Calcium entry in Toxoplasma gondii and its enhancing effect of invasion-linked traits

Abstract

During invasion and egress from their host cells, Apicomplexan parasites face sharp changes in the surrounding calcium ion (Ca(2+)) concentration. Our work with Toxoplasma gondii provides evidence for Ca(2+) influx from the extracellular milieu leading to cytosolic Ca(2+) increase and enhancement of virulence traits, such as gliding motility, conoid extrusion, microneme secretion, and host cell invasion. Assays of Mn(2+) and Ba(2+) uptake do not support a canonical store-regulated Ca(2+) entry mechanism. Ca(2+) entry was blocked by the L-type Ca(2+) channel inhibitor nifedipine and stimulated by the increase in cytosolic Ca(2+) and by the specific L-type Ca(2+) channel agonist Bay K-8644. Our results demonstrate that Ca(2+) entry is critical for parasite virulence. We propose a regulated Ca(2+) entry mechanism activated by cytosolic Ca(2+) that has an enhancing effect on invasion-linked traits.

Keywords: Calcium; Cell Invasion; Conoid Extrusion; Fluorescence; Gliding Motility; Nifedipine; Parasite; Protozoan; Signaling; Toxoplasma gondii.

FIGURE 1.

Ca²⁺ entry in extracellular tachyzoites. Measurements were made in low Ca²⁺ EB (see “Experimental Procedures”). A, addition of Ca²⁺ (1 mm) to Fura-2/AM-loaded tachyzoites leads to an increase in cytosolic Ca²⁺. Inset, no addition. B, addition of various extracellular Ca²⁺ concentrations leads to higher intracellular Ca²⁺ increase. At 100 s, varying concentrations of Ca²⁺ were added to the extracellular buffer: 2 mm (blue), 1.5 mm (red), 1 mm (green), and no addition (black). C, increase in extracellular Ca²⁺ concentration resulted in a large increase in cytosolic Ca²⁺ followed by a plateau at ∼200 nm. D, amplification of the boxed area shown in C. The error bars in all cases indicate means ± S.E. (n = 3).

FIGURE 2.

Ca²⁺ entry in tachyzoites is enhanced in the presence of thapsigargin but entry is not through a canonical store-regulated channel. Parasites (5 × 10⁷ cells) were loaded with Fura-2/AM and resuspended in 2.5 ml of EB in the presence of 100 μm EGTA. A, cytosolic Ca²⁺ changes upon addition of 1 μm TG at 100 s and then 1 mm CaCl₂ at 400 s (blue line). The red line represents the cytosolic Ca²⁺ changes after Ca²⁺ addition alone. The inset shows quantification of the initial rate of Ca²⁺ entry after adding Ca²⁺. At least three independent experiments were used for the quantification. B, Ca²⁺ entry measured by Mn²⁺ quenching. Fura-2/AM-loaded parasites were monitored at the Fura-2 isosbestic fluorescence point (excitation = 360 nm). The blue tracing is the tracing obtained after adding 1 μm TG. The inset shows quantification of the rate of MnCl₂ uptake from three or more independent experiments. 2 mm MnCl₂ was added where indicated. C, Ba²⁺ entry and binding of Fura-2. The blue line shows the increase in Fura-2 fluorescence after store depletion with 1 μm TG. The red line indicates increase in fluorescence without depletion of intracellular stores. There is no significant difference in the initial rate of fluorescence increase after Ba²⁺ addition (1 mm) under both conditions (inset). The inset shows the quantification of the initial rate of BaCl₂ uptake from a minimum of three independent experiments. D, Gd³⁺ inhibition. The blue line shows preincubation with 5 μm Gd³⁺ and addition of 1 μm TG and then 1 mm Ca²⁺. The red line shows increased Fura-2 fluorescence after store depletion with 1 μm TG alone. The inset shows quantification of initial rates of Ca²⁺ uptake. A minimum of three independent experiments was used for the quantification. E, Ca²⁺ entry enhanced by TG is not affected by 2-APB. 2-APB (30 μm) was added where indicated. The error bars in all cases indicate means ± S.E. (n = 3).

FIGURE 3.

