American Society of Hematology

Figure 5.

$Figure 5. K13C580Y, the major mutation of artemisinin resistance, amplifies PI3P tubovesicles, propagating them throughout the parasite and into the red cell. (A-B) Cryo-IEM of artemisinin-sensitive PfNF54K13WT (A) and artemisinin-resistant PfNF54K13C580Y (B) late trophozoite/schizont parasites probed for PI3P (10 nm gold). (C) Region of ER tubules/vesicles. Scale bars, 100 nm (bar sizes differ on basis of magnification). (D) Quantitation of gold particles associated per parasite vacuole for indicated numbers of parasites for each strain. (E-F) PfNF54K13WT and PfNF54K13C580Y ER dually probed for PI3P (6 nm gold) and the secretory ER marker BiP (15 nm gold) (E) or PI3P (6 nm gold) and K13 (15 nm gold) (F). Boxes in left-hand panels are shown magnified in the right-hand panels. Black arrows indicate luminal (space) PI3P; double yellow arrows indicate PI3P vesicular clusters adjacent to K13; red arrowheads indicate small PI3P clusters, and double red arrowheads indicate large PI3P clusters devoid of K13. Scale bars, 150 nm. (G-H) Stereological analyses of percentage of PI3P gold particle density in PfNF54K13WT or PfNF54K13C580Y with each strain normalized to itself (G) and comparative fraction of PI3P-gold particles in each strain (H), accounting for threefold increase in PfNF54K13C580Y in relation to PfNF54K13WT (total number of gold particles in PfNF54K13WT set to 100). (I) RNA sequence analyses for K13C580Y in PfNF54K13WT and PfNF54K13C580Y. Y-axis indicates arbitrary intensity units. (J) Western blots showing levels of K13, PfFKBP protein (a parasite cytosolic protein that serves as a loading control), and associated ratios, indicating no significant difference in levels of K13 protein expression in PfNF54K13C580Y in comparison with PfNF54K13WT. (K-L) PfNF54K13C580Y-infected red cells probed for PI3P (10 nm gold). Red arrows show PI3P at the parasite plasma and vacuolar membranes, in vesicles in the host red cell (scale bar, 500 nm) (K) and associated with “Maurer’s clefts,” intermediates in export to the red cell membrane (scale bar, 100 nm) (L). PI3P is not detected in the host in PfNF54K13WT-infected red cells. Experimental replicates, n = 2. IEM imaged in a Philips CM120 electron microscope (Eindhoven, The Netherlands) under 80 kV. PM, plasma membrane; WT, wild-type.$

K13C580Y, the major mutation of artemisinin resistance, amplifies PI3P tubovesicles, propagating them throughout the parasite and into the red cell. (A-B) Cryo-IEM of artemisinin-sensitive PfNF54K13WT (A) and artemisinin-resistant PfNF54K13C580Y (B) late trophozoite/schizont parasites probed for PI3P (10 nm gold). (C) Region of ER tubules/vesicles. Scale bars, 100 nm (bar sizes differ on basis of magnification). (D) Quantitation of gold particles associated per parasite vacuole for indicated numbers of parasites for each strain. (E-F) PfNF54K13WT and PfNF54K13C580Y ER dually probed for PI3P (6 nm gold) and the secretory ER marker BiP (15 nm gold) (E) or PI3P (6 nm gold) and K13 (15 nm gold) (F). Boxes in left-hand panels are shown magnified in the right-hand panels. Black arrows indicate luminal (space) PI3P; double yellow arrows indicate PI3P vesicular clusters adjacent to K13; red arrowheads indicate small PI3P clusters, and double red arrowheads indicate large PI3P clusters devoid of K13. Scale bars, 150 nm. (G-H) Stereological analyses of percentage of PI3P gold particle density in PfNF54K13WT or PfNF54K13C580Y with each strain normalized to itself (G) and comparative fraction of PI3P-gold particles in each strain (H), accounting for threefold increase in PfNF54K13C580Y in relation to PfNF54K13WT (total number of gold particles in PfNF54K13WT set to 100). (I) RNA sequence analyses for K13C580Y in PfNF54K13WT and PfNF54K13C580Y. Y-axis indicates arbitrary intensity units. (J) Western blots showing levels of K13, PfFKBP protein (a parasite cytosolic protein that serves as a loading control), and associated ratios, indicating no significant difference in levels of K13 protein expression in PfNF54K13C580Y in comparison with PfNF54K13WT. (K-L) PfNF54K13C580Y-infected red cells probed for PI3P (10 nm gold). Red arrows show PI3P at the parasite plasma and vacuolar membranes, in vesicles in the host red cell (scale bar, 500 nm) (K) and associated with “Maurer’s clefts,” intermediates in export to the red cell membrane (scale bar, 100 nm) (L). PI3P is not detected in the host in PfNF54K13WT-infected red cells. Experimental replicates, n = 2. IEM imaged in a Philips CM120 electron microscope (Eindhoven, The Netherlands) under 80 kV. PM, plasma membrane; WT, wild-type.

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