Calcium signalling in intracellular protist parasites

Abstract

Calcium ion (Ca²⁺) signaling is one of the most frequently employed mechanisms of signal transduction by eukaryotic cells, and starts with either Ca²⁺ release from intracellular stores or Ca²⁺ entry through the plasma membrane. In intracellular protist parasites Ca²⁺ signaling initiates a sequence of events that may facilitate their invasion of host cells, respond to environmental changes within the host, or regulate the function of their intracellular organelles. In this review we examine recent findings in Ca²⁺ signaling in two groups of intracellular protist parasites that have been studied in more detail, the apicomplexan and the trypanosomatid parasites.

Introduction

Trypanosomatids include two important genera: Trypanosoma and Leishmania, that cause important diseases in humans. Trypanosoma cruzi and Leishmania species have intracellular forms that cause Chagas disease, and different forms of leishmaniasis, respectively. Apicomplexan also include several human pathogenic genera like Plasmodium, Toxoplasma, and Cryptosporidum. With respect to Ca²⁺ signaling, the most studied apicomplexans with intracellular forms are Plasmodium spp., and Toxoplasma gondii, which cause malaria and toxoplasmosis, respectively.

T. cruzi life cycle includes two vector stages: epimastigotes and metacyclic trypomastigotes and two animal host stages: intracellular amastigotes and cell-derived trypomastigotes. Trypomastigotes are the host infective, non-replicative stages that can invade any nucleated cell, while amastigotes replicate in the cytosol of animal cells until they differentiate back to trypomastigotes to be released to the blood circulation. Leishmania spp. have only three life cycle stages: the promastigote found in the vector that transforms into metacyclic forms that infect the animal host, and the amastigote that lives surrounded by a parasitophorous vacuole (PV) within phagocytic cells. T. gondii has two main developmental stages in the animal intermediate host: the tachyzoite that replicates in any nucleated cell inside a parasitophorous vacuole, and the semi-dormant bradyzoite, which lives inside tissue cysts in brain and muscle controlled by the host immune response. Sporozoites released from sporulated oocysts can also reproduce within intestinal cells in the intermediate host when infection occurs through consumption of contaminated food or water with oocysts released in cat feces. The definitive hosts of T. gondii are members of the cat family and additional intracellular stages reproduce in their intestine. Two intracellular stages of Plasmodium spp. occur in the animal host: the liver stage initiated by the sporozoites released by the female mosquitos, which end up releasing merozoites that invade red blood cells to establish the asexual blood stages (ring, trophozoite, and schizont stages) and gametocyte stages that reproduce in the insect vector.

Intracellular life affects how Ca²⁺ levels are regulated. In most cells the cytosol has its resting Ca²⁺ concentration ([Ca²⁺]_i) determined by the extracellular Ca²⁺ concentration [Ca²⁺]_e, which is fixed, because the extracellular volume is effectively infinite, and by the properties of the plasma membrane alone [1]. However, while extracellular stages are in contact with Ca²⁺ concentrations around 1.8 mM in plasma, which are 10 to 20 thousand times their intracellular concentrations (~50–100 nM), intracellular parasites living in the cytosol of cells, like T. cruzi, or surrounded by a permeable PV, like T. gondii [2], are in contact with the Ca²⁺ concentration of the host cytosol (~50–100 nM). This resting host Ca²⁺ concentration likely fluctuates as a consequence of Ca²⁺ signaling and could reach micromolar concentrations. These Ca²⁺ levels are not very different from those of their intracellular Ca²⁺ stores, suggesting that both intracellular organelles and the host cytosol could have an important role in regulating steady state Ca²⁺ levels in intracellular parasites.

Ca²⁺ signaling in intracellular parasites may be used as a mechanism for attachment and internalization of their infective forms to host cells, to respond to changes in the host cytosolic environment, and to regulate the function of their own organelles. Within host cells, these intracellular parasite stages behave as organelles and are able to efficiently compete with the host organelles for the uptake of nutrients and ions. We will examine these functions in apicomplexan and trypanosomatid parasites.

Trypanosomatids

T.   cruzi

Recent developments on Ca²⁺ signaling in T. cruzi have been the characterization of most of the components of the inositol phosphate/diacylglycerol signaling pathway and the characterization of the functional role of this pathway in regulating cell bioenergetics [3].

The first enzyme of the pathway, the plasma membrane located phosphoinositide phospholipase C (TcPI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) with generation of inositol 1,4,5-trisphosphate (IP₃), which gates the IP₃ receptor (IP₃R) releasing Ca²⁺ to the cytosol, and diacylglycerol, which remains in the plasma membrane [4]. This TcPI-PLC is peculiar because it does not have a pleckstrin homology (PH) domain, like the mammalian counterparts, and is N-myristoylated in glycine in the second position and palmitoylated or stearylated in the cysteine in the fourth position of the enzyme [4,5]. TcPI-PLC is stimulated by Ca²⁺ at relatively low concentrations [4], its expression increases when trypomastigotes differentiate into amastigotes and when amastigotes differentiate back to trypomastigotes within host cells [6]. The increase during trypomastigote to amastigote differentiation can be reproduced in vitro, and this differentiation can be inhibited by antisense oligonucleotides targeting TcPI-PLC, and stimulated by TcPI-PLC overexpression [5]. Interestingly, in addition to its localization in the inner surface of the plasma membrane, TcPI-PLC is also expressed in the exoplasmic leaflet of intracellular amastigotes. This surface located TcPI-PLC could have a role in shedding of glycosylphosphatidylinositol (GPI) anchored proteins of the parasite, or in IP₃ signaling in the host cell, as its surface expression correlates with a decrease in host cell PIP₂ [6,7].

The target of IP₃, the IP₃ receptor (IP₃R) (Fig. 1), was found in the acidocalcisomes rather than in the endoplasmic reticulum of these parasites [3]. The channel was expressed in DT40 chicken lymphocytes knockout for the three animal IP₃Rs to study the effect of IP₃ on Ca²⁺ release from intracellular Ca²⁺ stores [3,8]. Downregulation of its expression by knockdown [8] or knockout [3] revealed the importance of this Ca²⁺ signaling pathway for growth, metacyclogenesis, and infectivity in vitro and in vivo.

Fig. 1. Trypanosoma cruzi IP₃ receptor structure.

(A) Schematic representation of TcIP₃R showing predicted functional domains in its primary sequence. Six key regions and five transmembrane domains (TMD), are highlighted with amino acid residue numbering that comprises them. (B) Ribbon representation of the TcIP₃R 3D model highlighting key domains architecture. Areas colored in purple, dark yellow, red and black correspond to the structurally conserved domains highlighted in the scheme of panel A. Predicted cytosolic, transmembrane and acidocalcisome lumen domains are indicated. Reproduced with permission from Chiurillo et al. (2020).

The functions of this signaling pathway are potentially two: 1) to stimulate trypomastigotes attachment and internalization within host cells, and 2) to regulate the bioenergetics of the parasites. Evidence for the role of Ca²⁺ signaling in host cell invasion is based on the detection of cytosolic Ca²⁺ increase in trypomastigotes loaded with chemical Ca²⁺ indicators, upon host cell attachment [9], and the inhibition of host cell invasion by preloading the parasites with intracellular Ca²⁺ chelators (BAPTA/AM, Quin 2/AM) [9]. Evidence of the participation of the inositol phosphate/diacylglycerol pathway and the acidocalcisomes in host cell invasion is that downregulation of the IP₃R expression inhibits host cell invasion [3,8], that inhibitors of the phospholipase C decreases the infectivity of the parasites [10], and that treatments that deplete Ca²⁺ from acidocalcisomes reduce the infectivity of trypomastigotes [10].

