Indeed, glutathione homeostasis has been shown to be differentially regulated in CQ- resistant and sensitive P. falciparum strains. This neverless differential regulation seems to be mainly effected by glutathione biosynthesis, by PfGR, and by GSSG efflux [40], [21]. If the difference observed between our two strains is specific for the parasites used or represents a general difference between drug-sensitive and drug-resistant parasites, this needs to be addressed in further studies. So far different concentrations (0.4 mM to 2.3 mM) of total cytosolic glutathione have been reported in P. falciparum [19], [27], [41]. These discrepancies may be due to real strain-specific differences and/or to the use of cell-disruptive methods, which can result in a loss of glutathione, in mixing of glutathione from different compartments, in oxidative processes (e.
g. enhancing glutathionylation reactions [42]), and in red blood cell contaminations. Many of these problems can be overcome by using the hGrx1-roGFP2 sensor. Notably, when estimating the basal EGSH in the cytosol on the basis of the fluorescence after maximal oxidation (1 mM diamide) and after full reduction (10 mM DTT) [31], [33], we obtained values of ?314.2��3.1 mV and ?313.9��3.4 mV for 3D7 and Dd2, respectively. These values did not differ significantly and are also supported by previous studies [43] using a pH-sensitive green fluorescent protein (pHluorin), in which no significant differences in the cytosolic pH were found between the CQ-sensitive HB3 (pH=7.03��0.09) and CQ-resistant Dd2 strain (pH=7.20��0.06).
Other studies using different methodological approaches reported pH values of 7.29��0.01 for the FAF-6 strain [44] and 7.31��0.02 for the FCR-3 strain [45]. Using the hGrx1-roGFP2 sensor systematically in parallel with other methodological approaches, it is now possible to approach the direct comparison of basal redox ratios, redox potentials, and pH values in various Plasmodium strains. The basal EGSH of Dd2 and 3D7 indicate that the parasites’ cytosol is more reducing than suggested in previous estimates [22]. Furthermore, our data are comparable to the cytosolic EGSH determined by roGFP in other organisms such as ?325 mV in HeLa cells [31], ?318 mV in Arabidopsis thaliana [33], and ?310 to ?320 mV in Saccharomyces cerevisiae [28], [46]. The dynamic range of hGrx1-roGFP2 was determined to be 6.
36��0.73 and 5.28��0.49 in 3D7 and Dd2, respectively. These values are also in agreement with previously reported dynamic ranges of hGrx1-roGFP2 in other organisms of 4.4 [32] Cilengitide and 4 to 8 [28]. The data suggest furthermore that the presence of higher GSH levels confers a greater redox buffering capacity in the Dd2 strain compared to the 3D7 strain. Treatment of cells with H2O2 is a common tool for probing the sensitivity of biosensors to oxidative stress [28].