Figure 7.
Figure 7. Effect of K13 mutation and drug exposure on export of VAR2CSA, cytoadherence, and models. (A) Antibodies to VAR2CSA detect ∼250 kDa in infected red cells but not in uninfected red cells by Western blots (left; see also supplemental Figure 6A). IFA and fluorescence quantitation (right) show inhibition of VAR2CSA (green) export to the red cell by 4nM DHA in artemisinin-sensitive CS2 strain, in relation to mock treatment. Red, Pf exported protein 2 marker of the parasitophorous vacuolar membrane; Hoechst (blue), parasite nucleus; dotted line, red cell periphery. (B) Parental CS2 parasites or transgenic CS2 expressing K13C580Y show similar levels of adherence to CSA; transgenic expression of K13R539T increases adherence slightly (see supplemental Figure 6C for construction of transgenic lines). (C) Potent inhibition of cytoadherence in parental CS2 by DHA was blocked by in trans expression of K13C580Y and K13R539T. Trans expression of K13WT in CS2 reduced cytoadherence, but DHA further decreased adherence (hatched box; see also supplemental Figure 6D; that HA-tagged K13WT and dominant-negative genes are functional was established by Mbengue et al in 201510). Means (±SDs) from 2 experimental replicates are shown (each with triplicate data points; P values as shown) (panels B-C). Imaged with a 100×, NA-1.4 objective on an Olympus IX inverted fluorescence microscope and quantified using DeltaVision Deconvolution microscopy software.25 (D) Western blots show 4 nM DHA does not block VAR2CSA expression in the CS2 strain (parental) or in transgenic CS2-expressing HA-tagged K13C580Y or K13R539T. Although transgenic K13WT reduces VAR2CSA levels by 50%, DHA does not cause further reduction. Experimental replicates: n = 2 (panels A-D). (E) Models integrate study findings of K13-dependent PI3P tubovesicular action in parasites sensitive and resistant to artemisinins. Key as shown. Green solid circles are apicoplasts; yellow solid circles are food vacuoles; orange solid circles are mitochondria. Scale bars, 5 µm. Exp-2, Pf exported protein 2 marker of the parasitophorous vacuolar membrane; IR, infected red cell; P, parasite; R, red cell; TriC, TCP1 ring complex; UR, uninfected red cell.

Effect of K13 mutation and drug exposure on export of VAR2CSA, cytoadherence, and models. (A) Antibodies to VAR2CSA detect ∼250 kDa in infected red cells but not in uninfected red cells by Western blots (left; see also supplemental Figure 6A). IFA and fluorescence quantitation (right) show inhibition of VAR2CSA (green) export to the red cell by 4nM DHA in artemisinin-sensitive CS2 strain, in relation to mock treatment. Red, Pf exported protein 2 marker of the parasitophorous vacuolar membrane; Hoechst (blue), parasite nucleus; dotted line, red cell periphery. (B) Parental CS2 parasites or transgenic CS2 expressing K13C580Y show similar levels of adherence to CSA; transgenic expression of K13R539T increases adherence slightly (see supplemental Figure 6C for construction of transgenic lines). (C) Potent inhibition of cytoadherence in parental CS2 by DHA was blocked by in trans expression of K13C580Y and K13R539T. Trans expression of K13WT in CS2 reduced cytoadherence, but DHA further decreased adherence (hatched box; see also supplemental Figure 6D; that HA-tagged K13WT and dominant-negative genes are functional was established by Mbengue et al in 201510 ). Means (±SDs) from 2 experimental replicates are shown (each with triplicate data points; P values as shown) (panels B-C). Imaged with a 100×, NA-1.4 objective on an Olympus IX inverted fluorescence microscope and quantified using DeltaVision Deconvolution microscopy software.25  (D) Western blots show 4 nM DHA does not block VAR2CSA expression in the CS2 strain (parental) or in transgenic CS2-expressing HA-tagged K13C580Y or K13R539T. Although transgenic K13WT reduces VAR2CSA levels by 50%, DHA does not cause further reduction. Experimental replicates: n = 2 (panels A-D). (E) Models integrate study findings of K13-dependent PI3P tubovesicular action in parasites sensitive and resistant to artemisinins. Key as shown. Green solid circles are apicoplasts; yellow solid circles are food vacuoles; orange solid circles are mitochondria. Scale bars, 5 µm. Exp-2, Pf exported protein 2 marker of the parasitophorous vacuolar membrane; IR, infected red cell; P, parasite; R, red cell; TriC, TCP1 ring complex; UR, uninfected red cell.

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