INTRODUCTION

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Trypanosoma cruzi is an obligate intracellular protozoan parasite that infects a wide variety of vertebrates and is the etiologic agent of Chagas' disease in humans. The life cycle of T. cruzi involves several different stages. The epimastigotes proliferate within the gut of reduviid insects and then transform into nondividing, but highly infective, metacyclic trypomastigote forms, which are released into the urine and feces and inoculated into the vertebrate host. In this host, the trypomastigotes invade different cell types, remain in an acidic parasitophorous vacuole for a few hours, and then disrupt the vacuolar membrane and gradually transform into amastigote forms, which actively reproduce in direct contact with the cytoplasm of the host cell. Subsequently, the amastigotes transform into trypomastigotes which are released from the host cells and reach the bloodstream, from which they are taken up by the vectors (12, 35).

Ca²⁺ signaling has been shown to play a key role in the process of mammalian cell invasion and the intracellular development of this parasite. An increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) of T. cruzi trypomastigotes occurs upon invasion (13, 31), and pretreatment of the trypomastigotes with intracellular Ca²⁺ chelatorsthe tetraacetoxymethyl esters of (bis)-o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 2-{[2-bis(carboxymethyl)-amino-5-methylphe- noxyl]-methyl}-6-methoxy-8-bis(carboxymethyl)-aminoquinoline (QUIN-2)to prevent the increase in [Ca²⁺]_i results in an inhibition of cellular invasion (31, 66).

Two large Ca²⁺ sinks, the extracellular space and the endoplasmic reticulum, are important sources of Ca²⁺ for calcium signaling in most eukaryotic cells (8). However, T. cruzi amastigotes are not in contact with the extracellular space because they live in the cytosol, where the free calcium concentration is very low (of the order of 0.1 µM) compared to that of the extracellular space (of the order of 1 mM). This dramatic difference in external free calcium suggests that intracellular stores must be very important in the regulation of Ca²⁺ homeostasis in the amastigotes.

In contrast to mammalian cells, the different stages of T. cruzi possess most of their intracellular Ca²⁺ in an acidic compartment named the acidocalcisome (14). The biochemical characterization of this organelle has provided evidence that it is acidified by a vacuolar-type proton-translocating (V-H⁺) ATPase and that it has a Ca²⁺/H⁺ countertransporting ATPase for Ca²⁺ uptake (14). Acidocalcisomes have also been found in other trypanosomatids, such as Trypanosoma brucei (43, 58, 59) and Leishmania mexicana amazonensis (28), and in Toxoplasma gondii (32). This organelle is in various aspects similar to the vacuole present in fungi and plant cells (62) but apparently has no counterpart in mammalian cells. The use of quick freezing, ultracryomicrotomy, and electron probe microanalysis to study the elemental composition of different compartments in T. cruzi epimastigotes with or without prior treatment with ionophores has recently provided evidence (44) that acidocalcisomes correspond to the electron-dense vacuoles previously described for these parasites (15).

In mammalian cells, Ca²⁺ has also been reported to be present in acidic organelles carrying vacuolar-type proton pumps, such as endosomes, lysosomes, and the trans-Golgi network, and secretory granules such as chromaffin, pancreatic zymogen, and atrium-specific granules (19, 37, 50, 61), but the functional significance of the high Ca²⁺ content of these organelles is unknown (37). Recent evidence has indicated, however, that second messengers such as inositol 1,4,5-trisphosphate and cyclic ADP ribose can release Ca²⁺ from pancreatic zymogen granules (19), although this conclusion is disputed by other investigators (57). The mechanism of Ca²⁺ uptake might not be the same in all these organelles. In fact, zymogen granules seem to acquire their Ca²⁺ together with the proteins from the Golgi complex, whereas chromaffin granules seem to be endowed with a specific Ca²⁺/Na⁺ antiport (37). Except for a Ca²⁺-ATPase that was purified from rat liver lysosomes (16) and a Ca²⁺-ATPase gene that was cloned from rat stomach tissue (21) and that exhibits 50% amino acid identity with the Golgi complex-located PMR1 gene product of Saccharomyces cerevisiae (2, 38), no studies have been carried out yet to investigate the presence of specific Ca²⁺-translocating ATPases in most of these organelles.

This study provides further evidence that acidocalcisomes are organelles distinct from lysosomes and other previously recognized organelles present in these parasites. It also reports the identification in T. cruzi of the tca1 gene, which encodes a protein with homology to mammalian plasma membrane Ca²⁺-ATPases (PMCA) but with characteristics that place it in a novel category of Ca²⁺-ATPases along with the vacuolar Ca²⁺-ATPases described for S. cerevisiae (9) and Dictyostelium discoideum (30). Indirect immunofluorescence and immunoelectron microscopy analysis suggest that the product of this gene (Tca1) is associated not only with the plasma membrane but also with the acidocalcisomes. The gene is expressed at a high level in the amastigote stages that contain an elevated Ca²⁺ concentration and a larger number of acidocalcisomes compared to other stages of the parasite and is able to functionally complement the PMC1 gene, encoding the vacuolar Ca²⁺-ATPase of S. cerevisiae.

	MATERIALS AND METHODS

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Culture methods. T. cruzi trypomastigotes and amastigotes (Y strain) were obtained from the culture medium of L₆E₉ myoblasts by a modification of the method of Schmatz and Murray (42) as we have described before (14). The final concentration of trypomastigotes and amastigotes was determined with a Neubauer chamber. The contamination of trypomastigotes with amastigotes and intermediate forms or of amastigotes with trypomastigotes and intermediate forms was always less than 5%. T. cruzi epimastigotes (Y strain) were grown at 28°C in a liquid medium consisting of brain heart infusion (37 g/liter), hemin chlorohydrate (20 mg/liter dissolved in 50% triethanolamine), and 10% heat-inactivated newborn calf serum (14). Five days after inoculation, cells were collected by centrifugation. The protein concentration was determined by the biuret assay (20) in the presence of 0.2% deoxycholate. L₆E₉ myoblasts were cultured as described before (33). Yeast strains K665 MATa (vcx1::hisG pmc1::TRP1) and K661 MATa (vcx1::hisG), kindly provided by Kyle W. Cunningham, Department of Biology, The Johns Hopkins University, Baltimore, Md. (10), were grown at 30°C in standard YPD medium (1% Difco yeast extract, 2% Bacto Peptone, 2% dextrose) or in YPD medium, pH 5.5 (adjusted with succinic acid), supplemented with 200 mM CaCl₂. Cell growth was assessed by measuring the optical density of the liquid cultures at 600 nm (see Fig. 5B) or by counting the number of colonies in plates (see Fig. 5A). Both strains are isogenic and harbor the following additional mutations: ade2-1, can1-100, his3-11, 15 leu2-3, 112 trp1-1, and ura3-1.

Chemicals. Fetal and newborn calf serum, Dulbecco's phosphate-buffered saline (PBS), Tween 20, Triton X-100, arsenazo III, EGTA, and poly-L-lysine (molecular weight, 70,000) were purchased from Sigma Chemical Co. Fluorescein- and rhodamine-labeled antibodies were from Molecular Probes, Inc., Eugene, Oreg. Trizol reagent and Taq polymerase were from Gibco BRL, Life Technologies Inc., Grand Island, N.Y. The Poly(A)Tract mRNA isolation system, lambda EMBL3 phage, restriction enzymes, and pGEM-T vectors were from Promega (Madison, Wis.). Sequenase was from United States Biochemical Corporation (Cleveland, Ohio). The pET-28a expression vector and the Escherichia coli DE3 strain were from Novagen (Madison, Wis.). The pYES2 vector was from Stratagene (La Jolla, Calif.). Monoclonal antibody N-2 against the 110-kDa accessory protein of the V-H⁺-ATPase was purchased from the Monoclonal Antibody Center of the University of Hawaii through Agnes Fok. Polyclonal antibodies against T. cruzi cruzipain were kindly provided by Juan Jose Cazzulo (University of San Martin, San Martín, Argentina). Gold-labeled goat anti-rabbit antibodies were obtained from Ted Pella, Inc., Reddington, Calif. Biotin succinimidyl ester and the enhanced chemiluminescence (ECL) detection kit were from Amersham Life Sciences, Inc., Arlington Heights, Ill. All other reagents were analytical grade.

Nucleic acid analysis. DNA was isolated by standard procedures (39). Total RNA was isolated with Trizol reagent according to the manufacturer's recommendations. The polyadenylated RNA was obtained with the Poly(A)Tract mRNA isolation system. DNA was run in 0.8% agarose gels with TBE (9.5 mM Tris-boric acid, 2.0 mM EDTA, pH 8.0) buffer. RNA was electrophoresed in 1% agarose gels with 2.2 M formaldehyde, 100 mM MOPS (morpholinepropanesulfonic acid), 40 mM sodium acetate, and 5 mM EDTA (pH 8.0) (39). Southern and Northern hybridizations were done by standard procedures (39). All the probes for hybridization were labeled with [³²P]dCTP by using random hexanucleotide primers. The hybridized filters were washed under high-stringency conditions (0.1% standard saline citrate-0.1% sodium dodecyl sulfate [SDS] at 65°C), unless otherwise indicated. Oligonucleotide primers were designed to recognize the ATP phosphorylation site and the ATP-binding site of cationic ATPase genes (1, 36), i.e., 5'CGGGATCCGTNATNTGYWSNGAYAA3' and 5'CGGAATTCGSRTCRTTNRYNCCR3' (where N is A + T, Y is C + T, W is A + T, S is G + C, and R is A + G) as the 5' primer and the 3' primer, respectively. PCR was performed in a PTC-100 programmable thermal controller (MJ Research, Inc., Watertown, Mass.) at 94°C for 1 min, 55 to 72°C for 2 min, and 72°C for 2 min/cycle (30 cycles) with Taq polymerase. PCR products were cloned into the pGEM-T vector according to the manufacturer's instructions. The cloned PCR products were sequenced, and the deduced amino acid sequences were compared with the database in GenBank. A ~1.0-kb PCR clone with identity to organelle-type Ca²⁺-ATPases was used to screen a genomic DNA library of T. cruzi. The library was constructed in lambda EMBL3 phage with 9- to 23-kb BamHI fragments of genomic DNA from T. cruzi epimastiotes according to the manufacturer's instructions. DNA sequencing was performed by the dideoxynucleotide chain termination method of Sanger et al. (40) either manually or with a 373A DNA automatic sequencer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). Internal oligonucleotide primers were designed to complete the DNA sequence in both directions. DNA and deduced amino acid sequence analyses were performed with the University of Wisconsin Genetics Computer Group software package (GCG program, version 8.0). Hydropathy analysis was done with the Gene Jockey sequence processor (Biosoft, Cambridge, United Kingdom). The TcP0 fragment used as a control in Northern blots (see Fig. 3) was obtained by amplifying T. cruzi genomic DNA by the PCR technique, with primers corresponding to nucleotides 3 to 54 and 918 to 936 in the sequence of the TcP0 gene (47). Densitometric analysis of Northern blots was done with an ISI-1000 digital imaging system (Alpha Inotech Corp.). Comparison of levels of tca1 transcripts between the different stages was done by taking as a reference the densitometric values obtained with the TcP0 transcripts and assuming a similar level of expression of this gene in all stages (47). Similar results were obtained when the densitometric values were compared by taking into account the amount of RNA added to each lane in four different experiments.

