The role of potassium and host calcium signaling in Toxoplasma gondii egress

Abstract

Toxoplasma gondii is an obligate intracellular parasite and replicates inside a parasitophorous vacuole (PV) within the host cell. The membrane of the PV (PVM) contains pores that permits for equilibration of ions and small molecules between the host cytosol and the PV lumen. Ca²⁺ signaling is universal and both T. gondii and its mammalian host cell utilize Ca²⁺ signals to stimulate diverse cellular functions. Egress of T. gondii from host cells is an essential step for the infection cycle of T. gondii, and a cytosolic Ca²⁺ increase initiates a Ca²⁺ signaling cascade that culminates in the stimulation of motility and egress. In this work we demonstrate that intracellular T. gondii tachyzoites are able to take up Ca²⁺ from the host cytoplasm during host cell signaling events. Both intracellular and extracellular Ca²⁺ sources are important in reaching a threshold of parasite cytosolic Ca²⁺ needed for successful egress. Two peaks of Ca²⁺ were observed in egressing single parasites with the second peak resulting from Ca²⁺ entry. We patched infected host cells to allow the delivery of precise concentrations of Ca²⁺ for the stimulation of motility and egress. Using this approach of patching infected host cells, allowed us to determine that increasing the host cytosolic Ca²⁺ to a specific concentration can trigger egress, which is further accelerated by diminishing the concentration of potassium (K⁺).

Keywords: Calcium signaling; GCaMP6f; GeneticallyEncoded Calcium Indicators; R-GECO; Toxoplasma egress; Toxoplasma gondii.

Figure 1:. Ca²⁺ entry in intracellular T. gondii tachyzoites.

A, Representative Images of HeLa cells stably expressing jRGECO1a that were infected with tachyzoites expressing jGCaMP7f in their PVs. Dashed white outlines indicates the area used for the green fluorescence (PVs) analysis and the red square indicates the region of the host cell used for the analysis of the jRGECO1a fluorescence shown on the graphs. B, Representative Images of HeLa cells transiently expressing the red GECIRGECO infected with tachyzoites expressing cytosolic GCaMP6f. Dashed white outlines show GCaMP6f expressing parasites used for the fluorescence analysis shown in the graphs and the dashed red square shows the region of the host cell used to analyze the RGECO fluorescence. C, Representative images of HeLa cells infected with tachyzoites expressing cytoplasmic GCaMP6f and RGECO in their PVs. Dashed white outlines show GCaMP6-expressing parasites and dashed red outlines indicate the PV region used to analyze the RGECO fluorescence. For the three parts, 1 mM carbachol was added 1 min after recording started. Numbers at the upper right of each panel indicate the time frame of the video. Tracings to the right of each panel shows green fluorescence (GCaMP6f or jGCaMP7f) and red fluorescence (jRGECO1a or RGECO) fluctuations with the scales for the green fluorescence shown on the left Y axis and for the red fluorescence on the right Y scale. Ca²⁺ fluctuations of PVs are shown in A (green) and C (red). Single parasites fluctuations are shown in B and C (green). Host cytosolic changes are shown in A and B (red). Bar graphs represent quantification of the average ΔF values of a minimum of three independent trials (red, jRGECO1a or RGECO) (green, GCaMP6f or jGCaMP7f) with Standard Error of the Mean (S.E.M.) represented as error bars.

Figure 2:. A threshold for the tachyzoite cytosolic Ca²⁺:

HeLa cells expressing the mitochondrial Ca²⁺ indicator LAR-GECO1.2 were infected with tachyzoites expressing cytosolic GCaMP6f for approximately 20 h. The host mitochondria surrounding each PV becomes labeled with LAR-GECO1.2 (red). Dashed white outlines show GCaMP6f-expressing parasites whose fluorescence were used for the analysis in B. Dashed red outlines indicate the region of the host cell that was used to analyze the LAR-GECO1.2 fluorescence in B. Numbers at the upper right of each panel indicate the time frame of the video. A, Addition of 1 μM ionomycin (IO) stimulated egress. B, Fluorescence tracings of single parasites shown in A (green) or the host cell area (red) after addition of IO. The left Y axis scale shows green fluorescence and the right Y axis scale shows red fluorescence changes. Inset bar graph represents ΔFluorescence values of IO response with S.E.M represented by error bars. C, Addition of 2 μM thapsigargin (TG) at 1 min leads to stimulation of red fluorescence (host cytosol) trailed by green fluorescence (parasite cytosol). D, Fluorescence tracings of areas indicated in B (green) or the host cell area (red) after addition of TG. Inset bar graph represents ΔFluorescence values of TG response with S.E.M represented by error bars. E and G, HeLa cells infected with GCaMP6f parasites and IO was added at the indicated concentration (1 and 0.005 μM, respectively). Dashed white outlines indicate the area used as a region of interest to analyze the fluorescence changes shown in F and H. Numbers at the upper right of each panel indicate the time frame of the video. F and H, fluorescence tracings of parasites shown in E and G respectively. I, Average ΔF of GCaMP6 fluorescence after stimulating cultures with 1, 0.1, 0.01, and 0.005 μM IO. The ΔF values of GCaMP6f expressing parasites were determined after the addition of IO. Data from 3 independent sets of experiments were combined and the average was calculated. Error bars represent the S.E.M. **** represent a p-value ≤ 0.0001 in a T-test between 1 μM and 0.005 μM IO average ΔF values. J, Comparison of ΔF values generated by different agonists.

