Calcium Entry in Toxoplasma gondii and Its Enhancing Effect of Invasion-linked Traits

Background: Toxoplasma gondii is exposed to large Ca²⁺ gradients during its lytic cycle.

Results: Ca²⁺ entry in T. gondii is a source of Ca²⁺ increase and is mediated by a nifedipine-sensitive pathway and not by a canonical store-operated Ca²⁺ entry (SOCE) pathway.

Conclusion: Ca²⁺ entry enhances parasite virulence traits.

Significance: This is the first study linking regulation of Ca²⁺ entry and virulence traits of T. gondii.

Abstract

During invasion and egress from their host cells, Apicomplexan parasites face sharp changes in the surrounding calcium ion (Ca²⁺) concentration. Our work with Toxoplasma gondii provides evidence for Ca²⁺ influx from the extracellular milieu leading to cytosolic Ca²⁺ increase and enhancement of virulence traits, such as gliding motility, conoid extrusion, microneme secretion, and host cell invasion. Assays of Mn²⁺ and Ba²⁺ uptake do not support a canonical store-regulated Ca²⁺ entry mechanism. Ca²⁺ entry was blocked by the L-type Ca²⁺ channel inhibitor nifedipine and stimulated by the increase in cytosolic Ca²⁺ and by the specific L-type Ca²⁺ channel agonist Bay K-8644. Our results demonstrate that Ca²⁺ entry is critical for parasite virulence. We propose a regulated Ca²⁺ entry mechanism activated by cytosolic Ca²⁺ that has an enhancing effect on invasion-linked traits.

Introduction

Elevation in cytoplasmic Ca²⁺ mediates a plethora of cellular responses in all cells. Protein function is dependent on shape and charge, and both of these features are affected by Ca²⁺ binding. Cells contain a sophisticated set of mechanisms to balance the cytosolic Ca²⁺ concentration, and the signals that increase Ca²⁺ in the cytosol are compensated by mechanisms that decrease it (1).

Ca²⁺ entry plays an important role in replenishing intracellular organelles and in activating signaling pathways that respond to elevated cytosolic Ca²⁺ (2). Five types of plasma membrane Ca²⁺ channels are present in vertebrate cells such as the store-operated channel (ORAI),⁵ which is linked to the endoplasmic reticulum sensor protein stromal interaction molecule (STIM), voltage-operated channels, ligand-operated channels, transient receptor potential (TRP) channels, and second messenger-operated channels (3), but little is known about the mechanisms involved in Ca²⁺ entry in the Apicomplexan parasite Toxoplasma gondii.

During its lytic cycle, T. gondii actively invades host cells, creating a parasitophorous vacuole, where it divides to finally exit in search of a new host cell. Parasite invasion is a highly coordinated and active process involving several discrete steps (4). Gliding motility, conoid extrusion, secretion of specific proteins, attachment to the host cell, and active invasion are all critical steps for invasion. Previous studies have shown that intracellular Ca²⁺ stores are important for initiation of gliding motility (5), microneme secretion (6), conoid extrusion (7), active parasite invasion (8–10), and egress (8) (reviewed in Ref. 11), but the role of extracellular Ca²⁺ in these processes was not studied in detail or was considered minor.

Considering the enormous Ca²⁺ concentration gradient (∼20,000-fold), to which tachyzoites are exposed upon egress (100 nm in the host cytosol, taking into account that the parasitophorous vacuole functions as a molecular sieve (12) versus ∼1.8 mm in the extracellular space), it seems a feasible hypothesis that the extracellular stage represents an opportunity to use extracellular Ca²⁺ as a mechanism to enhance Ca²⁺-dependent invasion processes as well as to replenish intracellular Ca²⁺ stores that are likely used during the process of parasite egress. In this work, we explore the mechanism of Ca²⁺ entry in extracellular tachyzoites and its role during the lytic cycle of the parasite. We present evidence for a regulated Ca²⁺ entry process and its enhancing effect on invasion-related traits. This is the first detailed study linking Ca²⁺ entry and its regulation directly to virulence traits of an eukaryotic microbe.

EXPERIMENTAL PROCEDURES

Cell Culture and Preparation

T. gondii tachyzoites (RH strain) were maintained in hTERT human fibroblasts using DMEM with 1% fetal bovine serum. Intracellular tachyzoites were isolated under intracellular conditions by scraping off infected fibroblasts and passing them through a 27-gauge needle. In this way, parasites were released from host cells while still at intracellular ionic conditions. Extracellular tachyzoites were collected from cultures with 50–75% of egressed parasites. Parasites (both intracellular and extracellular) were purified as described (13) and washed in either low Ca²⁺ extracellular buffer (EB) or intracellular buffer (IB) (see below).

