Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites

Abstract

Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²⁺ from intracellular stores to activate stage-specific Ca²⁺-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²⁺ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²⁺ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²⁺ effectors, PKG emerges as a unifying factor to control multiple cellular Ca²⁺ signals essential for malaria parasite development and transmission.

Figure 1. Role of PKG in regulating ookinete gliding.

(A) Gliding traces of ookinetes in matrigel recorded for 20 min from a representative field of view. Scale bar, 50 µm. The coloured tracks were created by superimposing individual images from a time series, each marking the tip of each ookinete. (B) Effect of C2 on the gliding speed of ookinetes. (C) Average gliding speed of ookinetes at increasing concentrations of C2. Error bars show standard deviations of 20 ookinetes from each of two independent biological replicates. (D) Gliding speeds of mutant ookinetes. The range of whisker plots in (B) and (D) indicates the 2.5 and 97.5 percentiles, the box includes 50% of all values, and the horizontal line shows median values obtained for 20 ookinetes from each of two independent biological replicates. Statistical analyses in (B) and (D) were carried out using a two-tailed t test.

Figure 2. Effects of perturbed cGMP synthesis and PKG inhibition on the ookinete phosphoproteome.

(A) Schematic illustrating of experiment 1 to compare global phosphorylation of proteins between wild-type and gcβ ookinetes by pulse-chase SILAC labelling with medium (D₄ L-lysine plus ¹³C₆ L-arginine) and heavy isotopes (¹³C₆,¹⁵N₂ L-lysine plus ¹³C₆,¹⁵N₄ L-arginine), respectively. Crude extracts from purified ookinetes were combined and analysed together by LC-MS/MS prior or after enrichment for phosphopeptides by IMAC purification. (B) Normalised phosphorylation ratios for all class I sites that were quantified in both wild-type and gcβ mutant ookinetes are plotted against the heavy and medium intensities for each site. Data points are coloured to indicate significance of regulation as determined from five biological replicates: blue circles show significantly regulated sites (p<0.01, ratio count ≥6, and fold change >3). Labelled sites are in enzymes linked to cGMP signalling (orange) or phosphoinositide metabolism (green). (C) Schematic illustrating experiment 2 to measure the effect of C2 on global protein phosphorylation using label-free quantification. Purified ookinetes expressing PKG-HA or PKG^T619Q-HA were snap-frozen after a 2 min exposure to C2. (D) Normalised phosphorylation ratios for all class I phosphorylation sites that were quantified in both lines in experiment 2 are plotted against the intensity for each site. Data points are coloured to indicate significance of regulation as determined across six biological replicates: red circles show significantly regulated sites (false discovery rate ≤0.05 and fold change ≥1.5). Proteins with likely roles in cGMP signalling and phosphoinositide metabolism are coloured as in (B). (E) Functional categories from the Malaria Parasite Metabolic Pathway database that were enriched among proteins with regulated phosphorylation sites in experiments 1 (blue bars) or 2 (red bars). The dashed line shows the chosen significance cutoff of p<0.05.

Figure 3. Phosphorylation sites in proteins with likely roles in phosphoinositide metabolism.

All class I phosphorylation sites are shown as squares next to the schematic illustrations of the relevant proteins and their annotated functional domains. PI-PLC was not detected and is shown in light green. Each phosphorylation site is represented by a divided square, the colour of which shows the degree of regulation upon inhibition of PKG by C2 or in the gcβ mutant. Failure to quantify a phosphorylation site with one of the two experimental designs is shown in white.

Figure 4. Phosphoinositide phosphorylation links PKG to gliding in P. berghei ookinetes.

(A) Ookinete gliding speed of PI4K^S534A and PI4K^S534A/S538A ookinete mutants. Values are representative of 20 individual ookinetes from two independent biological replicates. (B) Ratio of peripheral to cytosolic fluorescence intensity from optical sections taken of different ookinetes from the experiment shown in (C); n = 10 sections from different ookinetes. Statistical analysis was carried out using a two-tailed t test. (C) Confocal immunofluorescence images of fixed ookinetes showing the effect of 0.5 µM C2 on the cellular distribution of a C-terminally HA tagged PIP5K (PBANKA_020310) expressed from its endogenous promoter. Scale bar, 5 µm. (D) Relative quantification of PI, PIP, PIP₂, and PIP₃ levels after 10 min treatment with 0.5 µM C2 or DMSO in PKG-HA ookinetes. (E) As in (D) but for PKG^T619Q-HA ookinetes. Error bars in (D) and (E) show standard deviations of two biological replicates. The p values are from two-tailed t test. See also Figure S5.

Figure 5. PKG controls cytosolic Ca²⁺ levels in ookinete and upon gametocyte activation in P. berghei.

(A and B) Determination of relative cytosolic Ca²⁺ levels in purified ookinetes expressing PKG-HA or PKG^T619Q-HA using pericam, a dual excitation Ca²⁺ reporter. We added 0.5 µM C2 treatment (A) or DMSO treatment (B) at t = 20 s. Fluorescence was normalised as follows: ΔF = (F_n−F₂₀)/F₂₀, in which F_n is the fluorescence at t = n s; F₂₀ is the reference time before addition of C2 or DMSO, and was normalised to the baseline provided by the C2-resistant parasites expressing PKG^T619Q-HA. Error bars show standard errors from three independent replicates each using 10 ookinetes per condition. See Figure S6A and Figure S6B for a validation of the pericam reporter in P. berghei. (C) Luminescence responses of gametocytes expressing GFP-aequorin to different concentrations of C2. Gametocytes were stimulated with 50 µM XA at t = 0 s. Data are representative of at least four independent experiments.

Figure 6. PKG controls phosphoinositide metabolism and Ca²⁺ mobilisation in P. falciparum schizonts prior to merozoite egress.

(A) Relative fluorescence intensity of ∼10⁸ Fluo-4–loaded synchronised P. falciparum schizonts in response to simultaneous exposure to 100 µM zaprinast and increasing concentrations of C2. Data are representative of two independent experiments. (B) Relative abundance over time of PI, PIP (left panel), PIP₂, and PIP₃ (right panel, note different scale) after simultaneous inhibition of PDEs by zaprinast and PKG by C2. The response of P. falciparum 3D7 (dashed lines) is compared to a transgenic clone expressing the C2-resistant PKG^T618Q allele (solid lines).

Figure 7. Proteins with PKG-dependent phosphorylation sites and their hypothetical functions in gliding ookinetes.

(A) Proteins with phosphorylation sites that are significantly regulated in response to C2 (red dots) or deletion of gcβ (blue dots) and that belong either to known signalling pathways are linked to the glideosome, or which belong to the enriched functional groups of proteins with likely roles in vesicular trafficking. The numeric part of the PBANKA gene ID is shown in grey. The amino acid numbers for the regulated sites in each protein are stated next to a letter indicating if the phosphorylated residue is a serine (S), threonine (T), or tyrosine (Y). (B) Model illustrating hypothetical functions for the proteins in (A) in the molecular motor or during microneme biogenesis in a gliding ookinete.