Preparation of antibodies. A DNA fragment encoding the 174-amino-acid COOH-terminal domain of the Tca1 protein was removed by NdeI and BglII double digestion from plasmid ptca1 containing the complete tca1 gene. The fragment was subcloned into the NdeI and BamHI sites of the pET-28a expression vector, resulting in a construct that encoded the protein fused to a six-histidine tag that allowed its purification on nickel-agarose columns. This plasmid was checked by DNA sequencing to ensure that the correct construct had been obtained. The recombinant plasmid was transfected into the DE3 strain of E. coli, the fusion protein was induced, and the expressed protein of about 33 kDa, present in inclusion bodies, was solubilized and purified according to the manufacturer's instructions. Rabbits were injected subcutaneously with 100 µg of fusion protein emulsified in Freund's complete adjuvant, followed 2 weeks later by subcutaneous injection of 100 µg of fusion protein in Freund's incomplete adjuvant. At 6, 8, and 10 weeks following the initial injection, rabbits were boosted with 100 µg of fusion protein in PBS containing a 10-mg/ml suspension of A1(OH)₃. Serum was collected before the initial injection (preimmune serum) and 10 days after each boost. The antiserum was aliquoted and stored at 70°C. Affinity purification of anti-Tca1 antibody was performed as described elsewhere (39). Briefly, 500 µg of fusion protein was fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. After being blocked with 5% nonfat dry milk in TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween 20), the paper was incubated with the anti-Tca1 serum to bind the specific antibody to Tca1 protein. Then the paper was washed three times with TBST, and antibody was eluted from the nitrocellulose paper with elution buffer (0.2 M glycine [pH 2.8], 1 mM EDTA).

SDS electrophoresis and preparation of Western blots. The electrophoretic system used was essentially the same as that described by Laemmli (25). T. cruzi epimastiotes (2.5 × 10⁸), trypomastigotes (1 × 10⁹), or amastigotes (1 × 10⁹) were centrifuged at 1,000 × g for 10 min, resuspended in 300 µl of Dulbecco's PBS containing proteinase inhibitors (1 µg of aprotinin per ml, 1 µg of leupeptin per ml, 1 µg of pepstatin per ml, and 1 mM phenylmethylsulfonyl fluoride), and frozen at 70°C. Cells were thawed and homogenized with a Teflon pestle at 4°C. Aliquots of different stages of T. cruzi (10 µl; about 20 µg of protein) were mixed with 10 µl of 125 mM Tris-HCl (pH 7)-10% (wt/vol) -mercaptoethanol-20% (vol/vol) glycerol-4.0% (wt/vol) SDS and 4.0% (wt/vol) bromophenol blue as tracking dye and boiled for 5 min prior to application to SDS-7.5% polyacrylamide gels. Electrophoresed proteins were transferred to nitrocellulose by the method of Towbin et al. (56), with a Bio-Rad Laboratories (Richmond, Calif.) Transblot apparatus. Following transfer, the nitrocellulose was blocked in 5% nonfat dry milk in TBST overnight at 4°C. A 1:1,000 dilution of polyclonal antiserum in blocking buffer was then applied at room temperature for 60 min. The nitrocellulose was washed three times for 15 min each with TBST. Immunoblots were visualized on radiographic film (Kodak) with the ECL chemiluminescence detection kit and according to the instructions of the manufacturer (Amersham Life Sciences).

Functional complementation of the PMC1 gene of S. cerevisiae with tca1. We transformed the S. cerevisiae vcx1 pmc1 strain K665 with the yeast expression vectors pYES2 and pYES2-tca1 by electroporation. The Ura⁺ transformants were selected by plating on synthetic-complete Ura medium (10). The tca1 coding region was amplified by the PCR technique from a lambda clone containing the complete tca1 gene and a HindIII site created on the PCR primers. The tca1 coding region was placed at the HindIII site of pYES2 with the same orientation as the GAL1 promoter. The cultures were grown in YPD medium (pH 5.5) containing 200 mM CaCl₂ to identify Ca²⁺-tolerant transformants.