Experiments testing Ca²⁺ entry were conducted using EB (116 mm NaCl, 5.4 mm KCl, 1 mm MgSO_4, 5.5 mm glucose, 20 mm Hepes, pH 7.4), IB (10 mm NaCl, 130 mm KCl, 10 mm glucose, 0.8 mm MgCl₂, 1 mm EGTA, 0.62 mm CaCl₂, 20 mm Hepes, pH 7.4) (final free Ca²⁺, 117 nm) or buffer A (116 mm NaCl, 5.4 mm KCl, 0.8 mm MgSO₄, 5.5 mm d-glucose, and 50 mm Hepes, pH 7.4). For low Ca²⁺ EB, 1 mm EGTA and 0.62 mm CaCl₂ were added to bring the free Ca²⁺ to 110 nm. The final Ca²⁺ concentrations were determined by using CaCl₂-EGTA combinations and calculated using Maxchelator software.

Cytosolic Ca²⁺ Measurements

Parasites (intracellular or extracellular) were loaded with Fura-2/AM as described (14). Briefly, after harvesting the cells, they were washed twice at 500 × g for 10 min at room temperature in the buffer indicated in each figure legend. The cells were resuspended to a final density of 1 × l0⁹ cells/ml in loading buffer (similar to the isolation buffer plus 1.5% sucrose and 5 μm Fura-2/AM). The suspensions were incubated for 26 min in a 26 °C water bath with mild agitation. Subsequently, the cells were washed twice to remove extracellular dye. Cells were resuspended to a final density of 1 × 10⁹ cells/ml in the indicated buffer and kept in ice. Parasites were viable for several hours under these conditions. For fluorescence measurements, a 50-μl portion of cell suspension was diluted into 2.5 ml of the indicated buffer (final density, 2 × l0⁷ cells/ml) in a cuvette placed in a Hitachi F-4500 spectrofluorometer. Excitation was at 340 and 380 nm, and emission was at 510 nm. The Fura-2 fluorescence response to intracellular Ca²⁺ concentration ([Ca²⁺]_i) was calibrated from the ratio of 340/380-nm fluorescence values after subtraction of the background fluorescence of the cells at 340 and 380 nm as described by Grynkiewicz et al. (15). Ca²⁺ influx (ΔCa²⁺/s) was evaluated by measuring the rate of [Ca²⁺]_i change during the first 20 s after its addition (linear rate) when the mechanisms of compensation are not yet at full capacity. Manganese quenching experiments were conducted on Fura-2/AM-loaded parasites, and Fura-2 fluorescence was monitored at the isosbestic wavelength (excitation = 360 nm, emission = 510 nm). MnCl₂ was added to a final concentration of 2 mm, and the rate of Fura-2 fluorescence quenching was recorded. ΔFU was calculated by measuring the rate of change in fluorescence during the first 20 s after adding manganese (when the rate is linear). No Fura-2 leakage into the extracellular media was detected (data not shown).

Gliding Assays

Purified parasites were lightly adhered to polylysine-coated coverslips by placing them (2 × 10⁷ cells/ml) on a coverslip on ice for 4 min. Non-adhered cells were washed away, and coverslips were incubated at different conditions at 37 °C for 10 min, and immunofluorescence images were acquired as described previously (5). Images were acquired using an Olympus IX-71 inverted microscope with a Photometrix CoolSnap CCD camera operated by DeltaVision image reconstruction software (Applied Precision, Seattle, WA). Gliding trail length was quantified with ImageJ software (National Institutes of Health) by measuring trails (positively stained with SAG1 antibody) that were directly associated with a parasite. For all treatments, at least 50 parasite trails were measured within each experiment.

Conoid Extrusion

Conoid extrusion was tested following the protocol of Mondragon and Frixione (7). Parasites were kept in IB at 110 nm free Ca²⁺. Parasites were spun down and resuspended in IB or EB at different conditions (e.g. Ca²⁺ or in the presence of the calcium ionophore ionomycin). Experiments were initiated by placing the parasites at 37 °C and terminated by the addition of 4% formaldehyde. The percentage of parasites with their conoids extruded, reflecting the initiation of the invasion process, was assessed using an Olympus BX60 microscope. At least 100 parasites were evaluated for treatment within each independent experiment.

