Discussion

Leishmania spp. parasites must rapidly respond to environmental shifts within the insect vector and the mammalian host for successful progression toward human infective forms. Due to negligible trascriptional regulation in these parasites, trans-acting factors drive Leishmania spp. lifecycle progression. These trans-acting RNA binding proteins (RBPs) coordinate the location, translation and decay of RNAs within messenger ribonucleoprotein complexes (mRNPs) in response to shifting environmental cues. To isolate key trans-regulators implicit in virulent parasite stages, we pursued developmentally-regulated mRNA-associating proteins in the three most- characterised parasite lifecycle stages; procyclic promastigote (P), metacyclic promastigote (M) and amastigote (A) forms of Leishmania mexicana. These lifecycle stages were isolated and purified by optimised axenic culturing (P,M) and macrophage infections (A). Each stage was verified by molecular markers, FACS analysis and human complement susceptibility prior to an enhanced system-wide mRNA interactome capture approach. Preliminary data from this has confirmed known RBP expression from previous literature and identified novel RNA interactors. To examine candidate trans-regulator expression, we endogenously tagged specific RBPs whose RNAs exhibit developmental regulation. Distinctions in relative protein/RNA expression were confirmed, indicating stage-specific control of transcript stability and translation and supporting the role for these RBPs in developmental progression and parasite virulence. We are currently quantifying the L.mexicana lifecycle's mRNA-bound proteome while confirming RNA-binding activity and transcript target identity for select trans-regulator candidates. This work will provide novel insight into trans-regulatory mRNP complexes which drive the Leishmania spp. lifecycle. To examine candidate trans-regulator expression,we endogenously tagged specific RBPs