The Digestive Food Vacuole of the Malaria Parasite Is a Dynamic Intracellular Ca2+ Store
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m304193200
ISSN1083-351X
AutoresGiancarlo A. Biagini, Patrick G. Bray, David G. Spiller, Michael White, Stephen A. Ward,
Tópico(s)Mosquito-borne diseases and control
ResumoThe acidic food vacuole of Plasmodium falciparum has been the subject of intense scientific investigation in the 40 years since its role in the digestion of host hemoglobin was first suggested. This proposed role has important implications for the complex host-parasite inter-relationship and also for the mode of action of several of the most effective antimalarial drugs. In addition, adaptive changes in the physiology of this organelle are implicated in drug resistance. Here we show that in addition to these functions, the digestive food vacuole of the malaria parasite is a dynamic internal store for free Ca2+, a role hitherto unsuspected. With the aid of live-cell laser scanning confocal imaging, spatiotemporal studies revealed that maintenance of elevated free Ca2+ in the digestive food vacuole (relative to cytosolic levels) is achieved by a thapsigargin (and cyclopiazonic acid)-sensitive Ca2+-pump in cooperation with a H+-dependent Ca2+ transporter. Redistribution of free cytosolic and vacuolar Ca2+ during parasite growth also suggests that vacuolar Ca2+ plays an essential role in parasite morphogenesis. These data imply that the digestive food vacuole of the malaria parasite is functionally akin to the vacuole of plants (tonoplast) and the small electron-dense granules of some parasites (acidocalcisomes) whereby H+-coupled Ca2+ transport is involved in ion transport, Ca2+ homeostasis, and signal transduction. These findings have significant implications for parasite development, antimalarial drug action, and mechanisms of drug resistance. The acidic food vacuole of Plasmodium falciparum has been the subject of intense scientific investigation in the 40 years since its role in the digestion of host hemoglobin was first suggested. This proposed role has important implications for the complex host-parasite inter-relationship and also for the mode of action of several of the most effective antimalarial drugs. In addition, adaptive changes in the physiology of this organelle are implicated in drug resistance. Here we show that in addition to these functions, the digestive food vacuole of the malaria parasite is a dynamic internal store for free Ca2+, a role hitherto unsuspected. With the aid of live-cell laser scanning confocal imaging, spatiotemporal studies revealed that maintenance of elevated free Ca2+ in the digestive food vacuole (relative to cytosolic levels) is achieved by a thapsigargin (and cyclopiazonic acid)-sensitive Ca2+-pump in cooperation with a H+-dependent Ca2+ transporter. Redistribution of free cytosolic and vacuolar Ca2+ during parasite growth also suggests that vacuolar Ca2+ plays an essential role in parasite morphogenesis. These data imply that the digestive food vacuole of the malaria parasite is functionally akin to the vacuole of plants (tonoplast) and the small electron-dense granules of some parasites (acidocalcisomes) whereby H+-coupled Ca2+ transport is involved in ion transport, Ca2+ homeostasis, and signal transduction. These findings have significant implications for parasite development, antimalarial drug action, and mechanisms of drug resistance. Malaria remains one of the largest global health (and economic) problems, resulting in more than a million deaths, mostly in young African children (1World Health OrganizationRoll Back Malaria. World Health Organization, Geneva2002Google Scholar). With this in mind, it is astonishing how little is known about the basic physiology of the intraerythrocytic malaria parasite and its organelles. The food vacuole is a major digestive organelle of the malaria parasite and a proven chemotherapeutic target. This organelle has a role in degradation of host-derived hemoglobin, is the site of action of important classes of antimalarials, and harbors transporters associated with drug resistance (2Rudzinska M.A. Bray R.S. Trager W. J. Protozool. 