Prolonged PKA activity in newly invaded PfPDEβ-null parasites blocks ring stage development.

(A) The PDE inhibitor BIPPO phenocopies the PfPDEβ-null post-invasion phenotype. Giemsa-stained blood films showing the morphology of PfPDEβΔcatHA parasites at different time points in cycle 1, following either mock or RAP treatment of the parasites in cycle 0, compared to mock-treated parasites exposed to the PDE inhibitor BIPPO immediately following invasion. (B) The PKA inhibitor H89, but not the PKG inhibitor Compound 2, rescues BIPPO-treated early ring stages. The plots were generated by microscopic analysis of Giemsa-stained smears from ring stage cultures (18–22 hpi) treated with the indicated compounds. Kinase inhibitors (H89 and C2) were added at 1–5 hpi and BIPPO at 2–6 hpi. Scale bar, 5 μm. Data presented are mean counts (evaluated by three independent researchers) from two independent experiments, with >100 parasites counted per condition. Error bars, SEM. *, significant by unpaired t test (p-value = 0.0005); n.s., not significant (p-value = 0.8508). (C) The PDE inhibitor BIPPO induces PKA-dependent phosphorylation in ring stages. Western blot of total protein from ring stages (12–16 hpi) treated for 90 minutes with various inhibitors: BIPPO (2 μM), H89 (30 μM), C2 (2 μM). Lane 1 = no inhibitor control, lane 2 = BIPPO only, lane 3 = BIPPO + H89, lane 4 = BIPPO + C2. The blot was probed with an antibody to phosphorylated PKA substrate motif. The gel was stained for total protein prior to blotting to show equal loading (right panel). BIPPO, 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one; hpi, hours post invasion; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; pS/pT, phosphoserine or phosphothreonine; RAP, rapamycin; R/K, arginine or lysine.