Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii

Abstract

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca²⁺) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca²⁺ signaling is essential for the cellular and developmental changes that support T. gondii parasitism.

The characterization of the molecular players and pathways directly activated by Ca²⁺ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca²⁺ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca²⁺ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.

1. Calcium and the Toxoplasma lytic cycle

Toxoplasma gondii belongs to the phylum Apicomplexa, which includes a number of unicellular eukaryotes that infect humans and animals and cause diseases such as malaria (Plasmodium spp.), babesiosis (Babesia spp.), toxoplasmosis (Toxoplasma gondii), cryptosporidiosis (Cryptosporidium parvum), and cyclosporiasis (Cyclospora cayetanensis), among others. Apicomplexan are highly divergent from mammals and more closely related to ciliates and dinoflagellates [1]. The name of the phylum Apicomplexa denotes the presence of a unique set of secretory organelles at the apical complex of zoites. These specialized organelles secrete unique effectors, in a highly regulated manner, critical for entering into their host cell [2].

Toxoplasma gondii is an obligate intracellular parasite that infects approximately one third of the world population [3, 4]. Toxoplasma can be transmitted through contaminated food or water via oocyts released with the feces of infected cats, the definitive host [3, 5]. Intermediate hosts, such as animals and humans, can be infected via mature oocysts containing sporozoites or via contaminated meat with tissue cysts containing bradyzoites. Both sporozoites and bradyzoites transforms into tachyzoites, the fast replicating form, which change back into slow replicating bradyzoites within tissue cysts, which are characteristic of the chronic infection and usually found in the brain, eye, and striated muscle tissues [6]. Disseminated toxoplasmosis can cause severe complications in immunocompromised patients including HIV-infected individuals, fetuses, and organ transplant recipients [7, 8]. Some of the most devastating effects of toxoplasmosis are the result of the parasite lytic cycle (Fig. 1), which starts when the tachyzoite attaches and invades a host cells, replicates inside a parasitophorous vacuole and egresses to find another host cell to invade and re-start the cycle [9, 10].

Figure 1.

Toxoplasma tachyzoites attach and invade host cells and form a parasitophorous vacuole (PV) where they reside and replicate by endodyogeny. The PV is surrounded by a PV membrane (PVM) that allows small molecules to sieve through. After several rounds of replication, they rupture the PVM and the host cell membrane and egress followed by a short extracellular phase and invasion of another host cell. Christina Moore designed the Lytic Cycle

Invasion and egress are dynamic processes that are highly regulated and essential for the propagation of the infection. Toxoplasma and other Apicomplexan parasites like Plasmodium rely on gliding motility for host-cell invasion, egress and dissemination in the infected host. Gliding motility, vital to a successful lytic cycle, is a peculiar mode of substrate dependent motility, unique to Apicomplexan parasites, that facilitates entry into host cells and is powered by a macromolecular complex termed the glideosome [11].

During invasion, the parasite secretes proteins from specific apical organelles [12], and their contents mediate attachment to host cells and formation of the specialized parasitophorous vacuole (PV) occupied by the parasite [13]. Each step during the lytic cycle has to be precise, fast and effective and the parasite possesses specific molecules that become activated at the right moment. A strong body of evidence supports that intracellular calcium oscillations in the parasite precede the activation or stimulation of the specific steps of the lytic cycle [14]. Both extracellular and intracellular Ca²⁺ pools contribute to cytosolic Ca²⁺ increases resulting in downstream signaling pathways that are decoded into critical biological functions of the parasite lytic cycle.

Genomic analysis suggests the presence of a variety of calcium channels, which could potentially play a role in Ca²⁺ entry or release from intracellular stores [15]. There is evidence for Ca²⁺ release stimulated by IP₃ and cADP ribose [16, 17] but no receptors for these second messengers have been identified for any Apicomplexan parasite [18]. A number of Ca²⁺-binding proteins including calmodulins, calmodulin-like proteins, and an array of Ca²⁺-dependent protein kinases are predicted to be present in these parasites [19, 20]. A large number of recent studies have provided evidence of their function in the lytic cycle of Toxoplasma.

2. The Toxoplasma calcium signaling toolkit

The information on the elements that form part of the calcium signaling toolkit of Toxoplasma and other Apicomplexan parasites is fragmented. The main challenge is the evolutionary distance of the Apicomplexa from mammalian model systems for which most of the information is available, and making direct comparisons is not always informative. However, the characterization of the molecular pathways involved in calcium signaling in Toxoplasma and other pathogens is highly relevant for several reasons. First, it is known that calcium fluctuations control vital cellular processes essential for parasitic life. Second, it is very likely that new molecules with unique characteristics may result in the discovery of druggable targets. Lastly, as an early branching eukaryote, Toxoplasma occupies a unique phylogenetic position providing insights into the early origin of complex signaling pathways.

Toxoplasma possesses characteristic calcium compartments such as the endoplasmic reticulum (ER), Golgi apparatus and mitochondria, and additional compartments that could contribute to calcium signaling like acidocalcisomes [21–23] and endosome-like compartments like the plant-like vacuole [24].

The concentration of Ca²⁺ in the cytosol of Toxoplasma tachyzoites is around 50–100 nM [25], similar to the cytosolic concentration of other eukaryotic cells. Ca²⁺ signaling starts when the cytoplasmic levels of Ca²⁺ are raised due to Ca²⁺ entry or Ca²⁺ release from intracellular stores. Ca²⁺ entry has an important role in refilling intracellular organelles that participate in signaling pathways that respond to elevated cytosolic Ca²⁺ [26]. T. gondii does not appear to express the molecular elements of Store Operated Calcium Entry (SOCE). There is no genomic evidence for the presence of store-operated channels (ORAI), or for the ER sensor protein stromal interaction molecule (STIM). It appears that ligand-operated channels [26], or second messenger-operated channels are also missing in T. gondii. Gene sequences with similarity to voltage dependent Ca²⁺ channels are present in its genome [15, 19]. The role of the encoded proteins as functional Ca²⁺ channels has not been demonstrated although evidence has been presented for the presence of a nifedipine-sensitive Ca²⁺ entry pathway that supports the function of a voltage-gated Ca²⁺ channel [27]. Sequences with similarity to transient receptor potential (TRP) channels were also found in T. gondii [15] but have not been characterized. TRP channels are a superfamily of channels with diverse cellular functions [28] that can localize at the plasma membrane and allow Ca²⁺ influx or to intracellular stores and allow Ca²⁺ release into the cell cytosol.

