InsP₃ Signaling in Apicomplexan Parasites

Abstract

Background:

Phosphoinositides (PIs) and their derivatives are essential cellular components that form the building blocks for cell membranes and regulate numerous cell functions. Specifically, the ability to generate myo-inositol 1,4,5-trisphosphate (InsP3) via phospholipase C (PLC) dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to InsP3 and diacylglycerol (DAG) initiates intracellular calcium signaling events representing a fundamental signaling mechanism dependent on PIs. InsP3 produced by PI turnover as a second messenger causes intracellular calcium release, especially from endoplasmic reticulum, by binding to the InsP3 receptor (InsP3R). Various PIs and the enzymes, such as phosphatidylinositol synthase and phosphatidylinositol 4-kinase, necessary for their turnover have been characterized in Apicomplexa, a large phylum of mostly commensal organisms that also includes several clinically relevant parasites. However, InsP3Rs have not been identified in genomes of apicomplexans, despite evidence that these parasites produce InsP3 that mediates intracellular Ca2+ signaling.

Conclusion:

Evidence to supporting IP3-dependent signaling cascades in apicomplexans suggests that they may harbor a primitive or non-canonical InsP3R. Understanding these pathways may be informative about early branching eukaryotes, where such signaling pathways also diverge from animal systems, thus identifying potential novel and essential targets for therapeutic intervention.

1. Introduction

Phosphoinositides (PIs) and their derivatives are important for controlling a variety of vital cell functions including intracellular signaling cascades, regulated secretion, and cytoskeleton integrity [1]. The inositol containing phospholipids are abundant constituents of cell membranes of Archaea and all eukaryotes [2]. The importance of inositol signaling has been known for many years. However, the seminal discovery that ligation of cell surface receptors and PLC activity coupled to PI turnover and calcium release [3,4] paved the way for the discovery of the secondary messengers InsP₃ and diacylglycerol (DAG) and the downstream signaling cascades regulated by their generation and metabolism. InsP₃ formation depends on phospholipase C (PLC) that is activated by a variety of specific cell surface receptors. PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP₂), thus releasing soluble InsP₃ [5,6]. Pharmacological evidence supports InsP₃ signaling in unicellular eukaryotes such as members of the phylum Apicomplexa, a diverse group that includes Plasmodium, Toxoplasma and Cryptosporidium. The mobilization of intracellular Ca²⁺ level by InsP₃ in single cell pathogens was described in Trypanosoma, a Kinetoplastid member of the phylum Euglenozoa. Although the cell surface receptor mediated signaling cascades that lead to InsP₃-dependent, and potentially DAG-dependent, signaling cascades have not been fully delineated, there is mounting evidence to suggest these pathways are operative in apicomplexans.

Interest in Ca²⁺ signaling in these parasites is motivated by a history of studies demonstrating the requirement of Ca²⁺ signaling for infection by apicomplexan parasites, as recently reviewed [7]. Elevation of intracellular calcium regulates secretion of adhesins from microneme organelles, as well as activating actin-myosin dependent motility. As described below, parasite pathways that recruit proteins sufficiently divergent from their mammalian hosts may be exploited for development of novel therapeutic interventions (Fig. 1).

Fig. (1).

Schematic representation of the myo-inositol 1,4,5-triphosphate (InsP₃) turnover and its function in intracellular Ca²⁺ release. Step 1: Inositol incorporation into CDP-DAG (CDP = cytidine diphosphate DAG = diacylglycerol) catalyzed by phosphatidylinositol synthase generating phosphatidylinositol (PI). Step 2: Phosphorylation of PI by phosphatidylinositol 4-kinase forming phosphatidylinositol 4-phosphate (PIP). Step 3: Phosphorylation of PIP by phosphatidylinositol 4-phosphate 5-kinase forming phosphatidyl 4,5-bisphosphate (PIP₂). Step 4: PIP₂ is cleaved by phospholipase C (PLC) generating a soluble molecule, inositol 1,4,5-trisphosphate (InsP₃), that is capable to bind to a InsP₃ receptor (InsP₃R) present in the membranes of intracellular compartments. InsP₃ binding temporarily opens the InsP₃R that acts as a Ca²⁺-release channel, so that Ca²⁺ can flow into the cytoplasm. The insoluble fraction formed by cleavage of PIP₂ is diacylglycerol (DAG). Step 5: InsP₃ is dephosphorylated by inositolpolyphosphate 5-phosphatase forming inositol 1,4-bisphosphate (InsP₂). Step 6: InsP₂ is desphosphorylated by inositol polyphosphate 1-phosphatase generating inositol 1-monophosphate (InsP). Step 7: Dephosphorylation of InsP to inositol by inositol monophosphatase. Step 8: phosphorylation of InsP₃ by inositol 1,4,5-trisphosphate 3-kinase generating inositol 1,3,4,5-tetrakisphosphate (InsP₄).

