Calcium is relevant for several vital functions in apicomplexan parasites, including host cell invasion, parasite motility and differentiation. The ER (endoplasmic reticulum) and calcium-rich acidocalcisomes have been identified as major calcium stores. Other potential calcium-storage organelles include the Golgi, the mitochondrion, the apicoplast and the recently described plant-like vacuole in Toxoplasma gondii. Compared with most eukaryotic systems, apicomplexan parasites contain a reduced number of calcium-related genes, a vast majority of which remain uncharacterized. Several Ca2+-ATPases have been described in apicomplexans, several of which are annotated in the different genomes. There is experimental evidence for an IP3 (inositol 1,4,5-trisphosphate)-dependent calcium response in Plasmodium spp. and T. gondii, although no IP3 or ryanodine receptors have been identified. Genes encoding potential calcium channels are present in T. gondi, but not in Plasmodium spp. and Cryptosporidium spp. Effector calcium-binding proteins including calmodulins and CDPK (calcium-dependent protein kinase) genes mainly found in plants have also been described. The characterized CDPKs were found to play important roles in protein secretion, host cell invasion and parasite differentiation. Taken together, the available information on calcium storage and function in apicomplexans, although fragmented, suggest the existence of unique calcium-mediated pathways in these parasites. An in-depth functional characterization of the apicomplexan calcium-related genes could lead to the identification of novel therapeutic targets, and will improve our understanding of the role of calcium in parasite development and virulence.
Fluctuations of cytosolic free Ca2+ concentrations ([Ca2+]i) regulate a variety of cellular functions in all eukaryotes. The [Ca2+]i is maintained at very low levels (of the order of 10−7 M) compared with that in the extracellular medium (approximately 10−3 M). A variety of mechanisms, enzyme transporters, channels and CBPs (calcium-binding proteins) contribute to maintaining the [Ca2+]i at 10−7 M. The total calcium inside the cell is much higher than 10−7 M, but the bulk of this calcium is either bound to proteins, polyphosphate, membranes and/or other cellular constituents, or is sequestered inside intracellular organelles such as mitochondria, ER (endoplasmic reticulum), Golgi apparatus and nuclei . A key event in calcium signalling is the influx of calcium across the plasma membrane. Ca2+-storage organelles capable of both high-affinity uptake and rapid triggered release of Ca2+ are believed to be ubiquitous among eukaryotes .
Ca2+ homoeostasis and storage in apicomplexans
Apicomplexan parasites are a large group of protists, which include a number of pathogens of medical and agricultural relevance. These parasites are named for their peculiar apical end, which contains a number of unique organelles and structures.
Calcium homoeostasis and storage has been studied mainly in two groups of apicomplexan parasites: Toxoplasma gondii and Plasmodium spp. T. gondii is an important cause of congenital disease and infection in immunocompromised patients. Plasmodium spp. represent the causative agents of malaria, one of the most devastating human infectious diseases. Intracellular calcium measurements have been performed in apicomplexan parasites mainly using the fluorescent calcium indicator fura 2/AM (fura 2 acetoxymethyl ester). This reagent crosses the plasma membrane and is converted inside the cell into fura 2 by endogenous cytosolic esterases. This de-esterified form of the reagent is unable to cross membranes and becomes trapped in the cytoplasm of the cell, where it is able to monitor changes in the cytosolic calcium concentration. Using this methodology, the [Ca2+]i in T. gondii tachyzoites was measured at 70±6 nM in the absence of extracellular Ca2+ (with the Ca2+ chelator EGTA added to the medium) and 100±9 nM in the presence of 1 mM extracellular Ca2+ . In Plasmodium chabaudi and Plasmodium falciparum, the [Ca2+]i was also measured using fura 2-loaded free parasites and the values obtained were also at nanomolar levels in single-cell imaging experiments  or in parasite suspensions . When imaging P. falciparum-infected erythrocytes loaded with fura red, a ratiometric calcium indicator with a low sensitivity to pH, much higher levels of [Ca2+]i (289–352 nM), were measured . This high value may be the result of superposition of Ca2+ signals from the cytosol and the extensive ER compartment within these cells . The [Ca2+]i in the parasite is probably within the range of concentrations observed in other eukaryotic cells (i.e. 90–100 nM).