1960; 7: 24-25Google Scholar, 3Winstanley P. Lancet Infect. Dis. 2001; 1: 242-250Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4Warhurst D.C. Craig J.C. Adagu I.S. Lancet. 2002; 360: 1527-1529Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Hydrolysis of hemoglobin in the malaria parasite digestive food vacuole occurs by the integrated action of aspartic, cysteine, and metalloproteases (5Coombs G.H. Goldberg D.E. Klemba M. Berry C. Kay J. Mottram J.C. Trends Parasitol. 2001; 17: 532-537Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), resulting in the production of hemozoin (malaria pigment), a biocrystal of the toxic precursor ferriprotoporphyrin IX (FPIX) 1The abbreviations used are: FPIX, ferriprotoporphyrin IX; MOPS, 4-morpholinepropanesulfonic acid; CLSM, confocal laser scanning microscopy; AM, acetomethyl ester.1The abbreviations used are: FPIX, ferriprotoporphyrin IX; MOPS, 4-morpholinepropanesulfonic acid; CLSM, confocal laser scanning microscopy; AM, acetomethyl ester. (6Francis S.E. Sullivan D.J. Goldberg D.E. Annu. Rev. Microbiol. 1997; 51: 97-123Crossref PubMed Scopus (651) Google Scholar). The internal pH of the digestive food vacuole is lower than the parasite cytosol, although the precise pH value is under debate (7Spiller D.G. Bray P.G. Hughes R.H. Ward S.A. White M.R. Trends Parasitol. 2002; 10: 441-444Abstract Full Text Full Text PDF Scopus (26) Google Scholar). In collaboration with Prof. Kiaran Kirk's laboratory (8Saliba K.J. Allen R.J. Zissis S. Bray P.G. Ward S.A. Kirk K. J. Biol. Chem. 2003; 278: 5605-5612Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), we have recently described the function of two H+-pumping mechanisms in the digestive food vacuole; one is a bafilomycin-sensitive V-type H+-ATPase, and the other is a NaF-sensitive H+-pyrophosphatase showing a partial dependence on K+. The combination of a H+-ATPase and a H+-pyrophosphatase acting in the digestive vacuole is analogous to the situation in acidic tonoplasts of plant cells (9Sze H. Li X. Palmgren M.G. Plant Cell. 1999; 11: 677-690PubMed Google Scholar) and of the acidocalcisome of some protozoa (10Docampo R. Moreno S.N.J. Parasitol. Today. 1999; 15: 443-448Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). It should be noted that although there may be similarities with acidocalcisomes (which are small electron-dense granules/organelles that store Ca2+ as a phosphate precipitate) and the malarial parasite food vacuole, they are morphologically (and functionally, with regards to hemoglobin digestion) very distinct. Based on the known H+-coupled Ca2+ transport in these nonmalarial organelles, we hypothesized that an analogous coupled pathway would be present in the malaria parasite digestive food vacuole, thereby contributing to cellular Ca2+ trafficking and storage. By using live-cell confocal laser scanning microscopy and a judicious choice of Ca2+ probes, we have uncovered a novel function for this unique organelle. We show that the food vacuole of the malaria parasite is involved in dynamic Ca2+ storage. Evidence is presented showing that Ca2+ storage by the digestive food vacuole is linked directly to parasite cell signaling and asexual development. This novel discovery raises many new questions about the functions, physiology, and regulation of this critical malarial parasite organelle. As the target for the actions of a number of drugs, the requirement for tightly regulated Ca2+ movement and