Apicomplexans share many molecules pertinent to Ca²⁺ signaling with their closest relatives, the ciliates (phylum Ciliophora), despite these two groups being evolutionarily quite diverse [8]. Both groups are combined in the Alveolata, due to their endowment with subplasmalemmal flattened membranes, i.e. alveolar sacs in ciliates and inner membrane complex in apicomplexans. In ciliates, Ca²⁺ signaling mechanisms are known in some detail, including the molecular identity of intracellular Ca²⁺ channels involved in Ca²⁺ release from intracellular Ca²⁺ stores [9,10]. In contrast, identifying Ca²⁺-channels in apicomplexans such as Plasmodium and Toxoplasma has proved a major challenge. However, the recent development of transgenic parasites expressing genetically encoded sensors for detection of Ca²⁺ in P. falciparum and T. gondii may reveal new aspects of Ca²⁺ signaling [11,12] (Fig. 2).

Fig. (2).

Phylogenetic tree showing evolutionary relationships among organisms discussed in this review.

2. Evidence of InsP₃ turnover and InsP₃-induced Ca²⁺ release in Apicomplexa

Inositol and inositol phospholipids have been described in most Archaea and eukaryotes indicating this system developed in a common ancestor of the Archaea kingdom approximately 2 billion years ago [2,13]. Inositol phospholipids (PI) are ubiquitous components of cell membranes, there is not a unique PI found only in apicomplexan. Table 1 shows some important PI specific enzymes that have been reported in Apicomplexa and other unicellular parasites. The pathway by which PI is generated appears conserved amongst all eukaryotes [13]. The genes encoding the enzyme phosphatidylinositol synthase that catalyzes the formation of phosphatidylinositol (PI) are well characterized in P. falciparum and P. knowlesi [14], T. gondii [15] and T. brucei [16]. Phosphatidylinositol transfer protein encoding genes are also present, suggesting the existence of systems responsible for transporting PI from the ER to the plasmamembrane [1]. In Plasmodium, Gardner et al., 2002 [17] reported a gene encoding the enzyme phosphatidylinositol 4-kinase (PI4K) that catalyses the formation of phosphatidylinositol 4-phosphate (PIP) from phosphatidylinositol (PI). Thus, apicomplexans have the machinery to generate the precursor of InsP₃. Table 1 summarizes known and putative proteins from the InsP₃ pathway in model organisms and parasites. In mammalian systems the ability of InsP₃ to act as a second messenger requires the enzymes to rapidly release InsP₃ from PIP₂, as well as the ability to reduce InsP₃ levels to basal. Importantly, genetic evidence for phospholipase C enzymes can be found in all apicomplexans, except Sacrocysis (presumable due to a lack of genetic data), and Kinetoplastids (See 
Table 1). The presence of the archetypal PLCδ isoform in all genomes indicates the ability to generate InsP₃ and DAG in unicellular parasites arising from a common ancestor. The enzymes involved in InsP₃ metabolism are less well conserved. Plasmodium falciparum have an annotated inositol polyphosphate 5-phosphatase, an indication that they too can metabolize InsP₃, to InsP₂, whilst a specific inositol 1,4,5 trisphosphate 3-kinase has not been annotated. However, inositol polyphosphate kinase genes can be found in both P. falciparum and T. gondii genomes and may metabolize InsP₃ to InsP₄.

Table 1.

