**Abstract**

Unicellular eukaryotic pathogen *Leishmania donovani*, an intra-macrophage protozoan parasite, on exposure to phagolysosome conditions (PC) of mammalian macrophages, show increased cAMP level and cAMP-dependent protein kinase A (PKA) resulting in resistance to macrophage oxidative burst. In order to have a comprehensive understanding of cAMP signaling and their contribution to infectivity, studies were carried out on all the enzymes associated with cAMP metabolism such as adenylate cyclase, phosphodiesterase, pyrophosphatase and the regulatory and catalytic subunits of PKA. This chapter deals in detail the contribution of these components of cAMP signaling in cAMP homeostasis of the parasite as well as their role on successful host-parasite interaction leading to intracellular parasite survival and establishment of infection. Finally, a discussion is made about how these observations might be exploited for developing drug candidates targeting parasite specific features.

**Keywords:** *Leishmania*, parasite, cAMP, phosphodiesterase, pyrophosphatase, receptor adenylate cyclase, infectivity

### **1. Introduction**

Leishmaniasis, caused by protozoan parasite *Leishmania* is still endemic in many countries and is considered as one of the potent neglected tropical disease. There are three main forms of leishmaniases-visceral (also known as kala-azar and the most serious form of the disease), cutaneous (the most common), and mucocutaneous form of the disease. Though there are surveillance and control measures for leishmaniasis being used by the World Health Organization, the treatment regime of the disease is yet to be enough to eradicate the disease worldwide. There are continuous research on potential new treatments and possible vaccines for leishmaniasis, but adequate treatment is still unavailable.

Unicellular eukaryotic pathogen *Leishmania donovani*, when exposed to phagolysosome conditions (PC) of macrophages (37°C and pH 5.5); a pre-requisite for parasite survival and infectivity, showed to elevate cAMP level and cAMP-mediated protein kinase A (PKA) activation. In eukaryotes, several researches indicate that most of the cAMP mediated effects are due to the activation of the cAMP-regulated protein kinase A, and the subsequent phosphorylation of other substrates of PKA

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 (A1F4 <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

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

or cAMP-analogs but by the products of hydrolysis of the membrane permeable cAMP-analogs [14]. In *Leishmania*, previous studies also showed that cAMP causes G1 arrest in cell cycle which perhaps aids the parasite transformation [15]. Although cAMP seemed to induce cell cycle arrest in *Leishmania*, little is known about the intricate mechanism of the arrest. Though spatiotemporal regulation of cAMP and slight changes of it seemed important in the parasite, scanty data exist regarding the potential toxicity of *Leishmania* cells to pharmacologic elevation of cAMP levels. Moreover, in several mammalian systems, elevation of cAMP level is one of the stimuli that can induce growth arrest or cell death (or both) in many cultured lymphoid cells, including resting B cells, germinal center B cells, T lymphocytes, and thymocytes [16–20]. cAMP also induces cell death in cells derived from lymphoid malignancies, including murine lymphoma cell line S49.1, B-CLL cells, and multiple

To understand the importance of canonical cAMP signaling components, enzymes associated with cAMP metabolism were studied. cAMP is universally generated by adenylate cyclase in a G-protein coupled receptor signaling cascade, which catalyzes the cyclization of ATP to cAMP. In *Leishmania*, the absence of G-proteins made this signaling cascade a unique one. In many instances, adenylate cyclase is regulated by various molecules including bicarbonate, calcium, and hormones. Interestingly, our studies confirmed the importance of inorganic pyrophosphate pool (PPi), an energy storage compound and byproduct of cAMP synthesis, as one of the regulators of receptor adenylate cyclases in *Leishmania*. Also, amongst the stage specific receptor adenylate cyclases, LdRAC-A showed to regulate cAMP levels in the parasite when exposed to phagolysosome conditions. The PPi pool seemed to a stringent control by membrane bound pyrophosphatases of acidocalcisomes (ACms). Downstream, a differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) and another cytosolic cAMP-dependent PDE, LdPDED, seemed responsible for controlling cAMP homeostasis. Also, a functional cAMP-binding effector molecule from *L. donovani* (a regulatory subunit of PKA, LdPKAR) seemed important in parasite infectivity playing a substantial role in autophagy induction, an event important for parasite transformation in phagolysosome conditions. Protein phosphorylation in a cAMP-dependent manner is important in the life cycle of the parasite and in any trypanosomatids, the pattern of protein phosphorylation changes within the life cycle of the parasite [23–32].

This chapter will deal in detail, the components of cAMP signaling in the parasite and unequivocally demonstrate their contribution in cAMP homeostasis; an important event for parasite survival, successful host-parasite interaction, which might be

exploited for developing drug candidates targeting parasite specific features.

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

PPase) and acidocalcisomal soluble

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

proton transporting pyrophosphatase (V-H+

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

myeloma cells [21, 22].

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

or cAMP-analogs but by the products of hydrolysis of the membrane permeable cAMP-analogs [14]. In *Leishmania*, previous studies also showed that cAMP causes G1 arrest in cell cycle which perhaps aids the parasite transformation [15]. Although cAMP seemed to induce cell cycle arrest in *Leishmania*, little is known about the intricate mechanism of the arrest. Though spatiotemporal regulation of cAMP and slight changes of it seemed important in the parasite, scanty data exist regarding the potential toxicity of *Leishmania* cells to pharmacologic elevation of cAMP levels. Moreover, in several mammalian systems, elevation of cAMP level is one of the stimuli that can induce growth arrest or cell death (or both) in many cultured lymphoid cells, including resting B cells, germinal center B cells, T lymphocytes, and thymocytes [16–20]. cAMP also induces cell death in cells derived from lymphoid malignancies, including murine lymphoma cell line S49.1, B-CLL cells, and multiple myeloma cells [21, 22].

To understand the importance of canonical cAMP signaling components, enzymes associated with cAMP metabolism were studied. cAMP is universally generated by adenylate cyclase in a G-protein coupled receptor signaling cascade, which catalyzes the cyclization of ATP to cAMP. In *Leishmania*, the absence of G-proteins made this signaling cascade a unique one. In many instances, adenylate cyclase is regulated by various molecules including bicarbonate, calcium, and hormones. Interestingly, our studies confirmed the importance of inorganic pyrophosphate pool (PPi), an energy storage compound and byproduct of cAMP synthesis, as one of the regulators of receptor adenylate cyclases in *Leishmania*. Also, amongst the stage specific receptor adenylate cyclases, LdRAC-A showed to regulate cAMP levels in the parasite when exposed to phagolysosome conditions. The PPi pool seemed to a stringent control by membrane bound pyrophosphatases of acidocalcisomes (ACms). Downstream, a differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) and another cytosolic cAMP-dependent PDE, LdPDED, seemed responsible for controlling cAMP homeostasis. Also, a functional cAMP-binding effector molecule from *L. donovani* (a regulatory subunit of PKA, LdPKAR) seemed important in parasite infectivity playing a substantial role in autophagy induction, an event important for parasite transformation in phagolysosome conditions. Protein phosphorylation in a cAMP-dependent manner is important in the life cycle of the parasite and in any trypanosomatids, the pattern of protein phosphorylation changes within the life cycle of the parasite [23–32].

This chapter will deal in detail, the components of cAMP signaling in the parasite and unequivocally demonstrate their contribution in cAMP homeostasis; an important event for parasite survival, successful host-parasite interaction, which might be exploited for developing drug candidates targeting parasite specific features.
