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Experimental Parasitology 118 (2008) 2–9

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Acidocalcisomes in Apicomplexan parasites

Kildare Miranda a,b, Wanderley de Souza b, Helmut Plattner c, Joachim Hentschel c,Urara Kawazoe d, Jianmin Fang a, Silvia N.J. Moreno a,*

a Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, 350 Paul D. Coverdell Center,

University of Georgia, Athens, GA 30602, USAb Laboratorio de Ultraestrutura Celular Hertha Meyer, IBCCF, CCS, UFRJ, Rio de Janeiro, RJ, Brazil

c Fachbereich Biology, Universitat Konstanz, 78457 Konstanz, Germanyd Departamento de Parasitologia, IB, UNICAMP, Campinas, SP, Brazil

Received 21 June 2007; received in revised form 19 July 2007; accepted 21 July 2007Available online 3 August 2007

Abstract

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidicmatrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Theirmatrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase.The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomeshave been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and inToxoplasma gondii. In this paper, we review the structural, biochemical and physiological aspects of acidocalcisomes in Apicomplexanparasites and discuss their functional roles in the maintenance of intracellular ion homeostasis.� 2007 Published by Elsevier Inc.

Index Descriptors and Abbreviations: Toxoplasma gondii; Plasmodium falciparum; Eimeria acervulina; Apicomplexan; Acidocalcisomes

1. Introduction

Ion homeostasis in protozoan parasites has been the sub-ject of intense investigation in the last few years (Docampoand Moreno, 2001; Moreno and Docampo, 2003). Cell via-bility requires perfect functioning of these mechanisms sincedisruption of homeostasis of certain ions, such as Ca2+, canlead to cell death (Berridge et al., 2000). In addition, Ca2+ isinvolved in the invasion of host cells by different parasites, aprocess that is crucial for maintaining their life cycles(Moreno et al., 2007a,b; Lu et al., 1997; Vieira and Moreno,2000; Lovett and Sibley, 2003; Moreno et al., 1994).

Different intracellular organelles are involved in the reg-ulation of ion homeostasis in protozoan parasites, such asthe endoplasmic reticulum, mitochondria, and acidocalci-somes. The acidocalcisomes were first described in Trypan-

0014-4894/$ - see front matter � 2007 Published by Elsevier Inc.

doi:10.1016/j.exppara.2007.07.009

* Corresponding author. Fax: +1 706 583 0181.E-mail address: smoreno@cb.uga.edu (S.N.J. Moreno).

osoma brucei (Vercesi et al., 1994) and T. cruzi (Docampoet al., 1995), and, since then, have been identified in a vari-ety of microorganisms, including Apicomplexan parasitessuch as T. gondii and Plasmodium spp. (Docampo et al.,2005). In this review, we will examine the structural, chem-ical and physiological properties of acidocalcisomes fromApicomplexan parasites, as well as their functional rolein the cell biology of these parasites.

2. Acidocalcisomes in Apicomplexan parasites

They are characterized by their acidic internal milieu,high electron density, and high content of phosphorus inthe form of phosphate, pyrophosphate and polyphosphate(poly P) and cations such as calcium, magnesium, sodium,potassium, and zinc. Acidocalcisomes were first identifiedin Coccidian parasites in 1907 (Kunze, 1907), and inToxoplasma gondii in 1966 (Mira Gutierrez and Del ReyCalero, 1966) when they were named metachromatic or

K. Miranda et al. / Experimental Parasitology 118 (2008) 2–9 3

volutin granules, for their ability to stain red when treatedwith toluidine blue (metachromasia). In T. gondii they werealso named ‘black granules’ (Bonhomme et al., 1993). Thename acidocalcisome was first used in T. gondii when it wasshown that these organelles possess mechanisms for thetransport of protons and calcium (Moreno and Zhong,1996).

3. Acidocalcisomes in Toxoplasma gondii

Acidocalcisomes are the largest store of Ca2+ in T. gon-

dii (Bouchot et al., 1999; Luo et al., 2001; Moreno andZhong, 1996). Their acidity is easily demonstrated throughthe incubation of tachyzoites with the weak base AcridineOrange (AO) and subsequent observation by fluorescencemicroscopy. Cells incubated in the presence of AO showorange labeling of several acidocalcisomes, which are themost acidic compartments in the cell (Fig. 1). Other com-partments that have been shown to be acidic, such as rhop-tries (Shaw et al., 1998) and a vacuolar compartmentinvolved in microneme protein maturation (Harper et al.,2006), are not as evident by acridine orange staining.

