Peroxiredoxin-linked Detoxification of Hydroperoxides in Toxoplasma gondii
2004; Elsevier BV; Volume: 280; Issue: 1 Linguagem: Inglês
10.1074/jbc.m406367200
ISSN1083-351X
AutoresSusan E. Akerman, Sylke Müller,
Tópico(s)Sulfur Compounds in Biology
ResumoThe apicomplexan parasite Toxoplasma gondii is highly susceptible to oxidative stress caused by tert-butyl-hydroperoxide, juglone, and phenazine methylsulfate with IC50 in the nanomolar range. Using dichlorofluorescein diacetate, a detector of endogenous oxidative stress, it was shown that juglone and phenazine methylsulfate are potentially toxic to the parasites without affecting the host cells. These results demonstrate that T. gondii is vulnerable to oxidative challenge that results from disruption of its redox balance and so this could be an effective approach to therapeutic intervention. This study has characterized redox active and antioxidant peroxidases belonging to the class of 1-Cys and 2-Cys peroxiredoxins that play crucial roles in maintaining redox balance. The tachyzoite stages of T. gondii express thioredoxin (TgTrx), 1-Cys peroxiredoxin (TgTrx-Px2), and a 2-Cys peroxiredoxin (TgTrx-Px1) and immunofluorescent studies revealed that all three proteins are located in the cytosol of the parasite confirming previous studies on TgTrx-Px1 (Kwok, L.Y., Schlüter, D., Clayton, C., and Soldati, D. (2004) Mol. Microbiol. 51, 47-61). TgTrx-Px1 showed Km values for H2O2 and tert-butyl hydroperoxide in the nanomolar range, emphasizing the great affinity of the protein for theses substrates. Moreover, the catalytic efficiency of TgTrx-Px1 for these substrates at 106-107m-1 s-1 is unusually high, which qualifies the enzyme as an extremely potent antioxidant. Kinetic analyses revealed that TgTrx-Px1 is inhibited by tert-butyl hydroperoxide, and apparent inhibition constants were determined to be between 33 and 35.6 μm depending on the concentration of the non-inhibitory substrate thioredoxin. TgTrx-Px2 protected glutamine synthetase from inactivation by Fe3+/DTT, showing that it is an active peroxiredoxin. The apicomplexan parasite Toxoplasma gondii is highly susceptible to oxidative stress caused by tert-butyl-hydroperoxide, juglone, and phenazine methylsulfate with IC50 in the nanomolar range. Using dichlorofluorescein diacetate, a detector of endogenous oxidative stress, it was shown that juglone and phenazine methylsulfate are potentially toxic to the parasites without affecting the host cells. These results demonstrate that T. gondii is vulnerable to oxidative challenge that results from disruption of its redox balance and so this could be an effective approach to therapeutic intervention. This study has characterized redox active and antioxidant peroxidases belonging to the class of 1-Cys and 2-Cys peroxiredoxins that play crucial roles in maintaining redox balance. The tachyzoite stages of T. gondii express thioredoxin (TgTrx), 1-Cys peroxiredoxin (TgTrx-Px2), and a 2-Cys peroxiredoxin (TgTrx-Px1) and immunofluorescent studies revealed that all three proteins are located in the cytosol of the parasite confirming previous studies on TgTrx-Px1 (Kwok, L.Y., Schlüter, D., Clayton, C., and Soldati, D. (2004) Mol. Microbiol. 51, 47-61). TgTrx-Px1 showed Km values for H2O2 and tert-butyl hydroperoxide in the nanomolar range, emphasizing the great affinity of the protein for theses substrates. Moreover, the catalytic efficiency of TgTrx-Px1 for these substrates at 106-107m-1 s-1 is unusually high, which qualifies the enzyme as an extremely potent antioxidant. Kinetic analyses revealed that TgTrx-Px1 is inhibited by tert-butyl hydroperoxide, and apparent inhibition constants were determined to be between 33 and 35.6 μm depending on the concentration of the non-inhibitory substrate thioredoxin. TgTrx-Px2 protected glutamine synthetase from inactivation by Fe3+/DTT, showing that it is an active peroxiredoxin. Peroxiredoxins (Trx-Px) 1The abbreviations used are: Trx-Px, peroxiredoxin; DCF, 2′,7′-dichlorodihydrofluorescein diacetate; GS, glutamine synthetase; GSSG, glutathione disulfide; HFFs, human foreskin fibroblasts; PBS, phosphate-buffered saline; PfTrxR, P. falciparum thioredoxin reductase; t-BuOOH, tert-butyl hydroperoxide; TgTrx, T. gondii thioredoxin; TgTrx-Px1, T. gondii 2-Cys peroxiredoxin; TgTrx-Px2, Toxoplasma gondii 1-Cys peroxiredoxin; Trx, thioredoxin, TrxR, thioredoxin reductase; FITC, fluorescein isothiocyanate. 