A tryparedoxin-coupled biosensor reveals a mitochondrial trypanothione metabolism in trypanosomes
Trypanosomatids are the causative agents of African sleeping sickness ( Trypanosoma brucei gambiense and T . b . rhodesiense ) , Nagana cattle disease ( Trypanosoma b . brucei , T . congolense ) , South-American Chagas’ disease ( T . cruzi ) and the different manifestations of leishmaniasis ( Leishmania spp . ) . All these parasitic protozoa lack the nearly ubiquitous glutathione reductases ( GRs ) and thioredoxin reductases but , instead , have a trypanothione ( T ( SH ) 2 ) /trypanothione reductase ( TR ) system . The trypanothione system delivers reducing equivalents for a large number of vital processes ( for reviews see Krauth-Siegel and Leroux , 2012; Manta et al . , 2018 and Manta et al . , 2013 ) . Most of the reactions are mediated by tryparedoxin ( Tpx ) , a distant member of the thioredoxin family . For T ( SH ) 2 synthesis , two molecules of glutathione ( GSH ) are linked by one molecule of spermidine , with glutathionylspermidine ( Gsp ) as intermediate . Both steps are catalyzed by trypanothione synthetase ( TryS ) ( Comini et al . , 2004; Krauth-Siegel and Leroux , 2012; Oza et al . , 2002 ) . Difluoromethylornithine ( DFMO , Eflornithine ) , a drug against late-stage T . b . gambiense sleeping sickness , is an irreversible inhibitor of ornithine decarboxylase ( ODC ) , the enzyme generating putrescine , the precursor for spermidine synthesis . Treatment of T . brucei with DFMO decreases the levels of spermidine and T ( SH ) 2 and slightly increases the level of GSH ( Bellofatto et al . , 1987; Fairlamb et al . , 1987; Xiao et al . , 2009 ) . All enzymes involved in T ( SH ) 2 biosynthesis and also TR , which catalyses the NADPH-dependent reduction of trypanothione disulfide ( TS2 ) as well as glutathionylspermidine disulfide ( Gsp2 ) , appear to be restricted to the cytosol . African trypanosomes lack catalase . Hydroperoxides are detoxified by 2-Cys-peroxiredoxins ( Prxs ) ( Budde et al . , 2003; Tetaud et al . , 2001; Wilkinson et al . , 2003 ) and non-selenium glutathione peroxidase-type enzymes ( Pxs ) ( Hillebrand et al . , 2003; Schlecker et al . , 2005; Wilkinson et al . , 2003 ) . The Pxs preferably reduce lipid hydroperoxides ( Bogacz and Krauth-Siegel , 2018; Diechtierow and Krauth-Siegel , 2011; Hiller et al . , 2014; Schaffroth et al . , 2016 ) whereas the Prxs mainly detoxify hydrogen peroxide and peroxynitrite ( Thomson et al . , 2003; Trujillo et al . , 2004 ) . Both types of thiol peroxidases are reduced by the TR/T ( SH ) 2/Tpx system and thus act as tryparedoxin peroxidases ( Scheme 1; Castro and Tomás , 2008; Krauth-Siegel and Comini , 2008; Krauth-Siegel and Leroux , 2012; Manta et al . , 2013 ) . T . brucei encodes three virtually identical Pxs ( Hillebrand et al . , 2003 ) which occur in the cytosol ( Px I and II ) and the mitochondrion ( Px III ) . Depletion or deletion of the Pxs is lethal ( Diechtierow and Krauth-Siegel , 2011; Hiller et al . , 2014; Schaffroth et al . , 2016; Schlecker et al . , 2005; Wilkinson et al . , 2003 ) . However , survival and proliferation of cells lacking Pxs can be restored by supplementing the medium with an iron chelator or the vitamin E analogue Trolox [ ( ± ) −6-hydroxy-2 , 5 , 7 , 8-tetramethylchromane-2-carboxylic acid] ( Bogacz and Krauth-Siegel , 2018; Diechtierow and Krauth-Siegel , 2011; Hiller et al . , 2014; Schaffroth et al . , 2016 ) . Cell death of Px-deficient insect stage procyclic ( PC ) T . brucei closely resembles ferroptosis in mammalian cells ( Bogacz and Krauth-Siegel , 2018 ) . In the case of the Prxs , two different enzymes are expressed , one in the cytosol ( cPrx ) and one in the mitochondrion ( mPrx ) ( Tomás and Castro , 2013 ) . In bloodstream ( BS ) T . brucei , depletion of cPrx , but not of mPrx , is lethal ( Wilkinson et al . , 2003 ) . In Leishmania , mPrx is required for survival of the parasite in the mammalian host . However , this essential function is not related to its peroxidase activity but to the ability of the protein to act as a thiol-independent molecular chaperone ( Castro et al . , 2011; Teixeira et al . , 2015 ) . The thiol-disulfide redox state of roGFP2 ( reduction-oxidation-sensitive green fluorescence protein 2 ) can be monitored in real time , inside intact cells , in a ratiometric manner ( Meyer et al . , 2007; Meyer and Dick , 2010; Schwarzländer et al . , 2016 ) . Importantly , this approach allows to measure the degree of probe oxidation independently of absolute sensor concentration . Equilibration of the roGFP2 thiol-disulfide redox couple with the intracellular glutathione redox couple was found to be mediated , almost exclusively , by glutaredoxins ( Grxs ) ( Meyer et al . , 2007 ) . Thus , fusion of roGFP2 to human glutaredoxin 1 ( hGrx1 ) generated a probe for specifically measuring the redox potential of the glutathione redox couple ( EGSH ) which is a function of both the GSH:GSSG ratio and total glutathione concentration ( Gutscher et al . , 2008; Morgan et al . , 2013; Morgan et al . , 2011 ) . The hGrx1-roGFP2 fusion protein proved to be a valuable tool for measuring changes in EGSH in various model organisms and compartments ( Gutscher et al . , 2008; Kojer et al . , 2012; Morgan et al . , 2013; Radzinski et al . , 2018 ) . It has also been used to follow drug-induced changes of EGSH in the malaria parasite Plasmodium falciparum ( Kasozi et al . , 2013 ) and in T . brucei ( Franco et al . , 2017b; Franco et al . , 2017a ) . By genetically fusing roGFP2 to thiol-disulfide oxidoreductases other than Grx , additional biosensors , selective for other kinds of small thiols , were generated . For instance , fusion of roGFP2 to mycoredoxin-1 yielded a probe selectively responding to changes in the mycothiol redox potential in actinomycetes ( Bhaskar et al . , 2014; Reyes et al . , 2018; Tung et al . , 2019 ) . In yeast and human cells , GSH biosynthesis is confined to the cytosol . GSH must therefore be imported into both the intermembrane space ( IMS ) and the matrix of the mitochondrion ( Calabrese et al . , 2017 ) . In yeast , the hGrx1-roGFP2 probe revealed that the cytosol , IMS and mitochondrial matrix rapidly recover after exposure of cells to an oxidant ( Kojer et al . , 2015 ) . This indicates that all three compartments either harbor a GSSG reducing system or are connected to a compartment in which GSSG is reduced . Indeed , GR localizes to both the cytosol and the mitochondrial matrix ( Calabrese et al . , 2017 ) . Trypanosomatids contain both free GSH and T ( SH ) 2 ( Ariyanayagam and Fairlamb , 2001; Krauth-Siegel and Comini , 2008 ) . This finding , however , does not provide any information about the nature and concentration of the individual thiol ( s ) within distinct subcellular compartments . Trypanosomes have a single mitochondrion that spans the complete length of the cell . In BS T . brucei , the mitochondrion has a tubular cristae-poor morphology and the parasites produce ATP by glycolysis and possibly also by substrate-level phosphorylation reactions in the mitochondrial matrix ( Zíková et al . , 2017 ) . In contrast , PC cells have a highly reticulated , cristae-rich mitochondrion and generate ATP via oxidative phosphorylation . PC T . brucei have a high demand for mitochondrial iron sulfur cluster proteins ( Lukeš and Basu , 2015 ) , the synthesis of which