Figure 6. Polyprenyl synthase (PPS) (PF3D7_0202700) is essential for parasite viability and apicoplast function.
(A) Schematic depiction of the aptamer/TetR-DOZI system for ligand-dependent protein expression. (B) Immunofluorescence analysis of fixed parasites endogenously expressing PPS-HA-FLAG and stained with anti-acyl carrier protein (ACP) and anti-hemagglutinin (HA)-tag antibodies. The intensity plot displays the overlap in pixel intensity for ACP and HA signals as a function of distance along the white line in the merged image. (C) Western blot of endogenously tagged PPS-HA/FLAG showing detection of tagged PPS at the expected size for mature PPS of ~60 kDa for growth in +aTc conditions but diminished signal for parasites grown -aTc + isopentenyl pyrophosphate (IPP) for 5 days. Densitometry of the PPS signal relative to the EF1α loading control indicated a threefold signal reduction in +aTc versus -aTc/ + IPP conditions. (D) RT-qPCR analysis of PPS transcript levels (normalized to the average of two nuclear control genes) in biological replicate samples of synchronous parasites cultured for 72 hr ± aTc or -aTc/ + IPP. (E) Synchronous growth assay of Dd2 parasites tagged at the PPS locus with the aptamer/TetR-DOZI system and grown ±aTc and ± 200 µM IPP or 5 µM farnesol (FOH), geranylgeraniol (GGOH), or decaprenol (C50-OH). Parasitemia values for each condition are the average ± SD of three biological replicates.
Figure 6—figure supplement 1. Scheme for modification of the polyprenyl synthase (PPS) genomic locus to integrate the aptamer/TetR-DOZI system and Southern blot confirming correct integration.
Figure 6—figure supplement 2. Additional immunofluorescence microscopy images showing co-localization of endogenous polyprenyl synthase (PPS) and apicoplast acyl carrier protein (ACP).
Figure 6—figure supplement 3. Blood-smear images of Dd2 parasites tagged at the polyprenyl synthase (PPS) locus with the aptamer/TetR-DOZI system and grown ±aTc for 8 days.
