The role of extracellular calcium in the filling of intracellular stores.

A, scheme depicting the Fura-2-AM loading and the experimental set-up. B, T. gondii tachyzoites loaded with Fura-2 were in suspension in Ringer buffer with 100 µM EGTA. Thapsigargin (TG) was added at 100 seconds, at two different concentrations (1 and 2 µM). C, Similar experimental set-up to the one shown in B. 1.8 mM CaCl2 was added to the suspension at 100 sec, followed by 2 µM TG at 300 sec (dark blue trace). The light blue trace shows the same experiment without the addition of CaCl2. D, similar to C but using 40 µM GPN instead of TG. E, same experimental set-up to the one shown in C but adding the potassium ionophore nigericin, 10 µM. F, same experimental set-up to the one shown in C but using the mitochondrial uncoupler CCCP. G, quantification of the % increase of cytoplasmic calcium by adding extracellular calcium prior to each indicated reagent (TG, thapsigargin; CP, CCCP; NIG, nigericin). H, cytosolic calcium measurements following the addition of 1.8 mM extracellular calcium, 1 µM TG prior to the addition of GPN, 40 µM. I, cytosolic Calcium measurements following the addition of 1.8 mM extracellular Calcium, 1 µM TG prior to the addition of 10 µM NIG. The quantification shows the comparison of the cytosolic increase with and without the addition of TG. Data are presented as mean ± SD for G, H and I. p value: unpaired two tailed t test performed in all comparisons.

Ca2+ uptake by intracellular stores.

A, Scheme showing the loading with MagFluo4-AM followed by permeabilization with digitonin of a T. gondii tachyzoite (RH parental strain) suspension (IS, intracellular store). B, Fluorescence measurements (see Materials and Methods for specifics) of the suspension of parasites loaded with MagFluo4. MgATP was added at 500 µM at 50 seconds. The bar graph shows the quantification of the slope of the increase in fluorescence after adding the SERCA substrate MgATP. The concentration of free calcium was varied, and it is indicated. The calculation of free calcium was done with MaxChelator. C, similar experimental set-up to the one shown in B but the concentration of MgATP was varied at the concentrations indicated in the bar graph. Quantification of the slope of fluorescence increase after adding MgATP is shown in the bar graph. D, this experiment was done with 500 µM MgATP and 220 nM CaCl2. Thapsigargin (TG) was added to inhibit SERCA causing calcium to be released from the store. The concentrations used are indicated. The bar graph shows the negative slope after the addition of TG. E, similar to D, but adding ionomycin (IO). The concentrations used are indicated and the slopes were measured after the addition of IO. Data are presented as mean ± SD for B-D. p value: unpaired two tailed t test performed in all comparisons.

The sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) is essential for T. gondii replication.

A, Scheme showing the strategy used for the down regulation of the expression of TgSERCA by promoter insertion and regulation by 0.5 µg/ml Anhydrotetracyclin (ATc). The mutants generated were termed iΔTgSERCA or iΔTgSERCA-HA for the C-terminal tagged one. DHFR, Dihydrofolate reductase gene for selection with pyrimethamine; CAT, chloramphenicol acetyl transferase gene for selection with chloramphenicol. B, Western blots of iΔTgSERCA-HA lysates grown with and without ATc. The expression of TgSERCA is shown by the signal obtained with the anti-HA antibody. C, plaque assays comparing the size of plaques formed by tachyzoites (150/well) of the iΔTgSERCA mutant grown with and without 0.5 μg/ml ATc for 8 days. Plaques formed by the parental strain TatiΔku80 are shown for comparison. D, quantification of the size of the plaques presented in C. E, replication assays using the iΔTgSERCA_RFP mutant. Number of parasites per parasitophorous vacuole (PV) obtained 24 h after infection of fibroblast cells is reported and compared between the mutant grown with and without 0.5 μg/ml ATc. Data represented as mean ± SD. p value: unpaired two tailed t test performed in all comparisons. F, Average number of parasites per PV counted at 24 h after the initial infection. The graph shown to the right shows the number of parasites per PV of the iΔTgSERCA (+ATc) for 24 or 48 hours after the initial infection. G, Invasion assays with the iΔTgSERCA mutant treated with ATc for 24 h were done using the red-green assay. H, Egress assays with the iΔTgSERCA mutant previously grown with or without ATc. Egress was triggered with IO (100 or 50 nM) or saponin (0.01%). The protocol for natural egress uses compound 1 and it is detailed in the methods section.

Organellar calcium pools in the iΔTgserca mutant.

