A plasma membrane-type Ca(2+)-ATPase co-localizes with a vacuolar H(+)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii

Abstract

Ca(2+)-ATPases are likely to play critical roles in the biochemistry of Toxoplasma gondii, since these protozoa are obligate intracellular parasites and the Ca(2+) concentration in their intracellular location is three orders of magnitude lower than in the extracellular medium. Here, we report the cloning and sequencing of a gene encoding a plasma membrane-type Ca(2+)-ATPase (PMCA) of T.gondii (TgA1). The predicted protein (TgA1) exhibits 32-36% identity to vacuolar Ca(2+)-ATPases of Trypanosoma cruzi, Saccharomyces cerevisiae, Entamoeba histolytica and Dictyostelium discoideum. Sequencing of both cDNA and genomic DNA from T.gondii indicated that TgA1 contains two introns near the C-terminus. A hydropathy profile of the protein suggests 10 transmembrane domains. TgA1 suppresses the Ca(2+) hypersensitivity of a mutant of S.cerevisiae that has a defect in vacuolar Ca(2+) accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that TgA1 localizes to the plasma membrane and co-localizes with the vacuolar H(+)-pyrophosphatase to intracellular vacuoles identified morphologically and by X-ray microanalysis as the acidocalcisomes. This vacuolar-type Ca(2+)-ATPase could play an important role in Ca(2+) homeostasis in T.gondii.

Fig. 1. Alignment of different Ca²⁺-ATPases. CLUSTAL W alignment (Thompson et al., 1994) of Ca²⁺-ATPases from T.gondii (DDBJ/EMBL/GenBank accession No. AF151371), T.cruzi (U70620) and S.cerevisiae (U03060). Similar residues are shaded. Amino acid residues not present within other sequences are denoted by dashes. The ATP-binding and autophosphorylation domains employed in the design of degenerate oligonucleotides for PCR are underlined, the predicted transmembrane domains in the T.gondii sequence are indicated by dashed lines above the alignment. The potential N-glycosylation sites are indicated with a dash above the alignment.

Fig. 2. Southern and northern analysis of TgA1. (A) Southern blot analysis of the TgA1 gene in genomic DNA from T.gondii. Total genomic DNA (10 µg/lane) was digested with various restriction enzymes and analyzed as described in Materials and methods. Size markers are indicated. Trypanosoma gondii DNA digested with the following restriction enzymes: lane 1, EcoRI; 2, BamHI; 3, HindIII; 4, PstI; 5, BglII. Control BT cell’s DNA (10 µg/lane) was digested with EcoRI and BamHI (lane 6). The blots were hybridized with the ³²P-labeled 1.4 kb PCR product (TgA1.4) and washed at high stringency. (B) Expression of TgA1 mRNA in tachyzoite (lanes 1 and 2) and bradyzoite (lane 3) forms of T.gondii. Upper panel, poly(A)⁺ RNA (3.7 µg/lane) was electrophoresed, blotted, and probed at high stringency with a ³²P-labeled probe corresponding to the entire TgA1 ORF. Size markers are indicated on the right. Approximately equal amounts of RNA were observed in the three lanes under UV light. Lower panel, the membranes were stripped and reprobed with a ³²P-labeled PCR fragment of the TUB1 gene from T.gondii as control. Exposure times were 3 days (upper panel) and 4 h (lower panel) at –80°C. Lane 1, tachyzoites, RH strain; lane 2, tachyzoites, ME49 strain; lane 3, bradyzoites, ME49 strain.

Fig. 3. Western blot analysis of TgA1. Total cell lysates containing 20 µg of protein from tachyzoites (RH strain, lanes 1 and 4; ME49 strain, lanes 2 and 5) and bradyzoites (ME49 strain, lanes 3 and 6) and 3 µg of purified recombinant His₆-TgA1 fusion protein (lane 7) were subjected to SDS–PAGE on 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and probed with antibodies prepared as described in Materials and methods (lanes 4–7) or with pre-immune serum (lanes 1–3).