Occurrence of proteins important for the InsP₃ signaling pathway in some model organisms. The corresponding genes were searched for in the databases of Plasmodium, Toxoplasma, Homo sapiens, Trypanosoma and Paramecium (PlasmoDB, ToxoDB, PDB/NCBI, TriTrypDB and ParameciumDB, respectively). Phospholipase C of Paramecium tetraurelia is described in Klöppel et al., 2009 [27]. (1) Putative protein; (2) -like proteins; (3) encoded by pseudogene; (4) Leondaritis et al. (2013) [28], described 62 highly homologous phosphoinositide kinases genes; *genes that could not be found in databases.

	Phosphatidyl Inositol 4-kinase	Phosphatidyl Inositol 4-phosphate 5-kinase	Phospholipase C	Inositol Polyphosphate 5-phosphatase	Inositol 1,4,5-Trisphosphate 3-kinase	Inositol Triphosphate Receptor
Plasmodium falciparum 3D7	PF3D7_0509800; PF3D7_0419900(1)	PF3D7_0110600; PF3D7_1129600(1)	PF3D7_1013500	PF3D7_1354200(1)	*	*
Toxoplasma gondii ME49	TGME49_328200	TGME49_245730	TGME49_248830	*	*	*
Homo sapiens	AAA56839.1; BAA21661.1	CAD99242.1	AAA60112.1; BAA07688.1	CAA74743.1; CAA67071.1	AAH26331.1; CAC40650.1	NP_002214.2
Leishmania major Friedlin	LmjF.34.3590(1); LmjF.29.1450(1)	LmjF.36.0370(2); LmjF.34.3090(2); LmjF.35.0560(1,2)	LmjF.30.2950(2); LmjF.22.1680(1)	*	*	LmjF.16.0280(1)
Trypanossoma brucei gambiense	Tbg972.4.970(1)	Tbg972.10.4830(1)	Tbg972.11.6720(1)	*	*	Tbg972.8.2330(1)
Trypanossoma cruzi CL Brener	TcCLB.505987.70(1)	TcCLB.510289.30(1); TcCLB.511001.70(1)	TcCLB.508039.90(3)	*	*	BAM68694.1
Paramecium tetraurelia	XP_001346932.1	XP_001347030.1; XP_001347013.1	XP_001432600.1; XP_001426835.1	*	(4)	PTMB.445c

3. Pharmacological evidence for InsP₃ signaling in apicomplexa

Importantly, biochemical evidence to support PI turnover in apicomplexan parasites has also been reported in the literature. Despite the fact that mammalian RBCs have all the enzymes enabling the production of PIP₂ from PI [18], a non-infected RBC has limited biosynthesis of PIP₂ and other PIs [19]; in contrast, when a RBC is infected with Plasmodium knowlesi; P falciparum, [20] and Babesia bovis [21] PI turnover increases due parasite metabolism. Incubating red blood cells (RBC) infected with P. falciparum with radiolabeled myo-inositol [22] results in the biosynthesis of PI, PIP and PIP₂. Moreover, treatment with the Ca²⁺ ionophore, ionomycin, caused an increase in inositol phosphate production with Ins1,4,5P₃ levels being the highest. The increase of intracellular Ca²⁺ promoted by ionomycin presumably activates the Ca²⁺ sensitive PLC [23] to cleave PIP₂, thus generating InsP₃. Martin et al. [24] demonstrated the formation of InsP₃ and diacylglycerol (DAG) during exflagellation of P. falciparum gametocytes, an important event in the sexual cycle that takes place in the Anopheles mosquitos. Fang et al.,[25] identified a PI-PLCδ in T. gondii that cleaves PIP₂. Whilst recent bioinformatic approaches have failed to identify intracellular Ca²⁺ release channels such as IP₃Rs and Ryanodine receptors (RyRs) within the genomes of apicomplexans [7, 26-28], a large body of pharmacological evidence suggests that a Ca²⁺ release channel sensitive to InsP₃ does indeed exist in these species. Prompted by evidence that malaria parasites maintain intracellular Ca²⁺ stores, Passos et al. [29] published the first work demonstrating InsP₃ mobilized Ca²⁺ from an intracellular store in P. chabaudi using a permeabilized cell system. The response was blocked by heparin (an inhibitor of InsP₃Rs) providing pharmacological evidence for an InsP₃ dependent Ca²⁺ channel in P. chabaudi. Similarly, treatment of microsome preparations from T. gondii tachyzoites with InsP₃ initiated Ca²⁺ release which could be fully blocked by pharmacological inhibitors of InsP₃Rs (Xestospongin C and heparin) [30].

