Cytosolic Peroxidases Protect the Lysosome of Bloodstream African Trypanosomes from Iron-Mediated Membrane Damage

<div><p>African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream <i>Trypanosoma brucei</i> lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the <i>px I–II</i> knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. <i>T. brucei</i> acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the <i>px I–II</i> knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. <i>In vivo</i> experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with <i>px III</i> knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of <i>T. brucei</i>. The respective knockout of the cytosolic <i>px I–II</i> in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.</p></div

Lysosomal disintegration precedes damage of the mitochondrion.

<p>The <i>px I–II</i>^−/− BS cells were kept for the indicated times at 37°C in medium ± Trolox and then subjected to immunofluorescence analysis. <b>A.</b> MitoTracker staining (left), overlay of the signals for MitoTracker (red), p67 (green), and DAPI (blue) (middle), and phase contrast images (right). <b>B.</b> Quantitative analysis of the staining pattern of the cells (for details, see text). The phenotypes visible in the respective pictures in (<b>A</b>) are highlighted by bold numbers. For each time point, at least 194 cells were inspected. The data are representative of two independent sets of experiments giving very similar results. (Scale bar: 10 µm).</p

Withdrawal of Trolox results in morphological changes of the <i>px I–II</i>^−/− BS cells.

<p><b>A.</b> Living parasites were fed at 37°C with Alexa Fluor-488 conjugated dextran and then kept at RT in medium with (+) or without (−) 100 µM Trolox as outlined under <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004075#s4" target="_blank">Materials and Methods</a>. On the left site, representative cells for the three different phenotypes observed in the remaining intact and fluorescent parasites are shown. The pictures were taken from cells incubated (1) 1 h, + Trolox, (2) 1 and 2 h, - Trolox, and (3) 2 h, - Trolox. On the right site, the quantitative analysis is provided. At each time point, ≥60 parasites were analyzed in three independent experiments and the mean ± SD was calculated. <b>B.</b> LysoTracker Green staining of living parasites incubated for the indicated times at 37°C in the presence or absence of Trolox. <b>C.</b> Immunofluorescence analysis of cells stained with antibodies against p67 (green) and DAPI (blue) to visualize nuclear (large dot) and kinetoplast (small dot) DNA (upper panel) and the corresponding phase contrast pictures (lower panel). At each time point, the p67 signal of at least 130 parasites was analyzed in each of three independent experiments and the mean ± SD was calculated (below). (Scale bar: 10 µm).</p

Holo-transferrin induces, whereas apo-transferrin slows down lysis of the <i>px I–II</i>^−/− BS parasites.

<p>The mutant cells were incubated <b>A.</b> for 4 h at RT in standard medium ±100 µM Trolox in the presence and absence of 25 µM holo-transferrin (holo-TF), <b>B.</b> for 6 h at RT and then cultured overnight at 37°C in medium ±100 µM Trolox in the presence and absence of 320 µM apo-transferrin (apo-TF), <b>C.</b> for 5 h at RT followed by overnight cultivation at 37°C in standard medium as well as in FCS-free medium ±100 µM Trolox in the presence and absence of 5 µM and 25 µM holo-TF, and <b>D.</b> for 5 h at 20°C in standard medium as well as in transferrin-depleted medium (-TF medium) ±100 µM Trolox in the presence and absence of 5 µM and 25 µM holo-TF. The values represent the mean ± SD of three independent experiments.</p

Infectivity of <i>px III</i>^−/− and WT <i>T. brucei</i>.

<p><b>A.</b> and <b>B.</b> Female BALB/cJ mice (n = 6/group) were infected intraperitoneally with 10⁴ WT parasites or <i>px III</i>^−/− cells that, prior to infection, had been cultivated in medium supplemented with 100 µM Trolox, and <b>C.</b> and <b>D.</b> in medium without Trolox. <b>E.</b> and <b>F.</b> Mice (n = 3/group) were inoculated with <i>px III</i>^−/− and WT parasites isolated from infected animals (for details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004075#s4" target="_blank">Materials and Methods</a>). <b>A.</b>, <b>C.</b>, and <b>E.</b> Kaplan-Meyer plots for animal survival, <b>B.</b>, <b>D.</b>, and <b>F.</b> Average parasite load in blood samples taken from the animals at the indicated times. All data are the mean value ± SD.</p

Exogenous iron promotes lysis of the <i>px I–II</i>^−/− BS cells.