More recent work has provided evidence of the role of the IP₃R in the regulation of the parasite bioenergetics [3]. Acidocalcisomes form membrane contact sites (MCS) with the single mitochondrion of the parasite creating Ca²⁺ microdomains (Fig. 2). Mitochondrial Ca²⁺ uptake through the mitochondrial Ca²⁺ uniporter (MCU) stimulates the pyruvate dehydrogenase, the tricarboxylic acid cycle, and O₂ uptake with production of ATP (Fig. 2) [5]. Ablation of the IP₃R by CRISPR/Cas9 perturbs Ca²⁺ uptake by the mitochondrion, the regulation of the pyruvate dehydrogenase by Ca²⁺, and the mitochondrial oxygen consumption, leading to increase in ammonia production because of amino acid utilization, increase in the AMP/ATP ratio, and autophagy [3].

Fig. 2. Regulation of the mitochondrial bioenergetics by the T. cruzi acidocalcisome IP₃ receptor.

Acidocalcisomes and mitochondria have membrane contact sites. Ca²⁺ (black circles) is released from acidocalcisomes upon stimulation of the IP₃ receptor by IP₃, and after passing the outer mitochondrial membrane through the highly permeable voltage-dependent anion channel (VDAC), it is handled by the mitochondrial Ca²⁺ uniporter (MCU). When in the matrix Ca²⁺ stimulates the TCA function and oxidative phosphorylation (OXPHOS) with generation of ATP, preventing autophagy. Reproduced with permission from Chiurillo et al. (2020).

The increase in cytosolic Ca²⁺ is followed by its binding to Ca²⁺-binding proteins (calmodulin, calmodulin-like proteins, calcineurin, Ca²⁺-calmodulin dependent kinases) with transduction of the Ca²⁺ signal into physiological changes, and their return to basal levels by plasma membrane and organellar ATPases. T. cruzi lacks transcription factors that are the primary down-stream effectors of these Ca²⁺-binding proteins in other eukaryotes. Most of the known Ca²⁺ effectors have been reviewed recently [11].

Leishmania spp.

Early studies showed that intracellular Ca²⁺ increases during differentiation of promastigotes to amastigotes of L. donovani [12] and that Ca²⁺ increase occurs in amastigotes of L. mexicana amazonensis upon attachment to macrophages and this increase and invasion of host cells is prevented by preloading the cells with intracellular Ca²⁺ chelators (BAPTA/AM, Quin 2/AM) [13]. Some environmental factors like heat shock have also been shown to be associated to increase in Ca²⁺ levels and differentiation of promastigotes to amastigotes in vitro [12].

More recently, a Ca²⁺ signaling pathway probably involving Ca²⁺ influx, and activation of calcineurin, has been proposed to be operative in L. major [14]. Calcineurin (Cn) is the only Ca²⁺/calmodulin-regulated serine-threonine phosphatase and is a heterodimer of catalytic (CnA) and regulatory (CnB) subunits. The enzyme is autoinhibited by sequences in CnA (autoinhibitory domain) that are displaced when Ca²⁺ binds to CnB and Ca²⁺/calmodulin binds to CnA [15]. Sequence similarity searches of CnA in the T. cruzi, T. brucei and L. major genomes revealed that only L. major has the four domains commonly found in animal CnAs (catalytic, regulatory subunit, calmodulin binding and autoinhibitory domain), while those of all other trypanosomatids do not possess the regulatory and autoinhibitory domains [16]. CnB-KO promastigotes of L. major are more sensitive to heat stress, and perturbation of membrane composition and fluidity caused by inhibitors, like myriocin and ketoconazole, and their response to these environmental stresses is restored by complementation with CnB [14]. These CnB-KO promastigotes fail to proliferate within macrophages and are avirulent in susceptible Balb/c mice [14].

