*2.3.2 Role of PDED in cAMP homeostasis*

Apart from the membrane bound phosphodiesterases, a soluble, cytosolic phosphodiesterase (PDED) was cloned and characterized from *L. donovani*. Bioinformatic studies showed the presence of two pseudo-substrate sites and a putative PKA phosphorylation site at the C-terminus of PDED and PKA-mediated phosphorylation is important for the regulation of phosphodiesterase activity (**Figure 5**) [50]. It was observed that catalytic subunits of PKA (PKAC1 and PKAC2) interacts with the pseudo substrate sites of PDED after 3 hours of PC exposure. Moreover, inhibition of phosphodiesterase activity through PKA-mediated phosphorylation was observed at a further later time point of PC exposure [51]. The cytosolic localization of LdPDED was established by immunolocalization analysis using anti-LdPDED antibody which revealed its localization to be predominantly cytosolic. Interaction of LdPDED with the catalytic subunits of LdPKA within

#### **Figure 5.**

*LdPDED interacting with PKAC1 and PKAC2 resulting in the inhibition of kinase activity of PKA. PKA on the other hand phosphorylates threonine residue of PDED increasing its phosphodiesterase activity.*

**145**

maintenance.

*Role of cAMP Homeostasis in Intra-Macrophage Survival and Infectivity of Unicellular Parasites…*

3 hours of exposure to differentiation condition leads to the inhibition of LdPKA (short-term regulation). LdPKA-mediated phosphorylation of LdPDED is observed when parasites are exposed to differentiation condition for more than 6 hours. Hydrolytic property of LdPDED is enhanced due to this phosphorylation event and this enhancement in hydrolytic activity might play a pivotal role in the maintenance of cAMP homeostasis (long term regulation) when the total cytosolic PDE activity falls because of PDEA depletion during stress condition [49]. This role of PDED in maintaining the PKA activity which in turn regulates cAMP homeostasis in the parasite during initial exposure to stress condition, might be important in the life cycle of the parasite particularly in the infection establishment within the host.

Though the existence and functioning of cAMP-dependent protein kinase (PKA) is well pronounced in eukaryotes, very little is known about the functioning of PKA in cAMP signaling of this particular parasite. PKA acts as the immediate downstream effector of cAMP in the adenylate cyclase pathway, catalyzing the transfer of γ-P from ATP to specific serine/threonine residues on the substrate protein [52]. Studies on *S. cerevisiae* reveal that one of the three PKA catalytic subunits mediates stress-induced differentiation [53]. Researches in *Dictyostelium* have suggested that cAMP is not required for differentiation if sufficient levels of PKA activity are present [54, 55] indicating profound role of PKA in differentiation. Activation of PKA by a short-term cAMP pulse induces bradyzoite differentiation, whereas a prolonged cAMP pulse inhibits differentiation [56]. It is likely that there are distinct PKA signaling pathways in the tachyzoite with opposing effects on parasite differentiation. Inhibition of PKA signals by treatment with PKA catalytic subunit inhibitor H89 induces bradyzoite differentiation [57], suggesting that PKA catalytic subunit activity may be involved in cAMP-mediated tachyzoite

When *Leishmania* parasites were exposed to stress condition, PKA activity was significantly enhanced along with increased level of cAMP. Protein kinase activity of five different species of *Leishmania* was found to be quite high in both logarithmic and stationary phase promastigotes, being most active in *L. amazonensis* and least in *L. donovani* [58]. PKA catalytic subunits in the *Toxoplasma* genome were identified. PKA is the most important downstream effectors of cAMP signaling pathway and it exists as an holoenzyme in inactive state with the association of regulatory subunit [59–61]. In case of cAMP analog-treated cells and PC-exposed cells, substrate level phosphorylation on serine and threonine residues were also found to be increased. In most of the eukaryotic cells, PKA exist as an inactive tetrameric holoenzyme consisting of two catalytic and two regulatory subunits denoted as PKA-C and PKA-R respectively. The PKA-R subunit actually binds with cAMP causing a conformational change in the molecule resulting in the dissociation of the R and C subunits of the holoenzyme. This dissociation activates the catalytic C subunit of PKA which phosphorylates specific serine or threonine residues on

A 34 KD protein with similar properties of mammalian PKA-C was purified from *L. donovani* [63]. The effect of different activators and inhibitors on PKA activity was measured using promastigote lysates and fluorescent kemptide and it was found that though cAMP analogue treatment did not have any conspicuous effect on kemptide phosphorylation, treatment with PKA inhibitors like PKI and H89 profoundly decreased kemptide phosphorylation. On the other hand, PDEresistant PKA activators increased kemptide phosphorylation when compared to basal activity. Addition of PDE inhibitors like dipyridamole and rolipram also

**2.4 PKA as the downstream effector of cAMP in** *Leishmania*

substrate proteins in the cytoplasm and nucleus [62].