Immunofluorescence microscopy. Parasites fixed with 4% formaldehyde were allowed to adhere to poly-L-lysine-coated coverslips, permeabilized with 0.3% Triton X-100 for 3 min, blocked with 3% bovine serum albumin in PBS, and prepared for immunofluorescence with a 1:100 or 1:200 (see Fig. 8) dilution of the antibody against the 33-kDa expressed protein or a 1:25 dilution of the monoclonal antibody against the 110-kDa accessory protein of the V-H⁺-ATPase and a rhodamine- or fluorescein isothiocyanate-coupled goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1:80), respectively. Control preparations were incubated with preimmune serum or without the primary antibody. Immunofluorescence images were obtained with an Olympus BX-60 fluorescence microscope. The images were collected with a system consisting of a charge-coupled device camera (model CH250; Photometrics Ltd., Tucson, Ariz.), an electronic unit (model CE 200A, equipped with a 50-Hz 16-bit A/D converter), and a controller board (model NU 200; both from Photometrics Ltd.). Images were acquired and evaluated by a software package (Adobe Photoshop) on a Macintosh Quadra 840 AV computer (Apple Computer, Inc., Cupertino, Calif.). In experiments aiming at the colocalization of Ca²⁺- and H⁺-ATPases, the samples were examined in a Zeiss LSM10 laser scanning confocal microscope. Optical sections of 0.1 µm were used.

Electron microscopy. For imaging whole cells (see Fig. 12), trypomastigotes were treated exactly as described by Dvorak et al. (15). Amastigotes were suspended in Dulbecco's PBS (pH 7.2). Drops were applied to Formvar- and carbon-coated 200-mesh copper grids, and cells were allowed to adhere for 10 min and then carefully blotted dry and observed directly with a Hitachi 600 transmission electron microscope operating at 100 kV.

Cell surface labeling. Cell surface labeling was performed by a modification of the method of Hart et al. (22). Epimastigotes were washed three times with 10 mM sodium phosphate (pH 8.6)-150 mM NaCl-0.1 mM MgCl₂-0.1 mM CaCl₂ at room temperature and were then resuspended in 1 ml of the same ice-cold medium (4 × 10⁶ cells/ml). Biotin succinimidyl ester (in dry dimethyl formamide) was added to the cells according to the instructions provided by Amersham and incubated for 1 h at 4°C with rotation. Cells were then washed three times with ice-cold Dulbecco's PBS and lysed in 0.5 ml of ice-cold immune precipitation buffer (1% Nonidet P-40, 20 mM Tris-HCl [pH 7.6], 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg of pepstatin per ml, 2 µg of leupeptin per ml, 1 µg of soybean trypsin inhibitor per ml). Cells were kept at 4°C for 30 min with rotation, and the lysate was centrifuged for 20 min at 10,000 × g. The supernatant was diluted to 1.5 ml with immunoprecipitation buffer containing 150 mM NaCl and precipitated with polyclonal anti-Ca²⁺-ATPase antibody (1:300). After incubation for 16 h at 4°C, immunocomplexes were allowed to bind to protein A-Sepharose beads by incubating them for 1 h at 4°C. The beads were washed five times with immunoprecipitation buffer, and bound proteins were eluted in boiling SDS sample buffer. The proteins were separated on an SDS-10% polyacrylamide gel and blotted onto a nitrocellulose membrane as described above. The membrane was probed with avidin-peroxidase conjugate according to the protocol provided by Amersham. Proteins were visualized by ECL (Amersham).

Cell permeabilization. Variations in free Ca²⁺ concentrations in permeabilized cells were monitored by measuring the changes in the absorbance spectrum of arsenazo III (14), with the SLM Aminco DW2000 spectrophotometer at the wavelength pair of 675 and 685 nm.

Nucleotide sequence accession number. DNA sequence data was deposited in GenBank under accession no. U70620.

	RESULTS

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Cloning and characterization of a Ca²⁺-ATPase gene. Since acidocalcisomes are in various respects similar to the vacuoles present in fungi and plant cells (62), we looked for the presence of a gene encoding a Ca²⁺-ATPase with homology to the Ca²⁺-ATPase present in the vacuole of S. cerevisiae (9) and in D. discoideum (30). Degenerate oligonucleotides corresponding to two conserved domains, a phosphorylation site and a site involved in ATP binding (1, 36), were used to amplify, by the PCR technique, specific sequences from T. cruzi genomic DNA. The PCR products were cloned and sequenced. Analysis of the deduced partial amino acid sequence of these clones revealed that a ~1.0-kb PCR clone had the best scores of sequence identity (31 and 37%) and similarity (50 and 55%) with the vacuolar-type Ca²⁺-ATPases described for S. cerevisiae and D. discoideum, respectively.

View larger version (98K): [in this window] [in a new window]   View larger version (34K): [in this window] [in a new window]   FIG. 1.   (A) Nucleotide and predicted protein sequences of tca1. Amino acid residues are numbered in the left margin; nucleotides are numbered in the right margin. The amino acid sequences corresponding to the highly conserved catalytic autophosphorylation and ATP-binding domains employed in the design of degenerate oligodeoxyribonucleotides for PCR are in boldface. The 10 transmembrane domains identified by hydropathy analysis are underlined. The TAA stop codon for tca1 is marked by an asterisk. Potential N-glycosylation sites are in boldface italics. (B) Hydropathy plots of T. cruzi Tca1, S. cerevisiae Pmc1p (9), and D. discoideum PAT1 (30). Putative transmembrane domains are numbered. Hydropathy was computed and putative transmembrane domains were predicted according to the method of Hopp and Woods (23) over a running window of 20 amino acid residues. Numbers at the bottom of the figure denote positions of the amino acid residues from the NH2 terminus. Numbers on the ordinate are relative hydropathy values.

