H2O2 dynamics in the malaria parasite Plasmodium falciparum

Hydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H 2 O 2 dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H 2 O 2 sensors roGFP2-Orp1 and HyPer-3 in Plasmodium falciparum . Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H 2 O 2 in the lower micromolar range in vitro and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H 2 O 2 levels and detoxification in Plasmodium . None of the quinolines or artemisinins tested had detectable direct effects on the H 2 O 2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H 2 O 2 . The systematic evaluation and comparison of the two genetically encoded cytosolic H 2 O 2 probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity.