Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite <i>Plasmodium falciparum</i>

<div><p>In the malaria parasite <i>Plasmodium falciparum</i>, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (<i>E</i>_GSH) of drug-sensitive (3D7) and resistant (Dd2) <i>P. falciparum</i> parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects <i>E</i>_GSH changes induced by oxidative and nitrosative stress. The cytosolic basal <i>E</i>_GSH of 3D7 and Dd2 were estimated to be −314.2±3.1 mV and −313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in <i>E</i>_GSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of <i>P. falciparum</i>, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' <i>E</i>_GSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular <i>E</i>_GSH in <i>P. falciparum</i>. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in <i>Plasmodium</i> and might also stimulate redox research in other pathogens.</p></div

Immediate oxidation of the <i>P. falciparum</i> cytosol by methylene blue.

<p>Different concentrations of methylene blue (MB) were added to 3D7 (<b>A</b>, <b>B</b>) and Dd2 (<b>C</b>, <b>D</b>) trophozoite stage parasites of <i>P. falciparum</i> expressing hGrx1-roGFP2 and monitored for 9 min. Ratio images (405/488 nm) of the cells at different time points are provided (<b>A</b>, <b>C</b>). Furthermore, the ratios 405/488 nm were computed and plotted against time (<b>B</b>, <b>D</b>). For each concentration data from 3 trophozoites were analyzed. Standard errors of the mean were not greater than 15%.</p

<i>In vitro</i> interaction of recombinant hGrx1-roGFP2 with antimalarial drugs.

<p><b>A</b>. Oxidation of reduced hGrx1-roGFP2 by methylene blue (MB) and pyocyanin (PYO) at 1 mM in comparison to 10 mM H₂O₂ and 1 mM BSO. <b>B</b>. Excitation spectra of hGrx1-roGFP2 with different concentrations of MB. <b>C</b>. Ratio change of hGrx1-roGFP2 plotted against concentrations of MB, PYO, and MNA. <b>D</b>. Excitation spectra of hGrx1-roGFP2 with different concentrations of PYO. <b>E</b>. Oxidation of reduced hGrx1-roGFP2 with quinoline and (<b>F</b>) artemisinin-based antimalarial drugs at 100 µM.</p

Short-term effects of redox-active compounds and antimalarial drugs on the redox ratio of hGrx1-roGFP2 in living parasites.

<p>Values are given for representative time points and drug concentrations of particular interest. For complete dose or time/response curves please refer to the figures.</p

Changes in the glutathione redox potential in <i>P. falciparum</i> via 24 h incubation with antimalarial drugs.

<p>A ring stage culture (10 ml, 2.5% hematocrit, 3–4% parasitemia) of 3D7 (dark bars) or Dd2 (white bars) strains of <i>P. falciparum</i> expressing hGrx1-roGFP2 were treated with antimalarial drugs at a concentration of 4×IC₅₀ for 24 h. (<b>A</b>) Preliminary experiments established that 20 mM NEM (30 min) induces instant clamping of the hGrx1-roGFP2 redox state, which could not be reverted by adding 10 mM diamide. (<b>B</b>) Following 24 h incubation, cultures were treated with 20 mM NEM for 30 min <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003782#ppat.1003782-Gutscher1" target="_blank">[32]</a>, enriched by magnetic separation (Miltenyi Biotec, Germany), and fluorescence was immediately measured. The parasites were excited with 405 and 488 nm laser wavelengths, and the ratio of emissions (405/488 nm) in the green channel (500–530 nm) was calculated. All experiments included a negative control (no drug treatment, CTL) and a positive control (treatment with 1 mM diamide (DIA)). Results are shown for methylene blue (MB), pyocyanin (PYO), buthionine sulfoximine (BSO), artemisinin (ART), artemether (ATM), artesunate (ATS), chloroquine (CQ), amodiaquine (AQ), quinine (QN), mefloquine (MQ), sodium nitroprusside (SNP), and paraquat (PQT). For each drug, the data (mean ± SEM) represent 10–15 (3D7) and 25–30 (Dd2) trophozoites. The fluorescence ratio 405/488 nm values for the 3D7 strain were significantly different from those of the Dd2 strain as indicated (**, p<0.001; *, p<0.0001).</p

hGrx1-roGFP2 is suitable for monitoring the effects of oxidative and nitrosative stress on the glutathione redox potential.

<p>After 60<i>P. falciparum</i> parasites expressing hGrx1-roGFP2 were treated with different concentrations of H₂O₂ (<b>A</b>, <b>B</b>: 3D7; <b>C</b>, <b>D</b>: Dd2), <i>tert</i>-butyl hydroperoxide (TBHP: <b>E</b>, <b>F</b>: Dd2) and 3-morpholinosydnonimine hydrochloride (SIN1: <b>G</b>, <b>H</b>: Dd2) and monitored for 4 min. Ratio images (405/488 nm) of the cells at different time points are provided (<b>A</b>, <b>C</b>, <b>E</b>, <b>G</b>). Furthermore, the ratios 405/488 nm were computed and plotted against time (<b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>). For each concentration, data from 3 trophozoites were analyzed per data point. Mean and standard errors of the mean are shown.</p