Structure of the coding region and genomic organization of tca1. Analysis of the Tca1 amino acid sequence (Fig. 1A) showed that this gene product contains all the conserved subdomains and invariant residues found in other P-type ATPases, such as the phosphorylation and ATP-binding domains (1, 36). Hydropathy analysis (23) of the deduced amino acid sequence (Fig. 1B) revealed a profile very similar to those of other calcium pumps containing 10 transmembrane domains. As occurs with the vacuolar Ca²⁺-ATPases described for S. cerevisiae (9) and D. discoideum (30), a TFASTA search of protein databases showed that Tca1 was closely related to the PMCA, with 38% identity (58% similarity) to the PMCA from human erythrocytes (53). It also had 34 and 38% identity and 56 and 59% similarity to the vacuolar Ca²⁺-ATPases of S. cerevisiae (9) and D. discoideum (30), respectively, and had 23 to 27% identity with sarcoplasmic (endoplasmic) reticulum-type Ca²⁺-ATPases (SERCA) and 20 to 25% identity with Na⁺,K⁺-ATPases from different species (29, 46). Tca1 contains four potential N-glycosylation sites (indicated in boldface italics in Fig. 1A).

View larger version (81K): [in this window] [in a new window]   FIG. 2.   Southern blot analysis of the tca1 gene in genomic DNA from T. cruzi, T. brucei, L. mexicana amazonensis, and L. donovani. Total genomic DNA (10 µg/lane) was digested with various restriction enzymes and analyzed as described in Materials and Methods. Size markers (M) are indicated. (A) T. cruzi genomic DNA digested with the following restriction enzymes: lane 1, BamHI; 2, EcoRI; 3, HindIII; 4, PstI; 5, SacI; 6, PvuII. The blots were hybridized with the 32P-labeled 1.0-kb PCR product (probe 1) and washed at high stringency. (B) Digestion with PstI of 10 µg of genomic DNA from T. brucei (lane 7), L. mexicana amazonensis (lane 8), and L. donovani (lane 9). The blots were hybridized with the 32P-labeled ptca1 probe (probe 2) at 60°C and washed with 2× SSC at 60°C. The scheme shows the restriction sites in the tca1 gene (gray area) and the probes used. P, PstI; E, EcoRI; Pv, PvuII.

Higher expression of tca1 in intracellular forms of T. cruzi. Northern blot analysis showed a single ~4.3-kb transcript in each of the three life cycle stages of T. cruzi (Fig. 3, upper panel). Analysis of the ~4.3-kb band by densitometry indicated that the tca1 transcript is >6-fold more abundant in amastigotes and >3-fold more abundant in trypomastigotes than in epimastigotes. Bands obtained after hybridization with a PCR product for the TcP0 gene, which is expressed at similar levels in all stages of T. cruzi (47) (Fig. 3, lower panel), were used as a reference control.

View larger version (52K): [in this window] [in a new window]   FIG. 3.   Expression of tca1 mRNA. (Upper panel) Poly(A)+ RNAs isolated from epimastigotes (3 µg [lane 1]), amastigotes (1 µg [lane 2]), or trypomastigotes (3 µg [lane 3]) were electrophoresed, blotted, and probed at high stringency with the 32P-labeled 1.0-kb PCR fragment. Size markers correspond to a 0.24- to 9.5-kb RNA ladder (Gibco BRL). Approximately equal amounts of RNA were observed in lanes 1 and 3 under UV light. (Lower panel) The membrane was stripped and reprobed with a 32P-labeled PCR fragment of the TcP0 gene from T. cruzi (47) as a control.

View larger version (53K): [in this window] [in a new window]   FIG. 4.   Western blot analysis of Tca1. Homogenates containing 20 µg of protein from epimastigotes (lane 1), amastigotes (lane 2), and trypomastigotes (lane 3) were subjected to SDS-polyacrylamide gel electrophoresis on 7.5% polyacrylamide gels and transferred to nitrocellulose membranes. Lanes were probed with antibodies prepared as described in Materials and Methods. Lanes 4 and 5 show the proteolytic products obtained after incubation of epimastigote lysates in ice for 10 (lane 4) and 30 (lane 5) min in the absence of proteinase inhibitors. For lane 6, epimastigotes were incubated with biotin succinimidyl ester. After the cells were lysed, Ca2+-ATPases were immunoprecipitated and the immunoprecipitate was subjected to Western blot analysis. Visualization of biotinylation was by streptavidin-peroxidase conjugate and ECL. Several bands were recognized. Lane 7 shows a control with normal serum instead of anti-Ca2+-ATPase antibodies for immunoprecipitation. Migration positions of prestained molecular mass standards (Bio-Rad Laboratories, Hercules, Calif.) are shown to the left of the gels.

