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, Same conditions as in B. 1 µM Thapsigargin (TG) was added at 100 sec for T1 and 400 sec for T2. 1 µM ionomycin (IO) was added at 400 sec for trace 1 (T1) and at 100 sec for trace 2 (T2). Bar graph shows Ca2+ increase after adding TG before (T1) and after IO (T2). D, Same conditions as in B. 1.8 mM CaCl2 was added 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. E, similar to D but using 40 µM GPN instead of TG. F, same experimental set-up to the one shown in D but using the mitochondrial uncoupler CCCP. G, same experimental set-up to the one shown in D but adding the potassium ionophore nigericin (Nig), 10 µM. The quantification for D, E, F, and G shows the % increase of cytoplasmic calcium compared with the same condition without calcium. H, 1.8 mM CaCl2 was added at 100 sec, 1 µM TG was added at 300 sec followed by 40 µM GPN at 500 sec. The light blue trace shows the same experiment without the addition of TG. The quantification shows the Ca2+ increase after adding GPN ± previous addition of TG. I, Identical experiment to H but instead using 10 µM Nig at 500 sec. The quantification shows the Ca2+ increase after adding Nig ± previous addition of TG. J, 1 µM TG was added at 100 sec followed by 40 µM GPN at 300 sec. The light blue trace shows the same experiment without the addition of TG. The buffer contains 100 µM EGTA. Quantification shows Ca2+ increase after adding GPN ± previous addition of TG K, Similar conditions to J but with10 µM Nig at 300 sec instead. Data are presented as mean ± SD for all comparisons. p value: unpaired two tailed t test performed in all comparisons.

Ca2+ uptake by intracellular stores.

A, Scheme showing the loading with Mag-Fluo-4 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 Mag-Fluo-4. MgATP (500 µM), the SERCA substrate was added at 50 seconds. The bar graph shows the quantification of the slope of the increase in fluorescence after adding MgATP. The concentration of free calcium was varied, and it is indicated. The calculation of free calcium was done using MaxChelator. C, similar experimental set-up to the one shown in B with 220 nM free Ca2+, with varied concentrations of MgATP as indicated in the bar graph, which shows the quantification of the slope of fluorescence increase after adding MgATP. D, experiment was done with 500 µM MgATP and 220 nM free Ca2+. 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 various concentrations of 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. ns, not significant, p > 0.05. *, p ≤ 0.05. **, p ≤ 0.01. ***, p ≤ 0.001. ****, p ≤ 0.0001

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

A, Scheme showing the strategy used for generating conditional knock outs of TgSERCA by promoter insertion and regulation by 0.5 µg/ml Anhydrotetracyclin (ATc). The resulting mutants were named iΔTgSERCA or iΔTgSERCA-3HA (C-terminally HA-tagged). DHFR, dihydrofolate reductase gene (pyrimethamine selection); CAT, chloramphenicol acetyltransferase gene (chloramphenicol selection). B, Western blots of iΔTgSERCA-3HA parasites grown ± ATc. TgSERCA expression was detected using an anti-HA antibody, showing reduced levels with ATc treatment. C, plaque assays comparing the growth of iΔTgSERCA tachyzoites (150 parasites/well) cultured ± 0.5 µg/ml ATc for 8 days. Plaques formed by the parental TatiΔku80 strain are shown for comparison. D, quantification of the size of the plaques presented in C. E, Replication assay using the iΔTgSERCA-RFP mutant. The number of parasites per parasitophorous vacuole (PV) was quantified 24 h post-infection of fibroblast cells and compared between parasites grown ± 0.5 µg/ml ATc. F, Average number of parasites per PV counted at 24 h after the initial infection. The graph 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 assay of the iΔTgSERCA mutant following 24 h of ATc treatment, performed using the red-green assay described in Materials and Methods. H, Egress assays with fibroblast monolayers infected with iΔTgSERCA-RFP parasites for 24 or 48 h. Egress was triggered with ionomycin (IO; 100 nM or 50 nM) or saponin (0.01%). Natural egress was monitored following treatment with 1 μM compound 1 as described in the Methods section. % Vacuoles: 100 X Vacuoles egressed/total vacuoles. Data (D, E, F, G, H) are presented as mean from at least three biological replicates ± SD. Statistical significance was assessed using an unpaired two-tailed t-test.

Organellar calcium pools in the iΔTgSERCA mutant.

