Identification and overexpression of the A2 amastigote-specific protein in Leishmania donovani

Abstract

Leishmania protozoa must adapt rapidly to widely different environments and thus exist as promastigotes in their sandfly host and as amastigotes in their mammalian host. Promastigote differentiation into amastigotes is accompanied by both morphological and biological changes. The molecular mechanisms regulating the differentiation and survival of the different life cycle stages are poorly understood. We have therefore undertaken to identify and characterize amastigote-specific genes and their corresponding products based on the rationale that such products may be involved in the survival in the mammalian host. Previous studies in our laboratory have revealed that the A2 gene family-derived transcripts are abundant in L. donovani amastigotes but are barely detectable in promastigotes. In the present study, we have raised polyclonal and monoclonal antibodies against a recombinant A2 protein synthesized in Escherichia coli. These antibodies have been used to identify a family of A2 proteins ranging from 45 kDa to about 100 kDa which are specifically detected in L. donovani cells when they are cultured in 37°C, and pH 4.5 (conditions which mimic the macrophage phagolysosome) but not in promastigotes cultured at 26°C and pH 7.4. A2 protein therefore represents a unique amastigote-specific protein marker for L. donovani. It is also demonstrated that it was possible to overexpress the A2 protein specifically in amastigote-like cells using a plasmid construct containing the A2 coding and non-coding sequences. These advances set the foundation for defining the biological function of the A2 protein and other genes when specifically expressed in amastigotes.