Functional complementation of the PMC1 gene of S. cerevisiae with tca1. S. cerevisiae K665 with deletion of the genes encoding the high-affinity Ca²⁺-ATPase and low-affinity Ca²⁺/H⁺ antiporter (PMC1 and VCX1) is intolerant of high Ca²⁺ in the growth medium (10). Since the T. cruzi tca1 gene encodes a vacuolar-type Ca²⁺-ATPase with homology to PMC1, we investigated whether complementation of the vcx1 pmc1 yeast mutants with the tca1 gene could suppress their Ca²⁺ hypersensitivity. Figure 5 shows that transformation of the vcx1 pmc1 K665 strain with pYES2-tca1 restored growth on high Ca²⁺ almost completely, thus suggesting the function of tca1 as a vacuolar Ca²⁺-ATPase in these mutants. K665 was transformed with a control vector (pYES2 K665) or a vector containing the entire open reading frame of T. cruzi tca1 (pYES2-tca1 K665). Strain K661 has the PMC1 gene (10) and thus served as the positive control (Fig. 5).

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Suppression of the Ca2+-hypersensitivity of the S. cerevisiae vcx1 pmc1 mutant by T. cruzi tca1. S. cerevisiae vcx1 pmc1 strain K665 was transformed with a control vector (pYES2 K665) or a vector containing the entire open reading frame of T. cruzi tca1 (pYES2-tca1 K665). 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 200 mM CaCl2, and growth was estimated by measuring the optical density at 600 nm (B), to identify Ca2+-tolerant transformants.

Localization of T. cruzi Ca²⁺-ATPase. We investigated the localization of the Ca²⁺-ATPase in T. cruzi by immunocytochemistry with the antibodies described above. The reaction of these antibodies in the various developmental stages of T. cruzi as revealed with fluorescein-labeled secondary antibodies was of variable intensity. In epimastigotes, we observed labeling of cytoplasmic structures more frequently found in the posterior and central region and a weak labeling of the cell surface, including the flagellum (Fig. 6A and B). Live cells were not labeled with these antibodies (data not shown), in agreement with the cytoplasmic location of the C-terminal region of PMCA-type calcium pumps (7). Staining of the flagellum and the cell body was observed in trypomastigotes (Fig. 6C, thin arrows). Amastigotes were intensely stained when they were located either extracellularly (Fig. 6C, thick arrow) or intracellularly (Fig. 6D). This is in agreement with their higher calcium pump content (Fig. 4). No fluorescence was observed in control parasites incubated only in the presence of the secondary fluorescein-labeled goat anti-rabbit IgG (data not shown) or in the host cells (Fig. 6D).

View larger version (133K): [in this window] [in a new window]   FIG. 6.   Immunofluorescence microscopy showing the localization of Tca1 in epimastigote (B), trypomastigote (C), and extracellular (C) or intracellular (D) amastigote forms of T. cruzi. Panel A shows the same cells as in panel B by bright-field microscopy. Arrows indicate features discussed in the text. N, host cell nucleus. Bars, 20 µm.

View larger version (130K): [in this window] [in a new window]   FIG. 7.   Immunocytochemical localization of Ca2+-ATPase (A to F) and H+-ATPase (A, B, and F) in epimastigote (A, B, C, and F), amastigote (D), and trypomastigote (E) forms of T. cruzi. FP, flagellar pocket; F, flagellum; V, vacuole. Bars, 140 nm (A to C), 260 nm (D and E), and 100 nm (F). Note that 10-nm gold particles were used in panels B (thick arrows), C, and F (thick arrows) to localize the Ca2+-ATPase while 5-nm particles were used in all other cases (A, D, and E). Conversely, 5-nm particles were used to localize the V-H+-ATPase in panels B (arrowheads) and F (thin arrow), and 10-nm particles were used in panel A (no particles detected). Arrowheads in panel D show labeling of vacuoles, while arrows show surface labeling of amastigotes with antibodies against the Ca2+-ATPase.

View larger version (82K): [in this window] [in a new window]   FIG. 8.   Confocal laser scanning, microscopy showing the colocalization of V-H+-ATPase (A and B; green in panels E and F) and Ca2+-ATPase (C and D; red in panels E and F) in a mixture of amastigotes and epimastiotes (asterisks in panels E and F) of T. cruzi. Some of the vacuoles labeled with both antibodies are indicated with arrows in each picture and shown in yellow in panels E and F. Bar, 10 µm.

Localization of Ca²⁺-containing vacuoles. In previous studies, we have indicated that most of the intracellular Ca²⁺ in different stages of T. cruzi is located in the acidocalcisomes (14). The use of quick-freezing, ultracryomicrotomy, and electron probe microanalysis to study the elemental composition of different compartments in T. cruzi epimastigotes with or without prior treatment with ionophores has recently provided evidence (44) that acidocalcisomes correspond to the electron-dense vacuoles previously described for these parasites (15). Because the vacuoles (presumptive acidocalcisomes) giving a reaction with antibodies against both ATPases appear empty and in some cases are irregular in shape in cryosections (Fig. 7C), we used a cytochemical technique to detect these organelles with better preservation of their structure than is possible with immunoelectron microscopy. In addition, it was important to establish the difference between these vacuoles and the lysosomal vacuoles present in epimastigotes known as reservosomes (48), which were also found to be acidic (49).

View larger version (164K): [in this window] [in a new window]   FIG. 9.   Cytochemical localization of Ca2+ in trypomastigotes (A), amastigotes (B), and epimastigotes (C to E) incubated in the presence of potassium pyroantimoniate-osmium tetroxide solution. The electron-dense reaction product is restricted to cytoplasmic vacuoles (arrows in panels A and B and asterisks in panels C and D). The arrow in panel E shows the vacuole membrane. G, glycosome; K, kinetoplast; M, mitochondria; N, nucleus; R, reservosome. Bars, 250 (A), 200 (B), 80 (C), 170 (D), and 100 (E) nm.

