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2 Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103
3 Department of Biomedical Sciences, University of Padua, 35121 Padua, Italy
Address correspondence to to Célia R.S. Garcia, Department of Physiology, Institute of Biosciences, University of São Paulo, Rua do Matão, travessa 14, n321, São Paulo 05508-900, Brazil. Tel.: 5511-30917518. Fax: 5511-30917422. E-mail: cgarcia{at}usp.br
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Abstract |
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Key Words: parasitophorous vacuole; signal transduction; Plasmodium; calcium indicators; melatonin
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Introduction |
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Here, we propose that the solution to the above question, in the case of Plasmodia, resides in the nature and sidedness of the PVM. Specifically, if the PVM is derived at least in part (Dluzewski et al., 1995) from the RBC membrane, its sidedness is predicted to be inside-out (i.e., the former extracellular surface of the RBC plasma membrane facing the lumen of the PV). Thus, if the erythrocyte plasma membrane Ca2+ ATPase is still present in the PVM, it should pump Ca2+ into the PV, generating a high [Ca2+] microenvironment in the space between the PVM and the parasite plasma membrane. If this is the case, the Plasmodium during its intraerythrocytic life is not exposed to the low [Ca2+] of the cytosol, but rather to a [Ca2+] not very different from that experienced by any other eukaryotic cell. Last but not least, this model implies that the PVM should be less permeable to ions and other small molecules than predicted from some in vitro data (Desai et al., 1993).
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Results |
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The Ca2+ content of the Plasmodia intracellular stores depends on the [Ca2+] in the PV
As a final test to prove that parasites within intact RBCs are indeed exposed to a relatively high Ca2+ environment, the experiments presented in Fig. 5 were carried out. RBCs infected with P. chabaudi were first loaded with Fluo-3/AM, then after 90 min incubation in 1 mM CaCl2, the Ca2+ content of the parasite intracellular Ca2+ stores was verified by the addition of THG (Fig. 5 A). In a parallel experiment, after loading with Fluo-3/AM, the RBC plasma membrane was permeabilized with digitonin and then, after washing out the digitonin, the cells were incubated in a low [Ca2+] medium (100 nM) for 90 min (Fig. 5 C). The rationale of the experiment is as follows: digitonin completely permeabilizes the RBC plasma membrane and the PVM. On the other hand, the plasma membrane of the parasites is not affected by the detergent at these concentrations, as revealed by the maintenance of Fluo-3 fluorescent signal within the Plasmodium. However, under these conditions, the parasite is exposed not to the [Ca2+] of the PV, but to that of the extracellular medium. Addition of THG under these conditions resulted in no [Ca2+] increase, revealing that exposure of the parasite to a low [Ca2+] medium for 90 min results in almost complete emptying of its intracellular Ca2+ stores (Fig. 5 C). On the contrary, if the cells were permeabilized with digitonin (see previous paragragh), but incubated in a medium containing 100 µM CaCl2 (i.e., a concentration close to that calculated to be in the PV), the intracellular release caused by THG was similar to that observed in controls, i.e., without digitonin permeabilization (compare Fig. 5 E with Fig. 5 A). Results similar to those shown above for P. chabaudi were obtained in RBCs infected with P. falciparum (unpublished data).
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The Ca2+ levels in the PV affects the intraerythrocytic maturation of the parasites
The final question is whether the relatively high level of Ca2+ in the PVM is just accidental or whether it is essential for the proper development of the parasites within the RBC. The simplest direct test to answer this question is to artificially decrease the [Ca2+] in the PV and monitor the effect on parasite development. To this end, P. chabaudi or P. falciparum were allowed to invade RBCs in normal [Ca2+]-containing medium containing Fluo-3 (free acid, to monitor the [Ca2+] within the PV), and then they were incubated in Ca2+-free medium. As shown in Fig. 6 A, the Fluo-3 signal representing PV [Ca2+] for P. falciparum-infected RBC remained constant for a few minutes and then slowly decreased (similar results were obtained with P. chabaudi; not depicted). After 30 min under these conditions, the fluorescence of Fluo-3 was about 50% of the initial value. Continuous incubation for 20 h in EGTA completely prevented the maturation of the parasites, but many RBCs appeared damaged under these conditions. A less drastic protocol was thus adopted; the invaded RBCs were incubated for 2 h in an EGTA-containing medium and then were returned to the normal [Ca2+]-containing medium. Fig. 6 (B and C) shows that after 48 h, the number of infected RBCs (parasitemia) is identical in controls and in cells that have been treated for 2 h in EGTA (Fig. 6 C), indicating that the Ca2+ removal protocol did not damage the RBC. However, the percentage of immature forms was significantly increased in the Ca2+-depleted condition, and the mature form, the schizonts, was reduced by about 30%. This experiment demonstrates that maintaining a high [Ca2+] in the PV is necessary for a normal maturation of Plasmodia within the RBC, and that even a short depletion of [Ca2+] in the PV results in a substantial alteration in the maturation process.
