Expression of a Mitochondrial Peroxiredoxin Prevents Programmed Cell Death in Leishmania donovani

Cultivation of cells. L. donovani strain Lo8, a gift from D. Zilberstein (Department of Biology, Technicon, Israel Institute of Technology, Haifa, Israel), was used for all experiments. Promastigotes (day 0) frozen directly after passage through BALB/c mice were thawed and cultivated at 25°C in M199 medium supplemented with 25% fetal calf serum and 20 μg/ml gentamicin. In vitro differentiation to amastigotes was achieved as described previously (30). Briefly, promastigotes (day 0, early logarithmic stage, 2 × 10⁶ cells/ml) were heat shocked at 37°C for 24 h (day 1) and then cultivated for up to 5 days at 37°C in mildly acidic medium (pH 5.5, days 2 to 5).

PEC infection assay (intracellular amastigotes).Peritoneal exudate cells (PECs) from 4- to 6-week-old female C57black/6 mice were used for infection assays. Mice were treated with 5% thioglycolate in phosphate-buffered saline (PBS) given intraperitoneally 4 days prior to experiment. On day 4, mice were sacrificed and PECs were prepared by rinsing the peritoneum with 10 ml of sterile PBS. PECs were washed once and seeded at a density of 10⁶ cells per well in a 12-well plate on coverslips in RPMI medium supplemented with 10% fetal calf serum, 5 mM glutamine, and 50 μg/ml gentamicin. After incubation under 5% CO₂ at 37°C for 24 h, PECs were incubated with L. donovani parasites at a parasite-to-PEC ratio of 10:1 for 48 h. Nonengulfed parasites were washed away three times with warm RPMI medium, and cells on coverslips were used for immunofluorescence studies.

Genomic DNA isolation.Genomic DNA from L. donovani logarithmic phase promastigotes was prepared with the Puregene DNA purification system (Gentra Systems) according to the manufacturer's recommendations.

Cloning and sequencing of the LdmPrx gene.Two primers were designed on the basis of the sequence of peroxiredoxin from L. major (accession no. 6066432): sense primer Prx-S26(NdeI) (5′-GAGACATATGCTCCGCCGTCTTTCCA-3′) and antisense primer Prx-AS27(XhoI) (5′-GAGACTCGAGTCACATGTTCTTCTCGA-3′). Prx-S26(NdeI) and Prx-AS(XhoI) were used to PCR amplify L. donovani genomic DNA (95°C for 1 min, 50°C for 1 min, 72°C for 2 min; 30 cycles with a Perkin-Elmer DNA Thermal Cycler 480). The amplified product (679 bp) was gel purified and cloned into the pCR2.1-TOPO vector. The gene was sequenced with the Big Dye Terminator PCR cycle sequencing kit in accordance with the manufacturer's (Applied Biosystems) instructions.

Expression and purification of recombinant protein.The PCR-amplified DNA fragment coding for LdmPrx was cloned into prokaryotic expression plasmid pJC45, a derivative of pJC40 (16), with restriction enzymes NdeI and XhoI. Following transformation into Escherichia coli BL21(DE3)(pAPlacIQ), the protein was expressed according to standard procedures. Recombinant protein was isolated with Ni-nitrilotriacetic acid resin according to the manufacturer's (QIAGEN, Hilden, Germany) recommendations.

Generation of polyclonal antibodies.Two hundred micrograms of recombinant LdmPrx was injected subcutaneously into a chicken. The first injection was done in combination with complete Freund's adjuvant, and the following two booster injections were done in combination with incomplete Freund's adjuvant after 2 weeks. Antibodies were purified from eggs with increasing concentrations of polyethylene glycol 6000.

Western blot assays.Twelve percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions. Samples from promastigotes and in vitro-derived amastigotes were obtained by lysing the cells directly in hot SDS sample buffer (95°C, 125 mM Tris-HCl [pH 6.8], 20% glycerin, 20% SDS, 20 mM dithiothreitol [DTT], 0.001% bromophenol blue). Western blot analyses were carried out by the semidry blotting technique with electrophoresis buffer (0.25 M Tris, 0.5 M glycine, 1% SDS) as blotting buffer. Polyclonal chicken antisera (LdmPrx, 1:10,000) or monoclonal mouse antibodies (anti-β tubulin clone Tub2.1 [Sigma]) and alkaline phosphatase-conjugated anti-chicken immunoglobulin M (IgM) or anti-mouse IgG (Sigma) as the secondary antibody were used to detect the protein with the 5-bromo-4-chloro-3-indolylphosphate (BCIP)-Nitro Blue Tetrazolium color developmental substrate (Promega).

