Role of cAMP Signaling in the Survival and Infectivity of the Protozoan Parasite, Leishmania donovani

Abstract

Leishmania donovani, while invading macrophages, encounters striking shift in temperature and pH (from 22°C and pH 7.2 to 37°C and pH 5.5), which act as the key environmental trigger for differentiation, and increases cAMP level and cAMP-mediated responses. For comprehensive understanding of cAMP signaling, we studied the enzymes related to cAMP metabolism. A stage-specific and developmentally regulated isoform of receptor adenylate cyclase (LdRACA) showed to regulate differentiation-coupled induction of cAMP. The soluble acidocalcisomal pyrophosphatase, Ldvsp1, was the major isoform regulating cAMP level in association with LdRACA. A differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) might be related to infection establishment by shifting trypanothione pool utilization bias toward antioxidant defense. We identified and cloned a functional cAMP-binding effector molecule from L. donovani (a regulatory subunit of cAMP-dependent protein kinase, LdPKAR) that may modulate metacyclogenesis through induction of autophagy. This study reveals the significance of cAMP signaling in parasite survival and infectivity.

Figure 1

Enzymes intimately associated with cAMP metabolism in Leishmania. Cyclic adenosine monophosphate (cAMP) is formed from adenosine triphosphate (ATP) by adenylate cyclases where pyrophosphate (PPi) is also produced as one of reaction products which is hydrolyzed by pyrophosphatases to inorganic phosphate (Pi). Downstream to cAMP, leishmanial phosphodiesterases (PDE) hydrolyzes cAMP to 5′ adenosine monophosphate (5′AMP). There are 5 different PDEs in the parasite (PDEA, PDEB1, PDEB2, PDEC, and PDED). cAMP-dependent protein kinase A (PKA) exists as an inactive tetramer consisting of two catalytic subunits (PKAC) and two regulatory subunits (PKAR). Binding of cAMP to regulatory subunit releases catalytic subunit.

Figure 2

Model for comprehensive cAMP signaling in Leishmania parasites. Receptor adenylate cyclase A (LdRACA) and receptor adenylate cyclase B (LdRACB) are G-protein independent membranae bound adenylate cyclases (AC). LdRACA primarily converts ATP to cAMP along with the formation of PPi. This PPi pool provides an inhibition to AC towards the formation of cAMP. During stress, the PPi pool is hydrolyzed by vacuolar acidocalcisomal soluble pyrophosphatase (LdVSP1) which is released by membrane disintegration of acidocalcisomes releasing the inhibition on LdRACA to produce more cAMP. The increased level of cAMP stalls the cell cycle of the parasite at G1 stage and also elevates the expression of antioxidant genes like peroxidoxin, superoxide dismutase and tryparedoxin peroxidase. cAMP also downregulates a stage specific cytosolic PDE, LdPDEA leading to peroxide degradation due to trypanothione (TSH) pool utilization bias towards peroxide degradation instead of DNA synthesis by ribonucleotide reductase which helps in the survival of the parasite in macrophages. Moreover, LdPKAR might have a role in the infective metacyclic stage of the parasite as it induces the formation of autophagosome and process of autophagy initiating metacyclogenesis.