A, cytosolic calcium response to the addition of 1 µM TG. The iΔTgserca grown with and without ATc was previously loaded with Fura-2 and the cytosolic Ca2+ responses were measured. 1 µM TG was added at 200 sec. The purple trace shows the response of the parental cell line grown without ATc and the pink one shows the response of the same mutant grown with ATc for 24 h. The bar graph shows the analysis of the Δ[Ca2+] from three independent experiments. B, same experimental set-up as the one in A but adding 1.8 mM extracellular Ca2+ at 200 sec. C, SERCA activity measured with MagFluo4-loaded tachyzoites of iΔTgserca mutant previously grown with or without ATc. Parasites were collected and loaded with MagFluo4-AM as described in the Methods section. Digitonin permeabilized parasites were used in the experiment. The concentration of free calcium in the buffer is 220 nM and MgATP (0.125 mM) was added at 100 sec. The response of the control mutant grown without ATc is shown in purple. The other traces show the response of the mutant grown for either 24 or 48 h with ATc. TG was added at 1 µM TG. The bar graph shows the quantification of the slope after adding MgATP. D. Ca2+ entry measured in Fura-2 loaded iΔTgSERCA (±ATc). 1.8 mM extracellular Ca2+ was added at 200 sec. The inset shows the ΔF measurement from three independent experiments and showed no significant difference. E, Similar conditions to the ones used in A but adding 100 µM Zaprinast. The bar graph shows the analysis of the Δ[Ca2+] from three independent experiments. F, Similar conditions to the ones used in B but adding 1.8 mM extracellular calcium at 200 sec and 100 µM Zaprinast at 400 sec. The bar graph shows the analysis of the Δ[Ca2+] from 3 independent experiments. G, Same as A but adding 40 µM GPN. The bar graph shows the analysis of the Δ[Ca2+] from three independent experiments. H, Same set-up as in F but adding 1.8 mM Ca2+ at 200 sec followed by 40 µM GPN. The bar graph shows the analysis of the Δ[Ca2+] from three independent experiments. Data are presented as mean ± SD. p value: unpaired two tailed t test performed in all comparisons.

Mitochondrial Calcium uptake.

A, Fluorescence image of T. gondii tachyzoites expressing SOD2-GCaMP6f (pDT7S4H3-SOD2-GCaMP6f). The generation of this cell line is described in material and methods. B, T. gondii tachyzoites expressing SOD2-GCaMP6f (5×107) permeabilized with digitonin as described in the method’s section were tested in a buffer with 100 µM EGTA. Ca2+ was added at 100 sec at the final concentrations 1, 10, 20, 50, 100 and 200 µM. Maxchelator was used to calculate the amount of Calcium to be added to attain the indicated concentrations. C, ΔF was measured as the change in fluorescence between the baseline and the maximum obtained 20 sec after the addition of Ca2+ from three independent biological experiments. D, Fura-2 loaded tachyzoites of the SOD2-GCaMP6f expressing mutant in suspension. The experimental set-up was identical to the one described in Fig. 1A-B. 1.8 mM CaCl2 was added at 400 sec. Fluorescence measurements were done using the conditions for Fura-2. E, GCaMP6 fluorescence measurements of parasites expression SOD2-GCaMP6f in their mitochondrion. The fluorometer conditions were set to measure GCaMP6. F, 1 µM TG was added at 100 sec followed by 1.8 mM CaCl2 at 400 sec. Fura2 loaded parasites and Fura2 conditions were used. G, Same additions as in F but measuring fluorescence of GCaMP6. H, Response to the addition of 1 μM TG of the iΔTgserca-sod2-gcamp6f mutant (transfected with pCTH3-SOD2-GCaMP6f plasmid) previously grown in the presence (pink trace) or absence (blue trace) of ATc. The conditions for the fluorescence measurements are the same used in part G with intact parasites. The bar graph shows the ΔF measurements from three independent experiments. I, same as H but using 40 μM GPN. J, Same as H but using 100 μM Zaprinast. K, Same as H but using 1 μM Ionomycin. Data are presented as mean ± SD from 3 independent biological experiments. p value: unpaired two tailed t test performed in all comparisons.

ER membrane contacts with the mitochondrion and the plant like vacuolar compartment (PLVAC). A, Super-resolution IFAs of intracellular parasites with the mitochondrion labeled with the αTom40 (green, 1:20,000) antibody and the ER labeled with the αTgcalumenin antibody (an ER calcium binding protein) (red, 1:1000) or the TgSERCA (red 1:1000). B, Immunofluorescence (IFA) of extracellular tachyzoites with the same antibodies used for part A. The mitochondrion and ER membranes interact in several regions. C, The PLVAC was localized with the αVP1 antibody (green, 1:200) or the αTgCPL antibody (green, 1:500). The ER membranes were labeled with the αTgERC antibody (red). The spots of contact between the ER and the PLVAC are yellow. Scale bars in A-C represent 5 µm. D, Transmission Electron Microscopy imaging of the contact sites formed between ER and PLVAC, ER and Apicoplast, ER and mitochondrial. Size bars are 100 nm.

Model showing calcium entry through two different types of Ca2+ channels, uptake to the ER by SERCA and distribution to the other organelles via transfer from the ER to the mitochondria, PLVAC and apicoplast. In intracellular parasites, the mitochondria is in close contact with the ER which is constitutively leaking Ca2+ into the cytosol. The mitochondria take up Calcium from the ER through an unknown mechanism. VDAC could be involved in the transfer through the outer mitochondrial membrane (Mallo et al., 2021). The PLVAC interacts with the ER and may also interact with the mitochondrion and the apicoplast. TgA1, a calcium ATPase previously characterized may be the pump involved in uptake (Luo et al., 2001; Luo et al., 2005). The mechanism of release is unknown. The TPC was shown to be involved in the formation of contacts between the ER and the apicoplast (Li et al., 2021). Ca2+ could leak from the ER through the TgTRPPL-2 channel previously described (Marquez-Nogueras et al., 2021).