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Discussion |
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It may be argued that the 10-µM doses of THG used here are higher than those used in vertebrate cells to induce Ca2+ release from intracellular stores, and thus may reflect a nonspecific effect of the drug on intracellular [Ca2+] (Vercesi et al., 1993). Furthermore, it has been suggested that the SERCAs expressed in P. falciparum are insensitive to THG, but sensitive to cyclopiazonic acid (Alleva and Kirk, 2001). We would argue that (1) it is not surprising that the doses of THG necessary to completely inhibit the SERCA in Plasmodia are much higher than in mammalian cells, given the large evolutionary distance between these cells (Varotti et al., 2003); and (2) effects similar to those obtained with THG have been obtained with cyclopiazonic acid (another SERCA inhibitor) at the same doses effective in mammalian cells (520 µM).
The intracellular Ca2+ stores in malaria parasites appear to play a major role in the signaling pathway initiated by the host hormone melatonin via the production of InsP3 (Hotta et al., 2000). InsP3, in turn, is known to act in Plasmodia on both classical ER-like stores and on another Ca2+ store, characterized by an acidic lumen, the so-called acidic Ca2+ store (Docampo and Moreno, 1999; Garcia, 1999). Therefore, we investigated whether the [Ca2+] in the medium surrounding the Plasmodia (PV [Ca2+]) needs to be maintained in the 100-µM range for full response to the physiological agonist, melatonin.
Our experiments demonstrate that, at least as far as Ca2+ handling is concerned, the Plasmodia are surrounded by a microenvironment whose [Ca2+] is 40 µM, 1001,000-fold higher than that in the parasite and RBC cytoplasm (Adovelande et al., 1993; Garcia et al., 1996). This [Ca2+] in the PV is lower than that experienced in the extracellular fluid by cells of multicellular organisms. However, if the RBC plasma membrane is permeabilized and the parasites are exposed to an extracellular medium containing a [Ca2+] in this range, they preserved the Ca2+ content of their intracellular stores, a prerequisite for the use by Plasmodia of a Ca2+-based signaling mechanism (Wasserman et al., 1982, Passos and Garcia, 1998; Garcia, 1999; Hotta et al., 2000). Several not mutually exclusive hypotheses can be proposed to explain the relatively high [Ca2+] of the PV: (1) extracellular Ca2+ remains trapped within the PV during invasion; (2) the RBC plasma membrane Ca2+-ATPase continuously supplies Ca2+ to the vacuolar space. Consistent with this latter possibility is the localization on the PVM (Langreth 1977; Caldas and Wasserman, 2001) of a Ca2+-ATPase. The electronmicrographs suggest that the enzyme is located on the inner surface of the PVM membrane, i.e., with the sidedness required to continuously refill Ca2+ into the PV; or (3) an alternative explanation would be the diffusion of Ca2+ into the PV from the extracellular medium through specialized membrane structures. It has been shown that parasite maturation is accompanied by the development of tubular membrane structures connecting the PV to the extracellular medium. These membranes have been suggested to play a role in the uptake of nutrients and proteins into the developing Plasmodia (Pouvelle et al., 1991; Lauer et al., 1997). Although we cannot exclude that these membrane structures could contribute to the maintenance of Ca2+ homeostasis at later stages in Plasmodia development, it should be stressed that they are not observed during the first hours after infection (Gormley et al., 1992; Garcia et al., 1997).
The sequencing of the Plasmodium genome (Gardner et al., 2002) and several recent studies have identified in this parasite a number of signaling molecules related to those of vertebrate cells, including many proteins concerned with Ca2+ handling and signaling (Dyer and Day, 2000; Le Roch et al., 2000; Marchesini et al., 2000). The key question addressed here is how the parasite can use Ca2+-based signaling mechanisms while located within the RBC, where it might be expected to be exposed to a very low [Ca2+]. We have shown unambiguously that the PV provides a sufficiently high [Ca2+] to ensure the maintenance of the parasite Ca2+ stores (represented in Fig. 7), and thus the sensitivity to agents, such as melatonin, that use Ca2+ as a second messenger to regulate the Plasmodia cell cycle (Hotta et al., 2000). In addition, a prolonged decrease of the [Ca2+] of the PV appears to impair the maturation of the parasites, and eventually is incompatible with the survival of the Plasmodia within the RBC.