usually involves GSH ( Lill et al . , 2015 ) . However , virtually nothing is known about the low-molecular-weight thiols that reside in the mitochondrion of trypanosomatids ( Manta et al . , 2013; Tomás and Castro , 2013 ) . Here we studied PC T . brucei cell lines constitutively expressing a novel biosensor , Tpx-roGFP2 , in either the cytosol or mitochondrion . We show for the first time that the mitochondrial matrix of trypanosomes harbors a trypanothione-based redox metabolism . The disulfide reducing capacity in the mitochondrial matrix appears to be slightly weaker than in the cytosol , probably due to a lower T ( SH ) 2/TS2 ratio . Inhibition of T ( SH ) 2 biosynthesis by DFMO does not alter the redox steady state of the probe but impairs the ability of both the cytosol and mitochondrion to cope with exogenously applied oxidants , indicating a direct link between the trypanothione systems of the two compartments . Depletion of Tpx abolishes the response of cytosolic roGFP2-hGrx1 or roGFP2 to H2O2 but only partially affects the mitochondrial sensor response . This finding strongly suggests that the mitochondrion harbors low levels of Tpx and , in addition , another , as yet unidentified , oxidoreductase that is also able to transfer reducing equivalents from T ( SH ) 2 to the mitochondrial thiol peroxidases . Notably , our data also reveal that the high selectivity of roGFP2-hGrx1 for the GSH/GSSG redox couple is restricted to cells that do not convert GSH into additional closely related low molecular weight thiols such as Gsp and T ( SH ) 2 . In trypanosomes , roGFP2-hGrx1 predominantly equilibrates with the T ( SH ) 2/TS2 redox couple .
To generate a trypanothione-selective sensor , roGFP2 was fused to Tpx . Recombinant Tpx-roGFP2 , hGrx1-roGFP2 and roGFP2 were purified by metal affinity chromatography ( Figure 1—figure supplement 1 ) . The pre-reduced proteins were treated with GSSG , Gsp2 or TS2 and the degree of oxidation ( OxD ) of the probe was followed over time ( Figure 1—figure supplement 2 ) . Representative unprocessed fluorescence curves are shown in Figure 1—figure supplement 3 . At an equimolar concentration of 1 µM , Tpx-roGFP2 responded slightly faster to Gsp2 and TS2 than to GSSG ( Figure 1A–C ) . As previously published , hGrx1-roGFP2 was rapidly oxidized by GSSG ( Gutscher et al . , 2008; Figure 1D ) . The same sensor was also efficiently oxidized by Gsp2 ( Figure 1E ) , whereas a comparable reaction with TS2 required a 10-times higher concentration ( Figure 1F and Figure 1—figure supplement 2F ) , possibly due to the steric constrains imposed by the cyclic disulfide . Unfused roGFP2 did not react efficiently with any of the disulfides ( at 1 µM ) and at high concentrations was more efficiently oxidized by Gsp2 and TS2 than by GSSG ( Figure 1G–I and Figure 1—figure supplement 2G–I ) . In conclusion , the fusion to either Tpx or hGrx1 accelerated roGFP2 oxidation by all three disulfides . In the next step , we studied the reactivity of the oxidized probes towards GSH , Gsp , and T ( SH ) 2 ( Figure 2 and Figure 2—figure supplement 1 ) . Representative unprocessed fluorescence curves are shown in Figure 2—figure supplement 2 . Sensor proteins stored in the absence of reducing agent were fully oxidized and thus used without further treatment . Tpx-roGFP2 was readily reduced by 100 µM Gsp and 10 µM T ( SH ) 2 whereas 100 µM GSH did not cause any reaction ( Figure 2A–C ) . Reduction of Tpx-roGFP2 by even 1 mM GSH was very slow . In contrast , 500 µM T ( SH ) 2 resulted in almost complete probe reduction within five min ( Figure 2—figure supplement 1A and C ) . In comparison , hGrx1-roGFP2 showed virtually the same low reduction rate when treated with 100 µM GSH , 100 µM Gsp or 10 µM T ( SH ) 2 ( Figure 2D–F ) . Clearly , for significant reduction , ≥500 µM of either GSH or T ( SH ) 2 were required ( Figure 2—figure supplement 1D and F ) . This finding indicated that the selectivity of hGrx1-roGFP2 for GSH applies to cells with an exclusive glutathione system , but not to Kinetoplastida . Unfused roGFP2 was not even reduced by 15 mM GSH , but fully reduced by 7 . 5 mM T ( SH ) 2 ( Figure 2—figure supplement 1G and I ) . Taken together , all three sensors were most efficiently reduced by T ( SH ) 2 . The cellular concentrations of GSH and T ( SH ) 2 in PC T . brucei are approximately 100 µM and 500 µM , respectively ( see Figure 4B ) . Reduction of Tpx-roGFP2 and hGrx1-roGFP2 by 1 µM and 10 µM T ( SH ) 2 was accelerated by the presence of 100 µM or 500 µM GSH ( Figure 2—figure supplement 3A–D , compare with Figure 2—figure supplement 1C and F ) . However , even 500 µM GSH had a negligible additional effect in the presence of ≥100 µM T ( SH ) 2 . Unfused roGFP2 was only slightly reduced by 500 µM T ( SH ) 2 and the simultaneous presence of GSH had no additional effect ( Figure 2—figure supplement 3E and F ) . Finally , we followed the response of Tpx-roGFP2 and hGrx1-roGFP2 to small incremental additions of GSSG or TS2 when both GSH and T ( SH ) 2 were present at physiological concentrations . In the presence of 100 µM GSH and 500 µM T ( SH ) 2 , Tpx-roGFP2 and hGrx1-roGFP2 reached an OxD of 0 . 15 and 0 . 24 , respectively ( Figure 2—figure supplement 4 ) . This was in agreement with the data obtained for the individual thiols ( Figure 2—figure supplement 1A , C , D and F ) and supported the concept that T ( SH ) 2 is the superior disulfide reductant for both kinds of sensor . Addition of 1 µM GSSG or TS2 did not have any effect . At a starting OxD of about 0 . 2 , the incremental addition of 1 µM disulfide may be too small to cause any significant further sensor oxidation . When 25 µM disulfide was applied , TS2 oxidized both sensors to a higher degree than GSSG . Interestingly , when treated with 10 µM GSSG , hGrx1-roGFP2 displayed a small transient increase in OxD . Thus , hGrx1-roGFP2 appears to be able to sense GSSG before it rapidly equilibrates with the trypanothione redox couple . Nevertheless , a physiological situation that could give rise to an increase of GSSG , but not of TS2 , appears to be highly unlikely . Taken together , under in vitro conditions , Tpx-roGFP2 and hGrx1-roGFP2 displayed a very similar behavior and primarily responded to changes in the T ( SH ) 2/TS2 ratio . To follow probe redox changes in vivo , we generated cell lines constitutively expressing either Tpx-roGFP2 , roGFP2-hGrx1 or roGFP2 in the cytosol of the parasites ( Figure 3—figure supplement 1 ) . The domain order of the hGrx1 fusion protein was inverted , to ensure proper folding ( Figure 3—figure supplement 1C , see Materials and methods for details ) . Live cell imaging displayed an intense fluorescence that was equally distributed over the whole cell body in accordance with a cytosolic localization ( Figure 3A ) . Under normal culture conditions , all three sensors showed an OxD of close to zero indicating that they are almost fully reduced in the cytosolic environment ( Figure 3B–G ) . Exposure of the parasites to exogenous H2O2 resulted in a concentration-dependent oxidation of the probes . When 5 µM H2O2was applied , Tpx-roGFP2 was oxidized within seconds to an OxD of 0 . 8 ( Figure 3B ) . Oxidation of roGFP2-hGrx1 and roGFP2 was slightly slower and yielded a maximum OxD of about 0 . 6 and 0 . 