Role of cAMP signalling in the developmental transformation of Leishmania donovani

The main theme of the project is to understand molecular basis of transformation of promastigotes into amastigotes in terms of signalling events mediated by cAMP. The objectives of the project are (i) to study in vitro developmental transformation of Leishmania donovani in presence of activators and inhibitors of adenylyl cyclases, cAMP-phosphodiesterases (cAMP-PDEs), and protein kinase A (PKA), (ii) identification of adenylyl cyclase, cAMP-PDE, and PKA in different developmental stages of the parasite, and (iii) purification and characterization of adenylyl cyclase, cAMP-PDE and PKA from the parasite.

A pre-requisite of this project was to have consistent supply of amastigotes. We, thus, first attempted axenization of the parasite Leishmania donovani (DD8 strain). During axenization we chose four successive developmental stages viz., procyclic promastigote (4 days old promastigotes), metacyclic promastigote (7 days old promastigotes), temperature transformant (metacyclic promastigotes shifted to 350C at pH 7.2), and axenic amastigote (temperature transformants shifted to pH 5.5 at 350C). Initial attempt was made in medium 199 with HI-FBS. During the process, it was learnt that axenic amastigotes couldn’t survive after 3-4 days. Moreover, this medium was fortified with heat-inactivated FBS (HI-FBS) and hence was not suitable for biochemical manipulations. We then shifted to a completely defined medium, dDME, which is basically DMEM fortified with several growth factors and lipid-free BSA in place of HI-FBS. An acidic pH (5.5) was used for amastigote stage, which was close to the pH of phagolysosomal compartment. Amastigotes not only survived in this medium but also could be propagated for few generations at a higher split ratio. Light and electron microscopies, biochemical analyses, metabolic labeling, infectivity, and back transformation were used to characterize these stages.
D-[2-3H]mannose was used to metabolically label parasites and then lipophosphoglycans (LPGs) were isolated from these parasites. Radioactive counting revealed that promastigotes contained huge amounts of LPG and axenic amastigotes were basically devoid of any detectable LPG. The absence of LPG and the ultrastructural details from transmission microscopy confirmed a very high degree of similarity between axenic amastigotes and amastigotes obtained from infected macrophages. Both metacyclic promastigotes and axenic amastigotes were used to infect J774A.1 cells at 10 m.o.i. Both the stages could infect macrophage-like cells to almost similar degree. Sequential reversal of temperature and pH (first temperature shift and then pH shift but not the reverse) resulted into highly active promastigotes.

After achieving well-characterized amastigotes intracellular level of cAMP was measured in all the four stages using competitive EIA. Intrinsic phosphodiesterase activity was killed and intracellular cAMP was stabilized by using 0.1 M HCl. Estimation of intracellular concentration of cAMP revealed approximately eight-fold higher levels of the second messenger in axenic amastigotes than in procyclic promastigotes under normal conditions. In presence of a specific inhibitor of adenylyl cyclase, 22 ,52 -dideoxyadenosine (5 mM), the corresponding levels of cAMP were marginally different and the transformation of promastigotes to amastigotes seemed arrested.

In order to further probe the role of cAMP in the transformation process back transformation was done in presence of phosphodiesterase (PDE) inhibitors. 5 x 108 axenic amastigotes were back transformed with either IBMX or etazolate or rolipram. IBMX is a non-specific PDE inhibitor. At a concentration of 4 mM IBMX could inhibit transformation of amastigotes into promastigotes. Rolipram and etazolate are cAMP-specific PDE inhibitors (type IV cAMP-PDE). 0.5 mM rolipram and 1 mM etazolate were found sufficient to stop back transformation. Microscopic observations were made for accompanying morphological changes.

These data put together suggest that in the transformation of promastigotes to amastigotes cAMP level goes up and stays there. So long as this high cAMP level is maintained, the transformation process can’t be reversed. In other words higher cAMP levels favour transformation of promastigotes to amastigotes and lower levels of cAMP favour transformation of amastigotes to promastigotes. Also that the intracellular cAMP level drastically changes between metacyclic promastigotes and temperature transformant.

During the reporting year, we were also able to identify a kinase activity in the metacyclic promastigotes that was cAMP-dependent. This was made possible by using biotinylated Kemptide as substrate and a highly specific biotin capture membrane, and lysing the cells in presence of sodium orthovanadate and etazolate hydrochloride in addition to protease inhibitor cocktail. Under these conditions the kinase activity almost doubled in presence of cAMP. This is for the first time that PKA activity is being demonstrated in Leishmania parasite. Use of specific inhibitors of PKA would confirm this observed activity. Evaluation of status of PKA activity in various developmental stages is warranted.

During the reporting period we also tried to identify and purify PDE from metacyclic promastigotes. 1010 cells were lysed in presence of protease inhibitors, benzamidine, and NaBr. PDE activity in the cytosolic portion was assayed by measuring unused cAMP in the reaction mixture after a fixed interval of incubation of cAMP with it. After confirming the presence of PDE activity in the cytosol, purification was attempted on Cibacron Blue-F3GA-agarose column. PDE activity was eluted with a linear gradient of NaCl after recycling the cytosolic fraction through the column o/n and then thorough washing. The main peak was pooled and analysed for PDE activity. Further characterization of PDE activity is going on. Attempts are also being made to see the status of cytosolic PDE in different developmental stages.