Immunoelectron microscopy.Cells were harvested by centrifugation (10 min, 690 × g, 4°C), washed twice with PBS, and fixed for 24 h at 37°C in 200 mM sodium cacodylate buffer with 4% paraformaldehyde. Fixed cells were dehydrated in ethanol and embedded in LR-White. Ultrathin sections were prepared on an Ultracut E (Reichert) and placed on 200-mesh Ni grids.

Anti-LdmPrx antibodies (1:500) or preimmune antibodies were incubated with the grids for 1 h at 37°C and then overnight at 4°C. The sections were then treated with rabbit anti-chicken antibody (1:300; Jackson Immunolab) and with protein A-gold (10 nm, 1:100; Biocell). Electron micrographs were taken on a Philips CM-10 transmission electron microscope.

Immunofluorescence assay. L. donovani promastigotes were incubated with 1 nM MitoTracker red CMXROS (Molecular Probes) diluted in M199 medium for 30 min, washed once with medium alone, added to poly-l-lysine-covered glass slides, and air dried. Cells were fixed with 3.7% formaldehyde in M199 medium for 15 min, washed three times in PBS, and permeabilized in PBS-0.2% Triton X-100, followed by three washes in PBS. Subsequently, cells were incubated for 30 min in PBS containing 10% fetal calf serum (FCS). After blocking, cells were incubated with anti-LdmPrx, diluted 1:1,000 in PBS-10% FCS, following three washes in PBS. Slides were incubated with Cy2-conjugated donkey anti-chicken IgG antibody (Dianova), diluted 1:1,000 in PBS-10% FCS, and washed another three times in PBS. After incubation with Hoechst 33258 (Molecular Probes), 1:2,000 in PBS, cells were mounted in mounting medium (Dako Cytomation) and examined with a Zeiss Axioskop 2 plus immunofluorescence microscope and the improvision software.

Intracellular amastigotes on coverslips were incubated with 1 nM MitoTracker diluted in RPMI medium, washed once with medium alone, and fixed with 4% paraformaldehyde in PBS. After three washes in PBS, cells were permeabilized in absolute methanol at −20°C. Further staining steps were performed as described above.

Peroxide assay (ferrous ammonium sulfate-potassium thiocyanate).The ability of LdmPrx to remove H₂O₂ and t-BOOH was evaluated with the ferrithiocyanate system (50). Reaction mixtures (1-ml reaction volume) containing 25 mM HEPES (pH 7.0) and LdmPrx protein (10 to 100 μg) were preincubated with 3 mM DTT for 10 min at 37°C. After preincubation, 480 μM H₂O₂ or 350 μM t-BOOH (final concentration), respectively, was added and the reaction was allowed to proceed for another 30 min. The reaction was stopped by addition of 8% (vol/vol) trichloroacetic acid, and protein was removed by centrifugation (5 min, 12,000 × g). Subsequently, 0.2 volume of 10 mM ferrous ammonium sulfate and 0.1 volume of 2.5 M potassium thiocyanate were added and the absorbance was measured at 480 nm. The amounts of H₂O₂ and t-BOOH were determined spectrometrically by using known amounts of H₂O₂ and t-BOOH as standards.

Nicking assay.The ability of LdmPrx to protect DNA from hydroxyl radical-induced nicking was determined as previously described (34, 48). Reaction mixtures containing 0.1 mM HEPES (pH 7.2), 3.3 μM FeCl₃, 10 mM DTT, 5 mM EDTA (pH 8), and various concentrations of LdmPrx were incubated in a total volume of 50 μl at 37°C for 3 h. After incubation, 1 μg of supercoiled pUC18 plasmid DNA (Invitrogen) was added to the reaction mixture and the mixture was incubated at 37°C for an additional 3 h. The DNA was separated on a 1% agarose gel at a 100-V constant voltage. All solutions were made fresh immediately before use.

