**2. cAMP and associated enzymes in** *Leishmania*

In eukaryotes, cAMP a second messenger, is an essential molecule playing a vital role in intracellular signaling which control a vast array of cellular events like cytoskeletal modeling, proliferation, virulence, differentiation and apoptosis [33]. cAMP is formed from adenosine triphosphate (ATP) by receptor adenylate cyclases (RAC). In *Leishmania*, there are reports of several isoforms of both membrane bound receptor adenylate cyclases [34] as well as soluble adenylate cyclases. When cAMP is produced, inorganic phosphate (Pi) is also produced as one of by-product of the reaction. Regulation of the pyrophosphate (PPi) pool formed by the accumulation of Pi, is hydrolysed by pyrophosphatase. In *Leishmania*, there are three isoforms of pyrophosphatases: Inorganic pyrophosphatase (IoPPase), vacuolar proton transporting pyrophosphatase (V-H+ PPase) and acidocalcisomal soluble

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

which act as transcription factors, or metabolic enzymes such as lipases, phosphorylase kinase or glycogen synthase. In unicellular eukaryotes, there are many reports which implicates cAMP as one of the major environmental sensing machineries associated with stress response in *Plasmodium*, *Trypanosoma*, *Toxoplasma* and others. In malarial parasite, *Plasmodium falciparum*, cAMP is one of the main molecules responsible for the formation of sexual precursor, gametocytes from the asexual forms [1]. *P. falciparum* produces its own cAMP requirement by receptor adenylate cyclase (AC) which seemed to be unaffected by the well-known mammalian RAC activator Forskolin or heteromeric G-protein activators fluoroaluminate

<sup>−</sup>). Moreover, cAMP signaling effector molecule protein kinase A (PKA) plays an important role in conductance of anions across the host cell membrane of *Plasmodium*-infected RBC [2]. Moreover, recent researches showed that PKAR (PKA regulatory subunit) is functionally associated with the activation of anion conductance channel in *P. falciparum*-infected RBC [3]. cAMP-dependent signaling pathway activation and PKC activation in *Entamoeba histolytica* triggers the phosphorylation of proteins involved in actin rear-arrangements necessary for its movement and adhesion. Moreover, cAMP-response elements could play an important role in regulating actin expression and organization in signaling processes activated during tissue invasion. However, there are several other reports of mechanisms of cAMP action, such as the direct regulation of ion channels in olfactory cells, or the activation of chemotactic receptors in the slime mould, *Dictyostelium*. In unicellular eukaryotes like *Toxoplasma gondii*, both cyclic GMP (cGMP) and cyclic AMP (cAMP) can induce bradyzoite formation. These effects could be due to an increase in host or parasite cyclic nucleotides. Host cell environments including cAMP elevations contribute to the bradyzoite differentiation process in *T. gondii*, which has a receptor or sensor for cyclic nucleotides [4]. In *Dictyostelium*, cAMP secreted into the environment binds to cAMP receptors to regulate the differentiation program of cells within the fruiting body [5]. In *Leishmania*, the mechanism of action of cAMP signaling represent a particularly intriguing question since the major pathway of cAMP signaling in eukaryotes, the regulation of transcription, does not seem to be applicable because kinetoplastid parasites like *Trypanosoma* and *Leishmania* exhibit obscure transcriptional regulation. An attempt to understand cAMP signaling in *Leishmania* was undertaken by Seebeck and his group and initial studies in *L. major* where they identified five PDE genes, PDEA, PDEB1, PDEB2, PDEC and PDED encoding class I enzymes similar to those found in higher eukaryotes [6].

The protozoan parasite *Leishmania donovani*, when exposed to stress condition in the mammalian macrophages, encounter an oxidative burst as the first line of defense, offered by the macrophages by producing reactive oxygen species and reactive nitrogen intermediates [7, 8]. Still, a subset of the parasites can survive and transforms into amastigotes leading to disease manifestation [9, 10]. In *Leishmania*, cAMP is one of the major players driving the transformation of the parasite from promastigotes to amastigotes and allowing survival of parasites in macrophages [11]. Not only in the differentiation of *Leishmania*, cAMP also an important role in the differentiation of *Trypanosoma* from slender form to short stumpy form [11]. In kinetoplastid parasite *Trypanosoma*, cAMP levels are modulated all through the different stages of the cell cycle plays a significant function in transformation from slender forms to stumpy forms [12]. Also a stumpy induction factor (SIF) has been reported in *Trypanosoma* which triggers cell cycle arrest in G1/G0 phase and induces differentiation with high efficiency and elicits an immediate two- to three-fold elevation of intracellular cAMP content upon addition to slender forms [13]. Membrane-permeable derivatives of cAMP or the phosphodiesterase inhibitor etazolate perfectly mimic SIF activity in *Trypanosoma*. Moreover, it was also shown that the transformation in *Trypanosoma* was not mediated directly by cAMP

