In eukaryotes, effective calcium homeostasis is critical for many key biological processes. There is an added level of complexity in parasites, particularly multicellular helminth worms, which modulate calcium levels while inhabiting the host microenvironment. Parasites ensure efficient calcium homeostasis through gene products, such as the calmodulin-dependent kinases (CaMK), the main focus of this review. The importance of CaMK is becoming increasingly apparent from recent functional studies of helminth and protozoan parasites. Investigations on the molecular regulation of calcium and the role of CaMK are important for both supplementing current drug regimens and finding new antiparasitic compounds. Whereas calcium regulators, including CaMK, are well characterised in mammalian systems, knowledge of their functional properties in parasites is increasing but is still in its infancy.
Calcium (Ca2+) functions as a central molecule in signal transduction and is one of the most widely used second messengers in cell signalling . Ca2+ signalling is important in many cellular processes, including regulation of gene transcription, cell division and growth, muscle contraction, neuronal transmission, secretion and ion transport. Ca2+ is also the most important ion in maintaining the integrity of the endo- or exo-skeleton in many higher order animals, which also functions in Ca2+ storage.
Ca2+, being a crucial element in vital cellular processes, has had its signalling pathways studied extensively in mammals and other organisms [2–7]. Protein members of the Ca2+ signalling pathway have been identified for the development of various therapeutic targets [8–12]. Components of the Ca2+ signalling pathways have, been targeted for example in the treatment of cardiovascular diseases, notably hypertension . Ca2+ and its regulators also play an important role in the pathogenesis and treatment options of major infectious disease agents. Ca2+ has been shown to be an important molecule in host pathogen interactions in bacterial diseases [14,15], protozoan diseases including malaria , disease caused by ticks  and helminthiases such as schistosomiasis [18–20]. In this review we focus on the importance of the Ca2+ signalling pathway molecules, Ca2+/calmodulin dependent Kinases (CaMKII) in helminths and their potential value as control targets.
Calcium regulation in cell biology
Ca2+ is highly reactive and hence is toxic to cells . It binds strongly to phosphates, compared to water, thus resulting in precipitation of salt complexes, with potential cytotoxic consequences for the cell [1,2]. To minimise this potential damage, cells act to exclude Ca2+ from the cytosol and maintain it at a low concentration (50–100 nM)  by its chelation, compartmentalisation or exclusion .
The free Ca2+ concentration within cells increases either by entry of extracellular Ca2+ into the cell or by the release of intracellular stored Ca2+ from the endoplasmic (ER) or sarcoplasmic reticulum (SR) . The concentration of this free Ca2+ is controlled either as ions entering the cell or are retained within endoplasmic/sarcoplasmic reticulum ER/SR.
There are many proteins that participate in the regulation of the free cellular Ca2+ concentration. These proteins assert their effect either by binding free intracellular Ca2+ ions simply for buffering or for the enacting of processes such as cell signalling . Calbindin and parvalbumin are Ca2+ binders and transporters that do not undergo significant conformational changes following Ca2+ binding. These proteins act as buffers through attaching and sequestering cellular Ca2+ whereas proteins that undergo significant conformational changes following Ca2+ binding, such as troponin-C and calmodulin, function as sensors to make Ca2+ signals useful in cellular functions .
CaMKs comprise substrate specific kinases (e.g. CaMKIII) and multifunctional kinases (e.g. CaMKI, CaMKII, CaMKIV), all of which have many downstream targets . There are multiple cellular and biological functions related to the CaMKs due to the fact they display broad substrate specificity and are ubiquitous . CaMK has been studied in great detail with regard to neuronal [23,24] and cardiac function in mammals . An excellent generalised review of CaMK enzymes is available .
In this review, we focus primarily on the biological and regulatory roles of CaMKII, with an emphasis on its potential as an anti-parasitic target. The scale of human infections with helminth parasites and the ever present spectre of drug resistance strengthen the case for research into novel compounds. Specific protein targets, such as the Ca2+/Calmodulin-dependent kinases discussed here, must be identified and interrogated using biochemical, cellular and animal infection strategies, to demonstrate their importance to the parasite and efficacy as a future therapy.