4 , respectively ( Figure 3D and F ) . Also , the re-reduction was fastest in the case of Tpx-roGFP2 . Nine min after treating the cells with 10 µM H2O2 , the sensor was again fully reduced , whereas roGFP2-hGrx1 and roGFP2 required 15 min and 22 min , respectively . The unprocessed fluorescence curves are provided in Figure 3—figure supplement 2 . Both Tpx-roGFP2 and roGFP2-hGrx1 showed a stronger response than roGFP2 , confirming that the detection of transient redox changes by the probes is facilitated by the fused oxidoreductase . In contrast , the response of roGFP2 requires the interaction with an endogenous oxidoreductase , most probably Tpx . Making the assumption that the thiol concentration in the cytosol is comparable to that in the total cell lysate ( see Figure 4B ) , we conclude that both fusion probes respond primarily to changes in the T ( SH ) 2/TS2 redox couple . This is supported by the efficient reduction of Tpx-roGFP2 by low micromolar concentrations of T ( SH ) 2 in vitro ( Figure 2C ) and the inability of 100 µM GSH to reduce Tpx-roGFP2 as well as hGrx1-roGFP2 ( Figure 2A and D ) . To rule out direct oxidation of the sensors by H2O2 , we incubated the pre-reduced recombinant sensors with 50 µM or even 100 µM H2O2 for 11 min which resulted in an only marginal increase in OxD comparable with the non-treated controls ( Figure 3—figure supplement 3 ) . Upon subsequent addition of TS2 , all three sensors were oxidized with the same rate and to the same degree as the respective non-treated probe ( Figure 3—figure supplement 3 compare with Figure 1—figure supplement 2C , F andI ) . The data demonstrated that also the Tpx-roGFP2 sensor is insensitive towards micromolar H2O2 concentrations and confirmed previous studies on hGrx1-roGFP2 and roGFP2 ( Gutscher et al . , 2008; Kasozi et al . , 2013; Müller et al . , 2017 ) . All three sensors completely recovered when cells were treated with up to 20 µM H2O2 . A second H2O2 bolus elicited an almost identical response , demonstrating full reversibility of the response . In contrast , after exposing the cells to 50 µM H2O2 , the sensors remained partially oxidized , and a second H2O2 bolus did not trigger any further oxidation ( Figure 3B , D and F ) . However , when diamide was used as a second bolus treatment , all sensors were again oxidized , ruling out irreversible probe inactivation by hyperoxidation ( Figure 3—figure supplement 4 ) . Most likely , the cPrx was hyperoxidized and inactivated by the high H2O2 concentration , thus abrogating the Prx-catalyzed generation of TS2 responsible for oxidizing the roGFP2-based probes . When the cells were challenged with a single bolus of 10 µM or 30 µM diamide , Tpx-roGFP2 was rapidly oxidized , but to a lower maximum OxD than cells expressing roGFP2-hGrx1 or roGFP2 ( Figure 3C , E G ) . All sensors became fully oxidized upon exposure of the cells to 100 µM diamide , suggesting that they were directly oxidized . In general , probe recovery ( from comparable OxD maxima ) was slower in diamide- than in H2O2-treated cells . As observed in BS T . brucei ( Ulrich et al . , 2017 ) , diamide treatment of the PC cells may primarily lead to the formation of protein mixed disulfides which , most likely , are more slowly reduced than TS2 . After both H2O2- and diamide-stress , re-reduction of Tpx-roGFP2 was fastest , followed by roGFP2-hGrx1 and roGFP2 , corroborating the kinetic superiority of the Tpx-coupled sensor also under in vivo conditions . Treatment of T . brucei with DFMO results in impaired de novo synthesis of T ( SH ) 2 ( Bellofatto et al . , 1987; Fairlamb et al . , 1987 ) . Wildtype ( WT ) parasites and Tpx-roGFP2-expressing cells were cultured in the presence or absence of 5 mM DFMO . After 48 hr , DFMO-treated cells displayed a significant proliferation defect ( Figure 4A ) and growth almost stopped after 72 hr . No difference was observed between WT parasites and cells expressing Tpx-roGFP2 , ruling out any effect of the sensor on the parasites’ sensitivity towards DFMO . Cellular free thiol concentrations were determined by preparing total lysates , labeling the thiols with monobromobimane ( mBBr ) , and quantifying the fluorescent derivatives by HPLC analysis ( Figure 4B ) . When untreated WT cells were lysed in the presence of TCEP , we obtained total GSH and T ( SH ) 2 concentrations of 134 ± 8 µM and 521 ± 34 µM , respectively . In cells cultured for 48 hr in the presence of DFMO , the GSH and T ( SH ) 2 concentrations were 322 ± 24 µM and 210 ± 17 µM , respectively ( Figure 4B ) . Thus , in DFMO treated cells , the GSH concentration was doubled , whereas the T ( SH ) 2 concentration was halved , in agreement with published data ( Bellofatto et al . , 1987 ) . When the parasites were disintegrated in the absence of the reducing agent TCEP , the values in all samples were 10–20% lower , indicating that the cellular thiols are mainly in the reduced state as previously shown for BS T . brucei ( Ulrich et al . , 2017 ) . This finding matches the highly reduced state of the sensors expressed in the cytosol . Clearly , DFMO treatment did not cause any shift of intracellular thiol/disulfide ratios . The basal OxD of the Tpx-roGFP2 sensor was almost zero , independently if the cells had been cultured in the absence or presence of DFMO ( Figure 4C and D ) , indicating that the altered GSH and T ( SH ) 2 concentrations had no effect on the steady state thiol/disulfide ratios in the cytosol . The rate and degree of sensor oxidation triggered by exogenously applied H2O2 was also unaffected . However , recovery of reduced Tpx-roGFP2 appeared to be slightly delayed in cells that had been cultured in the presence of DFMO ( Figure 4C ) . Upon diamide stress , reductive recovery was clearly delayed in the drug-treated cells ( Figure 4D ) . Cells that were grown with DFMO for 72 hr showed an identical behavior ( Figure 4—figure supplement 1 ) . In summary , parasites with diminished cellular T ( SH ) 2 content exhibit decreased reductive capacity when exposed to an oxidative insult . PC T . brucei cell lines constitutively expressing roGFP2-hGrx1 or roGFP2 were transfected with a construct that allowed the Tet-inducible depletion of Tpx . Tpx-roGFP2-expressing cells could not be analyzed since induction of RNA interference ( RNAi ) against Tpx also depleted the sensor ( not shown ) . After 24 hr of RNAi , Tpx was significantly down-regulated ( Figure 5A and B ) , but proliferation was not yet affected . After 72 hr the cells died in accordance with the fact that Tpx is essential ( Figure 5—figure supplement 1A ) . The same behavior was previously observed for BS T . brucei , and attributed to a transient increase in the free thiol levels ( Comini et al . , 2007 ) . For Tpx-depleted PC cells , we obtained 10–20% higher GSH and T ( SH ) 2 concentrations compared to the non-induced controls ( Figure 5—figure supplement 1B ) . As expected , the sensor response towards exogenous H2O2 in non-induced cells was identical to that in the parental cell line ( Figure 5C and D , left graphs and Figure 3D and F ) . In the induced cells , the sensors still showed a basal OxD of almost zero indicating that Tpx depletion as such did not affect the cytosolic T ( SH ) 2/TS2 steady state ratio . However , both sensors reacted only marginally when the cells were challenged with H2O2 ( Figure 5C and D , right graphs ) . A direct effect on sensor sensitivity can be ruled out because only unfused roGFP2 , but not roGFP2-hGrx1 , requires the presence of Tpx to equilibrate with the T ( SH ) 2/TS2 redox couple . In the absence of Tpx , the parasite peroxidases are unable to reduce H2O2 . Under these conditions , H2O2 can only be reduced by a direct reaction with T ( SH ) 2 ( Hillebrand et al . , 2003 ) . The spontaneous formation of TS2 in this reaction is probably too slow ( and the subsequent reduction of TS2 by TR too fast ) to result in measurable probe oxidation . PC parasites lacking Px I-III undergo a ferroptosis-like cell death that starts at the mitochondrion . The cells are , however , fully viable and proliferative if the medium is supplemented with Trolox ( Bogacz and Krauth-Siegel , 2018 ) . PC T . brucei harboring Tpx-roGFP2 ( Figure 6A ) , roGFP2-hGrx1 ( Figure 6B ) or roGFP2 ( Figure 6C ) were transfected with a construct for Tet-inducible depletion of Px I-III ( Schlecker et al . , 2005 ) . RNAi was induced while keeping the cells in medium with Trolox . When control cells that were kept without Trolox showed a growth defect ( usually between 18 and 43 hr ) , the + Trolox cells were transferred into Trolox-free medium . 