Tryparedoxin peroxidase-deficiency commits trypanosomes to ferroptosis-type cell death

  1. Marta Bogacz
  2. R Luise Krauth-Siegel  Is a corresponding author
  1. Biochemie-Zentrum der Universität Heidelberg, Germany
8 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Ferroptosis inhibitors protect the Px I-III KO cells.

The mutant cells were either kept in Trolox-supplemented medium or transferred into medium containing 100 µM Dfx, 100 nM ferrostatin-1 or 200 nM liproxstatin-1 or no addition. (A) Every hour viable …

https://doi.org/10.7554/eLife.37503.003
Figure 1—figure supplement 1
Protection of Px I-III KO cells by Trolox, Dfx and α-Toc.

The cells were incubated in the presence of (A) different concentrations of Trolox, (B) various concentrations of Dfx, (C) in medium supplemented with 10 µM Trolox or 20 µM Dfx alone or in …

https://doi.org/10.7554/eLife.37503.004
Figure 2 with 1 supplement
Px I-III KO cells are affected in mitochondrial matrix – but not outer membrane – immuno-staining.

The parasites were incubated for 1 or 2 hr in medium ± Trolox, treated with MitoTracker (red) and subjected to immunofluorescence microscopy using antibodies against (A) mtTXNPx (green) or (B) VDAC …

https://doi.org/10.7554/eLife.37503.005
Figure 2—figure supplement 1
Mitochondrial damage precedes plasma membrane leakage.

The Px I-III KO cells were incubated ± Trolox for up to 4 hr, stained with MitoTracker or propidium iodide and subjected to flow cytometry. (A) Forward- (FSC) and side-scatter (SSC) DotPlots of …

https://doi.org/10.7554/eLife.37503.006
Figure 3 with 1 supplement
PC Px I-III KO T. brucei undergo strinking ultrastructural changes at their single mitochondrion.

Parasites kept in the presence or absence of Trolox were fixed, processed and subjected to transmission electron microscopy as described under Materials and methods. Electron micrographs of …

https://doi.org/10.7554/eLife.37503.007
Figure 3—figure supplement 1
Short-term incubation of Px I-III KO cells in Trolox-free medium results in mitochondrial alterations without changes at other organelles, whereas prolonged incubation can finally lead to plasma membrane blebs.

Electron micrographs of Px I-III KO parasites kept for (A) 1 hr and (B) 2 hr in Trolox-free medium. (A) The intact elongated mitochondrion is darker than the cytosol. Other subcellular structures …

https://doi.org/10.7554/eLife.37503.008
Figure 4 with 1 supplement
MitoSOX Red exerts a dual effect on the Px I-III KO cells, sensing oxidant production and acting as protecting agent.

(A) The cells were pre-loaded with MitoSOX in Trolox-containing medium, transferred into medium ± Trolox, incubated for 2 hr, and stained with MitoTracker Green. Nuclear (large dot) and kinetoplast …

https://doi.org/10.7554/eLife.37503.009
Figure 4—figure supplement 1
The P2 sub-population displays the strongest increase in MitoSOX fluorescence.

The Px I-III KO cells were loaded with MitoSOX for 10 min in Trolox-supplemented medium, washed with PBS and re-suspended in medium ± Trolox. After 5 hr incubation, the cells were stained with DAPI …

https://doi.org/10.7554/eLife.37503.010
Cellular ATP levels rapidly decrease when Px I-III KO cells are in medium lacking a protecting agent.

The cells were incubated ± Trolox in (A) SDM-79 medium or (B) MEM-Pros medium. After 0 to 4 hr, aliquots of each sample were removed and (A) cells with normal morphology or only highly motile cells …

https://doi.org/10.7554/eLife.37503.011
Lipid peroxidation in the Px I-III KO cells originates at the mitochondrion.