storage within this organelle may represent a new chemotherapeutic target. Parasite Culture—Plasmodium falciparum TM6 strain was kindly provided by Dr. P. Tan-Ariya (Mahidol University, Bangkok, Thailand) and maintained in continuous culture. Cultures contained a 2% suspension of O+ erythrocytes in RPMI 1640 (R8758) medium supplemented with 10% pooled human AB serum, 25 mm HEPES (pH 7.4), and 20 μm gentamicin sulfate (11Trager W. Jenson J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6040) Google Scholar). Effect of FPIX on the Fluorescence Quantum Yield of Ca2 + Indicators—Fluorescence emission spectra for pentapotassium salts (1 μm) of Fura-2 (excitation 340 nm) and Fluo-4 (excitation 488 nm) in 39 μm free Ca2+ buffer (10 mm CaEGTA in 100 mm KCl, 30 mm MOPS, pH 7.2) with 0, 10, 20, and 50 μm ferriprotoporphyrin IX was performed in a Shimadzu RF-5001PC spectrofluorophotometer. Real Time Ca2 + Confocal Laser Scanning Microscopy—For experimentation, suspensions (1%) of infected erythrocytes in HEPES-buffered RPMI medium (no serum) were loaded with Fluo-4/AM (3 μm, Molecular Probes BV) for 20 min at 37 °C. For imaging, malaria parasite-infected erythrocytes were immobilized using polylysine-coated coverslips in a Bioptechs FCS2 perfusion chamber and maintained at 37 °C in growth medium (no serum). Inhibitors were added to the perfusate, and the Ca2+-dependent fluorescence responses were monitored in real time. Fluorescence signals from Fluo-4 were collected on a Zeiss LSM510 confocal microscope through a Plan-Apochromat 63× 1.4 N.A. oil objective using the "tracking mode." Excitation of Fluo-4 was performed using an argon ion laser at 488 nm. Emitted light was reflected through a 505–550-nm band pass filter from a 540-nm dichroic mirror. Photobleaching (the irreversible damage of Fluo-4/AM producing a less fluorescent species) was assessed by continuous exposure (5 min) of loaded cells to laser illumination. For each experiment, the laser illumination setting that gave a minimal reduction in cytosolic and food vacuole fluorescence was used (this varied according to dye loading and confocal laser scanning microscopy (CLSM) settings). Data capture and extraction was carried out with LSM510 version 3 software (Zeiss). Cytosolic and vacuolar [Ca2+] was calibrated in situ by the addition of known amounts of [Ca2+]free in the presence of nigericin (0.8 μm) and the Ca2+ ionophore A23187 (1 μm). Using Calcium Calibration Buffer Kit 1 solutions (Molecular Probes), the [Ca2+]free was calculated from the K d of EGTA for Ca2+ (107.9 nm at 37 °C (12Tsien R. Pozzan T. Methods Enzymol. 1989; 172: 230-262Crossref PubMed Scopus (392) Google Scholar)) using the equation [Ca2+]free = K dEGTA × [CaEGTA/K2EGTA]. Effect of FPIX on the Fluorescence Quantum Yield of Ca2 + Indicators—Heme (FPIX) is a major component of the digestive food vacuole of P. falciparum. As the fluorescence quantum yield of some food vacuole targeted probes can be reduced dramatically by heme (FPIX), we decided to test the effect of monomeric heme E on a number of Ca2+-specific UV- and visible light-excitable fluorescent probes. Titration of FPIX to solutions of Fura-2 or Fluo-4 resulted in a progressive reduction of fluorescence signal (Fig. 1). However, this reduction was more pronounced in the case of the UV-excitable probe (e.g. Fura-2) than for visible light-excitable probes (e.g. Fluo-4 and Fluo-3 (not shown)); Fluo-4 retained ∼60% of its fluorescent emission signal at FPIX levels, which eliminated the Fura-2 signal. Fluo-4 was therefore used as the probe of choice to measure cytosolic and food vacuole Ca2+ fluctuations. The Digestive Food Vacuole of P. falciparum Contains Elevated Free Ca2 + —With the use of CLSM and the cell-permeant AM ester of Fluo-4 to probe for Ca2+ in trophozoites of infected red blood cells, we observed a strong fluorescence signal deriving from the digestive food vacuole (e.g. Fig. 3e). Some Ca2+ indicators have a tendency to compartmentalize, and differences in apparent Ca2+ affinities in different compartments/organelles within a cell can sometimes lead to the formation of artifactual Ca2+ gradients (13Thomas D. Tovey S.C. Collins T.J. Bootman M.D. Berridge M.J. Lipp P. Cell Calcium. 