In addition to known eukaryotic calcium stores, including acidocalcisomes, the ER, Golgi apparatus and mitochondria, apicomplexan parasites contain several unique organellar compartments that potentially could contribute to diverse calcium transients necessary for vital functions within the parasites. These include the apicoplast, a remnant plastid derived from a secondary endosymbiotic event, and various acidic organelles including the recently described PLV (plant-like vacuole).
The largest store of Ca2+ in cells is usually found in the ER (Figure 1), with the local concentration reaching millimolar levels. The ER possesses independent pathways for calcium influx and efflux. The influx is catalysed by the very well known SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), which actively translocates two Ca2+ molecules for the hydrolysis of one ATP molecule. SERCA-type Ca2+-ATPases have been characterized in T. gondii (Figure 1, b)  and in P. falciparum . Thapsigargin, a specific inhibitor of SERCA-type Ca2+-ATPases of other eukaryotes produces an increase in the cytosolic Ca2+ levels of T. gondii and of Plasmodium spp. at high concentrations [4,9]. Release of Ca2+ from the ER occurs through an IP3 (inositol 1,4,5-trisphosphate)-stimulated calcium channel. An IP3/ryanodine-sensitive store has been postulated to be present in T. gondii on the basis of pharmacological studies (Figure 1, f) , and the release of Ca2+ from intracellular stores of malaria parasites was also shown to respond to IP3 [9,11]. The enzyme that catalyses the production of IP3, PI-PLC [phosphoinositide PLC (phospholipase C)], has been characterized in T. gondii (TgPI-PLC) . However, there is no genetic evidence to indicate the presence of an IP3 receptor in any of the apicomplexan parasites. This means that the parasite may use a different mechanism (probably responsive to IP3) to release calcium from the ER.
Acidocalcisomes are organelles that contain large amounts of calcium in an acidic environment. This calcium is probably not free but bound to other molecules as short- and long-chain polyphosphate . Acidocalcisomes were first studied in trypanosomes and apicomplexan parasites and later found to be similar to the previously described ‘volutin granules' in bacteria or polyphosphate bodies in algae . These organelles were initially found because the stored Ca2+ could be released into the cell cytoplasm with nigericin (a K+/H+ exchanger) or the weak base NH4Cl . This compartment was characterized further and named the acidocalcisome (Figure 1). More recent work in Leishmania suggests that acidocalcisomes are lysosome-related organelles . Acidocalcisomes have also been found in Plasmodium spp.  and more recently in Eimeria parasites .
The calcium inside the acidocalcisome is probably pumped in by a Ca2+-ATPase, which in T. gondii has been named as TgA1 (Figure 1, b in the acidocalcisome) . Recent work with a purified acidocalcisome fraction from T. gondii tachyzoites shows that calcium uptake is sensitive to vanadate (a Ca2+-ATPase inhibitor), supporting the idea of calcium being stored in this organelle .
Two enzymes with proton pumping activity have been found to localize to acidocalcisomes. The vacuolar-H+-pyrophosphatase is an enzyme that hydrolyses pyrophosphate, and the energy released is used to pump protons towards the lumen of the organelle where it localizes. This enzyme was described and characterized in T. gondii (TgVP1) (Figure 1, c) and also in P. falciparum (PfVP1) . The second enzyme is the vacuolar-H+-ATPase (Figure 1, d), which pumps protons and uses the energy from the hydrolysis of ATP. Acidification of the acidocalcisome by these pumps is important to maintain organellar calcium, as alkalinizing agents such as NH4Cl release calcium into the cytoplasm. This is postulated to occur through a Ca2+/H+ exchanger (Figure 1, e in the acidocalcisome).
The vacuolar-H+-pyrophosphatase also localizes to other compartments, for example the PLV in T. gondii, the food vacuole in Plasmodium spp., the plasma membrane in both parasites and also to other endocytic compartments. This is unique because, in mammalian cells, acidic compartments are maintained by the vacuolar-H+-ATPase. One possible explanation for this difference is that the vacuolar-H+-pyrophosphatase uses pyrophosphate as an energy source, which is quite abundant in these parasites. Some of the developmental stages of the parasites might run short of an ATP supply because of their suboptimal mitochondrial function as, for example, in the blood stages of Plasmodium spp. and extracellular tachyzoites of T. gondii .