In vertebrate cells binding of a ligand to a surface receptor like a G-protein linked receptor (GPCR) may result in the stimulation of the phosphoinositide-signaling pathway via the activation of a phosphatidylinositol phospholipase C (PI-PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) to form inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). Binding of IP₃ to an IP₃ receptor usually present in the membrane of the ER, will result in its opening allowing Ca²⁺ to be released into the cytosol [29]. A diversity of cell signaling pathways can be generated through this mechanism [29]. Cyclic ADP ribose (cADPR) is also able to release intracellular Ca²⁺ acting through ryanodine receptors usually located in the ER [30]. Some of the elements of this signaling pathway have been found and characterized in Toxoplasma but a large number of them remains to be identified. The T. gondii phosphoinositide phospholipase C (TgPIPLC) has been studied [31] and characterization of conditional mutants of the Tgpiplc gene highlighted its importance for parasite survival and its participation in a vast and currently incompletely understood signaling cascade [32]. It is not clear, yet what is the target of the IP₃ generated by TgPIPLC because there is no genomic support for the presence of IP₃ receptors in any Apicomplexan parasite. However, IP₃ stimulates Ca²⁺ release from isolated microsomes of T. gondii [17] and cyclic ADP ribose does as well, presumably by stimulating a ryanodinetype receptor [17]. Ca²⁺ release by IP₃ is inhibited by the IP₃ receptor inhibitor xestospongin C [17], which also inhibited secretion of micronemes, secretory proteins involved in host invasion [16]. T. gondii possesses ADP ribosyl cyclase and hydrolase [17] but there is no molecular evidence of a ryanodine receptor.

The ER plays critical roles in calcium signaling in eukaryotic cells. Ca²⁺ influx into the ER is driven by SERCA-type Ca²⁺ ATPases, which have been characterized in T. gondii [33]. Inhibition of the T. gondii SERCA-type Ca²⁺-ATPase by thapsigargin [34], results in increase of cytosolic Ca²⁺ in T. gondii [25], due to Ca²⁺ leakage from the ER through an unknown pathway. Ca²⁺ leakage is the passive calcium efflux from the ER that is thought to prevent ER calcium overload and thus allow cytosolic Ca²⁺ signaling [35]. In mammalian cells it has been proposed that leak channels in the membrane of the ER play a role in the steady state concentration of the ER luminal Ca²⁺ [36]. Several types of membrane proteins have been proposed to be involved in the ER calcium leak pathway, Bcl-2, pannexin 1, presenilins and TRPC1. The Toxoplasma genome contains ortholog genes for a presenilin (TGME49_204040) and two TRP channels (TGME49_247370 and TGME49_310560) [15]. These Toxoplasma molecules have not been characterized except for a recent study using high-affinity tags, which localized TGME49_247370 to the ER [37]. An active ER Ca²⁺ release channel, termed transmembrane and coiled-coil domains 1 (TMCO1) or Ca²⁺ load-activated Ca²⁺ (CLAC) channel has been shown to assemble as a homotetramer upon ER Ca²⁺ overload [38]. The main function of the channel is to prevent ER Ca²⁺ overload. A small portion of TMCO1 exists as homotetramer even under normal levels of ER Ca²⁺, which could also cause passive leak of Ca²⁺ from the ER. The genome of Toxoplasma shows evidence for the presence of a TMCO1 ortholog (TGME49_310870), predicted to have a signal peptide at its N-terminus. This Toxoplasma gene has not been characterized.

Acidocalcisomes are acidic organelles that store large amounts of Ca²⁺, which is mostly bound to polymers of phosphate [39]. Acidocalcisomes of T. gondii possess a plasma membrane-type Ca²⁺-ATPase for Ca²⁺ uptake named TgA1 [40]. Purified acidocalcisome fractions from T. gondii tachyzoites show vanadate-sensitive Ca²⁺ uptake supporting for the presence of this enzyme [23].

A vacuolar compartment with lysosomal characteristics (Plant-Like Vacuole or PLV) that expresses several orthologs of pumps and transporters usually found in the plant vacuole has been characterized in Toxoplasma and shown to store Ca²⁺ [24]. An increase in cytosolic Ca²⁺ in response to glycyl-L-phenylalanine-naphthylamide (GPN) indicated the presence of Ca²⁺ in the PLV where lytic activities are also present. GPN-dependent Ca²⁺ release was independent from release from other Ca²⁺ stores, such as the ER [24].

The mitochondrion of T. gondii maintains a membrane potential and carries out energy-linked functions like respiration coupled to oxidative phosphorylation [41]. However, it was not possible to demonstrate Ca²⁺-uptake reliant on its membrane potential (unpublished results). In addition, there is no genomic evidence for the presence of a mitochondrial Ca²⁺ uniporter (MCU) in the inner membrane of any apicomplexan parasite [42]. A Ca²⁺/H⁺ antiporter (CAX) was localized to the mitochondria of P. falciparum [43] but the Toxoplasma ortholog did not localize to the mitochondrion [44]. The potential role of the Toxoplasma mitochondria in cytoplasmic Ca²⁺ homeostasis or even the role of mitochondrial Ca²⁺ on the regulation of mitochondrial enzymes is unknown [45].

Downstream to Ca²⁺, the signaling pathways that control essential and specific biological functions of Toxoplasma are not clearly defined. However, the information available is growing and several important players are emerging (Fig. 2). A role for a cyclic GMP (cGMP)-activated Protein Kinase G (PKG) in the activation of Toxoplasma egress, motility and differentiation, has been reported [46, 47]. Plant like Ca²⁺ dependent proteins kinases (CDPKs) are expanded across Apicomplexa and play pivotal roles in Ca²⁺ signaling throughout the lytic cycle [20]. CDPKs have been implicated in a range of processes in Toxoplasma, like invasion [48], egress [49] [50, 51] and microneme secretion [20, 48]. A recent study linked the central regulating role of PKG on the Ca²⁺ signals that precede egress to the activity of Protein kinase A. It was proposed that PKA down-regulates PKG-dependent signaling leading to egress by potentially activating a phosphodiesterase that would hydrolyze cGMP [52]. In a second study on Toxoplasma PKA the authors proposed that PKA is in the pathway that leads to suppression of cytosolic Ca²⁺ right after invasion promoting a decrease in motility and the beginning of intracellular replication [53]. It is clear that Ca²⁺, PKG and PKA signaling pathways crosstalk but we still do not know all the elements involved and the order in which they interact (Fig. 2). However, the expanse of information during the last three years about signaling pathways involved in the regulation of Toxoplasma parasitism is encouraging and it is very likely that we will soon know how those elements interact.

Figure 2.

Ca²⁺ signaling in T. gondii tachyzoites. Ca²⁺ enters the parasite likely through a Ca²⁺ channel that is sensitive to nifedipine. Inside the cell, Ca²⁺ is pumped either outside by a plasma membrane type Ca²⁺ ATPase or into organelles like the SERCA-Ca²⁺-ATPases localized to the endoplasmic reticulum (ER). The plasma membrane type Ca²⁺-ATPase in Toxoplasma also localizes to acidic stores (AS) like acidocalcisome or PLV. A signaling cascade that starts by an increase in cGMP level would lead to activation of a cGMP-protein kinase G that would result in cytosolic Ca²⁺ fluctuations that would activate biological responses like microneme (MIC) secretion. In this hypothetical scenario PKA could phosphorylate an unidentified phosphodiesterase (PDE) resulting in its inhibition and leading to an increase in cGMP levels. Downstream to PKG the pathway is hypothetical because the evidence is incomplete but CDPK3 could be part of it together with PI-PLC. The hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP₂) by PI-PLC forms two second messengers inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ would act on an unknown release channel in the ER allowing Ca²⁺ to be released into the cytosol, which could activate CDPK1 stimulating microneme secretion. Changes in DAG levels would activate DAGK1 (DAG kinase 1), which converts DAG into phosphatidic acid (PA), which is sensed by the micronemal protein APH (acylated pleckstrin homology domain-containing protein) stimulating secretion. A Ca²⁺ /H⁺ exchanger that was characterized in the PLV could use the proton gradient to take-up Ca²⁺ into the acidic store [140]. How Ca²⁺ is released into the cytoplasm is not known. The bar at the upper right represents a Ca²⁺ concentration scale by blue color gradient (light to dark). Known pathways are shown by continuous lines, while hypothetical ones are represented by dashed lines.

3. Calcium binding proteins in T. gondii

Intracellular Ca²⁺ increase activates a variety of cellular processes, including the mechanisms that return cytosolic Ca²⁺ concentration to its steady-state level, like the membrane transporters that sequester Ca²⁺ into intracellular organelles or actively pump it out of the cell. Most signaling responses, however, involve Ca²⁺-binding proteins (CaBPs). One of the most common motifs that bind Ca²⁺ and transduces these signals is the helixloop-helix motif known as EF-hand. EF-hands frequently occur in pairs (also called EFhand domains) that allow for the cooperative binding of two Ca²⁺ ions per domain. CaBPs with single or odd number of EF hands sometimes function via dimerization mechanisms. Analysis of the T. gondii genome has revealed approximately 68 EF-hand domaincontaining proteins encoded by its genome (ToxoDB, http://toxodb.org/toxo/). The majority of these genes have not been characterized.

EF-hand containing proteins can be grouped into the calmodulin (CaM) family, the calcineurin (CN) family, and the Ca²⁺-dependent protein kinase (CDPK) family. Both CaM and CN family members lack effector domains and are thought to act by regulating other proteins. The CaM group includes classical CaMs (with > 75% sequence identity to human CaM), calmodulin-like (CML) proteins, which include myosin light chains (sequence identity with human CaM < 75%), and centrins or calcactrins. Calmodulins comprise two globular domains (each with a pair of EF hands) linked by a flexible helical region [54, 55]. The T. gondii genome encodes a single prototypical CaM and a variable number of CML proteins. The T. gondii prototypical CaM binds Ca²⁺ in vitro [56] and was localized to the apical domain [57] and more recently to the conoid where it may interact with calcineurin [58]. No studies have addressed directly the role of CaM in the specific steps of the parasite lytic cycle.

Toxoplasma contains plant-like features because of its early branching prior to the animal-plant separation [1] and also because of the acquisition of an endosymbiont derived from an algal cell [59]. Because of this, signaling pathways and molecules present in Toxoplasma will likely be very unique. For example, as in plants, Toxoplasma possesses a large number of CML proteins with low sequence identity and with degenerate EF-hand motifs [60]. CML proteins can have diverse subcellular localizations broadening the potential substrates for their interaction [61]. Three of the CaM-like proteins, named CaM1, CaM2, and CaM3, are important for motility and invasion and localize to the conoid where they overlap with the motor protein myosin H (MyoH). These proteins were shown to interact with MyoH and its light chain MLC7 [62, 63]. CaM1 has two conserved EF hands, while CaM2 has one conserved and one degenerate EF hand. Analysis of mutants unable to bind Ca²⁺ was consistent with Ca²⁺ binding to the conserved EF hands for the regulation of function of CaM1 and CaM2 [62]. CaM3 is essential for growth and it was proposed that it may replace CaM1 and CaM2 in the activation of MyoH [64]. In agreement with the function of CaMs in invasion, the CaM inhibitors calmidazolium and trifluoperazine were shown to inhibit T. gondii host cell entry [65].