**136**

(A1F4

pyrophosphatase (VSP1). Downstream to cAMP, leishmanial phosphodiesterases (PDE) hydrolyzes cAMP to 5′ adenosine monophosphate (5′AMP). There are five different PDEs in the parasite (PDEA, PDEB1, PDEB2, PDEC, and PDED). cAMPdependent protein kinase A (PKA) exists as an inactive tetramer consisting of two catalytic subunits (PKAC) and two regulatory subunits (PKAR). Binding of cAMP to PKAR releases PKAC subunit.

### **2.1 Receptor adenylate cyclase in** *Leishmania*

cAMP signaling cascade is activated only when local cAMP concentration reaches a level high enough to activate a cAMP responsive respective effector protein/s. It has been observed that mostly, the activation threshold lies around 1 ± 10 mm. The increase of cAMP from a basal level can be brought about either by the activation of one or several RACs, or by the inactivation of the PDEs. In eukaryotic cells, cAMP is predominantly generated at the plasma membrane since most of the known RACs are integral membrane proteins. From the site of its generation, the cAMP diffuses until it hits the respective effector molecule, or until it is hydrolysed by PDEs (**Figure 1**). The cAMP signal can take the form of a diffusioncontrolled concentration gradient [35], it can be delivered in the form of time- and space-controlled spikes of cAMP concentration or consists of a sustained increase or decrease in intracellular cAMP concentration. Adenylate cyclase-cAMP pathway is also involved in the internalization process of the parasite by the host cells [36].

Studies have confirmed that cAMP is involved in signal transduction events occurring during transformations in *Leishmania* and other related kinetoplastid protozoa. Different life cycle stages contain different intracellular concentrations of cAMP in *Trypanosome brucei* [37] and in *T. cruzi* [23]. Furthermore, cAMP analogs and phosphodiesterase inhibitors promote *in vitro* differentiation of non-infectious epimastigotes of *T. cruzi* into infectious metacyclic trypomastigotes [38]. This major

#### **Figure 1.**

*Receptor adenylate cyclase in* Leishmania*: PPi inhibiting adenylate cyclase. In normal condition, when the parasites are not exposed to stress, receptor adenylate cyclase synthesizes cAMP from ATP and PPi is produced as by-product. This PPi interacts with the receptors and inhibits further synthesis of cAMP. On the other hand, PDEs present in the cell also helps in maintaining the concentration of cAMP by hydrolyzing cAMP to 5′-AMP. So, both PPi produced and the PDEs help in the regulation of cAMP level in the parasite.*

**139**

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

In *T. brucei*, genes showing homology with yeast adenylate cyclase were identified and they were termed expression-site associated genes (ESAGs). Many more copies of these putative adenylate cyclases were identified and were named GRESAG4.1 and GRESAG4.2 [41]. Related adenylate cyclase genes which have actually been proved to code for functional adenylate cyclase enzymes were also identified in *T. congoense*, *T. mega*, *T. brucei gambiense*, *T. vivax* and *T. equiperdum* [42, 43]. Similar families of multigene having high homology with ESAG4 and GRESAG4.1 have been identified in *T. cruzi* and *L. donovani* are said to be sharing the common protein architecture [39]. In kinetoplastids, the cellular localization of adenylate cyclases is consistent where they act as receptors as proved by binding of antibodies against ESAG4,

