A forward genetic screen identifies a negative regulator of rapid Ca2+-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii

Abstract

Toxoplasma gondii, like all apicomplexan parasites, uses Ca²⁺ signaling pathways to activate gliding motility to power tissue dissemination and host cell invasion and egress. A group of "plant-like" Ca²⁺-dependent protein kinases (CDPKs) transduces cytosolic Ca²⁺ flux into enzymatic activity, but how they function is poorly understood. To investigate how Ca²⁺ signaling activates egress through CDPKs, we performed a forward genetic screen to isolate gain-of-function mutants from an egress-deficient cdpk3 knockout strain. We recovered mutants that regained the ability to egress from host cells that harbored mutations in the gene Suppressor of Ca²⁺-dependent Egress 1 (SCE1). Global phosphoproteomic analysis showed that SCE1 deletion restored many Δcdpk3-dependent phosphorylation events to near wild-type levels. We also show that CDPK3-dependent SCE1 phosphorylation is required to relieve its suppressive activity to potentiate egress. In summary, our work has uncovered a novel component and suppressor of Ca²⁺-dependent cell egress during Toxoplasma lytic growth.

Keywords: Ca2+ signalling; Toxoplasma gondii; apicomplexa; forward genetic screen; host cell egress; molecular genetics; parasite; proteomics; signaling.

Figure 1.

Forward genetic strategy for the enrichment of Δcdpk3_rev mutants. A, outline of the strategy for the enrichment of Δcdpk3_rev mutants from chemically mutagenized egress-deficient Δcdpk3. ν, host cell nucleus. B, egress of wild-type (RH:Δku80), Δcdpk3, Δcdpk3_EMS.7.1, Δcdpk3_EMS.7.2, Δcdpk3_EMS.10.1, and Δcdpk3_EMS.10,2 parasites stimulated with A23187 for 3 min. C, protein features of SCE1 (TGME49_269260), including 5× transmembrane (TM) domains, and point mutations seen in mutagenized parasites from population 7 and population 10. Error bars show mean ± S.D. *, adjusted p < 0.0001 using one-way ANOVA and Tukey test to correct for multiple comparisons. There is no statistical difference in egress capacity between any of the EMS-generated lines. The column data were derived from three independent biological replicates.

Figure 2.

Characterization of the role of SCE1 in Toxoplasma egress. Ai, localization of SCE1-Ty and staining with α-Ty and DAPI merged with a phase-contrast image. Aii, reducing Western blot of SCE1-Ty, staining with α-Ty antibody, and α-GAP45 as loading control. B, egress of wild-type, Δsce1, Δcdpk3, and Δcdpk3Δsce1 parasites stimulated with A23187 for 3 min. Error bars show mean ± S.D. ns, not significant. All other comparisons, adjusted p = < 0.0001 using one-way ANOVA. Ci, localization of ectopically expressed SCE1_wt in Δsce1 and staining with α-HA and α-GAP45. Cii, egress of Δsce1, Δcdpk3, Δcdpk3Δsce1, Δcdpk3_EMS7.2, and Δcdpk3_EMS10.2 with ectopically expressed SCE1_wt-HA, stimulated with A23187 for 3 min. Error bars show mean ± S.D. All pairwise comparisons not labeled, p < 0.0001 using one-way ANOVA and Tukey test to correct for multiple comparisons. The column data were derived from three independent biological replicates.

Figure 3.

Sensitivity of SCE1 mutants to egress agonists. Shown is egress of wild-type, Δsce1, Δcdpk3, and Δcdpk3Δsce1 parasites stimulated with either A23187 or BIPPO. A, egress assay measured at 2.5, 5, and 15 min. There was no difference in egress capacity between the wild type and Δsce1 at any time point. B, egress at 5 min over a concentration gradient of A23187. The only statistically significant differences observed are by comparison between all strains and with Δcdpk3 for the two highest concentrations of A23187. C, egress in response to BIPPO, measured at 2.5, 5, and 15 min. There was no statistically significant difference between the wild type and Δsce1 at any time point. D, egress in response to BIPPO at 5 min over a concentration gradient. The only statistically significant differences observed are by comparison between all strains and Δcdpk3 for the two highest concentrations of A23187. Error bars show mean ± S.D. All statistical tests were performed using two-way ANOVA and Tukey correction for multiple comparisons. The data were derived from three independent biological replicates.

