H(2)O(2) 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.