1The abbreviations used are: Trx-Px, peroxiredoxin; DCF, 2′,7′-dichlorodihydrofluorescein diacetate; GS, glutamine synthetase; GSSG, glutathione disulfide; HFFs, human foreskin fibroblasts; PBS, phosphate-buffered saline; PfTrxR, P. falciparum thioredoxin reductase; t-BuOOH, tert-butyl hydroperoxide; TgTrx, T. gondii thioredoxin; TgTrx-Px1, T. gondii 2-Cys peroxiredoxin; TgTrx-Px2, Toxoplasma gondii 1-Cys peroxiredoxin; Trx, thioredoxin, TrxR, thioredoxin reductase; FITC, fluorescein isothiocyanate. are a family of antioxidant enzymes that protect cells from oxidative damage by hydroperoxides (1Hofmann B. Hecht H.J. Flohe L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (763) Google Scholar, 2Wood Z.A. Schröder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2085) Google Scholar). The enzymes are peroxidases that exert their reductive activity via active site cysteine residues. This group of proteins can structurally be distinguished into three classes: the typical 2-Cys peroxiredoxins, the atypical 2-Cys peroxiredoxins, and the 1-Cys peroxiredoxins (2Wood Z.A. Schröder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2085) Google Scholar). Atypical 2-Cys peroxiredoxins have only been identified recently and they differ from typical 2-Cys peroxiredoxins by forming an intramolecular disulfide bridge during their catalytic cycle rather than an intermolecular disulfide bridge like typical peroxiredoxins (2Wood Z.A. Schröder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2085) Google Scholar, 3Choi H.J. Kang S.W. Yang C.H. Rhee S.G. Ryu S.E. Nat. Struct. Biol. 1998; 5: 400-406Crossref PubMed Scopus (327) Google Scholar). The abundance of typical 2-Cys peroxiredoxins can be extraordinarily high, possibly compensating for their rather low catalytic efficiency when compared with other peroxidases such as catalase and glutathione-dependent peroxidases (1Hofmann B. Hecht H.J. Flohe L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (763) Google Scholar, 2Wood Z.A. Schröder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2085) Google Scholar). In addition to their protective antioxidant role, it has been suggested that 2-Cys peroxiredoxins are involved in redox signaling and other regulatory processes (2Wood Z.A. Schröder E. Robin Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2085) Google Scholar, 4Delaunay A. Pflieger D. Barrault M.B. Vinh J. Toledano M.B. Cell. 2002; 111: 471-481Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 5Georgiou G. Cell. 2002; 111: 607-610Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6Wood Z.A. Poole L.B. Karplus P.A. Science. 2003; 300: 650-653Crossref PubMed Scopus (1122) Google Scholar). Thus this class of enzymes has a wide variety of functions that are vital for metabolism and cellular integrity. The role of the 1-Cys peroxiredoxins is less clear. Their catalytic mechanism has hardly been studied to date, and the nature of their endogenous reducing partner is controversial (3Choi H.J. Kang S.W. Yang C.H. Rhee S.G. Ryu S.E. Nat. Struct. Biol. 1998; 5: 400-406Crossref PubMed Scopus (327) Google Scholar, 7Chen J.W. Dodia C. Feinstein S.I. Jain M.K. Fisher A.B. J. Biol. 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Med. 1974; 290: 1110-1116Crossref PubMed Scopus (444) Google Scholar) affecting the central nervous system and leading to a variety of severe disorders. Recently the antioxidant systems of T. gondii have started to attract attention as these might be essential for the adaptation and survival of the parasites in macrophages and other immune effector cells, which probably generate reactive oxygen species to combat the parasites (12Denkers E.Y. Kim L. Butcher B.A. Cell. Microbiol. 