Figure 2. Inhibiting isoprenoid precursor biosynthesis prevents apicoplast inheritance by daughter parasites.
(A) Schematic summary of delayed mevalonate rescue of fosmidomycin (FOS) treatment. PfMev parasites were synchronized with 5% D-sorbitol and cultured in 10 µM FOS (washed out after 48 hr in second-cycle rings) without or with addition of 50 µM DL-mevalonate (Mev) at 0, 30, 34, or 38 hr after synchronization. Clonal parasites from all growth conditions were isolated at 60 hr post-synchronization by limiting dilution and growth in 50 µM Mev. (B) Parasite growth was monitored for 6 days by flow cytometry using acridine orange staining (FOS = treated only with FOS, UT = untreated, 0–38 hr time delay of Mev addition after synchronization and initiation of FOS treatment). (C) Bright-field (BF) and fluorescence microscopy images of representative live clonal parasites with disrupted apicoplast (if observed) isolated after 60 hr of growth under the conditions described in panel A. Images of all clones are shown in Figure 2—figure supplements 1–4. Parasite nuclei were visualized using 1 µg/mL Hoechst 33342. To the right of each clonal image panel is a gel image showing the result of PCR analysis to amplify a (Nu) nuclear (PPS, PF3D7_0202700) and (Api) apicoplast (SufB, PF3D7_API04700) gene and a growth assay to monitor the ability of each indicated clone to grow in the presence or absence of 50 µM Mev. Data points are the average± SD of three biological replicates and were normalized to the parasitemia on day 3 of growth in +Mev conditions. (D) Graphical representation of the number of clones isolated under each growth condition and the clonal percentage with an intact or disrupted apicoplast (determined by microscope analysis of ACPL-GFP signal and genomic PCR).