Cloning and Characterization of Three Differentially Expressed ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 36, Issue of September 7, pp. 34279 –34287, 2001 Printed in U.S.A.

Cloning and Characterization of Three Differentially Expressed Peroxidoxin Genes from Leishmania chagasi EVIDENCE FOR AN ENZYMATIC DETOXIFICATION OF HYDROXYL RADICALS* Received for publication, May 15, 2001, and in revised form, July 2, 2001 Published, JBC Papers in Press, July 3, 2001, DOI 10.1074/jbc.M104406200

Stephen D. Barr and Lashitew Gedamu‡ From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

Antioxidants have been implicated in protecting cells from oxygen radicals produced as a result of aerobic metabolism and in response to foreign pathogens by phagocytic cells. The mechanisms allowing pathogens to withstand the toxic prooxidant environment within the phagolysosome are poorly understood. We have cloned and characterized three antioxidant genes belonging to the 2-Cys family of peroxidoxins from Leishmania chagasi that may prove to provide these parasites with an enhanced defense mechanism against toxic oxidants. The 5ⴕ-untranslated regions and coding regions of each gene are highly conserved, whereas the 3ⴕ-untranslated regions have diverged significantly. L. chagasi peroxidoxin 1 (LcPxn1) is predominantly expressed in the amastigote stage, whereas LcPxn2 and LcPxn3 are expressed mainly in the promastigote stage, with LcPxn3 being far less abundant than LcPxn2. LcPxn2 and LcPxn3 possess a nine-amino acid extension at the carboxyl terminus, which LcPxn1 lacks. LcPxn1 appears to exist as high molecular weight multimers in vivo, and recombinant LcPxn1 was shown to detoxify hydrogen peroxide and alkyl hydroperoxides. We also present strong evidence that recombinant LcPxn1 can enzymatically detoxify hydroxyl radicals, an activity never before clearly demonstrated for a protein.

protein at amino acid position 170. Peroxidoxins exist predominantly as homodimers arranged in a head-to-tail orientation; however, evidence exists of multimeric forms of peroxidoxins (2–5). So far, the substrates of peroxidoxin include hydrogen peroxide, alkyl hydroperoxides (e.g. cumene and t-butyl hydroperoxides) (6), and peroxynitrite (ONOO⫺) (7). The mechanism of action for the detoxification of these substrates involves the oxidation of the Cys47 residue to form a sulfenic acid intermediate, which then reacts with the adjacent thiol group of the opposing subunit (Cys47 for 1-Cys peroxidoxins and Cys170 for the 2-Cys peroxidoxins) to form an intermolecular disulfide bond (6). Recently, a third type of peroxidoxin protein has been reported that resembles the 1-Cys peroxidoxins, where instead of forming an intermolecular disulfide bond with the adjacent monomer, it forms an intramolecular disulfide bond with a cysteine residue whose surrounding residues do not share homology with any of the conserved cysteine motifs found in other peroxidoxins from other species (8). The reported substrates of peroxidoxins suggest that they may play key roles in the defense against oxidative stress. H2O2, hydroperoxides, and ONOO⫺ are all extremely reactive by-products from the molecular reduction of O2 during normal cell metabolism and within the phagolysosomes of phagocytic cells during the respiratory burst. These molecules can be produced in many reactions such as Reactions 1– 4.

Peroxidoxins (also known as peroxiredoxins and thiol-specific antioxidants) comprise a family of antioxidants that have been recently discovered in numerous prokaryotes and eukaryotes. These proteins do not possess antioxidant activities found in other well known antioxidants. Peroxidoxins do not contain metal ions as seen in superoxide dismutases, they do not contain selenium-like glutathione peroxidases, nor do they contain heme like catalases (1). The gene sequence and protein functions of peroxidoxins are highly conserved among organisms and are currently classified into two groups: 1-Cys and 2-Cys. The 1-Cys peroxidoxins contain a conserved cysteine residue in the amino-terminal region of the protein at amino acid position 47, whereas the 2-Cys peroxidoxin, in addition to the conserved cysteine 47 residue, also contains a second conserved cysteine residue in the carboxyl-terminal region of the

O2 ⫹ Fe2⫹ ¡ O2. ⫹ Fe3⫹ 2O2. ⫹ 2H⫹ ¡ H2O2 ⫹ O2 O2 ⫹ NO⫺ ¡ ONOO⫺ NO䡠 ⫹ O2. ¡ ONOO⫺ REACTIONS 1– 4

A molecule that is of great concern to all cells is the hydroxyl radical (䡠OH). Due to its large reduction potential, 䡠OH is the most biologically reactive molecule known to exist. 䡠OH can be produced as a result of numerous reactions such as Reactions 5–9. O2. ⫹ H2O2 ¡ 䡠 OH ⫹ O2 ⫹ ⫺OH

* This work was supported by a grant from the Canadian Institutes of Health Research (to L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF134161 (for LcPxn1), AF312397 (for LcPxn2), AF312398 (for LcPxn3), and AF205887 (for LdPxn1). ‡ To whom correspondence should be addressed. Tel.: 403-220-5556; Fax: 403-289-9311; E-mail: [email protected]. This paper is available on line at http://www.jbc.org

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O2. ⫹ HOCl ¡ 䡠 OH ⫹ O2 ⫹ Cl⫺ H2O2 ⫹ UV ¡ 2 䡠 OH H2O2 ⫹ Fe2⫹ ¡ 䡠 OH ⫹ ⫺OH ⫹ Fe3⫹ ONOO⫺ ⫹ H⫹ ¡ 䡠 OH ⫹ NO2 ⫹ NO2⫹ REACTIONS 5–9

