The first evidence of the cAMP signaling in malaria parasite arose through an experimental study in which the addition of external cAMP to Plasmodium culture was shown to positively affect the exflagellation or gametocyte formation during the ring stage of the parasite (20). However, the parasitic AC differs biochemically from that of host counterpart. Forskolin and Alf4, the activators of mammalian AC, and GTPγs, the activator of G protein, are unable to cause stimulation in parasitic AC (21). Moreover, expression of G stimulatory α have been demonstrated in early asexual stage and mature sexual stage. So, it was assumed that G protein might be implicated in the signaling during gametogenesis. However, this finding leads to a dilemma because the Plasmodium genome is devoid of gene corresponding to the G protein (22).

Adenylyl cyclase and cAMP signaling were demonstrated to play an important role during the infection of hepatocytes by sporozoites. The migration of sporozoites across the host hepatocytes results in their activation and triggering of apical regulated exocytosis. The sporozoites can be activated externally by calcium ionophore, which are then able to infect liver cell without migration (23). The gene knockout experiment of ACα in Plasmodium berghei explained the prevention of the exocytosis along with the reduced infectivity. However, the results were reciprocated after the reintroduction of the ACα gene into the mutant. Thus, the involvement of cAMP-mediated signaling in the initial phase of infection was confirmed. Besides this, the ACα also share homology with K⁺ channels, which are required for exocytosis in sporozoites (24). Not only sporozoites but also merozoites invasion process also involves cAMP-dependent signaling. During invasion, there occurs the formation of tight junctions with host cells, which leads to the secretion of apical organelle containing several proteins like gliding-associated protein 45 and apical membrane antigen 1 (AMA-1) (25, 26). The whole event of invasion is regulated by the cAMP-dependent phosphorylation of protein AMA-1 mediated by PfPKA (Figure 1). The mutational analysis of AMA-1 showed the hampering of the invasion process due to a change in phosphorylation site (serine 610) (27). It was evidenced from another study that merozoite proteins, particularly localized to the microneme and rhoptry organelle, get secreted and interact with receptors on fresh erythrocytes. The secretion of proteins involves a stepwise signaling cascade initiated due to the exposure of the low K⁺ extracellular environment (28–32). Low K⁺ triggers activation of PfACβ followed by an increase in cAMP. The cAMP activates Epac (exchange protein activated by cAMP) pathway, which subsequently cause an increase in PfPKA coupled with elevation of Ca²⁺ level (33). In the Epac pathway, Ras-proximate-1 (Rap1) converted to Rap1-GTP and further activates phospholipase C (PLC). The activated PLC induces calcium-dependent protein kinase 1 and calcineurin, which eventually leads to the secretion of microneme and rhoptry proteins (33–35) (Figure 1). Intense analysis of the pathway and its regulating components rendered a better understanding of overall mechanism, which can be implemented for inhibiting parasitic growth, invasion, and malaria prevention.

The parasitic infection remodels the membrane of host erythrocytes for the nutrient acquisition and maintaining the balance of electrolytes. The electrophysiology-based studies indicated a peak of conductance of anionic channels during Plasmodium infection (36). The conductance of anions across the infected host cell membrane is mainly regulated by cAMP signaling. The experimental addition of PKA and ATP to the uninfected erythrocyte caused the upregulation of anion conductance (26), while the process overturned on the addition of alkaline phosphatase (ALP) or by dephosphorylation (37, 38). In this contest, the dependency of anion channel regulation on cAMP was further confirmed through an experiment with PfPKI (H89 cAMP-dependent protein kinase inhibitor) or by developing transgenic parasite with higher expression of PfPKA-R (phosphokinase A-regulatory subunit), which binds to cAMP, thereby downregulating the process and directly affecting the parasitic growth (39, 40). The signaling cascade either involves parasitic components or host is still controversial. Also, little is known about the actual substrates of PKA. The above discussion presents the essentiality of cAMP. Cyclic nucleotides are produced by AC and GC on hydrolysis, which requires PDE (19, 41). Therefore, PDE regulates the production and functioning of cAMP and cGMP. In P. falciparum, PfPDE1 was the first-reported PDE, which is specific for cGMP (42). Among various PDE types, PDE4 is predominant in the immune cells. Implementation of PDE4 inhibitors was found to enhance chemokines production and elicit inflammatory response. To delineate the regulatory mechanism of cyclic nucleotide, parasite-specific PDE inhibitors were developed. Zaprinast, a PDE5 inhibitor, and 5-benzyl-3-isopropyl-1H-pyrazolo[4, 3-d] pyrimidin-7(6H)-one, inhibitor of PfPDEα, were potentially most effective inhibitors blocking the parasite proliferation (43). PDE inhibitors can be explored in developing antimalarial therapy (44).

