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. 2020 Feb 25;11(1):e02918-19. doi: 10.1128/mBio.02918-19

FIG 2.

FIG 2

AP-2μ is required for asexual replication and schizont maturation. (A) Schematic for the integration of loxP recombination elements into the endogenous pfap2μ locus of a parasite line constitutively expressing a split-Cre recombinase (43). The addition of rap initiates Cre dimerization and excision of the loxP-flanked (floxed) region of pfap2μ on chromosome 12. (B) PCR confirmation of rap-induced excision of floxed region by PCR using the primers P4 and P5 (see panel A). (C) Western blot confirmation that excision of floxed pfap2μ causes a loss of AP-2μ-3xHA protein (within 24 h) but has no effect on levels of CDC48 protein. Molecular weight is presented in kDa. (D) Parasite multiplication in the 3D7-AP-2μ-floxed-3xHA line across 2.5 cell cycles, with or without rap induction of Cre. The mean parasitemia (normalized to 0.25% starting parasitemia) with the standard error is shown at each time point. Each data point represents the average of at least three biological replicates (different cultures, different days). (E) PCR confirmation of rap-mediated pfap2μ excision in 3D7-AP-2μ-floxed-3xHA parasites transfected with an episome encoding cam-AP-2μ-GFP. The construction of this complementation plasmid is described in Fig. S5. (F) Western blot confirmation that excision of chromosomal pfap2μ from 3D7-AP-2μ-floxed-3xHA/cam-AP-2μ-GFP parasites causes a loss of AP-2μ-3xHA protein, but it does not prevent episomal expression of AP-2μ-GFP. (G) Parasite multiplication in the 3D7-AP-2μ-floxed-3xHA/cam-AP-2μ-GFP line across 2.5 cell cycles, with or without induction of Cre by rap. Means and standard errors are shown as in panel D. (H) Giemsa staining of 3D7-AP-2μ-floxed-3xHA schizonts, without rap treatment at 48 h postinfection and with rap treatment at 48 and 60 h postinfection. (I) Electron micrograph of 3D7-AP-2μ-floxed-3xHA schizonts, with or without 1-h ring-stage treatment with 10 nM rap. Micronemes at the apical end of developing merozoites are labeled with arrowheads; asterisks indicate membrane separation (see Fig. S7). FV, food (digestive) vacuole; H, hemozoin; L, lipid body; M, merozoite; R, rhoptry. Scale bar, 500 nm.