In living cells, other thiol-reactive dyes can be applied to assess redox homeostasis, this is, selleck chemicals llc however, usually limited to detecting cellular levels of e.g. reduced glutathione [29]. To overcome the limitations of these redox potential measurements, genetically encoded biosensors have been developed, including redox-sensitive green fluorescent protein (roGFP) and yellow fluorescent protein (YFP) [30], [31], which enable non-invasive and dynamic redox measurements in vivo. Recently, a highly specific and sensitive glutathione biosensor consisting of human glutaredoxin-1 (hGrx1) fused to roGFP2 was described [32]. Interestingly, hGrx1-roGFP2 was reported to detect nanomolar changes in GSSG concentrations against a backdrop of millimolar concentrations of reduced GSH on a scale of seconds to minutes [32].

This detection method is based on the ratiometric imaging of the hGrx1-roGFP2 sensor by using two different excitation wavelengths (405 nm and 488 nm) and fluorescence measurements in the green channel (500�C530 nm). Transition from reduction to oxidation changes the state of the disulfide bond in the redox sensor, increases the fluorescence intensity of the 405 nm emission peak, and decreases the intensity in the 488 nm peak. This allows one to measure the EGSH independently from different expression levels of hGrx1-roGFP2, photo bleaching, or autofluorescence in the cells [28]. As described here, we successfully transfected CQ-sensitive and resistant P. falciparum parasites with the hGrx1-roGFP2 probe and characterized the functionality of the redox sensor.

Furthermore we provide the first data on the effects of antimalarial drugs on the cytosolic glutathione redox potential in Plasmodium using the redox probe in combination with confocal live cell imaging. Based on our data we propose hGrx1-roGFP2 as a suitable and powerful tool to study redox-related changes in malaria parasites. Results hGrx1-roGFP2 as a sensor of the glutathione redox potential in P. falciparum The reliable responsiveness of the Grx-roGFP probe to various GSH/GSSG ratios and GSH concentrations has been investigated in great detail and verified in previous studies [31], [32], [33]. To gain insight into the redox-sensing properties of hGrx1-roGFP2 [32] in P. falciparum, we transfected the hGrx1-roGFP2 gene cloned into the pARL-1a+ expression vector [34] into the CQ-sensitive 3D7 and the CQ-resistant Dd2 strains of the parasite.

Following 3�C4 weeks of selection with 2 nM WR99210, the stable transfectants showed a strong hGrx1-roGFP2 fluorescence signal in the parasite’s cytosol when visualized by confocal live cell microscopy (Fig. 1A). Furthermore, we confirmed the expression of the full-length fusion hGrx1-roGFP2 AV-951 protein with the predicted size of 47 kDa in parasite lysates of 3D7 (3D7hGrx1-roGFP2) and Dd2 (Dd2hGrx1-roGFP2) strains via western blotting (Fig. S1).