SPARK interacts with an Elongin C-like protein.

(A) Protein abundances from immunopurified SPARK-mNG lysates or an untagged control strain. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 104.5. (B) TGGT1_291010 SPARK elongin-like protein (SPARKEL) gene model. (C) Neighbor-joining phylogenetic tree of SKP1/BTB/POZ domains of TGGT1_291010 orthologs in apicomplexan species along with human and arabidopsis proteins as outgroups. Bootstrap values determined from 1000 replicates. (D) Protein abundances from immunopurified SPARKEL-Ty lysates or an untagged control strain. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 104.5. (E) Protein abundances following biotinylation and streptavidin enrichment of samples derived from parasites expressing mNG- or SPARKEL-TurboID fusion constructs. A pseudocount of 104.5 was assigned to proteins identified in only one sample. Point colors correspond to significance thresholds. Dotted lines correspond to one median absolute deviation. (F) Immunofluorescence microscopy of intracellular parasites co-expressing SPARKEL-mNG and SPARK-mCherry. Parasites and nuclei were stained with GAP45 and Hoechst 33342, respectively.

Proteins discussed in the text.

SPARKEL depletion phenocopies the loss of SPARK.

(A) Plaque assays of 500 TIR1 or SPARKEL-AID parasites infected onto host cell monolayers and allowed to undergo repeated cycles of invasion, replication, and lysis for 7 days in media with or without 500 µM IAA. (B) Immunoblot of parasites expressing SPARKEL-V5-AID and SPARK-V5 after the indicated hours with IAA. TUB1 serves as a loading control. (C) Immunoblot of parasites expressing SPARK-V5-AID and SPARKEL-V5 after the indicated hours with IAA. CDPK1 serves as loading control. (D) The number of parasites per vacuole measured for SPARKEL-AID and the parental strain after 24 hours of 500 µM IAA treatment. Mean counts (n = 8) are expressed as a percentage of all vacuoles counted. SEM is shown as shaded area. No comparisons yielded significant p-values using ANOVA and Tukey’s test. (E) Invasion assays SPARKEL- AID, SPARK-AID or TIR1 parental strains treated with IAA or vehicle for 3 or 24 hours. Parasites were incubated on host cells for 20 minutes prior to differential staining of intracellular and extracellular parasites. Parasite numbers were normalized to host cell nuclei for each field. Different shapes correspond to means of n = 3 biological replicates. For clarity, only significant comparisons (Welch’s one-sided t-test) are shown. (F) The time to egress of individual intracellular vacuoles following zaprinast treatment. Points correspond to different vacuoles; shapes different replicates. Black shapes are the mean for each replicate. P-values were calculated from a one-tailed t-test. (G, H) Selected frames from live video microscopy of zaprinast-treated SPARK-AID and SPARKEL-AID parasites, respectively, expressing the calcium indicator GCaMP6f, and the corresponding parental strain, 25 hours after infection and with the indicated IAA treatment period. See also Video S1 and Video S2. (I, J) Relative GCaMP fluorescence of SPARK-AID or SPARKEL-AID vacuoles, respectively, 60 s following zaprinast treatment. Points correspond to different vacuoles; shapes different replicates. Black shapes are the mean for each replicate. P-values were calculated from a one-tailed t-test.

Depletion of SPARK or SPARKEL leads to downregulation of AGC kinases and upregulation of chronic-stage markers.