Microneme Secretion

Purified intracellular parasites were collected in IB and resuspended in the same buffer with varying amounts of free calcium. Microneme secretion was measured at 37 °C for 15 min following published protocols (6). Rabbit anti-GRA1 at 1:10,000 was used as secretion control.

Host Cell Invasion

Red-Green assay was modified from the assay described previously (16). Intracellular RH tachyzoites were collected in IB, centrifuged, and resuspended in the following concentrations of free calcium: 160 nm, 99.3 μm, 1 mm, and 2 mm at a parasite concentration of 1 × 10⁸ cells/ml. 2.5 × 10⁷ tachyzoites were added to subconfluent hTERT monolayers, allowed to settle for 15 min on ice, and then incubated for 2 min at 37 °C. Subsequently, the medium was aspirated and fixed with 2.5% formaldehyde for 20 min. Subsequent steps were done as published (16). Cell counting data from three independent experiments were collected; each one by triplicate, counting eight randomly selected fields per individual coverslip. The number of invaded parasites was calculated as the difference between the total number of parasites (green) and the number of attached parasites (red). Nifedipine inhibition of invasion was tested in invasion media (DMEM, 10 mm Hepes, pH 7.4, and 3% fetal bovine serum).

RESULTS

Evidence for a Regulated Ca²⁺ Entry Pathway in T. gondii Tachyzoites

Upon egress from their host cell, T. gondii tachyzoites face a large change in extracellular Ca²⁺ concentration from nanomolar levels, when in contact with the host cytoplasm through the parasitophorous vacuole, to ∼1.8 mm, in the extracellular milieu. Our hypothesis is that tachyzoites allow for some of this Ca²⁺ to enter but in a regulated manner. To simulate conditions present upon exit of tachyzoites from the host cells, we suspended parasites obtained under intracellular conditions (see “Experimental Procedures”) and loaded them with Fura-2/AM at a Ca²⁺ concentration usually present in the cytosol (110 nm; low Ca²⁺ EB; see “Experimental Procedures”). The addition of extracellular Ca²⁺ (∼1 mm) elicited an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (Fig. 1A) that slowly returns to the basal level. Higher extracellular Ca²⁺ concentrations (1.5 and 2 mm) led to higher cytosolic Ca²⁺ (Fig. 1B). We measured [Ca²⁺]_i over a wide range of extracellular Ca²⁺ concentrations (Fig. 1C), consistently observing an increase in final [Ca²⁺]_i (after return to basal levels) up to 0.6 μm (Fig. 1D). Higher extracellular Ca²⁺ concentrations (1.5–2 mm) led to minor changes in intracellular levels (Fig. 1C). Although there was significant entry of Ca²⁺, its final concentration was orders of magnitude lower than the extracellular concentration (Fig. 1C). The curve in Fig. 1C shows a biphasic phenomenon, which could be due to the participation of more than one Ca²⁺ permeation pathway. Tachyzoites isolated under extracellular conditions (see “Experimental Procedures”) also showed this phenomenon of Ca²⁺ entry upon addition of Ca²⁺ (data not shown) (see “Experimental Procedures” for buffer compositions).

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).

This phenomenon of saturable [Ca²⁺]_i increase with increasing extracellular Ca²⁺, and the maintenance of cytosolic Ca²⁺ levels orders of magnitude below that of the extracellular Ca²⁺ concentration, support the conclusion that entry of extracellular Ca²⁺ is regulated by the parasite and probably occurs through the coordination of specific entry channels (probably more than one) and compensatory mechanisms operated by Ca²⁺ pumps.

Calcium Entry Is Not Stimulated by Store Depletion but Blocking the SERCA-type Ca²⁺ ATPase Leads to Higher Cytosolic Ca²⁺ Levels

We next investigated whether Ca²⁺ entry could be further enhanced by depletion of Ca²⁺ from intracellular stores (store-operated Ca²⁺ entry, or SOCE), a mechanism proposed to operate in the related Apicomplexan Plasmodium falciparum (17). To test for SOCE, we used thapsigargin (TG), an inhibitor of the SERCA-type Ca²⁺-ATPase, which results in leakage of Ca²⁺ from the endoplasmic reticulum as a consequence of inhibiting the uptake pathway. The addition of TG 300 s prior to adding CaCl₂ resulted in a reproducible higher influx, which can be observed by an increase in [Ca²⁺]_i as compared with the control in the absence of TG (Fig. 2A).

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).