Centrins are Ca²⁺ binding proteins usually present with centrosomes. Apicomplexan centrins have been shown to associate with various cytoskeletal components including the centriole and conoid [66, 67], but none have been demonstrated to be Ca²⁺ regulated. Interestingly, a recent finding showed that TgCentrin2 co-localized with the TgPIPLC [37]. This localization was secondary to the plasma membrane localization of the Tg PIPLC and it was only possible to observe with high-affinity tags. The significance of this finding is intriguing and deserves further investigation.

Myosin light chains (MLCs) are calmodulin (CaM)-related proteins containing four EF-hand motifs and are important for the stability of the myosin motor complex [68–70]. It is unlikely that these CaM-like proteins will bind Ca²⁺ given that most of their EF-hands are degenerate. Two myosin regulatory light chain proteins, essential light chains 1 and 2 (ELC1, ELC2), which form part of the glideosome, have been studied and ELC1 has been shown to bind Ca²⁺ as estimated by modeling studies and thermal shift assays [71, 72]. Their affinity for Ca²⁺ of this protein is low (37 ± 9 μM) suggesting that for this interaction to be physiologically relevant it would have to be in a high Ca²⁺ concentration microdomain. It is interesting that four CML proteins localize to the conoid. Considering what occurs with plant CML proteins and CaMs, which can activate a variety of target proteins that regulate metabolism, transcription and ion transports, it is very likely that the Toxoplasma CML proteins that co-localize could have more than one target. The redundancy of these proteins, suggest that they play more than one role in Ca²⁺ signaling.

The CN family members have been identified only in some protist genomes. Calcineurin is a Ca²⁺ regulated-phosphatase that consists of a catalytic subunit (CnA) and a Ca²⁺-binding regulatory subunit (CnB). Calcineurin phosphatase activity is usually regulated by changes in intracellular Ca²⁺ [73]. The role of calcineurin in T. gondii was studied by analyzing conditional CnA mutants and found to be important for host cell attachment, invasion and stage-specific development of the parasite [58]. The next challenge will be to determine which are the substrates for T. gondii calcineurin.

A less prominent Ca²⁺-binding motif is the C2 domain, which was originally identified as the domain responsible for the Ca²⁺-dependent phospholipid binding of protein kinase C (PKC). Although no PKC can be found in apicomplexan genomes, various C2-domain containing proteins have been identified. Among them, DOC2 has been shown to be required for the Ca²⁺-regulated secretion of micronemes in T. gondii [74]. Apicomplexan PIPLC, which is thought to mediate intracellular Ca²⁺ store release through the production of IP₃, is also known to contain a C2 domain, in addition to an EF-hand, although it is unknown how Ca²⁺ impacts its regulation [31].

In plants there is a unique group of Ca²⁺ sensors and regulators known as Calcium Dependent Protein Kinases (CDPKs). Canonically, CDPKs are composed of a kinase domain followed by a CaM-like domain, also known as the CDPK activation domain (CAD) [75, 76]. The activity of these kinases is directly regulated by Ca²⁺ binding to the CAD. The important role of CDPKs in apicomplexans is reflected by their overrepresentation in the parasite genome. T. gondii CDPKs regulate a variety of events in the parasites like egress, replication, motility and protein secretion.

4. Ca²⁺-dependent protein kinases

The classical calmodulin-dependent kinases (CamK) typical of animal cells [77] are absent in Apicomplexan. Instead, they contain calcium-dependent protein kinases (CDPKs), a family of serine/threonine kinases that is present in plants and protists including ciliates [20, 78]. In plants, CDPKs regulate a diversity of pathways like cell cycle progression, stress response, stomatal closure and root-nodule formation [75]. In Apicomplexan parasites CDPKs represent the best characterized link between Ca²⁺ signaling and parasite biological functions like invasion, motility, egress and differentiation [20].

The typical structure of CDPKs comprises an N-terminal serine/threonine kinase domain (KD) connected by a junction region to a calcium-binding regulatory domain with four EF-hands forming a calmodulin-like domain. The regulatory domain is termed the CDPK-activation domain (CAD) [76]. The structures of T. gondii CDPK1 in the presence and absence of Ca²⁺ revealed the molecular details of their activation [76, 79]. In the absence of Ca²⁺, the junction region lodges in the substrate-binding region, blocking the catalytic pocket of the enzyme keeping it inactive. Upon binding of Ca²⁺ to the EF-hands a dramatic structural rearrangement leads to a segmentation of the autoinhibitory helix around the calmodulin like-domain altering the interaction and relieving the inhibition [76]. A study with CDPK1 showed that upon binding calcium the CAD domain rotates with respect to the kinase domain and in addition to relieving autoinhibition stabilizes the active form of the enzyme [80]. The Toxoplasma CDPK1 is essential and conditional knockdown mutants of TgCDPK1 are defective in microneme secretion, motility, host-cell invasion and egress [48]. These studies highlighted the role of CDPKs in transducing Ca²⁺ signals.

A remarkable characteristic of TgCDPK1 is the presence of a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents. Because of this, it is possible to inhibit TgCDPK1 with bulky ATP-analogues designed for kinases with extended ATP-binding pockets [48, 79, 81], as well as with similar compounds designed for their improved antiparasitic properties [82–84].

TgCDPK3 has been shown to have a more specific function in parasite egress, which was shown by characterizing a direct knockout of the gene as well as with a chemicalgenetic approach [49–51]. CDPKs represent attractive drug targets because of their absence from the mammalian genome [20]. Several recent screens have targeted CDPKs for the development of new antiparasitic compounds [85–87].

T. gondii also expresses a number of noncanonical CDPKs with different numbers of EF-hands, N-terminal EF hands, or with additional domains. Much less is known about the function of these noncanonical CDPKs with the exception of TgCDPK7, which contains the kinase domain preceded by a pleckstrin homology (PH) domain and two or more incomplete EF-hands. Conditional knockout of TgCDPK7 showed a defect in cell division [88]. Another study developed seven individual mutants of noncanonical CDPKs, plus double and triple mutants [89]. Only one of these mutants, ΔCDPK6, showed a mild growth phenotype. There is not a clear link between Ca²⁺ signaling and the potential function of these noncanonical CDPKs.

