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

锥虫在脊椎动物和一种食血昆虫之间穿梭，采采虫在热带非洲。在寄生生活史中，对寄主环境突变的快速适应对其生存至关重要。寄主环境提供的信号触发寄生虫基因表达的协调变化，导致分化到适应的发育阶段。我们以前的工作表明，温度变化(冷休克)、培养液中的柠檬酸或顺式酸根以及细胞周期停滞对于启动分化到苍蝇中肠期是重要的。我们建议确定一条尚未探索的传递寄主线索、温度和柠檬酸的途径的分子成分。将采取两种互补的方法：(1)在诱导分化的培养模型中使用RNAi筛选一组207个候选基因的生物信息，包括布鲁氏锥虫基因组中的所有蛋白激酶；(2)在2D蛋白质电泳中检测宿主提示诱导的蛋白质磷酸化变化，并通过磷蛋白质组学鉴定其他途径组件；(3)鉴定已发现的冷诱导蛋白激酶。鉴于锥虫早期的系统发育分支，我们期待着不寻常的和新的信号蛋白。因此，该项目可能会深入了解信号和组织间控制机制的演变，并为目前治疗重要热带疾病的不足发现潜在的药物靶点。

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.