A tryparedoxin-coupled biosensor reveals a mitochondrial trypanothione metabolism in trypanosomes

Abstract

Trypanosomes have a trypanothione redox metabolism that provides the reducing equivalents for numerous essential processes, most being mediated by tryparedoxin (Tpx). While the biosynthesis and reduction of trypanothione are cytosolic, the molecular basis of the thiol redox homeostasis in the single mitochondrion of these parasites has remained largely unknown. Here we expressed Tpx-roGFP2, roGFP2-hGrx1 or roGFP2 in either the cytosol or mitochondrion of Trypanosoma brucei. We show that the novel Tpx-roGFP2 is a superior probe for the trypanothione redox couple and that the mitochondrial matrix harbors a trypanothione system. Inhibition of trypanothione biosynthesis by the anti-trypanosomal drug Eflornithine impairs the ability of the cytosol and mitochondrion to cope with exogenous oxidative stresses, indicating a direct link between both thiol systems. Tpx depletion abolishes the cytosolic, but only partially affects the mitochondrial sensor response to H2O2. This strongly suggests that the mitochondrion harbors some Tpx and, another, as yet unidentified, oxidoreductase.

Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening

“…The transgenic cell line expresses a GFP-based redox biosensor (roGFP2) 28 that allows monitoring non-invasively, and in a dynamic fashion, perturbations in the pool of reduced and oxidised low molecular weight thiols (i.e. GSH/GSSG and T(SH) 2 /TS 2 ) 36 . For instance, a high fluorescence intensity of roGFP2 excited at 488 nm indicates a reducing intracellular milieu (i.e.…”

Evaluation of the Anti-Leishmania mexicana and -Trypanosoma brucei Activity and Mode of Action of 4,4′-(Arylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol)

“…Nowadays, the perturbation of the intracellular pool of low-molecular-weight thiols (i.e., glutathione, trypanothione, bacillithiol, and mycothiol) in different pathogens can be monitored using genetically encoded fluorescent biosensors [ 42 , 44 , 52 , 53 ]. In T. brucei , a redox sensitive version of GFP, namely roGFP2, fused to human glutaredoxin (hGrx) allowed detecting changes in the ratio of oxidized and reduced glutathione and bis-glutathionyl-spermidine (trypanothione).…”

“…Phleomycin and hygromycin were added to keep the constitutive expression of the tetracycline repressor protein and the tet-inducible hGrx1-roGFP2 gene, respectively. The biosensor hGrx1-roGFP2 was designed by others [ 41 ] and its capacity to non-invasively detect perturbations in the redox state of intracellular low-molecular-weight thiols has been demonstrated in T. brucei [ 42 , 43 , 44 , 45 ].…”

Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei

“…The redox biosensor (hGrx1-roGFP2) [ 47 ] consists of a redox sensitive green fluorescent protein (roGFP2) fused to human glutaredoxin 1 (hGrx1) that enables a rapid redox equilibration between roGFP2 and the pool of reduced and oxidized glutathione and trypanothione [ 46 , 48 , 49 ].…”