Genetically encoded biosensors for monitoring redox changes in the trypanothione-based thiol metabolism of trypanosomes

锥虫和利什曼原虫缺乏谷胱甘肽还原酶和硫氧还蛋白还原酶，这两种酶几乎普遍存在，负责维持细胞内的还原环境。寄生原生动物的巯基氧化还原稳态是基于锥虫硫酮，一种由多胺亚精胺和两个谷胱甘肽分子组成的缀合物。参与锥虫硫酮生物合成和还原的所有酶都位于细胞的胞质溶胶中。在这里，通过巯基/二硫键交换，锥虫硫酮还原谷胱甘肽二硫化物以及氧化还原蛋白，如tryparedoxin，硫氧还蛋白和谷氧还蛋白。总谷胱甘肽，谷胱甘肽亚精胺和锥虫硫酮的测量结果表明，细胞低分子量的硫醇池经历了非常明显的变化时，血液形式的布氏锥虫，非洲昏睡病和Nagana牛疾病的病原体，暴露于外源性和内源性氧化应激。本项目的目的是建立能在T细胞胞浆、膜间隙和基质中表达多种氧化还原活性绿色荧光蛋白（roGFPs）的细胞系。布氏杆菌，并因此允许在完整寄生虫中在稳态和硫醇转换条件下对硫醇氧化还原状态进行隔室特异性分析。将解决的主要问题是：全细胞巯基状态是否反映了胞质溶胶中的条件？在细胞质和线粒体的硫醇氧化还原代谢之间是否存在交叉作用？巯基氧化还原状态在昆虫阶段的充分阐述的细胞器中与在感染性血流寄生虫的基本细胞器中相比如何？锥虫硫酮生物合成或还原的下调如何影响细胞溶质和线粒体巯基氧化还原状态？这两个谷氧还蛋白，分别位于细胞质和线粒体膜间隙，作为巯基氧化还原开关？计划中的工作应该给非洲锥虫的细胞溶质和线粒体硫醇代谢的深刻见解。此外，所产生的稳定细胞系有望成为未来研究寄生虫中的氧化还原调节过程如分化的有吸引力的工具。

Trypanosomes and Leishmania lack both glutathione reductases and thioredoxin reductase, nearly ubiquitous enzymes that are responsible for maintaining the reducing intracellular milieu. The thiol redox homeostasis of the parasitic protozoa is based on trypanothione, a conjugate composed of the polyamine spermidine and two molecules of glutathione. All enzymes involved in the biosynthesis and reduction of trypanothione are located in the cytosol of the cell. Here, by thiol/disulfide exchange, trypanothione reduces glutathione disulfide as well as redox proteins such tryparedoxin, thioredoxin and glutaredoxins. Measurements of total glutathione, glutathionylspermidine and trypanothione revealed that the cellular low molecular mass thiol pool undergoes highly distinct changes when the bloodstream form of Trypanosoma brucei , the causative agent of African sleeping sickness and Nagana cattle disease, is exposed to exogenous and endogenous oxidative stresses. This type of analysis, however, cannot provide any information about the specific situation in the different cell compartments.Aim of this project is to generate cell lines that express various redox active green fluorescent proteins (roGFPs) in the cytosol as well as intermembrane space and matrix of the single mitochondrion of T. brucei and thus allow a compartment-specific analysis of the thiol redox status under steady state and thiol switch conditions in the intact parasites. The main questions that will be addressed are: Does the whole cell thiol status reflect the conditions in the cytosol? Is there a cross-talk between the thiol redox metabolism of the cytosol and the mitochondrial compartments? How does the thiol redox status in the fully elaborated mitochondrion of the insect stage compare with that in the rudimentary organelle of the infectious bloodstream parasite? How does down-regulation of the trypanothione biosynthesis or reduction affect the cytosolic and mitochondrial thiol redox status? Do the two glutaredoxins, localized in the cytosol and mitochondrial intermembrane space, respectively, act as thiol redox switches?The planned work should give a profound insight into the cytosolic and mitochondrial thiol metabolism of African trypanosomes. In addition, the stable cell lines generated are expected to be attractive tools for future studies of putatively redox regulated processes in the parasites such as differentiation.