The PLV, recently described in T. gondii, was also found to contain calcium. This was demonstrated by adding the compound GPN (glycyl-l-phenylalanine-naphthylamide) to intact parasites loaded with the Ca2+ indicator fura 2/AM. GPN is specifically hydrolysed in the lysosome of a variety of different cell types by a CPC (cathepsin C) protease, which results in swelling and loss of membrane integrity of the lysosome, leading to calcium leaking out to the cytoplasm . The presence of a CPC inside the PLV was demonstrated by proteomic data of enriched fractions and IFA (immunofluorescence assay) analysis of cells expressing a C-terminal tagged CPC gene (S.N.J. Moreno and V.B. Carruthers, unpublished work). This GPN-dependent calcium release was shown to be independent of other calcium stores, such as the ER .
Further evidence supporting the presence of Ca2+ inside the PLV is the detection of a vacuolar PMCA (plasma membrane Ca2+-ATPase)-type calcium ATPase (TgA1). This was shown by proteomic analysis of subcellular fractions and by IFAs. TgA1 has been characterized previously and shown to have a role in intracellular Ca2+ homoeostasis as well as in parasite virulence. The specific role of the PLV in Ca2+ homoeostasis or Ca2+-related function is still not known.
Mitochondria possess a high capacity to sequester Ca2+ although, under physiological conditions, total mitochondrial Ca2+ levels and free Ca2+ reflect and parallel cytosolic Ca2+. The inner mitochondrial membrane possesses a uniport carrier for Ca2+, which allows the electrogenic entry of the cation driven by the electrochemical gradient generated by respiration or ATP hydrolysis (Figure 1, Ψ in the mitochondrion). Ca2+ efflux, on the other hand, takes place by a different pathway, which appears to catalyse the electroneutral exchange of internal Ca2+ by external sodium or protons. Experiments performed with malaria parasites using digitonin to measure mitochondrial activity in situ showed Ca2+ uptake from the incubation medium by a mechanism associated with depolarization of the membrane potential. These results support the presence of a Ca2+ uniport similar to that of mammalian mitochondria . A Ca2+/H+ antiporter was recently localized to P. falciparum mitochondria . Sensitivity to ruthenium red and ruthenium 360 suggested the presence of a Ca2+ uniport in these mitochondria . Unlike the mammalian mitochondria, where intracellular Ca2+ regulates the activity of several dehydrogenases, no such Ca2+-regulated dehydrogenases have been reported in apicomplexan parasites.
Calcium signalling and functions in apicomplexan parasites
Ca2+ signalling involves the mobilization of Ca2+ from two sources: intracellular stores and the extracellular medium. Mechanisms to introduce Ca2+ into the cytoplasm are compensated by a co-ordinated set of removal mechanisms consisting of buffers, pumps and exchangers. Free Ca2+ binds a number of effectors, which are responsible for stimulating numerous Ca2+-dependent processes. Each cell type expresses a unique set of mechanisms and effectors, which create a Ca2+ signalling system with the appropriate spatial and temporal properties.
In excitable mammalian cells, such as in muscle cells, the primary signal (membrane depolarization) activates Ca2+ entry across the plasma membrane, and this Ca2+ signal is amplified and propagated by a mechanism of CICR (Ca2+-induced Ca2+ release) from the sarcoplasmic reticulum . In non-excitable mammalian cells, such as endothelial cells, activation by a hormone or growth factor receptor coupled to a PLC results in the hydrolysis of PIP2 (phosphatidylinositol 4,5-bisphosphate) to generate IP3 and diacylglycerol. IP3 diffuses to the intracellular stores (ER) and causes the release of Ca2+ into the cytoplasm through IP3 receptors . Some mammalian cells, such as smooth muscle and neuroendocrine cells, can utilize both of these pathways.