Inhibitors of the InsP₃ signaling pathway, including agents that block InsP₃R, such as heparin, xestospogin C and 2-aminoethoxydiphenyl borinate (2-APB), as well as inhibitors of PLC, such as U73122, have also been used to study Ca²⁺ signaling in Apicomplexa [31]. In T. gondii, the release of adhesins that mediate parasite attachment to host cells is an event regulated by Ca²⁺-mediated microneme secretion [32,33]. Lovett et al., (2002) also reported that T. gondii mobilizes Ca²⁺ when exposed to ryanodine or caffeine, both RyR ligands [34]. Addition of xestospongin C inhibited the caffeine- or ethanol-induced increase of intracellular Ca²⁺, thus preventing microneme secretion in T. gondii [35], and suggesting a role for InsP₃R. In Paramecium this pathway controls stimulated trichocyst exocytosis, as found by energy-dispersive X-ray microanalysis and fluorochrome analysis [36], via activation of RyR-type Ca²⁺ release channels in alveolar sacs [10,37]. RyRs share common features and an evolutionary history with InsP₃Rs [9,38,39], yet RyRs are activated by the intracellular messenger cADPR (cyclic ADP-ribose) [40]. In fact, cADPR has been shown to activate Ca²⁺ signaling pathways in T. gondii and P. falciparum [30,41].

In malaria melatonin dependent signaling via calcium is well established [29,42,43] enabling the investigation of a natural ligand to induce intracellular signaling. Hotta et al., [42] reported that intracellular Ca²⁺ mobilization induced by melatonin in P. falciparum and P. chabaudi is abolished by the PLC inhibitor U73122, but not by its inactive analogue, indicating that melatonin may activate signaling via PLC/InsP₃. Enomoto et al., [44] blocked spontaneous Ca²⁺ mobilization in the ring and trophozoite forms of intraerythrocytic stage of P. falciparum using the InsP₃R inhibitor, 2-APB [45]. The presence of 2-APB during intraerythrocytic development of P. falciparum compromised the asexual replication of this parasite suggesting that blocking the InsP₃R can be a potential target for antimalarial treatment. Raabe et al., [46] investigated the role of PLP₂/PLC/InsP₃ during P. berghei gametocyte exflagellation induced by xanthurenic acid (XA), a small metabolic intermediate found in Anopheles mosquito gut. Under XA stimulus, addition of the PLC inhibitor U73122 inhibits Ca²⁺ mobilization in this model. Together this work suggested a vital role of the InsP₃/PLC signaling pathway at different points during the Plasmodium life cycle, and the important nature of this pathway suggests it may contain potential antimalarial targets expressed during all life cycle phases.

Evidence for a ryanodine sensitive store in T. gondii [35] motivated Raabe et al., [46] to investigate the role of Ca²⁺ release via the RyR in P. berghei during gametocyte maturation. In the presence of RyR inhibitors, dantrolene and ruthenium red (RR), XA mediated Ca²⁺ increases were attenuated. Remarkably, RR treatment decreased InsP₃ levels and dantrolene inhibited gametocyte maturation during XA stimulation. These data also suggest the existence of ryanodine sensitive channels in P. berghei. The development of cell permeant caged Ins1,4,5P₃,[47], enabled the investigation of InsP₃ dependent calcium signaling without compromising membrane integrity. With this tool, Alves et al. [43] reported that P. falciparum trophozoites within intact RBCs release Ca²⁺ from thapsigargin-sensitive stores due to liberation of caged Ins1,4,5P₃. This observation was the first demonstration of a Ca²⁺-increase induced by exogenous InsP₃ under physiological conditions in Apicomplexa. Furthermore, the authors reported an increase of InsP₃ in infected RBC treated with melatonin. These data further support the concept of PIP₂ hydrolysis to InsP₃ as a signaling pathway activated by melatonin in P. falciparum, as reported previously [42,48].

Mossaad et al. [49] first reported a complete reversal of chloroquine resistance in malaria parasites after treatment with 2-APB in vitro and in vivo, and this effect was justified by disturbance of Ca²⁺ homeostasis in the parasite cell. This result showed that 2-APB and other related compounds that block the InsP₃ pathway might be promising candidates in the search for new-resistance reversing agents that aid in treatment of the disease.