Figure 4.

Phosphoproteomic investigation of CDPK3 and SCE1 egress. A, overview of the strategy for phosphoproteomic investigation of CDPK3-regulated egress. Wild-type, Δcdpk3Δsce1, and Δcdpk3 parasites were grown in light, medium, or heavy SILAC labeling medium, respectively. After labeling, parasites were treated with A23187 for 1.5 min to trigger egress-signaling events, parasites were harvested by needle passage, and host cell debris was removed by differential centrifugation. SILAC-labeled samples were mixed 1:1:1 before tryptic digestion. Peptides were fractionated by strong cation exchange (SCX) chromatography, phosphopeptides were enriched on TiO₂ beads, and the resulting samples were analyzed by MS/MS. B, the number of high-confidence Toxoplasma and human proteins, peptides, and phosphopeptides identified following MaxQuant analysis. C, histograms of median-centered log₂ SILAC ratios, Δcdpk3Δsce1/wild type (M/L), Δcdpk3/wild type (H/L), and Δcdpk3/Δcdpk3Δsce1 (H/M). D, the number of high-confidence phosphosites that are significantly up- or down-regulated in Δcdpk3 and Δcdpk3Δsce1 (relative to wild-type parasites) and restored to near wild-type levels in Δcdpk3Δsce1 (relative to Δcdpk3).

Figure 5.

GO term enrichment of phosphopeptide datasets. Shown is an analysis of GO term enrichment in phosphoprotein datasets. Phosphopeptide data “test” sets were compared with a “reference” set comprising the complete Toxoplasma genome. A, all phosphosites showing up- or down-regulation in Δcdpk3. B, all Δcdpk3-misregulated phosphosites restored in Δcdpk3Δsce1. C, all Δcdpk3-misregulated phosphosites not restored in Δcdpk3Δsce1. D, all phosphosites showing no change in Δcdpk3. The color coding indicates the ontology source of GO terms: green, biological process; yellow, molecular function; purple, cellular component.

Figure 6.

Phosphorylation controls the activity of SCE1. A, schematic of SCE1 and position of identified phosphorylation sites. TM, transmembrane. B, localization of SCE1_M1, SCE1_M2, SCE1_N1, and SCE1_N2 and staining with α-HA and α-GAP45. C, Western blot of complemented lines and staining with α-HA and α-GAP45 as a loading control. D, SCE1-phosphomutant complementation egress assay, stimulated with A23187 for 3 min. Δsce1 (i), Δcdpk3 (ii), and Δcdpk3Δsce1 (iii) backgrounds were complemented with SCE1_wt and two sets of phosphomimetic (SCE1_M1, SCE1_M2) or phospho-null (SCE1_N1, SCE1_N2) mutants. Error bars show mean ± S.D. *, p < 0.05; **, p < 0.001. All statistical tests were performed using two-way ANOVA and Tukey correction for multiple comparisons. The data were derived from three independent biological replicates.

Figure 7.

SCE1 does not control resting concentration or amplitude of cytosolic Ca²⁺. A, the [Ca²⁺]_cyt in extracellular WT parasites (gray), Δcdpk3 parasites (green), Δse1 parasites (blue), and Δcdpk3/Δse1 parasites (red) in the presence of varying [Ca²⁺]_o values. Data are averaged from three to four independent experiments (performed on different days) for each parasite line. B, relative levels of cytosolic Ca²⁺ in intracellular parasites as measured by GCaMP6/mCherry ratio as described previously (25). C and D, the rate of rise in GCaMP6/mCherry fluorescence (C) and peak fluorescence level upon stimulation of egress with either A23187, BIPPO or saponin (D). Error bars show mean ± S.D.

Figure 8.

A hypothetical model of CDPK3- and SCE1-dependent egress. The yellow lightning bolt represents stimulation with A23187 or BIPPO. Arrows represent downstream events, including direct and indirect phosphorylation events, where the thickness of the line represents the hypothetical strength of the signal. A, model of CDPK3/SCE1-dependent egress in wild-type parasites. B, egress upon loss of CDPK3. C, egress upon deletion of both CDPK3 and SCE1. D, suppression of egress upon complementation of with the SCE1 phospho-null mutant. E, egress upon complementation of an SCE1 phosphomimetic mutant.