(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. In total, 4474 proteins were quantified, 3847 of which registered more than one peptide. (B) Schematic of the phosphoproteomics experiment following SPARK depletion. Intracellular parasites expressing SPARK-AID were treated with 500 µM IAA for 24, 8, 3, or 0 h and were harvested simultaneously with the TIR1 parental strain as a control. Samples were multiplexed with tandem mass tags (TMT). The same experimental design was used for SPARKEL-AID proteomics. Each experiment included two biological replicates. (C) Protein abundances of PKG, PKA-R, and PKA-C1 relative to the TIR1 parental strain after the indicated period of SPARK (thick lines) or SPARKEL (dotted lines) depletion. (D) Protein abundances of up-regulated bradyzoite genes relative to the TIR1 parental strain after the indicated period of SPARK (blue lines) or SPARKEL (purple lines) depletion. Up-regulated bradyzoite proteins were defined as those significantly increased in parasites overexpressing BFD1 (Waldman et al., 2020) and two modified Z-scores above the median in the SPARK depletion proteome. Rank-ordered plots of (E) Phosphopeptide basal dysregulation score (peptide abundance in the SPARK-AID strain without IAA treatment relative to the TIR1 parental strain) or (F) IAA-dependent score (summed peptide ratios relative to the SPARK-AID t0 peptide abundance). Dotted lines correspond to 3.5 modified Z scores. Colored points correspond to phosphosites discussed in the main text. (G) Gene ontology (GO) enrichment of phosphoproteins exhibiting SPARK-dependent regulation. Gene ratio is the proportion of proteins with the indicated GO term divided by the total number of proteins. Significance was determined with a hypergeometric test; only GO terms with p < 0.05 are shown. Redundant GO terms were removed. Categories discussed in the main text are highlighted with colored text. (H) Gaussian mixture modeling of SPARK-dependent peptides identified by more than one PSM revealed seven kinetically resolved clusters. Individual peptides or the median ratios in each cluster are depicted by light and dark gray lines, respectively. Clusters are numbered according to their discussion in the main text. (I) Protein abundances following biotinylation and streptavidin enrichment of samples derived from parasites expressing mNG- or SPARK-TurboID fusion constructs. A pseudocount of 104.5 was assigned to proteins identified in only one sample. Point colors correspond to significance thresholds. Dotted lines correspond to one median absolute deviation.

PKA C1 levels are down-regulated upon SPARK depletion.

(A–B) Protein and phosphopeptide abundances of PKA C1 (A) and PKA R (B) following SPARK depletion. (C) Schematic of the genetic strategy used to monitor PKA C1 and PKA R abundances following SPARK (or SPARKEL) down-regulation with IAA. (D) Immunofluorescence microscopy of parasites expressing SPARK-AID, PKA C1-mNG, and PKA R-mCherry after 0 or 24 hours of IAA treatment to degrade SPARK. Parasites and nuclei were stained with GAP45 and Hoechst 33342, respectively. GAP45 staining and mNG or mCherry staining were normalized to vehicle-treated tagged samples. (E, F) Flow cytometry analysis of parasites expressing PKA C1-mNG, PKA R-mCherry, and SPARK-AID or SPARKEL-AID, respectively, after the indicated period of IAA treatment. The dotted line centers the mode of the vehicle-treated sample. Traces were normalized by unit area. (G) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKA C1 targets, as defined in the text and methods, are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (H) Heat map of the abundance ratios of candidate PKA C1 targets following SPARK depletion. PKA R depletion results in up-regulation of PKA C1, and candidate PKA C1 targets therefore have positive abundance ratios following PKA R down-regulation.

Characterization of PKG function during SPARK depletion.

(A) Protein and phosphopeptide abundances of PKG following SPARK depletion. (B) A23187-stimulated egress assays performed at different concentrations of compound 1 after TIR1 and SPARK-AID parasites had been treated with IAA for 24 hours. Curves were fit to the average values of six replicates and were compared with an extra sum of squares F test. (C) The IC50 values of each strain for compound 1; each point represents a biological replicate. Significance was assessed with a two-tailed t-test. (D) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKG targets are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (E) Heat map of the abundance ratios of candidate PKG targets following SPARK depletion.

SPARK, SPARKEL, and PKA C3 are negative regulators of differentiation.