2003; 5: 75-83Crossref PubMed Scopus (61) Google Scholar, 13Kwok L.Y. Schluter D. Clayton C. Soldati D. Mol. Microbiol. 2004; 51: 47-61Crossref PubMed Scopus (101) Google Scholar). Thus Toxoplasma needs to have effective antioxidant systems to maintain the intracellular redox homeostasis even when the parasite is under oxidant challenge. In previous studies it has been shown that T. gondii possesses a whole array of antioxidant proteins including three superoxide dismutases, catalase, and a variety of putative glutathione- and thioredoxin-dependent peroxidases of the peroxiredoxin family (13Kwok L.Y. Schluter D. Clayton C. Soldati D. Mol. Microbiol. 2004; 51: 47-61Crossref PubMed Scopus (101) Google Scholar, 14Kaasch A.J. Joiner K.A. J. Biol. Chem. 2000; 275: 1112-1118Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Ding M. Clayton C. Soldati D. J. Cell Sci. 2000; 113: 2409-2419Crossref PubMed Google Scholar, 16Brydges S.D. Carruthers V.B. J. Cell Sci. 2003; 116: 4675-4685Crossref PubMed Scopus (36) Google Scholar). The presence of catalase is unusual as other apicomplexan parasites such as Plasmodium (17Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Pain A. Nelson K.E. Bowman S. Paulsen I.T. James K. Eisen J.A. Rutherford K. Salzberg S.L. Craig A. Kyes S. Chan M.S. Nene V. Shallom S.J. Suh B. Peterson J. Angiuoli S. Pertea M. Allen J. Selengut J. Haft D. Mather M.W. Vaidya A.B. Martin D.M. Fairlamb A.H. Fraunholz M.J. Roos D.S. Ralph S.A. McFadden G.I. Cummings L.M. Subramanian G.M. Mungall C. Venter J.C. Carucci D.J. Hoffman S.L. Newbold C. Davis R.W. Fraser C.M. Barrell B. Nature. 2002; 419: 498-511Crossref PubMed Scopus (3375) Google Scholar), and the members of the kinetoplastida lack this protein and other antioxidants appear to compensate for its absence (18Müller S. Liebau E. Walter R.D. Krauth-Siegel R.L. Trends Parasitol. 2003; 19: 320-328Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 19Müller S. Mol. Microbiol. 2004; 53: 1291-1305Crossref PubMed Scopus (240) Google Scholar). As the disruption of the catalase gene in T. gondii was found to be non-lethal, the enzyme presumably does not have a vital role in the parasite (13Kwok L.Y. Schluter D. Clayton C. Soldati D. Mol. Microbiol. 2004; 51: 47-61Crossref PubMed Scopus (101) Google Scholar). It is feasible that the parasite peroxiredoxins compensate for the loss of catalase and indeed the non-essentiality of catalase suggests that this group of proteins represents the major antioxidant defense system in Toxoplasma. Indeed, it has been shown that the thioredoxin-dependent reduction of hydroperoxides is of vital importance in the related apicomplexan parasite Plasmodium falciparum as thioredoxin reductase is essential for survival of this parasite (20Krnajski Z. Gilberger T.W. Walter R.D. Cowman A.F. Müller S. J. Biol. Chem. 2002; 277: 25970-25975Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In this study we have characterized the peroxiredoxin proteins of T. gondii, and the results suggest that the systems involving these proteins play key roles in protecting the parasite against oxidative damage. Materials—FITC-labeled secondary anti-rat and anti-rabbit antibodies were obtained from Molecular Probes, Leiden, The Netherlands. Polyclonal antibodies were raised against T. gondii thioredoxin (TgTrx) in a rat, T. gondii 1-Cys peroxiredoxin (TgTrx-Px2) and 2-Cys peroxiredoxin (TgTrx-Px1) in rabbits by Eurogentec (Belgium). Genomic and cDNA libraries of T. gondii were obtained from the National Institutes of Health. [3H]Uracil (48 Ci/mmol) was purchased from Amersham Biosciences. P. falciparum thioredoxin reductase was expressed and purified according to Gilberger et al. (21Gilberger T.W. Walter R.D. Müller S. J. Biol. Chem. 1997; 272: 29584-29589Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). P. falciparum thioredoxin was expressed and purified according to Krnajski et al. (22Krnajski Z. Gilberger T.W. Walter R.D. Müller S. Mol. Biochem. Parasitol. 