Control of stage differentiation of Trypanosoma brucei by the host environment: a large scale RNAi screen of the kinome and phosphoproteomic analysis

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Trypanosomes shuttle between vertebrates and a blood feeding insect, the tsetse in tropical Africa. In the parasitic life cycle, rapid adaptation to sudden changes of the host environments are essential for survival. Signals provided by the host environment trigger coordinate changes in parasite gene expression that result in differentiation to adapted developmental stages. Our previous work shows that coincidence of temperature change (cold shock), citrate or cisaconitate in the medium, and cell cycle arrest is important to initiate differentiation to the fly midgut stage. We propose to identify molecular components of a yet unexplored pathway that transmits the host cues temperature and citrate. Two complementary approaches will be pursued: (1) screening of a bioinformatically identified set of 207 candidate genes, including all protein kinases in the T. brucei genome, using RNAi in a culture model of induced differentiation; (2) survey of protein phosphorylation changes induced by the host cues in 2D protein electrophoresis, and identification of additional pathway components by phosphoproteomics; (3) characterization of an already discovered cold-induced protein kinase. We expect unusual and novel signaling proteins, given the early phylogenetic branching of trypanosomes. Consequently, insights into the evolution of signaling and interorganismic control mechanisms, and discovery of potential drug targets for presently insufficient therapy of important tropical diseases may result from this project.