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䡠OH reacts with all biological targets at diffusion-limited rates and like other prooxidants can lead to the peroxidation of lipids, lethal damage to DNA, and the oxidation of sugars and protein thiols (9). To date, no clear evidence for an enzymatic defense against 䡠OH has been demonstrated. Most intracellular pathogens are heavily equipped with antioxidant defenses such as superoxide dismutase, catalases, and glutathione peroxidases in order to withstand the toxic prooxidant environment of the phagolysosome. However, many intracellular pathogens such as Leishmania lack detectable catalases and glutathione peroxidases. Leishmania is an obligate intracellular protozoan parasite of mammalian macrophages. The parasites exist in two forms during their life cycle. The extracellular promastigote form is found to survive in the gut of its sandfly vector and is inoculated into its host during the bite of the sandfly. It is phagocytosed by cells such as macrophages and transforms into the intracellular amastigote form. Phagocytosis of these parasites is accompanied by an oxidative burst that results in the production of all of the reactive prooxidants aforementioned in Reactions 1–9 (10). Clearly, Leishmania parasites and other intracellular pathogens must evade the toxic effects of these prooxidants in order to survive and establish an infection. We have previously reported that Leishmania chagasi possesses iron superoxide dismutases, which act as a first line of defense against O2. (11), but no efficient defense against H2O2, hydroperoxides, ONOO⫺, or 䡠OH has been demonstrated. This paper presents the isolation and characterization of three different peroxidoxins from L. chagasi and is the first report of the differential expression of multiple peroxidoxins in Trypanosomatids. We demonstrate that one of the recombinant L. chagasi peroxidoxins (LcPxn1)1 can detoxify both H2O2 and alkyl hydroperoxides in vitro. In addition, we present for the first time clear evidence that LcPxn1 can enzymatically detoxify 䡠OH in vitro. EXPERIMENTAL PROCEDURES

Materials—All enzymes, [␣-32P]dCTP, Hybond N⫹, Rapid-Hyb buffer, and protein molecular weight markers were purchased from Amersham Pharmacia Biotech. DNA and RNA molecular weight markers, agarose and parasite medium, and medium supplements were obtained from Life Technologies, Inc. All other chemicals unless stated otherwise were of the highest purity and purchased from Sigma. Nucleic acid quantifications were done using the Beckman DU 640 Spectrophotometer. Parasites—L. chagasi (MHOM/BR/00/1669) and Leishmania donovani (1S2D kindly provided by Dr. Greg Matlashewski) promastigote parasites were cultured at 26 °C in minimal essential medium supplemented with L-glutamine, sodium pyruvate, minimal essential medium, essential and nonessential amino acids, glucose, sodium bicarbonate, 10% fetal calf serum (inactivated at 58 °C for 30 min), 20 ␮g/ml Hemin, and 35 mM Hepes sodium salt (hemoflagellate minimal essential medium). Promastigotes were seeded at 1 ⫻ 106/ml and harvested at logarithmic or stationary phase, defined according to morphological characteristics as previously described (12). Genomic DNA Isolation—Genomic DNA was isolated from L. chagasi and L. donovani logarithmic promastigotes by lysing parasites in lysis buffer (10 mM Tris-Cl, pH 8.3, 50 mM EDTA, 1% SDS). RNase A (100 ␮g ml⫺1) (Sigma) was added to the suspension and incubated overnight at 37 °C, after which 100 ␮g ml⫺1 proteinase K (Sigma) was added and incubated at 42 °C overnight. The DNA was extracted with a phenolchloroform treatment and ethanol-precipitated. Cloning and Sequencing of Peroxidoxin cDNA and Genomic Clones— Two primers were designed based on the sequences surrounding the two conserved cysteine residues (Cys52 and Cys173) of the 2-Cys family 1 The abbreviations used are: LcPxn1–3, L. chagasi peroxidoxins 1–3; LdPxn1, L. donovani peroxidoxin 1; PCR, polymerase chain reaction; GST, glutathione S-transferase; IPTG, isopropyl-1-thio-␤-D-galactoside; bp, base pair(s); kb, kilobase pair(s); UTR, untranslated region; NEM, N-ethylmaleimide; BSA, bovine serum albumin; MFO, mixed function oxidation; UTR, untranslated region; Fe-SOD, iron-containing superoxide dismutase; SA, specific activity.