CaMKII and calcium regulation/signalling
CaMKII is a multifunctional kinase that has specific isoforms present in different tissues and organs. In mammals, CaMKII is found in highest concentration within cardiac muscle where it is involved in excitation contraction coupling . Mammalian CaMKII has four isoforms: α, β, γ and δ, all encoded by distinct genes . CaMKs are activated by the binding of Ca2+ bound calmodulin which opens up the substrate binding site. The binding of Ca2+/CaM is associated with auto-phosphorylation of CaMKII, leading to prolonged action even after binding dissociation. Auto-phosphorylation of CaMKII can also significantly increase its affinity for Ca2+/CaM . CaMKII has to be dephosphorylated and inactivated by phosphatases, such as PP2A and PP1 .
CaMKII regulates other proteins in the Ca2+ signalling pathway such as Ryanodine receptor (RYR2), L-type voltage gated Ca2+ channels (LTCC), phospholambin (PLN), and K+ and Na+ channels (Figure 1). When dephosphorylated, CaMKII inhibits the SR Ca2+ pump reducing intra cellular Ca2+ . Both CaMKII and Protein Kinase A (PKA) have the ability to phosphorylate and activate PLN which, in turn, activates the SR Ca2+ pump to import Ca2+ into ER .
General features of the role of CaMK in cellular Ca2+ regulation.
CaMKII phosphorylates RYR2 resulting in the increase of SR Ca2+ leakage [9,11,29,30]. LTCC is also directly modulated by CaMKII which allows it to increase Ca2+ influx into the cell [9,31,32]. CaMKII is also thought to have a negative feedback effect on LTCC protein levels when the Ca2+ concentration is elevated in the cardiac myocyte . A recent study using murine endothelial cells has explained this negative feedback effect of CaMKII on cellular Ca2+ increases through an impact on inositol-1,4,5-triphosphate (IP3) . Therefore, CaMKII inhibition results in the dysregulation of free Ca2+ and Ca2+ signalling in the cell. An overview of the interaction between calmodulin and CaMKII in Ca2+ homeostasis is presented in Figure 1.
CaMKII structural elements
CaMKII is a dodecameric (12-meric) holoenzyme [35,36]. Each subunit has an N terminal catalytic/kinase domain, a regulatory domain and a C terminal association- or a hub-domain [1,9,35]. The catalytic domain is bi-lobed and has an ATP-binding site and a substrate-binding site (Figure 2). The regulatory segment consists of an auto-inhibitory domain and a CaM-binding domain. Comparison of human, mouse and Schistosoma mansoni (blood fluke) CaMKII isoforms (Figure 3) emphasises the highly conserved nature of the catalytic and the regulatory domains among distinct animal clades. Autophosphorylation sites are found in the auto-inhibitory domain at Thr286or287 depending on the enzyme isoform (Figure 3). The hub domain forms a central hub to organise the twelve subunits together [1,9,35–37]. The secondary structure of S. mansoni CaMKII, predicted with 100% confidence by the single highest scoring template (human CaMKII), is shown in Figure 4.
CaMKII secondary structure.
Protein sequence alignment of S. mansoni CaMKII with human CaMKIIδ and mouse CaMKIIα.
Tertiary structure model of Schistosoma mansoni CaMKII.
CaMKII and other Ca2+ regulators of helminths
As mentioned in earlier paragraphs, the importance of Ca2+ in a multitude of cellular processes cannot be overstated. In helminths, Ca2+ is found to be involved in many important functions including neuronal activity, motility, fecundity, host immune response  and as a structural component. The fact that Ca2+ is a crucial element in cellular processes makes the proteins involved in its regulation become potential therapeutic targets for parasitic helminths. The metabolism of calcium has been investigated for many medically important organisms , but while calcium regulatory proteins have been identified as putative therapeutic targets, knowledge of their function in helminths is limited.
