2Y1344STOP-GFP fusion protein with an episomally expressed mCherry-K13 fusion protein. Foci from rings and trophozoite stage parasites were categorized into ‘overlap’ (black), ‘partial overlap’ (dark grey) and ‘no overlap’ (light grey) and shown as frequencies in the pie chart (n = 46 cells were scored from a total of three independent experiments). Scale bar, 5 μm. (D) Live cell microscopy images of parasites expressing the truncated MCA2Y1344STOP-GFP fusion protein with the IMC marker protein PhIL1mCherry. Zoom, enlarged region (factor 400%). Nuclei were stained with Hoechst-33342; scale bars, 5 μm and for zoom 1 μm. (E) Relative growth of synchronised MCA2Y1344STOP compared with 3D7 wild type parasites after two growth cycles. Each dot shows one of four independent experiments. P-values determined by one-sample t-test. (F) Parasite survival rate (% survival compared with control without DHA) 66 h after 6 h DHA treatment in standard RSA. Two (3D7) or three (MCA2Y1344STOP) independent experiments, P-value determined by unpaired t-test. Green dashed line indicates 1% ART resistance cut-off [26]; 3419–3828 (mean 3632) erythrocytes for control and 10364–11734 (mean 11254) cells for DHA treated samples were counted.

Schmidt S, Wichers-Misterek JS, Behrens HM, Birnbaum J, Henshall IG, Dröge J, Jonscher E, Flemming S, Castro-Peña C, Mesén-Ramírez P, Spielmann T. The Kelch13 compartment contains highly divergent vesicle trafficking proteins in malaria parasites. PLoS Pathog. 2023 19(12):e1011814.