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Acidocalcisomes are acidic organelles with a high concentration of phosphorus present as pyrophosphate (PPi) and polyphosphate (poly P) complexed with calcium, and other cations. The acidocalcisome membrane contains a number of pumps (Ca2+-ATPase, V-H+-ATPase, H+-PPase), exchangers (Na+/H+, Ca2+/H+), and channels (aquaporins), while its matrix contains enzymes related to PPi and poly P metabolism. Acidocalcisomes have been observed in pathogenic, as well as non-pathogenic prokaryotes and eukaryotes e.g. Chlamydomonas reinhardtii, and Dictyostelium discoideum. Some of the potential functions of the acidocalcisome are the storage of cations and phosphorus, the participation of phosphorus in PPi and poly P metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis, and osmoregulation. In addition, acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. For example, we found that platelet dense granules, which are LROs, are very similar to acidocalcisomes. They share a similar size, acidic properties, and both contain PPi, poly P and calcium. Recent work that indicates that they also share the system for targeting of their membrane proteins through adaptor protein 3 (AP-3) reinforces this concept. The fact that acidocalcisomes interact with other organelles in parasitic protists, e.g. the contractile vacuole in Trypanosoma cruzi, and other vacuoles observed in Toxoplasma gondii, suggests that these cellular compartments may be associated with the endosomal/lysosomal pathway.
ACIDOCALCISOMES are acidic calcium-storage organelles found in a diverse range of organisms, including trypanosomes in which they were first defined (Docampo et al. 1995; Vercesi, Moreno, and Docampo 1994). Beginning as early as 1895, acidic calcium-storage organelles have been variously defined as “metachromatic granules” (Babes 1895), “volutin granules” (Meyer 1904) or “polyphosphate bodies” (Wiame 1947). The high concentrations of both calcium and polyphosphate (poly P) in prokaryotes was first succinctly described by Kornberg (Kornberg 1995). Subsequently, when these highly acidic calcium-storage organelles were discovered in eukaryotes, the trypanosomatids and apicomplexans, they were biochemically redefined as “acidocalcisomes” (Docampo et al. 2005). The presence of enzymes and transporters in the surrounding membranes of these organelles was fundamental in understanding their potential function and origin (Docampo et al. 2005). More recent work from our laboratory reveals that the previously described “volutin granules” in prokaryotes are acidocalcisome-like organelles (Seufferheld et al. 2003, 2004). Interestingly, the dense granules from human platelets also have characteristics similar to acidocalcisomes (Ruiz et al. 2004). The fact that acidocalcisome-like organelles occur both in bacteria and humans suggests that this organellar compartment was established before prokaryotic and eukaryotic lineages diverged, and that these organelles have been conserved over evolutionary time.
In trypanosomatids and other protists, acidocalcisomes are easily identified because they stain with dyes such as acridine orange (Docampo et al. 1995; Miranda et al. 2008; Vercesi et al. 1994) or cycloprodigiosin (Scott and Docampo 2000) that accumulate within acidic compartments, or they appear as cytoplasmic granules in Giemsa-stained preparations. The morphology of acidocalcisomes varies among different species and methods of cultivation. In general these organelles are spherical, with average diameters of 0.2 μm in Trypanosoma cruzi (Miranda et al. 2000) and Trypanosoma brucei (Rodrigues, Scott, and Docampo 1999a), which can reach 0.6 μm in some Leishmania species (Rodrigues, Scott, and Docampo 1999b). However, the morphology of these organelles may vary from spherical to elongated and polymorphic as in some Leishmania and Phytomonas species (Docampo et al. 2005). They are typically randomly distributed within the cell (Fig. 1 and 2).