The challenge has been to characterize the biological targets of CDPKs. Several studies have addressed this question by searching for widespread changes in the phosphorylation state of diverse proteins following an increase in intracellular Ca²⁺ [70, 90]. These studies have uncovered interesting molecules but it is still not clear what are the specific effectors that are controlled by Ca²⁺. A comparison of the phosphoproteomes of wild type and TgCDPK3-defficient parasites uncovered hundreds of changes in diverse cellular pathways including proteins associated with gliding motility [90]. Several components of the motor complex are heavily phosphorylated and this has been documented [91]. Mutation of the identified phosphorylation sites was undertaken to identify which one was relevant for motor function [91]. The central player of the glideosome, a multisubunit motor complex, is T. gondii myosin A (TgMyoA), an unconventional Myosin that is essential for motility, invasion and egress [69, 92]. An increase in cytoplasmic Ca²⁺ resulted in increased MyoA phosphorylation and mutation of the major sites for TgMyoA phosphorylation altered the parasite response to cytoplasmic Ca²⁺ elevation by ionophores and other drugs [93]. The expression of MyoA bearing phosphoro-mimetic mutations rescued the response to ionophores [93]. A more recent study using the proximity-based protein interaction trap BioID identified 13 targets of CDPK3, among them TgMyoA [94]. In this study, a peptide array approach identified serines 21 and 743 as the targets for CDPK3 phosphorylation. CDPK3 phosphorylation of MyoA was important for initiation of motility and egress providing a mechanistic link between CDPK3 regulation and the lytic cycle [94]. A study on egress and the role of CDPKs on Ca²⁺ signaling showed that CDPK1 and CDPK3 play a role in the Ca²⁺ signal recovery prior to egress [95]. A forward-genetic screen to isolate gain-of-function CDPK3 mutants [96] followed this study, which uncovered a new component that was characterized as suppressor of Ca²⁺-dependent cell egress [96].

It is very likely that the essential phenotypes attributed to CDPKs are the result of their important regulatory functions but it is clear that more work is needed to clarify the signaling network that is triggered upon cytoplasmic Ca²⁺ increase and its downstream stimulation of kinases, phosphatases or other targets. Deciphering the specific targets of CDPKs will be an essential task to understand the signaling pathways that lead to the biological functions that are essential for Toxoplasma parasitism.

5. Microneme secretion, motility, host cell invasion and egress

Toxoplasma is an obligate intracellular parasite that multiplies inside host cells. The process of invasion is actively driven by the parasite and is fast, precise and involves gliding motility, conoid extrusion, and microneme secretion [97–99]. An increase in cytosolic Ca²⁺ artificially elevated with ionophores like ionomycin or A23187 stimulated conoid extrusion of T. gondii tachyzoites, an effect that was prevented if the parasites were pre-loaded with BAPTA-AM [98]. Conoid extrusion also responded to other cytosolic Ca²⁺ triggers like ethanol [100] and extracellular Ca²⁺ enhanced and extended the extrusion time [27]. BAPTA-AM also prevented gliding motility [97] and host cell invasion [101] by T. gondii supporting a role for Ca²⁺ in these essential parasite functions. Conditional downregulation of the expression of a conoid protein hub 1 (CPH1) caused the conoid to collapse and become shorter [64]. In this work, the stimulation of conoid extrusion or microneme secretion by ionophore was not affected even though CPH1 was essential for invasion and conoid structure [64]. The specifics about how Ca²⁺ increase leads to extension of the conoid are not clearly defined. Several Ca²⁺ binding protein have been localized to the structure [62]. A recently described conoid ring protein, RNG2, was shown to change its orientation in response to stimulation of conoid extrusion with Ca²⁺ ionophores [102]. Based on this result, a role for RNG2 in secretion was proposed.

Toxoplasma gondii contains micronemes, specialized secretory organelles important for gliding motility and host cell invasion [103, 104]. Micronemes are small apical organelles with an elongated shape and electron-dense structure [104]. Micronemes store adhesins that upon secretion participate in the interactions with the host-cell surface and participate in the early stages of host-cell invasion. Secretion of micronemes can be triggered by elevating cytosolic Ca²⁺ with ionophores and can be blocked by chelating intracellular Ca²⁺ with BAPTA-AM [103, 105]. A number of studies have shown that multiple external stimuli can trigger microneme secretion such as a drop in extracellular K⁺ [106], an increase in H⁺ [107], serum albumin [108] and extracellular Ca²⁺ [27]. The precise signaling pathway that leads to microneme exocytosis, and especially how Ca²⁺ stimulates it, is not well defined but some participating molecules have been identified. One study on the characterization of Ca²⁺-ATPase (TgA1) [40] knockout mutants, which showed altered levels of intracellular Ca²⁺, examined the role of Ca²⁺ in the lytic cycle of Toxoplasma. These mutant parasites are deficient in microneme secretion, invasion, and showed reduced virulence in vivo [109].

A study looking at the mechanism of action of the antimalarial drug artemisinin, showed that thapsigargin and artemisinin triggered Ca²⁺-dependent secretion of micronemes. Artemisinin treatment altered Toxoplasma intracellular Ca²⁺ and increased the periodicity of Ca²⁺ oscillations as imaged in parasites loaded with the Ca²⁺ fluorescent indicator Fluo4 [33]. Previous reports had shown in Plasmodium, that the SERCA orthologue PfATPase6 could be the target of artemisinin [110] and considering that artemisinin altered Ca²⁺ signaling in Toxoplasma [111] it was proposed that the drug could inhibit TgSERCA, which was further supported by the sensitivity of yeast expressing TgSERCA to artemisinin. Presently, the mechanism (s) of action of artemisinin, still remains unclear because Toxoplasma artemisinin-resistant mutants did not show apparent mutations or alterations in the expression levels of TgSERCA or other Ca²⁺-ATPase genes [111].