2000; 28: 213-223Crossref PubMed Scopus (317) Google Scholar). To overcome these possible artifacts, free Ca2+ was calibrated independently in situ in both the parasite cytosolic and food vacuole compartments (Fig. 2). The differing slopes (and fluorescence quantum yields in response to Ca2+) of the parasite cytosol and food vacuole calibration curves revealed that there was a difference in the respective apparent Ca2+ binding affinities, probably resulting from differences in the Ca2+ buffering capacity of the different intracellular milieus and possibly from a degree of dye compartmentalization. However, taking into account the differences in Fluo-4 Ca2+ affinities (by using the respective in situ calibration curves), the digestive food vacuole was measured nevertheless as having elevated free Ca2+ relative to the cytosol, with levels ranging between 250 and 300 nm (n = 27), some 5 to 6 times higher than the cytosol (measured at ∼50 nm, n = 27).Fig. 2Cytosol (•) and digestive food vacuole (○) in situ free Ca2+ calibration of Fluo-4/AM-loaded P. falciparum-infected erythrocytes. Data points represent the mean ± S.E. of ≥19 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Intracellular Distribution of Free Ca2 + during the Intraerythrocytic Development of the Malaria Parasite—The distribution of free Ca2+ in the malaria parasite was observed to change during asexual development. As described, early trophozoites were observed as having a Ca2+ gradient between the digestive food vacuole and cytoplasmic compartments (Fig. 3e). However, as parasites underwent schizogony, free Ca2+ was observed to leave the digestive food vacuole and move to the intermembrane space surrounding each segment (Fig. 3, f and g). On rupture and release of merozoites from the erythrocytic host, there was a sustained elevation in free Ca2+; however the resolution of the CLSM did not allow us to determine the specific intracellular distribution of the signal (Fig. 3h). This increase was lost shortly after reinvasion of new red blood cells (i.e. the early ring stage), which may imply an important role for elevated merozoite free Ca2+ in the invasion process. Spatiotemporal Dynamics of Free Ca2 + in the Malaria Parasite and Digestive Food Vacuole—Fluo-4/AM is a BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid)-based indicator with a large dynamic range (R f ≈ 50–200 (14Maravall M. Mainen Z.F. Sabatini B.L. Svoboda K. Biophys. J. 2000; 78: 2655-2667Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar)), making it very suitable for the monitoring of both elementary and global Ca2+ signals (13Thomas D. Tovey S.C. Collins T.J. Bootman M.D. Berridge M.J. Lipp P. Cell Calcium. 2000; 28: 213-223Crossref PubMed Scopus (317) Google Scholar). Using this indicator, we were able to determine spatiotemporal Ca2+ fluctuations in malaria parasite-infected erythrocytes that were immobilized using polylysine coated coverslips in a Bioptechs FCS2 perfusion chamber, in response to a variety of inhibitors and agents. As described, trophozoites were shown to posses elevated Ca2+ in the digestive vacuoles (Fig. 3e). The addition of thapsigargin (a plant sesquiterpene lactone that interacts irreversibly with sarcoplasmic reticulum-like Ca2+ ATPases (15Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar)) to trophozoites caused a rapid depletion of vacuolar Ca2+ (Figs. 4A (and Fig. 4B, QuickTime movie) and 5A). The addition of cyclopiazonic acid, another known selective irreversible inhibitor of sarcoplasmic reticulum-like Ca2+-ATPases (16Goeger D.E. Riley R.T. Biochem. Pharmacol. 