2001; 112: 219-228Crossref PubMed Scopus (43) Google Scholar). Minimal essential medium (MEM), Dulbecco's modified Eagle's medium, RPMI 1640, penicillin/streptomycin, and fetal calf serum were from Invitrogen. Glutamine synthetase from Escherichia coli was purchased from Sigma. Human foreskin fibroblasts (HFFs) were from LGC Promochem (Teddington, UK). T. gondii Culture and Determination of IC50 of Oxidative Stressors—T. gondii tachyzoites (RH strain) were maintained on monolayers of HFFs in MEM containing 1% fetal calf serum at 37 °C in 5% CO2 according to Ref. 23Roos D.S. Donald R.G.K. Morrissette N.S. Moulton L.C. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (498) Google Scholar. In order to determine the susceptibility of T. gondii to the oxidative stressors tert-butyl-hydroperoxide (t-BuOOH), 5-hydroxy-1,4-naphthoquinone (juglone; Fluka) and the superoxide-generating compound phenazine methylsulfate (Fluka) a monolayer of HFFs was grown in 24-well plates, infected with 1 × 105 parasites per well, and incubated with increasing concentrations of the stressors (0.1 nm to 100 μm). After 24 h, the medium was supplemented with [3H]uracil (2.5 μCi/well) and incubated for an additional 4 h. Then the cells were treated with 0.6 m trichloric acid for 1 h and subsequently washed extensively with excess water overnight. Then the cells were solubilized for 1 h with 500 μl of 0.1 m NaOH before 250 μl of each well are transferred into scintillation vials containing 3 ml of Picofluor scintillation fluid (Packard Bioscience) and the incorporation of [3H]uracil into the ribonucleic acids of the parasites determined by scintillation counting (Beckman LS6500). In a control experiment, non-infected HFFs were treated with increasing concentrations of the three stressors under the same conditions as described for the parasitized cells, and their viability was determined using a Live/Dead assay (Molecular Probes) and subsequent analysis by fluorescent microscopy with an Axiovert 200 M microscope (Zeiss). 2′,7′-Dichlorodihydrofluorescein Diacetate (DCF) Assay—HFFs grown on chamber slides (Nunc) were infected with 0.5 × 104T. gondii and after 24 h exposed to 10 μm juglone or 10 μm phenazine methylsulfate for 15 min. After washing the slides, the cells were incubated for 3-5 min with 0.5 μm DCF, washed extensively with phosphate-buffered saline (PBS), and subsequently fixed using 4% paraformaldehyde. After mounting the slides, the parasites were analyzed using an Axiovert 200 m fluorescence microscope (Zeiss) equipped with AxioCam HRC digital camera (Zeiss). Isolation of Nucleic Acids and Proteins—In order to isolate genomic DNA and total RNA from T. gondii tachyzoites, they were grown to high density and isolated from lysed HFFs using 0.2 μm Nucleopore Track-Etch membranes (Whatman). Parasites were pelleted and either resuspended in TE buffer (10 mm Tris, pH 7.5, 1 mm EDTA) (for DNA isolation) or TRIzol (Invitrogen) for RNA isolation. In order to obtain parasite proteins, the isolated parasites were resuspended in PBS containing EDTA-free protease mixture (Roche Applied Science) and subsequently lysed by several cycles of freeze-thawing followed by sonication. Cloning of TgTrx-Px1 (2-Cys Peroxiredoxin), TgTrx-Px2 (1-Cys Peroxiredoxin), and TgTrx—Searching the T. gondii genome data base (www.ToxoDB.org) with the translated amino acids sequences of P. falciparum Trx, PfTrx-Px1 (2-Cys peroxiredoxin) and Pf1-CysTrx-Px (1-Cys peroxiredoxin) revealed the presence of homologues of the three Plasmodium proteins. Oligonucleotides TgTrx-1sense and TgTrx-1antisense (5′-GCGCCATATGCCGGTCCATCACGTCACC-3′ and 5′-GCGCGGATCCCTACAAATCCCCGAGACTGTTTGTC-3′), TgTrx-Px1-1 sense and antisense (5′-GCGCCATATGCCGGCCCCGATGGTGTCTCAG-3′ and 5′-GCGCCTCGAGAATGAACGAAGGCTCTATGAGG 3′) and TgTrx-Px2-1 sense and antisense (5′GCGCCATATGTTGGTCCTCGGCAGCACG3′ and 5′GCGCCTCGAGCTACGCCGACGGATCCGGAGC3′) were used to amplify the coding regions of the three genes with Pfu polymerase and a T. gondii cDNA library as template. The conditions for the polymerase chain reaction (PCR) were as follows: 1 cycle at 95 °C for 