Role of [Ca²⁺]_i in Ca²⁺ entry as detected by varying the time interval between TG and Ca²⁺ additions. Fura-2/AM loaded parasites were treated with 1 μm TG and Ca²⁺ added back at different times. A, the red line shows a representative tracing where Ca²⁺ (1 mm) was added 50 s after TG (at maximum TG response, red arrow). Green line shows the response to addition of Ca²⁺ 100 s after TG (green arrow). The blue line represents a time interval of 150 s (after TG response, blue arrow). Inset, quantified responses for four independent experiments measuring Ca²⁺ entry for Ca²⁺ alone (Ca²⁺) and Ca²⁺ addition at different time points (in seconds) after TG addition. Rates of Ca²⁺ entry were measured in the first 20 s. Rate of Ca²⁺ entry was significantly higher at 50 s when the cytosolic Ca²⁺ was highest (ANOVA, p < 0.05). B, Ca²⁺ entry in T. gondii after the addition of TG. Fura-2/AM loaded parasites in EB were treated with 1 μm TG at 50 s, and then the concentrations of Ca²⁺ indicated were added at 100 s. For reference, the increase in cytosolic Ca²⁺ upon addition of TG alone is also shown (gray line). Inset, quantification of the initial Ca²⁺ entry rates of three or more independent experiments. Gray bars represent increase in cytosolic Ca²⁺ after adding only extracellular Ca²⁺, colored bars represent increase when the same amount of Ca²⁺ addition is preceded by 1 μm TG. Error bars in all cases indicate means ± S.E. (n ≥ 3).

FIGURE 4.

Ca²⁺ entry inhibition and stimulation. Conditions as in Fig. 2. A, Ca²⁺ entry (1 mm) is inhibited in parasites preincubated for 2 min with nifedipine (Nif) ranging from 1 to 10 μm. Inset, quantification of initial rates measure from at least three independent experiments. B, Mn²⁺ quenching inhibition by nifedipine. Fura-2/AM-loaded parasites were monitored at the Fura-2 isosbestic fluorescence point (excitation = 360 nm). 2 mm MnCl₂ was added where indicated. Inset, rate of Mn²⁺ quenching was significantly decreased (ANOVA of slope: n = 4, p < 0.05) by nifedipine. C, Bay K-8644 enhances Ca²⁺ entry. Fura-2/AM-loaded parasites in EB were analyzed in the presence of 100 μm EGTA, and 1 mm Ca²⁺ was added at the indicated time. Inset, quantification of initial rates ± S.E. after adding Ca²⁺ of at least three independent experiments. D, Mn²⁺ entry in tachyzoites in Fura-2/AM-loaded parasites (5 × 10⁷ cells in EB). Quenching by Mn²⁺ was measured in the presence of Bay K (5 μm) (red line) or 10 μm nifedipine (maroon line). Mn²⁺ (2 mm) was added where indicated (100 s). The conditions were 2 mm MnCl₂ + DMSO (control entry, gray line), 2 mm MnCl₂ + 5 μm BayK (red line), and 2 mm MnCl₂ + 10 μm nifedipine (maroon line). Inset, rates of change in Fura-2 fluorescence at the isosbestic point ± S.E. (n = 4) during the first 20 s after addition of manganese alone or plus 1, 2.5, and 5 μm Bay K. **, p < 0.01 (one-way ANOVA).

FIGURE 5.

Nifedipine inhibits Ca²⁺ entry enhanced by thapsigargin. A, Ca²⁺ accumulation enhanced by TG (1 μm), which was added at 50 s followed by 2 mm Ca²⁺ at 100 s (red line), is inhibited by preincubating parasites with 10 μm nifedipine (blue line). The black line is the baseline cytosolic Ca²⁺ level, the gray line shows addition of TG, and the light blue line shows the response to Ca²⁺ addition alone. Inset, quantification of rate of Ca²⁺ entry after nifedipine inhibition. One-way ANOVA of regression model yielded a significant relationship between inhibition of Ca²⁺ entry and nifedipine concentration: p < 0.001. B, Bay K enhances Ca²⁺ entry. TG (1 μm), Ca²⁺ (2 mm) and Ca²⁺ + Bay K (5 μm) were added where indicated. Blue line, 2 mm Ca²⁺ + TG + BayK; red line, Ca²⁺ + TG; light blue line, baseline Ca²⁺ addition, green line is the response to TG (1 μm) alone; black line, cytosolic Ca²⁺ with no additions. Inset shows the quantification of the rate of Ca²⁺ entry for multiple replicate measurements of TG + Ca²⁺ (red bar) and TG + Ca²⁺ + Bay K (blue bar). ***, p < 0.01 (one-way ANOVA). Conditions as in Fig. 3.

FIGURE 6.