1989; 38: 3995-4003Crossref PubMed Scopus (112) Google Scholar), also caused a rapid reduction in vacuolar free Ca2+ (Figs. 4C (and Fig. 4D, QuickTime movie) and 5B). Unlike the case of thapsigargin, however, the reduction of food vacuolar Ca2+ upon addition of cyclopiazonic acid was accompanied by a conspicuous transient increase in cytosolic Ca2+. Maneuvers that caused a perturbation of the trans-vacuolar membrane pH gradient (the food vacuole is acidic relative to the cytosol), such as the addition of the selective V-type ATPase inhibitor, bafilomycin A1 (Fig. 5C), and of NH4Cl (QuickTime movie, Figs. 4E and 5C) to the perfusate, also caused a rapid reduction of vacuolar Ca2+. A transient rise in cytosolic Ca2+ was also evident upon addition of NH4Cl to the perfusate (Fig. 5C). The membrane integrity of the digestive food vacuole was maintained on the addition of the irreversible inhibitors thapsigargin, cyclopiazonic acid, and bafilomycin A1 (see QuickTime movies, Fig. 4, B, D, and E) and the malaria pigment crystals were observed to continue to move inside the vacuoles. Washing infected erythrocytes with HEPES-buffered RPMI (pH 7.4) or Ringer solution after treatment with NH4Cl restored the food vacuole Ca2+ gradient of the cells (data not shown).Fig. 5Effect of thapsigargin (1 μm) (A), cyclopiazonic acid (10 μm) (B), NH4Cl (80 mm) (C), and bafilomycin A1 (200 nm) (D) on the concentration of free Ca2+ in the malaria parasite cytosol (○) and digestive food vacuole (•). Values shown are the mean ± S.E. (n ≥ 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) As the digestive food vacuole of the malaria parasite contains a P-glycoprotein pump (PGH1), which can affect the compartmentalization of some fluorescence indicators, verapamil, a known PGH1 modulator (17Reed M.B. Saliba K.J. Caruana S.R. Kirk K. Cowman A.F. Nature. 2000; 403: 906-909Crossref PubMed Scopus (705) Google Scholar), was added to the perfusate of the single cell chamber system. Verapamil (10 μm) was not shown to effect any change in the fluorescence signal deriving from either the food vacuole or the cytoplasm, suggesting that there was no interference from PGH1 in our system. It has been suggested previously that the malaria parasite contains an acidic Ca2+ storage organelle (18Garcia C.R. Ann S.E. Tavares E.S. Dluzewski A.R. Mason W.T. Paiva F.B. Eur. J. Cell Biol. 1998; 76: 133-138Crossref PubMed Scopus (50) Google Scholar, 19Marchesini N. Luo S. Rodrigues C.O. Moreno S.N. Docampo R. Biochem. J. 2000; 347: 243-253Crossref PubMed Scopus (110) Google Scholar, 20Alleva L. Kirk K. Mol. Biochem. Parasitol. 2001; 117: 121-128Crossref PubMed Scopus (75) Google Scholar). However, negative staining of the digestive vacuole by UV-excitable Ca2+ fluorescent probes precluded its involvement (20Alleva L. Kirk K. Mol. Biochem. Parasitol. 2001; 117: 121-128Crossref PubMed Scopus (75) Google Scholar). We have shown recently that FPIX can interact with fluorescent probes such as acridine orange, resulting in a dramatic reduction in fluorescent emission, probably occurring from the absorption by the FPIX Soret peak (21Bray P.G. Saliba K.J. Davies J.D. Spiller D.G. White M.R. Kirk K. Ward S.A. Mol. Biochem. Parasitol. 2002; 119: 301-304Crossref PubMed Scopus (38) Google Scholar). We therefore investigated whether the fluorescent emission of previously used UV-excitable Ca2+-specific probes such as Fura2 (19Marchesini N. Luo S. Rodrigues C.O. Moreno S.N. Docampo R. Biochem. J. 2000; 347: 243-253Crossref PubMed Scopus (110) Google Scholar, 20Alleva L. Kirk K. Mol. Biochem. Parasitol. 