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
Unicellular eukaryotic pathogen
The protozoan parasite
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
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.
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
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 diffusion-controlled 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
In
The existence of receptor adenylate cyclase has also been discovered in
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
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.
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
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
Identification of different isoforms of phosphodiesterases in
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
2.3.1 Effect of PDEA on peroxide resistance and TSH pool
In
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
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
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
When
A 34 KD protein with similar properties of mammalian PKA-C was purified from
In spite of the discovery of the role played by adenylate cyclases and phosphodiesterases in cAMP homeostasis of
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
Autophagy is one of the survival strategies of
3. Conclusion
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
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
On the other hand, several known PDE inhibitors were tested against
Acknowledgments
We thank DST-INSPIRE Project grant (IFA-12 LSBM-22), PRG-University of Kalyani and NASI Senior Scientist Platinum Jubilee Fellowship for this work.
Acronyms and abbreviations
AC | acidocalcisomes |
cAMP | cyclic adenosine monophosphate |
PDE | phosphodiesterase |
PKA | protein kinase A |
PPi | inorganic pyrophosphate |
RAC | receptor adenylate cyclase |
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