1 . 5 hr after Trolox withdrawal , all three cell lines still displayed a basal OxD of zero and responded to a bolus of H2O2 like the parental cell line , yet sensor reduction was slightly delayed ( Figure 6 , left graphs and Figure 3B , D and F ) . After 3 hr in the absence of Trolox , the basal OxD of all three sensors increased during the course of the experiment . The response to an exogenous H2O2 bolus was weaker and the sensors were only partially re-reduced when cells were treated with 20 µM H2O2 ( Figure 6 , central graphs ) . After keeping cells without Trolox for 4 or 4 . 5 hr , the sensors reported a basal OxD of 0 . 1–0 . 3 which further increased to 0 . 4–0 . 6 towards the end of the measurement ( Figure 6 , right graphs ) . At this time point , the cultures already contained dead cells , and only Tpx-roGFP2 still responded to an H2O2 bolus ( Figure 6A , right graph ) . Taken together , in the absence of Trolox , the cytosolic probes were increasingly oxidized over time . These findings demonstrate that cells that are unable to reduce lipid hydroperoxides suffer a decrease in the cytosolic T ( SH ) 2/TS2 ratio , associated with an impaired ability to cope with exogenously applied oxidants . Oxidation of the cytosolic trypanothione pool precedes lysis of Px-depleted cells . To identify the so far unknown mitochondrial thiol system of trypanosomes , we generated cell lines that express roGFP2-based sensors in the mitochondrial matrix ( Figure 7—figure supplement 1 ) . Live cell fluorescence microscopy confirmed the specific localization of the probes in the single mitochondrion of the parasite ( Figure 7A ) . The matrix-targeted mito-roGFP2-Tpx , mito-roGFP2-hGrx1 , and mito-roGFP2 reported a steady state OxD of about 0 . 1 ( Figure 7B–G ) . Unprocessed fluorescence curves are shown in Figure 7—figure supplement 2 . These data revealed that the mitochondrion has a trypanothione-based reducing system , but with a slightly lower reducing capacity than the cytosol . When the parasites were challenged with exogenous H2O2 , the Tpx-coupled probe displayed the fastest and strongest response ( Figure 7B , D and F ) . The overall sensitivity of the parasites towards H2O2 was unaffected by the presence of the sensor ( Figure 7—figure supplement 3 ) . All three mitochondrial sensors were oxidized to a higher degree and recovery of the reduced form was slower relative to the respective cytosolic probes ( Figure 3B , D and F ) . Interestingly , unfused mito-roGFP2 was able to respond to an exogenously applied H2O2 bolus ( Figure 7F ) . This strongly suggested the presence of an oxidoreductase which uses T ( SH ) 2 to reduce mitochondrial thiol peroxidases ( see also next section ) . In contrast to the situation in the cytosol , the mitochondrial probes responded to a second 50 µM H2O2 pulse ( Figure 7B , D and F and Figure 3B , D and F ) and diamide treatment led to stronger oxidation compared to the cytosolic sensors ( Figure 7C , E and G and Figure 3C , E and G ) . Again , reductive recovery was fastest for mito-roGFP2-Tpx . The mitochondrial sensors recovered from both oxidants more slowly than the respective cytosolic sensors . To investigate the possible influence of heat on the cellular thiol redox status , PC parasites expressing the roGFP2-based sensors , either in the cytosol or mitochondrial matrix , were cultured for 24 hr at 37°C . The cells multiplied 4- to 6-times compared to the 10-times under standard culture conditions . They did not reveal any significant difference in the basal OxD values nor in the response to exogenous H2O2 when grown at either 27°C or 37°C ( Figure 7—figure supplement 4 , compare with Figure 3B , D and F and Figure 7B , D and F ) . The proliferation defect of the parasites at high temperature does not appear to be related to any significant change in their cytosolic or mitochondrial T ( SH ) 2/TS2 redox state . In vitro , lipoamide has been shown to be able to replace T ( SH ) 2 as a reductant for L . infantum mPrx , leading to speculations about a putative involvement of 2-oxo acid dehydrogenase complexes in the mitochondrial hydroperoxide metabolism ( Castro et al . , 2008 ) . To further corroborate that the mitochondrial matrix harbors a trypanothione-based thiol system , cells expressing mito-roGFP2-Tpx were treated with DFMO ( Figure 7—figure supplement 5 ) . Inhibition of the cytosolic de novo synthesis of T ( SH ) 2 affected the recovery of the mitochondrial sensor from exogenously applied diamide ( Figure 7—figure supplement 5 ) . Taken together , we provide evidence in favor of the notion that the single mitochondrion of trypanosomes harbors a trypanothione-based thiol system , with a reducing capacity slightly lower than that in the cytosol . Based on the finding that Tpx depletion abolished the ability of cytosolic roGFP2-hGrx1 and roGFP2 to respond to an exogenous H2O2 bolus , we asked if Tpx affects also the response of the mitochondrial probes . PC T . brucei expressing mito-roGFP2-hGrx1 or mito-roGFP2 were transfected with the construct for inducible RNAi against Tpx as described above . After 24 hr of Tet-induction , the cells showed a strong depletion of the oxidoreductase ( Figure 8A and B ) , but not yet a proliferation defect . The response of the non-induced cells towards exogenous H2O2 corresponded to that of the respective parental cell line ( Figure 8C and D , left graphs and Figure 7D and F ) . Notably , in Tpx-depleted cells , both mito-roGFP2-hGrx1 and mito-roGFP2 were still able to respond to an exogenous H2O2 bolus ( Figure 8C and D , right graphs ) . However , oxidation of mito-roGFP2-hGrx1 by 5 µM or 10 µM H2O2 was slow and weak , and re-reduction of the sensor was delayed as compared to non-induced cells . When 20 µM H2O2 was applied , sensor oxidation was still increasing at the end of the experiment ( Figure 8C , right graph ) . In the case of mito-roGFP2 , oxidation by 10 µM and 20 µM H2O2 did not reach a maximum within the observation time ( Figure 8D , right graph ) . Taken together , in cells lacking Tpx , the mitochondrial sensors are still able to respond to exogenously applied H2O2 , in sharp contrast to the situation in the cytosol ( Figure 5C and D , right graphs ) . Nevertheless , the response was significantly weaker than in cells proficient