Effects of redox-active compounds and antimalarial drugs on the redox ratio of isolated recombinant hGrx1-roGFP2 <i>in vitro</i> and IC₅₀ values of the antimalarial drugs on the <i>P. falciparum</i> strains 3D7 and Dd2.

a<p>In this column the absolute change in the fluorescence ratio 405/488 nm of isolated recombinant hGrx1-roGFP2 after incubation with the compounds at given concentrations and time points is shown. The ratio 405/488 nm of reduced recombinant hGrx1-roGFP2, which served as starting point for the experiments, was 0.49±0.01.</p>b<p>In this column the fold change in the fluorescence ratio 405/488 nm of isolated recombinant hGrx1-roGFP2 after incubation with the compounds at given concentrations and time points is shown.</p>c<p>Please see also references <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003782#ppat.1003782-Meierjohann1" target="_blank">[21]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003782#ppat.1003782-Kasozi1" target="_blank">[36]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003782#ppat.1003782-Akoachere1" target="_blank">[38]</a>, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003782#ppat.1003782-Desjardins1" target="_blank">[67]</a> for details on IC₅₀-values.</p

Effect of a 4<i>P. falciparum</i>.

<p>Trophozoite stage parasites (26–30 h) of the 3D7 and Dd2 strains of <i>P. falciparum</i> expressing hGrx1-roGFP2 were magnetically enriched (Miltenyi Biotec, Germany), then counted using the improved Neubauer hemocytometer (Brand GmbH, Germany), and returned to cell culture (at ∼5.0×10³ trophozoites/µl) for at least 2 h to recover. After the 2 h recovery period, the parasites were treated with the drugs at concentrations ranging from ∼1×IC₅₀ to 100×IC₅₀ for 4 h. Each drug concentration had 5 ml with ∼5.0×10³ trophozoites/µl. The parasites were excited with 405 and 488 nm lasers, and the ratio of emissions in the green channel (500–530 nm) was calculated. The ratio of emissions after excitation at 405 and 488 nm (ratio 405/488 nm) was plotted against the drug concentration. Results are shown for (<b>A</b>) methylene blue (MB), (<b>B</b>) pyocyanin (PYO), (<b>C</b>) buthionine sulfoximine (BSO), (<b>D</b>) artemisinin (ART), (<b>E</b>) artesunate (ATS), (<b>F</b>) artemether (ATM), (<b>G</b>) mefloquine (MQ), (<b>H</b>) quinine (QN), (<b>I</b>) chloroquine (CQ), (<b>J</b>) amodiaquine (AQ), (<b>K</b>) sodium nitroprusside (SNP), and (<b>L</b>) paraquat (PQT). Each data point comprises at least 8 trophozoites. Mean and standard errors of the mean are shown.</p

Real-time imaging of the glutathione redox potential in <i>P. falciparum</i>.

<p><b>A.</b> Confocal live cell images of 3D7 and Dd2 parasites showing the expression of hGrx1-roGFP2 localized in the cytosol. <b>B</b>. hGrx1-roGFP2 is a fusion protein with human glutaredoxin 1 (hGrx1, black) fused to the N-terminal end of roGFP2 (green) through a linker (red) comprising 30 amino acids (Gly-Gly-Ser-Gly-Gly)₆. Live cell imaging of the trophozoite stages of (<b>C</b>) 3D7^hGrx1-roGFP2 and (<b>D</b>) Dd2^hGrx1-roGFP2. After 60 s, 3D7 (<b>C</b>) and Dd2 (<b>D</b>) parasites were treated with 1 mM diamide followed 4 min later by addition of 10 mM dithiothreitol (DTT). 405 nm, 488 nm, merge (405/488 nm), and false color ratio images at different time points are shown. <b>E</b>. The ratio of emissions (ratio 405/488 nm) after excitation at 405 and 488 nm was computed for both strains and plotted against time. Data from 5 trophozoites for each strain were analyzed per data point. <b>F</b>. Fluorescence ratio as a function of time. The ratio 405/488 nm remained stable over a period of 10 min. Data from 5 trophozoites for each strain were analyzed per data point. Mean and standard errors of the mean are shown.</p

Effect of heat shock on compartmental <i>E</i>_GSH and pH.

<p>A) Wild-type cells with the HSE2-lacZ (heat-shock response induction reporter) construct were pregrown in SC_URA (48 h; 30°C; 600 rpm) and then inoculated in SC_URA (A₆₀₀ = 0.001) and grown (25°C; 600 rpm) until exponential phase (A₆₀₀ = ∼0.5). Cells were then shifted to 42°C for 60 min and harvested. β-galactosidase and total protein (Bradford) assays were carried out on cell pellets and specific activity determined. B-C) Cells were pregrown in SC_URA (48 h; 30°C; 600 rpm), inoculated in SC_URA (A₆₀₀ = 0.001) and grown (25°C; 600 rpm) until exponential phase (A₆₀₀ = ∼0.5). Cells were then shifted to 42°C for 60 min and <i>E</i>_GSH B) and pH C) analyzed via flow cytometry. 10,000 cells were counted for each condition. Each experiment was conducted in triplicate with error bars representing the standard deviation of three experiments.</p