CDPK1 conditional knockdowns highlighted the role of the Ca²⁺-stimulated kinase in attachment and secretion of micronemes [48]. The authors characterized the inhibition of TgCDPK1 by 3-methylbenzyl-PP1 (3-MB-PP1), an ATP analog that inhibited the enzyme due to the presence of the aminoacid glycine in the gate-keeper position allowing the inhibitor to block the ATP binding site. The PP1 inhibitor blocked microneme secretion and related functions. Mutation of G to a methionine (G for M) made the enzyme insensitive to the inhibitor [48].

A chemical genetic study with the inhibitor WRR-086, identified an homologue of the human redox chaperone protein, T. gondii DJ-1, with specific function in parasite motility and microneme secretion [112] even when stimulating secretion with ionophore or ethanol. This suggested that the role of TgDJ-1 is downstream of Ca²⁺ signaling [112] as was also shown for TgCDPK1. It was proposed that the regulation of microneme secretion by CDPK1 was in part due to its interaction with TgDJ-1 in a Ca²⁺ and H₂O₂dependent manner [113]. The role of the cGMP-dependent protein kinase (PKG) in microneme exocytosis was first demonstrated by its inhibition with compound 1 (Cpd1), a trisubstituted pyrrole derivative with in vivo activity against Toxoplasma [114]. The validation of PKG as target of Cpd1 was done with transgenic parasites complemented with mutated versions of TgPKG (T761Q), which are insensitive to the inhibition of microneme secretion by Cpd1 [46]. Increase of cytosolic Ca²⁺ with ionophores did not reverse the inhibition of microneme secretion by Cpd1 suggesting a downstream role for PKG in this process [46]. Toxoplasma possesses a single gene for PKG, which is essential, but expresses two isoforms with identical regulatory and catalytic domains, PKG^I is membrane bound and PKG^II is cytosolic [115]. Recent work on PKG differentiated the contribution of each PKG isoform to Toxoplasma biology using the auxin-inducible degron (AID) tagging system for conditional depletion of PKG protein followed by complementation with mutated versions of PKG [116]. PKG^I, the membrane associated isoform is essential and sufficient for microneme secretion and parasite survival. PKG^II, the cytosolic version is dispensable but its function became relevant when the gene is modified to localize to the plasma membrane [116]. These results support a role for PKG downstream or independently of the events that lead to intracellular Ca²⁺ increase [46, 116]. However, studies with parasites loaded with the Ca²⁺ indicator Fura2-AM showed that accumulation of cGMP by inhibiting its phosphodiesterases (PDE) with zaprinast, resulted in an increase in cytosolic Ca²⁺, which is dependent on PKG activity because this Ca²⁺increase was almost 80–90% inhibited by Cpd1 [117].

It is interesting that serum albumin acts synergistically with ethanol to increase cytosolic Ca²⁺ and microneme secretion [108]. The stimulation of microneme secretion was proposed to occur through a Ca²⁺-independent pathway on the basis of Ca²⁺ measurements using the genetic indicator GCaMP6f. However, it remains to be determined if the Ca²⁺ increase was below the GCaMP6 sensitivity as this indicator exhibits different kinetics and affinities for Ca²⁺ than Fura2 [118]. Early studies evaluating microneme secretion of Toxoplasma were performed in the presence of bovine serum, which has been shown to stimulate microneme secretion in related apicomplexan [16, 99, 105, 108]. More recently it was determined that serum albumin, the major component of serum, was sufficient to induce microneme secretion, while γ-globulins, the second major component of serum had no effect [108]. It is well characterized that Ca²⁺ ionophores such as ionomycin or A23187 induce microneme secretion, however in a study characterizing the role of extracellular Ca²⁺, microneme secretion was shown to correlate with the concentration of extracellular Ca²⁺ supporting an enhancing role for Ca²⁺ influx [27]. This study did not use ionophores or other triggers, or serum and parasites were kept in low Ca²⁺ buffer until the microneme assay was performed. It is possible that Ca²⁺ influx leads to an increase of Ca²⁺ at specific peripheral domains of the parasite and bypasses the intracellular signaling triggered by PKG. Additional studies are needed to clarify the link between Ca²⁺ and PKG signaling in T. gondii.

Most evidence linking Ca²⁺ signaling to parasite replication is indirect. Conditional knock-downs of CDPK7, a potentially Ca²⁺ regulated kinase, results in parasites with replication phenotypes [88]. Reduction of CDPK7 alters centrosome duplication and positioning. CDPK7 appears to play a role in the positioning of secretory organelles like rhoptries and micronemes. The miss-positioning of these organelles in the mutants implicated CDPK7 in the trafficking of these organelles to daughter cells [88]. GRA1, is a dense granule protein shown to be involved in the formation of the tubulovesicular network [119, 120]. Using ⁴⁵Ca²⁺, it was demonstrated that GRA1 binds Ca²⁺ [119]. The role of host Ca²⁺, specifically Ca²⁺ entry, was shown with the inhibitor L-651,582, which apparently blocks Ca²⁺ entry in the host cell affecting parasite replication [121]. L-651,582 inhibited Toxoplasma and Eimeria growth in a variety of mammalian host cells [121]. This result suggests that during its intracellular replication phase the parasite might need to take up Ca²⁺ from the host to keep its stores replenished. It is also possible that a number of Ca²⁺ regulated proteins are involved in the replication of the parasite. There is evidence for the presence of a higher concentration of Ca²⁺ within the PV [122] and host Ca²⁺ oscillation do cause oscillations in the parasite [123]. It is still not clear how host cytosolic Ca²⁺ impacts the biological functions of the parasite. Parasites mutants on a dense granule protein secreted to the PV, GRA41, showed alteration in their cytosolic Ca²⁺ levels and egress [124].

