Characterization of Leishmania Soluble Exo-Antigen

Abstract : Leishmaniasis is a debilitating disease that afflicts more than 350 million people worldwide. It occurrence in many US soldiers deployed to the endemic area emphasizes its importance as a health problem for the military. Vaccine development is the ultimate solution for this problem. Our previous research indicates that Leishmania parasites secrete, excrete, or shed antigens into the medium during in vitro culture. These exo-antigens possess the features of eliciting strong protective immune response in animal models. This project aims at defining the molecular properties of these soluble exo-antigens. In this research period, we have thoroughly characterized a nucleoside hydrolase (NH) and its gene from L. donovani, the causative agent of severe visceral leishmaniasis. This enzyme is implicated as an important catalytic activity of purine salvage in parasites belong to the family Trypanosornatidae. We showed that NH is encoded by a single copy gene in the parasite, and its homologues are present in members representing other Leishmania species complexes, suggesting that chemotherapy targeting this molecule will have a broad spectrum for all Leishmania parasite species. We have also shown that this enzyme and its mRNA are constitutively expressed during the parasite growth in vitro. Enzyme kinetic study revealed that it is a nonspecific nucleoside hydrolase based on its substrate specificity. To this end, we have mass-produced this antigen for the evaluation of its antigenic potential. We have further screened an expression library and purified 10 clones for sequencing analysis. The result showed that only one clone encodes a hydrophilic protein, and the rest of the clones all encode the proteins of the surface glycoprotein GP46 complex. Our analysis of the GP46 protein complex allowed us to completely sequenced three genomic clones representing different GP46 genes, and partially characterized 6 additional clones.