Construction of expression vectors. Leishmania-specific expression vector pX63pol (kindly provided by Martin Wiese, Bernhard Nocht Institute, Hamburg, Germany) was used to express LdmPrx in L. donovani promastigotes. Primers Prx-S26(NdeI) and Prx-AS27(XhoI) were used to PCR amplify the coding region of LdmPrx. The product was digested with NdeI and XhoI, and the 5′overlapping ends were filled in by using Klenow polymerase to create blunt ends. The vector was digested with EcoRV and ligated with the prepared insert. The correct orientation and sequence were reconfirmed by nucleotide sequencing.

Transfection of L. donovani promastigotes.Plasmid DNA was purified with a Nucleobond AX PC2000 Maxiprep Kit (Macherey & Nagel), and 100 μg of DNA was used per transfection. Parasites were transfected by electroporation. Cells were harvested during the late log phase of growth, washed twice in ice-cold PBS and once in prechilled electroporation buffer (21 mM HEPES [pH 7.5], 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM glucose), and suspended at a density of 10⁸/ml in electroporation buffer. Chilled DNA was mixed with 0.4 ml of the cell suspension, which was immediately used for electroporation with a Bio-Rad Gene Pulser apparatus. Electrotransfection was carried out with a 4-mm electroporation cuvette at 3,750 V/cm and 25 μF.

After electroporation, cells were kept on ice for 10 min before being transferred into 10 ml of drug-free medium. After 24 h, transfectants were selected with 7.5 μg/ml bleomycin (Calbiochem).

Viability assays.Promastigotes (5 × 10⁶) harvested at early log phase and transfected either with LdmPrx/pX63pol or pX63pol in 100 μl of Hanks balanced salt solution were exposed in triplicate to various concentrations of different oxidative stress inducers in 96-well plates at 25°C. H₂O₂ and t-BOOH (Merck) were used as peroxide donors. MAHMA NONOate, PAPA NONOate, spermine NONOate, NOC-7, NOC-9, NOC-12 (Calbiochem), and GSNO (S-nitroso-l-glutathione; Alexis Biochemicals) were used as nitric oxide donors. Sin-1 (Calbiochem) yields NO and superoxide anion radicals. NOR-3 (Calbiochem) is cell permeating and releases NO intra- and extracellularly. DMNQ (2,3-dimethoxy-1,4-naphthoquinone; Alexis Biochemicals) induces intracellular superoxide anion formation. The investigated concentrations ranged from 0 to 5 mM. After incubation (1 h), the stress inducers were removed and parasite viability was measured by monitoring incubation in Cell Proliferation Reagent WST-1 (Roche) for 3 h. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium salt to formazan. Formazan was detected on a microplate reader at 440 nm. Percent viability was calculated from the ratio of optical density readings in wells with stress inducers to those in control wells × 100. In order to compare the ratio of control viabilities to viabilities of overexpressing parasites more easily, control values were equated to 1.

In situ labeling of DNA fragments.Cells undergoing apoptosis generate abundant DNA fragments in their nuclei. In situ detection of DNA fragments by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) was performed with an in situ cell death detection kit, TMR red (Roche), according to the manufacturer's instructions. Briefly, 10⁷ promastigotes harvested at early log phase and transfected with either LdmPrx/pX63pol or pX63pol or untransfected cells from the early (2 × 10⁶) and late (6 × 10⁷) logarithmic phase in 1 ml of M199 medium were exposed to 1 to 3 mM H₂O₂ for 3 h. Control cells were incubated in PBS. Cells were used to coat poly-l-lysine-covered slides and fixed with 4% paraformaldehyde. Permeabilization was done with 0.1% Triton X-100-0.1% sodium citrate for 2 min on ice, followed by incubation in a TUNEL reaction mixture containing TdT and TMR red-labeled nucleotides for 1 h. The samples were counterstained with Hoechst 33258 (Molecular Probes), diluted 1:2,000 in PBS, mounted in mounting medium (Dako Cytomation), and visualized with a Zeiss Axioskop 2 plus immunofluorescence microscope. At least 500 cells in two independent experiments were counted. All counts were done with coded samples to prevent bias.

