An Extracellular Redox Signal Triggers Calcium Release and Impacts the Asexual Development of Toxoplasma gondii

Abstract

The ability of an organism to sense and respond to environmental redox fluctuations relies on a signaling network that is incompletely understood in apicomplexan parasites such as Toxoplasma gondii. The impact of changes in redox upon the development of this intracellular parasite is not known. Here, we provide a revised collection of 58 genes containing domains related to canonical antioxidant function, with their encoded proteins widely dispersed throughout different cellular compartments. We demonstrate that addition of exogenous H₂O₂ to human fibroblasts infected with T. gondii triggers a Ca²⁺ flux in the cytosol of intracellular parasites that can induce egress. In line with existing models, egress triggered by exogenous H₂O₂ is reliant upon both Calcium-Dependent Protein Kinase 3 and diacylglycerol kinases. Finally, we show that the overexpression a glutaredoxin-roGFP2 redox sensor fusion protein in the parasitophorous vacuole severely impacts parasite replication. These data highlight the rich redox network that exists in T. gondii, evidencing a link between extracellular redox and intracellular Ca²⁺ signaling that can culminate in parasite egress. Our findings also indicate that the redox potential of the intracellular environment contributes to normal parasite growth. Combined, our findings highlight the important role of redox as an unexplored regulator of parasite biology.

Keywords: calcium; egress; redox; signaling; toxoplasma.

Figure 1

Schematic representation of a T. gondii tachyzoite displaying 58 redox-associated genes, and their primary protein location as determined by HyperLOPIT (Barylyuk et al., 2020). Gene ID accession numbers are provided, and genes categorized into five groups: black (Trxs); red (PDIs); green (Prxs); blue (Grx-GSHs) and orange (metabolic genes). Further details are provided in Supplementary Table 1.

Figure 2

H₂O₂ induces Ca²⁺ release in intracellular parasites and triggers CDPK3-dependent egress. (A) Representative trace of 100 μM H₂O₂ induction of Ca²⁺ flux in intracellular RH-GFP-T2A-jRCaMP1b parasites. a – c: widefield microscopy images depicting changes in parasite fluorescence signal intensity for both GFP and the Ca²⁺ sensor at baseline (a at 30s), the peak of Ca²⁺ after H₂O₂ addition (b at 270s), and peak of Ca²⁺ after 1 μM A23187 addition (c at 460s). Data are representative of 27 infected vacuoles from four independent experiments. (B) Representative trace of 100 μM H₂O₂ induction of Ca²⁺ flux following pre-treatment of infected host cells with 50 μM α-tocopherol. a: baseline at 30s, b: trace after H₂O₂ addition at 270s and c: peak of Ca²⁺ triggered by A23187 at 520s. Data are representative of 14 infected vacuoles from three independent experiments. For (A, B) black arrows indicate the time of compound addition. Scale bar: 5 μM. (C) Intensity of parasite Ca²⁺ signal increase in RH-GFP-T2A-jRCaMP1b parasites (expressed as a percentage over baseline) following addition of: 1 μM A23187 alone, 1 μM of A23187 following 100 μM H₂O₂ pre-treatment, and 100 μM H₂O₂ alone. Gray dots indicate vacuole data points where parasite egress was observed during the measurement period, blue dots represent vacuoles where egress was not observed. (D) Intensity of parasite Ca²⁺ signal increase (%) over the baseline upon addition of 100 μM H₂O₂ in RH-GFP-T2A-jRCaMP1b versus RH-GFP-T2A-jRCaMP1bΔCDPK3 parasites. For (C, D) histograms present data mean ± SEM of three independent experiments (five vacuoles measured in each experiment), with individual vacuole data points also shown. (E, F) Egress assay measuring tachyzoite release after compound treatment in RH-GFP-T2A-jRCaMP1b and RH-GFP-T2A-jRCaMP1bΔCDPK3, respectively. Data represent the mean ± SEM of three independent experiments (except for DMSO that has two independent experiments), with six technical replicates for each. All data were normalised to the water control. Significance was calculated using one-way Anova, Bonferroni’s multiple comparisons test. P values: *< 0.05; **< 0.01, ***< 0.001 and ****< 0.0001.

Figure 3

Oxidation induced by exogenous H₂O₂ results in a redox change within the PV and parasite cytosol. (A) Schematic representation of the redox sensor based on the catalytic domain of glutaredoxin (GRX1) fused with ro-GFP2 depicting how this sensor interacts with GSH/GSSG and the excitation and emission values of the reduced and oxidized states. (B, C) Proof-of-principle tracking dynamic changes of GSH/GSSG during an oxidation event in RH-GRX1-roGFP2 parasites. (B) Representative signal trace from the GRX1-roGFP2 sensor monitoring both fluorescence channels for reduced (red line) and oxidized (orange line) readouts over time following treatment with 10 mM H₂O₂. Microscopy images of an infected vacuole presented in pseudocolor (orange for oxidation, red for reduction) at a: baseline (45s), b: at the peak of the oxidation event (100s) and c: the return to baseline (280s). This is a representative trace from two independent experiments, with eight vacuoles. Scale bar: 5 µm. (C) Presents the oxidation/reduction ratio of normalized signal from graph (B) depicting the intensity changes in redox compared to baseline upon addition of 10 mM H₂O₂. (D) Representative trace from RH-GRA8-GRX1-roGFP2 parasites depicting 100 μM H₂O₂ induced redox change within the PV. Microscopy images of an infected vacuole highlighting the presence of the sensor within PV (left image), alongside the brightfield image of the infected host cell. (E) A representative trace from RH-GRX1-roGFP2 parasites depicting 100 μM H₂O₂ induction of redox change within the parasite cytosol. Microscopy images depicting an infected vacuole highlighting the presence of the sensor on cytosol (left panel image) alongside the brightfield image of the infected host cell. (D, E) Representative traces from three independent experiment, nine vacuoles for each group. Scale bar: 5 µm. (F, G) Representative trace of redox fluctuations in intracellular parasite following 1 μM ionomycin treatment for RH-GRA8-GRX1-roGFP2 (F), and RH-GRX1-roGFP2 parasites (G). (F, G) red arrows indicate the moment of parasite egress. Representative traces from three independent experiments, nine vacuoles for each group.

Figure 4

The GRX1-roGFP redox sensor affects T gondii asexual replication. (A) Bar graphs presenting plaque count data from a six-day plaque assay using RH-GFP-Luc parasites as a reference control group. (B) Representative images of plaques formed. Small plaques are indicated by white arrows. Scale bar: 2 mm. (C) Violin plot presenting the distribution of plaque areas (mm²) for parasites expressing the redox sensor within the cytosol. (D) Histogram presenting the effect of the GRX1-roGFP sensors and NAC on parasite intracellular replication. (A–D) All obtained from three independent experiments, with three technical replicates. Significance was calculated using one-way Anova, Bonferroni’s multiple comparisons test for (A, B). P values: **< 0.01, **< 0.001 and ****< 0.0001. The significance analyse for (D) is provide on Supplementary Figure 6.