in Tpx . Based on these data we hypothesize that a small portion of cytosolic Tpx may be present in the mitochondrial matrix , as suggested previously by immune-gold labeling ( Tetaud et al . , 2001 ) , and that the mitochondrion harbors another oxidoreductase that is able to transfer electrons between the trypanothione redox couple and mitochondrial thiol peroxidases . Depletion of mPrx in PC T . brucei over three days results in undetectable levels of the protein . At this time point , the cells still proliferate like WT cells , whereas after seven days of depletion , proliferation is severely affected and the parasites display a highly elongated shape ( Bogacz et al . unpublished data ) . Cells expressing Tpx-roGFP2 or uncoupled roGFP2 in either the mitochondrial matrix or the cytosol were transfected with a construct for Tet-inducible depletion of mPrx . RNAi was induced for three or seven days . The altered morphology of the mPrx-depleted cells had no effect on the expression level and subcellular targeting of the sensors ( Figure 9A ) . Non-induced cells displayed the same sensor response as the respective parental strain ( Figure 9B–E , left graphs compare with Figure 7B , D and F and Figure 3B , D and F ) . In mPrx-depleted cells , the basal OxD of all sensors was unchanged ( Figure 9B–E ) indicating that neither the mitochondrial nor the cytosolic T ( SH ) 2/TS2 ratio was affected . However , depletion of mPrx had a strong impact on the sensor response when the cells were exposed to exogenous H2O2 . In cells subjected to RNAi for three days , the mitochondrial sensors showed virtually no oxidation when 5 µM or 10 µM H2O2 was applied . Addition of 20 µM H2O2 resulted in a slow and minor increase in OxD , together with a delayed reductive recovery ( Figure 9B and C , middle graphs ) . After seven days of mPrx depletion , oxidation of the mitochondrial sensors was again increased and re-reduction even more delayed ( Figure 9B and C , right graphs ) . Interestingly , RNAi against mPrx had also a strong effect on the response of the cytosolic sensors . In cells subjected to mPrx depletion for three days and exposed to a bolus of 20 µM H2O2 , re-reduction of cytosolic Tpx-roGFP2 was slowed down and roGFP2 showed a slightly increased OxD peak ( Figure 9D and E , middle graphs ) . In cells induced for seven days and treated with 20 µM H2O2 , Tpx-roGFP2 displayed an OxD peak lasting for at least 10 min . At all H2O2 concentrations used , re-reduction of cytosolic Tpx-roGFP2 was markedly attenuated relative to non-induced cells ( Figure 9D , right and left graphs ) . At this time point , also roGFP2 was oxidized to a higher degree and for a longer time ( Figure 9E , right graph ) . Thus , depletion of mPrx affected the response of mito-roGFP2-Tpx and mito-roGFP2 to a challenge with exogenous H2O2 . This indicates that mPrx is involved in the reduction of exogenously applied H2O2 and is the main mitochondrial H2O2-metabolising thiol peroxidase . However , the mitochondrial sensor response was not completely lost . Probably , H2O2 can still be reduced by Px III ( Bogacz and Krauth-Siegel , 2018; Diechtierow and Krauth-Siegel , 2011; Schaffroth et al . , 2016 ) . In cells that were subjected to long-term mPrx depletion , reduction of exogenously applied H2O2 in the cytosol was strongly delayed . One may speculate that this is due to a general impairment of metabolism in these proliferation arrested cells .
The expression of Tpx-roGFP2 , a novel biosensor , in African trypanosomes allowed for the first time to follow real-time changes of the trypanothione redox state and , most importantly , to demonstrate that the parasites harbor a mitochondrial T ( SH ) 2/TS2 system . Remarkably , hGrx1-roGFP2 also equilibrated with the parasite thiol/disulfide couples , albeit less efficiently than the Tpx-roGFP2 sensor . As expected , unfused roGFP2 showed an overall low reactivity . Nevertheless , T ( SH ) 2 was the most efficient reductant for all three sensors . This finding does not appear to be related to the redox potential as the E0’ of T ( SH ) 2 has been reported to be only slightly more negative ( −242 mV ) than that of GSH ( −230 mV ) ( Fairlamb and Cerami , 1992 ) . However , formation of an intramolecular disulfide ( TS2 ) can be expected to be kinetically more favorable than the formation of intermolecular disulfides ( GSSG , Gsp2 ) . Another crucial point is that the thiol pK-value of T ( SH ) 2 is at least one pH unit lower than that of GSH . Thus , at physiological pH values , a significantly higher portion of T ( SH ) 2 is present in the reactive deprotonated form ( Manta et al . , 2013; Moutiez et al . , 1994 ) . When treated with small increments of TS2 or GSSG in the presence of cellular GSH and T ( SH ) 2 concentrations , both Tpx-roGFP2 and hGrx1-roGFP2 were oxidized , with TS2 yielding a slightly faster response . As trypanosomes lack glutathione-dependent peroxidases and GSH is regenerated from GSSG by thiol-disulfide exchange with T ( SH ) 2 , an in vivo situation that could lead to the specific formation of GSSG is not known . In the physiological context of trypanosomes , both Tpx-roGFP2 and hGrx1-roGFP2 equilibrate with the T ( SH ) 2/TS2 redox couple . The reactivity of Grxs towards T ( SH ) 2 has been observed in previous studies showing that the parasite dithiol reduces E . coli Grx1 as efficiently as dithioerythritol ( Ceylan et al . , 2010 ) . Tpx-roGFP2 , roGFP2-hGrx1 and roGFP2 expressed in PC trypanosomes reported a fully reduced cytosol , similar to other organisms ( Gutscher et al . , 2008; Kasozi et al . , 2013; Loi et al . , 2017; Morgan et al . , 2013 ) and the reduced state was rapidly restored after a challenge with H2O2 . The sensor response to exogenous H2O2 is most likely mediated by cellular thiol peroxidases ( Meyer and Dick , 2010 ) . The parasite enzymes use the T ( SH ) 2/Tpx system for re-reduction and thus enzymatically generate TS2 ( Castro and Tomás , 2008; Krauth-Siegel and Comini , 2008 ) which is then reported by the biosensors . Tpx-roGFP2 showed the fastest and strongest response , but also roGFP2-hGrx1 and even unfused roGFP2 reversibly responded to exogenous oxidants . This strongly suggested that all three sensors reacted to changes of the T ( SH ) 2/TS2 ratio in vivo . BS T . brucei expressing hGrx1-roGFP2 in the cytosol show sensor oxidation in response to treatment with different anti-parasitic compounds which was proposed to reflect a decrease in the GSH/GSSG ratio ( Franco et al . , 2017b; Franco et al . , 2017a ) . However , as shown here , it is more likely that the compounds affected the cytosolic T ( SH ) 2/TS2 redox state . When PC cells were treated with diamide , Tpx-roGFP2 displayed a lower maximum OxD and shorter lag phase compared to roGFP2-hGrx1 and roGFP2 , in accordance with the efficient in vitro reduction of the sensor by low micromolar concentrations of T ( SH ) 2 . In BS T . brucei , H2O2-treatment results primarily in the formation of free disulfides , whereas in the case of diamide , the main products are protein-bound thiols ( Ulrich et al . , 2017 ) . The generally slower probe re-reduction after diamide- relative to H2O2-treatment may be due to a slower reduction of protein-mixed disulfides than of TS2 by TR . In Staphylococcus aureus and Corynebacterium glutamicum , depletion of bacillithiol and mycothiol , respectively , leads to basal