of peroxidoxins previously isolated from Trypanosoma brucei rhodesiense (GenBankTM accession number U26666): sense primer 1 (5⬘-TATTCGGATCCTCTCGACTTCACGTTTGTGTGCCCC-3⬘; antisense primer 2 (5⬘-TCCAGGGATCCAGTTAGCGGGGCACACCTCACCGTG-3⬘ (underlined sequence refers to T. brucei peroxidoxin sequence). The amino acid sequence surrounding the conserved cysteine residues in the primers are as follows: sense primer 1, 45PLDFTFVCP53; antisense primer 2, 169HGEVCPAN176 (underlined C represents Cys52 and Cys173, respectively). Primer 1 and primer 2 were used to PCR-amplify L. chagasi and L. donovani genomic DNA (94 °C for 5 min; 40 cycles of 94 °C for 1 min, 40 °C for 2 min, 72 °C for 2 min; 72 °C for 10 min) using a PHC-2 Dri-Block Cycler (Techne, Cambridge, UK). The amplified product (⬃350 bp) was gel-purified and sequenced using a PCR cycle sequencing kit as per the manufacturer’s instructions (Amersham Pharmacia Biotech). L. chagasi genomic DNA was digested to completion. Restriction fragments ⬃9 –25 kb in size were extracted using standard low melting point-agarose protocol (13). Purified BamHI fragments were cloned into Lambda DASHII DNA and packaged as per the manufacturer’s instructions (Stratagene). The ⬃350-bp PCR fragment isolated above was randomly labeled with [␣-32P]dCTP using the T7 Quick Prime Kit (Amersham Pharmacia Biotech) and used as a probe to screen the Lambda DASHII BamHI library for an individual clone containing the peroxidoxin cluster. ⬃105 plaque-forming units were plated, and duplicate filters were lifted. Potential positive clones were treated to secondary screening to recover isolated clones. Isolated clones were amplified using standard protocols (13), and DNA was extracted using a Lambda Maxi Kit (Qiagen). The ␭ DNA was digested with SalI, and a pGEM-2 plasmid library was constructed using the SalI-restricted fragments (Promega). Several potential positive clones were characterized by restriction digestion analysis and sequencing using T3, T7, and various internal primers (University of Calgary DNA Sequencing Facility, Calgary, Alberta, Canada). Similarly, amastigote cDNA libraries from L. chagasi and L. donovani were screened to obtain full-length cDNAs. DNA from the isolated clones was recovered into pBluescript (pBS) vector using an in vivo excision procedure utilizing R408 helper phage and Escherichia coli XL-1 Blue cells (Stratagene, Palo Alto, CA). The sequences of all isolated clones have been deposited in GenBankTM with the following accession numbers: LcPxn1, AF134161; LcPxn2, AF312397; LcPxn3, AF312398; LdPxn1, AF205887 (L. donovani clone). Southern and Northern Blots—Genomic DNA was restricted with various enzymes and separated on a 1% agarose gel. The DNA was transferred to Hybond N⫹ via capillary action (13) and fixed with long wave ultraviolet light for 5 min. The blots were blocked with Rapid-Hyb buffer and incubated at 65 °C with [␣-32P]dCTP-labeled DNA probes for 2 h, after which the blots were washed successively with decreasing salt concentrations at 65 °C for 15 min each wash. Total RNA was isolated from promastigotes using the acid guanidinium isothiocyanate method (14). Varying amounts of total RNA was separated in a 1.2% formaldehyde-containing agarose gel, transferred onto Hybond N⫹ membrane via capillary action, and baked at 80 °C for 2 h. The blots were blocked with Rapid-Hyb buffer (Amersham Pharmacia Biotech) and incubated at 62 °C with [␣-32P]dCTP random prime-labeled DNA probes for 2 h, after which the blots were washed with decreasing salt concentrations at 62 °C for 15 min each wash. All RNA blots were hybridized with [␣-32P]dCTP-labeled ␣-tubulin as a loading control (kindly provided by Dr. M. E. Wilson (University of Iowa, Iowa City). Blots were exposed to film at ⫺80 °C with an intensifying screen (X-Omat; Eastman Kodak Co.). Expression and Purification of Recombinant Peroxidoxin Protein— The prokaryotic expression vector pGEX-2T (Amersham Pharmacia Biotech) was used to express LcPxn1 as a GST fusion in E. coli DH5␣ cells. The coding region of LcPxn1 was amplified by PCR using the sense primer (Pxn1ATG) 5⬘-ACCAGGGATCCATGTCCTGCGGTGACGCC-3⬘ and the antisense primer (Pxn1TAA) 5⬘-ACATCGGATCCTTACTTATTGTGATCGACCTTCAGGCC-3⬘. Both primers contained a BamHI site (underlined) to facilitate insertion of the amplified product into the pGEX-2T vector using standard techniques (13). pGEX-2T vector alone and the recombinant pGEX-2T/LcPxn1 vector were used to transform E. coli DH5␣ cells. Transformed cells were grown shaking at 37 °C in Luria-Burtani broth containing 100 ␮g ml⫺1 ampicillin for 8 h, after which 0.2 mM IPTG was added to the culture and shaken overnight. Fusion protein (GST-LcPxn1) and GST protein alone were harvested by sonication and passing over a glutathione-agarose resin column (Sigma) as described by Smith and Johnson (15). The GSTLcPxn1 fusion protein was cleaved with thrombin (50 cleavage units/ml of bed resin) overnight at 24 °C and passed over a glutathione-agarose column to remove excess GST protein. Protein purity was determined