1 min and 30 cycles of 1 min at 95 °C, 1 min 50 to 60 °C, and 1 min at 72 °C followed by an extension cycle at 72 °C for 7 min. The PCR products were cloned into TOPO blunt (Invitrogen), and their sequences were determined by The Sequencing Service (School of Life Sciences, University of Dundee, Scotland, www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. In order to recombinantly express the three T. gondii proteins the inserts of the TOPO clones were isolated by restriction digest using NdeI, BamHI, and XhoI as required and subcloned into pJC40 previously digested with the respective restriction enzymes. The sequence of the expression constructs pJC40-TgTrx, pJC40-TgTrx-Px1, and pJC40-TgTrx-Px2 was verified and they were subsequently transformed into E. coli BLR (DE3)-pLys (Novagen). Recombinant Expression of TgTrx-Px1, TgTrx-Px-2, and TgTrx—Bacteria harboring the expression plasmids were used to inoculate overnight cultures in Luria-Bertani medium containing 50 μg ml-1 ampicillin. The cultures were diluted 1:50 and bacteria grown at 37 °C until their OD600 reached 0.5 before recombinant expression was induced using 1 mm isopropyl-β-thio-d-galactoside. The bacteria were cooled to 27 °C after induction and grown for an additional 18 h before they were harvested at 3500 × g (Beckman J6). Bacterial pellets were resuspended in 50 mm sodium phosphate buffer pH 8.0 containing 300 mm NaCl and 10 mm imidazole (lysis buffer) and lysed by adding 50 μg ml-1 lysozyme, freeze-thawing, and by using a French Press (1000 pounds inch-2; American Instrument Company). The bacterial extract was cleared by centrifugation for 1.5 h at 50,000 × g (Beckman, Avanti, JA-25). The resulting supernatants were applied to Ni2+-agarose, and the subsequent purification of the recombinant proteins was performed in a batch procedure according to the manufacturer's instructions (Qiagen). Protein concentrations were determined using the Bradford method (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) with bovine serum albumin as standard. Enzyme Assays for Thioredoxin—To test whether T. gondii Trx is a competent substrate of P. falciparum thioredoxin reductase (PfTrxR), the TrxR activity was determined spectrophotometrically using three different assay systems (1Hofmann B. Hecht H.J. Flohe L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (763) Google Scholar). In the insulin assay, the reduction of thioredoxin was coupled to the reduction of insulin as previously described (25Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar, 26Luthman M. Holmgren A. Biochemistry. 1982; 21: 6628-6633Crossref PubMed Scopus (503) Google Scholar). The second assay system was performed using a fast kinetics instrument (SFA-20, Hi-Tech Scientific) connected to the spectrophotometer (Shimadzu, 2401 PC) in order to determine the initial rates of Trx reduction (27Akerman S.E. Müller S. Mol. Biochem. Parasitol. 2003; 130: 75-81Crossref PubMed Scopus (48) Google Scholar). The assay mixture contained 200 μm NADPH, 100 mm HEPES pH 7.6, 1 mm EDTA, 1 μg of PfTrxR, and increasing concentrations of TgTrx or PfTrx (1-20 μm). Time points were taken every 10 ms, and the initial rates were used to determine the steady-state kinetic parameters for the reduction of both parasite Trxs (3Choi H.J. Kang S.W. Yang C.H. Rhee S.G. Ryu S.E. Nat. Struct. Biol. 1998; 5: 400-406Crossref PubMed Scopus (327) Google Scholar). The third assay system was performed in order to test whether TgTrx also reduces glutathione disulfide (GSSG) as has been previously reported for the proteins from P. falciparum and Drosophila melanogaster (28Kanzok S.M. Schirmer R.H. Turbachova I. Iozef R. Becker K. J. Biol. Chem. 2000; 275: 40180-40186Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 29Kanzok S.M. Fechner A. Bauer H. Ulschmid J.K. Müller H.M. Botella-Munoz J. Schneuwly S. Schirmer R. Becker K. Science. 