The protein sequence analysis of PfPKG with respect to vertebrate counterpart revealed certain differences between their sequence features. For instance, the presence of three cAMP/cGMP binding motif, a degenerated cGMP binding motif and lack of leucine zipper motif required for dimerization and insensitivity towards cGMP analogue (45, 46). Along with its role in gametogenesis, cGMP has been demonstrated to express its functionality during the ring and schizogony stage (45). A known PKG inhibitor, 4-[2-(4-fluorophenyl)-5-(1-methylepiperidine-4-yl)-1H pyrrol-3-yl] pyridine (compound 1), has a retarding effect on parasitemia level (47, 48). In contrast, the mutant strain with genetically modified PfPKG showed normal development in the presence of inhibitor compound 1. These findings established the key role of cGMP signaling during the asexual phase of parasite development.

Conditional knockout study carried on P. berghei PKG (PfPKG) at late liver sporozoites (LS) stage depicted that parasite can infect hepatic HePG2 cell lines, but failed to get released from hepatocytes as merosome. Sporozoites at this stage elicited protective immune response in the host (49). Thus, it reflects an essentiality of cGMP signaling during the formation and release of merosome. So far, the precise role of PKG and trigger for its stimulation is not clear. A deep insight into the signaling involved in LS would be opportune to control disease pathologies at the erythrocytic stage and can be advantageous in search of prospective medication of malaria and preerythrocytic vaccine development (49).

During the egress cascade, PKG is required in proteolytic processing of proteins such as proteolytic processing of P. falciparum serine-like repeat antigen (PfSERAs) and PfMSP-1 by P. falciparum subtilisin-like protease (PfSUB1) (Figure 1), which in turn are involved in merozoite egress and secretion of apical organelle (50, 51). The proteolytic processing of PfMSP-1 was prevented with the inhibitor of PfPKG, compound 1 without affecting the activity of SUB1. Noticeable, in an experiment involving schizonts deficient in PfCDPK5 showed normal processing of PfMSP-1 in the presence of compound 1. Therefore, it indicates the role of PfCDPK5 downstream to PKG (52). The phosphoproteomics and chemical genetic approach involving the use of PKG inhibitor, compound 2 4-[7-[(dimethylamino) methyl]-2-(4-fluorophenyl)imidazo[1,2-α]pyridin-3-yl]pyrimidin-2-amine along with strain containing PKG mutant allele (PfPKG_T618_Q), resistant to compound 2 further demonstrated the role of PKG in the signaling event. Comparative analysis of the effect of treatment with compound 2 in wild-type and PfPKG mutant brought out various cellular targets of PfPKG involved in egress and invasion (53). The significance of PfPKG upstream to calcium signaling was indicated by a study in which phosphorylation of PfCDPK1 occurred in a PKG-dependent manner (50, 53). Moreover, PKG plays a significant role in regulating the cytosolic calcium signaling both during egress and at the asexual stage (54). The key importance of PfPKG in phosphorylation of the substrates gives a reflection that both PfPKG and its substrates are the therapeutic candidates in malaria treatment and in transmission blocking (55).