Differentially Expressed Peroxidoxin Genes from L. chagasi on a SDS-polyacrylamide gel, and the concentration was determined using the BCA protein assay kit (Pierce). Generation of Polyclonal Antibodies—⬃200 ␮g of recombinant GSTLcPxn1 protein was purified from a denaturing SDS-polyacrylamide gel, crushed in liquid nitrogen, resuspended in 1⫻ phosphate-buffered saline, and injected subcutaneously into New Zealand White rabbits. The first injection was done in combination with complete Freund’s adjuvant; monthly injections thereafter were done in combination with incomplete Freund’s adjuvant. Bleeds were taken 7 days after boosters until an acceptable titer was obtained for Western blot analysis. Peroxide Assays—Hydrogen peroxide metabolism was measured as described by Thurman et al. (16). Briefly, the reaction mixture contained 50 mM Tris-HCl (pH 8.0), 0.2 mM dithioerythritol, 50 ␮M H2O2 (or 50 ␮M t-butylhydroperoxide or 50 ␮M cumene hydroperoxide), and various concentrations of protein. Immediately prior to the addition of the peroxides to the reaction, the recombinant peroxidoxin protein was incubated with 0.2 mM dithioerythritol for 30 min at 37 °C in order to reduce the disulfide bridges formed between the catalytic cysteine residues. The reaction was stopped at various time intervals with the addition of 1 ml of trichloroacetic acid (10% final concentration). After the precipitated protein was removed with a quick spin, 0.2 ml of 10 mM ferrous ammonium sulfate and 0.1 ml of 2.5 M potassium thiocyanate were added to the reactions and mixed thoroughly. Peroxide concentrations were determined spectrophotometrically at 480 nm using known amounts of peroxide (1–50 ␮M) as a standard. All solutions were made fresh immediately before use. Hydroxyl Radical-DNA Nicking Assay—3 ␮M FeCl3 and 0.1 mM EDTA were allowed to react for 10 min at 37 °C to generate 䡠OH as described by Lim et al. (17). 0.5 mg/ml recombinant protein (preincubated with 0.2 mM dithioerythritol for 30 min at 37 °C) was then added to the mixture and incubated at 37 °C for 30 min. Peroxidoxin was inactivated with 5 mM N-ethylmaleimide (Sigma) for 30 min at 30 °C immediately before adding it to the experimental assay. 2 ␮g of pGEM-2 plasmid (Promega) was then added to each tube and incubated at 37 °C for 4 h. The DNA was separated on a 1% agarose gel at 100-V constant voltage. All solutions were made fresh immediately before use. Hydroxyl Radical-Deoxyribose Assay—The production of 䡠OH and the 䡠OH-induced damage of 2-deoxy-D-ribose were measured using the protocol described by Halliwell and Gutteridge (18). A 50-␮l reaction mixture was set up to contain the following components to give the final concentrations as stated: 10 mM potassium phosphate buffer (pH 7.4); 63 mM NaCl, 0.8 mM 2-deoxy-D-ribose; 0.2 mM dithioerythritol; 0.125 ␮g/␮l peroxidoxin (preincubated in 0.2 mM dithioerythritol for 30 min at 37 °C). 21 ␮M ferrous ammonium sulfate was added to initiate the reaction, and the tubes were incubated at 37 °C for 15 min. 100 ␮l of thiobarbituric acid (1% w/v) and 100 ␮l of trichloroacetic acid (2.8%, w/v) were then added to the mixture and boiled for 10 min. Tubes were quick spun, and the fluorescence was then measured in a 96-well plate using a SpectraMax Gemini plate reader (Molecular Devices) with six reads/well (excitation ⫽ 532 nm; emission ⫽ 553 nm). All solutions were made fresh immediately before use. RESULTS

Southern Blot Hybridization Analysis of the Peroxidoxin Gene Locus—The amino acid sequences surrounding the two cysteine residues of 2-Cys peroxidoxins from a variety of organisms are highly conserved (6). Two primers were designed containing some of the conserved amino acids surrounding Cys52 and Cys173 from the 2-Cys peroxidoxin from Trypanosoma brucei rhodesiense. The Cys52 and Cys173 residues are equivalent to the conserved Cys47 and Cys170 residues observed in other 2-Cys peroxidoxins. Primer 1 and primer 2 were used to PCR-amplify genomic DNA from L. chagasi. A single ⬃350-bp PCR product was obtained, and sequence analysis of the PCR fragment revealed 90.0% identity to the corresponding amino acid sequence between the two conserved cysteine residues of the Leishmania major peroxidoxin cDNA (19, 20). A Southern blot hybridization using the ⬃350-bp PCR product as a probe was performed on total DNA from L. chagasi and L. donovani that was subjected to restriction digestion with BamHI, PstI, SacII, and SalI enzymes (Fig. 1). The hybridization pattern of the peroxidoxin probe appears to be very similar between L. chagasi and L. donovani. The hybridization pattern observed in Fig. 1 also reveals that the peroxidoxins are part of

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FIG. 1. Southern blot of L. chagasi and L. donovani. Total DNA (500 ng) of each species was isolated; restricted with BamHI (BHI), PstI (P), SacII (Sc), and SalI (SI); resolved on a 1% agarose gel; and transferred to nitrocellulose. The membrane was hybridized with a radiolabeled peroxidoxin-specific coding region probe.