The results described above (Fig. 2A) seemed to indicate the existence of SOCE in T. gondii. However, the increases in [Ca²⁺]_i evoked by restoring Ca²⁺ with and without prior TG treatment differed only in that the former had a transient overshoot, but thereafter the “sustained” phases were similar (after the initial peak). These results seem most easily explained by a larger increase in [Ca²⁺]_i because of the inhibition of SERCA, which can no longer contribute to sequester incoming Ca²⁺. We next tested this possibility by different strategies. The surrogate ion Mn²⁺ is known to permeate through Ca²⁺ channels, effectively quenching Fura-2 fluorescence. In addition, Mn²⁺ is a poor substrate for most Ca²⁺-dependent enzymes/processes so that any quenching observed in Fura-2/AM-loaded parasites would be due to Mn²⁺ entry. An abrupt quenching of cytosolic Fura-2 fluorescence was observed at the Ca²⁺-independent excitation wavelength of 360 nm (Fig. 2B). However, the rate of quenching was not increased by thapsigargin, suggesting that this flux of Mn²⁺ was not stimulated through store depletion (Fig. 2B, inset). To further investigate this phenomenon, we used Ba²⁺, which enters cells through Ca²⁺ channels and binds Fura-2 modifying its fluorescence in a similar way to Ca²⁺ (18). We measured the initial rate of entry under identical conditions used to measure Ca²⁺ entry after thapsigargin (Ca²⁺ “add back”), and rates were similar in the presence and absence of thapsigargin (Fig. 2C, inset). This is in agreement with the Mn²⁺ quenching experiment and indicates that the phenomenon of Ca²⁺ entry is likely not store-regulated. We also tested two inhibitors of SOCE channels, gadolinium (Gd³⁺) at 5 μm (Fig. 2D) and 2-aminomethoxydiphenyl borate (2-APB) at 30 μm (Fig. 2E). We did not observe any effect on Ca²⁺ entry with any of these inhibitors. Although it appears as if Gd³⁺ inhibits the amount of Ca²⁺ entry, initial rates (see “Experimental Procedures” for explanation of calculations) were not significantly different to the initial rate of Ca²⁺ entry after adding Ca²⁺ alone (Fig. 2D, inset). It is possible that Gd³⁺ could partially block a plasma membrane voltage-gated calcium Ca²⁺ channel (VGCC) as reported previously (19), limiting the amount of Ca²⁺ entering, but the initial rate is not affected.

In summary, our results do not support the presence of a Ca²⁺ entry channel regulated by store depletion. The absence of store regulation of Ca²⁺ entry is supported by the lack of genomic evidence for the presence of SOCE molecular players (STIM and ORAI) in any Apicomplexan parasite (EuPath.DB).

The Influence of Cytosolic Ca²⁺ on Ca²⁺ Entry

The increased Ca²⁺ accumulation after TG addition as a result of SERCA inhibition could result in opening of a plasma membrane Ca²⁺ channel through a Ca²⁺ induced Ca²⁺ uptake mechanism. To study whether this was the case, we investigated whether there was a correlation between the [Ca²⁺]_i levels and the rate of Ca²⁺ entry. The addition of TG leads to an increase in cytosolic Ca²⁺ and a decrease after reaching a maximum caused by reuptake or efflux of released Ca²⁺. We added Ca²⁺ back at different times after the addition of TG (50, 100, and 150 s) at points when the cell reached different levels of cytosolic Ca²⁺ (Fig. 3A, inset). If Ca²⁺ entry was modulated by cytosolic Ca²⁺, then as [Ca²⁺]_i decays, so too would the rate of influx of extracellular Ca²⁺. Calcium “add back” at different times after TG showed that there is a significant difference between the rate of Ca²⁺ entry at 50 s versus 100 and 150 s after TG addition (Fig. 3A, inset). Ca²⁺ entry rate at 100 and 150 s was ∼30% lower than the rate at 50 s, which is when [Ca²⁺]_i is highest.

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).

We also observed that the rate of Ca²⁺ entry after addition of 1, 1.5, and 2.0 mm Ca²⁺ after TG addition increased significantly when compared with the rates of entry with the same Ca²⁺ concentrations without TG (Fig. 3B) (compare gray and color bars of Fig. 3B, inset). We added Ca²⁺ back 50 s after the addition of TG, and we observed that the rate of Ca²⁺ entry was consistently 2.5 times higher in the presence of TG for all the treatments (Fig. 3B, inset). This enhancing effect of TG was independent of the ionic conditions used for purification of parasites and measurements (data not shown). In summary, Ca²⁺ release from intracellular stores results in an increase in cytosolic Ca²⁺, and this elevated Ca²⁺ could directly modulate a Ca²⁺ entry channel. This result agrees with the results shown in Fig. 2A where a higher level of Ca²⁺ entry was observed after blocking the SERCA with TG.