2001; 117: 121-128Crossref PubMed Scopus (75) Google Scholar) as well as a range of visible light-excitable Ca2+ probes were also affected by FPIX. Titration of FPIX to solutions of Fura-2 or Fluo-4 resulted in a progressive reduction of fluorescence signal (Fig. 1). However, this reduction was more pronounced in the case of Fura-2 than Fluo-4, which retained ∼60% of its original fluorescent emission signal at FPIX levels that eliminated the Fura-2 signal. Importantly, the probe remained Ca2+-sensitive within this organelle, albeit with an altered apparent Ca2+ affinity (Fig. 2). FPIX is reported to achieve mm concentrations in the food vacuole (22Loria P. Miller S. Foley M. Tilley L. Biochem. J. 1999; 339: 363-370Crossref PubMed Scopus (222) Google Scholar), which would fully explain the lack of food vacuole-derived Fura-2 fluorescence reported by other investigators. With the use of CLSM and Fluo-4/AM (which is less susceptible to FPIX fluorescence quenching and has a good dynamic range) to probe for Ca2+ in mature trophozoites of infected red blood cells, we observed a free Ca2+ pool in the digestive food vacuole (Fig 3e) generating a Ca2+ gradient some 5–6-fold greater than that measured in the cytoplasm (at rest). It was clearly observed that the sequestration of free Ca2+ altered during the asexual intraerythrocytic development of the malaria parasite (Fig 3). For example, as parasites underwent schizogony, free Ca2+ was observed to leave the digestive food vacuole and move to the intermembrane space surrounding each segment (Fig. 3, f and g) producing "wagon wheel"-looking Ca2+ distributions. It has been accepted for more than 20 years that Ca2+ in the culture medium is essential for the complete asexual development of the malaria parasite in red blood cells in culture (23Wasserman M. Alarcon C. Mendoza P.M. Am. J. Trop. Med. Hyg. 1982; 31: 711-717Crossref PubMed Scopus (74) Google Scholar). The results presented here lend further support to a role for Ca2+-dependent processes in the asexual development of the parasite. Our findings showing Ca2+ storage by the digestive food vacuole have allowed us to shed light on a recent controversy relating to the pharmacology of the sarco/endoplasmic reticulum Ca2+-ATPases of P. falciparum. A recent study demonstrated that the malaria parasite contains an endoplasmic reticulum-like Ca2+ store that is controlled by a thapsigargin-insensitive but cyclopiazonic acid-sensitive Ca2+-pump (20Alleva L. Kirk K. Mol. Biochem. Parasitol. 2001; 117: 121-128Crossref PubMed Scopus (75) Google Scholar), a finding that was at odds with previous studies indicating a thapsigargin-sensitive mechanism (19Marchesini N. Luo S. Rodrigues C.O. Moreno S.N. Docampo R. Biochem. J. 2000; 347: 243-253Crossref PubMed Scopus (110) Google Scholar, 24Garcia C.R. Dluzewski A.R. Catalani L.H. Burting R. Hoyland J. Mason W.T. Eur. J. Cell Biol. 1996; 71: 409-413PubMed Google Scholar). It is now apparent that both of these studies adopted methods that were ineffective for the measurement of vacuolar Ca2+ and therefore that their fluorescence signals originated from cytosolic Ca2+ fluctuations. Using our live cell CLSM system to monitor Fluo-4-loaded infected red blood cells, we observed that upon addition of thapsigargin (1 μm) or cyclopiazonic acid (10 μm) to the perfusate, Ca2+ from the digestive food vacuole was rapidly (∼60 s) released (Fig. 4, A and C, and Fig. 5, A and B (QuickTime movies, Fig. 4, B and D)). In the case of cyclopiazonic acid (but undetectable for thapsigargin), the vacuolar release of Ca2+ was accompanied by a small transient increase in cytosolic Ca2+ (Figs. 4C and 5B). These results help explain previous observations of transient elevations of Ca2+ upon addition of cyclopiazonic acid (20Alleva L. Kirk K. Mol. Biochem. Parasitol. 