a multigene family and that the entire peroxidoxin cluster is contained within a single ⬃23-kb BamHI restriction fragment in both strains. Partial restriction digestions with SacII, pulsed field gel electrophoresis, and Southern blot analyses using various different probes suggest that there are approximately six peroxidoxin genes all present on one chromosome in both L. chagasi and L. donovani (data not shown). Cloning and Sequence Analysis of Peroxidoxin Genes—From Fig. 1 it was apparent that the entire L. chagasi peroxidoxin cluster is contained within a ⬃23-kb BamHI restriction fragment. In order to obtain full-length peroxidoxin clones, a Lambda DASHII library was constructed containing large BamHI fragments. Using the ⬃350-bp PCR product as a probe, a ␭ clone containing the peroxidoxin cluster was isolated. The ␭ clone was digested with SalI, and a pGEM-2 plasmid library was constructed and screened for bacterial clones containing peroxidoxin fragments. Sequence analysis of several clones revealed three different peroxidoxin genes: LcPxn1, LcPxn2 and LcPxn3 (Fig. 2, A and B). In parallel, amastigote cDNA libraries from L. chagasi and L. donovani were screened. The sequence of several potential positive clones from each library corresponded to the sequence of LcPxn1 that was obtained from the genomic clones. The 5⬘-UTR, coding region, and 3⬘-UTR sequences of the clone LdPxn1 obtained from the L. donovani amastigote library were 100% identical to the LcPxn1 clone. Blast searches of LcPxn1, LcPxn2, and LcPxn3 amino acid sequences revealed significant homology to several other 2-Cys peroxidoxins from other organisms, particularly the amino acid sequence surrounding Cys52 and Cys173 (Fig. 2C). LcPxn1 consists of 190 amino acids with a predicted molecular mass of 21.3 kDa and theoretical pI ⫽ 6.31. Both LcPxn2 and LcPxn3 consist of 199 amino acids each with predicted molecular masses of 22.1 kDa and a theoretical pI of 7.6. The coding region of LcPxn1 is 89.4% similar to the coding regions of LcPxn2 and LcPxn3, whereas LcPxn2 and LcPxn3 are 99.5% identical to each other, the difference being a single amino acid change at amino acid position 199 (Gln in LcPxn2; Leu in LcPxn3). The carboxyl ends of LcPxn2 and LcPxn3 have diverged significantly from LcPxn1. The carboxyl terminus of LcPxn2 and LcPxn3 contains a nine-amino acid extension that is not present in LcPxn1. At the end of this extension in LcPxn3 is a putative glycosomal targeting signal sequence (SKL) (21). Interestingly, the one amino acid substitution (Leu for Gln) at

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FIG. 2. Sequence comparisons of several different peroxidoxins. A, amino acid comparison of LcPxn1, LcPxn2, and LcPxn3 coding regions. B, nucleic acid comparison of the first 90 bases of the 3⬘-UTRs after the stop codons of LcPxn1, LcPxn2, and LcPxn3. C, sequence comparisons of LcPxn1, LcPxn2, and LcPxn3. Conserved cysteines that are characteristic of 2-Cys peroxidoxins are at amino acids 87 and 209. Gray boxes denote

Differentially Expressed Peroxidoxin Genes from L. chagasi the carboxyl terminus of LcPxn2 does not appear to code for a putative glycosomal targeting sequence or any other targeting sequence (21). LcPxn1 lacks the nine-amino acid extension found in LcPxn2 and LcPxn3 and does not appear to possess any putative organelle targeting signal sequences. The 239 nucleotides immediately upstream of the start codons of each peroxidoxin gene are virtually identical (data not shown), whereas the 3⬘-UTRs are very different (Fig. 2B).2 These results suggest that there are at least three different, yet highly conserved, 2-Cys peroxidoxins in L. chagasi. Differential Expression of Peroxidoxin mRNAs—To study the expression pattern of peroxidoxin mRNA as the parasites progress through their life cycle, we isolated total RNA from parasites at early logarithmic phase, stationary phase, and amastigote stage (mimicked by infecting U937 macrophagelike cells). The amount of RNA loaded in each lane was normalized with a constitutively expressed ␣-tubulin probe (Fig. 3E). Northern blot analysis of this total RNA using the LcPxn1 coding region as the probe revealed the presence of two different sized transcripts (⬃1.6 and ⬃2.4 kb) that were differentially expressed (Fig. 3A). A 3⬘-UTR-specific probe from LcPxn1 hybridized to a ⬃1.6-kb transcript and, after taking into account the loading controls, revealed a visually significant increase in intensity from the early logarithmic phase to the amastigote phase (Fig. 3B). A 3⬘-UTR-specific probe from LcPxn2 hybridized to a ⬃2.4-kb sized transcript but conversely demonstrated a significant decrease in intensity from the early logarithmic to the amastigote phase (Fig. 3C). A 3⬘-UTR-specific probe from LcPxn3 hybridized to a ⬃1.6-kb transcript and revealed a similar pattern of expression to the LcPxn2 transcript with a significant decrease in intensity from the early logarithmic phase to the amastigote phase (Fig. 3D). LcPxn3 transcripts appeared to be significantly less abundant than LcPxn2 after taking into account the specific activity of the probe and that the blot had to be exposed to film for 3 days to obtain a signal with intensity close to LcPxn1 and LcPxn2. The presence of the LcPxn3 band in the Northern blot using the coding region of LcPxn1 was evident after a 3-day exposure (data not shown). These results clearly demonstrate that LcPxn1 is predominantly expressed in the amastigote stage, while LcPxn2 and LcPxn3 are expressed predominantly in the promastigote stage of L. chagasi. Similar studies with RNA isolated from L. donovani revealed an identical pattern of peroxidoxin mRNA expression (data not shown). Expression and Purification of Recombinant L. chagasi Peroxidoxin Protein—The coding region of LcPxn1 was cloned into the E. coli expression vector pGEX-2T and used to transform E. coli DH5␣ cells. Induction of the protein expression system with 0.2 mM IPTG at 24 °C overnight resulted in a majority of the fusion protein being expressed in the soluble form (Fig. 4, lanes 1 and 2). Recombinant GST-LcPxn1 fusion protein was isolated by passage over glutathione-Sepharose beads (Fig. 4, lane 3), cleaved with thrombin, and further purified by passage over fresh glutathione-Sepharose beads (Fig. 4, lane 4). Purified LcPxn1 protein migrated with a molecular mass of ⬃22 kDa under denaturing conditions, which correlates well with the predicted molecular mass of 21.3 kDa. To ascertain whether LcPxn1 mRNA is translated into protein within L. chagasi parasites, stationary phase parasite extract was sepa-

