Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites

Abstract

In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites.

Fig. 1

A genetic interaction screen identifies a synthetic interaction between PbPKG and PbCDPK4 in asexual blood stages. a Schematic overview of the genetic interaction screen, definitions and analysis. b Interaction coefficients plotted against p values. Interactions selected for validation (red) required ε ≤ −0.25 or ≥ 0.25 and p value < 0.05 (shaded areas, two-tailed t-test). c Effect of cdpk4 deletion and pkg mutagenesis on the growth of asexual blood stage parasites (error bars show standard deviations from the mean; 3 independent infections). d Complementation of cdpk4 gene deletion or pkg mutagenesis with non-tagged wild-type alleles of cdpk4 or pkg (error bars show standard deviations from the mean; 3 independent infections; two-way analysis of variance (ANOVA)). e Number of merozoites per schizont in control and PKG^T619Q-3xHA parasites (data shown is from 3 independent in vitro cultures). f Parasitaemia observed 1 h after the intravenous injection of control or PKG^T619Q-3xHA/CDPK4-KO mature segmented schizonts (data shown are from technical duplicates of four independent schizont cultures; error bars show standard deviations from the mean; two-tailed t-test). g Electron microscopy analysis of mature WT, CDPK4-KO, PKG^T619Q-3xHA, and PKG^T619Q-3xHA/CDPK4-KO schizonts (data from duplicates; error bars show standard deviations from the mean; two-way ANOVA, n_WT = 140, n_CDPK4-KO = 103, n_PKGT619Q_-3xHA = 48, n_PKGT619Q_{-3xHA/CDPK4-KO} = 200). h Representative electron microscopy pictures of mature WT and PKG^T619Q-3xHA/CDPK4-KO schizonts. White arrows indicate gaps in the IMC that were more frequently observed in the transgenic line. Scale bar 1 µm (low magnification) and 200 nm (high magnification)

Fig. 2

The CDPK4/PKG functional interaction is conserved to control P. falciparum merozoite invasion. a Difference in ring and schizont parasitaemia after 3 h of C2 treatment added on synchronised segmented schizonts in the control 3D7, PKG^T618Q, and CDPK4-KO lines (error bars show standard deviations from the mean; 2 independent cultures; two-tailed t-test). b Difference in ring and schizont parasitaemia after 3 h of C2 treatment added on synchronised segmented schizonts in the CDPK4-KO line and in daughter lines complemented with P. berghei CDPK4 or CDPK4^S147M alleles (error bars show standard deviations from the mean; 2 independent cultures; two-tailed t-test). c Difference in ring and schizont parasitaemia after 3 h of Compound A treatment added on synchronised segmented schizonts in the control 3D7, PKG^T618Q, and CDPK4-KO lines (error bars show standard deviations from the mean; 2 independent cultures; two-tailed t-test). d Determination of the ATP Km for recombinant PfPKG and PfPKG^T618Q. e Fluorescence of the cell permeable calcium probe Fluo-4-AM in non-synchronised 3D7 and PKG^T618Q parasites labelled with the DNA dye Vybrant Green (error bars show standard deviations from the mean; 2 biological replicates). f Relative fluorescence response of Pf3D7 and PfPKG^T618Q lines loaded with the calcium indicator Fluo-4-AM in response to the phosphodiesterase inhibitors Zaprinast and BIPPO as well as the ionophore A23187 (error bars show standard deviations from the mean; 3 or 4 biological replicates, two-tailed t-test)

Fig. 3

CDPK4 co-immunoprecipitates with components of the glideosome and the IMC. a Western blots and immunofluorescence analysis of schizonts expressing endogenously 3xHA tagged cdpk4, gap40, cdpk1 and soc6 alleles. Scale bars are 1 µm. b Protein interactions between IMC or glideosome proteins identified from GAP40-3xHA, SOC6-3xHA, MyoE-3xHA, CDPK4-3xHA and CDPK1-3xHA immunoprecipitates. Thick bars indicate that the interaction was identified from both immunoprecipitates. Blue and green filled circles denote the number of residues phosphorylated by CDPK4 ex vivo and by recombinant CDPK1 in vitro, respectively. The colour of the circle around the protein name indicates the requirement of its encoding gene for growth in asexual blood stages: red is essential, green is redundant; black denotes that the gene essentiality was not tested. c Effect of gap40 mutagenesis on the growth of asexual blood stage parasites (error bars show standard deviations from 2 independent infections). d Electron microscopy analysis of mature WT and GAP40^S448/449A-3xHA schizonts (error bars show standard deviations from the mean; duplicates; two-tailed t-test; n_WT = 140, n_GAP40S448/449A_-3xHA= 132)