Using transmission electron microscopy (TEM), acidocalcisomes typically contain electron-dense material, although this may vary with the method used for sample preparation. Using standard methods for transmission electron microscopy part of the dense material can be lost, leaving an empty vacuole, or a thin layer of dense material that adheres to the inner face of the membrane, prompting the name “inclusion bodies” (Fig. 4 and 5). A useful method for observing acidocalcisomes is to dry entire protist cells (Fig. 1, 2) or fractions (Fig. 3) onto carbon- and formvar-coated grids and then view them by TEM. When unfixed cells of the apicomplexan Toxoplasma gondii or the trypanosomatid Leptomonas collosoma are treated in this manner, the acidocalcisomes are clearly delineated as electron-dense spheres within the cytoplasm (Fig. 1 and 2).
By light microscopy, acidocalcisome-like organelles in prokaryotes and eukaryotes can be stained by a number of dyes that accumulate either in polyphosphate or acidic compartments eg. 4′-6′-diamino-2-phenylindole (DAPI), cycloprodigiosin or Lysosensor blue DND-167, respectively (Marchesini et al. 2002; Miranda et al. 2000; Ruiz et al. 2001b, 2004; Seufferheld et al. 2003, 2004).
In addition to analyzing the elemental composition of acidocalcisomes in trypanosomatids and apicomplexans using TEM, both 31P NMR, and biochemical techniques were employed to demonstrate the presence of O, Na, Mg, P, K, Ca and Zn in these organelles (Docampo et al. 2005). Elemental iron was also detected in several trypanosomatids cultivated in complex media, as well as in T. cruzi trypomastigotes, Leishmania amazonensis promastigotes, and promastigotes of the plant kinetoplastid Phytomonas francai (Docampo et al. 2005). The presence of P and Ca in similar organelles led some authors to propose the presence of hydroxyapatite in some apicomplexan (Lukes and Stary 1992).
In protists, both structural and elemental composition of acidocalcisomes can be modulated by varying culture conditions. In semi-defined medium promastigotes of L. amazonensis contain spherical organelles lacking iron, but when these cells are transferred into a complex, iron-rich medium, the acidocalcisomes accumulate iron and became dispersed throughout the cytoplasm (Miranda et al. 2004). On the other hand, changes in the content of acidocalcisomes appear to have diverged among various lineages of trypanosomatids. i.e. under identical conditions of culture, the elemental composition varies. This is congruent with the idea that the function of this cellular compartment has become tailored for species within each lineage.
As mentioned previously, it is well established that the acidocalcisomes from eukaryotes concentrate phosphorus both as inorganic pyrophosphate (PPi) and polyphosphate (poly P). Poly P is a linear chain of a few to many hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds that occurs across a wide spectrum of diverse organisms (Kornberg, Rao, and Ault-Riche 1999). It is proposed that acidocalcisomes accumulate poly P as one mechanism to reduce the osmotic effect of large pools of cellular phosphate. Trypanosomatids are especially rich in short-chain poly P such as poly P3, poly P4, and poly P5 (Moreno et al. 2000). 31P NMR spectra of purified acidocalcisomes in the kinetoplastids T. cruzi, T. brucei and L. major have indicated that the average chain length of poly P is 3.2 Pi residues. On the basis of its total concentration in different stages of T. cruzi, and the relative volume of the acidocalcisomes in these cells (2.3%, 0.86%, and 0.26% of amastigote, epimastigote, and trypomastigote cell volume, respectively (Miranda et al. 2000), and assuming that poly P is mostly concentrated in acidocalcisomes, the calculated concentration in these organelles is about 3–5 M (Docampo et al. 2005). This is congruent with the detection of solid-state condensed phosphates by magic-angle spinning NMR techniques, as well as by electron density measurements in situ using TEM (Moreno et al. 2002). It has recently been proposed that carbohydrates and/or lipids might also help maintain the physical configuration of poly P in these organelles (Salto et al. 2008).
Compartmentalization of amino acids is also apparent in trypanosomatids. In T. cruzi epimastigotes, 90% of the 1,250 nmol per mg total protein in acidocalcisomes consisted of the basic amino acids arginine and lysine, whereas whole cell extracts chiefly contained neutral and acidic amino acids (Rohloff, Rodrigues, and Docampo 2003).