Beraldo et al. [50] showed that increase in cytosolic Ca²⁺ concentration in melatonin induced P. falciparum is abolished with the use of 2-APB, U73122 (PLC inhibitor) and luzindole (a melatonin antagonist). In the same work, capacitative calcium entry was also reported in malaria parasites.

Since then, other pharmacological agents have further been applied to investigate this pathway. It should be noted that most drugs used to interfere with signaling in protozoa are used solely on the basis of effects observed in mammalian cells [31], and all these drugs have potential off target effects. Despite this caveat, the combined evidence of a number of studies suggest the presence of an InsP₃R and/or RYR calcium release channels in Plasmodia and Toxoplasma.

4. InsP₃Rs and RyRs in Protozoans

The history of InsP₃R discovery and the impact of this receptor on mammalian cell signaling has been previously reviewed [51]. Knowledge of receptor sequences and functional proprieties in mammals, together with increasing information and access to genome databases for different organisms, made bioinformatics tools such as BLAST searches a major strategy to identify orthologs to putative InsP₃R and RyRs in metazoans.

Until recently no information about such channels has been available from protozoa. After successful identification of InsP₃Rs and RyR-like proteins [9,37,52] in the ciliated protozoan, Paramecium, partial sequences suggested the occurrence of InsP₃Rs in several protozoan phyla, including trypanosomatids [37,39]. Similarity with established InsP₃Rs is often scattered throughout the protein, with highest similarity in the carboxy-terminal region containing the pore domain. Prole and Taylor [53] also used conserved regions of InsP₃R and RyR, specifically the amino-terminal RIH (RyR and InsP₃R homology) domain and the N-terminal InsP₃-binding domain, to successfully identify candidates for InsP₃R in T. cruzi and T. brucei. Such work supported the identification of an InsP₃R-type protein in T. cruzi [54,55] and T. brucei [56]. Hashimoto et al. [54] demonstrated that TcInsP₃R is essential for T. cruzi epimastigote survival, contributes to parasite invasiveness in mammalian cells, mediates Ca²⁺ release in trypomastigotes upon attachment to host cells, modulates parasite development and contributes to parasite virulence. These findings are consistent with previous evidence for a InsP₃/DAG pathway in T. cruzi (49) and T brucei [47].

The identification of an InsP₃R candidate in apicomplexan parasites is a much more challenging task. When the same sequences of mammalian InsP₃Rs were used for BLAST searches, no InsP₃R orthologs were identified for Plasmodium, Toxoplasma, Cryptosporidium or Babesia species [53]. Previously, Naganume et al. [58], using a collection of Ca²⁺-associated protein orthologs to identify proteins participating in Ca²⁺ signaling, were also unable to find either any orthologs of InsP₃Rs, or of protein kinase C (PKC), a classic target for DAG activation [59]. Ladenburger et al., (2009) [37] were also unable to detect any InsP₃R or RyR orthologs in genomes of apicomplexans, in contrast to ciliates where they are readily found.

Recently, it was reported that, when TcInsP₃R expression is decreased by two thirds compared to wildtype, the invasion of trypomastigotes of T. cruzi is blocked, indicating TcInsP₃R as a potential therapeutic target [60]. After treatment of trypomastigotes with specific antisense oligonucleotides of TcInsP₃R, a reduction in infectivity, thus suggesting that the suppression of transcription of this gene led to reduced levels of TcInsP₃R protein. In Chagas disease, Ca²⁺ signaling mediated by InsP₃R plays a key role in multiple parasite differentiation steps [61]. A therapeutic approach using antisense transcripts is particularly feasible in the acute phase of infection in which trypomastigotes predominate in the bloodstream. It is possible that a treatment with antisense transcripts is effective in preventing the development of the disease by blocking the proliferation of trypomastigotes by inhibition of production of TcInsP₃R [62].

The presence of dense mitochondria in T. cruzi with nonfunctional InsP₃R provides new insights for the contribution of InsP₃Rs to organelle integrity and the fact that 2-APB does not completely inhibit the development of trypomastigotes [63] suggest structural difference to mammalian InsP₃Rs [64]. On the other hand, at high concentrations, 2-APB might influence functions of proteins other than InsP₃Rs.