Sequencing of a mitochondrial peroxiredoxin gene of L. donovani and analysis of its amino acid sequence.In the course of a proteome analysis of in vitro stage differentiation of L. donovani, a mitochondrial peroxiredoxin was found to be expressed in a stage-specific manner (7). Primers deduced from the coding region of a homologous L. major peroxiredoxin gene (accession no. CAJ03825) were used to amplify the corresponding DNA by PCR with L. donovani genomic DNA as the template. A product of 679 bp was obtained; it was 98% identical to its L. major homologue. Southern blot analysis indicated that the LdmPrx gene is a single-copy gene (data not shown).

The gene for the L. donovani peroxiredoxin encodes a protein of 226 amino acid residues with a calculated M_r of 25,000 and a pI value of 6.4. LdmPrx contains a 27-amino-acid N-terminal mitochondrial targeting sequence, as predicted by the program MitoProtII (http://ihg.gsf.de/ihg/mitoprot.html ). The amino acid sequence displays 90% identity to the mitochondrial targeting sequences of peroxiredoxins of L. infantum and L. major and 47% identity to that of Trypanosoma brucei. The overall similarities of the whole sequence range from 55 to 60% (higher eukaryotic organisms like Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens) to more than 97% identity to the other Leishmania peroxiredoxins. LdmPrx is a 2-Cys peroxiredoxin (for alignment, see the supplemental material). Classical 2-Cys peroxiredoxins are characterized by two highly conserved cysteine residues (25). The N-terminal cysteine is embedded in a VCP motif as is typical for peroxiredoxins. It appears to be the one that is attacked by the hydroperoxide and has been shown to be essential for the activity in several peroxiredoxins. The second redox-active cysteine, the one that interacts with the reductant, is located near the C terminus and is also part of a VCP motif in most of the 2-Cys peroxiredoxins (25). The C-terminal conserved cysteine residue of LdmPrx is different from the VCP motif found in other peroxiredoxins but identical to the IPC motif found in the mitochondrial peroxiredoxins of T. brucei, T. cruzi, L. major, and L. infantum. This indicates that LdmPrx belongs to a peroxiredoxin subfamily, together with the mitochondrial homologues of T. cruzi, T. brucei, L. major, and L. infantum, distinct from the cytosolic enzymes from these organisms, which also display a VCP motif within the C-terminal cysteine.

Biochemical characterization of recombinant LdmPrx.The full-length LdmPrx protein was expressed in E. coli as an N-terminally His-tagged protein. The purified protein (rLdmPrx) was about 28 kDa in size, which corresponds to the predicted M_r of mature LdmPrx of 25,000, taking the His tag with its molecular mass of approximately 3 kDa into account (Fig. 1A). Matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis of the product after digestion with trypsin confirmed the identity of rLdmPrx (data not shown). With a specific antibody generated against rLdmPrx, a protein with a molecular mass of 26 kDa was detected by Western blot analysis of promastigote cells grown to stationary phase. Under nonreducing conditions, the molecular mass of the native protein shifted to about 50 kDa, suggesting dimerization of the protein (Fig. 1B). The capacity of peroxiredoxins to form dimers is mediated by the cysteines within the two conserved domains (12, 20).

The antioxidant activity of rLdmPrx was characterized in a thiol mixed-function oxidation system (Fig. 1C). Peroxiredoxins are known to prevent DNA damage by ROS. ROS can be produced by incubating DTT with Fe³⁺, which catalyzes the reduction of O₂ to H₂O₂. The latter is further converted by the Fenton reaction to hydroxyl radicals (34, 35). It has been demonstrated that these hydroxyl radicals can induce strand breaks in DNA, as well as chemical changes in the bases and deoxyriboses (17). In the presence of DTT and Fe³⁺, part of plasmid pUC18 was converted into the nicked form after 3 h of incubation (Fig. 1C, lane 3). Addition of rLdmPrx completely abolished the conversion of the DNA into the nicked form (Fig. 1C, lane 7), while bovine serum albumin had no effect (Fig. 1C, lane 8). The degree of protection correlated with the amount of rLdmPrx added to the assay (Fig. 1C, lanes 4 to 7). Several recombinant peroxiredoxins from different organisms like Plasmodium falciparum, Chlamydomonas reinhardtii, Brugia malayi, Onchocerca volvulus, and Entamoeba histolytica were also tested for their DNA-protective effects. For most of them, 1 to 16 μM recombinant protein is necessary for complete DNA protection. It was shown that 5 μM rLdmPrx inhibits nicking of DNA completely, which is in good correlation with the properties of the other peroxiredoxins (14, 23, 24, 27, 36).