Interestingly, elemental sulfur was only detected in small amounts, suggesting that very few proteins are present in this cellular compartment. Nevertheless, a number of rather critical enzymes have been detected in trypanosomatid acidocalcisomes. In T. cruzi these include a polyphosphate kinase which responds to cell growth, differentiation and environmental stress (Ruiz, Rodrigues, and Docampo 2001a), an a Zn+-sensitive soluble exopolyphosphatase affecting osmoregulation (Fang et al. 2007b) which is also present in L. major (Rodrigues et al. 2002), a soluble inorganic pyrophosphatase in T. brucei that regulates poly P metabolism and which is essential for virulence in mice (Lemercier et al. 2004), a metacaspase in L. donovani which may play a role in cell death (Lee et al. 2007), and an acid phosphatase in T. rangeli epimastigotes detected by cytochemistry (Gomes et al. 2006).
The membranes of acidocalcisomes differ from other types of organellar membranes. Highly purified acidocalcisomes from T. cruzi epimastigotes primarily contain phospholipids, one of which is a glycoinositolphospholipid (GIPL) quite distinct from microsomal GIPLs (Salto et al. 2008). These phospholipids are also accompanied by low concentrations of 3b-hydroxyesterols.
As in other organisms, the membranes of acidocalcisomes are known to have several pumps, exchangers and at least one channel (Docampo et al. 2005). Ca2+-ATPase activities have been observed in isolated acidocalcisomes of T. cruzi (Scott and Docampo 2000) and T. brucei (Rodrigues et al. 1999a). Not only have the genes encoding these enzymes been identified in T. cruzi (tca1) (Lu et al. 1998), T. brucei (TbPMC1) (Luo et al. 2004), and Toxoplasma gondii (TgA1) (Luo et al. 2001), but each of these can complement yeast deficient in the vacuolar Ca2+-ATPase, PMC1. Furthermore, the acidocalcisomal Ca2+-ATPases of T. cruzi, T. brucei, T. gondii and Dictyostelium discoideum, as well as the vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Entamoeba histolytica belong to a subcluster of conserved core sequences within the family of plasma membrane calcium ATPases (PMCA). In contrast to other PMCA-Ca2+-ATPases a common feature of these pumps is the apparent lack of a typical calmodulin-binding domain (Docampo et al. 2005). A scheme of all the transporters identified in the acidocalcisomes of diverse organisms is depicted in Fig. 2.
Two proton pumps have been detected in acidocalcisomes of different microorganisms. One is a vacuolar-type H+-ATPase and the other is a vacuolar-type H+-pyrophosphatase (V-H+-PPase). The V-H+-ATPase was first identified in permeabilized trypomastigotes of T. brucei and epimastigotes of T. cruzi by its sensitivity to bafilomycin A1, a specific inhibitor at low concentrations of this proton pump (Docampo et al. 1995; Vercesi et al. 1994). Using intact trypanosomatids loaded with the fluorescent calcium indicator Fura 2, bafilomycin A1 was able to release calcium from an intracellular compartment of T. cruzi, T. brucei, and L. amazonensis, implying its presence in the acidocalcisome (Docampo et al. 2005). In T. cruzi a V-H+-ATPase was also shown, by immunofluorescence and immunoelectron microscopy, to co-localize to acidocalcisomes with the vacuolar-type Ca2+-ATPase, and to be absent in the endocytic pathway (Lu et al. 1998).