Ca²⁺ entry enhances gliding motility and conoid extrusion. A, gliding trails visualization by SAG1 immunolocalization. Representative micrographs showing the SAG1 signal after different treatments (see label details below). These micrographs were used for the quantification number of motile parasites in B and the average trail length in C. Scale bar, 5 μm. B, average percentage (n = 5 ± S.E.) of extracellular tachyzoites that were directly associated with a gliding trail. C, the average length (n = 100 ± S.E.) of parasites directly associated with a gliding trail. TG, TG (2 μm); Ca²⁺ = 1.6 mm free Ca²⁺; TG + Ca²⁺, 2 μm TG and 1.6 mm free Ca²⁺; Nifedipine, same as TG + Ca²⁺ with a 60-s preincubation with 10 μm nifedipine; IO = 1 μm ionomycin. D, role of extracellular Ca²⁺ on conoid extrusion. Representative images obtained after the treatments: extracellular parasites were collected and exposed to Ca²⁺ (2 mm, EB), ionomycin (1 μm IO), or both (IO + Ca²⁺) for 5 min at 37 °C. E, quantification of conoid extrusion response. Parasites were collected at specified times, fixed, and assessed for the percentage of parasites with an extruded conoid. Inset, kinetic analysis of conoid extrusion in extracellular parasites. Kinetic monitoring of conoid extrusion shows that when ionomycin-treated parasites were also given extracellular Ca²⁺ (2 mm), they responded with a greater percentage of extruded conoids and for extended amount of time (red symbols) as evidenced by a statistically nonsignificant slope value (p > 0.05). Parasites treated with ionomycin in buffer with cytosolic levels of Ca²⁺ (110 nm) responded with an initial increase in conoid extrusion that displayed exponential decay kinetics (green symbols). F, effect of nifedipine (10 μm, Nif) and Bay K (2.5 μm) on conoid extrusion. Parasites were treated as described under “Experimental Procedures,” and the percentage of parasites with their conoid extruded was evaluated at 300 s. The average ± S.E. of three experiments of parasites with their conoids extruded under extracellular buffer with Ca²⁺ (1 mm) was 21.3%. Incubation with nifedipine reduced this number to 12% (p = 0.0062) and in the presence of Bay K; 23.7% of the parasites had their conoids extruded. Measurements were done using parasites suspended in extracellular buffer.

FIGURE 7.

Extracellular Ca²⁺ entry enhances microneme secretion and host cell invasion. A, Western blot analysis of a representative MIC2 secretion experiment (upper panel) under different extracellular Ca²⁺ concentrations. Lower panel is secretion control with anti-GRA1. Where indicated, 1 μm TG was added. MIC2 signal was below the level of detection at 0 mm Ca²⁺. B, quantification of gel band intensity of MIC2 secretion (± S.E.) from three independent experiments (one of which is shown in A) with (blue bars) and without (red bars) TG addition. Experiments were standardized to loading controls and quantified using ImageJ (National Institutes of Health). C, relationship of extracellular Ca²⁺ concentration and number of parasites attached to or invaded into host cells as determined by a red-green invasion assays. Both attachment and invasion showed significant relationships with Ca²⁺ concentration (p < 0.01 for both attachment and invasion). D, inhibition of invasion and attachment by nifedipine. IM, invasion medium; DMSO, = vehicle control; Nifedipine, 10 μm nifedipine. **, p < 0.01.

FIGURE 8.

Model for the role of Ca²⁺ entry in the lytic cycle of T. gondii. The proposed model shows Ca²⁺ changes in the cytosol and intracellular stores (IS) of the parasite when exposed to changes in extraparasite Ca²⁺ concentrations during the lytic cycle of T. gondii. Step 1, cytoplasmic Ca²⁺ [Ca²⁺]_i increases in the tachyzoite (darker orange) before egress because of Ca²⁺ release from intracellular stores. Step 2, once outside the parasite, [Ca²⁺]_i returns to nanomolar levels (lighter orange). Step 3, because of large electrochemical gradient, extracellular Ca²⁺ (represented by darker blue) enters parasite cytosol through the regulated mechanisms observed in this study (darker orange in tachyzoite). Step 4, according to our data, release of Ca²⁺ from intracellular stores could contribute to this increase by an unknown mechanism. Step 5, an increase in cytosolic Ca²⁺ would lead to initiation of invasion. Step 6, gliding, conoid extrusion, microneme secretion, and invasion. Step 7, the parasite [Ca²⁺]_i (lighter orange) decreases by the action of Ca²⁺ pumps at the plasma membrane and the SERCA at the ER. This last event would result in replenishment of the intracellular stores. Step 8, tachyzoite invades its host cell and forms a parasitophorous vacuole where it resides and replicates exposed to host [Ca²⁺]_i (∼50–100 nm). The blue color bar represents relative extraparasite Ca²⁺ concentration. The orange color bar represents parasite Ca²⁺ concentration. The arrows show Ca²⁺ fluxes. The black lines and dots on invading parasites represent conoid extrusion and microneme secretion, respectively. (Christina A. Moore drew the model.)