<p><b>A.</b> The mutant parasites were incubated for up to 5.5°C in standard medium ±100 µM Trolox in the presence and absence of 100 µM Fe³⁺. WT cells behaved like the mutant cells in the presence of Trolox. <b>B.</b> The <i>px I–II^−/−</i> cells were incubated for 5 h at 22°C and then cultured overnight at 37°C in medium ±100 µM Trolox in the presence and absence of 100 µM deferoxamine (dfx). WT behaved like the mutant cells in the presence of Trolox. <b>C.</b> The <i>px I–II^−/−</i> cells were incubated for 5 h at 19°C and then cultured overnight at 37°C in medium ±10% FCS and/or 100 µM Trolox in the presence and absence of 100 µM Fe³⁺. The behavior of WT cells in the absence of FCS was identical to that of the mutant cells. At the different time points, living cells were counted. The values represent the mean ± SD of three independent experiments.</p

Lysis of the <i>px I–II</i>^−/− BS cells is temperature-dependent.

<p>The <i>px I–II^−/−</i> parasites were seeded at a density of 5–9×10⁵ cells/ml in standard HMI-9 medium (which contains 10% FCS) supplemented ±100 µM Trolox. The cells were incubated at 37°C, 21°C, and 9°C, respectively. At the indicated time points, living cells were counted. The values represent the mean ± SD of three independent experiments.</p

Screening work flow.

<p>The different steps, the most relevant assay conditions and the go/no-go criteria of the screening campaign are indicated in boxes. The figures on the right refer to the number of compounds screened and that subsequently advanced during the campaign. From 144 compounds, 22 compounds lowered assay signal ≥ 45% for at least one TryS. From these 22, 7 BDA were false positive and the remaining 15 compounds were confirmed as enzyme inhibitors. Two of them are <b>AI</b> with potency in the submicromolar range against <i>Li</i>TryS. AI (P), 4,5-dihydroazepino[4,5-<i>b</i>]indol-2(1<i>H</i>,3<i>H</i>,6<i>H</i>)-one derivatives (P, paullone); APPDA, 6-arylpyrido[2,3-<i>d</i>]pyrimidine-2,7-diamine derivatives; BZ, benzofuroxan derivatives; BDA, <i>N</i>,<i>N'</i>-bis(3,4-substituted-benzyl) diamine derivatives.</p

Kinetic parameters for recombinant tritryp trypanothione synthetase (TryS).

<p>Kinetic parameters for recombinant tritryp trypanothione synthetase (TryS).</p

Biological activity of compounds against infective <i>Trypanosoma brucei brucei</i> with downregulated expression of trypanothione synthetase (TryS).

<p><b>A)</b> Western blot analysis of cell extracts from 2x10⁷ <i>T</i>. <i>b</i>. <i>brucei</i> from the wildtype (WT), 48 h tetracycline-induced (+) and non-induced (-) TryS-RNAi cell line. Two hundred ng of recombinant <i>Tb</i>TryS was loaded as control. Bands from the molecular weight marker are indicated on left. The picture at the bottom shows the abundance of TryS for each condition as estimated by densitometry and expressed relative to the level of the WT cell line. <b>B)</b> Ponceau staining of the Western blot membrane that served as normalization control of protein load for each condition. <b>C)</b> Cytotoxicity (%) ± S.D. (n = 2) for tetracycline-induced (+) and non-induced (-) TryS-RNAi <i>T</i>. <i>b</i>. <i>brucei</i> treated with 5 μM nifurtimox or 100 nM EAP1-47.</p