Most Leishmania species, including Leishmania chagasi, possess on their surface an abundant, highly conserved, glycoprotein called GP46 or PSA-2. Different molecular weights for GP46 have been reported within and among different Leishmania species. The function of GP46 is unknown, but GP46 has been shown to confer partial protection of mice against challenge with Leishmania amazonensis. In those Leishmania species where it has been studied, the GP46 gene organization is complex and consists of multiple, non-identical, genes arranged in clusters. In Preliminary studies and as described in reference 1, we found that in L. chagasi the abundance of 44 and 66 kDa versions of GP46 increases 30-fold as virulent promastigotes develop from less infectious to highly infectious forms, but in attenuated promastigotes the GP46 abundance does not increase during development. Two species of GP46 RNA exist (2.8 and 4.8 kb), and both also increase 30-fold during virulent promastigote development. However, whereas the abundances of the 44 and 66 kDa GP46 are within 2-fold of each other, the abundance of the 2.8 kb RNA is 40-fold more than that of the 4.8 kb RNA. Nuclear run-on experiments indicate that the GP46 mRNA levels are regulated post- transcriptionally. Experiments using promastigotes stably transfected with plasmids containing various regions of one of the L. chagasi GP46 genes showed that its 3' UTR confers a developmentally regulated abundance to a reporter gene/RNA. Thus, the specific aims of this project are to: (1) determine the relationships between the GP46 genes and the abundances of the different GP46 mRNAs and proteins, (2) determine the molecular basis for the loss of regulated GP46 expression in attenuated promastigotes, (3) identify and characterize cis- and trans-acting elements that participate in the developmental regulation of GP46 mRNA abundance, and (4) determine whether pre-processing, co- processing or post-processing of the RNA mediates the increased abundance of GP46 mRNA in the infectious form. These studies are intended to identify the molecular processes that regulate GP46 expression, fundamental processes that we expect will require proteins which will be targets for future research aimed at new treatments and prevention of visceral leishmaniasis.