2

S. D. Barr and L. Gedamu, unpublished results.

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rated on a polyacrylamide gel under denaturing conditions. Western blot analysis using crude LcPxn1 polyclonal antibody resulted in a diffuse band ⬃22 kDa in size (Fig. 4B). The appearance of a diffuse band could be indicative of a high abundance of LcPxn1 protein and/or cross-reactivity of the polyclonal antibody with other peroxidoxin proteins. Peroxidoxins are known to exist predominantly as homodimers in many organisms (6), so we wanted to determine if this was the case with L. chagasi peroxidoxins. Recombinant LcPxn1 protein was separated on an 8% polyacrylamide gel under nondenaturing conditions. A Coomassie stain of this gel revealed three distinct bands ranging from ⬃80 to 140 kDa (Fig. 4C, lane 1). To determine whether this was an artifact from purification of the protein, we performed a Western blot using crude recombinant LcPxn1 antibody and parasite extract separated under nondenaturing conditions. A diffuse band was observed ranging from ⬃100 to 180 kDa (Fig. 4D, lane 2). Taken together, these results suggest that L. chagasi peroxidoxins may exist as high molecular weight multimers in vivo, a finding also observed for peroxidoxins in Crithidia fasciculata (2), human erythrocytes (3), and Thermus aquaticus (5). Peroxides Are Enzymatically Detoxified by Recombinant LcPxn1—The highly conserved amino acid sequence of peroxidoxins among organisms suggests a conserved function in peroxidase metabolism for all peroxidoxin proteins. We have analyzed the ability of recombinant LcPxn1 to detoxify hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide in vitro. Table I shows the peroxide-detoxifying activities of recombinant LcPxn1. Under our conditions, LcPxn1 removed hydrogen peroxide at a rate of 307.4 ⫾ 22.1 nmol min⫺1 ␮g⫺1, cumene hydroperoxide at a rate of 352.0 ⫾ 72.2 nmol min⫺1 ␮g⫺1, and t-butyl hydroperoxide at a rate of 126.8 ⫾10.9 nmol min⫺1 ␮g⫺1. In each case, boiled LcPxn1 did not remove any of the peroxides at a significant rate (Table I). NEM-treated LcPxn1, GST alone, and 0.2 mM dithioerythritol alone also did not significantly remove any of the peroxides (data not shown). These results strongly support the highly conserved peroxide/ hydroperoxide detoxification activities observed in most peroxidoxins isolated from other organisms. Hydroxyl Radicals Are Enzymatically Detoxified by Recombinant LcPxn1—Reactive oxygen species have been implicated in a number of cytotoxic events such as lethal damage to the genetic blueprint of cells. Although considered reactive oxygen species, it appears that O2. and H2O2 are not capable of producing DNA strand breaks at physiological concentrations (22, 23). All of the toxicity of O2. and H2O2 in vivo arises from their conversion into 䡠OH (Reactions 5 and 8) (24 –26). It has been demonstrated that 䡠OH can induce strand breaks in DNA as well as chemical changes to the bases and deoxyribose (27). Using a thiol-mixed function oxidation (MFO) system to generate 䡠OH in vitro (28), we have studied the ability of L. chagasi peroxidoxins to protect intact supercoiled plasmid DNA and deoxyribose from 䡠OH-induced nicking and degradation, respectively. In the absence of the thiol-MFO system, the majority of the pGEM-2 plasmid DNA stayed in the supercoiled form (Fig. 5, lane 1). Recombinant LcPxn1 protein alone had no effect on the plasmid in the absence of the thiol-MFO system (Fig. 5, lane 2). In the presence of the thiol-MFO system, most of the plasmid was converted into the nicked form after the 4-h incubation (Fig. 5, lane 3). The addition of 0.5 mg/ml recombinant LcPxn1

amino acid differences. GenBankTM accession numbers are as follows: LcPxn1 (AF134161), LcPxn2 (AF312397), LcPxn3 (AF312398), Leishmania major (AF069386, AF044679), Trypanosoma brucei (U26666), Entamoeba histolytica (X70996), Fasciola hepatica (U88577), Onchocerca vulvulus (AF029247), Brugia malayi (U34251), Candida albicans (AF149421), Saccharomyces cerevisiae (L14640), and Homo sapiens (X82321). Gray shading represents nucleic acid differences.

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FIG. 3. Northern blot analyses. 10 ␮g of total RNA extracted from early log phase L. chagasi parasites (lane 1), stationary phase L. chagasi parasites (lane 2), U937 cells infected with L. chagasi parasites representing amastigotes (lane 3), and U937 uninfected cells was extracted, resolved on a 1.2% agarose gel under denaturing conditions, blotted, and probed with coding region of LcPxn1 (SA ⫽ 3.2 ⫻ 108 cpm/␮g) (A), 3⬘-UTR of LcPxn1 (SA ⫽ 3.4 ⫻ 108 cpm/␮g) (B); 3⬘-UTR of LcPxn2 (SA ⫽ 3.0 ⫻ 108 cpm/␮g) (C), 3⬘-UTR of LcPxn3 (SA ⫽ 4.5 ⫻ 108 cpm/␮g) (D), or ␣-tubulin from L. chagasi (loading control) (SA ⫽ 3.5 ⫻ 108 cpm/␮g) (E). F, ethidium bromide stain of the agarose gel. The RNA band corresponding to the 28 S RNA (F, lane 4) is absent in the U937-infected RNA (F, lane 3) because the infection was allowed to proceed for 4 days, resulting in nearly complete lysis of the infected U937 cells. A–C, exposure times of 21 h; D, 72 h; E, 2 h at room temperature.

