Noninvasive, Fluorescence-Based Detection of Perturbations in the Thiol-Redox Homeostasis of Bloodstream Trypanosoma brucei brucei.

Here, we describe a method to detect the oxidation of the pool of low molecular weight thiols (LMWT) in the infective stage of Trypanosoma brucei brucei in situ and non-invasively. The redox reporter cell line was generated by transfecting the parasites with a DNA construct coding for a redox-sensitive green fluorescent protein fused to human glutaredoxin 1 (hGrx1-roGFP2). The reporter gene is expressed in a tetracycline-inducible manner in the parasite's cytosol. roGFP2 displays a ratiometric and reversible change in fluorescence emission at 510 nm when excited at 405 and 488 nm, which is proportional to the changes in the ratio of oxidized vs. reduced LMWT trypanothione and glutathione. The role of hGrx1 is to catalyze a rapid equilibration of the LMWT pool with roGFP2 redox state, thereby allowing a biosensor response within seconds. The dynamic response of the biosensor enables the monitoring of cellular events in response to drugs or other stimuli in real time. The assay was adapted to a 96-well plate format for flow cytometry-based analysis. The fluorescent readout can be intensiometric or ratiometric, depending on the flow cytometer features, and the use of calibration controls is recommended for quantitative analysis. This bioassay can be applied to study fundamental questions of trypanosomatids' redox biology, as go/no-go criteria in drug discovery campaigns, and to investigate drug mode of action.