The existence of receptor adenylate cyclase has also been discovered in *L. donovani* and a membrane bound RAC-A is found to be functional during exposure to phagolysosome condition (PC) which actively catalyze cAMP generation [39]. Expression of receptor adenylate cyclase mRNAs (RAC-A and RAC-B) was also found to be developmentally regulated in *Leishmania* as their expression was only found in promastigotes but not in amastigotes [39]. It has been reported that promastigotes exposed to PC shown elevated level of cAMP after 60 minutes of PC exposure which was decreased when treated with DDA (di-deoxy adenosine), an adenylate cyclase inhibitor [44]. Expression of both LdRAC-A and LdRAC-B were analyzed by immunoblot technique using anti-RAC-A and anti-RAC-B antibodies raised against *Leishmania* and their expressions were revealed in both plasma membrane and flagella. Interestingly, the expression of LdRAC-A increased significantly in PC-exposed cells after 60 minutes of exposure despite the unchanged expression profile of LdRAC-B under such condition. This result suggests that in spite of the presence of two developmentally regulated isoforms of adenylate cyclases in *Leishmania*, LdRAC-A is only functionally active during stress condition. Inducible anti-sense knock-down strategy was adopted to downregulate RAC-A and RAC-B in *L. tarentolae*, which had been successfully implemented for a number of genes earlier. Ldrac-A knocked-down parasites generated in Lt.T7TR strain of *L. tarentolae* showed no change or no elevation in intracellular cAMP level after exposure to PC. Moreover, there was a 10.2% decrease in cAMP level when RAC-A knockeddown PC-exposed parasites were further treated with DDA. Ldrac-B knocked-down parasites behaved as control parasites showing elevated cAMP levels on PC exposure. However, there was a significant decrease of cAMP level when RAC-B knockeddown cells were treated with DDA. The results indicate that LdRAC-A plays a conspicuous role in triggering cAMP response in the parasites during stress condition.

role of cAMP in the transformation of kinetoplastid protozoa has led to the investigation of adenylate cyclases in *L. donovani*. A family of five clustered genes in *L. donovani* was identified which encodes signal transduction receptors [39]. The coding region of these genes was sequenced and have been shown to code for proteins with an NH2-terminal hydrophilic domain, an intervening transmembrane segment and a carboxylic terminal domain having high sequence similarity with the catalytic domain of adenylate cyclases from other eukaryotes [39]. These genes are designated as rac-A and rac-B. One of these genes is expressed in *Xenopus* oocytes and have been shown to function as an adenylate cyclase. Another interesting observation was that of rac-A and rac-B mRNAs expression which was only found in promastigotes by Northern blot technique but was not detectable in amastigotes, which proves that these are developmentally regulated mRNAs [39]. So, these proteins might be involved in developmental transitions where they can function in the switch between non-infectious procyclic and infectious metacyclic promastigotes [40], or they can also interact with ligands present in the host macrophages and initiate a signal cascade

leading to the differentiation of promastigotes to amastigotes.

specifically to the cell surface along the flagella in trypanosomes [43].

*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*

role of cAMP in the transformation of kinetoplastid protozoa has led to the investigation of adenylate cyclases in *L. donovani*. A family of five clustered genes in *L. donovani* was identified which encodes signal transduction receptors [39]. The coding region of these genes was sequenced and have been shown to code for proteins with an NH2-terminal hydrophilic domain, an intervening transmembrane segment and a carboxylic terminal domain having high sequence similarity with the catalytic domain of adenylate cyclases from other eukaryotes [39]. These genes are designated as rac-A and rac-B. One of these genes is expressed in *Xenopus* oocytes and have been shown to function as an adenylate cyclase. Another interesting observation was that of rac-A and rac-B mRNAs expression which was only found in promastigotes by Northern blot technique but was not detectable in amastigotes, which proves that these are developmentally regulated mRNAs [39]. So, these proteins might be involved in developmental transitions where they can function in the switch between non-infectious procyclic and infectious metacyclic promastigotes [40], or they can also interact with ligands present in the host macrophages and initiate a signal cascade leading to the differentiation of promastigotes to amastigotes.

In *T. brucei*, genes showing homology with yeast adenylate cyclase were identified and they were termed expression-site associated genes (ESAGs). Many more copies of these putative adenylate cyclases were identified and were named GRESAG4.1 and GRESAG4.2 [41]. Related adenylate cyclase genes which have actually been proved to code for functional adenylate cyclase enzymes were also identified in *T. congoense*, *T. mega*, *T. brucei gambiense*, *T. vivax* and *T. equiperdum* [42, 43]. Similar families of multigene having high homology with ESAG4 and GRESAG4.1 have been identified in *T. cruzi* and *L. donovani* are said to be sharing the common protein architecture [39]. In kinetoplastids, the cellular localization of adenylate cyclases is consistent where they act as receptors as proved by binding of antibodies against ESAG4, specifically to the cell surface along the flagella in trypanosomes [43].