The V-H+-PPase activity first noted in T. cruzi (Scott et al. 1998), and later in T. brucei (Rodrigues et al. 1999a) and L. donovani (Rodrigues et al. 1999b) has also been localized within acidocalcisomes of L. amazonensis, P. francai, T. gondii and P. falciparum (Docampo et al. 2005). The T. cruzi enzyme could be functionally expressed in yeast (Hill et al. 2000), whereas this gene in T. brucei has not yet been tested (Lemercier et al. 2002). This enzyme is a member of the K+-stimulated group of V-H+-PPases (type I), and serves as a marker for purified acidocalcisomes because it localizes to and is concentrated in them. Interestingly, the V-H+-PPase of trypanosomatids and apicomplexans can also occur within one or two additional compartments. In T. cruzi this enzyme may localize within the Golgi and plasma membrane (Martinez et al. 2002), whereas in species of Plasmodium the pyrophosphatase has been observed, in addition to the acidocalcisomes (Luo et al. 1999, Marchesini et al. 2000), in the plasma membrane (McIntosh et al. 2001) and digestive vacuoles (Saliba et al. 2003), and in T. gondii within a microneme maturation vacuolar compartment (Harper et al. 2006).
There is also evidence for the presence of Na+/H+ and Ca2+/H+ exchangers in the acidocalcisomes of procyclic and promastigote forms of T. brucei (Vercesi and Docampo 1996; Vercesi, Grijalba, and Docampo 1997)and L. donovani (Vercesi et al. 2000), respectively. Although the Na+/H+ exchanger is unable to transport lithium, it is sensitive to 3,5-dibutyl-4-hydroxy toluene (BHT) and insensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) (Vercesi et al. 1997). It was suggested that the Ca2+/H+ exchanger might serve as a mechanism for Ca2+ release, because Ca2+ is released from acidocalcisomes both in situ and in vitro when Na+ is present, whereas other known second messengers such as inositol trisphosphate (InsP3), are unable to do so ( Docampo et al. 1993; Moreno et al. 1992a, 1992b). Isolated organelles of trypanosomatids also differ remarkably in the type and functions of exchangers. Although T. brucei procyclic trypomastigotes and L. donovani promastigotes both possess an Na+/H+ exchanger (Rodrigues et al. 1999a), only in procyclic forms of T. brucei it is stimulated by ADP (Vercesi and Docampo 1996; Vercesi et al. 1997, 2000). Furthermore, trypomastigotes of T. cruzi entirely lack this exchanger, but their acidocalcisomes posses an unique water channel or aquaporin (Montalvetti, Rohloff, and Docampo 2004) which appears to participate in osmoregulation because it is translocated to the contractile vacuole complex (Rohloff, Montalvetti, and Docampo 2004).
Protist acidocalcisomes are a major storage compartment for phosphorus (Pi, PPi and poly P). Of these, PPi is a byproduct of the biosynthesis of nucleic acids, coenzymes, and proteins, activation of fatty acids and biosynthesis of isoprenoids. Almost nothing is known about how PPi is transported across acidocalcisomes or why it is stored. On the other hand, quite a lot is known about poly P, which occurs across a wide spectrum of prokaryotes and eukaryotes (Kornberg 1995; Kulaev and Kulakovskaya 2000). In trypanosomatids, changes in the concentration of short and long chain poly P have been detected during the life cycle and differentiation of T. cruzi. These concentrations rapidly decrease when cells are exposed to hyposmotic stress, whereas concentrations increase after hyperosmotic stress (Ruiz et al. 2001). This might indicate a role for acidocalcisomes in the stress response to environmental changes, and may potentially be linked to changes in the concentration of Pi. The concentration of poly P has been linked to virulence both in prokaryotes (Kornberg et al. 1999) and eukaryotes eg. decreased virulence in the apicomplexan T. gondii (Luo, Ruiz, and Moreno 2005).
