Role of leishmanial acidocalcisomal pyrophosphatase in the cAMP homeostasis in phagolysosome conditions required for intra-macrophage survival.

Exposure of Leishmania donovani to macrophage phagolysosome conditions (PC) (37°C and pH 5.5) led to increased intracellular cAMP and cAMP-mediated responses, which help in intra-macrophage survival pre-requisite for infectivity. In the absence of typical orthologs for G-proteins and G-protein coupled receptors, we sought to study the precise mechanisms for positive modulation of cAMP production during exposure to PC. Amongst two promastigote-stage specific membrane bound receptor adenylate cyclases (LdRAC-A and LdRAC-B), LdRAC-A appeared to function as a major cAMP generator following PC exposure. Pyrophosphate (PPi), an energy storage compound as well as a by-product of cAMP biosynthesis by adenylate cyclise, was found to be decreased following PC exposure. This may be due to microtubule and microfilament-driven translocation of acidocalcisomes near plasma membrane vicinity with concomitant increase of acidocalcisome membrane pyrophosphatase (LdV-H+ PPase) and acidocalcisomal soluble pyrophosphatase (LdVSP1). Episomal over-expression and conditional silencing demonstrated regulatory role of V-H+ PPase on cAMP trigger and consequent induction of resistance to macrophage-derived pro-oxidants and parasite killing. Furthermore, immunofluorescence analysis revealed possible co-localization of LdV-H+ PPase and LdRAC-A during PC exposure. Collectively, these results suggest that translocation of acidocalcisome in membrane vicinity functions as a trigger for LdRAC-A-driven cAMP generation through depletion of PPi pool by LdV-H+ PPase.