The existence of receptor adenylate cyclase has also been discovered in *L. donovani* and a membrane bound RAC-A is found to be functional during exposure to phagolysosome condition (PC) which actively catalyze cAMP generation [39]. Expression of receptor adenylate cyclase mRNAs (RAC-A and RAC-B) was also found to be developmentally regulated in *Leishmania* as their expression was only found in promastigotes but not in amastigotes [39]. It has been reported that promastigotes exposed to PC shown elevated level of cAMP after 60 minutes of PC exposure which was decreased when treated with DDA (di-deoxy adenosine), an adenylate cyclase inhibitor [44]. Expression of both LdRAC-A and LdRAC-B were analyzed by immunoblot technique using anti-RAC-A and anti-RAC-B antibodies raised against *Leishmania* and their expressions were revealed in both plasma membrane and flagella. Interestingly, the expression of LdRAC-A increased significantly in PC-exposed cells after 60 minutes of exposure despite the unchanged expression profile of LdRAC-B under such condition. This result suggests that in spite of the presence of two developmentally regulated isoforms of adenylate cyclases in *Leishmania*, LdRAC-A is only functionally active during stress condition. Inducible anti-sense knock-down strategy was adopted to downregulate RAC-A and RAC-B in *L. tarentolae*, which had been successfully implemented for a number of genes earlier. Ldrac-A knocked-down parasites generated in Lt.T7TR strain of *L. tarentolae* showed no change or no elevation in intracellular cAMP level after exposure to PC. Moreover, there was a 10.2% decrease in cAMP level when RAC-A knockeddown PC-exposed parasites were further treated with DDA. Ldrac-B knocked-down parasites behaved as control parasites showing elevated cAMP levels on PC exposure. However, there was a significant decrease of cAMP level when RAC-B knockeddown cells were treated with DDA. The results indicate that LdRAC-A plays a conspicuous role in triggering cAMP response in the parasites during stress condition.

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

to PKAR releases PKAC subunit.

**2.1 Receptor adenylate cyclase in** *Leishmania*

pyrophosphatase (VSP1). Downstream to cAMP, leishmanial phosphodiesterases (PDE) hydrolyzes cAMP to 5′ adenosine monophosphate (5′AMP). There are five different PDEs in the parasite (PDEA, PDEB1, PDEB2, PDEC, and PDED). cAMPdependent protein kinase A (PKA) exists as an inactive tetramer consisting of two catalytic subunits (PKAC) and two regulatory subunits (PKAR). Binding of cAMP

cAMP signaling cascade is activated only when local cAMP concentration reaches a level high enough to activate a cAMP responsive respective effector protein/s. It has been observed that mostly, the activation threshold lies around 1 ± 10 mm. The increase of cAMP from a basal level can be brought about either by the activation of one or several RACs, or by the inactivation of the PDEs. In eukaryotic cells, cAMP is predominantly generated at the plasma membrane since most of the known RACs are integral membrane proteins. From the site of its generation, the cAMP diffuses until it hits the respective effector molecule, or until it is hydrolysed by PDEs (**Figure 1**). The cAMP signal can take the form of a diffusioncontrolled concentration gradient [35], it can be delivered in the form of time- and space-controlled spikes of cAMP concentration or consists of a sustained increase or decrease in intracellular cAMP concentration. Adenylate cyclase-cAMP pathway is also involved in the internalization process of the parasite by the host cells [36]. Studies have confirmed that cAMP is involved in signal transduction events occurring during transformations in *Leishmania* and other related kinetoplastid protozoa. Different life cycle stages contain different intracellular concentrations of cAMP in *Trypanosome brucei* [37] and in *T. cruzi* [23]. Furthermore, cAMP analogs and phosphodiesterase inhibitors promote *in vitro* differentiation of non-infectious epimastigotes of *T. cruzi* into infectious metacyclic trypomastigotes [38]. This major

*Receptor adenylate cyclase in* Leishmania*: PPi inhibiting adenylate cyclase. In normal condition, when the parasites are not exposed to stress, receptor adenylate cyclase synthesizes cAMP from ATP and PPi is produced as by-product. This PPi interacts with the receptors and inhibits further synthesis of cAMP. On the other hand, PDEs present in the cell also helps in maintaining the concentration of cAMP by hydrolyzing cAMP to 5′-AMP. So, both PPi produced and the PDEs help in the regulation of cAMP level in the parasite.*

**138**

**Figure 1.**

### **2.2 Enzymes regulating receptor adenylate cyclase function in** *Leishmania***: pyrophosphatase**

Pyrophosphates (PPi) are produced as by-product during the conversion of ATP to cAMP by receptor adenylate cyclase, the product accumulation of which inhibits adenylate cyclase reaction toward the formation of cAMP. PPi is found to be stored in a specialized compartment like acidocalcisomes in kinetoplastid parasites [45]. The concentration of PPi is equivalent to that of ATP in the cell in spite of its huge confinement in the acidocalcisomes of *Leishmania*. There were speculations that this high concentration of PPi (in millimolar range) might be responsible for the inhibition of cAMP production in the parasites by modulation adenylate cyclase reaction in subcellular micro domains [46]. There are at least three different pyrophosphatases present in *L. major* as revealed by genome sequence analysis. These are: membrane associated H<sup>+</sup> -translocating pyrophosphatase (V-H+ PPase), soluble acidocalcisomal pyrophosphatase (VSP1) and an inorganic pyrophosphatase (IoPPase) and are responsible for maintaining the cAMP levels in the parasite.