Active egress of Toxoplasma from host cells requires rupture of the parasitophorous vacuole membrane (PVM) and the host cell membrane [125]. Egress is essential for the dissemination of the infection and it has been known for several years that Ca²⁺ ionophores can trigger egress [126]. However, it was the use of Genetically Encoded Ca²⁺ Indicators (GECIs) that provided the final and conclusive evidence that there was a cytosolic Ca²⁺ increase right before egress [123]. The use of these indicators has vastly impacted the studies of Ca²⁺ in intracellular parasites [95, 117, 123, 127]. Secretion from the micronemes of the perforin-like protein TgPLP1, assists in the permeabilization of the PVM and host cell membrane [128]. Initiation of parasite motility results in mechanical pressure and final rupture of the host cell allowing the release of the parasites. Both, secretion of the micronemal protein TgPLP1 and initiation of motility during egress are stimulated by an increase in cytosolic Ca²⁺. A role for a change in extracellular potassium concentration was proposed to stimulate Ca²⁺ signaling leading to egress [106]. The presence of extracellular Ca²⁺ also affected the rate of egress, which was blocked with the Ca²⁺-channel blocker nifedipine [123]. A plant-like pathway involving the phytohormone abscisic acid (ABA) was proposed to be the pathway leading to the production of the natural signal for Toxoplasma egress [129]. The initial trigger that starts the Ca²⁺ signaling leading to egress is still a mystery.

The isolation of parasite mutants resistant to ionophore-induced egress (or delayed response) identified mutants defective in Ca²⁺ signaling. A mutant defective in a Na⁺/H⁺ exchanger (TgNHE1), was isolated in this way and found to have elevated intracellular Ca²⁺ levels and reduced pathogenicity [130]. A similar screen identified TgCDPK3 as a mediator of ionophore-induced egress [50]. TgCDPK3 likely controls rapid exit from the host by phosphorylating proteins involved in the activation of motility. Another CDPK3 substrate that plays a role in egress is the Suppressor of Ca²⁺ Egress 1 or SCE1, and its phosphorylation relieves the suppression of Ca²⁺-dependent host cell egress [96].

Extracellular tachyzoites exhibit three types of motility (Fig. 3): circular (counterclockwise circular motion), helical (forward corkscrew motion), and twirling (attachment to posterior end and spinning vertically) [131], though within a 3D matrix, motility is one continuous irregular corkscrew motion [132]. Helical gliding is considered the most effective and essential for invasion [131]. Ca²⁺ oscillations were first observed in actively motile parasites loaded with Fluo-4 that preceded periods of microneme secretion and bursts of speed [97, 133]. Cytosolic Ca²⁺ fluctuations were further characterized with parasites expressing GECIs and a direct correlation between distance traveled and amplitude of the oscillations was exposed [95, 123]. Cytosolic Ca²⁺ levels are reduced at the moment of invasion and stay low right after invasion. The oscillation pattern of cytosolic Ca²⁺ in motile parasites suggests that Ca²⁺ influx channels and reuptake mechanisms are highly active in gliding parasites and necessary for effective lytic cycle events [95, 123, 133]. Similar perturbations of Ca²⁺ signaling also affect gliding motility and microneme secretion [134], which has been used to deduce our current understanding of this form of parasite movement. For example, increasing the frequency and lowering the amplitude of Ca²⁺ oscillations with calmidazolium enhanced microneme secretion and gliding motility [97]. Calmidazolium stimulated gliding and this was proposed to depend on MIC2 secretion. The increase of cytosolic Ca²⁺ levels was shown to occur through release from intracellular stores and entry from the extracellular milieu. The calmidazolium study highlighted the importance of Ca²⁺ oscillations vs. constant Ca²⁺ increase for efficient gliding of T. gondii [97]. It is possible that Ca²⁺ oscillations enable the parasite to traverse longer distances over a longer period of time, via coordinating the release of micronemes only when necessary instead of constitutively [95, 123]. Whether Ca²⁺ regulates aspects of gliding motility beyond secretion of adhesins remains to be determined.

Figure 3.

T. gondii Motility Types. (From left to right) Representative stills of Circular (counter clockwise circular motion), Helical (forward corkscrew motion), and Twirling (attachment on posterior end with windmill-like motion) motility post extracellular Ca²⁺ addition. T. gondii tachyzoites are expressing cytosolic GCaMP6f [123] and exposure to extracellular Ca²⁺ stimulates motility and fluorescence of the indicator. Size bar: 5 μM

Release of intracellular Ca²⁺ stores was proposed to be sufficient to initiate gliding motility and host-cell invasion by use of ionophores or intracellular Ca²⁺ chelators like BAPTA [16]. However, extracellular Ca²⁺ influx impacted and enhanced invasion-linked traits like motility, conoid extrusion, microneme secretion and invasion [27]. T. gondii replicates through endodyogeny, and its lytic cycle is complex and parasites commonly invade and exit with little or no replication [135, 136]. This complex behavior, all of which depends on Ca²⁺, likely requires replenishing intracellular Ca²⁺ stores with extracellular Ca²⁺. It would be advantageous for the parasite to utilize the high concentration of extracellular Ca²⁺ to enhance invasion-related processes and to resupply intracellular stores for use during subsequent rounds of invasion and egress. Experimental evidence supports a general mechanism of Ca²⁺ entry, which may not be sensitive to the filling state of the ER, as is the case in mammalian cells, but which could work in parallel with intracellular store release to elevate intracellular Ca²⁺, and activate downstream events like invasion and motility [27].

The glideosome is the molecular machinery that generates the mechanical force needed for completion of the lytic cycle [137]. Inhibition (using pharmacological agents) or disruption (using knockouts or conditional depletion of core components) of the glideosome has been known to adversely affect the lytic cycle events of egress, extracellular motility, and invasion thus highlighting its significance across the lytic cycle [10, 125]. Egress results from the initiation of the glideosome and parasites will continue to be actively motile until invasion of new host cells suggesting that extracellular motility serves as the bridge connecting egress and invasion [10]. As an intracellular parasite, T. gondii must replicate inside host cells; and as such, glideosome activation must be tightly regulated across time and space [91]. Several studies have demonstrated that glideosome activation occurs in a Ca²⁺-dependent manner, through multiple, interconnected and crosstalking mechanism(s) [51, 94, 125] (Fig. 4A). Furthermore, a recent study identified that the force and direction of gliding motility occurs in a Ca²⁺-dependent manner. At basal levels the net mechanical force of the parasite is random and disorganized. Post increases in cytosolic Ca²⁺, the net mechanical force of the parasite becomes polarized and oriented across the long axis of the parasite thus sufficient for productive motility [138].

