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.
Highlights
Despite strong worldwide efforts to combat malaria, the spread of parasites resistant to effective and affordable antimalarials including chloroquine (CQ) has worsened the situation
P. falciparum is exposed to substantial oxidative stress during its intraerythrocytic life cycle, in part due to the oxidative burst of macrophages activated by the host immune system during malaria infection[5, 6]
H2O2-induced conformational changes of the probe can be detected in cells via confocal laser scanning microscopy (CLSM) after excitation at 405 and 488 nm, with emission set at 510 nm[23]
Summary
Despite strong worldwide efforts to combat malaria, the spread of parasites resistant to effective and affordable antimalarials including chloroquine (CQ) has worsened the situation (reviewed in ref. 1). We reported on the transient expression of the genetically encoded fluorescent H2O2 redox probes roGFP2-Orp[1] and HyPer-3 in the cytosol of P. falciparum, which paved the way for non-disruptive, ratiometric, real-time, and dynamic measurements of H2O2 levels[22]. Both sensors were found to be highly selective and sensitive in detecting submicromolar concentrations of H2O222. Stable integration of the sensor reduces the heterogeneous fluorescence signals that can arise in episomal expression systems[22] Using this integrated reporter line, we were able to systematically analyze by CLSM the effects of oxidative and pharmacological stress on the cytosolic and mitochondrial H2O2 metabolism of P. falciparum. Results, presented below, demonstrate that the stably integrated roGFP2-Orp[1] sensor provides a robust tool for differentially studying H2O2 homeostasis in subcellular compartments of P. falciparum
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