Inhibition of Ca²⁺ Entry

To investigate the mechanism of Ca²⁺ entry we tested a range of channel blockers and inhibitors like 2-APB (30 μm) (20), nifedipine (1–10 μm), verapamil (1–100 μm), and diltiazem (10–100 μm) (21). Only nifedipine, an inhibitor of L-type voltage-activated Ca²⁺ channels, was effective in blocking Ca²⁺ entry (Fig. 4A). 2-APB at 30 μm had no effect on Ca²⁺ entry enhanced by thapsigargin, supporting the lack of participation of a SOCE Ca²⁺ channel (Fig. 2E). Nifedipine also decreased Mn²⁺ quenching in a dose-dependent manner (Fig. 4B), suggesting that entry of extracellular Ca²⁺ is through a channel-mediated process and that nifedipine is an effective blocker of such Ca²⁺ entry.

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).

Taking into account the inhibition of Ca²⁺ entry by nifedipine, we tested Bay K-8644 (Bay K), a Ca²⁺ channel agonist that acts by opening L-type channels (Ca_V1 family), increasing the time the channel is open and causing a large increase in the channel macroscopic response (22). Bay K (2.5–5 μm) stimulated Ca²⁺ entry when tested in Fura-2-loaded cells (Fig. 4C, inset). Using the Mn²⁺-quenching technique (Fig. 4D), we found that Ca²⁺ entry in extracellular tachyzoites was significantly enhanced when parasites were treated with 5 μm Bay K (Fig. 4D). The inset in Fig. 4D shows the quantification of rates obtained after adding 1, 2.5, and 5 μm Bay K.

We also tested nifedipine and Bay K after blocking the SERCA with TG to obtain higher cytosolic Ca²⁺ levels (Fig. 5). We observed a significant inhibition by nifedipine of Ca²⁺ entry measuring the initial rate (Fig. 5A, inset). The addition of TG followed by the simultaneous addition of CaCl₂ and 5 μm Bay K (blue trace) elicited a greater accumulation of Ca²⁺ over that of TG and Ca²⁺ alone (Fig. 5B, red trace). The quantification of these relative differences in these measurements demonstrated that Bay K addition resulted in a significant increase (ANOVA comparison of slopes: p < 0.001) in Ca²⁺ accumulation (Fig. 5B, inset).

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.

The Role of Ca²⁺ Entry in Virulence-related Traits: Gliding Motility, Conoid Extrusion, Microneme Secretion, and Host Cell Invasion

Previous work (6–10) has shown that [Ca²⁺]_i increase is involved during all these steps of the tachyzoite lytic cycle, but the source of this Ca²⁺ increase has been proposed to be mostly Ca²⁺ release from intracellular Ca²⁺ stores on the basis of experiments using high concentrations of extracellular EGTA to prevent Ca²⁺ entry. Our hypothesis is that upon egress, the parasite encounters a ∼20,000-fold higher concentration of Ca²⁺, and this gradient should result in a strong electrochemical driving force favoring Ca²⁺ influx. We therefore investigated whether this Ca²⁺ influx plays a role in gliding motility, conoid extrusion, microneme secretion, and host cell invasion. We isolated parasites using both intra- and extracellular ionic concentrations and exposed them to higher, physiologically relevant Ca²⁺ concentrations to quantify these processes.

Parasites treated with different reagents to promote high cytosolic Ca²⁺ concentration were stained to determine whether they possessed a SAG1 positive gliding trail and the length of the trail. Parasites kept in standard buffer conditions showed only minimal movement and relatively small gliding trails (Fig. 6A, no additions). Parasites that were placed in the physiological context of Ca²⁺ entry by addition of extracellular Ca²⁺ or that had an increase in Ca²⁺ accumulation by addition of TG and extracellular Ca²⁺ (Fig. 6A, TG + Ca²⁺) or ionomycin (Fig. 6A, IO) showed an increase in both the percentage of gliding parasites and the length that the parasites moved (Fig. 6, B and C). Fig. 6A shows representative images used to obtain the quantification shown in Fig. 6 (B and C), which show the percentage of motile parasites and the trail length, respectively. Ionomycin (IO, 1 μm) was used as a positive control because this reagent releases all Ca²⁺ stored in neutral compartments and also increases Ca²⁺ entry evoking a maximal response. Importantly, the addition of nifedipine (10 μm) (in the presence of TG + Ca²⁺) caused a significant decrease in gliding response (Fig. 6, B and C, maroon bars). Interestingly, motility, as expressed as the percentage of parasites associated with a trail, did not get reduced beyond the levels of Ca²⁺ alone (Fig. 6B), whereas gliding motility, expressed as average trail length, did (Fig. 6C), suggesting that nifedipine is inhibiting gliding motility rather than other types of motility.