Figure 4.

A) Signaling and activation of Motility. Activation of Ca²⁺ signaling will translate into the activation of CDPK3 which will phosphorylate proteins from the molecular machinery and activating motility. The glideosome is the main molecular motility machinery and is anchored to the underlying cytoskeleton termed the inner membrane complex (IMC). Adapted from [14], with permission. (B) The glideosome of T. gondii. MLC1 and/or ELC1 or ELC2 interact with Ca²⁺ directly or via Ca²⁺ dependent phosphorylation events to stimulate motility. The glideosome was adapted from [71].

The glideosome is a multi-subunit actomyosin motor complex that resides at the periphery of the parasite; confined to the space below the plasma membrane (PM) and above an underlying structure termed the inner membrane complex (IMC) (Fig. 4A) [11, 125]. The complex (Fig. 4B) is anchored to the IMC via the scaffolding complex of glideosome-associated proteins (GAP’s), and members of the GAPM family [11]. TgGAP45 serves as a molecular tether that links the IMC to the PM through lipid modification links [11, 70]. TgMyoA, an important component of the glideosome [70, 92], is an unconventional myosin of the class XIVa that interacts with its associated light chain, TgMLC1 and either the essential light chain 1 or 2 (ELC1 or ELC2) [70, 72]. Though MyoA serves as the primary source for the generation of force needed for motility, it is the interaction between the MyoA neck with ELC1/2 that supports force transduction throughout the motor complex and more broadly throughout the body of the parasite as GAP proteins are anchored to the cytoskeleton [11, 72, 139]. Within the glideosome MLC1, ELC1 and ELC2, interact either directly with Ca²⁺ or via Ca²⁺ dependent phosphorylation to stimulate motility and microneme secretion [14, 51, 70].

Though several Ca²⁺-dependent phosphorylation sites of the glideosome are dispensable, mutations of S20, S21, and S29 of MyoA have been determined to be important for MyoA activity, whereas S21 appears to be the primary site for phosphorylation [70, 91, 93]. A recent study determined that disruption of the phosphorylation state(s) of MyoA by expressing mutated versions of MyoA serine residues, altered the motility behavior of gliding parasites as compared to wild type cells [93]. These results suggest that the summation of Ca²⁺ oscillations may coordinate the dynamic cycles of phosphorylation/dephosphorylation of MyoA, needed for effective gliding motility/invasion [51, 93, 94].

An in vitro assay utilized to monitor actin displacement of purified MyoA, MLC1, and ELC1 identified that when ELC1 was added to complexed MyoA-MLC1, MyoA moved actin filaments at more than twice the speed (and more in line with motor complexes isolate from parasites) as compared to MyoA-MLC1 alone [139]. Later it was determined that in addition to enhancing the thermostability of ELC1 in the presence of Ca²⁺, ELC1 had an enhanced affinity for MyoA and is recruited by MLC1 [70, 72]. Given the low affinity (μM) of ELC1 (and presumably ELC2) for Ca²⁺, it is reasonable to hypothesize that ELC1(/2) may have evolved to optimally bind Ca²⁺ at high concentrations and/or rapidly release Ca²⁺ in continuous succession [72]. Perhaps through rapidly binding and releasing Ca²⁺, during the Ca²⁺ oscillations that govern parasite motility, ELC1(/2) may serve as a “molecular throttle” to enhance MyoA activity, thus highlighting its potential role in decoding the Ca²⁺ signals that govern parasite motility.

Outlook

In the last 3 years since our last review of the Ca²⁺ research literature [14] we have witnessed how new genetic tools have advanced our knowledge on Ca²⁺ signaling in Toxoplasma. Some of these advances include the use of CRISPR-Cas9 for characterization of Toxoplasma genes, the expression of genetically encoded Ca²⁺ indicators in Toxoplasma tachyzoites, the use of a biotin ligase fusion protein to identify proximal and interacting proteins, and the adaptation of tools to down-regulate protein levels allowing studies of more immediate phenotypic differences without previous adaptation. The field is moving leap wise and it is expected that we will see more and more discoveries of new elements and pathways components.

During these last three years we have finally been able to see Ca²⁺ oscillations prior to exit of the parasite from its host cells. This was years after Endo [126] showed that ionophores trigger egress and assumed that it was because of a Ca²⁺ response. These studies were possible thanks to the use of GECIs that allowed to specifically detect Ca²⁺ changes in live intracellular parasites. We still need to know how intracellular vs extracellular Ca²⁺ crosstalk to deliver the threshold needed for increase in motility and egress.

We have also seen evidence of crosstalk between second messengers like Ca²⁺, cGMP and cAMP. The picture is still not very clear, especially about which one is upstream of which, but there are no doubts that information is flowing and we will see this mystery uncovered in the near future.

There is no doubt about the significance of Ca²⁺ for gliding motility, microneme secretion, host cell invasion and egress of T. gondii. How Ca²⁺ regulates aspects of gliding motility beyond secretion of adhesins remains to be determined. The same argument can be extended to our understanding of parasite invasion and egress from host cells, although certain cellular components may affect the different phenotypes to different extents. Ca²⁺ signaling is controlled by its uptake and release from different cellular compartments and there are important differences with the processes that control Ca²⁺ homeostasis in other eukaryotic cells, providing opportunities for finding novel drug targets. It is also apparent that the more we learn about Ca²⁺ signaling in Toxoplasma the more we know about the basic mechanisms that regulate how Toxoplasma, and other related pathogens, cause disease.

Highlights.

• Ca2+ signaling is essential for cellular and developmental changes that support T. gondii parasitism.

• Ca2+ signaling is important for gliding motility, microneme secretion, host cell invasion and egress of T. gondii.

• The Toxoplasma genome reveals a large number of genes with potential functions in calcium signaling that have not been characterized

• Toxoplasma express a number of plant-like calcium-dependent protein kinases, which link Ca2+ signaling to parasite biological functions

• The discovery of new Ca2+ players represents potential novel targets for antitoxoplasma drugs.