Figure 3:. Two peaks of Ca²⁺ precede egress:

A and D, HeLa cells infected with tachyzoites expressing cytosolic GCaMP6f were stimulated to egress with 100 μM Zaprinast. They were bathed in Ringer buffer supplemented with either 2 mM Ca²⁺ (+ Ca²⁺, A) or 100 μM EGTA (− Ca²⁺, D). The numbers at the upper right of each panel indicate the time frame of the video. B and E, Representative fluorescence tracings of single parasites under Zaprinast simulation in the presence of high or low Ca²⁺, respectively. Note that under low Ca²⁺ conditions (100 μM EGTA) the second peak is reduced. C, Quantification of GCaMP6f peak numbers preceding egress. Graph represents the combined summation of 3 independent experiments. F, Amplitude of the second Ca²⁺ peak after stimulating egress with Zaprinast in the presence of high (+) and low Ca²⁺ (−). G, Average time of egress comparing Zaprinast induced egress from cells resuspended in extracellular media supplemented with either 2 mM Ca²⁺ (+) or 100 μM EGTA (−). Error bars represent the S.E.M. in which **** represents a p-value ≤ 0.0001. H, Intracellular GCaMP6f parasites were treated with 0.01% (w/v) saponin to permeabilize the host cells in Ringer buffer with 2 mM Ca²⁺ (SAP + Ca²⁺). Dashed white outlines represent the parasites that were used for the analysis. Numbers at the upper right of each panel indicate the time frame of the video. I, Representative fluorescence tracings of egressing parasites treated with saponin in +Ca²⁺ conditions taken from H. J, Intracellular GCaMP6f parasites were exposed to Saponin 0.01% (w/v) in Ringer buffer with 1 mM EGTA (SAP – Ca²⁺). Dashed white outlines represent the parasites that were used in the analysis. Numbers at the upper right of each panel indicate the time frame of the video. K, Representative fluorescence tracings of non-egressing parasites treated with saponin in Ca²⁺-free conditions as shown in J. L, amplitude of the ΔF changes from fluorescent parasites in H and J.

Figure 4:. Cytosolic Ca²⁺ during natural egress:

Intracellular parasites expressing cytosolic GCaMP6f were synchronized for natural egress with Compound 1 (cpd1). A, still images of HeLa cells expressing jRGECO1a infected with tachyzoites expressing GCaMP6f and treated with 1 μM cpd1 for 24 hours. After washing off cpd1, parasites egressed within 3-5 min. Dashed white outlines show fluorescent parasites used for the analyses in C and D. Dashed red outlines indicate the region of the host cell used to analyze the jRGECO1a fluorescence. Numbers at the upper right of each panel indicate the time frame of the video. White stars indicate “leader” parasites whose fluorescence begins the egress process. B, still images of HeLa cells expressing jRGECO1a infected with ΔPLP1 tachyzoites expressing cytosolic GCaMP6f and treated with 1 μM cpd1 for 24 hours. Washing off cpd1, results in egress within 3–5 mins. Dashed regions show the ΔPLP1 fluorescent parasites used for the analysis shown in E. Dashed red outlines indicate the region of the host cell that was used to analyze the jRGECO1a fluorescence. Numbers at the upper right of each panel indicate the time frame of the video. White stars indicate “leader” parasites, which shows the highest increase in cytosolic Ca²⁺. C, Fluorescence tracings of host cell jREGO1a (red tracing, scale shown on the right Y axis) and GCaMP6f parasites (green tracings, left Y axis); D, Fluorescence tracings of host cell jREGO1a (red tracing) and a single parasite expressing GCaMP6f (green tracing). Note that parasites that egress show two fluorescence peaks; Inset, Quantification of 3 independent trials of ΔF of jRGECO1a (red bar) and GCaMP6f (green bar) after cpd1 washout. Error bars represent the S.E.M. E, Fluorescence tracings of host cell jREGO1a (red tracing, right Y axis) and ΔPLP1 GCaMP6f parasites (green tracings, Y left axis). Size bars are 5 μm.