View larger version (118K): [in this window] [in a new window]   FIG. 10.   Electron micrographs of control epimastigotes (A) and trypomastigotes (B). Cells were fixed as described in Materials and Methods. Arrowheads show empty vacuoles with an electron-dense material in their periphery in an epimastigote (A) and electron-dense vacuoles of similar size in a trypomastigote (B). N, nucleus; K, kinetoplast DNA; R, reservosomes. Bars, 1 µm (A) and 0.5 µm (B).

View larger version (141K): [in this window] [in a new window]   FIG. 11.   Localization of cruzipain in thin sections of epimastigotes embedded in Unicryl. R, reservosomes. Arrowheads show labeling of the cell surface. Stars show the acidocalcisomes. Bars, 250 nm (A) and 330 nm (B and C).

Detection of acidocalcisomes in whole parasites. We recently identified the electron-dense vacuoles present in epimastigotes as the acidocalcisomes (44), but similar experiments were not done with other cell types. Whole unfixed amastigotes and trypomastigotes were deposited on Formvar- and carbon-coated grids and examined by transmission electron microscopy. Electron-dense spherical structures appeared in large numbers in amastigotes (about 30 to 40 per cell [Fig. 12A and B]) and in lower numbers in trypomastigotes (about 15 to 20 per cell [Fig. 12C and D]). In amastigotes, these vacuoles were occasionally arranged in rows and preferentially located towards the cell periphery, whereas in trypomastigotes they were located in the anterior region in close proximity to the flagellum. When they were submitted to the electron beam, we could observe changes in their internal structure leading to the appearance of a sponge-like structure (Fig. 12B).

View larger version (57K): [in this window] [in a new window]   FIG. 12.   Visualization of electron-dense vacuoles in whole unfixed amastigote and trypomastigote forms allowed to adhere to a Formvar- and carbon-coated grid and then observed in the transmission electron microscope. A large number of dense vacuoles can be seen in amastigotes, preferentially located towards the cell periphery (A and B), and a lower number can be seen in slender (C) and broad (D) trypomastigotes, preferentially located in the anterior region, close to the flagellum. F, flagellum. Bars, 1 µm (A, C, and D) and 0.3 µm (B).

Ca²⁺ content in different stages of T. cruzi. Because amastigotes express the vacuolar Ca²⁺-ATPase to a greater extent (Fig. 4, 7D, and 8C and D) and have more electron-dense vacuoles or acidocalcisomes (Fig. 12) than the other stages, we investigated whether this was correlated with their intracellular Ca²⁺ content. Addition of 0.04% Triton X-100 to amastigotes (Fig. 13A), epimastigotes (Fig. 13B), or trypomastigotes (Fig. 13C) caused an immediate Ca²⁺ release. The total releasable Ca²⁺ of amastigotes (247 ± 20 nmol/mg of protein) was 6.2-fold higher than that of epimastigotes (40 ± 4 nmol/mg of protein) and 13.8-fold higher than that of trypomastigotes (19 ± 2 nmol/mg of protein) (mean ± SD from three different experiments).

View larger version (11K): [in this window] [in a new window]   FIG. 13.   Endogenous Ca2+ release from Triton X-100-permeabilized cells. Triton X-100 (TX-100; 0.04%) was added where indicated by the arrow to T. cruzi amastigotes 55 µg of protein/ml [A]), epimastigotes (90 µg of protein/ml [B]), or trypomastigotes (60 µg of protein/ml [C]) in a buffer containing 130 mM KCl, 1 mM MgCl2, 2 mM K2HPO4, 20 mM Tris-HCl (pH 7.4), and 40 µM arsenazo III at 30°C. Results shown are from representative experiments.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this work, we have shown that T. cruzi intracellular amastigotes possess a higher Ca²⁺ content than the extracellular stages of the parasite. This correlates firstly with the higher expression of a calcium pump, the gene for which was cloned and sequenced, and secondly with an abundance of acidocalcisomes or electron-dense vacuoles.

Comparison of the sequence of tca1 from T. cruzi with other P-type ATPases indicates that this ATPase gene is closely related to the family of plasma membrane calcium (PMCA) pumps. The expression of tca1 in a yeast mutant deficient in vacuolar Ca²⁺ accumulation (K665) provides genetic evidence that tca1 encodes a vacuolar Ca²⁺ pump. This calcium pump was shown to be localized to intracellular vacuoles, and the plasma membrane of T. cruzi, as indicated by immunofluorescence (Fig. 6 and 8), immunoelectron microscopy (Fig. 7), and biotinylation experiments (Fig. 4). This pump apparently lacks the calmodulin-binding domain present in other PMCA pumps (52). This characteristic places this enzyme in a novel category together with the vacuolar Ca²⁺-ATPases described for S. cerevisiae (9) and D. discoideum (30). However, it is interesting to note that in a previous work (4) we were able to purify a plasma membrane-located Ca²⁺-ATPase from T. cruzi epimastigotes by calmodulin affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal (5F10) antibodies raised against human erythrocyte Ca²⁺-ATPase, had a molecular mass of 140 kDa, and formed Ca²⁺-dependent hydroxylamine-sensitive phosphorylated intermediates (4). These results could indicate either the presence of two Ca²⁺-ATPases in the plasma membrane of these parasites, perhaps with different activities in different stages, or the existence of a calmodulin-binding domain different from that previously identified in other plasma membrane Ca²⁺-ATPases (53). As in the case of D. discoideum PAT1 (30), we cannot rule out the possibility that calmodulin regulates Tca1 activity by interacting with as-yet-unidentified sequences on the enzyme. Alternatively, Tca1 could be present in the plasma membrane of epimastigotes due to normal membrane recycling or targeting mechanisms. In addition, the strong reactivity of the antibody against Tca1 in the flagellar region of trypomastigotes (Fig. 6C and 7E) could suggest a different role for this pump in this stage of the parasite.