2001; 291: 643-646Crossref PubMed Scopus (333) Google Scholar). The assay conditions were as follows: 5-10 μg of PfTrxR, 1 to 10 μm TgTrx or PfTrx, and 0.025-2 mm GSSG in 100 mm HEPES pH 7.6, 1 mm EDTA, and 200 μm NADPH. The change in absorbance was monitored spectrophotometrically at 340 nm, and the results were used to calculate the second order rate constant k2 according to Equation 1. v=k2⋅[Trx(SH) 2]⋅[GSSG] Further whether TgTrx and PfTrx are reduced by dihydrolipoamide dehydrogenase (LipDH) and dihydrolipoamide was tested. The assay systems contained 200 μm NADH, 5 units of dihydrolipoamide dehydrogenase (Sigma), 25 μm lipoamide, 1 mm EDTA, 100 mm Hepes pH 7.6, 10 to 50 μm of TgTrx and PfTrx, and 0.2 mg ml-1 insulin, and the change in absorbance was monitored at 340 nm on a Shimadzu 2401 PC spectrophotometer. The second order rates for the reduction of both parasite Trxs by dihydrolipoamide were calculated according to Equation 2. v=k2⋅[TrxS2]⋅[lipoamide disulfide] Peroxiredoxin Assays—The T. gondii 2-Cys peroxiredoxin kinetic parameters were determined according to Akerman and Müller (27Akerman S.E. Müller S. Mol. Biochem. Parasitol. 2003; 130: 75-81Crossref PubMed Scopus (48) Google Scholar) using a spectrophotometric assay employing stopped-flow fast kinetics with 15 μg of PfTrxR, 1-25 μm TgTrx, 0.2 μm TgTrx-Px1, 100 mm Hepes pH 7.6, 1 mm EDTA, 200 μm NADPH, and 0.5-15 μm hydroperoxide substrate. Steady-state kinetic parameters for the interaction TgTrx-Px1 with hydrogen peroxide and tert-butyl hydroperoxide (0.5-50 μm) were determined, and the resulting data were fitted using Grafit 5.0. Substrates for TgTrx-Px2—To establish the catalytic activity of TgTrx-Px2 the glutamine synthetase (GS) protection assay was utilized as described by Kim et al. (30Kim K. Kim I.H. Lee K.Y. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1988; 263: 4704-4711Abstract Full Text PDF PubMed Google Scholar). Because the endogenous reducing partner of T. gondii 1-Cys peroxiredoxins is elusive, a number of intracellular thiols, glutathione and dihydrolipoamide and dithiol-containing peptides, thioredoxin and glutaredoxin from P. falciparum, were tested for their ability to act as a reductant for TgTrx-Px2. The assay system consisted of 1 mm GSH, 200 μm NADPH, 100 mm HEPES pH 7.6, 1 mm EDTA, 8-10 μg of P. falciparum glutathione reductase prepared according to Ref. 31Gilberger T.W. Schirmer R.H. Walter R.D. Müller S. Mol. Biochem. Parasitol. 2000; 107: 169-179Crossref PubMed Scopus (47) Google Scholar, 0.5-5 μm TgTrx-Px2 and 100 μm H2O2. The glutaredoxin assay system essentially contained the same components as described above with the exception that 1-10 μmP. falciparum glutaredoxin, prepared according to Ref. 32Rahlfs S. Fischer M. Becker K. J. Biol. Chem. 2001; 276: 37133-37140Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, was included into the assay before the reaction was initiated by addition of H2O2. In the third assay system it was assessed whether lipoic acid acts as a reductant for both parasite 1-Cys peroxiredoxins using the following assay conditions, 200 μm NADH, 5-50 μm lipoamide, 5 units of dihydrolipoamide dehydrogenase (Sigma), 0.5-20 μm peroxiredoxin, and 100 μm H2O2. To test the ability of thioredoxin to act as a reducing thiol, a similar assay system as shown above with TgTrx-Px1 was performed where TgTrx-Px 1 was replaced with TgTrx-Px2. Western Blotting—Polyclonal antibodies were raised against TgTrx in rats and against TgTrx-Px1 and TgTrx-Px2 in rabbits (Eurogentec). The antibodies were purified according to standard protocols (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and used to detect the three proteins in Western blots of T. gondii tachyzoite protein extracts. The parasite protein extract was separated on a 4-12% SDS-PAGE (Invitrogen) and subsequently blotted onto nitrocellulose. The blots were probed with the primary anti-TgTrx rat antibody at 1:1000 dilution, the anti-TgTrx-Px1 at 1:250 dilution, and with the anti-TgTrx-Px2 antibody at 1:2000 dilution according to Sambrook et al. (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Anti-rat or anti-rabbit horseradish peroxidase-conjugated antibodies (DaKo; Scottish Antibody Production