Role of cAMP signalling in the developmental transformation of Leishmania donovani

Understanding of molecular events, or more precisely signalling events, during developmental transformation of Leishmania donovani – aetiological agent of visceral leishmaniasis or kala-azar – holds the key to understand as to how and why these parasites survive intracellularly and lead to pathogenesis. Transformation from promastigotes to amastigotes in humans is crucial from the viewpoint of pathogenesis and is accompanied by wide variations in the surrounding pH and temperature of the parasite which undergoes rapid and extensive morphological, physiological, and biochemical changes. Available evidences indicated toward an operational but hitherto poorly understood role of cAMP signalling during this transformation process. Thus the main theme of the project is to understand signalling events mediated by cAMP during transformation of promastigotes into amastigotes. 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.

During the reporting period we attempted to assay PKA activity in the four developmental stages. In each case equal number of cells (1010) were taken. Cells were lysed in presence of sodium orthovanadate and etazolate hydrochloride in addition to protease inhibitor cocktail. Using biotinylated kemptide as substrate and a highly specific biotin capture membrane PKA activity was measured both in presence and absence of cAMP. We found that PKA activity was maximum in temperature transformants. In order to confirm the specificity of observed kinase activity, we used a highly specific PKA inhibitor (PKI). In presence of PKI the observed kinase activity in all the four developmental stages was inhibited to almost basal levels. Use of specific substrate (kemptide), enhancement of a kinase activity in presence of cAMP and its inhibition to basal values in presence of PKI confirmed the presence of PKA activity in the parasite. It was also very clear from these experiments that the activity was maximum in Temperature Transformants.

We also purified and characterized a PDE activity in the cytosolic portion. 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. Purification was done 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. The PDE activity was dependent on Mg2+ dependent. The Km obtained from reciprocal plots was found to be 1.67x10-3 M. Relative rate of hydrolysis of cAMP and cGMP was in the ratio100:33. Various compounds such as caffein, theophylline, etazolate hydrochloride, rolipram, and IBMX caused varying degree of inhibition of the observed activity.

During the reporting period we were successful in identifying a Gas of about 45 kDa in these developmental stages. For this cell membranes were prepared from parasites in different developmental stages. Membrane proteins were electrophoresed under reducing conditions, blotted and probed with sequence-specific antibodies. In order to confirm the functional identity of these protein bands, ADP ribosylation in presence of CTX-A, CT, PT was performed.