Further studies have been conducted to elaborately decipher the role that RAC plays along with various molecules associated with it. PPi formed as by-product of cAMP biosynthesis inhibits adenylate cyclase function and this inhibition is reversed when PPi is hydrolysed by acidocalcisomal LdV-H+ PPase which is translocated to plasma membrane on exposure to phagolysosome condition (**Figure 2**).

Apart from the direct role of LdRAC-A in the production of cAMP during stress condition, intracellular PPi and pyrophosphatases also play a major role in regulation of cAMP concentration in the cell. *L. donovani* promastigotes were treated with foscarnet, a pyrophosphate analogue that acts as an adenylate cyclase inhibitor [47] under PC-exposed condition. PC induced cAMP generation was inhibited by foscarnet treatment after 60 minutes of PC exposure [44]. Furthermore, in PC exposed cells, total pyrophosphate pool was markedly reduced. Presence of three pyrophosphatases have been detected in *L. donovani*, namely, soluble acidocalcisomal pyrophosphatases (LdVSP1), vacuolar proton transporting pyrophosphatase (LdV-H<sup>+</sup> PPase) and inorganic pyrophosphatase (LdIoPPase) which collectively maintain the intracellular pyrophosphate pool. Co-localization analysis with cells expressing GFP-fusion proteins of the three pyrophosphatases and acidocalcisometargeted dye DND-lysotracker, showed little localization of LdIoPPase which was localized in cytoplasm but significant co-localization was observed for LdVSP1 and LdV-H+ PPase they were predominantly localized in the acidocalcisomes.

As revealed by immune-electron microscopic analysis, the acidocalcisomes localize in the vicinity of the cell membrane on PC exposure. PC exposure resulted in gradual decrease in intraluminal pH because of enhanced proton import by LdV-H+ PPase indicating translocation of acidocalcisome that actively imports proton, in the cell periphery following PC exposure (**Figure 2**). The translocation of acidocalcisome to membrane vicinity was further explored to find the mechanism behind such stress driven translocation. Studies clearly indicated that the movement of acidocalcisomes during stress is a microtubule and microfilament-dependent process. Pre-treatment with F-actin inhibitor, cytochalasin D, and stress exposure showed absence of acidocalcisomal translocation toward membrane. Nocodazole pre-treatment, an inhibitor of microtubule, and subsequent stress exposure also resulted in inhibition of acidocalcisomal translocation [44].

Moreover, presence of putative actin/tubulin binding proteins in *Leishmania* might provide significant clues and insight on interlinking of cytoskeletal rearrangement. One such protein has been cloned from *L. donovani* (cyclase-associated protein, LdCAP1) (Bhattacharya et al. personal communication). Unravelling the function of the same might throw light on cytoskeletal protein rearrangement

**141**

acidocalcisome (LdV-H+

*hydrolysed by acidocalcisomal V-H<sup>+</sup>*

*also elevates cAMP level.*

**Figure 2.**

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

and acidocalcisomal translocation. With the translocation of acidocalcisomes in membrane vicinity, a possible co-proximal localization of membrane bound

*Role of receptor adenylate cyclase when parasites are exposed to phagolysosomal conditions. Generally PPi inhibits adenylate cyclase function in normal conditions, but when cells are exposed to stress, the PPi are*

*adenylate cyclase to synthesize more cAMP. In addition to this, PDE level decreases in stress-induced cells which*

No such co-localization was detected with LdRAC-B. Episomal over-expression

be established by the study of Biswas et al. [44], the use of PPi analogue foscarnet and the decrease in the PPi level during PC exposure indicate toward the regulation of PPi pool by this pyrophosphatase isoform. LdRAC-A, PPi pool and LdV-H<sup>+</sup>

