Comparative proteome analysis of Leishmania donovani at different stages of transformation from promastigotes to amastigotes

Abstract.

To pass through its life cycle, protozoan parasites of the genus Leishmania have to differentiate from promastigotes to amastigotes. The molecular basis underlying this major transformation is poorly understood. One way to study this phenomenon is to isolate and characterize proteins that are specifically expressed in one of the two stages of the life cycle or during the stage differentiation. Using two-dimensional gel electrophoresis, we mapped the Leishmania donovani proteome during stage differentiation to identify stage-specific proteins and regulons. A protocol for extracting proteins of both promastigote and amastigote L. donovani cells was developed, which is compatible with isoelectric focusing. Up to 400 L. donovani protein spots were visualized on a silver-stained gel. Metabolic labeling of the cells was used to compare directly the protein synthesis pattern with the protein level pattern. The silver-stained images of L. donovani cells harvested on different days of stage differentiation were compared to the corresponding autoradiographs. A marked decrease in protein synthesis during stage differentiation from promastigotes to amastigotes was observed. The stained protein pattern as well as the protein pattern on the autoradiograph changed dramatically, especially after day 3 (about 24 h after pH shift) of transformation.