Stable Integration and Comparison of hGrx1-roGFP2 and sfroGFP2 Redox Probes in the Malaria Parasite Plasmodium falciparum

Abstract

Studying redox metabolism in malaria parasites is of great interest for understanding parasite biology, parasite-host interactions, and mechanisms of drug action. Genetically encoded fluorescent redox sensors have recently been described as powerful tools for determining the glutathione-dependent redox potential in living parasites. In the present study, we genomically integrated and expressed the ratiometric redox sensors hGrx1-roGFP2 (human glutaredoxin 1 fused to reduction-oxidation sensitive green fluorescent protein) and sfroGFP2 (superfolder roGFP2) in the cytosol of NF54- attB blood-stage Plasmodium falciparum parasites. Both sensors were evaluated in vitro and in cell culture with regard to their fluorescence properties and reactivity. As genomic integration allows for the stable expression of redox sensors in parasites, we systematically compared single live-cell imaging with plate reader detection. For these comparisons, short-term effects of redox-active compounds were analyzed along with mid- and long-term effects of selected antimalarial agents. Of note, the single components of the redox probes themselves did not influence the redox balance of the parasites. Our analyses revealed comparable results for both the hGrx1-roGFP2 and sfroGFP2 probes, with sfroGFP2 exhibiting a more pronounced fluorescence intensity in cellulo. Accordingly, the sfroGFP2 probe was employed to monitor the fluorescence signals throughout the parasites' asexual life cycle. Through the use of stable genomic integration, we demonstrate a means of overcoming the limitations of transient transfection, allowing more detailed in-cell studies as well as high-throughput analyses using plate reader-based approaches.

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor studies

“…Recently, the cytosolic glutathione redox sensor was stably integrated into NF54- attB parasites [52]. The direct response of NF54 [hGrx1–roGFP2] to mid- and long-term exposures were determined using the time-efficient plate reader (Clariostar).…”

“…The in vitro interaction of the pharmacologically used anti-malarial drugs ATS and CQ with the purified hGrx1–roGFP2 protein had been determined previously and did not have an effect at the concentrations used in this study [35]. The characterization of the in vitro interaction of hGrx1–roGFP2 with ML304 was performed according to Schuh et al [52].…”

Stable integration of the Mrx1-roGFP2 biosensor to monitor dynamic changes of the mycothiol redox potential in Corynebacterium glutamicum

“…Thus, expression of roGFP2 fusions from strong constitutive promoters should circumvent the problem of low roGFP2 fluorescence intensity after genomic integration. The advantage and utility of a stably integrated Grx1-roGFP2 biosensor has been also recently demonstrated in the malaria parasite Plasmodium falciparum which can circumvent low transfection frequency of plasmid-based roGFP2 fusions [55] . Moreover, quantifications using the microplate reader are more reliable, less time-consuming and reproducible with strains expressing genomic biosensors compared to measurements using confocal microscopy [55] .…”

“…The advantage and utility of a stably integrated Grx1-roGFP2 biosensor has been also recently demonstrated in the malaria parasite Plasmodium falciparum which can circumvent low transfection frequency of plasmid-based roGFP2 fusions [55] . Moreover, quantifications using the microplate reader are more reliable, less time-consuming and reproducible with strains expressing genomic biosensors compared to measurements using confocal microscopy [55] . Thus, stably integrated redox biosensors should be the method of the choice for future applications of roGFP2 fusions to monitor redox changes in a greater cellular population.…”

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

“…R represents the ratio of the fluorescence intensity measured at 405 nm and 488 nm (R ¼ 405 nm 488 nm ); R red and R ox are the ratios of the fluorescence intensity of fully reduced or fully oxidized parasites, respectively; I 488ox is the fluorescence intensity at 488 nm for fully oxidized parasites; and I 488red is the fluorescence intensity at 488 nm for fully reduced parasites [89]. Graphs were plotted using GraphPad Prism version 8.…”