That acidocalcisomes contained very high concentrations of calcium was their first distinguishing characteristic (Vercesi et al. 1994). This and other cations such as magnesium, sodium, potassium, and others are combined with poly P for storage (Moreno et al. 2007). In addition, heavy metals can accumulate with poly P in acidocalcisomes when present in the environment (Kulaev and Kulakovskaya 2000). Furthermore, host cell invasion is inhibited by depletion of calcium from within acidocalcisomes by pretreatment of metacyclic (Neira, Ferreira, and Yoshida 2002) and extracellular amastigote (Fernandes et al. 2006) stages of T. cruzi with ionomycin in combination with nigericin or ionomycin to which NH4Cl has been added (Docampo et al. 1995). Additional evidence that acidocalcisome calcium plays a role in invasion has been observed in T. gondii tachyzoite knockouts of TgA1, the enzyme necessary for pumping calcium into the organelles. This results in the deregulation of cytosolic calcium, which then alters micronemal secretion leading to less virulent tachyzoites (Luo et al. 2005).
In addition to osmoregulation, stress response and virulence, poly P appears to help regulate intracellular pH. For example, the generation of H+ from the hydrolysis of poly P has been shown to neutralize pH changes as much as 2.5 units in S. cerevisiae (Castro, Koretsky, and Domach 1999). RNA interference (RNAi) experiments to reduce acidocalcisome V-H+-PPase activity confirm this regulatory function (Lemercier et al. 2002). The phenotypic changes induced in T. brucei procyclic trypomastigotes by RNAi resulted in the loss of the cell’s ability to maintain pH homeostasis after exposure to an external basic pH > 7.4. These cells were also slower to recover from intracellular acidification, and failed to reach their more neutral, starting intracellular pH (Lemercier et al. 2002).
It is well established that trypanosomatids encounter extreme changes in their osmotic environments as they move from insect guts into the mammalian bloodstream (Rohloff et al. 2004). Because of this, it is assumed that mechanisms for osmoregulation play an essential role in their survival during transmission from invertebrate into vertebrate host. In fact it has been shown that the cellular release of ions and osmolytes, including amino acids and potassium, does occur and that this enables metacyclic trypomastigotes to adjust their volume following the hyposmotic stress induced during transmission (Rohloff et al. 2004). Nevertheless, this mechanism is unable to account for the total recovery of volume observed, and our hypothesis is that acidocalcisomes, in conjunction with the contractile vacuole complex, might aid this osmoregulatory process (Rohloff et al. 2004). Evidence supporting this hypothesis in trypanosomatids has been documented in epimastigotes of T. cruzi by the rapid hydrolysis or synthesis of acidocalcisomal poly P during hypo- or hyperosmotic stress (Ruiz et al. 2004), and by the change in sodium and chloride concentrations in acidocalcisomes of L. major promastigotes in response to acute hyposmotic stress (LeFurgey, Ingram, and Blum 2001).
Interestingly, the aquaporins of T. cruzi, which are located in both the acidocalcisomes and contractile vacuole complex (Montalvetti et al. 2004), have also been implicated in osmoregulation. Aquaporins are vacuolar and plasma membrane water channel proteins that occur among a diverse spectrum of organisms (Kjellbom et al. 1999) which passively permit water molecules to move along a gradient. In T. cruzi, the aquaporin TcAQP1 is translocated from the acidocalcisome to the contractile vacuole when cyclic AMP stimulates the fusion of these two organellar compartments (Rohloff et al. 2004). Additional evidence that acidocalcisomes participate in trypanosomatid osmoregulation was observed when RNAi knockdowns of the T. brucei acidocalcisomal soluble pyrophosphatase TbVSP1 markedly reduced the concentration of organellar poly P and the subsequent ability of cells to respond to hyposmotic stress (Lemercier et al. 2004). Furthermore, the ablation by RNAi of the T. brucei vacuolar transporter chaperone TbVTC1 resulted in abnormal morphology of acidocalcisomes, decrease in their poly P content, and a deficient response to hyposmotic stress (Fang et al. 2007a).
Lysosome-related organelles (LROs) are a heterogeneous group of organelles that both share some physiological features with lysosomes, and yet are distinguished from them by structural and functional diversity (Cutler 2002) eg. melanosomes, lytic granules, major histocompatibiliy complex (MHC) class II compartments, platelet dense granules, basophil granules, and neutrophil azurophil granules (Dell’Angelica et al. 2000). Their sharing of traits suggests that there may be a common origin of biogenesis for lysosomes and LRO.