In thin sections, the acidocalcisomes of T. gondii appearas empty vesicles sometimes with an electron dense mate-rial adjacent to the inner face of their membrane (Fig. 2).By electron spectroscopic imaging of whole cells directlydried onto Formvar-coated grids it is possible to see severalacidocalcisomes per cell (Fig. 3A and B). Acidocalcisomesappear as spherical electron dense organelles randomlyspread throughout the cell body. Approximately 10 acido-calcisomes, with diameters varying between �150 and�400 nm, are observed per cell. X-ray microanalysis ofintact cells (Luo et al., 2001) has revealed considerableamounts of oxygen, sodium, magnesium, phosphorus,chlorine, potassium, calcium and zinc concentrated in theacidocalcisomes (Fig. 3F), similarly to what has beenreported previously in the acidocalcisomes of trypanoso-matids (LeFurgey et al., 2001; Miranda et al., 2000, 2004;Rodrigues et al., 1999; Scott et al., 1997). The low sulfurcontent detected by elemental analysis suggests a lowcontent of proteins within acidocalcisomes.

Fig. 1. Fluorescence detection of acidic compartments in isolated tachyzoites25 lg/ml Acridine Orange in Dulbecco’s phosphate buffer saline (PBS) for 10 mAxioplan fluorescence microscope equipped with a 488 nm excitation filter setCCD camera (Model C5810) and an image analysis system attached to the micfluorescence image shown in (B). Scale bar for (A) and (B): 3 lm.

Toxoplasma gondii acidocalcisomes have a plasma mem-brane type Ca2+-ATPase (PMCA), involved in Ca2+

uptake, with similarity to vacuolar Ca2+-ATPases of otherunicellular eukaryotes (Bouchot et al., 2001; Luo et al.,2001). In addition, there occur two proton pumps, a vacu-olar H+-ATPase (V-H+-ATPase) and a vacuolar H+-pyro-phosphatase (V-H+-PPase), both contributing to theacidification of acidocalcisomes (Moreno et al., 1998;Rodrigues et al., 2000; Drozdowicz et al., 2003; Luoet al., 2001). Ca2+ release from acidocalcisomes of T. gondii

has only been detected upon their alkalinization (Morenoand Zhong, 1996) or after poly P hydrolysis (Rodrigueset al., 2002a) (Fig. 4).

The gene encoding the acidocalcisomal Ca2+-ATPase(TgA1) was able to complement yeasts deficient in the vac-uolar Ca2+-ATPase gene PMC1, providing genetic evi-dence for its function (Luo et al., 2001). This calciumpump is closely related to other acidocalcisomalCa2+-ATPases such as those present in Trypanosoma cruzi,

T. brucei, and Dictyostelium discoideum, as well as to thevacuolar Ca2+-ATPases of yeast, and Entamoeba histolytica

(Luo et al., 2001). These pumps lack a calmodulin-binding domain, in contrast to other PMCA-typeCa2+-ATPases. T. gondii mutants deficient in TgA1 havedecreased virulence in vitro and in vivo due to their deficientinvasion of host cells (Luo et al., 2005). Poly P content intachyzoites is drastically reduced in these mutants, and basalCa2+ levels are increased and unstable. Microneme secretionis also affected. Complementation of null mutants with TgA1

restores most of these defects in T. gondii (Luo et al., 2005).The V-H+-ATPase was first detected in T. gondii by its

sensitivity to bafilomycin A1, a specific inhibitor of thispump when used at low concentrations (Bowman et al.,1988). Bafilomycin A1 is able to release calcium from aci-docalcisomes of intact tachyzoites loaded with the fluores-cent calcium indicator Fura 2 (Moreno and Zhong, 1996).The V-H+-ATPase also localizes to the plasma membranewhere it has a role in regulating intracellular pH homeosta-sis (Moreno et al., 1998).

A V-H+-PPase activity is also detected in T. gondii

(Rodrigues et al., 2000). This enzyme localizes in acidocal-

of Toxoplasma gondii. (A and B): Cells were incubated in the presence ofin, washed twice, mounted on a microscope slide and observed in a Zeiss

. Emission signal (above 500 nm) was detected with a Hamamatzu digitalroscope. (A) Differential interferential contrast image corresponding to the

Fig. 2. Transmission electron microscopy of Toxoplasma gondii acidocalcisomes in isolated tachyzoites and intracellular parasites. (A–E), thin sections ofisolated T. gondii tachyzoites (A) and intracellular parasites (B) showing several acidocalcisomes (arrows) (C–D), high magnification of portions oftachyzoites showing several profiles of acidocalcisomes. They appear as empty vesicles containing some residual electron dense material as a result of theprocedure used for routine transmission electron microscopy. Note that some organelles seem to fuse with each other or to undergo fission (D–E). Scalebars: (A) 500 nm; (B) 3 lm; (C) 200 nm; (D) 100 nm; (E) 200 nm. (A, C–E) from Moreno et al. (2007a), with permission.