(A) Immunofluorescence differentiation assays following knockdown of the indicated AID strains for 48 h under standard growth conditions (unstressed) or alkaline stress. GAP45 was used to stain parasite vacuoles. Differentiated vacuoles were stained with biotinylated DBA/streptavidin-APC. Nuclei were stained with Hoechst. (B) Quantification of the number of DBL+ vacuoles expressed as a percentage of the total stained vacuoles is shown for parasites grown under unstressed or stressed conditions. One-sided t-test, n = 3 biological replicates. (C) Fixed, intracellular PKA C3-mNG-AID parasites visualized by immunofluorescence microscopy using the mNG epitope after 1 h of vehicle or IAA treatment. The mNG signal was internally normalized to the TIR1 parental strain. (D) Immunoblot of PKA C3-AID parasites following the addition of vehicle or 500 µM IAA for 1, 3, or 24h. TIR1 was included as an untagged control. PKA C3-AID was detected with V5, and ALD1 was probed as a loading control. (E) Immunofluorescence differentiation assays following knockdown of the indicated ME49/AID strains under unstressed conditions for 48 hours. Staining was performed as described above, except CDPK1 was used as a parasite vacuole marker. (F) Quantification of the number of DBL+ vacuoles expressed as a percentage of the total stained vacuoles in (E). Two-sided t-test, n = 5 biological replicates. (G) Immunofluorescence differentiation assays of parasite strains with or without BFD1, depleted of PKA C3 or SPARK with 500 µM IAA and grown under unstressed conditions or alkaline stress for 48 h. (H) The percentage of DBL+ vacuoles corresponding to (G). One-sided t-test. (I) Effects of 24 hours of PKA C3 knockdown on the transcriptome relative to the untagged strain. Dotted lines correspond to an absolute log2 change of 1. Genes significantly affected by BFD1 overexpression (p-value < 0.001 as previously defined (Waldman et al., 2020)) are colored according to log2 change in the chronic-stage transcriptome. Highlighted points are discussed in the text.

SPARK and PKA C3 physically and genetically interact.

(A) Protein abundances from immunopurified PKA C3-mNG-AID lysates or an untagged control strain. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 104.5. Point colors correspond to significance thresholds. (B) Immunoblot of parasites expressing SPARK-V5-AID-HA and PKA C3-mNG after 24 hours of IAA treatment. ALD1 serves as a loading control. Band intensity normalized to the dual-tagged strain is shown in (C) from 3 replicates. (D) Flow cytometry analysis of SPARK-AID parasites expressing PKA C3-mNG treated with IAA for the indicated number of hours. Traces are representative of two biological replicates. The dotted line centers the mode of the vehicle-treated sample. Traces were normalized by unit area. (E–F) Fixed, intracellular SPARK-AID/PKA C3-mNG parasites visualized by immunofluorescence microscopy using the mNG epitope after the indicated period of IAA treatment (E). The mNG signal was internally normalized to the SPARK-AID parental strain. Quantification of the PKA C3 signal intensity of three replicates is shown in (F). (G) Phosphopeptide IAA-dependent score (summed peptide ratios relative to the PKA C3-AID t0 peptide abundance). Dotted lines correspond to 3.5 modified Z scores. Colored points correspond to phosphosites discussed in the main text. (H) Gaussian mixture modeling of PKA C3-dependent peptides identified by more than one PSM revealed three kinetically resolved clusters. Individual peptides or the median ratios in each cluster are depicted by light and dark gray lines, respectively. Clusters are numbered according to their discussion in the main text. (I) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKA C3 targets are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (J) Heat map of the abundance ratios of candidate PKA C3 targets following SPARK depletion. (K) Heat map of the abundance ratios of dense granule proteins following SPARK depletion, as discussed in the text.

Additional data supporting the interaction between SPARK and SPARKEL.

(A) Protein abundances from immunopurified SPARK-mNG-mAID (Smith et al., 2022) lysates or an untagged control strain. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 104.5. (B) Protein abundances following biotinylation and streptavidin enrichment of samples derived from parasites expressing mNG- or TurboID-SPARKEL fusion constructs. A pseudocount of 104.5 was assigned to proteins identified in only one sample. Point colors correspond to significance thresholds. Dotted lines correspond to one median absolute deviation.

(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.

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.

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).

(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 Δbfd1::dTom parasites showing red fluorescence arising from the knockout cassette.

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.