Ca2+ release from the ER is followed by the entry of Ca2+ across the plasma membrane. This process is known as capacitative Ca2+ entry or SOCE (store-operated Ca2+ entry) . The connection between the ER and the plasma membrane was only discovered in recent years and is orchestrated mainly by two protein families: Stim (stromal interaction molecule; Stim1 and 2), which appear to function as Ca2+ sensors within the ER, and Orai proteins (Orai1, 2 and 3), which function as SOCE channels in the plasma membrane . In addition, a number of newly discovered second messengers, such as cADPr (cyclic adenosine diphosphate ribose), sphingosine 1-phosphate and NAADP (nicotinic acid–adenine dinucleotide phosphate) have been observed to release or modulate the release of intracellular Ca2+ from different cells. The plasma membrane of eukaryotic cells contains a number of channels through which calcium gains access into the cytoplasm. Some of these channels respond to changes in the membrane potential (voltage-gated Ca2+ channels), others are under the control of receptors (receptor-operated Ca2+ channels), and the rest are controlled by the content of intracellular stores (store-operated Ca2+ channels). The active export of calcium from eukaryotic cells is accomplished by the action of an Na+/Ca2+ exchanger or the PMCA.
The information available on calcium signalling components in apicomplexan parasites is still fragmentary, although important features of their life cycle, such as motility, host cell invasion and egress from infected cells, are linked to calcium. A sequence with general similarity to a voltage-dependent Ca2+ channel has been found in the T. gondii genome (TDR Targets database number TGME49_005260). The demonstration that this gene product is functional as a Ca2+ channel awaits further work (Figure 1, a). There is no direct evidence for receptor-operated Ca2+ influx, and none of the genes that correspond to the SOCE machinery, Stim and Orai , are present in any of the apicomplexan genomes. A Ca2+ channel has been postulated to be inserted in the plasma membrane of erythrocytes infected with P. falciparum . There are several reports of PMCA-type-Ca2+-ATPases in apicomplexan parasites . Biochemical evidence for CaM (calmodulin) stimulation of this pump has been reported , although TgA1 appears to lack a typical CaM-binding domain. This might suggest the presence of a different domain able to bind CaM. A gene encoding a second putative PMCA has been found in the T. gondii genome (TDR Targets database number TGME49_033770) . The deduced amino acid sequence (1200 amino acids) shows 45% identity with TgA1 . No homologues to this enzyme are found in the Plasmodium genomes.
CBPs in apicomplexan parasites
Once inside the cell, Ca2+ can either interact with so-called soluble CBPs or become sequestered into intracellular organelles. CBPs are characterized by the presence of a highly conserved helix–loop–helix structural motif known as an EF hand. Typically, EF-hand motifs occur in pairs (also called the EF-hand domain) and facilitate the co-operative binding of two Ca2+ ions per domain. However, CBPs with a single or odd number of EF-hand motifs have been reported in both bacteria and eukaryotes and are believed to function via dimerization mechanisms. At least 69 EF-hand-domain-containing proteins are encoded by the P. falciparum genome (PlasmoDB), 55 in T. gondii (ToxoDB) and 45 in Cryptosporidium parvum (CryptoDB). As with most apicomplexan protein families, a majority of these EF-hand-domain-containing proteins are hypothetical with no known functions.
CBPs are generally classified into three main families: the CaM family, the CBL (calcineurin B-like) family, and the CDPK (calcium-dependent protein kinase) family. The CaM family includes classical CaMs (sequence identity with human CaM >75%), CML (CaM-like) proteins (sequence identity with human CaM <75%) and all other CaM-related proteins (presence of at least one non-CaM domain structure). Structurally, CaMs are acidic proteins composed of two globular domains (each with a pair of EF hands) linked by a flexible helical region. Most apicomplexan genomes encode single prototype CaMs and a variable number of CMLs/CaM-related proteins. Of these proteins, only the T. gondii CaM has been cloned and shown to bind Ca2+ in vitro . Additional evidence in support of a functional CaM in T. gondii has been provided by in vitro experiments using the CaM inhibitors calmidazolium and trifluoperazine . These drugs significantly reduce host cell entry by T. gondii tachyzoites, suggesting a role for TgCaM (T. gondii CaM) in host cell invasion. CaM inhibitors are equally toxic to P. falciparum, affecting parasite development and erythrocyte invasion by merozoites [32,33]. Unlike CaM proteins, CBLs have been identified only in higher plants and, to a limited extent, in some protist genomes. Inspection of representative apicomplexan genomes reveals single gene sequences that potentially may encode functional CBLs in T. gondii and P. falciparum.
