IDENTIFICATION AND OVEREXPRESSION OF THE A2 AMASTIGOTE-SPECIFIC PROTEIN IN LEISHMANIA-DONOVANI

Leishmania protozoa must adapt rapidly to widely different environment s and thus exist as promastigotes in their sandfly host and as amastig otes in their mammalian host. Promastigote differentiation into amasti gotes is accompanied by both morphological and biological changes. The molecular mechanisms regulating the differentiation and survival of t he different life cycle stages are poorly understood. We have therefor e undertaken to identify and characterize amastigote-specific genes an d their corresponding products based on the rationale that such produc ts may be involved in the survival in the mammalian host. Previous stu dies in our laboratory have revealed that the A2 gene family-derived t ranscripts are abundant in L. donovani amastigotes but are barely dete ctable in promastigotes. In the present study, we have raised polyclon al and monoclonal antibodies against a recombinant A2 protein synthesi zed 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 3 7 degrees C, and pH 4.5 (conditions which mimic the macrophage phagoly sosome) but not in promastigotes cultured at 26 degrees C and pH 7.4. A2 protein therefore represents a unique amastigote-specific protein m arker for L. donovani. It is also demonstrated that it was possible to overexpress the A2 protein specifically in amastigote-like cells usin g a plasmid construct containing the A2 coding and non-coding sequence s. These advances set the foundation for defining the biological funct ion of the A2 protein and other genes when specifically expressed in a mastigotes.