Fig. 3. Structure and composition of Toxoplasma gondii acidocalcisomes as seen by energy-filtering transmission electron microscopy and electron probeX-ray microanalysis and elemental mapping. (A and B), electron spectroscopic imaging (ESI) of whole tachyzoites adhered to formvar-coated electronmicroscope grids. Note the presence of several electron dense organelles enriched in oxygen, sodium, magnesium, phosphorus, calcium and zinc, as shownby X-ray microanalysis (C). (D–F) Elemental mapping of the cells shown in (B) revealing the co-localization of phosphorus, oxygen (mainly present in theform of short chain and long chain poly Ps) and calcium. Scale bars: (A) 5 lm; (B, D–F) 1 lm.

4 K. Miranda et al. / Experimental Parasitology 118 (2008) 2–9

cisomes (Drozdowicz et al., 2003; Luo et al., 2001; Rodri-gues et al., 2000), as well as in a vacuole involved in micro-

neme protein maturation (Harper et al., 2006). Thisenzyme is also located in the plasma membrane and Golgi

Fig. 4. Schematic representation of a typical acidocalcisome. Ca2+ uptakeoccurs in exchange for H+ by a reaction catalyzed by a vacuolar Ca2+-ATPase. A H+ gradient is established by a vacuolar H+-ATPase and avacuolar H+-pyrophosphatase (V-H+-PPase). Ca2+ release could occur inexchange for H+ and is favored by sodium-proton exchange. Anaquaporin allows water transport. Other transporters (for example, forMg, Zn, inorganic phosphate (Pi) and pyrophosphate (PPi), basic aminoacids) are probably present. The acidocalcisome is rich in pyrophosphate,short- and long-chain polyphosphate (poly P), magnesium, calcium,sodium and zinc. An exopolyphosphatase (PPX), a pyrophosphatase(PPase) and a polyphosphate kinase (PPK) may also be present. Aquestion mark was added to indicate the lack of biochemical evidence fortheir presence. Adapted from Moreno et al., 2007b, with permission.

Fig. 5. Acidocalcisomes in Plasmodium falciparum merozoites. Transmis-sion electron microscopic image of whole unstained and unfixed merozo-ites showing numerous acidocalcisomes spread throughout the cells. Scalebar: 0.5 lm. From Ruiz et al. (2004), with permission.

K. Miranda et al. / Experimental Parasitology 118 (2008) 2–9 5

complex of other cells (Docampo et al., 2005), althoughthere is no evidence at present of communication ortrafficking between acidocalcisomes and the plasma mem-brane. A truncated version of the enzyme (lacking theN-terminal region) was functionally expressed in yeast(Drozdowicz et al., 2003). Interestingly, the V-H+-PPase-of T. gondii changes its intracellular localization duringinvasion of host cells as shown by specific staining usingantibodies raised against a conserved domain of the T. gon-

dii V-H+-PPase (Drozdowicz et al., 2003). A chimaera ofthe T. gondii V-H+-PPase with the N-terminal extensionof T. cruzi V-H+-PPase at its N-terminus or the completegene were recently found to complement yeast cells defi-cient in the soluble pyrophosphatase (Drake et al., 2004).The acidocalcisomal enzyme belongs to the K+-stimulatedgroup of V-H+-PPases (type I) (Drozdowicz et al., 2003;Rodrigues et al., 2000). It is concentrated and it could beused as a marker for acidocalcisome purification in cellfractionation experiments (Rodrigues et al., 2002a).

