title: Comparison of methods probing the intracellular redox milieu in Plasmodium falciparum.
abstract: Glutathione plays a crucial role in the redox regulation of the malaria parasite Plasmodium falciparum and is linked to drug resistance mechanisms, especially in resistance against the antimalarial drug chloroquine (CQ). The determination of the glutathione-dependent redox potential was recently established in living parasites using a cytosolically expressed biosensor comprising redox-sensitive green fluorescent protein coupled to human glutaredoxin 1 (hGrx1-roGFP2). In order to further elucidate redox changes induced by antimalarial drugs and to consolidate the application spectrum of the ratiometric biosensor we systematically compared it to other methods probing thiol and redox metabolism. Among these methods were cell disruptive and non-disruptive approaches including spectrophotometric assays with Ellman's reagent and naphthalene dicarboxyaldehyde as well as molecular probes such as ThiolTracker™ Violet and the dichlorofluorescein-based probe CM-H2DCFDA. To directly compare the methods, blood stages of the CQ-sensitive P. falciparum 3D7 strain were challenged with the oxidative agent diamide and the antimalarial drugs artemisinin and CQ for 1h, 4h, and 24h. For all conditions, dose-dependent changes in the different redox parameters could be monitored which are compared and discussed. We furthermore detected slight differences in thiol status of parasites transiently transfected with hGrx1-roGFP2 in comparison with control 3D7 cells. In conclusion, ThiolTracker™ Violet and, even more so, the hGrx1-roGFP2 probe reacted reliably and sensitively to drug induced changes in intracellular redox metabolism. These results were substantiated by classical cell disruptive methods.
authors.affiliation: Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University Giessen, 35392 Giessen, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Mohring
authors.affiliation: Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University Giessen, 35392 Giessen, Germany.
authors.forename: Esther
authors.initials: E
authors.lastname: Jortzik
authors.affiliation: Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University Giessen, 35392 Giessen, Germany. Electronic address: katja.becker@uni-giessen.de.
authors.forename: Katja
authors.initials: K
authors.lastname: Becker
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D037621:Artemisinins / Q000494:pharmacology; D001681:Biological Assay; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003958:Diamide / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004351:Drug Resistance; D004912:Erythrocytes / Q000187:drug effects* / Q000469:parasitology; D005452:Fluoresceins / Q000737:chemistry; D017930:Genes, Reporter; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009281:Naphthalenes / Q000737:chemistry; D010084:Oxidation-Reduction; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D005452:Fluoresceins; D054477:Glutaredoxins; D009281:Naphthalenes; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D003958:Diamide; D049452:Green Fluorescent Proteins; C037631:2',7'-dichlorofluorescein; C027717:2,3-naphthalenedicarboxaldehyde; D002738:Chloroquine; D004228:Dithionitrobenzoic Acid; C031327:artemisinin; D005978:Glutathione
keywords: Glutathione redox potential; Malaria; Oxidative stress; Plasmodium falciparum; Thiols; roGFP