Fig. 4. Suppression of the Ca²⁺ hypersensitivity of the S.cerevisiae vcx1 pmc1 mutant by T.gondii TgA1. Saccharomyces cerevisiae vcx1 pmc1 strain K665 was transformed with a control vector (K665pYES2) or a vector containing the entire ORF of T.gondii TgA1 (K665pYES2-TgA1). Strain K661 has the PMC1 gene and thus served as the positive control. The cultures were streaked on YPD (1% Difco extract, 2% Bacto-Peptone, 2% dextrose pH 5.5) plates containing 200 mM CaCl₂ (A), or were inoculated into YPD pH 5.5 with 0, 50, 100, 200 and 400 mM CaCl₂, and growth was estimated by measuring the optical density at 600 nm (B), to identify Ca²⁺-tolerant transformants.

Fig. 4. Suppression of the Ca²⁺ hypersensitivity of the S.cerevisiae vcx1 pmc1 mutant by T.gondii TgA1. Saccharomyces cerevisiae vcx1 pmc1 strain K665 was transformed with a control vector (K665pYES2) or a vector containing the entire ORF of T.gondii TgA1 (K665pYES2-TgA1). Strain K661 has the PMC1 gene and thus served as the positive control. The cultures were streaked on YPD (1% Difco extract, 2% Bacto-Peptone, 2% dextrose pH 5.5) plates containing 200 mM CaCl₂ (A), or were inoculated into YPD pH 5.5 with 0, 50, 100, 200 and 400 mM CaCl₂, and growth was estimated by measuring the optical density at 600 nm (B), to identify Ca²⁺-tolerant transformants.

Fig. 5. Immunofluorescence microscopy showing the localization of TgA1 in tachyzoites (A and B) and bradyzoites (E and F) of T.gondii. (C) Tachyzoites incubated with pre-immune serum (1:100). (D, G and H) Tachyzoites (D) and bradyzoites (G and H) incubated with monoclonal antibody against BAG-5 (1:1000). (B), (E) and (G) show the same cells as in (A), (F) and (H), respectively, by bright-field microscopy. Bar, 10 µm.

Fig. 6. Confocal laser scanning microscopy analysis showing the localization of the Ca²⁺-ATPase (green in B, D and E) and BAG-5 (red in C, D and F) in bradyzoites (A–D) and tachyzoites (E and F) of T.gondii ME49 strain. (D) An overlay of (B) and (C) with no evidence of co-localization. Bar, 10 µm.

Fig. 7. Immunocytochemical localization of Ca²⁺-ATPase (A–C), GRA2 (D) and V-H⁺-PPase (A–D) in tachyzoites of T.gondii, and western blot analysis of the V-H⁺-PPase (E). Note that 20 (A and D) and 15 (B and C) nm particles were used to localize the V-H⁺-PPase (arrowheads) and 10 nm particles were used for the Ca²⁺-ATPase (arrows, B and C), or GRA2 (D). V, vacuole; DG, dense granule. Bars, 500 (A), 100 (B), 120 (C) and 220 nm (D). (E) Total cell lysates containing 20 µg of protein from T.cruzi epimastigotes (Ep) and tachyzoites (RH strain, lane T) were subjected to SDS–PAGE on 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and probed with the antibody prepared as described in Materials and methods.

Fig. 8. Transmission electron microscopy of whole tachyzoites (A–C). Whole, unfixed and unstained cells were suspended in 0.25 M sucrose. Drops were applied to Formvar-coated grids, cells were allowed to adhere for 10 min, and then carefully blotted dry and observed directly with the Hitachi-600 electron microscope. Note the numerous electron-dense vacuoles of different sizes. Bar, 1 µm.

Fig. 9. X-ray microanalysis of the electron-dense organelles. (A and B) The X-ray spectra recorded from an electron-dense vacuole (A) or the background (B) in whole tachyzoites.