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

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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.