Purified rLdmPrx was also able to detoxify H₂O₂, as well as t-BOOH, in vitro in a concentration-dependent manner. Removal of the two peroxides required the presence of DTT (3 mM), indicating that LdmPrx possesses thiol peroxidase activity (Fig. 1D). It was also shown for the recombinant L. infantum mitochondrial peroxiredoxin that it reduces H₂O₂, as well as t-BOOH (10). The enzymatic kinetics observed for the removal of H₂O₂ is comparable to that of peroxiredoxins from P. falciparum, C. elegans, human neutrophils, and Pisum sativum, where similar peroxidase assays were used (Fig. 1D) (8, 26, 27, 32).

Expression and localization of LdmPrx in L. donovani promastigotes and amastigotes.A polyclonal antiserum against rLdmPrx was used to detect the protein in Western blot assays of L. donovani cellular extracts of all days of stage differentiation from the promastigote to the amastigote form under reducing conditions (Fig. 2A). No protein could be detected in the promastigote stage (day 0 of stage differentiation). A single and specific polypeptide band of about 26 kDa could be detected directly after the heat shock (day 1 of stage differentiation), and the highest intensity was observed from day 3 of the stage differentiation process (Fig. 2A). This result was consistent with the result obtained from the proteomic approach to L. donovani stage differentiation (7). To study if the differential expression of LdmPrx is specific for the differentiation process or can also be induced in the presence of other culture conditions, Western blot assays of promastigote parasites harvested at different time points from the early logarithmic to the late logarithmic phase of culture were performed (Fig. 2B). As shown also in Fig. 2A for promastigotes grown to the early logarithmic phase (2 × 10⁶ cells/ml), no detectable amount of LdmPrx could be observed by Western blot analysis. At a cell density of 5 × 10⁶/ml, a peroxiredoxin signal could be detected which showed an increase in intensity when cells were cultivated to the stationary growth phase (6 × 10⁷/ml). Exposing the promastigote cells to sublethal doses of H₂O₂ and t-BOOH, on the other hand, did not have any effect (data not shown). Therefore, the increase in expression may occur in response to differentiation-induced signals.

Immunoelectron microscopy and immunofluorescence assays were used to determine the subcellular localization of LdmPrx. Promastigote cells showed labeling only in the kinetoplast area (Fig. 3A, magnifications). In amastigotes, labeling of the kinetoplast area was also observed. In addition, the amastigotes exhibited extensive labeling of the whole mitochondrion (Fig. 3B, magnifications), with a strong increase in the amount compared to that of the promastigotes. These results indicate correct targeting of LdmPrx into the mitochondrion and confirm the differences in protein amounts between the two stages detected by Western blot assay and the proteomic approach (7).

As suggested by the presence of a mitochondrial targeting sequence and indicated by the electron microscopic studies, immunofluorescence analysis also corroborated that this protein localizes to the single mitochondrion of the parasite. The anti-LdmPrx antibody staining perfectly colocalized with the MitoTracker dye, a marker for mitochondria (Fig. 3C and D). Late logarithmic phase promastigotes and in vivo-derived amastigotes (Fig. 3C and E) revealed anti-LdmPrx staining all over the mitochondrion. As shown in Fig. 3E, Mitotracker dye stains the Leishmania mitochondrion, as well as the mitochondria of the macrophages. As supposed from the electron microscopy studies and Western blot analyses, early logarithmic phase promastigotes showed only weak anti-LdmPrx staining (Fig. 3C). The immunofluorescence studies also indicate that promastigotes transformed with the expression vector LdmPrx/pX63pol, which led to overexpression of the peroxiredoxin in early logarithmic phase promastigotes, showed distinct staining only in the mitochondrial area. Clear colocalization with the MitoTracker dye was observed (Fig. 3D). Therefore, overexpression of LdmPrx leads to correct targeting of the molecule. No mislocalization could be detected.