sensor oxidation ( Loi et al . , 2017; Tung et al . , 2019 ) . When PC T . brucei were grown in the presence of DFMO , the cellular GSH was almost doubled and T ( SH ) 2 dropped to 40% of the concentration in untreated cells . Nevertheless , the basal OxD of Tpx-roGFP2 remained close to zero . This is consistent with data from BS trypanosomes , where depletion of TryS also causes an increase of GSH and a decrease of T ( SH ) 2 ( Comini et al . , 2004 ) without affecting the cellular thiol-disulfide ratio ( Ulrich et al . , 2017 ) . Notably , in the DFMO-treated cells , the total thiol concentration ( GSH plus 2 x T ( SH ) 2 ) was lowered by 63% . This further corroborates that the cytosolic redox potential is unaffected by these changes in thiol concentration . Indeed , the total acid-soluble thiol content can vary by up to 20-fold between life cycle stages , yet intact trypanosomes and Leishmania differ only 2-fold in their capacity to metabolize H2O2 between stages ( Ariyanayagam and Fairlamb , 2001 ) . In the DFMO-treated cells , recovery of reduced Tpx-roGFP2 after an exogenous oxidative challenge was slowed down whereby the effect was less pronounced with H2O2 compared to diamide . Thus , the cytosolic peroxidases were still able to efficiently reduce H2O2 , even at the significantly lower T ( SH ) 2 level . In an Arabidopsis thaliana mutant with 20% GSH compared to WT cells , the cellular GSH/GSSG ratio is unaffected . However , cytosolic roGFP2 is slightly oxidized because the lowered glutathione level shifts the glutathione redox potential to less negative values ( Meyer et al . , 2007 ) . The redox state of Tpx-roGFP2 was unaffected in the DFMO-treated parasites . Oxidation of T ( SH ) 2 results in the formation of an intramolecular disulfide . This distinguishes trypanosomatids from all other organisms . Independently of the nature of the individual thiol , mammals , yeast , plants and bacteria employ a monothiol for regulating the cellular redox homeostasis which renders the redox potential sensitive to changes in the overall thiol concentration ( Loi et al . , 2017; Meyer et al . , 2007; Tung et al . , 2019 ) . In the case of the T ( SH ) 2/TS2 couple , the redox potential and corresponding steady state sensor response are only determined by the thiol-disulfide ratio . P . falciparum strains with elevated GSH levels display a lower sensor OxD ( Kasozi et al . , 2013 ) reflecting an increased reducing capacity . Depletion of Tpx resulted in a 10–20% increase of the intracellular GSH and T ( SH ) 2 levels in PC T . brucei . As the steady state OxD of the cytosolic sensors was almost zero already in the non-induced cells , a further decrease in the Tpx-depleted cells could not be observed and was also not expected . In the absence of Tpx , both roGFP2 and roGFP2-hGrx1 did not respond to exogenous H2O2 . Tpx depletion abolishes the electron transfer from T ( SH ) 2 onto the parasite peroxidases and thus the enzymatic production of TS2 . This provided additional evidence that in trypanosomes , roGFP2-hGrx1 senses changes in the T ( SH ) 2/TS2 system . In cells in which the Px-type peroxidases were depleted , all three biosensors showed a dramatic increase of the steady state OxD . Thus , lipid peroxidation rapidly translates into the oxidation of the cytosolic trypanothione pool . When targeted to the mitochondrion , the redox sensors revealed an overall similar response as the cytosolic probes . This was strong evidence that the mitochondrial matrix harbors a trypanothione-based thiol metabolism . However , the slightly elevated basal OxD of the mitochondrial probes indicated that the mitochondrion is less reducing than the cytosol , in accordance with data reported for yeast cells ( Kojer et al . , 2012 ) . When the parasites were exposed to exogenous oxidants , all three mitochondrial probes were oxidized to a higher degree and were more slowly re-reduced than the respective cytosolic sensors . Also in yeast cells , the matrix glutathione pool was found to be significantly more sensitive to H2O2-induced oxidation than the cytosolic glutathione pool even though the H2O2 concentration in the matrix is lower than in the cytosol ( Calabrese et al . , 2019 ) . This indicates that the reducing capacity in the single mitochondrion of trypanosomes is comparably low which could be due to a diminished T ( SH ) 2/TS2 ratio and/or low total thiol concentration . In contrast to the cytosolic sensors , the mitochondrial probes responded to a second pulse of 50 µM H2O2 . This suggested that the mPrx is less prone to over-oxidation than the cPrx as shown for mammalian Prx3 ( Cox et al . , 2010 ) ; or less H2O2 reaches the mitochondrion due to the buffering capacity of the cytosol ( Calabrese et al . , 2019; Morgan et al . , 2016 ) . To analyze this point in more detail , future studies should include probes that directly record changes in the H2O2 concentration ( Morgan et al . , 2016 ) . The de novo synthesis of T ( SH ) 2 and the reduction of TS2 are confined to the cytosol ( Oza et al . , 2005; Smith et al . , 1991 ) . Therefore , the question arises how T ( SH ) 2 reaches the mitochondrial matrix and is then kept in the reduced state . So far , specific transporters for T ( SH ) 2/TS2 have not been identified . In yeast and human cells , the biosynthesis of GSH is also cytosolic , but GR occurs in both the cytosol and mitochondrial matrix ( Calabrese et al . , 2017 ) . In rabbit kidney mitochondria and mitoplasts , the dicarboxylate carrier ( DIC ) and oxoglutarate carrier ( OGC ) were identified as GSH transporters in the inner mitochondrial membrane ( Chen et al . , 2000 ) , whereas transport of GSH in Lactococcus lactis is independent of the two anion carriers ( Booty et al . , 2015 ) . In T . brucei , the mitochondrial carrier protein 12 ( MCP12 ) was shown to act as carboxylate and tricarboxylate carrier , thereby fulfilling the functions of DIC and OGC ( Colasante et al . , 2009; Colasante et al . , 2018 ) . Due to its overall positive charge , it is highly unlikely that T ( SH ) 2 is imported by these transporters . Regeneration of reduced mitochondrial hGrx1-roGFP2 in oxidatively challenged yeast cells requires the matrix-localized GR ( Kojer et al . , 2012 ) . This is not the case in trypanosomes . The basal OxD of the sensors in the matrix was only slightly higher than in the cytosol and the probes reversibly responded to exogenous oxidative challenges . One may thus speculate that trypanosomes possess a protein that mediates an exchange of TS2 and T ( SH ) 2 over the inner mitochondrial membrane . The finding that unfused mito-roGFP2 responded to exogenous H2O2 pointed to the presence of an oxidoreductase that facilitates the equilibration of the sensor with the mitochondrial T ( SH ) 2/TS2 couple and is capable of transferring reducing equivalents from T ( SH ) 2 onto the mitochondrial peroxidases . Nevertheless , re-reduction of mito-roGFP2 was slow when compared to cytosolic roGFP2 . The catalytic capacity of the mitochondrial oxidoreductase appears to be limited due to either low reactivity or low concentration . Tpx-depletion affected the response of mito-roGFP2 and mito-roGFP2-hGrx1 . One putative explanation may be that a minor fraction of Tpx resides in the mitochondrion . This would be in agreement with immuno-electron microscopy data which revealed some gold particles in the mitochondrion ( Tetaud et al . , 2001 ) although immunofluorescence studies and fractionated cell lysis displayed a cytosolic localization of Tpx ( Currier et al . , 2019; Ebersoll et al . , 2018; Tetaud et al . , 2001 ) . Notably , Tpx-depletion did not fully abolish the response of the mitochondrial sensors to exogenous H2O2 . Probably , another oxidoreductase can mediate the electron transfer between T ( SH ) 2 and the mitochondrial peroxidases . With respect to the identity of this protein we can only speculate . A second T . brucei Tpx is located in the outer mitochondrial membrane facing the cytosol ( Castro et al . , 2010 ) . Other proteins that might catalyze the electron transfer from T ( SH ) 2 are Grxs and Trxs . T . brucei Grx1 is a cytosolic protein . Grx2 is located in the IMS and is unable to replace Tpx in the Px-catalyzed peroxidase assay ( Ebersoll et al . , 2018; Ceylan et al . , 2010 ) . T . brucei Trx1 lacks a mitochondrial pre-sequence , but a genome-wide localization study revealed the N-terminally mNeonGreen-tagged protein in the mitochondrion ( Dean et al . , 2017 ) . Trx1 reduces Px and cPrx albeit with much lower efficiency than Tpx ( Hillebrand et al . , 2003; Schmidt and Krauth-Siegel , 2003 ) and was inactive in an mPrx assay ( unpublished data ) . A recently characterized mitochondrial Trx2 lacks reductase activity ( Currier et al . , 2019 ) . In addition , human Trx1 does not engage in thiol-disulfide exchange with roGFP2 , not even when genetically fused to the redox sensor ( Gutscher et al . , 2008; Meyer and Dick , 2010 ) . An importome study identified 21 new mitochondrial proteins with annotated oxidoreductase activity ( Peikert et al . , 2017 ) . Only three proteins appear to be possible candidates for the transfer of electrons from the trypanothione redox couple to the mitochondrial tryparedoxin peroxidases . These are Trx1 , Grx2 and a putative glutathione-S-transferase/glutaredoxin ( Tb927 . 7 . 3500 ) . The latter protein harbors a mitochondrial targeting sequence and a CxxC motif but has not yet been characterized . Depletion of mPrx affected neither the mitochondrial nor the cytosolic T ( SH ) 2/TS2 steady state , similar to various peroxidase deletion mutants of C . glutamicum which also show no alteration in the basal mycothiol redox potential ( Tung et al . , 2019 ) . After three days of RNAi against mPrx , the protein was strongly depleted but the cells still proliferated like WT parasites . When these cells were treated with H2O2 , mitochondrial roGFP2-Tpx and roGFP2 displayed a markedly diminished response . Thus , in PC T . brucei , mPrx is the key peroxidase that couples H2O2 reduction to trypanothione oxidation in the mitochondrial matrix . This contrasts with the situation in promastigote Leishmania where the enzyme neither plays a role for proliferation nor for detoxification of H2O2 ( Castro et al . , 2011; Teixeira et al . , 2015 ) , but compares with yeast cells where H2O2-induced matrix glutathione oxidation is dependent on the presence of the mitochondrial 1-Cys-peroxiredoxin ( Calabrese et al . , 2019 ) . After seven days of mPrx-depletion , the parasites adapted a highly elongated shape and virtually stopped proliferation . At this time point , the cytosolic sensors displayed an extremely long lag phase before they recovered from H2O2-induced oxidation . Apparently , the capacity of the cytosol to cope with exogenous oxidants was severely hampered in these growth-arrested cells , in agreement with the notion that there is a cross-talk between the two compartments . Future work should focus on the identification of the proteins that allow the transport/exchange of T ( SH ) 2 and TS2 in and out of the mitochondrion .
Cystatin , diamide , GSH , hemin , penicillin/streptomycin , pepstatin , phleomycin , phenylmethylsulfonyl fluoride ( PMSF ) , Tet , and Trolox were from Sigma-Aldrich , Munich , Germany . Geneticin disulfate ( G418 ) , hygromycin B , and NADPH were purchased from Carl Roth , Karlsruhe , Germany and GSSG from Serva , Heidelberg , Germany . DFMO was obtained from Cayman Chemicals , Ann Arbour , Michigan , dithiothreitol ( DTT ) from Biomol , Hamburg , Germany , fetal calf serum ( FCS ) from Biochrome , Berlin , Germany and DNaseI , H2O2 , lysozyme , and mBBr from Merck Millipore , Darmstadt , Germany . Isopropyl-β-D-thiogalacto-pyranoside ( IPTG ) was ordered from Peqlab , Erlangen , Germany and Bond Breaker tris ( 2-carboxyethyl ) phosphine ( TCEP ) solution from Thermo Scientific , Schwerte , Germany . T ( SH ) 2 and Gsp as well as TS2 and Gsp2 ( Comini et al . , 2009; Leroux et al . , 2013 ) , tag-free recombinant T . brucei TR ( Persch et al . , 2014 ) , and human GR ( Nordhoff et al . , 1993 ) were prepared as described . Polyclonal rabbit antibodies against T . brucei Tpx ( Schlecker et al . , 2005 ) and guinea pig antibodies against mPrx ( Ebersoll et al . , 2018 ) were generated previously . HRP-conjugated goat antibodies against rabbit IgGs were from Thermo Scientific , Heidelberg , Germany , and donkey antibodies against guinea pig IgGs from Merck . The pQE-60_rogfp2 ( Addgene #65046 ) , pQE-60_hgrx1-rogfp2 ( Addgene #64799; Gutscher et al . , 2008 ) , and pCaSpeR4_mito-rogfp2-hgrx1 ( Addgene #65000; Albrecht et al . , 2014 ) plasmids were obtained as described . The pHB 136 . 1 TbTXNH6 ( Budde et al . , 2003 ) and pHD vectors were kindly provided by Drs Leopold Flohé , Montevideo , Uruguay , and Christine Clayton , Heidelberg , Germany , respectively . Three liter ( 6 × 500 ml ) cultures of recombinant BL21 ( DE3 ) E . coli in 2 x YT medium with 100 µg/ml carbenicillin were grown overnight at 37°C and 180 rpm . At an OD600 of about 0 . 5–0 . 6 , expression of rogfp2 , hgrx1-rogfp2 , or tpx-rogfp2 was induced by 200 µM IPTG . After overnight cultivation at 18°C , the bacteria were harvested by centrifugation , resuspended in 15 ml buffer A ( 50 mM sodium phosphate , 300 mM NaCl , pH 7 . 5 ) per liter of culture , containing 50 µM PMSF , 150 nM pepstatin , 4 nM cystatin , 1 . 5 mg lysozyme and DNase , and disintegrated by sonification . After centrifugation , the supernatant was kept on ice and the extraction repeated . The combined supernatants from the 3 l culture were loaded onto an 8 ml Ni-NTA Superflow matrix ( Qiagen , Venlo , Netherlands ) equilibrated in buffer A . The column was washed with 48 ml buffer A containing 10 mM imidazole , 32 ml buffer A with 45 mM imidazole , and a 40 ml gradient to 90 mM imidazole . The proteins were eluted with 125 mM imidazole in buffer A . Their purity was assessed by SDS-PAGE and Coomassie staining . Fractions containing pure protein were combined , the buffer was exchanged to 100 mM Tris , 200 mM NaCl , 50 µM EDTA , pH 7 . 5 and the proteins were stored at −80°C . The parasites used in this work are PC T . brucei brucei of the 449 cell line , a descendant of the Lister strain 427 ( Cunningham and Vickerman , 1962 ) . These cells stably express the Tet-repressor ( Biebinger et al . , 1997 ) . The cells were cultured in MEM-Pros medium supplemented with 10% ( v/v ) heat-inactivated FCS , 7 . 5 µg/ml hemin , 50 U/ml penicillin , 50 µg/ml streptomycin , and 0 . 