Hydrogen peroxide dynamics in subcellular compartments of malaria parasites using genetically encoded redox probes

Abstract

Redox balance is essential for the survival, growth and multiplication of malaria parasites and oxidative stress is involved in the mechanism of action of many antimalarial drugs. Hydrogen peroxide (H2O2) plays an important role in redox signalling and pathogen-host cell interactions. For monitoring intra- and subcellular redox events, highly sensitive and specific probes are required. Here, we stably expressed the ratiometric H2O2 redox sensor roGFP2-Orp1 in the cytosol and the mitochondria of Plasmodium falciparum (P. falciparum) NF54-attB blood-stage parasites and evaluated its sensitivity towards oxidative stress, selected antimalarial drugs, and novel lead compounds. In both compartments, the sensor showed reproducible sensitivity towards H2O2 in the low micromolar range and towards antimalarial compounds at pharmacologically relevant concentrations. Upon short-term exposure (4 h), artemisinin derivatives, quinine and mefloquine impacted H2O2 levels in mitochondria, whereas chloroquine and a glucose-6-phosphate dehydrogenase (G6PD) inhibitor affected the cytosol; 24 h exposure to arylmethylamino steroids and G6PD inhibitors revealed oxidation of mitochondria and cytosol, respectively. Genomic integration of an H2O2 sensor expressed in subcellular compartments of P. falciparum provides the basis for studying complex parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity, and sets the stage for high-throughput approaches to identify antimalarial agents perturbing redox equilibrium.

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

“…Using a genetically-encoded, mitochondria-targeted GFP-fusion redox probe, we found substantial oxidation following 4h or 24h of DHA treatment, which greatly exceeded any oxidative effect of the cytochrome bc1 inhibitor ATQ or the hemozoin inhibitor CQ. These results agree with earlier studies that also showed an impact of ART derivatives on redox potential in the parasite cytosol [56,83,84]. Further studies are merited to elucidate the role of the mitochondria in both DHA action and K13-mediated resistance.…”

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor studies

“…Recently, it was shown that the dynamics of hydrogen peroxide are also affected by ML304. Employing stable integration of the H 2 O 2 redox probe roGFP2-Orp1 into blood stages of NF54- attB , ML304 was found to significantly increase the cytosolic H 2 O 2 levels [48]. It can, therefore, be deduced that inhibition of the G6PD reaction, likely followed by impairment of NADPH fluxes, markedly affects antioxidant capacity in Plasmodium parasites.…”

“…The CQ-sensitive P. falciparum 3D7 and NF54- attB strains were cultured as described in [47, 48], respectively. Briefly, the strains were propagated in red blood cells (RBCs) (A+) in RPMI 1640 medium supplemented with 0.5% w/v Albumax, 9 mM glucose, 0.2 mM hypoxanthine, 2.1 mM l -glutamine, 25 mM HEPES, and 22 µg mL −1 gentamycin at 3.3% haematocrit and 37 °C in a gaseous mixture consisting of 3% O 2 , 3% CO 2 , and 94% N 2 .…”

Efficient Active Oxygen Free Radical Generated in Tumor Cell by Loading-(HCONH2)·H2O2 Delivery Nanosystem with Soft-X-ray Radiotherapy

“…However, one important factor to limit radiotherapeutic efficacy is that intracellular microenvironment of most solid tumors is significantly hypoxic [ 6 , 7 , 8 ]. In order to overcome tumor hypoxia, many researches depend on transporting radiosensitizers, such as O 2 , reactive oxygen species (ROS), and radiosensitive drugs (such as bleomycin [ 9 ], topotecan [ 10 ], artemisinin [ 11 ], etc.) to the tumor hypoxic regions by the drug delivery system (DDS) [ 12 ].…”