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

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.