Figure 4. Phosphosite mutations render P. falciparum PKG inactive.

(A) Cartoon of the P. falciparum PKG x-ray crystal structure (PDB ID: 5DYK) in its apo form with rainbow colouring (N terminus in dark blue; C terminus in red). Cyclic nucleotide-binding domains A (dark blue), B (cyan), C (green), and D (lime) are shown, whereas the central kinase domains are in yellow/orange/red. Phosphosites identified by mass spectrometry are indicated and shown as sticks within colour-matching transparent spheres. The image was ray-traced in the PyMOL Open-Source Molecular Graphic System (https://pymol.org/2/). (B) Schematic of the modified pfpkg locus in the pkg:cKOR-mutGFP parasite line. Upon DiCre induction with RAP, recombination event 1 leads to conditional gene disruption (ΔPKG_mCherry), whilst recombination event 2 leads to replacement of the endogenous allele with a partially synthetic full-length allele containing Ala substitutions of all seven phosphosites (asterisks), fused to GFP (recombination event 2; PKGmut_GFP). (C) Diagnostic PCR results showing detection of the two distinct recombination events after DiCre activation. The amplicon specific for ΔPKG_mCherry (denoted by the black and red arrows) is ∼1 kb in the RAP-treated sample and ∼4.9 kb in the mock-treated (non-excised) sample. The amplicon specific for PKGmut_GFP is ∼4 kb in the RAP-treated sample. Amplification from mock-treated samples was unsuccessful, likely because of the large size of the predicted fragment. (D) Quantification of the ratio between PKGmut_GFP and ΔPKG_mCherry schizonts in the RAP-treated parasite population at the end of cycle 0. Data shown are from five independent experiments; individual and mean values are shown. Error bars ± SD (n = 5). (E) Giemsa-stained images of Percoll-enriched schizonts isolated at the end of cycle 0 of DMSO- and RAP-treated pkg:cKOR-mutGFP parasites, showing no discernible morphological differences. Scale bar, 10 μM. (F) Replication of DMSO- and RAP- treated pkg:ckoR_mutGFP parasites over three erythrocytic cycles. Parasitaemia values shown (obtained by flow cytometry) are averages of three independent experiments. Error bars ± SD (n = 3). (G) Both PKGmut_GFP and ΔPKG_mCherry schizonts are defective in egress. Still images of the first and final frame of a 30-min time-lapse video of mock-treated (grey) or RAP-treated (green and red) pkg:ckoR_mutGFP parasites. Schizonts were synchronised by incubation with the reversible PKG inhibitor compound 2, then washed, mixed in equal proportions, and monitored for egress over a period of 30 min. Neither the red (ΔPKG_mCherry) nor the green (PKGmut_GFP) schizonts underwent egress, whereas most of the parental mock-treated pkg:ckoR_mutGFP schizonts ruptured. Scale bar, 10 μΜ. (H) Western blot showing that no SERA5 P50 was released into culture supernatants of RAP-treated pkg:ckoR_mutGFP schizonts, consistent with impaired egress in both the PKGmut_GFP and ΔPKG_mCherry schizonts.