Toxoplasma gondii acidocalcisomes have high levels ofphosphorus in the form of inorganic pyrophosphate (PPi)and polyphosphate (poly P). It is not known why PPi

and poly P are concentrated in acidocalcisomes. Only threereactions use PPi in T. gondii, one is catalyzed by the phos-phofructokinase (Peng et al., 1995), another one is cata-lyzed by the V-H+-PPase responsible for acidification ofacidocalcisomes (Rodrigues et al., 2000; Drozdowicz

et al., 2003), and the third one is catalyzed by an inorganicpyrophosphatase (Fang, J. and Moreno, S., unpublishedresults). All these enzymes are apparently cytosolic, and atransporter would be required to transfer PPi from the aci-docalcisomes to the cytosol. T. gondii is sensitive in vitro

and in vivo to PPi analogs. Two main targets have beendescribed for these drugs. One is the acidocalcisomal vacu-olar proton translocating pyrophosphatase (V-H+-PPase),which is sensitive to aminomethylenediphosphonate(AMDP) and imidodiphosphate (IDP) (Rodrigues et al.,2000). The second is an enzyme of the isoprenoid synthesispathway, i.e., farnesyl diphosphate synthase, which is sen-sitive to inhibition by aminobisphosphonates (Ling et al.,2005).

Poly P is a polymer of a few to many hundreds of inor-ganic phosphate (Pi) residues, which are linked by high-energy phosphoanhydride bonds. They have been foundin every category of organism examined, from bacteria tohumans (Kornberg, 1999). In different organisms poly Pis accumulated in very large amounts in acidocalcisomes(Docampo and Moreno, 2001). Although poly P wasthought to have a role as an energy source, this has beendisputed on the basis of its low metabolic turnover as com-pared to that of ATP (Chapman and Atkinson, 1977). Animportant regulatory role has been suggested instead (Raoand Kornberg, 1996). However, it has been shown thatpoly P kinase can transfer in a reverse reaction the terminalphosphate residue of poly P to ADP to yield ATP, so thatpoly P could participate in the global maintenance of theintracellular nucleotide pool (Ishige and Noguchi, 2000).In addition, long-chain poly P has been shown to be essen-tial for adaptation to various types of stress and for sur-vival of bacteria in stationary phase (Rao and Kornberg,1996). Similar studies have been reported in yeast (Castroet al., 1995). It has been demonstrated that in bacteria polyP can also function as a phosphate reserve under condi-tions of phosphate starvation, in cation sequestration andstorage, in cell membrane formation and function, in gene

6 K. Miranda et al. / Experimental Parasitology 118 (2008) 2–9

activity control, in regulation of enzyme activities, and as acomponent of channels and pumps (Kornberg, 1999;Kulaev and Kulakovskaya, 2000). Most of these studieshave been done with bacteria, and little is known aboutthe functions of poly P in eukaryotes. The enzymesinvolved in poly P metabolism are the polyphosphatekinase (synthesis) and the endo- and exo-polyphosphatases(degradation) (Kornberg, 1999; Kulaev and Kulakovs-kaya, 2000). Genes encoding an exopolyphosphatase(Wurst et al., 1995) and an endopolyphosphatase (Sethur-aman et al., 2001) from Saccharomyces cerevisiae, together

Fig. 6. Acidocalcisomes in Eimeria acervulina. (A) Transmission electron micracidocalcisomes (arrows). Acidocalcisomes partially (white arrow) of totally (bof polyphosphate-bound cations (not shown), are observed. RB, refractile bodyacervulina. Several acidic organelles (stained in orange) are observed in the cybody (green). Scale bar: 3 lm. (C) and (D) Immunofluorescence of E. acervu

thaliana V-H+PPase. Staining of small intracellular structures (presumably theDifferential interferential contrast microscopy corresponding to the fluorescen

with a gene encoding an acidocalcisomal exopolyphospha-tase from Leishmania major (Rodrigues et al., 2002b) andan exopolyphosphatase from Trypanosoma brucei (Lemer-cier et al., 2003) are the only ones from eukaryotic organ-isms that have been cloned and expressed, while two poly Pkinase genes have been found in the genome of D. discoid-eum (Gomez-Garcia and Kornberg, 2004; Kornberg,1999). Acidocalcisomes from T. gondii also contain apolyphosphatase activity (Rodrigues et al., 2002a).

Short and long chain poly P levels rapidly decreasedupon exposure of tachyzoites to agents that mobilize

oscopy of sporozoites of Eimeria acervulina, showing the fine structure oflack arrow) filled by the electron dense content, mainly present in the form, N, Nucleus. Scale bar, 500 nm. (B) Acridine orange of a sporozoite of E.

toplasm. Note binding of acridine orange to the nucleus and the refractilelina using antibodies raised against a conserved sequence of Arabidopsis

acidocalcisomes) can be seen in the anterior region of the parasite (D). (C)ce image shown in (D). Scale bar: 3 lm.