and the 䡠OH scavengers mannitol and catechol to the system completely abolished the conversion of the DNA into the nicked form (Fig. 5, lanes 4, 13, and 14, respectively). Boiled LcPxn1, NEM-treated LcPxn1, NEM alone, and nonspecific proteins GST and BSA conferred no observable protection against nicking of the DNA (Fig. 5, lanes 5, 6, 7, 11, and 12). To determine whether the protection observed by LcPxn1 was due to its ability to remove H2O2, 0.5 mg/ml LcPxn1 in the presence of a 10 mM excess of H2O2 did not hinder LcPxn1’s ability to protect the DNA (Fig. 5, lane 8). Furthermore, the addition of 1 unit of catalase to the system did not protect the DNA from nicking (Fig. 5, lane 9), whereas the addition of both recombinant LcPxn1 and catalase was able to protect the DNA (Fig. 5, lane 10). In the presence of a thiol-MFO system, deoxyribose undergoes 䡠OH-induced degradation to yield a chromogenic product that emits fluorescence at 553 nm after heating under acidic conditions with thiobarbituric acid (18, 29). The addition of 䡠OH scavengers such as catechol and mannitol to the system results in 90.3 and 88.0% protection of the deoxyribose from degradation (Fig. 6, bars 1 and 2, respectively). In the presence of 0.125 mg/ml recombinant LcPxn1, 90.4% of the deoxyribose was protected (Fig. 6, bar 3). 0.125 mg/ml boiled LcPxn1 (Fig. 6, bar 4), NEM-treated LcPxn1 (Fig. 6, bar 5), and NEM alone (Fig. 6, bar 6) resulted in a significant decrease in protection, 68.8, 65.1, and 21.5%, respectively. To determine how much of the protection could be contributed to the presence of protein nonspecifically, we used equal

FIG. 4. Protein analysis of recombinant LcPxn1. A, recombinant LcPxn1/GST protein was induced with 0.2 mM IPTG at 24 °C overnight. Cell-sonicated supernatant was passed over a GST column to purify the fusion protein. Recombinant LcPxn1/GST protein was cleaved with thrombin and further purified through a GST column. Aliquots of the various supernatant fractions were separated on a 15% polyacrylamide gel under reducing conditions. A, lane 1, uninduced E. coli fraction; lane 2, E. coli fraction induced with IPTG; lane 3, purified recombinant LcPxn1/GST protein; lane 4, LcPxn1 protein after cleavage with thrombin and purification. B, Western blot analysis using crude LcPxn1 polyclonal antibody and whole cell extract from 5 ⫻ 106 stationary phase parasites separated on a 15% polyacrylamide gel electrophoresis under reducing conditions. C, aliquots of recombinant purified LcPxn1 (lane 1), whole cell extract from parasites (lane 2), GST (lane 3), and recombinant L. chagasi Fe-SODB1 were separated on an 8% polyacrylamide gel under nonreducing conditions and stained with Coomassie Brilliant Blue. D, Western blot analysis of gel (C) using crude LcPxn1 polyclonal antibody. No bands were observed below 46 kDa. TABLE I Peroxide-detoxifying activities of recombinant LcPxn1 Units of activity are measured in nmol/min/␮g of recombinant protein.

LcPxn1 Boiled LcPxn1 a b

H2 O2

Cumene hydroperoxide

t-Butyl hydroperoxide

307.4 ⫾ 22.1a 96.8 ⫾ 34.4b

352.0 ⫾ 72.2b 90.7 ⫾ 5.3b

126.8 ⫾ 10.9b 30.4 ⫾ 4.2b

Activity is the mean ⫾ S.E. of six independent trials. Activities are the means ⫾ S.E. of three independent trials.

amounts of GST and BSA (0.125 mg/ml) in the assay. GST (Fig. 6, bar 7) and BSA (Fig. 6, bar 8) did not confer significant protection, 63.0 and 71.0%, respectively. 0.14 units of catalase, which removed hydrogen peroxide at a similar rate to 0.125 mg/ml recombinant LcPxn1 under the assay conditions, conferred 13.3% protection to the deoxyribose (Fig. 6, bar 9). We found no evidence that LcPxn1 interfered with the formation of the chromogenic adduct, and peroxidoxins have been shown not to bind iron ions, which would prevent 䡠OH formation in the reaction mixture (1). These results correlate well with the DNA nicking results and strongly suggest that LcPxn1 can enzymatically detoxify 䡠OH.