Apart from pyrophosphatases that regulate the formation of intracellular cAMP by receptor adenylate cyclases, it is also important to study another dimension of cAMP regulation. Phosphodiesterases (PDEs), ubiquitous enzymes responsible for the termination of cyclic nucleotide signaling pathway by hydrolyzing cAMP to 5′-AMP or cGMP to 5′-GMP, the sole means by which the cell gets rid from the cAMP produced for controlling different cellular processes [48]. PDEs can be divided into three categories based on their catalytic properties namely, class I, class II and class III and 21 genes have been found in mammals for PDE and several in *Drosophila* and *Dictyostellium*. Though various isoforms of class I PDE have been identified in *T. brucei* and *T. cruzi*, only two PDEs have been cloned from *L. major* [48]. In *L. major* five different isoforms of PDE have been identified. Isoforms PDEB1 and PDEB2 are highly specific for cAMP and only poorly inhibited by

and conditional silencing demonstrated regulatory role of V-H+

production. Though the direct decrease in the level of PPi by V-H<sup>+</sup>

control intracellular cAMP level in the parasite during PC exposure.

**2.3 Phosphodiesterases and intracellular cAMP signaling in** *Leishmania*

PPase) and LdRAC-A was studied during PC exposure.

*PPase and translocated toward membrane vicinity allowing receptor*

PPase on cAMP

PPase could not

PPase

*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*

#### **Figure 2.**

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

reversed when PPi is hydrolysed by acidocalcisomal LdV-H+

resulted in inhibition of acidocalcisomal translocation [44].

**pyrophosphatase**

These are: membrane associated H<sup>+</sup>

**2.2 Enzymes regulating receptor adenylate cyclase function in** *Leishmania***:**

Pyrophosphates (PPi) are produced as by-product during the conversion of ATP to cAMP by receptor adenylate cyclase, the product accumulation of which inhibits adenylate cyclase reaction toward the formation of cAMP. PPi is found to be stored in a specialized compartment like acidocalcisomes in kinetoplastid parasites [45]. The concentration of PPi is equivalent to that of ATP in the cell in spite of its huge confinement in the acidocalcisomes of *Leishmania*. There were speculations that this high concentration of PPi (in millimolar range) might be responsible for the inhibition of cAMP production in the parasites by modulation adenylate cyclase reaction in subcellular micro domains [46]. There are at least three different pyrophosphatases present in *L. major* as revealed by genome sequence analysis.

soluble acidocalcisomal pyrophosphatase (VSP1) and an inorganic pyrophosphatase (IoPPase) and are responsible for maintaining the cAMP levels in the parasite. Further studies have been conducted to elaborately decipher the role that RAC plays along with various molecules associated with it. PPi formed as by-product of cAMP biosynthesis inhibits adenylate cyclase function and this inhibition is

cated to plasma membrane on exposure to phagolysosome condition (**Figure 2**). Apart from the direct role of LdRAC-A in the production of cAMP during stress condition, intracellular PPi and pyrophosphatases also play a major role in regulation of cAMP concentration in the cell. *L. donovani* promastigotes were treated with foscarnet, a pyrophosphate analogue that acts as an adenylate cyclase inhibitor [47] under PC-exposed condition. PC induced cAMP generation was inhibited by foscarnet treatment after 60 minutes of PC exposure [44]. Furthermore, in PC exposed cells, total pyrophosphate pool was markedly reduced. Presence of three pyrophosphatases have been detected in *L. donovani*, namely, soluble acidocalcisomal pyrophosphatases (LdVSP1), vacuolar proton transporting pyrophosphatase

PPase) and inorganic pyrophosphatase (LdIoPPase) which collectively maintain the intracellular pyrophosphate pool. Co-localization analysis with cells expressing GFP-fusion proteins of the three pyrophosphatases and acidocalcisometargeted dye DND-lysotracker, showed little localization of LdIoPPase which was localized in cytoplasm but significant co-localization was observed for LdVSP1 and

PPase they were predominantly localized in the acidocalcisomes. As revealed by immune-electron microscopic analysis, the acidocalcisomes localize in the vicinity of the cell membrane on PC exposure. PC exposure resulted in gradual decrease in intraluminal pH because of enhanced proton import by LdV-

PPase indicating translocation of acidocalcisome that actively imports proton, in the cell periphery following PC exposure (**Figure 2**). The translocation of acidocalcisome to membrane vicinity was further explored to find the mechanism behind such stress driven translocation. Studies clearly indicated that the movement of acidocalcisomes during stress is a microtubule and microfilament-dependent process. Pre-treatment with F-actin inhibitor, cytochalasin D, and stress exposure showed absence of acidocalcisomal translocation toward membrane. Nocodazole pre-treatment, an inhibitor of microtubule, and subsequent stress exposure also