The third class of CBPs comprises several enzymes that are modulated through direct interactions with Ca2+. These include a variety of kinases [e.g. CDPKs and CCaMKs (calcium/CaM-dependent protein kinases)], proteases, phosphatases, synthases and NTPases. Most apicomplexan genomes encode several CDPKs and serine/threonine protein phosphatases.
The fourth group of CBPs encoded by the apicomplexan genomes includes several hypothetical proteins, heat-shock proteins, centrin/troponin C-like proteins, and variants of the Plasmodium-specific virulence factor PfEMP1. These proteins, which have barely been characterized in some apicomplexans, are likely to exhibit vital functions, including a role in buffering and species-specific signalling processes.
Functional studies of calcium and CBPs in apicomplexan parasites
Microneme secretion, invasion and egress
T. gondii and Plasmodium spp. contain micronemes (Figure 1), specialized apical secretory organelles that appear to play an important role in the early phase of the invasion process. These organelles contain protein complexes or adhesins, which are discharged and participate in the interaction with the host cell surface. A large number of studies support the relevance of microneme secretion during invasion of the host cell.
The secretion of micronemes can be induced artificially by treating parasites with calcium ionophores . This effect can be blocked with the intracellular Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)], demonstrating that the secretion of micronemes is triggered by an increase in intracellular Ca2+. BAPTA-AM was also used to demonstrate the role of calcium in conoid extrusion , gliding motility  and invasion . The stimulation of microneme secretion by Ca2+ has also been demonstrated in C. parvum  and Plasmodium berghei .
The relevance of intracellular Ca2+ homoeostasis was also highlighted by the phenotype of the TgA1-null mutant parasites. These cells have their intracellular calcium levels altered, are unable to maintain it at a physiological level under the experimental conditions tested and are deficient in microneme secretion. In addition, these cells have an invasion defect and reduced virulence in vivo .
T. gondii replicates inside its host cell, but at some point needs to exit to be able to infect other cells, and this egress process is still poorly understood. T. gondii egress is rapid and results in lysis of the infected host cell. It is also known that calcium ionophores such as A23187 can stimulate this process . Parasite mutants with a defect in egress (delayed egress) have been isolated and found to have their intracellular calcium levels elevated  and in addition have reduced pathogenicity .
Calcium and motility
Secretion of microneme proteins is also important for motility of T. gondii. Microneme secretion is triggered by an increase in intracellular calcium (see above), meaning calcium may have a role in motility. Several pieces of information available in the literature support this statement. T. gondii parasites were loaded with the Ca2+ indicator Fluo-4 and analysed by live imaging, and it was observed that periodic oscillations in the intracellular Ca2+ levels were linked to gliding of the parasites . The exit from the host cell is also a process dependent on motility of the parasite.
The effect of Ca2+ ionophores on egress could be linked to the effect on the motility of the parasite. Changes in extracellular Ca2+ have not been evaluated as a possible trigger of microneme secretion, and the natural agonist responsible for stimulating intracellular Ca2+ increase and subsequent microneme release is yet to be identified.
Role of CDPK
The CDPK family constitutes a group of kinases that are only found in plants and protists. In plants, CDPKs are a required response mechanism that allows external Ca2+ signals to regulate a diverse number of pathways including cell-cycle progression and stress responses. The typical CDPK is composed of an N-terminal serine/threonine kinase domain, followed by a junction domain (also known as the auto-inhibitory domain) that connects to the C-terminal CML domain. The CML domain typically consists of multiple calcium-binding domains (i.e. four EF-hand domains).
Phylogenetic analyses of CDPKs in apicomplexans show that there is a large diversity of these kinases present in the Plasmodium, Toxoplasma and Cryptosporidium genera. In addition to the typical CDPKs described above and found in plants, apicomplexan parasites contain four additional structural variants. The primary source of this variation is the number of EF-hand domains and the length of the N-terminus preceding the kinase domain. P. falciparum possesses seven annotated CDPKs, whereas T. gondii possesses 12 CDPKs .