Unit) were applied at 1:5000, and the blots were developed using the ECL+ system (Amersham Biosciences). Immunofluorescence—HFFs grown on chamber slides were infected with 0.5 × 104T. gondii, and after 24 h the cells were fixed with 4% paraformaldeyde for 5 min at 37 °C and then permeabilized with 0.1% Triton X-100 for 5 min at room temperature. The wells were washed twice with PBS between each stage and blocked with 3% bovine serum albumin in PBS containing 0.05% Tween-20 and incubated overnight at 4 °C in a humidity chamber. Subsequently the wells were washed three times with PBS, 0.05% Tween-20 prior to incubation with primary rat anti-TgTrx or purified rabbit anti-TgTrx-Px1 and purified rabbit anti-TgTrx-Px2 antibodies, respectively, at dilutions between 1:200 and 1:2000 for at least 1 h at room temperature. After several washes with PBS, 0.05% Tween-20 the cells were incubated with a FITC-labeled anti-rat or anti-rabbit IgG (Molecular Probes) at a dilution of 1:500, incubated for 1 h, and after several washes slides were mounted using the slowfade light antifade kit containing 1.5 μg ml-1 DAPI (Molecular Probes) to stain the nucleic acids. Susceptibility to Oxidative Stress—T. gondii tachyzoites were exposed to increasing concentrations of the exogenous oxidant tert-butyl hydroperoxide and inducers of oxidative stress (juglone, phenazine methylsulfate) to determine their IC50 values (concentrations that caused 50% inhibition of viability). The latter two compounds cause endogenous oxidative stress either by generating reactive oxygen species by redox cycling or by non-enzymatic generation of superoxide anions in the presence of NADH or NADPH (34Hebbel R.P. Leung A. Mohandas N. Blood. 1990; 76: 1015-1020Crossref PubMed Google Scholar, 35Kampkötter A. Volkmann T.E. de Castro S.H. Leiers B. Klotz L.O. Johnson T.E. Link C.D. Henkle-Dührsen K. J. Mol. Biol. 2003; 325: 25-37Crossref PubMed Scopus (48) Google Scholar). T. gondii were found to be highly sensitive to all three compounds with IC50 values in the range between 100 and 500 nm (Fig. 1). They are most susceptible to tert-butyl hydroperoxide (IC50: 100 ± 8 nm) followed by juglone (IC50: 148 ± 16 nm) and phenazine methylsulfate (IC50: 406 ± 134 nm). At these concentrations, the viability of the host cells (HFFs) was not affected, as shown by incubating them by themselves under the same conditions as the parasite-infected HFFs and monitoring their viability with a Live/Dead assay system (data not shown). Indeed, the HFFs only start to show some response to the three stressors at concentrations 1 to 2 orders of magnitude higher than those affecting parasite growth. Further investigation revealed that parasite death was very likely caused by increased oxidative stress. Incubation of T. gondii-infected HFFs with the stressors and subsequent monitoring of the occurrence of oxidative stress using DCF, which enters the cell and is converted into a fluorescent compound after reaction with H2O2 (36Jakubowski W. Bartosz G. Cell Biol. Int. 2000; 24: 757-760Crossref PubMed Scopus (236) Google Scholar), revealed that juglone and phenazine methylsulfate cause strong fluorescence in the parasites, whereas no fluorescence was detected in the control cells (Fig. 2). Interestingly, the host cells did not show any increased oxidative stress, suggesting that the stressors specifically affected the parasite. Sequence Analyses of TgTrx-Px1, TgTrx-Px-2, and TgTrx—The genes encoding TgTrx-Px1 (2-Cys peroxiredoxin), TgTrx-Px2 (1-Cys peroxiredoxin), and TgTrx were amplified by PCR using a cDNA library as a template. The TgTrx-Px1 cDNA encodes a polypeptide of 196 amino acids and a theoretical molecular size of 21.7 kDa with 52.0% identity to P. falciparum 2-Cys peroxiredoxin PfTrx-Px1 and 55.6% identity to Trx-Px from Magnetoccocus (ZP_00043412), 53.6% identity to Trx-Px II from H. sapiens (P32119). TgTrx-Px1 contains two VCP motifs at positions 50-52 and 170-172, which is typical for the class of 2-Cys peroxiredoxins (
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