Malaria, caused by the apicomplexan parasite Plasmodium falciparum (P. falciparum), is still one of the world s most severe human infectious diseases and threatens the health and life of millions of people, mainly those living in tropical and subtropical regions, despite strong eradication efforts. P. falciparum depends on its complex antioxidative system based on thioredoxin and glutathione for survival. The intraerythrocytic parasite lives in a pro-oxidant environment, and the host immune response increases the oxidative burden on the parasite, which can cause major oxidative damage. Interfering with the parasites essential redox system is a promising target for malaria-eradicating drugs. Several antimalarial drugs are supposed to mediate their effects at least partially by increasing reactive oxygen species (ROS) in the parasite. ROS are highly reactive and damaging towards DNA, lipids and proteins. Hydrogen peroxide (H2O2) is one of the most important cellular ROS. Until now, neither molecular targets nor regulatory mechanisms and dynamic changes of H2O2-mediated signaling in P. falciparum are known. The development of the genetically encoded H2O2 sensors roGFP2-Orp1 and HyPer paved the way for non-disruptive, ratiometric, real-time, dynamic, and specific measurements of changes in H2O2 concentrations within a living cell. Studying the dynamics of H2O2 signaling in parasites exposed to antimalarial drugs can lead to a better comprehension of their molecular mode of action and give further insights into the development of future chemotherapeutic agents. In this work, the ratiometric H2O2 redox sensors roGFP2-Orp1 and HyPer-3 and the pH-insensitive version of HyPer (SypHer) were successfully transiently expressed in the cytosol of blood stage P. falciparum parasites and their functionality was systematically characterized in vitro and in cell culture. Both redox probes showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in cell culture. Due to the pH sensitivity of HyPer-3, parasites expressing roGFP2-Orp1 were used for evaluating the short, medium, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium in combination with confocal live-cell imaging. None of the quinolines or artemisinins tested had significant effects on H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. Based on the data, both roGFP2-Orp1 and HyPer-3 probes are reliable and valuable tools for studying H2O2 metabolism in living malaria parasites. However, the necessity to use a pH probe in parallel makes utilizing HyPer-3 more challenging and time consuming.Determination of the effects of oxidative and pharmacological stress on the H2O2 homeostasis was optimized by stably integrating the redox sensor roGFP2-Orp1 into the genome of P. falciparum using the attB/attP integration method. Stable genomic integration overcomes limitations of transient transfection of the probes, allowing more detailed in-cell studies. For the first time H2O2 dynamics in the mitochondrial subcellular compartment could be determined using the stably integrated Mito-roGFP2-Orp1 redox probe. In both cytosol and mitochondrion, the sensors 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 G6PD inhibitors affected the cytosol; 24 h exposure to an arylmethylamino steroid and G6PD inhibitors revealed oxidation of mitochondria and cytosol, respectively. Furthermore, the redox sensors hGrx1-roGFP2 (glutathione sensor) and sfroGFP2 were expressed in the cytosol of NF54-attB blood-stage P. falciparum parasites. Prior to stable integration, studies with the episomal transfected hGrx1-roGFP2 strain were performed. Both sensors were evaluated with regard to their sensitivity towards oxidative stress in cell culture. The results showed that G6PD inhibitors and the arylmethylamino steroid disturb GSH (reduced glutathione) redox ratio in either 4 h or 24 h incubations. The redox sensors hGrx1-roGFP2 and sfroGFP2 are both reliable tools for studying redox metabolism in malaria parasites with comparable oxidation/reduction sensitivities, at which sfroGFP2 appeared to exhibit a more pronounced fluorescence intensity. Microscopic and plate reader-based detection methods were directly compared in order to evaluate plate reader-based measurement of bulk cell cultures as an alternative for single live-cell imaging. It is now possible to investigate quickly and efficiently with high reproducibility direct responses and long-term effects of compounds on H2O2/GSH homeostasis in cell populations.
Verknüpfung zu Publikationen oder weiteren Datensätzen