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.

Experiments were also conducted to directly visualize the role of Ca²⁺ accumulation caused by Ca²⁺ entry in tachyzoite motility. Using the same conditions as seen in Fig. 1C parasites were treated with Ca²⁺ alone, thapsigargin alone, or with both. Time lapse microscopy showed that although the addition of Ca²⁺ or thapsigargin alone had a small effect on motility, the addition of both caused a substantial increase in motility (supplemental Videos S1 and S2).

Before invasion, tachyzoites extend their conoids (conoid extrusion), a highly dynamic organelle situated at the apical end of the parasite, which has been shown to be stimulated by an increase in [Ca²⁺]_i (7). We measured the ability of parasites to extrude their conoid over a relevant period of time (Fig. 6, D–F). Parasites kept in extracellular buffer conditions but at low Ca²⁺ concentration (Ca²⁺ ∼ 110 nm) for the 5-min experiment showed no significant increase in conoid extrusion (Fig. 6E, EB). The addition of IO (1 μm), which has been used previously to stimulate conoid extrusion (7), increased conoid extrusion when compared with control parasites. Extracellular Ca²⁺ (1 mm) produced a minor increase in conoid extrusion. A high percentage of parasites treated with both IO and extracellular Ca²⁺ extruded their conoids. Importantly, this increase in conoid response was maintained throughout the 5-min experiment, whereas the IO-treated parasites exhibited a higher initial increase in conoid extrusion followed by a significant reduction for the remainder of the experiment (Fig. 6E, inset). The difference obtained between IO treatment alone and in the presence of extracellular Ca²⁺ shows the importance of extracellular calcium in enhancing conoid extrusion and also that in the absence of extracellular Ca²⁺ (Fig. 6E, inset), the response is not sustained. In agreement with the enhancing effect of Ca²⁺ entry, nifedipine inhibited conoid extrusion (Fig. 6F) in the presence of extracellular Ca²⁺. We did not find a significant difference on the percentage of parasites with their conoids extruded when preincubated in the presence of Bay K. This could be because compensatory mechanisms in the parasite (i.e. plasma membrane Ca²⁺-ATPase) would not allow cytosolic Ca²⁺ to stay elevated for extended lengths of time. As with other experiments, switching the ionic environment from an extracellular environment to an intracellular environment resulted in similar patterns of conoid extrusion (data not shown).

T. gondii tachyzoites contain micronemes, which are secretory organelles involved in host cell invasion. Microneme secretion is stimulated naturally upon host cell attachment. Although microneme proteins are secreted at a low rate in extracellular tachyzoites (23), the process can be accelerated 10–100-fold by elevating the intracellular Ca²⁺ concentration (6). We studied the secretion of MIC2, a protein used as a marker to study microneme secretion (24). Parasites were exposed to increasing amounts of extracellular calcium (0–2.4 mm) after a short preincubation in a Ca²⁺-free buffer to measure secretion of MIC2 (Fig. 7A). This treatment resulted in a significant increase in the amount of MIC2 protein secreted from the parasite into the medium, as detected by Western blot analysis (Fig. 7A). The amount of MIC2 secretion in the same cohort of parasites when treated with TG (1 μm) followed by the same Ca²⁺ concentrations resulted in MIC2 secretion that was significantly higher than after the addition of Ca²⁺ alone (Fig. 7A, compare ± TG). Fig. 7B is the quantification of three independent experiments and displays the consistency of this phenomenon. Importantly, MIC2 secretion was significantly higher when parasites were exposed to TG (blue bars). These results show the previously documented importance of intracellular stored Ca²⁺ while demonstrating the significant enhancement that occurs in microneme secretion when Ca²⁺ accumulates under Ca²⁺ entry conditions.

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.

The cumulative effect of Ca²⁺ on invasion-linked traits was assessed by the direct measurement of host cell attachment and invasion of parasites. Red-green assays demonstrated that parasites exhibited higher frequencies of host cell attachment and invasion when incubated in increasing concentrations of extracellular Ca²⁺ (Fig. 7, C and D). Additionally, when access to extracellular Ca²⁺ was blocked through the use of nifedipine (10 μm), there was a significant decrease in parasite attachment and invasion in host cells relative to DMSO vehicle controls (Fig. 7D).

DISCUSSION

This study provides evidence for a highly regulated mechanism of Ca²⁺ entry in T. gondii extracellular tachyzoites. Ca²⁺ levels were enhanced after depleting intracellular Ca²⁺ stores with TG, a SERCA-type Ca²⁺-ATPase inhibitor. Our results do not support the activation of a SOCE channel. We assayed the rate of Mn²⁺ entry and its response to the degree of store filling (2). The rate of Mn²⁺ quenching was not stimulated by the preaddition of TG, indicating that store depletion did not activate Ca²⁺ entry in tachyzoites. We also could not demonstrate store-operated Ba²⁺ influx. The higher accumulation of cytosolic Ca²⁺ in the presence of TG, could be the result of the SERCA pump inhibition rather than of direct stimulation of the entry channel by store depletion.

This highly regulated Ca²⁺ entry was stimulated by [Ca²⁺]_i, inhibited by the L-type Ca²⁺ channel blocker nifedipine, and stimulated by the L-type Ca²⁺ channel agonist Bay K. Fura-2 fluorescence quenching by Mn²⁺ entry was also inhibited by nifedipine and significantly stimulated by Bay K, suggesting a similar mechanism of entry.

Our data support the presence of a Ca²⁺ channel in tachyzoites probably voltage-gated, modulated by Ca²⁺ itself. Certain voltage-gated Ca²⁺ channels (e.g. L-type channels) upon becoming permeable to Ca²⁺ (i.e. gate opening) will respond to cytosolic Ca²⁺ by becoming more permeable, a process called Ca²⁺-dependent facilitation (25). We tested this possibility by exploring the correlation between cytosolic Ca²⁺ concentration and extent of Ca²⁺ influx. We manipulated the intracellular Ca²⁺ concentration using TG to block uptake by the ER and allow other uptake mechanisms to operate for different lengths of times, resulting in different cytosolic Ca²⁺ concentrations prior to the addition of extracellular Ca²⁺. Under these conditions, the rate of Ca²⁺ entry was significantly higher at higher cytosolic Ca²⁺ concentration, indicating a possible enhancement effect of cytosolic Ca²⁺ itself on the putative Ca²⁺ entry channel.

Calcium ions impact nearly every aspect of cellular life. SOCE mechanisms are activated specifically in response to depletion of Ca²⁺ from intracellular organelles. This Ca²⁺ is used to replenish those organelles and to activate Ca²⁺-sensitive signaling pathways. The molecular players (STIM1 and ORAI) involved in SOCE in some eukaryotes were discovered only a few years ago, almost 20 years after the phenomenon was described in mammalian cells (26). Specific searches in the T. gondii genome, as well as in other Apicomplexan genomes, have revealed no evidence for the existence of STIM or ORAI (27, 28), in agreement with our results on the absence of SOCE in T. gondii. It is interesting to note, however, that evidence for the presence of STIM and ORAI orthologs has been reported in other unicellular protists, like the choanoflagellate Monosiga brevicollis, which belongs to the supergroup Ophisotkonta, which also includes animals and fungi (11, 29).

A critical question emerging from our data is the molecular identity of the Ca²⁺ permeation pathways. The dihydropyridine analog, nifedipine, an L-type calcium channel inhibitor, showed consistent inhibition of Ca²⁺ entry, whereas the use of an agonist of L-type Ca²⁺ channels, Bay K, demonstrated the ability to enhance Ca²⁺ and Mn²⁺ entry. Other members of the VGCC group (N, P/Q, and R) are insensitive to the effects of these modulators (30). The genome of T. gondii predicts the presence of a putative VGCC (31) (TGME49_205265), the type of channel expected to be nifedipine-sensitive. In addition, a recent evaluation by Prole and Taylor (27) found a second gene with homology to VGCCs (Ca_v), TgME49_267720, which may encode a subunit of a Ca²⁺ channel as the sequence predicts for the presence of six transmembrane domains. An attractive hypothesis is that the Ca²⁺ entry observed in this study involves one or more of these Ca²⁺ channels. Interestingly, Ca²⁺ entry in response to various extracellular Ca²⁺ concentrations showed a biphasic mode in support of the participation of more than one channel or permeation pathway. The use of T. gondii as a model basal eukaryote may provide very useful information for understanding the evolution of Ca²⁺ regulation and signaling. Further exploration of these genes is warranted given the supporting evidence in ToxoDB.org. Interestingly, Pezzella et al. (32) reported an effect on invasion when using dihydropyridine Ca²⁺ channel inhibitors similar to nifedipine, the reagent used in this study to inhibit Ca²⁺ entry and invasion-linked responses.