(A) Px I-III KO parasites in Trolox-supplemented medium were incubated for 1.5 hr with MitoPerOx, stained with MitoTracker Green and subjected to life cell fluorescence microscopy. Representative …

https://doi.org/10.7554/eLife.37503.012
Px I-III KO cells that overexpress mitochondrial SODA require less Dfx for survival.

(A) Total lysates of 5 × 106 Px I-III KO/SODA-myc cells cultured for 18 hr ± tetracycline (Tet) were subjected to Western blot analysis using antibodies against myc as well as aldolase for loading …

https://doi.org/10.7554/eLife.37503.013
Mitochondrial iron is involved in the death phenotype of Px I-III-deficient cells.

(A) Cells were pre-loaded with 150 nM RPA or 10 nM RPAC in PBS, re-transferred into Trolox-supplemented medium, stained with MitoTracker Green and subjected to life cell imaging. Merge, overlay of …

https://doi.org/10.7554/eLife.37503.014

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Gene
(Trypanosoma
brucei brucei)
sodATriTrypDatabase ID:
Tb427.05.3350
Cell line
(Trypanosoma
brucei brucei)
WT PC and BSPMID: 9108552WT PC and BS 449
Lister strain 427
Culture-adapted T. brucei
strains stably expressing
the tetracycline repressor
Cell line
(Trypanosoma
brucei brucei)
PC Px I-III KOPMID: 26374473PC WT cells in which both
alleles of the complete
px locus are replaced by
resistance casettes
(blasticidin and puromycin)
Cell line
(Trypanosoma
brucei brucei)
PC Px I-III KO/SODA-mycthis workPC Px I-III KO cells that
contain a Tet-inducible
construct for SODA-c-myc2
overexpression
AntibodyRabbit anti-cTXNPxPMID: 20826821IF (1:1000)
AntibodyGuinea pig anti-mTXNPxPMID: 29413965IF (1:1000)
AntibodyRabbit anti-aldolaseChristine Clayton,
Heidelberg, Germany
WB (1:20000)
AntibodyRabbit anti-VDACAndré Schneider,
Bern, Switzerland
IF (1:500)
AntibodyMouse anti-c-Myc
(monoclonal)
Santa Cruz
Biotechnology
sc-40,
RRID:AB_627268
WB, IF (1:200)
AntibodyHRP-conjugated
goat anti-mouse IgGs
Santa Cruz
Biotechnology
sc-2005,
RRID:AB_631736
WB (1:5000)
AntibodyHRP-conjugated
goat anti-rabbit IgGs
Santa Cruz
Biotechnology
sc-2004,
RRID:AB_631746
WB (1:10000)
AntibodyAlexa Fluor 488
goat anti-guinea pig IgGs
Thermo Fisher
Scientific
A11073,
RRID:AB_142018
IF (1:1000)
AntibodyAlexa Fluor 488
goat anti-mouse IgGs
Thermo Fisher
Scientific
A11001,
RRID:AB_2534069
IF (1:250)
AntibodyAlexa Fluor 488
goat anti-rabbit IgGs
Thermo Fisher
Scientific
A11008,
RRID:AB_143165
IF (1:1000)
AntibodyAlexa Fluor 594
goat anti-rabbit IgGs
Thermo Fisher
Scientific
A11012,
RRID:AB_141359
IF (1:1000)
Recombinant
DNA reagent
pHD1700/grx2-
c-myc2 (plasmid)
PMID: 20826822
Recombinant
DNA reagent
pHD1700/sodA-
c-myc2 (plasmid)
this work
Sequence-based
reagent
5'CGATAAGCTTATG
AGGTCTGTCATGATGC3'
this workPrimer for amplification
of sodA from genomic
T. brucei DNA
Sequence-based
reagent
5'CGATGGATCCCTT
CATAGCCTGTTCATAC3'
this workPrimer for amplification
of sodA from genomic
T. brucei DNA
Peptide,
recombinant
protein
T. brucei
trypanothione
reductase (TR)
PMID: 24788386
Peptide,
recombinant
