Development of redox reporter cell lines of pathogenic trypanosomatids and insights into the mechanism of action of natural sesquiterpene lactones

SARDI F; SCHMIDT T; ELSO O; COMINI M; SÜLSEN V

Simposio; ResNet NPND - 10th anniversary online celebration-symposium; 2021

Research Network Natural Products Neglected Diseases (ResNet NPND)

The thiol-dependent redox metabolism of trypanosomatids has been shown to be critical for parasite survival and persistence in the mammalian host, and for contributing to (pro)drug activation and resistance mechanisms. The quantitative monitoring of intracellular redox changes in real-time demands the development of reliable reporter cell lines with redox-sensitive and -specific fluorescent biosensors. Here we report the generation of stable transgenic cell lines of pathogenic trypanosomatids expressing a novel redox biosensor consisting of a glutaredoxin (Grx) that catalyzes the oxidation-reduction of a C-terminally linked redox-sensitive GFP (roGFP2). Furthermore, these cell lines were employed to investigate the mode of action of a small subset of sesquiterpene lactones (SL). This type of compounds is present in several plants and corals, and is a rich source of drugs. Recombinant Grx-roGFP2 proved to be sensitive and specific to detect changes in the amount of reduced and oxidized low molecular weight thiols. Transgenic cell lines of infective Trypanosoma brucei brucei and Leishmania infantum expressing Grx-roGFP2 were characterized by flow cytometry. The reporter cell lines showed a concentration-dependent response to different oxidants, which was reversible by incubation with DTT, a permeant reducing agent. Seven SL were tested against T. brucei and L. infantum, with the most potent displaying EC50 values of 0.3 and 6 µM, respectively. SL harbour a α,β-unsaturated carbonyl group that is highly electrophilic and prone to react with the thiol group present in cysteine residues. The modification of redox active cysteines may have important consequences in cell redox homeostasis. Interestingly, the redox reporter parasites revealed that not all SL with antiproliferative activity against trypanosomatids induced oxidative stress as part of their killing mechanism. Although of different magnitude, three SL generated intracellular oxidation in both parasite species, suggesting common molecular target(s). We demonstrated the potential of redox reporter trypanosomatids for targeted-drug discovery studies.