Although acidocalcisomes and genes homologues to those encoding the inositol phosphate/diacylglycerol pathway components are present in Leishmania spp. [17], this Ca²⁺ signaling pathway has not been investigated. Intriguingly, the L. major IP₃ receptor (LmjF.16.0280) could possibly be a substrate for calcineurin as it possesses short linear motifs (SLiMs) that are docking sites for the enzyme [15] but this needs to be further investigated.

Apicomplexan parasites

T.   gondii

T. gondii is an exceptional model organism for the study of Ca²⁺ signaling, as Ca²⁺ is involved in most steps of its lytic cycle from motility to conoid extrusion, microneme secretion, host cell internalization, and host cell egress [18]. The genetic tractability and the number of tools available to work with Toxoplasma facilitates the advancement of the field of Ca²⁺ signaling in this parasite. The recent introduction of newly developed genetically encoded Ca²⁺ indicators (GECIs) allowed the detection of Ca²⁺ signals through the lytic cycle including the intracellular stages of the parasite [19,20]. GECIs address many of the limitations of the previously used chemical dyes [21–23] to detect Ca²⁺ changes in real time. One of the problems with the use of chemical indicators is the compartmentalization of the dyes in organelles, limiting the length of time that loaded parasites can be studied. In addition, studies of intracellular stages cannot be performed because, when loading the parasites, the dye also enters host cells. Other issues are toxicity and the highly invasive nature of loading that can be damaging to cells. In contrast, GECIs can be targeted to specific cell types, or subcellular compartments. The delivery of GECIs into intact organisms is minimally invasive and therefore compatible with long term, in vivo measurements. The characteristics of different GECIs as well as their use in T. gondii have been reviewed recently [24].

The use of GECIs has vastly impacted the studies of Ca²⁺ in intracellular parasites [25–27]. Recent findings were able to elucidate the role of Ca²⁺ in parasite egress from the host cells. Egress of tachyzoites from the host cells was attributed to Ca²⁺ increase in the parasite based on indirect methods such as the use of Ca²⁺ ionophores [28], or permeabilizing agents, like saponin [29], or thiols, like DTT [30], to artificially increase intracellular Ca²⁺ and facilitate egress, or use of intracellular and extracellular chelators to prevent egress [29]. The use of GECIs and video imaging allowed the detection of Ca²⁺ oscillations in the parasite preceding their increase in motility needed for invasion [19]. Disruption of the PV, probably by a secreted perforin [31], was followed by nifedipine-sensitive Ca²⁺ entry [32], which further increased intracellular Ca²⁺ and facilitated egress [19]. Recent work [20] described two peaks of Ca²⁺ increase in the parasite that occur during egress, the first attributed to Ca²⁺ release from intracellular stores and the second to Ca²⁺ entry from the extracellular medium and both are important to reach the threshold needed for egress. Patching the infected host cells to deliver precise Ca²⁺ and K⁺ concentrations revealed that increasing cytosolic Ca²⁺ triggers egress, which is accelerated by decreasing K⁺ levels [20]. The use of GECIs was important to show that intracellular parasites can take up Ca²⁺ from their host cytosol. This uptake is essential for maintaining the parasite intracellular stores replenished, as they are used for initiating egress [20]. Changes in host cytosolic Ca²⁺ by physiological (histamine) or pharmacological (thapsigargin, Fig. 3) agents are able to stimulate Ca²⁺ increase in the parasite but not to a sufficiently high level as to trigger parasite egress. This is significant because it indicates that although Ca²⁺ changes in the cytosol of the host cells can be transmitted to the parasite, as the PV is permeable to Ca²⁺ [19], they are highly regulated so they do not cause their premature egress.

Fig. 3. In vivo imaging of HeLa cells expressing GECO infected with GCaMP6f expressing T. gondii tachyzoites.