Fig. 4

The CDPK4 substrate SOC6 is important for the IMC stability in merozoite and ookinete. a Effect of soc6 deletion on the growth of asexual blood stage parasites (error bars show standard deviations from 2 independent infections). b Number of merozoites per schizont in control and SOC6-KO parasites (data shown is from 4 independent in vitro cultures). c Parasitaemia observed 1 h after the intravenous injection of control or SOC6-KO mature segmented schizonts (data shown is from four independent schizont cultures, two-tailed t-test). d Electron microscopy analysis of mature SOC6-KO and WT schizonts (error bars show standard deviations from duplicates, n_WT = 140, n_SOC6-KO = 267). e Ookinete conversion rate of 2.34 and SOC6-KO lines (error bars are standard deviations from three independent ookinete cultures, two-tailed t-test). f Ultrastructural analysis of WT and SOC6-KO highlighting the integrity of the IMC in WT and SOC6-KO p28-expressing cells (error bars are standard deviations from the mean, two ookinete cultures, n_WT = 34, n_SOC6-KO = 27). g Representative images of IMC defects observed in SOC6-KO parasites. Scale bar: 2.34, 1 µm (lower magnification) and 200 nm (higher magnification); SOC6-KO, 1 µm (lower magnification) and 500 nm (higher magnification)

Fig. 5

PKG, CDPK4 and CDPK1 functionally interact to control asexual growth and ookinete motility. a Effect of cdpk3 or cdpk6 gene deletion in the PKG^T619Q-3xHA/CDPK4-KO background on the growth of asexual blood stage parasites (error bars show standard deviations from the mean; 2 independent infections). Note that a PKG^T619Q-3xHA/CDPK4-KO/CDPK1-KO line could not be generated. b Average gliding speed of 2.34 or PKG^T619Q-3xHA ookinetes ± 0.5 µM C2 (data show the average speed of single ookinetes from two independent biological replicates, two-way ANOVA). c Detection of CDPK1-AID-HA in ookinete lysate following a 30-min treatment ± auxin (IAA). d Average gliding speed of CDPK1-AID-HA ookinetes ± 1 µM 1294 and/or auxin (data show the average speed of single ookinetes from three independent biological replicates, two-way ANOVA). e Detection of the CelTOS-3xHA microneme protein and the cytosolic actin protein in the pellet and supernatant (SN) of 2.34 and PKG^T619Q-3xHA cultures ± 0.5 µM C2. The histogram shows quantification of the SN/pellet ratio for CelTOS-3xHA of each condition normalised to the SN/pellet ratio obtained in the 2.34 control in the absence of C2 (error bars show the standard deviations from the mean, three independent ookinete cultures, two-way ANOVA). f Detection of the CelTOS-3xHA microneme protein and the cytosolic actin protein in the pellet and supernatant of CDPK1-AID-HA cultures ± 1 µM 1294 and/or auxin. The histogram shows quantification of the SN/pellet ratio as in (e). g Average gliding speed of 2.34 and CDPK3-KO ookinetes  ± 1 µM 1294 (data show the average speed of single ookinetes from the mean, two independent biological replicates, two-way ANOVA). h Detection of the CelTOS-3xHA microneme protein and the cytosolic actin protein in the pellet and supernatant of 2.34 or CDPK3-KO cultures ± 1 µM 1294. The histogram shows quantification of the SN/pellet ratio for CelTOS-3xHA as in (e)

Fig. 6

Schematic representation of the CDPK networks downstream of PKG-mediated calcium signals. PKG acts as a stage-transcending calcium regulator that activates multiple CDPKs with stage-specific functions. In merozoites, calcium activates both CDPK4 and 1 to support the activity of the acto-myosin motor. In ookinetes, PKG-dependent calcium signals are required for efficient gliding through CDPK4 and 1 activation and microneme secretion via CDPK3 regulation. Under physiological activation of PKG, CDPK1 and CDPK4 may functionally complement each other to sustain the acto-myosin motor function, while hyperactivation of PKG by chemical or genetic inhibition of phosphodiesterase or adaptation to gene deletion may allow further complementation by CDPK3 in ookinetes. Note that other kinases such as CDPK5 or the protein kinase A (PKA) are possibly part of this network but the exact links with PKG and these kinases could not be revealed by this study and remain unmapped