*DOI: http://dx.doi.org/10.5772/intechopen.86360*

*Role of cAMP Homeostasis in Intra-Macrophage Survival and Infectivity of Unicellular Parasites… DOI: http://dx.doi.org/10.5772/intechopen.86360*

3 hours of exposure to differentiation condition leads to the inhibition of LdPKA (short-term regulation). LdPKA-mediated phosphorylation of LdPDED is observed when parasites are exposed to differentiation condition for more than 6 hours. Hydrolytic property of LdPDED is enhanced due to this phosphorylation event and this enhancement in hydrolytic activity might play a pivotal role in the maintenance of cAMP homeostasis (long term regulation) when the total cytosolic PDE activity falls because of PDEA depletion during stress condition [49]. This role of PDED in maintaining the PKA activity which in turn regulates cAMP homeostasis in the parasite during initial exposure to stress condition, might be important in the life cycle of the parasite particularly in the infection establishment within the host.

#### **2.4 PKA as the downstream effector of cAMP in** *Leishmania*

Though the existence and functioning of cAMP-dependent protein kinase (PKA) is well pronounced in eukaryotes, very little is known about the functioning of PKA in cAMP signaling of this particular parasite. PKA acts as the immediate downstream effector of cAMP in the adenylate cyclase pathway, catalyzing the transfer of γ-P from ATP to specific serine/threonine residues on the substrate protein [52]. Studies on *S. cerevisiae* reveal that one of the three PKA catalytic subunits mediates stress-induced differentiation [53]. Researches in *Dictyostelium* have suggested that cAMP is not required for differentiation if sufficient levels of PKA activity are present [54, 55] indicating profound role of PKA in differentiation. Activation of PKA by a short-term cAMP pulse induces bradyzoite differentiation, whereas a prolonged cAMP pulse inhibits differentiation [56]. It is likely that there are distinct PKA signaling pathways in the tachyzoite with opposing effects on parasite differentiation. Inhibition of PKA signals by treatment with PKA catalytic subunit inhibitor H89 induces bradyzoite differentiation [57], suggesting that PKA catalytic subunit activity may be involved in cAMP-mediated tachyzoite maintenance.

When *Leishmania* parasites were exposed to stress condition, PKA activity was significantly enhanced along with increased level of cAMP. Protein kinase activity of five different species of *Leishmania* was found to be quite high in both logarithmic and stationary phase promastigotes, being most active in *L. amazonensis* and least in *L. donovani* [58]. PKA catalytic subunits in the *Toxoplasma* genome were identified. PKA is the most important downstream effectors of cAMP signaling pathway and it exists as an holoenzyme in inactive state with the association of regulatory subunit [59–61]. In case of cAMP analog-treated cells and PC-exposed cells, substrate level phosphorylation on serine and threonine residues were also found to be increased. In most of the eukaryotic cells, PKA exist as an inactive tetrameric holoenzyme consisting of two catalytic and two regulatory subunits denoted as PKA-C and PKA-R respectively. The PKA-R subunit actually binds with cAMP causing a conformational change in the molecule resulting in the dissociation of the R and C subunits of the holoenzyme. This dissociation activates the catalytic C subunit of PKA which phosphorylates specific serine or threonine residues on substrate proteins in the cytoplasm and nucleus [62].

A 34 KD protein with similar properties of mammalian PKA-C was purified from *L. donovani* [63]. The effect of different activators and inhibitors on PKA activity was measured using promastigote lysates and fluorescent kemptide and it was found that though cAMP analogue treatment did not have any conspicuous effect on kemptide phosphorylation, treatment with PKA inhibitors like PKI and H89 profoundly decreased kemptide phosphorylation. On the other hand, PDEresistant PKA activators increased kemptide phosphorylation when compared to basal activity. Addition of PDE inhibitors like dipyridamole and rolipram also

*Vector-Borne Diseases - Recent Developments in Epidemiology and Control*

some condition as compared to normal cells [49].

*2.3.2 Role of PDED in cAMP homeostasis*

arginine by arginase, ornithine decarboxylase and other enzymes, which converts it into spermidine and is then conjugated with GSH. No significant change in arginine and ornithine transporter was detected in PDE inhibitor treated cells and also in PDEA knocked down cells. On the contrary, when the expression of arginase and ornithine decarboxylase, the enzymes responsible for TSH biosynthesis was checked in control and PDEA inhibitor-treated cells, an increase in the expression of these enzymes was observed indicating that PDEA inhibition might have a role in TSH biosynthesis. When total thiol or intracellular TSH content was analyzed, not much alteration was observed. TSH pool is generally utilized by the parasite either for DNA replication by ribonucleotide reductase or for peroxide degradation by peroxidoxin, ascorbate peroxidase and superoxide dismutase. The expressions of enzymes responsible for peroxide degradation like peroxidoxin, superoxide dismutase and ascorbate peroxidase were elevated in PDEA-inhibited cells (**Figure 4**). Cells overexpressing PDEA also showed reduced resistance to pro-oxidants when exposed to phagolyso-