protein
T. brucei
tryparedoxin (Tpx)
PMID: 22275351
Peptide,
recombinant
protein
T. brucei
peroxidase (Px)
PMID: 18684708
Chemical
compound, drug
trypanothione
and trypanothione
disulfide
PMID:19477177
Chemical
compound, drug
Chlorhexidine
diacetate
Monohydrate
Fluka24800
Chemical
compound, drug
(1S,3R)-RSL3José Pedro Friedmann
Angeli and Marcus
Conrad, Munich,
Germany and Cayman
Chemical
CAS: 1219810-16-8
Chemical
compound, drug
Racemic
mixture of RSL3
José Pedro Friedmann
Angeli and Marcus
Conrad, Munich,
Germany
Chemical
compound, drug
Ferrostatin-1Sigma-AldrichSML0583
Chemical
compound, drug
Liproxstatin-1Sigma-AldrichSML1414
Chemical
compound, drug
TroloxSigma-Aldrich238813
Chemical
compound, drug
Iron(III) chloride
hexahydrate,
FeCl3 × 6H2O
Merk31232
Chemical
compound, drug
Deferoxamine
mesylate
Sigma-AldrichD-9533
Chemical
compound, drug
8-HydroxyquinolineSigma-Aldrich252565
Chemical
compound, drug
(±)-α-TocopherolSigma-AldrichT3251
Chemical
compound, drug
ATPlite 1step solutionPerkin Elmer6016731
Chemical
compound, drug
Hoechst 33342Walter Nickel,
Heidelberg,
Germany
Chemical
compound, drug
4’,6-Diamidino-2
-phenylindole (DAPI)
Sigma-AldrichD-8417
Chemical
compound, drug
Rhodamine
B-[(1,10-phenanthroline-5-yl)-
aminocarbonyl]benzyl
ester (RPA)
Squarix
Biotechnology
ME043.1
Chemical
compound, drug
Rhodamine
B-[(phenanthren-9-yl)
-aminocarbonyl]-
benzylester (RPAC)
Squarix
Biotechnology
ME046.1
Chemical
compound, drug
BODIPY 581/591 C11Molecular ProbesD3861
Chemical
compound, drug
MitoPerOxMike Murphy,
Cambridge, UK
Chemical
compound, drug
MitoTracker Red CMXRosMolecular ProbesM7512
Chemical
compound, drug
MitoTracker Green FMMolecular ProbesM7514
Chemical
compound, drug
MitoSOX RedMolecular ProbesM36008
Chemical
compound, drug
Propidium iodide (PI)Molecular ProbesP3566
Chemical
compound, drug
2',7'-Dichlorodihydro
fluorescein-diacetate
(H2DCFDA)
Molecular ProbesD399

Additional files

Supplementary file 1

Trypanocidal activity and in vitro inhibitory potency of RSL3.

(A) Bloodstream T. brucei were cultured for 1, 2 or 3 days in the presence of 100 µM Trolox, 200 nM Liproxstatin-1 or 100 nM Ferrostatin-1 as well as different RSL3 concentrations and then subjected to plate reader-based ATPlite measurements. Chlorhexidine, a trypanocidal compound and known inhibitor of trypanothione reductase (TR) (Meiering et al., 2005; Beig et al., 2015), served as positive control. 1The data are the mean of at least two independent series of experiments each conducted in triplicate with standard deviations (SD). 2The values are the mean of an experiment conducted in triplicate with SD. (B) NADPH, T(SH)2, TR, and Tpx ± Px were incubated with 40 µM RSL3. After different times, the assays were started by adding (a) 100 µM H2O2 or (b) Px and H2O2. The data were derived from at least double determinations which varied by ≤ 10%.

https://doi.org/10.7554/eLife.37503.015
Transparent reporting form
https://doi.org/10.7554/eLife.37503.016

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