Figure 5:. Pharmacological inhibition of Ca²⁺ influx leads to inhibition of egress:

A, HeLa cells infected with tachyzoites expressing cytosolic GCaMP6f were exposed to 2 mM Ca²⁺ (+ Ca²⁺) containing 0.01% (w/v) saponin. B, Fluorescence tracings of egressing parasites (dashed outlines in A) after the addition of saponin in media with 2 mM Ca²⁺; C, HeLa cells infected with tachyzoites expressing GCaMP6f were pretreated for 5 min with 10 μM nifedipine (NIF) in the presence of 2 mM Ca²⁺ (+NIF +Ca²⁺) containing 0.01% (w/v) saponin. Note that parasites did not egress under these conditions; D, Tracings obtained following the fluorescence signal from the parasites highlighted in C. E, quantification and statistical analysis of the ΔF + and − NIF calculated from three independent experiments (p< 0.001). F, HeLa cells infected with tachyzoites expressing cytosolic GCaMP6f were stimulated with 100 μM Zaprinast; G, Tracings obtained following the fluorescence signal from the parasites highlighted in F. G, HeLa cells infected with tachyzoites expressing cytosolic GCaMP6f were pretreated with 1 μM of cpd1 for 5 mins prior to stimulation with 100 μM Zaprinast. Cytosolic Ca²⁺ slightly and briefly oscillated but no egress was evident; I, Tracings obtained following the fluorescence signal from the parasites highlighted in H. Dashed outlines indicate the area used as a region of interest for the analysis of the fluorescence changes shown in the tracings. Numbers at the upper right of each panel indicate the time frame of the video. J, quantification and statistical analysis of the ΔF with and without cpd1 calculated from three independent experiments (p< 0.001). Size bars are 5 μm.

Figure 6:. Role of host cytosolic Ca²⁺ and K⁺ studied by patching the host plasma membrane.

HeLa cells infected with T. gondii tachyzoites were whole-cell patched and egress was monitored. A, whole-cell patch allowed the exposure of PVs to defined Ca²⁺ concentrations by exchanging the cytosol of the host cell with the composition of the buffer inside the patch pipette. B, Representative still images of infected host cells patched under high potassium conditions (140 mM K⁺). Various concentrations of free Ca²⁺ were tested to monitor egress. The percentage of egressing vs non-egressing parasites is shown in the upper left-hand corner. C, Representative still images of infected hosts cells patched under low potassium conditions (10 mM K⁺ and 130 mM choline chloride) and egress monitored under the same experimental conditions as in A. D, Percentage of egressing parasites presented as pie charts of increasing Ca²⁺ concentration. Purple, 140 mM K⁺, pink, 10 mM K⁺. E, Violin Plots of the average time to egress under high (140 mM K⁺) and low potassium conditions (10 mM K⁺). Note that under low K⁺ conditions the percentage of egressing parasites increases, and parasites egress faster. N.S. was used to represent non-significant results and ** was used to represent p-values ≤ 0.01 of T-tests between the different Ca²⁺ concentrations.

Figure 7:. Model of host Ca²⁺ influx during intracellular growth.

Top left: An infected host cell with tachyzoites both with cytosolic Ca²⁺ at resting levels. Intracellular Stores (IS) are filled. Top right: A host cell Ca²⁺ signaling event triggers an increase in cytosolic Ca²⁺. Given that the PV is in equilibrium with the host cell cytosol, PV Ca²⁺ rises simultaneously. Bottom: Rise in PV Ca²⁺ is followed by Ca²⁺ influx into the parasite via a plasma membrane Ca²⁺ channel causing a rise in the cytosolic Ca²⁺ of the parasite. Host cytosolic Ca²⁺ returns to resting level and the tachyzoite cytosolic Ca²⁺ is pumped into intracellular stores (IS). Parasites will continue replicating within the host cell while utilizing the Ca²⁺ influx from the host cell to maintain IS Ca²⁺ filled to be eventually utilized and released during egress. The red scale estimates the concentration of Ca²⁺ in the host cell and PV and the green scale in the parasite cytosol.