Moreover, presence of putative actin/tubulin binding proteins in *Leishmania* might provide significant clues and insight on interlinking of cytoskeletal rearrangement. One such protein has been cloned from *L. donovani* (cyclase-associated protein, LdCAP1) (Bhattacharya et al. personal communication). Unravelling the function of the same might throw light on cytoskeletal protein rearrangement

PPase),

PPase which is translo-

**140**

(LdV-H<sup>+</sup>

LdV-H+

H+

*Role of receptor adenylate cyclase when parasites are exposed to phagolysosomal conditions. Generally PPi inhibits adenylate cyclase function in normal conditions, but when cells are exposed to stress, the PPi are hydrolysed by acidocalcisomal V-H<sup>+</sup> PPase and translocated toward membrane vicinity allowing receptor adenylate cyclase to synthesize more cAMP. In addition to this, PDE level decreases in stress-induced cells which also elevates cAMP level.*

and acidocalcisomal translocation. With the translocation of acidocalcisomes in membrane vicinity, a possible co-proximal localization of membrane bound acidocalcisome (LdV-H+ PPase) and LdRAC-A was studied during PC exposure. No such co-localization was detected with LdRAC-B. Episomal over-expression and conditional silencing demonstrated regulatory role of V-H+ PPase on cAMP production. Though the direct decrease in the level of PPi by V-H<sup>+</sup> PPase could not be established by the study of Biswas et al. [44], the use of PPi analogue foscarnet and the decrease in the PPi level during PC exposure indicate toward the regulation of PPi pool by this pyrophosphatase isoform. LdRAC-A, PPi pool and LdV-H<sup>+</sup> PPase control intracellular cAMP level in the parasite during PC exposure.

#### **2.3 Phosphodiesterases and intracellular cAMP signaling in** *Leishmania*

Apart from pyrophosphatases that regulate the formation of intracellular cAMP by receptor adenylate cyclases, it is also important to study another dimension of cAMP regulation. Phosphodiesterases (PDEs), ubiquitous enzymes responsible for the termination of cyclic nucleotide signaling pathway by hydrolyzing cAMP to 5′-AMP or cGMP to 5′-GMP, the sole means by which the cell gets rid from the cAMP produced for controlling different cellular processes [48]. PDEs can be divided into three categories based on their catalytic properties namely, class I, class II and class III and 21 genes have been found in mammals for PDE and several in *Drosophila* and *Dictyostellium*. Though various isoforms of class I PDE have been identified in *T. brucei* and *T. cruzi*, only two PDEs have been cloned from *L. major* [48]. In *L. major* five different isoforms of PDE have been identified. Isoforms PDEB1 and PDEB2 are highly specific for cAMP and only poorly inhibited by

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

#### **Figure 3.**

*PDE isoforms in* Leishmania*. PDE in* Leishmania *can be categorized into membrane-bound and cytosolic PDE. On exposure to phagolysosome condition, there is no change in the expression of membrane-bound PDEs (PDEC and PDEB); but there is a significant decrease in the expression of cytosolic PDEs (PDEA and PDED).*

most inhibitors of human PDEs [48]. Crystal structure of LmjPDEB1 showed that catalytic domain of LmjPDEB1 complexed with a general PDE inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX) show significant differences in binding of this inhibitor when compared to human PDEs.

Identification of different isoforms of phosphodiesterases in *L. major* indicated PDEB and PDEC to be membrane bound and PDEA and PDED to be predominantly cytosolic (**Figure 3**). LdPDEA and LdPDED were also cloned in *L. donovani* [49]. From the observation of the studies of Bhattacharya et al. [49], it has been found that the activity of cytosolic PDEs decrease during stage differentiation but the activity of membrane bound PDEs remained unchanged. From this observation, it can be inferred that PDEs might play an essential role as a controlling factor during stage differentiation of the parasites.

When cAMP-PDE activity was studied, it was found that the activity of cytosolic fraction was diminished gradually as the parasite started to differentiate into axenic amastigote stage from log phase promastigote. Protein level expression of different forms of PDEs in different stages of life cycle of *L. donovani* revealed depletion of PDEA expression in late stationary-phase promastigotes and axenic amastigotes as compared to log phase promastigotes but the expression of other PDEs such as PDEC and PDEB remain unaltered. Gradual decrease in PDEA level and its differential expression in the course of the differentiation of the parasites from promastigotes and amastigotes was observed by several experimental techniques.