During malaria, extensive modification in erythrocytes is accompanied by changes in membrane permeability (56, 57). It has been well documented that hypoxic signals or surface deformation of RBC accounts for the release of ATP (58, 59). An elevated level of ATP has been recorded both in the extracellular environment and in the cytoplasm of host and parasite (60, 61). Parasitemia is proved to be directly linked to level of ATP (62). Any diminution of ATP in the medium rendered the Plasmodium ineffective to infect the fresh erythrocytes (60, 63, 64). The essentiality of ATP in the invasion process of parasite was confirmed by thwarting the entry of the parasite through inhibitor-mediated blocking of purinergic receptor and the addition of apyrase (65). The underlying mechanism of this inhibition involved the boosting up of the cytoplasmic calcium levels by extracellular ATP (65). The effect of purinergic signaling is mediated through phosphorylation of skeleton protein like spectrin present in erythrocyte (64, 65). Purinoceptor signaling is linked to induction of the new permeation pathway. Therefore, purinoceptors antagonist, suramin, reduces the membrane permeability and leads to deterioration in parasitic growth, both in vivo and in vitro (66). Purinoceptor blocker, suramin, and pyridoxal phosphate-6-azophenyle-2′,4′-disulphonic acid alter the proteolytic processing of protein like MSP-1, which is involved in the invasion, while the addition of ATP was found to trigger the intracellular proteolytic event in P. berghie and Plasmodium yoelii (67). It suggests the involvement of purinergic receptors in ATP signaling. The purinergic receptors that are expressed on the parasite surface are phylogenetically distinct from human counterparts. Thus, these receptors can be exploited as targets so as to design inhibitors of parasite invasion process (68, 69). ATP is also thought to be involved in the host-induced inflammation following the malaria infection (70).

Live cell fluorescent video microscopy involving the use of fluo-4 Ca²⁺ probe, Ca²⁺ chelators, and inhibitors of Ca²⁺ ATPase revealed the differential role of various effector molecules in egress and the invasion of the merozoites. It has been shown that before egress of merozoites there is a constant increase in Ca²⁺ level in the cytoplasm of both parasite and infected RBC (iRBC) independent of extracellular Ca²⁺ ions.

Inhibition of Ca²⁺ ATPase from ER inhibitors and addition of Ca²⁺ ionophore enhanced the process of egress, while chelating agents like bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra acetic acid inhibited the process of egress (64, 86). The mechanism of inhibition involved the prevention of permeabilization of host membrane. The Ca²⁺ is found to regulate the release of perforin-like protein 1 (PLP1) from microneme to the membrane where it begins its lytic activity. Likewise, the expression of PLP2 also occurs during the asexual blood stage, but its role is not clear. The reverse genetic studies would be helpful in disclosure of the details of PLP1 and PLP2(86). P. falciparum subtilisin-like protease (PfSUB1) is another effector of calcium signaling, which is found to be required for the PfSERAs. The inhibition of PfSUB1 by the use of Ca²⁺ chelators prevented the rupture of the parasitophorous vacuole and consequently the egress of merozoites (86).

The invasion process requires the discharge of rhoptry and microneme, which is dependent on the elevated levels of Ca²⁺. On exposure to low K⁺ level, cytosolic Ca²⁺ level increases through PLC and it triggers the export of erythrocyte-binding antigen 175 (EBA175) and AMA-1 from microneme to merozoite surface (Figure 1). The interaction with the receptors of RBC brings the increased level of calcium to the basal level, which induces the release of rhoptry protein (29). P. falciparum reticulocyte-binding protein homologs-1 (PfRH1), present in trace amounts in rhoptry neck, was found to trigger the release of calcium ions, which further initiates the cascade. The use of PfRH1 antibody inhibited the invasion process by blocking the calcium signaling and halting the interaction of EBA175 with host receptors. Therefore, it is assumed that an alternative pathway, namely, K⁺ ion-dependent pathway for the release of protein from apical organs, might exist. In spite of the above findings, the mechanism of triggering of signals to release calcium ions is yet to be probed (87). A study indicated the role of PfCDPK1 in the discharge of microneme protein for invasion. The mutational analysis further analyzed the inhibition of invasion process upon mutating its active site residues (88, 89). Microneme secretion and thereby invasion was also found to be impaired on deletion of another calcium lipid-binding Doc 2 protein, PfDoc2. However, the actual mechanism of action is unclear (47, 90).