(A, B) still-images obtained from videos showing changes of GCaMP6f (tachyzoites) and GECO (HeLa cells) fluorescence after exposure to 2 μM thapsigargin (TG). (C) overlay of (A, B). (D) fluorescence tracings of host cells (red) and tachyzoites (green) after exposure to thapsigargin (TG). There is an increase in Ca²⁺ in both host cells and parasites but the increase is not sufficient to stimulate parasite egress. Reproduced with permission from Vella et al. (2020).

GECIs have also been used to show the requirement of a guanylyl cyclase (TgGC) expression to detect cytosolic Ca²⁺ increases in tachyzoites stimulated by the cGMP-dependent phosphodiesterase inhibitor 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO), and to respond to changes in K⁺ and pH, suggesting that cGMP production is required for sensing these changes [33].

Recent studies [34] identified a transient receptor potential (TRP) channel, TgTRPPL-2, that is important for both Ca²⁺ entry at the plasma membrane and Ca²⁺ efflux from the endoplasmic reticulum of T. gondii tachyzoites. TgTRPPL-2 was expressed in HEK-3KO cells and was shown to be a non-selective cation channel that conducts Ca²⁺. Ablation of TgTRPPL-2 affected both invasion and egress of T. gondii, and resulted in a growth defect. The results suggest that TgTRPPL-2 may be involved in the Ca²⁺ pathways that stimulate invasion and egress. Another recent study [35] revealed that the apicoplast of T. gondii possesses a two-pore channel (TPC) that has a critical role in the maintenance of the functional organelle. Using GECIs targeted to the apicoplast the authors demonstrated that there is uptake of Ca²⁺ released from the ER into the apicoplast and that this depends on the presence of a functional TPC.

Several Ca²⁺ signaling pathways have been described in T. gondii. There is evidence for the presence of an inositol phosphate/diacylglycerol pathway: 1) a plasma membrane-located phosphoinositide phospholipase C is able to generate IP₃ and diacylglycerol [36]; 2) IP₃ can release Ca²⁺ from ⁴⁵CaCl₂-loaded T. gondii microsome vesicles [37]; and 3) ethanol, which stimulates microneme secretion [38], also increases IP₃ formation, while the IP₃R antagonist xestospongin C inhibits microneme secretion, parasite attachment, and host cell invasion [21]. However, genomic evidence of the presence of a typical IP₃R is missing. There is also evidence that a cyclic ADP ribose (cADPR) signaling pathway controls Ca²⁺-mediated microneme secretion based on: 1) detection of cADPR cyclase and hydrolase activities, enzymes involved in synthesis and degradation of cADPR, in lysates of T. gondii; 2) presence of endogenous levels of cADPR in parasite extracts; and 3) Ca²⁺ release from ⁴⁵CaCl₂-loaded microsome vesicles by cADPR and block of this response by 8-bromo-cADPR and ruthenium red, which are inhibitors of the ryanodine receptor (RyR). These compounds also inhibited parasite motility and protein secretion [37]. There is, however, no genomic evidence for a RyR in T. gondii.

A number of proteins are the targets of Ca²⁺ increase in T. gondii. Some of them, like Ca²⁺-ATPases in the plasma membrane or intracellular organelles, return cytosolic Ca²⁺ concentration to its physiological level, while others, like Ca²⁺-binding proteins (calmodulin, calcineurin B, and the calcium-dependent protein kinases [CDPK]), are involved in transduction of the Ca²⁺ signal into physiological responses. These effector proteins have been the subject of recent reviews [18,39].

Plasmodium spp.

GECIs have also been recently used in Plasmodium falciparum but their use has been limited to the study of the Ca²⁺ response of trophozoites to the sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid [40,41]. Ca²⁺ is also involved in several processes in the malaria parasite, such as gliding motility [42], microneme secretion [43], host cell internalization [44], and host cell egress [45].

There is also evidence for the presence of an inositol phosphate/diacylglycerol pathway: 1) a phosphoinositide phospholipase C with all the domains of typical PIP-PLCs is present although its enzymatic characteristics have not been studied; 2) uncaging of IP₃ in trophozoites results in an increase in [Ca²⁺]_i [46]; 3) melatonin increases IP₃ formation [46,47]; 4) A protein kinase G controls Ca²⁺ signals needed for merozoite egress from the red blood cells, through the production of lipid precursors of PIP₂, which are necessary for IP₃ formation [48]. However, as it occurs with other apicomplexans, genomic evidence of the presence of a typical IP₃R or a RyR is missing.

The targets of Ca²⁺ signaling are, as occurs with T. gondii, the mechanisms that return [Ca²⁺]_i to basal levels and proteins that bind to Ca²⁺ resulting in physiological responses, such as calmodulin, calmodulin-like proteins, calcineurin, and Ca²⁺-dependent protein kinases. The functions of these effectors have been reviewed recently [18,49,50].

Conclusions

Host cell invasion by several intracellular protist parasites requires Ca²⁺ signaling. How these microorganisms sense the presence of a host cell to start this process is largely unknown and further work is needed to identify potential receptors. Intracellular life adds the additional challenge of surviving and replicating in an environment of very different ionic and nutrient composition as compared to the extracellular medium. One of these challenges is that the intracellular Ca²⁺ concentration of the parasites is not very different from the [Ca²⁺]_i of their hosts and the parasites need to be protected from sudden Ca²⁺ increases in the hosts cytosol due to Ca²⁺ signaling. The PV of some strains of T. gondii are surrounded by host cell mitochondria and it has been suggested that these mitochondria buffer these Ca²⁺ increases in the cytosol of the host [19]. Another protection in T. gondii is the need of a threshold for Ca²⁺ increase to stimulate egress [20]. Trypanosomatids possess an IP₃R with structural similarity to the animal IP₃Rs. Despite the physiological evidence of Ca²⁺ increase by IP₃ there is no similar channel in apicomplexans and further work is needed to investigate the nature of the different receptor. The use of GECIs in trypanosomatids and malaria parasites, as they were used in T. gondii, will be important to define the role of Ca²⁺ signaling in their intracellular stages. Many effectors of Ca²⁺ signaling have been identified and most responses appear to depend on phosphorylation/dephosphorylation of proteins, which is an active field of research. Fig. 4 shows the presence of homologs to human genes involved in Ca²⁺ homeostasis and signaling in the four parasitic protists discussed.

Fig. 4. Species possessing homologs to human genes involved in Ca²⁺ homeostasis and signaling.

Red indicates evidence of the presence of a homolog of a human gene family and black indicates the lack of an apparent homolog. PMCA, plasma membrane Ca²⁺-ATPase; SERCA, sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase; VGCA, voltage gated Ca²⁺ channel; PKD, polycystic kidney disease Ca²⁺ channel; TRP, transient receptor protein channel; IP₃R, Inositol 1,4-trisphosphate receptor; MCU, mitochondrial Ca²⁺ uniporter; TPC, two pore channel; CAKC, Ca²⁺ activated K⁺ channel; LETM1, mitochondrial Ca²⁺/H⁺ exchanger; PLC, phospholipase C; CaM, calmodulin; CALNA, calcineurin A; CALNB, calcineurin B; CALR, calreticulin; CDPK, Ca²⁺-dependent protein kinase (absent in humans); CCAMDPK, Ca²⁺-calmodulin-dependent protein kinase

Highlights.

• Intracellular protist parasites have similar basal Ca²⁺ levels than their host cells

• Ca²⁺ signaling is important to facilitate their invasion of host cells, to adapt to their intracellular environment, and to regulate their organelles

• Trypanosomatids possess well defined IP₃ receptors

• There is no genomic evidence for typical IP₃ or ryanodine receptors in apicomplexan parasites

• Genetically encoded Ca²⁺ indicators and video microscopy are being used to follow signaling in intracellular parasites