5 µg/ml phleomycin at 27°C . Cells constitutively expressing the sensors were kept in the presence of 30 µg/ml G418 . Cell lines harboring an inducible RNAi construct were grown in the presence of 50 µg/ml hygromycin . RNAi was induced by addition of 1 µg/ml Tet . Proliferation was monitored starting with 5 × 105 cells/ml and diluting the cultures to the initial density every 24 hr . For experiments with Px-depleted cells , the cultures contained 100 µM Trolox . The effect of Px-depletion ( see next section ) on the sensor response was analysed after removal of the antioxidant . To study the effect of DFMO , a stock solution of 10 mg/ml DFMO was prepared in PBS . 2–3 × 105 cells/ml were treated with 5 mM DFMO and proliferation was followed . Living cells were counted in a Neubauer chamber . To obtain parasites that constitutively express the roGFP2 probes in the cytosol or mitochondrion , 4 × 107 PC cells were transfected with 10 µg of the respective NotI-linearized and ethanol-precipitated pHD1991 plasmid by electroporation . After overnight cultivation without selecting antibiotic , 50 µg/ml G418 was added and clones were selected for resistance by serial dilutions as outlined previously ( Musunda et al . , 2015 ) . Expression and correct subcellular localization of the fluorescent redox sensors was verified by live cell imaging . Cell lines with inducible RNAi against Px I-III , Tpx , or mPrx were obtained by transfecting WT or roGFP2-sensor-expressing cells with NotI-linearized pHD678_pxIII ( Schlecker et al . , 2005 ) , pHD678_tpx ( this work ) , or pHD678_mprx ( unpublished ) . Selection was done with 150 µg/ml hygromycin . 1 . 2 × 106 cells were collected and washed with PBS . The roGFP2 fluorescence was examined under a Carl Zeiss Axiovert 200 M microscope equipped with an AxioCam MRm digital camera using the AxioVision software ( Zeiss , Jena , Germany ) . The sensor-expressing Tpx RNAi cells cultivated in the presence or absence of Tet were harvested and boiled for 5 min in reducing SDS sample buffer . Cell lysates equivalent to 2 × 106 cells per lane were subjected to SDS-PAGE on 12% gels . Proteins were transferred onto PVDF membranes and developed with antibodies against T . brucei Tpx ( 1:2 , 000 ) and mPrx ( 1:10 , 000 ) and secondary HRP-conjugated donkey anti rabbit and goat anti guinea pig antibodies ( 1:20 , 000 ) , respectively . The reduced and oxidized forms of roGFP2 have distinct fluorescence excitation maxima ( Dooley et al . , 2004 ) . After excitation at corresponding wavelengths ( 400 and 485 nm ) , the emission at 520 nm is measured . The measurements were conducted in 96-well black/clear bottom imaging plates ( BD Falcon 353219 ) in a PHERAstar FS plate reader with build-in injectors ( BMG Labtech , Ortenberg , Germany ) and optimal focus and gain adjustment . The degree of sensor oxidation ( OxD ) was calculated using the following formula ( Meyer and Dick , 2010 ) and plotted against time . OxD=I400sample*I485red-I400red*I485sampleI400sample*I485red-I400sample*I485ox+I400ox*I485sample-I400red*I485sample I represents the fluorescence emission at 520 nm after excitation at the indicated wavelength , red , fully reduced control and ox , fully oxidized control . The purified recombinant roGFP2-proteins were diluted in assay buffer ( 100 mM sodium phosphate , 100 mM NaCl , 50 µM EDTA , pH 7 . 0 ) to a final concentration of 1 µM in a total volume of 100 µl per well . To ensure full reduction or oxidation of the control samples , 10 mM DTT or 1 mM diamide was added to the respective well . For oxidation experiments , proteins were pre-treated with 20 mM DTT for 30 min at room temperature and DTT was removed by Zeba Spin Desalting Columns ( Thermo Scientific ) . Except stated otherwise , reduction experiments contained NADPH , TR , and/or hGR in excess to achieve full reduction of the respective thiol . The measurements were done at 25°C . Parasites constitutively expressing roGFP2 , roGFP2-hGrx1 , or Tpx-roGFP2 in the cytosol or roGFP2 , roGFP2-hGrx1 , or roGFP2-Tpx in the mitochondrial matrix were harvested , washed and resuspended in medium without hemin and phenol red . A 200 µl suspension corresponding to 1 × 107 cells , was transferred into each well and measured at 27°C . For full reduction and oxidation of the sensors , 1 mM DTT and 3 mM diamide , respectively , was added . The respective cells without sensor were used as fluorescence background control . Cells were subjected to exogenous oxidative stresses by injecting different concentrations of H2O2 or diamide . Untreated cells served as control . The total cellular free thiols and thiols plus disulfides were determined as described previously for BS T . brucei ( Ulrich et al . , 2017 ) . In short , 1 × 108 PC cells were harvested , washed with PBS , and proteins were precipitated by tri ( chloro ) acetic acid . The supernatant was split in two samples . The sample for free thiols was directly used for mBBr derivatization . The other one was treated with TCEP followed by labeling with mBBr . The samples were subjected to HPLC analysis and fluorescence detection using a PerfectSil 300 ODS C18 column . Quantification of the thiols was done by comparing integrated peak areas of the sample and respective standards . For calculation of the cellular concentration , values were normalized to the cell number and cell volume , assuming a volume of 96 fl for PC parasites ( Roldán et al . , 2011 ) . All experiments were performed three times on separate days as independent biological replicates , except stated otherwise . The data shown represent the mean ± SD and were evaluated using GraphPad Prism ( GraphPad Software , La Jolla , CA ) .
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 .
Trypanosoma brucei are single-celled parasites that cause human sleeping sickness and animal diseases . Like in other organisms , the parasite contains different compartments , each having several specific roles . The mitochondrion is the compartment that provides most of the energy needed to keep the cell alive . Many cellular processes , such as those that happen in the mitochondrion , produce compounds including hydrogen peroxide that can cause ‘oxidative damage’ . To counteract this , cells make small molecules called thiols . These thiols provide ‘reducing’ power to chemically balance out the oxidative damage . Trypanosomes have an unusual thiol system that relies on a molecule called trypanothione . Trypanosoma brucei cells make trypanothione in the cytosol , the fluid which surrounds all cellular compartments; here it is also used up with the help of a protein called tryparedoxin . However , it was not known which thiols are present in the mitochondrion . Ebersoll et al . have now made a molecular sensor that can detect trypanothione . The sensor includes a fluorescent protein , which changes its brightness based on its oxidation state , fused to the tryparedoxin protein . This probe could either be put in the cytosol or mitochondrion of Trypanosoma brucei cells . Treating the cells with hydrogen peroxide changed the fluorescence of the biosensor . Trypanosoma brucei cells without tryparedoxin protein in their cytosol still responded to an oxidative challenge in the mitochondrion . The experiments reveal that trypanosomes do have a mitochondrial trypanothione system . This new fluorescent biosensor will be used to study how other cellular compartments deal with oxidative conditions . The tests will reveal how different compartments communicate with each other to counteract the stress . The sensor could also be used to determine how anti-parasite drugs affect the cells’ trypanothione system .