SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites.


Figure 2 -
Figure 2-figure supplement 1. (A) Primers specific to the 3' terminus of SPARK and the V5-mAID-HA tagging payload amplified a product in the SPARK-V5-mAID-HA strain but not the untagged parental strain.(B) Confirmation of SPARK-V5-mAID-HA depletion via immunoblot using the V5 epitope.ALD1 was used as a loading control.(C) SPARK-V5-mAID-HA depletion was visualized in formaldehyde-fixed intracellular parasites using the HA epitope and GAP45 staining as a parasite marker.DNA was visualized with Hoechst.HA signal intensity was normalized relative to the TIR1 parental strain.(D) Uncropped immunoblots corresponding to Figure 2B.(E) Uncropped immunoblots corresponding to Figure 2C.(F, G) Normalized GCaMP6f fluorescence of individual SPARK-AID and SPARKEL-AID vacuoles, respectively, after zaprinast treatment and prior to egress (transparent lines) for the indicated period of IAA treatment.The solid line represents the mean normalized fluorescence of all vacuoles across n = 3 biological replicates.

Figure 3 -
Figure 3-figure supplement 1. Extended analysis of the SPARK-AID depletion phosphoproteome.(A) Protein abundance ratios of SPARK-or SPARKEL-AID parasites treated with IAA for 24 hours relative to the untreated samples.Enriched proteins identified as up-regulated in alkaline-induced bradyzoites (Waldman et al., 2020) are shown in blue.The points corresponding to PKA C1, PKA R, and PKG are highlighted in pink, orange, and green, respectively.(B, C) Principal component analysis of the SPARK-AID depletion phosphoproteome.Plots show the three components accounting for the greatest proportion of the variance (D) Abundances of SPARK and SPARKEL peptides detected by mass spectrometry.

Figure 4 -
Figure 4-figure supplement 1. Overlap between the SPARK and PP1 phosphoproteomes.Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion.The distributions of candidate PP1 targets, as defined in the text and methods, are shown in blue.The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset.PP1 proteome data was obtained from (Herneisen et al., 2022).

Figure 6 -
Figure 6-figure supplement 1. (A) Fixed, intracellular RH/3HA-mAID-SPARKEL parasites visualized by immunofluorescence microscopy using the HA epitope after 1h of vehicle or IAA treatment.The HA signal was normalized to the vehicle-treated sample.(B) Quantification of the number of DBL+ vacuoles expressed as a percentage of the total stained vacuoles following 48h of AID-SPARKEL knockdown.Two-sided t-test.(C) Fixed, intracellular ME49/PKA C3-mNG-AID parasites visualized by immunofluorescence microscopy using the mNG epitope after 3h of vehicle or IAA treatment.The mNG signal was normalized to the vehicle-treated sample.(D) Amplification of the SPARKEL-AID and PKA C3-AID genomic loci using tag-specific primers to confirm correct integration of the tagging payload.The integration was confirmed with Sanger sequencing between the 3' gene junction and CDPK3 3'UTR from the tag (Smith et al., 2022).(E) The strategy used to knock out BFD1 with a dTomato cassette containing homology to sequenced flanking the BFD1 locus (Waldman et al., 2020).Amplification of sequences specific to the intact locus or dTom knockout for the indicated strains are shown below the schematic.Oligonucleotide sequences are listed in Supplementary Table 5. (F) Live microscopy images of intact or ∆bfd1::dTom parasites showing red fluorescence arising from the knockout cassette.

Figure 7 -
Figure 7-figure supplement 1. Extended analysis of the PKA C3 depletion proteome.(A) Volcano plot displaying the protein abundance ratios of SPARK-AID parasites treated with IAA or vehicle for 24 hours and adjusted p-values.Proteins identified as up-or down-regulated in parasites overexpressing the driver of differentiation (BFD1) (Waldman et al., 2020) are shown in blue and vermilion, respectively.(B, C) Principal component analysis of the PKA C3-AID depletion phosphoproteome.Plots show the three components accounting for the greatest proportion of the variance.