Apart from the membrane bound phosphodiesterases, a soluble, cytosolic phosphodiesterase (PDED) was cloned and characterized from *L. donovani*. Bioinformatic studies showed the presence of two pseudo-substrate sites and a putative PKA phosphorylation site at the C-terminus of PDED and PKA-mediated phosphorylation is important for the regulation of phosphodiesterase activity (**Figure 5**) [50]. It was observed that catalytic subunits of PKA (PKAC1 and PKAC2) interacts with the pseudo substrate sites of PDED after 3 hours of PC exposure. Moreover, inhibition of phosphodiesterase activity through PKA-mediated phosphorylation was observed at a further later time point of PC exposure [51]. The cytosolic localization of LdPDED was established by immunolocalization analysis using anti-LdPDED antibody which revealed its localization to be predominantly cytosolic. Interaction of LdPDED with the catalytic subunits of LdPKA within

**144**

**Figure 5.**

*LdPDED interacting with PKAC1 and PKAC2 resulting in the inhibition of kinase activity of PKA. PKA on the other hand phosphorylates threonine residue of PDED increasing its phosphodiesterase activity.*

increased kemptide phosphorylation [64]. These results suggest that cAMP has some direct role in the activation of PKA during transformation in *Leishmania*. Treatment of promastigotes with PKA activators also resulted in growth arrest in the parasite [64]. Parasite survival in the peritoneal macrophages of Balb/c mice was examined using PKA-inhibitor treated parasites and there was a significant reduction in macrophage infection [64].

In spite of the discovery of the role played by adenylate cyclases and phosphodiesterases in cAMP homeostasis of *Leishmania*, existence of no specific cAMPbinding effector molecule was known. Bhattacharya et al. [65], in their studies, have identified a regulatory subunit of cAMP-dependent protein kinase (Ldpkar1) in *L. donovani* which was found to be homologous to class I cAMP-dependent protein kinase regulatory subunit of mammals. Studies proved beyond doubt that this regulatory subunit interact with both the catalytic subunits of PKA, thus inhibiting PKA activity. When co-immunoprecipitation assay was performed for both normal and Sp-8-Br-cAMP-pretreated cells, much weaker signal was detected for treated cells as compared to normal cells suggesting Sp-8-Br-cAMP-mediated activation of PKA. Moreover, when activity was analyzed in LdPKAR1-LdPKAC1 and LdPKAR1-LdPKAC2 immunoprecipitated complexes in the presence or absence

#### **Figure 6.**

*cAMP-dependent PKA activity in* Leishmania*: cAMP level increases on phagolysosome condition exposure and cAMP binds with the regulatory subunit of PKA enabling its dissociation from the catalytic subunit rendering the catalytic subunit active. cAMP associated regulatory subunit promotes metacyclogenesis and induces autophagy whereas activated catalytic subunit phosphorylates other proteins downstream to this signaling cascade.*

**147**

*Role of cAMP Homeostasis in Intra-Macrophage Survival and Infectivity of Unicellular Parasites…*

of excess cAMP, kemptide phosphorylation was increased significantly in the presence of cAMP indicating the pivotal role of cAMP in the dissociation of regulatory subunit from the catalytic subunit rendering the latter active. Moreover, the studies of Bhattacharya et al. [65] also establishes the functional importance of PKAR other than working as a cAMP effector molecule. LdPKAR1 expression was also found to be increased in late stationary phase promastigotes kept in nutrient deprived/ starvation condition and metacyclogenesis, which is a pre-requisite for successful macrophage infection, was significantly induced in starved cells as compared to normal cells. Cells overexpressing LdPKAR1 also showed increased metacyclogenesis, enhanced intra-macrophage survival suggesting that LdPKAR1 overexpressed cells had greater infectivity. It can be inferred that LdPKAR1 overexpression leads to

acceleration in the process of metacyclogenesis in *L. donovani* (**Figure 6**).

