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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 (H 2 O 2 ) 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 H 2 O 2  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 H 2 O 2  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 H 2 O 2  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 H 2 O 2  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.

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