K. Miranda et al. / Experimental Parasitology 118 (2008) 2–9 7

Ca2+ such as calcium ionophores (ionomycin), alkalinizingagents (NH4Cl) or inhibitors of the V-H+-ATPase (bafilo-mycin A1) (Rodrigues et al., 2002a). This would be compat-ible with a role for poly P in the adaptation of the parasitesto environmental stress.

4. Acidocalcisomes in malaria parasites

The presence of calcium-containing acidic compart-ments similar to acidocalcisomes in different species ofPlasmodium spp. is well established (Garcia et al., 1998;Marchesini et al., 2000; Alleva and Kirk, 2001). A signifi-cant amount of Ca2+ is stored in these acidic compart-ments, as indicated by the increase in intracellular Ca2+

concentration induced by bafilomycin A1, nigericin (aK+/H+ exchanger), monensin (a Na+/H+ exchanger),aminomethylenediphosphonate (AMDP; a specific inhibi-tor or the vacuolar H+-pyrophosphatase), or the weak baseNH4Cl, in the nominal absence of extracellular Ca2+. Thisassumption is supported by the effect of the ionophoreionomycin, which becomes effective only after alkaliniza-tion of acidic compartment by addition of bafilomycinA1, AMDP, nigericin, monensin, or NH4Cl (Marchesiniet al., 2000).

Acidocalcisomes of similar fine structure and chemicalcomposition to those of other unicellular eukaryotes havebeen found, especially in merozoites of P. falciparum (Ruizet al., 2004) (Fig. 5). In addition, large amounts of shortand long chain poly P are detected in different blood stagesof P. falciparum. Concentrations for short-chain poly P are10 times higher than those of long chain poly P. Both shortand long chain poly P increase during differentiation of theblood stages, reaching maximal concentrations in the tro-phozoite stages and then decreasing in the schizonts (Ruizet al., 2004). Incubation of these cells with chloroquine,which has an alkalinizing effect, was shown to decreasethe poly P content of their acidocalcisomes (Ruiz et al.,2004).

Plasmodium falciparum merozoites show several acido-calcisomes per cell. The acidocalcisomes have a diameterof about 50 ± 11 nm, and some of them have an irregularform. X-ray microanalysis of these acidocalcisomes showedpeaks of phosphorus, calcium, carbon and oxygen (Ruizet al., 2004). While some attempts to identify acidocalci-somes in intact forms of other P. falciparum stages wereunsuccessful due to the high electron-density of the para-sites (Ruiz et al., 2004), a study using soft X-ray micros-copy reported the presence of ‘‘dense spheres’’ of similarsize and shape in the cytoplasm of trophozoites (Magowanet al., 1997) that are morphologically identical to acidocal-cisomes. In addition, another recent study revealed thepresence of round organelles rich in phosphorus in tropho-zoites of P. falciparum (Rohrbach et al., 2005).

Taken together, these results demonstrate that, thoughwith minor morphological differences, acidocalcisomes ofsimilar characteristics to those present in other unicellulareukaryotes are present in P. falciparum (Ruiz et al., 2004).

5. Acidocalcisomes in other Apicomplexan parasites

Little is known about the presence of acidocalcisomesin other Apicomplexan parasites. The V-H+-PPase, amarker for acidocalcisomes, has not been found in thegenome of Cryptosporidium parvum for which also nomorphological evidence of acidocalcisomes has been pre-sented. However, other Apicomplexan parasites, such asSarcosystis neurona, Eimeria tenella and Eimeria acervuli-

na (Miranda, K., unpublished) have acidic organellesthat morphologically and physiologically resemble theacidocalcisomes of T. gondii (Fig. 6). The apparently dif-ferent staining observed after acridine orange or anti-body labeling in Fig. 6 could be due to the labeling ofthe nucleus and refractile body with acridine orangewhich makes difficult the visualization of acidocalcisomespresent in different focal planes. This does not occurwhen antibodies against the V-H+-PPase are used sincethey specifically label the acidocalcisomes.

Acknowledgments

We thank Dr. Renato DaMatta for kindly providing theEimeria parasites. Work in our laboratories was funded bythe U.S. National Institutes of Health (AI-43614 toS.N.J.M.), Programa de Nucleos de Excelencia (PRO-NEX)/FAPERJ/CNPq-Brazil to W.S., by grants fromthe Deutsche Forschungsgemeinschaft to H.P. and Progra-ma de Nanociencia e Nanotecnologia MCT/CNPq-Brazilto K.M.

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