PKA activity assay in the presence and absence of cAMP and cGMP analogs and PKA inhibitors in both soluble fraction (SF) and membrane fraction (MF) of infective promastigotes of *L. amazonensis* showed increase in phosphorylative activity of the kinase in cAMP-analog-treated cells, and not in cGMP-analog-treated cells, was conspicuous, particularly in the SF of the promastigotes. On the contrary, PKA activity of both SF and MF of axenic amastigotes was found to be much lower as compared to that of both SF and MF of infective promastigotes under same experi-

Autophagy is one of the survival strategies of *Leishmania* in mammalian macrophages. Since LdPKAR1 has a direct role in the process of metacyclogenesis, its relation to autophagy was studied in the parasite. ATG8 is a marker for autophagosome formation and ATG8 tracking was done in both starved cells and in normal cells by western blot technique using polyclonal anti-LmATG8 antibody. Cells under starvation condition showed much higher level of ATG8-PE, a cleaved form of ATG8, indicating the formation of autophagosome in starved condition. When a conditional knock-down system of LdPKAR1 was constructed in *L. tarentolae*, both mRNA and protein level expression of LdPKAR1 was found to be diminished after tetracycline induction. Uninduced cells showed higher percentage of ATG8-positive structures as compared to tetracycline-induced cells. This suggested the role of PKAR1 in autophagosome formation. LdPKAR not only acts as a cAMP binding molecule in the parasite, but induce metacyclogenesis and autophagy. Studies are further required to confirm whether the process is an autophagy-induced metacy-

To conclude we can say that the leading researches in the recent past has enriched our knowledge on the importance of cAMP signaling in kinetoplastid parasites like *Leishmania* and their association with parasite infectivity. These findings provide insight on the functioning of different enzymes associated with cAMP metabolism (**Figure 7**). These studies point toward the fact that modulation of cAMP level in the parasite might be one of the mechanisms to control leishmaniasis and the molecules associated with the same might be tested as potent drug targets against the disease. Presently, PDE inhibitors are potent drug targets against various human diseases. Study of human PDEs in cAMP signaling pathway has revealed their druggability in various human pathologies leading to various marketed drugs [67]. Moreover, there is a similarity between human and protozoan enzymes and in addition, the availability of human PDE inhibitors as therapeutics has thrown some light on the discovery of some specific protozoan PDE inhibitors as potential drug targets [68]. In kinetoplastid parasites like *Trypanosoma*, PDE inhibitors are being screened as potential drug targets

*DOI: http://dx.doi.org/10.5772/intechopen.86360*

mental conditions [66].

**3. Conclusion**

clogenesis or a metacyclogenesis-induced autophagy.

#### *Role of cAMP Homeostasis in Intra-Macrophage Survival and Infectivity of Unicellular Parasites… DOI: http://dx.doi.org/10.5772/intechopen.86360*

of excess cAMP, kemptide phosphorylation was increased significantly in the presence of cAMP indicating the pivotal role of cAMP in the dissociation of regulatory subunit from the catalytic subunit rendering the latter active. Moreover, the studies of Bhattacharya et al. [65] also establishes the functional importance of PKAR other than working as a cAMP effector molecule. LdPKAR1 expression was also found to be increased in late stationary phase promastigotes kept in nutrient deprived/ starvation condition and metacyclogenesis, which is a pre-requisite for successful macrophage infection, was significantly induced in starved cells as compared to normal cells. Cells overexpressing LdPKAR1 also showed increased metacyclogenesis, enhanced intra-macrophage survival suggesting that LdPKAR1 overexpressed cells had greater infectivity. It can be inferred that LdPKAR1 overexpression leads to acceleration in the process of metacyclogenesis in *L. donovani* (**Figure 6**).

PKA activity assay in the presence and absence of cAMP and cGMP analogs and PKA inhibitors in both soluble fraction (SF) and membrane fraction (MF) of infective promastigotes of *L. amazonensis* showed increase in phosphorylative activity of the kinase in cAMP-analog-treated cells, and not in cGMP-analog-treated cells, was conspicuous, particularly in the SF of the promastigotes. On the contrary, PKA activity of both SF and MF of axenic amastigotes was found to be much lower as compared to that of both SF and MF of infective promastigotes under same experimental conditions [66].

Autophagy is one of the survival strategies of *Leishmania* in mammalian macrophages. Since LdPKAR1 has a direct role in the process of metacyclogenesis, its relation to autophagy was studied in the parasite. ATG8 is a marker for autophagosome formation and ATG8 tracking was done in both starved cells and in normal cells by western blot technique using polyclonal anti-LmATG8 antibody. Cells under starvation condition showed much higher level of ATG8-PE, a cleaved form of ATG8, indicating the formation of autophagosome in starved condition. When a conditional knock-down system of LdPKAR1 was constructed in *L. tarentolae*, both mRNA and protein level expression of LdPKAR1 was found to be diminished after tetracycline induction. Uninduced cells showed higher percentage of ATG8-positive structures as compared to tetracycline-induced cells. This suggested the role of PKAR1 in autophagosome formation. LdPKAR not only acts as a cAMP binding molecule in the parasite, but induce metacyclogenesis and autophagy. Studies are further required to confirm whether the process is an autophagy-induced metacyclogenesis or a metacyclogenesis-induced autophagy.
