Effect of K13 mutation and drug exposure on PfEMP1 expression and export. (A) Schematic of parasite drug exposure. (B) RNA sequence analyses of PfEMP1 expression in 0- to 3-hour PfNF54K13WT and PfNF54K13C580Y rings exposed to 4 nM DHA (positive, red) or mock treated (negative, blue) for 6 hours. Intensity units are as follows: y-axis, PfEMP1 gene id; x-axis, black arrows indicate a major transcript seen in WT parasites. The main PfEMP1 transcript 1200600 (var2csa) was expressed in both wild-type and mutant parasites +/−DHA. Second and third K13WT transcripts (0425800, 0712300) were also expressed in DHA. In C580Y, 0425800, 0400400, 0600200, 0800200, and 0300300 were expressed +/−DHA. PfEMP1 transcript levels were sustained an hour after DHA was washed out (supplemental Figure 5A-B), and parasites successfully matured through subsequent stages of the asexual life cycle (not shown). (C-D) DHA 4 nM potently inhibits PfEMP1 export (green; detected by ATS antibodies in IFA) to the red cell in artemisinin-sensitive PfNF54K13WT but not resistant PfNF54K13C580Y (C). Quantitative analyses of 400 optical sections through 30 infected red cells (D). (E-F) Quantitative analyses of sensitivity of PfEMP1 export (green) to DHA in the artemisinin-sensitive clinical strain ANL-1 but not its resistant clinical counterpart ANL-2 (C580Y). Western blots reveal equivalent PfEMP1 protein levels with or without DHA. For all samples, pixel intensity at 100% exceeded 6.5 × 106 (AU) (panels C-F). Experimental replicates: n = 2 (panels A-B); n = 2 (panels C-F). PfExp2 (Pf-exported protein 2, red), a parasitophorous vacuolar membrane maker, was used to stain the parasite periphery (panels C,E). Scale bars, 5 µm. Imaged with a 100×, NA-1.4 objective on an Olympus IX inverted fluorescence microscope using DeltaVision Deconvolution microscopy software.25 P, parasite; R red cell.