Recent results from several laboratories have shed some light on the origin of acidocalcisomes, and their relatedness with LROs. Endocytic tracers such as transferrin (Scott et al. 1997), horseradish peroxidase (Coppens et al. 1993), and FM4-64 (Mullin et al. 2001), do not accumulate in these organelles. However, if parasites are treated with an inhibitor of the sterol biosynthetic pathway, their acidocalcisomes do accumulate endocytic markers (Vannier-Santos et al. 1999), suggesting that there is some association of acidocalcisomes with the endosomal/lysosomal pathway. In L. major, a mutant deficient in sphingolipid synthesis was shown to be defective in the biogenesis of both multivesicular bodies (or late endosomes) and acidocalcisomes, which suggests that these compartments have a common origin (Zhang et al. 2005). Besteiro et al. (Besteiro et al. 2008) recently found that Adaptor protein 3 (AP-3), the system known to be involved in transport of membrane proteins to lysosomes and LROs in other cells, has a similar function with respect to acidocalcisomes in Leishmania major, providing support for a close similarity between acidocalcisomes and the endo/lysosomal system. Furthermore, mutants of T. brucei deficient in an orthologue of the vacuolar sorting protein 41 (VSP41p), which is known to interact with the δ subunit of AP-3-coated carrier vesicles (Rehling et al. 1999), and is involved in the biogenesis of lysosome-related organelles (Dell’Angelica et al. 2000) were shown to have large numbers of small intracellular vesicles similar to acidocalcisomes (Lu et al. 2007). Finally, acidocalcisomes of L. donovani deficient in ADP-ribosylation factor-like protein (ARL-1), which controls vesicle traffic and vacuole formation in yeast, were deficient in V-H+-PPase (Sahin et al. 2008).
Acidocalcisomes also resemble LROs in many of their properties. For example, platelet dense granules, which are LROs, are very similar to acidocalcisomes (Ruiz et al. 2004). They share a similar size, acidic properties, and both organelles contain PPi, poly P and calcium. The finding that they also share the system for targeting of their membrane proteins reinforces this concept (Besteiro et al. 2008). The finding that acidocalcisomes and LROs are related implies that further studies of the acidocalcisomes could shed light on conserved functions in similar organelles of other cells of medical relevance. In turn, an analysis of the features of various LROs is likely to highlight as-yet-undiscovered functions of acidocalcisomes (Besteiro et al. 2008).
Acidocalcisomes were found in bacteria more than one hundred years ago but an investigation of these organelles, and of their main constituent, poly P, has been neglected for many years. The conservation of this organelle in bacteria and eukaryotes indicates that it has important functions that await discovery. Further studies are necessary to understand the biogenesis and function of acidocalcisomes in different organisms, why they have been conserved and how widely the organelle is distributed. Phylogenetic relationships of various acidocalcisomal enzymes need to be established, as sequence comparisons are important indicators of the evolution of these organelles. We do not know how acidocalcisomes are distributed in daughter cells after cell division or why morphological changes occur in acidocalcisomes of some trypanosomatids. Intracellular PPi, poly P, cations and basic amino acids are accumulated in large amounts in acidocalcisomes, but the mechanisms by which these compounds are transported into the organelle and the reasons for their accumulation are largely unknown. This is an exciting area of research, not only because these organelles have different characteristics in diverse eukaryotes, but because they might serve as new targets for drugs as recently reviewed by us (Docampo and Moreno 2008).
This work was supported in part by grants AI68467 (to SNJM) and AI68647 (to RD) from the National Institutes of Health.
1Presentation delivered at the symposium: Cellular Compartmentalization: Protists Do It Their Way, 21—26 July 2008, The International Society of Evolutionary Protistology and The International Society of Protistologists, Dalhousie University, Halifax, NB, Canada.