Leishmania parasites exist as flagellated promastigote forms in the sandfly vector and as obligate intracellular amastigotes within the acidic environment of host phagolysosomal vacuoles. The environmental habitats of these two developmental forms are extremely different and adaptation to the shift in environment requires a dramatic morphological and physiological transformation. The molecular basis for regulation of this event as well as the molecular process of gene transcription in Leishmania is poorly understood. This proposal examines the control of transcript abundance in a tandemly linked pair of developmentally regulated ion transporting ATPase genes as a model for Leishmania gene transcription and molecular regulation during transformation. Two in vitro culture systems for Leishmania differentiation are used; the genesis of an infective promastigote form in late stationary phase cultures of L. donovani, and the transformation of a xenically grown L. m. pifanoi amastigotes to promastigotes. Initial work will focus on the organization, sequence and expression of the ATPase locus in both species. The 5' and 3' ends of ATPase transcripts will be mapped and sequenced using RNase protection, primer extension and PCR techniques. The effect of mRNA stability on transcript abundance will be measured by mRNA half life determinations (pulse chase) and the size of the ATPase transcription unit estimated by nuclear run-on and UV inactivation assays. ATPase sequences implicated in transcript control will be joined to reporter genes, transfected into Leishmania, and reporter gene expression assayed during transformation. Successive deletion of the ATPase sequences and measurement of the effects on reporter gene expression in differentiating cultures will define regions involved in control of transcription in Leishmania developmental forms. This data will be confirmed with gel retardation experiments and DNA footprinting which can localize DNA binding proteins to specific ATPase sequences. Leishmania is a significant human pathogen. Better understanding of the mechanisms controlling gene transcription during parasite development will potentiate new strategies for the design of anti-leishmanial drugs.
