PDEβ disruption has no impact on parasite development in the excision cycle but is lethal to long-term growth.

(A) Images of Giemsa-stained blood films showing the morphology of RAP-treated (+RAP) and mock-treated (DMSO) PfPDEβΔcatHA parasites at different points in the excision cycle (cycle 0). Highly synchronised parasite cultures were treated whilst at ring stage. Scale bar, 5 μm. (B) DNA content analysis by flow cytometry of cycle 0 PfPDEβ-null schizonts (red plots) compared with untreated schizonts (blue plots). Data representative of three experiments, each performed in triplicate, are shown for each of the three time points. (C) Mean number of PfPDEβΔcatHA merozoites per schizont in cycle 0 following mock or RAP treatment. Data were derived from five independent experiments; a total of 100 schizonts were examined on DAPI-stained blood films. Error bars, 1 SD. (D) Growth curves of the PfPDEβΔcatHA and 1G5DC parental lines following mock or RAP treatment, derived by measuring parasite DNA content using SYBR Green fluorescence. Each data point is a mean value derived from two biological replicate experiments, each performed in triplicate. Error bars, 1 SD. (E) Diagnostic PCR analysis of the PfPDEβ locus in mock and RAP-treated PfPDEβΔcatHA cultures over six erythrocytic cycles of parasite replication, showing rapid loss of the signal diagnostic of the excised (PfPDEβ-null) parasites (EXC). Primers used were as described in Fig 2a and 2b. CTRL, control; EXC, excised; hpi, hours post invasion; INT, integration; n.s., not significant by unpaired t test (p-value = 0.9895); PDEβ, phosphodiesterase β; PfPDEβ, Plasmodium falciparum phosphodiesterase β; RAP, rapamycin; RFU, relative fluorescence unit.