A, The iΔTgSERCA mutant was grown ± ATc and was loaded with Fura-2 for cytosolic Ca2+ measurements. 1 µM TG was added at 200 sec to a suspension of tachyzoites. The purple trace shows the response of the parental cell line grown without ATc and the pink trace shows the response of the same mutant grown with ATc for 24 h. The bar graph shows the analysis of the Δ[Ca2+]cyt from three biological 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 in Mag-Fluo-4 loaded iΔTgSERCA tachyzoites grown ± ATc. Parasites were collected, loaded with Mag-Fluo-4 AM, and permeabilized with digitonin as described in the Methods section. Free Ca²⁺ in the buffer was set at 220 nM, and MgATP (0.125 mM) was added at 100 s. The purple trace represents the control (no ATc), while the other traces correspond to parasites treated with ATc for 24 or 48 h. TG (1 µM) was added as indicated. The bar graph shows the quantification of the initial slope after adding MgATP. D, Ca²⁺ entry measured in Fura-2–loaded iΔTgSERCA parasites grown ± ATc. Extracellular Ca²⁺ (1.8 mM) was added at 200 s. The inset shows ΔF values from three independent experiments, indicating no significant differences. E, Similar conditions to the ones used in A but adding 100 µM Zaprinast. The bar graph shows the quantification of the D[Ca2+] from three biological 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 quantification of the Δ[Ca2+] from 3 biological experiments. G, Same as A but adding 40 µM GPN. The bar graph shows the analysis of the Δ[Ca2+] from three biological replicates. H, Same set-up as in F but adding 1.8 mM Ca2+ at 200 sec followed by 40 µM GPN at 400 sec. The bar graph shows the quantification of the Δ[Ca2+] from three biological replicates. 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 of the RH strain expressing SOD2-GCaMP6f (pDT7S4H3-SOD2-GCaMP6f). The generation of this cell line is described in the methods section. B, Ca²⁺ uptake in digitonin-permeabilized T. gondii tachyzoites expressing SOD2-GCaMP6f. Parasites (5 × 10⁷) were permeabilized as described in the Methods section and suspended in buffer containing 100 µM EGTA. Ca²⁺ was added at 100 s to reach final free concentrations of 0.25, 0.5, 1, 10, 50, 100, and 200 µM, calculated using Maxchelator. C, ΔF was measured as the change in fluorescence between the baseline and the maximum value obtained 20 s after Ca²⁺ addition. Data represent the average of three independent biological experiments. D, Fura-2-loaded T. gondii tachyzoites expressing SOD2-GCaMP6f in suspension. The experimental setup was identical to that described in Fig. 1A-B. CaCl₂ (1.8 mM) was added at 400 s, and fluorescence measurements were performed under Fura-2 conditions. E, GCaMP6f fluorescence measurements of intact T. gondii tachyzoites expressing SOD2-GCaMP6f targeted to the mitochondrion. Fluorescence was recorded using optimized settings for GCaMP6 detection. F, 1 µM TG was added at 100 sec followed by 1.8 mM CaCl2 at 400 sec. Fura-2 loaded parasites and Fura-2 conditions were used. G, Same additions as in F but measuring fluorescence of GCaMP6f. H, Response to 1 µM TG of iΔTgSERCA-SOD2-GCaMP6f parasites (transfected with the pCTH3-SOD2-GCaMP6f plasmid), grown with (pink trace) or without (blue trace) ATc. Fluorescence measurements were performed under the same conditions as in panel G using intact parasites. The bar graph shows ΔF values from three independent biological replicates. 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, IFAs of extracellular tachyzoites with the same antibodies used for part A. The mitochondrion and ER membranes interact in several regions. C, The PLVAC was labeled with the αTgVP1 antibody (green, 1:200) or the αTgCPL antibody (green, 1:500). The ER was labeled with the αTgcalumenin antibody (red). The points of contact between the ER and the PLVAC are yellow. Scale bars in A-C is 5 µm. D, Transmission Electron Microscopy imaging of the contact sites formed between ER and PLVAC, ER and Apicoplast, ER and mitochondria. Size bars are 100 nm.

Hypothetical model showing calcium entry through two different types of Ca2+ channels, uptake by TgSERCA into the ER and distribution to the other organelles via transfer from the ER to the mitochondria, PLVAC and apicoplast.

The mitochondrion is shown in close contact to the ER which constitutively leaks Ca2+ into the cytosol. Ca2+ could leak from the ER through the TgTRPPL-2 channel previously described (Marquez-Nogueras et al., 2021). The mitochondria take up Ca2+ 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 Ca2+ uptake (Luo et al., 2001; Luo et al., 2005). The mechanism of release is unknown. The Two Pore Channel (TgTPC) was shown to be involved in the formation of contacts between the ER and the apicoplast (Li et al., 2021) and could form part of the mechanism of uptake. Question marks point to molecules or mechanism partially or not yet identified.