In this study, we quantitatively correlated Ca²⁺ entry with invasion-linked behavior. This strategy proved to be very useful in highlighting how extracellular Ca²⁺ enhances invasion-linked traits. We designed our experiments so that free Ca²⁺ concentrations were always defined and tightly controlled (∼110 nm range). We looked at motility, conoid extrusion, microneme secretion, and invasion, and Ca²⁺ entry enhanced all of these traits. The enhancement was more dramatic when cytosolic levels were elevated with ionophores or with the SERCA inhibitor TG. The use of ionophores in the presence of extracellular Ca²⁺ led to higher intracellular Ca²⁺ levels, suggesting a potential association. This can be appreciated in further examination of the conoid extrusion data. Release of stored Ca²⁺ by ionophore treatment results in a large response in conoid extrusion. However, the combined use of ionophore with extracellular Ca²⁺ results not only in a higher proportion of cells extruding their conoids, but the population also maintains extruded conoids for longer times, an advantageous trait for increasing invasion efficiency. The inhibition of conoid extrusion by nifedipine agrees with the Ca²⁺ entry enhancement effect on this trait.

Other studies have found some evidence for the role of extracellular Ca²⁺ in the lytic cycle of T. gondii. Lovett and Sibley (33) demonstrated that release of Ca²⁺ via ryanodine-sensitive stores was enhanced in the presence of extracellular Ca²⁺. Importantly, without extracellular Ca²⁺, ryanodine was incapable of stimulating microneme secretion. Treatment of parasites with caffeine (an agonist of ryanodine receptors) also elicited release of stored Ca²⁺. Although this release was enhanced in the presence of extracellular Ca²⁺, it was not necessary for stimulating microneme secretion. These results support the assertion that extracellular Ca²⁺ enhances virulence-related traits. Such enhancement was also observed in previous experiments on gliding motility (34) and conoid extrusion. Mondragon and Frixione (7) observed enhanced conoid extrusion in parasites that were treated with ionomycin. As observed in this study, ionomycin was able to initiate a significant response in conoid extrusion, but when combined with extracellular Ca²⁺, the response was more robust. Likewise, previous publications have established that although Ca²⁺ ionophores were sufficient to induce Ca²⁺ mobilization, through their probable effect on intracellular stores, the presence of extracellular Ca²⁺ resulted in greater microneme secretion (6).

The lytic cycle of T. gondii is complex, and the parasite does not simply invade a host cell, replicate until host cell integrity is lost, and then egress and begin the process anew. In fact, studies show that parasites commonly invade and exit with little or no replication (35, 36). Such complex invasion behavior, all of which is dependent on Ca²⁺, likely requires the replenishing of intracellular stored Ca²⁺ with extracellular Ca²⁺. Similarly, the process of egress is dependent on Ca²⁺ (8). A parsimonious feature for the parasites is to utilize the extracellular Ca²⁺ available to supplement invasion-dependent processes and to also resupply intracellular stores to use during reinvasion or to initiate egress.

Our model of invasion events, timing of intracellular Ca²⁺ release, and extracellular Ca²⁺ entry is shown in Fig. 8. Intracellular stores are depleted upon egress, and the parasites need to replenish them when they come in contact with the high Ca²⁺ of the extracellular environment. This wave of Ca²⁺ influx enhances invasion-related traits. Active Ca²⁺ pumping to intracellular compartments and to the extracellular space will allow control of the cytosolic Ca²⁺ levels after signaling events.

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.)

In conclusion, our data points toward a general mechanism of Ca²⁺ entry, which may not be sensitive to the filling state of the ER but that could work in parallel to elevate intracellular Ca²⁺ to activate downstream events essential for initiating biological processes like invasion-linked traits. Our thorough analysis of Ca²⁺ entry and its role during the lytic cycle of T. gondii will set the precedent for future discoveries of the molecules involved in this crucial process. To our knowledge, this is the first study that links regulation of Ca²⁺ entry to virulence traits of a unicellular pathogen.