Caenorhabditis elegans (C. elegans), which is a free-living nematode, is among one of the most commonly studies helminths and is an useful model to investigate calcium metabolism in helminths. Plasma membrane and intracellular Ca2+ channels are most important in maintaining cellular Ca2+ concentrations. NCX (sodium-calcium exchange channels) which are important in the transport of Ca2+ across cell membranes have been found in C. elegans . Tanimoto et al. (2017) found that CaMKII is found to be an important intracellular Ca2+ regulator which is involved in the control of movement through its effect on motor neuron synaptic activity [40,41]. It has also been shown to regulate the life span of C. elegans through phosphorylating a protein called DAF-16 . C. elegans CaMKII gene (unac-43) mutations are also associated with defects in egg laying, brood size and defecation .
Different groups of sodium Ca2+ exchangers, including NCX, involved in the transport of Ca2+ across cell membranes, have been found in many other nematode species. Sharma et al.  showed the variable expression of these channels in C. elegans, Ascaris suum, Trichuris trichiura and Loa loa. This variability in the different nematodes is to meet the unique Ca2+ regulatory requirements of the individual species . The need for tight Ca2+ homeostasis lies particularly in modulation of the host immune response against these parasites , and the diversity associated with each of their lifecycle stages, and the differing environments, they inhabit.
Ca2+ also contributes as a structural element in helminths. Formation of mineral concretions known as calcareous corpuscles have been described in tapeworms (phylum Cestoda). These corpuscles are thought to protect cestode larvae against calcification in host tissue sites and to prevent digestion of adult worms by gastric acids of the host . The trematode parasite Schistosoma has numerous homologues of mammalian proteins involved in cellular Ca2+ metabolism. SR calcium transport ATPase (SERCA) is one such membrane channel that has been identified in S. mansoni (Figure 5) [3,45]. Other possible Ca2+ modulating channels, including those gated by Famide-related peptide (FMRF) and serotonin, are important in Ca2+-dependent muscle contraction in this parasite. The presence of RYR channels in S. mansoni has also been demonstrated in cellular assays using isolated individual muscles fibres from adult worms .
Schistosoma mansoni Ca2+ signalling pathway.
Prole and Taylor  summarised some of the key intracellular and plasma membrane Ca2+ channel homologues found in schistosomes and other parasites. In addition to IP3R/RYR homologues, which are present in Schistosoma, Trypanosoma and Leishmania, Two-Pore channels (TPC) homologues, ORAI Ca2+ channels and STIM (stromal interactic molecules) Ca2+ sensor homologues have been identified in S. mansoni . ORAI-1 calcium channels have specifically been found in S. mansoni ovary and vitelline glands which illustrates the involvement of Ca2+ in the egg production process. Ca2+ binding protein, Calmodulin 4 has also been found in the vitelline ducts and ootype . Figure 5 shows the Ca2+ signalling pathway proposed for S. mansoni, from the KEGG Pathway database [48,49]. However, overall knowledge on CaMKII activity in worms is very limited and further research is required for a more complete understanding.
Calcium and the action of anthelmintic drugs
Praziquantel (PZQ) was introduced in the 1970s as an anthelmintic with broad efficacy against trematodes and cestodes [50,51]. Although the precise mode of action of PZQ against these worms is unknown, it is thought the drug exerts its action by affecting Ca2+ homeostasis [52,53], possibly by targeting the voltage gated Ca2+ channels [54,55]. Although not effective against immature schistosomes, PZQ is the treatment of choice for schistosomiasis . In humans, 80% of the oral dose is absorbed, with a plasma half-life of 1–2 h, corresponding to a peak plasma concentration of 0.2–1 µg/ml for an oral dosage of 20–50 mg/kg [50,51].
Intracellular Ca2+ is clearly involved in the initiation of PZQ action on schistosomes, as shown by the lack of drug efficacy in parasites maintained in Ca2+-depleted media . PZQ is also known to cause Ca2+-dependent blebbing of the schistosome tegument [19,56]. High concentrations of PZQ have no effect on the activities of schistosome Na+/K+ ATPase or Ca2+/Mg2+ ATPase .