2001; 117: 121-128Crossref PubMed Scopus (75) Google Scholar). In addition these results suggest the operation of more than one P-type Ca2+ ATPase, one that is sensitive to both thapsigargin and cyclopiazonic acid and one that is only sensitive to cyclopiazonic acid. Significantly, and in support of our findings, the recent functional characterization of P. falciparum transporters expressed in Xenopus laevis oocytes have revealed the presence of both a cyclopiazonic-sensitive Ca2+-ATPase (PfATPase4 (25Krishna S. Woodrow C. Webb R. Penny J. Takeyasu K. Kimura M. East J.M. J. Biol. Chem. 2001; 276: 10782-10787Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar)) and a thapsigargin-sensitive Ca2+-ATPase (PfATPase6). 2S. Krishna, personal communication. At this early stage in the pharmacological analysis of intracellular Ca2+ signaling in the malaria parasite, however, it should be noted that thapsigargin and cyclopiazonic acid have previously been shown to have different additional nonselective effects on Ca2+ signaling pathway components, e.g. capacitative Ca2+ entry (26Taylor C.W. Broad L.S. Trends Pharmacol Sci. 1998; 19: 370-375Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). However, it is unlikely that capacitative Ca2+ entry is responsible for the observed increase in cytoplasmic Ca2+ on treatment with cyclopiazonic acid, as this effect was observed by Alleva and Kirk (20Alleva L. Kirk K. Mol. Biochem. Parasitol. 2001; 117: 121-128Crossref PubMed Scopus (75) Google Scholar) to occur in freed P. falciparum parasites suspended in Ca2+-free solutions. The generation of a H+-gradient across the parasite vacuolar membrane by the action of V-type H+ ATPases and H+-pumping pyrophosphatases (8Saliba K.J. Allen R.J. Zissis S. Bray P.G. Ward S.A. Kirk K. J. Biol. Chem. 2003; 278: 5605-5612Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) suggests that, as with plant tonoplasts and acidocalcisomes of some parasites, the digestive food vacuole of the malaria parasite may also drive Ca2+ accumulation by H+-coupled transport mechanisms. This hypothesis is supported by our findings that a collapse of the transmembrane H+ gradient by the addition of NH4 (Fig. 5C (QuickTime movie, Fig. 4E)) or bafilomycin A1 (a selective V-type H+-ATPase inhibitor; Fig. 5C) results in a rapid discharge of vacuolar Ca2+. Given the essential role of Ca2+ as a secondary messenger molecule and the fact that physiological studies in plants have established a role for vacuolar (tonoplast) H+/Ca2+ exchange activity in ion transport, Ca2+ homeostasis, and signal transduction (27Hirschi K. Trends Plant Sci. 2001; 6: 100-104Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), further elucidation of the analogous H+-dependent mechanisms underlying Ca2+ homeostasis in the malaria parasite will be of significant importance. Here we add another critical transport function to the malarial parasites repertoire of vacuolar transport mechanisms (Fig. 6). It will be important to determine how the interplay of Ca2+ transport and H+ transport via either the H+-pyrophosphatase or the V-type H+-ATPase contributes to organelle function. The rapid redistribution of vacuolar Ca2+ at schizogony (asexual multiplication) must implicate communication between the parasite nucleus and the food vacuole. In addition this organelle harbors transporters implicated in drug resistance (notably PfCRT and PGH1 (reviewed in Ref. 4Warhurst D.C. Craig J.C. Adagu I.S. Lancet. 2002; 360: 1527-1529Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar)). The potential for proton-coupled Ca2+ transport to influence the function of these resistance transporters could have significant implications for malaria chemotherapy in the age of the quinoline-resistant parasite. Download .zip (2.12 MB) Help with zip files
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