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Materials and methods |
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The intraerythrocytic stages of parasites were determined by Giemsa stain smears. All the experiments were carried out with 107 cells in a final volume of 2 ml in a buffer containing (mM): 116 NaCl, 5.4 KCl, 0.8 MgSO4, 5.5 D-glucose and 50 Mops, pH 6.8. As indicated in the figure legends, the medium was either supplemented with 1 mM CaCl2 or without added CaCl2 with or without the addition of 100 µM or 10 mM EGTA. The parasitemia was about 25% at schizont stage both in the case of P. chabaudi and of P. falciparum. The culture was synchronized with sorbitol treatment (Lambros and Vanderberg, 1979). The infected RBCs were washed twice with RPMI 1640 medium (GIBCO BRL) supplemented with 10% calf serum (P. chabaudi) or 10% human serum (P. falciparum), and finally resuspended in the medium described above.
Loading with the Ca2+ indicators
In order to monitor the [Ca2+] in the PV the invasion was carried out at 37°C in the presence of the calcium indicators (acid form) Fluo-3 or Mag-Fura-2 (10 µM). At the end of invasion, cells were stored at 4°C to prevent the possibility of endocytosis of the indicator by the parasite. In order to label the parasite cytoplasm with acetoxymethyl ester forms of the dyes, we used infected cells immediately after invasion by following with Giemsa-staining the stage of the infection. In brief, segmented schizonts were monitored up to the moment of invasion, and early ring stages were used for loading with 10 µM Fluo-3/AM in the presence of 2 mM Probenecid (Sigma-Aldrich). Before analyzing the cells in the confocal microscope or in the spectrofluorimeter, the RBCs were washed twice with PBS and finally resuspended in the standard buffer. The relatively low pH (6.8) of the medium was chosen to reduce the transport of Ca2+ by ionomycin from the medium into the RBCs, while its efficacy on intracellular membrane is unaffected (Fasolato and Pozzan, 1989). In a few experiments, loading with Fluo-3/AM was carried out at the segmented schizont stage, i.e., before invasion of new RBCs, with no appreciable difference in the response of the Ca2+ indicator to the different treatments. In the experiments where the [Ca2+] of both cytoplasm and PV was measured simultaneously, the RBCs were invaded in the presence of 10 µM Mag-Fura-2, and after invasion, Fluo-3/AM was added to load the parasite cytoplasm.
Ca2+ measurements
Single-cell confocal microscope (model LSM-510; Carl Zeiss MicroImaging, Inc.) experiments were carried out at RT. The infected cells were plated on slides previously incubated for 1 h with L-polylysine (Sigma-Aldrich). The dyes were excited sequentially at 488 nm (for Fluo-3), 351 nm, and 375 nm (for Mag-Fura-2), and the emitted fluorescence was collected using band pass filters: 505530 nm (Fluo-3) and 475525nm (Mag-Fura-2). Experiments in cell suspension were carried out at 37°C in a spectrofluorimeter (model F-4500; Hitachi) as described previously (Garcia et al., 1996). The emission and excitation wavelengths for Mag-Fura-2 were 345/380 nm and 510 nm, respectively. A Kd of 53 µM for the Mag-Fura-2 Ca2+ complex was assumed (Hofer et al., 1998).
The fluorescence traces represent the average signal in the region of interest (ROI). Usually, the ROI covered the whole cell, on the assumption that the dye was distributed only in the PV (Fig. 2) or in the whole cytoplasm (Fig. 3 and Fig. 6). Also, in the case of double labeling with Fluo-3 and Mag-Fura-2 (Fig. 4), the ROI covered the whole cell because the spectra of the two dyes are well separated so that no signal from Fluo-3 was visible when exciting in the UV, and vice versa, no signal from Mag-Fura-2 was detected at 488 nm (i.e., the excitation wavelength of Fluo-3). In a few experiments, we used smaller ROIs centered on the PV or on part of the parasite cytoplasm, but the results were not significantly different from those obtained with the larger ROIs. Each frame (512 x 512 pixels) was acquired with a scan time of 784 ms/image and an interval of 200 ms between consecutive images. Experiments were carried out with both P. chabaudi (and mouse RBC) and P. falciparum (and human RBC) with no appreciable difference. For simplicity, for each specific experiment, only the results obtained with either P. falciparum or P. chabaudi are presented. All representative traces are typical of experiments carried out with similar results in at least in three independent trials.
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Acknowledgments |
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We thank Fundação de Amparo à Pesquisa de São Paulo (FAPESP) for funding C.R.S. Garcia (FAPESP 98/00410-2). This work was also partially supported by a United Nations Educational, Scientific, and Cultural Organization grant to T. Pozzan (UVO-Roste 875.635.1).
Submitted: 20 December 2002
Revised: 13 February 2003
Accepted: 24 February 2003
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References |
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