Studies on both S. japonicum and S. mansoni have shown up-regulation of genes involved in the Ca2+ signalling pathway and drug resistance after exposure to PZQ [18,58]. In vivo exposure of S. japonicum worms to PZQ identified some differences between male and female worms in the initial gene transcriptional profile, with males having a more pronounced up-regulation of genes associated with tegument/muscle repair and lipid/ion regulation, whereas genes involved in Ca2+ signalling were upregulated in female worms . Homologues of genes that have been shown to be associated with ion homeostasis and drug resistance were shown to be upregulated at a later time in males compared with females ; furthermore, CaMKII was one of the key Ca2+ regulator genes shown to be up-regulated. An in vitro PZQ exposure study on juvenile and sexually mature S. mansoni showed post-treatment up-regulation of drug transporters, Ca2+ regulatory genes and stress and apoptosis-related genes . The low efficacy of PZQ on juvenile parasites compared with adult worms has been attributed to their greater transcriptomic flexibility . However, in a recent study, PZQ treatment of juvenile S. mansoni worms resulted in significantly upregulated expression of both the β subunit of the VOC voltage gated Ca2+ channel and calmodulin after four days, observations not evident when examining adult parasites . Another in vitro study, using a lower dose of PZQ resulted in up-regulation of Ca2+ regulation-related genes including PMCA, SERCA and RYR but not CaMKII in paired parasites; PKC was down-regulated in unpaired mature females .
Ca2+ channel blockers (CCBs), commonly used to treat cardiovascular disease including hypertension in humans , have also been shown to have antischistosomal activity. In-vitro antischistosomal activity has been reported with several CCBs including nifedipine, amilodipine, verapamil and diltiazem [20,61]. Nifedipine, which is a known antagonist of LTTC, was tested at different concentrations on S. mansoni schistosomula and adult worms in vitro, in the presence or absence of PZQ . Light microscopy showed that both adult and immature parasites sustained tegumental injuries following the nifedipine treatment and had increased worm mortality; nifedipine also reduced the viability of schistosomula . Notably, no egg laying was observed in females treated with nifedipine, similar to results obtained by Bonn et al. , in response to verapamil, a different CCB.
The characterisation of Ca2+ signalling and regulation in parasites can provide novel, unique and fundamental biochemical and pharmacological information, and could facilitate future rational structure-activity-guided modifications of existing anti-parasite drugs and new compounds.
Mass drug administration (MDA), which is widely used in schistosomiasis control programs, has been shown to reduce the genetic diversity of schistosomes under this therapeutic selective pressure and may lead to the evolution of PZQ-resistant parasites . Reliance on a single drug, PZQ, to control a disease afflicting ∼200 million people worldwide continues to raise serious concerns in the schistosomiasis scientific community and for global public health. If schistosomes resistant to PZQ do inevitably eventuate, then the identification of novel molecular targets with important cellular functions in schistosomes will be key for future drug design and development. Further characterisation of the Ca2+ signalling and regulatory pathways in schistosomes can provide unique and fundamental biochemical and pharmacological information to facilitate future rational structure-activity-guided modification of PZQ and the development of new compounds. There is already evidence showing that the addition of CaMKII increases the efficacy of PZQ against adult schistosomes  and can provide a promising adjunct therapy against schistosomiasis and possibly other helminthiases. Therefore, the future targeting of CaMKII, a key molecule in the Ca2+ signalling pathway in helminth parasites, could provide a new direction for the development of effective treatment options.
G.G., S.N., H.Y. identified the topic for review. S.N. formulated the first draft. Additional drafts were constructed by all authors (S.N., H.Y., D.M., M.J., G.G.).
The support of the National Health and Medical Research Council of Australia (NHMRC) is acknowledged (Program APP1132975 and Projects APP1098244, APP1080007). DPM is a NHMRC Senior Principal Research Fellow (APP1102926) and Senior Scientist at QIMR Berghofer.
The Authors declare that there are no competing interests associated with the manuscript.