The identification of the intracellular vacuoles labeled with antibodies to the calcium pump as acidocalcisomes is suggested by the following lines of evidence. Prior biochemical data has indicated that acidocalcisomes are acidified by a vacuolar-type proton-translocating V-H⁺-ATPase, that they possess a Ca²⁺/H⁺ countertransporting ATPase for Ca²⁺ uptake, and that they contain most of the parasite's intracellular Ca²⁺ (14). In this work, we show that calcium and proton pumps colocalize to intracellular vacuoles (Fig. 7 and 8). These vacuoles have the same cellular distribution as and are morphologically indistinguishable from the vacuoles that give a strong reaction with potassium pyroantimoniate in combination with osmium (Fig. 9), a method that has been widely used for the localization of calcium in different cells (3, 5, 64). In addition, they have the same cellular distribution as and are similar in size to the electron-dense vacuoles that we previously demonstrated are the acidocalcisomes (44). In conventional thin sections, these intracellular vacuoles may appear empty or may contain more or less electron-dense material (Fig. 10). Observation of whole unfixed parasites by transmission electron microscopy (Fig. 12) showed that these electron-dense vacuoles have a uniform inner structural organization that changes following exposure to the electron beam, as previously reported for isolated sarcoplasmic reticulum vesicles loaded with calcium phosphate or oxalate (11).

We also provided evidence that acidocalcisomes are distinct from other organelles previously recognized in these parasites. In contrast to the appearance of the acidocalcisomes, the lysosomal compartment known as the reservosome, present only in T. cruzi epimastigotes, contains intravacuolar inclusions (48, 49). Reservosomes were not labeled with antibodies against the Ca²⁺-ATPase (data not shown) or by the potassium pyroantimoniate-osmium technique (Fig. 9). Furthermore, the morphology of the electron-dense vacuoles (acidocalcisomes) is clearly different from that of typical lysosomes (27). This evidence is also supported by previous subcellular fractionation and gold-labeled transferrin studies of T. cruzi epimastigotes which provided evidence that acidocalcisomes are different from lysosomes, endosomes, and reservosomes (44).

Dvorak et al. (15) first reported the presence of calcium-rich organelles in T. cruzi epimastigotes following energy-dispersive X-ray microanalysis. These cells were shown to possess electron-dense vacuoles identified by scanning transmission electron microscopy of whole cells. These vacuoles contain large amounts of magnesium, potassium, calcium, phosphorus, and zinc (15) and are morphologically indistinguishable from the electron-dense vacuoles that we detected in unfixed amastigotes and trypomastigotes (Fig. 12). Similar calcium-containing electron-dense vacuoles have also been described for other trypanosomatids such as Trypanosoma cyclops (60), Trypanosoma rhodesiense (65), and Leishmania major (26) and were named polyphosphate (60) or electron-dense (26, 60) vacuoles. These vacuoles were examined by energy-dispersive X-ray microanalysis (26, 60, 65) and in some cases were also observed by scanning transmission electron microscopy of whole cells (26), as were the electron-dense organelles found in T. cruzi (15). Acidocalcisomes have been found in all trypanosomatids examined to date (14, 28, 43, 58, 59), which further supports the suggestion that they correspond to the electron-dense vacuoles described for all these parasites.

The concentration of Ca²⁺ in the cytosol of vertebrate cells is known to be about 0.1 µM, which is dramatically different from the concentration of Ca²⁺ to which extracellular parasites are exposed (about 1 mM). The higher amount of Ca²⁺ in amastigotes would appear to indicate an adaptation to an intracellular environment. In this regard, we have shown previously that amastigotes of L. mexicana amazonensis also possess a higher amount of releasable Ca²⁺ and exhibit greater expression of a SERCA-type Ca²⁺-ATPase than do the extracellular promastigotes (28). Although SERCA-type Ca²⁺-ATPases are usually located in the endoplasmic (sarcoplasmic) reticulum, some SERCA-type Ca²⁺-ATPases have been shown elsewhere to be localized to plant vacuoles (17). It is possible either that a SERCA-type Ca²⁺-ATPase replaces a Tca1 homolog in Leishmania or that both calcium pumps are highly expressed in intracellular forms of these parasites, thus explaining their higher calcium content.

It has been demonstrated that the Ca²⁺ content of intracellular stores exerts a profound control over cell growth and the progression of cells through the cell cycle and that growth changes can result from the inability of Ca²⁺ to be pumped into intracellular stores (45). Our results provide further support for the link between intracellular Ca²⁺ pool content, expression of Ca²⁺-ATPases, Ca²⁺ signaling, cell growth in eukaryotic cells (45, 63), parasite virulence (28), and intracellular adaptation.