The cell cycle of parasite is known to be regulated by the coordinated release of calcium ions. The efflux of calcium ions from ER occurs through inositol 1,4,5-trisphosphate (IP3) channels. It was demonstrated that exogenous addition of IP3 also causes the release of ions. Melatonin hormone was found to induce the production of IP3 through PLC, which further opens the Ca²⁺ channels in the ER (91–93). An increase in calcium level on incubation with tumor necrosis factor (TNF)-α caused the downregulation of P. falciparum’s proliferating cell nuclear antigen-1 and ultimately the retardation of parasite growth (94). Calcium mediates its activity through PfCDPK7. The role of PfCDPK7 was confirmed by knockout studies in which PfCDPK7 mutant showed drastic retardation in growth. It can be assumed that the role of calcium might be correlated with protein kinase, PfPK7 and cdc2-related protein kinase, because the inhibition of these genes also demonstrated to retard parasitic growth, but this hypothesis needs further validation (95). Expression of PfCDK2 was also indicated to be at peak during the ring and trophozoite stage. However, the function of PfCDK2 has not been deciphered yet (96). A different group of Ca²⁺-binding orthologs of cytoskeletal-binding protein centrin (PfCEN), expressed during asexual and the gametocyte stage of parasite, was found to be colocalized with the centrosome. The role of centrin in cell division of Plasmodium yet concealed, while the knockout studies in Leishmania donovani revealed their involvement in the growth and cytokinesis (97).

There is an extensive involvement of calcium signaling in various important pathways of parasite. Any interruption would be deleterious for invasion, egress, and ultimately the growth of parasite. On these grounds, components of calcium signaling are considered for therapeutic interventions.

During malaria, TLR9 is involved in providing protective immune response in a MyD88-dependent manner (113). Hemozoin (HZ) was indicated to stimulate this response (114–116). In a study, the ligand property of purified HZ against TLR was studied. The studies performed in TLR9-deficient mice showed the inadequate functioning of HZ in the production of chemokine, cytokine, and costimulatory signals (117). Parroche et al. (118) identified that natural HZ but not purified hemin can induce TLR9. Experimentally, it was explored that the stimulation was forbidden due to the lack of binding to TLR9 in the presence of nuclease. Later, it was confirmed that DNA present on the surface of HZ interacts with TLR9 (118). Another piece of information supporting the role of TLR signaling during malaria was derived from the finding, which has demonstrated the activation of regulatory T (Treg) cells in a TLR9-dependent manner. During the P. yoelii infection, an unknown protein of parasite stimulates the TLR9 present on dendritic cells. These signals ultimately trigger off the Treg cells, which in turn suppress the effector T cells. Evidence suggests that mice deficient in TLR9 are more resistant to malaria infection due to the activation of the effective response of T cells (15).

The pro-inflammatory response is not restricted to be mediated through TLR9, but in recent past, natural HZ has been shown to bind with host fibrinogen, which interacts with fibrinogen receptor TLR4 on monocytes, and this interaction leads to downstream activation of NF-κB and MAPK, thereby arousing the oxidative burst and elevated expression of TNF and other pro-inflammatory cytokines (119). Similarly in case of murine malaria, 2-Cys peroxiredoxin antigenic protein of Plasmodium stimulates TLR4 on mast cell and macrophages to produce TNF-α (120) (Figure 2).

A study carried out in Plasmodium chabaudi elucidated the role of TLR7 in the production of IFN-1, IL-12, and IFN-γ. Earlier, TLR9 was reported as the key sensor of infection. But the experiments conducted in the absence of TLR7 and MyD88 showed the remarkable reduction in the pro-inflammatory cytokine-like IFN-1. Contrary to it, no influence on IFN production was observed in mice deficient in TLR2, TLR4, TLR9, interleukin-1 receptor, or IL18R. Rational for this disparity is that the activation of TLR9 or TLR 7 depending on the time or stage of infection and alteration in the available ligand (16). Although the parasitic ligand triggering TLR7 has not yet been proven, however, based on finding that single-stranded RNA is required for TLR7-dependent production of IFN-I during viral infection (121), it was hypothesized that the RNA of parasite might work as a ligand against the receptor (16). Despite this speculation, the actual receptor–ligand interaction needs to be elucidated.

TLR2 recognize glycosylphosphatidylinositol (GPI) with TLR1 and TLR6 in a heterodimeric form (110) and induces the inflammatory cytokine production (122) (Figure 2). Severity of malaria was found to be correlated to allelic variation in TLR1 (123). TLR2 signaling in liver stage initiates the production of pro-inflammatory response, which hampers the parasite’s development (124).