**143**

**Figure 4.**

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

In *Leishmania*, anti-oxidant machinery plays a vital role in regulating the sustenance of the parasites in mammalian macrophages where they are exposed to oxidative stress. cAMP level elevation is linked with such phenomenon. In order to find out the functional significance of LdPDEA in such defense mechanism, LdPDEA gene was silenced using tetracycline-inducible knock-down system [49]. When PDE inhibitors were used, the parasites exhibited enhanced viability against peroxides and peroxynitrite. When cells were treated with PDE inhibitors like etazolate and trequinsin, higher resistance against peroxide and peroxynitirite was observed as compared to untreated promastigotes. Since these inhibitors are not specific for PDEA, the result of the treatment might be due to inhibition of some other forms of PDEs in the promastigotes. To ascertain the exact role of PDEA, a knock down construct was prepared to build up a tetracycline-inducible PDEA knock down system. PDEA expression was strongly reduced in both RNA and protein level after tetracycline induction and they also showed enhanced resistance

Peroxide neutralization is one of the major strategies of leishmanial parasite, which makes their survival possible inside the mammalian macrophage and it is done by anti-oxidant machinery of the parasite which lacks catalase. In *Leishmania*, peroxide neutralization is mainly based on trypanothione (TSH), a glutathionespermidine conjugate, as they lack glutathione (GSH). TSH is biosynthesized from

*Role of PDEA to control local cAMP gradient and shifting of TSH pool to peroxide neutralization.*

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

against peroxide and peroxynitrite.

*2.3.1 Effect of PDEA on peroxide resistance and TSH pool*

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

## *2.3.1 Effect of PDEA on peroxide resistance and TSH pool*

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

most inhibitors of human PDEs [48]. Crystal structure of LmjPDEB1 showed that catalytic domain of LmjPDEB1 complexed with a general PDE inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX) show significant differences in binding of this inhibitor

*PDE isoforms in* Leishmania*. PDE in* Leishmania *can be categorized into membrane-bound and cytosolic PDE. On exposure to phagolysosome condition, there is no change in the expression of membrane-bound PDEs (PDEC and PDEB); but there is a significant decrease in the expression of cytosolic PDEs (PDEA and PDED).*

Identification of different isoforms of phosphodiesterases in *L. major* indicated PDEB and PDEC to be membrane bound and PDEA and PDED to be predominantly cytosolic (**Figure 3**). LdPDEA and LdPDED were also cloned in *L. donovani* [49]. From the observation of the studies of Bhattacharya et al. [49], it has been found that the activity of cytosolic PDEs decrease during stage differentiation but the activity of membrane bound PDEs remained unchanged. From this observation, it can be inferred that PDEs might play an essential role as a controlling factor during

When cAMP-PDE activity was studied, it was found that the activity of cytosolic fraction was diminished gradually as the parasite started to differentiate into axenic amastigote stage from log phase promastigote. Protein level expression of different forms of PDEs in different stages of life cycle of *L. donovani* revealed depletion of PDEA expression in late stationary-phase promastigotes and axenic amastigotes as compared to log phase promastigotes but the expression of other PDEs such as PDEC and PDEB remain unaltered. Gradual decrease in PDEA level and its differential expression in the course of the differentiation of the parasites from promastigotes and amastigotes was observed by several

when compared to human PDEs.

**Figure 3.**

stage differentiation of the parasites.

experimental techniques.

**142**

In *Leishmania*, anti-oxidant machinery plays a vital role in regulating the sustenance of the parasites in mammalian macrophages where they are exposed to oxidative stress. cAMP level elevation is linked with such phenomenon. In order to find out the functional significance of LdPDEA in such defense mechanism, LdPDEA gene was silenced using tetracycline-inducible knock-down system [49]. When PDE inhibitors were used, the parasites exhibited enhanced viability against peroxides and peroxynitrite. When cells were treated with PDE inhibitors like etazolate and trequinsin, higher resistance against peroxide and peroxynitirite was observed as compared to untreated promastigotes. Since these inhibitors are not specific for PDEA, the result of the treatment might be due to inhibition of some other forms of PDEs in the promastigotes. To ascertain the exact role of PDEA, a knock down construct was prepared to build up a tetracycline-inducible PDEA knock down system. PDEA expression was strongly reduced in both RNA and protein level after tetracycline induction and they also showed enhanced resistance against peroxide and peroxynitrite.

Peroxide neutralization is one of the major strategies of leishmanial parasite, which makes their survival possible inside the mammalian macrophage and it is done by anti-oxidant machinery of the parasite which lacks catalase. In *Leishmania*, peroxide neutralization is mainly based on trypanothione (TSH), a glutathionespermidine conjugate, as they lack glutathione (GSH). TSH is biosynthesized from

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 phagolysosome condition as compared to normal cells [49].