Ca2+ is known to have a key role in critical features of apicomplexan parasites as a second-messenger system, and CDPKs have been implicated as a mechanistic link between Ca2+ signalling and differentiation, motility, invasion and egress. The rodent malarial parasite P. berghei requires CDPKs for developmental differentiation. Genetic disruption of CDPK4 in P. berghei gametocytes (sexual stages located in the mammalian bloodstream) inhibits calcium-dependent pathways that are required for microgamete differentiation . Additionally, knockout of a different kinase, CDPK3, in P. berghei severely inhibits the ability of ookinetes to traverse the peritrophic membrane in the mosquito gut, thereby stopping oocyst production in the mosquito gut and short-circuiting the vector pathway of this parasite .
Essential attributes of CDPKs have also been observed in the related apicomplexan T. gondii. The protein kinase inhibitor KT5926, which inhibits microneme secretion required for host cell attachment of the parasite, has been shown to target a CDPK in T. gondii . More specifically, down-regulation of T. gondii CDPK1 resulted in loss of parasite motility and host cell invasion and egress abilities, further demonstrating the essential nature of CDPKs in this important apicomplexan parasite .
The essential nature of CDPKs in regulating critical pathways of apicomplexan parasites has been clearly established. However, further efforts in determining the specific substrates of CDPKs will provide a more mechanistic understanding of their control of invasion and differentiation. Additionally, Ca2+-dependent protein kinases exhibit a significant level of cross-talk with other protein kinases, most notably the cyclic nucleotide-dependent kinases. Taking this into account along with the large number and structural diversity of Ca2+-dependent kinases, it is apparent that further research on the CDPKs in apicomplexans is likely to be very rewarding in terms of cell biology and development of potential drug targets for clinical use.
Ca2+ has important roles in secretion, motility, cell invasion and differentiation of apicomplexan parasites. Ca2+ regulation is controlled by a variety of systems for uptake and release that differ in several aspects from the processes that occur in other eukaryotic cells, providing excellent opportunities for targeting them for new therapies.
Apicomplexan parasites contain several P-type Ca2+-ATPases, including a SERCA-type that is important for Ca2+ uptake in the ER, as well as Ca2+/H+ antiporters. However, a PMCA-type Ca2+-ATPase and voltage-dependent Ca2+ channels are only present in T. gondii. There is evidence for mechanisms of Ca2+ release stimulated by IP3 and cADPr in some of these parasites, but no receptors for these second messengers have been identified. A number of CBPs, including CaMs, CML proteins and an array of CDPKs, are present in these parasites. Acidocalcisomes are present in Plasmodium spp., T. gondii, and Eimeria spp., but are absent in Cryptosporidium spp. Other acidic organelles containing Ca2+ include the digestive vacuole of malaria parasites and the PLV of T. gondii. Because of their situation at an early branch point in eukaryotic evolution, studies of Ca2+ storage and signalling in these parasites could shed light about the origins of complex signalling networks in eukaryotes. Several apicomplexan genomes have been completed, making it possible to look for conserved pathways through sequence-based phylogenetic comparisons. Additionally, the continuing availability and enhancement of experimental tools for genetic manipulation and molecular investigation of apicomplexan parasites (especially T. gondii and Plasmodium spp.) should allow for significant advances in deciphering calcium signalling pathways in these important eukaryotes.
• Ca2+ is important for motility, secretion, invasion, egress and differentiation of apicomplexan parasites.
• There is insufficient evidence for the presence of some mechanisms for Ca2+ uptake and release, for example IP3 and cADPr receptors, PMCA-type Ca2+-ATPases or voltage-dependent Ca2+ channels, in many of these organisms.
• Acidic calcium stores, such as the acidocalcisome, PLV and digestive vacuole, appear to play important roles as Ca2+ stores.
• Apicomplexans contain a diversity of calcium-dependent kinases, which are commonly found in plants.
S.N.J.M. is supported by a grant from the U.S. National Institutes of Health (NIH) [grant number AI-079625]. D.A.P. is supported in part by NIH T32 training grant AI-060546 to the Center for Tropical and Emerging Global Diseases.
- © The Authors Journal compilation © 2011 Biochemical Society