Overexpression of LdmPrx did not decrease the sensitivity of L. donovani promastigotes to exogenously produced oxidative stress.To investigate the role of LdmPrx in protection against oxidative and nitrosative stress, we overexpressed the protein within the parasites by using Leishmania-specific expression vector pX63pol. LdmPrx/pX63pol-transformed parasites and control cells transfected with plasmid pX63pol showed no substantial growth rate alterations compared to wild-type parasites (Fig. 4A). Overexpressing of LdmPrx in early logarithmic phase promastigotes revealed a significant increase in expression of the protein compared to that in control cells (Fig. 4B).

To see if overexpression of the protein in L. donovani promastigotes protects parasites against oxidative stress, we measured the viability of pX63pol- and LdmPrx/pX63pol-transfected parasites after incubation with different oxidative stress inducers. Table 1 shows the relationship of the viability of overexpressing cells to that of control cells after exposure to oxidative stress. The same amount of viable cells in both populations was defined as 1. Treatment with neither peroxides (H₂O₂, t-BOOH, Sin-1) nor nitric oxides (MAHMA NONOate, PAPA NONOate, spermine NONOate, GSNO, NOC-7, NOC-9, NOC-12, Sin-1) had any effect on the viability of LdmPrx-overexpressing cells compared to that of control cells. Overexpression of the protein did not even show a protective effect after exposure to cell-permeating ROS producers like DMNQ and NOR-3, which generate superoxide anion radicals and nitric oxides intracellularly. These results are consistent with the observation that, after exposure to oxidative stress, no upregulation of LdmPrx occurred. In contrast to the missing resistance to ROS of L. donovani overexpressing LdmPrx, for L. infantum overexpressing LimTXNPx, significant resistance to t-BOOH, but not to H₂O₂ also, was observed (11).

Overexpression of LdmPrx prevents DNA breakdown in L. donovani promastigotes after exposure to H₂O₂.Exposure to H₂O₂ triggers apoptosis in numerous mammalian cells and yeast (15), leading to membrane blebbing, cytoplasmic and nuclear condensation, and chromatin aggregation with accompanying DNA breakage (52). Das and colleagues studied the death-associated phenotype in L. donovani promastigotes that occurs after exposure to H₂O₂ (18). They demonstrated that parasites undergo necrosis-like death with doses greater than 4 mM H₂O₂. Lesser doses, on the other hand, precipitate apoptosis-like death, resulting in breakdown of DNA material detected by TUNEL staining. We also were able to generate detectable DNA fragments in our control parasites transfected with pX63pol (Fig. 5A and B) after 3 h of treatment with 1 to 3 mM H₂O₂. About 60% of the cells counted became TUNEL positive. In contrast to that, only approximately 8% of the LdmPrx-overexpressing parasites became TUNEL positive after the appropriate treatment (Fig. 5A and B), even though no difference in the survival rate between peroxiredoxin-overexpressing Leishmania and the respective controls was observed (Table 1).

The same resistance to PCD was observed when cells grown to the early and late logarithmic phase were compared. For cells grown to the late logarithmic phase, which also showed an increase in the amount of peroxiredoxin, approximately 5% TUNEL-positive cells were counted. Cells from the early logarithmic growth phase were 30 to 90% TUNEL positive (Fig. 5B).

It is known that H₂O₂ is toxic for Leishmania. At the concentrations used for the TUNEL assay, all cells die after 3 h of exposure to H₂O₂ but only those cells having an increased amount of mitochondrial peroxiredoxin are protected from PCD. Nevertheless, H₂O₂ is stable enough to diffuse through the whole cell and is not only involved in induction of PCD. It also has dramatic cytoplasmic effects leading, for example, to disruption of biochemical pathways and therefore to necrosis-like death. Since LdmPrx is localized only in the mitochondria, the necrosis-like death is not affected.