5. Lessons from other systems

Clues about the composition of Ca²⁺ signaling pathways in apicomplexans can be drawn from their closest ancestral group, free living ciliates. It is estimated that ciliates originated between ~800 and 850 million years [65,66]. Apicomplexa are assumed to have arisen ~550, and the genus Plasmodium ~400 million years ago [65]. These events occurred much before Apicomplexa became parasites of vertebrates and then of mammalian species, which occurred only ~13 million years ago [67]. Paramecium tetraurelia contains several genes encoding InsP₃Rs and RyR-like channels that have been identified at a genomic and proteomic level [9,37,52]. Considering the finding of InsP₃Rs and of RyR-like channels in Paramecium [10], the argument that the old age of some phyla may explain the failure to detect Ca²⁺ release channels in apicomplexans appears unlikely.

The lack of conservation at the gene level, suggests that InsP₃R in apicomplexans may diverge significantly from mammalian channels and those found in ciliates; one may ask whether they adopted a different, distinct protein or a complex of proteins for InsP₃R function. If this is the case, successful identification of InsP₃R in apicomplexans may require a different strategy focused on biochemical and physiological criteria. Specifically one should consider that any Ca²⁺ release channel requires a pore domain with six transmembrane stretches in InsP₃Rs and RyRs, from ciliates to humans, and a rather conserved, though slightly variable selectivity filter [10].

Currently the lack of identified Ca²⁺ release channels in Apicomplexa leaves a significant gap in our understanding of PI and calcium signaling mechanisms. There are clear physiological effects of stimulating production of InsP₃, and of inhibiting putative channels, and yet the molecular machinery in Apicomplexa is clearly diverse from other cells. Ultrastructurally the inner membrane complex of Apicomplexa looks very much like the alveolar sacs of ciliates; however, whereas the sacs of ciliates are well established Ca²⁺ stores [36], they appear largely independent of such function in Apicomplexa [8]. The third enigma is the presence of well defined InsP₃R- and RyR-type Ca²⁺ release channels in ciliates [9,37,52], in contrast to their absence in their closest relatives, the Apicomplexa [68,69].

Similar to apicomplexans, plant genomes lack recognizable InsP₃Rs in despite of evidence for an InsP₃ signaling pathway [70]. Despite responding to InsP₃ to regulate Ca²⁺ increases, land plants typically lack an InsP₃R or RyR, leading to the suggestion that these channels were animal-specific, and perhaps evolved during the vertebrate lineage [70]. However, recent evidence from green algae Chlamydomonas indicates that chlorophyte plants contain InsP₃R, suggesting they were present in the ancestral eukaryotes prior to the plant-animal split [71]. Why InsP₃R channels appear to have been lost in land plants remains a mystery, but it is intriguing to consider that like apicomplexans, they may have evolved different mechanism to release internal Ca²⁺ stores in response to InsP₃.

Conclusion

Differences in Ca²⁺ signaling between parasites and their hosts may present unique targets for developing interventions. For example, McNamara et al., 2013 [72] reported PI4K as a target of imidazopyrazine, a class of new antimalarial drug that inhibits all stages of malaria parasite infection of the vertebrate host and also prevents transmission by mosquitos to a murine malaria model. Additionally, as mentioned above, agents that disrupt InsP₃R channels such as 2-ABP block malaria growth in vitro [44]. Given the apparent divergent mechanism by which InsP₃ is sensed in parasites, identification of the molecular basis for this pathway might also identify novel targets for intervention.

The pharmacological literature and functional studies with exogenous InsP₃ discussed herein seem to support the occurrence of InsP₃R-type Ca²⁺-channels in ciliates, trypanosomatids and apicomplexans. InsP₃R is the major missing piece of the PI signaling puzzle in Apicomplexa. Although InsP₃R have been found in ciliates and tryponosomatids, the lack of ³H-InsP₃ binding assays in parallel to a vast unknown protein function in apicomplexans genome has hampered the identification of potential candidates for InsP₃R. Although identification of these channels will certainly not be an easy task, it will be a rewarding engagement for groups willing to accept the challenge.

Consent for Publication

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CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.