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FIG. 5. 䡠OH DNA nicking assay. 3 ␮M FeCl3 was added to the reaction mixture and incubated at 37 °C for 10 min to generate 䡠OH. 0.5 mg/ml test proteins were then incubated with the mixture for an additional 30 min at 37 °C. 2 ␮g of pGEM-2 plasmid was then added to the reaction and incubated for a further 4 h at 37 °C during which 䡠OH-induced DNA nicking occurred. Samples were then loaded immediately onto a 1% agarose gel and run at 100-V constant voltage. Lane 1, no FeCl3; lane 2, no FeCl3 ⫹ LcPxn1; lane 3, FeCl3; lane 4, FeCl3 ⫹ LcPxn1; lane 5, FeCl3 ⫹ boiled LcPxn1; lane 6, FeCl3 ⫹ 5 mM NEM-treated LcPxn1; Lane 7, FeCl3 ⫹ 5 mM NEM; lane 8, FeCl3 ⫹ LcPxn1 ⫹ 10 mM H2O2; lane 9, FeCl3 ⫹ 1 unit of catalase; lane 10, FeCl3 ⫹ LcPxn1 ⫹ 1 unit of catalase; lane 11, FeCl3 ⫹ GST; lane 12, FeCl3 ⫹ BSA; lane 13, FeCl3 ⫹ 10 mM mannitol; lane 14, FeCl3 ⫹ 0.1 mM catechol. Results are representative of three independent trials.

FIG. 6. LcPxn1 protection of 䡠OH-induced degradation of deoxyribose. Ferrous ammonium sulfate (21 ␮M) was added to the reaction mixture containing deoxyribose (0.8 mM) and incubated at 37 °C for 15 min. The amount of deoxyribose degradation was measured by heating the mixture at 100 °C in the presence of thiobarbituric acid and trichloroacetic acid for 10 min. The amount of chromogen adduct formed under these conditions was determined by fluorescence (excitation ⫽ 532 nm; emission ⫽ 553 nm). Bar 1, 0.1 mM catechol; bar 2, 10 mM mannitol; bar 3, 0.125 mg/ml LcPxn1; bar 4, 0.125 mg/ml boiled LcPxn1; bar 5, 0.125 mg/ml NEM-treated LcPxn1; bar 6, 5 mM NEM alone; bar 7, 0.125 mg/ml GST; bar 8, 0.125 mg/ml BSA; bar 9, 0.14 units of catalase. Data shown are the mean ⫾ S.E. percentage protection of deoxyribose of at least three independent trials. Statistical analyses were done by t test. *, p ⬍ 0.01. DISCUSSION

Despite possessing the usual immuno-arsenal of T-cells, Bcells, natural killer cells, and antibodies, mice with a genetic deficiency in phagocyte oxidase and nitric-oxide synthase 2 are incapable of mounting a defense against commensal organisms (31). This finding strongly suggests that reactive oxygen products, such as those produced during the respiratory burst of phagocytes, are key mediators in the destruction of foreign pathogens. Intracellular pathogens such as L. chagasi that survive and replicate unhindered within these environments make them unequivocally one of the toughest cells known. Phagocytes themselves generally are not tough enough to withstand the reactive products they produce; however, they do possess several antioxidant mechanisms such as superoxide dismutase (32), catalase (33), and glutathione-dependent systems (34) that preserve their lives long enough to discharge a volley of oxidants in hopes that the foreign pathogen will eventually succumb to their toxicity. The molecular mechanism underlying L. chagasi survival within macrophages is poorly understood. We believe that L. chagasi possesses a strong antioxidant defense against the oxidants released by the macrophage, which preserves the lives of the parasites long enough to replicate and establish an infection. We have previously shown that L. chagasi possesses iron superoxide dismutases that act as a first line of defense against O2. (11). Recent findings suggest that a one-allele knockout of Fe-SODB in these parasites results in a 50% reduction in survival in macrophage cells,3 suggesting that an antioxidant defense is crucial for survival within macrophages. Recently, a peroxidoxin gene has been cloned and characterized from L. major, 3

S. D. Barr and L. Gedamu, unpublished data.

which is responsible for causing the self-healing, cutaneous form of leishmaniasis (19, 20). This L. major peroxidoxin has been shown to be constitutively expressed in these parasites, and no evidence of other peroxidoxins has been found (20). To determine whether peroxidoxins are present within L. chagasi and whether there are any differences in the armament of peroxidoxins between a nonlethal and lethal strain of the Leishmania family, we isolated and characterized three peroxidoxins from L. chagasi and present some unique and important features that deserve attention. It is important to note that the three peroxidoxins that we present here are completely different in their antioxidative function from the previously isolated antioxidants LcFe-SODA and LcFe-SODB1 from L. chagasi (11), therefore giving the parasites a very interesting defensive system. RNA transcripts of LcPxn1 increase significantly from the promastigote to the amastigote stage, whereas the transcripts of LcPxn2 and LcPxn3 decrease significantly over the same time period. This interesting expression pattern contrasts that of the peroxidoxin from L. major, which appears to be constitutively expressed in moderate abundance. This raises the question of why two L. chagasi peroxidoxin transcripts would decrease in abundance toward the amastigote stage while another peroxidoxin transcript increases, as opposed to maintaining a constant peroxidoxin transcript level like that apparently observed in L. major. The differential expression of the peroxidoxin genes in L. chagasi suggests that there may be a differential function for the different peroxidoxins. Leishmania parasites exist in very different environments throughout their life cycle. As the promastigotes divide in the gut of the sandfly, they may face nutrient limitations and overcrowding as they wait to be inoculated into their new host. Perhaps LcPxn2 and

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FIG. 7. Cellular reactions leading to the formation of various highly reactive intermediates. O2 undergoes a oneelectron reduction to produce O2. , which can interact with NO䡠 to produce ONOO⫺. O2. can also dismutate spontaneously or via superoxide dismutase into H2O2. H2O2 can in turn react with O2. or Fe2⫹ or ultraviolet light to produce 䡠OH, which can react with cellular lipids, proteins, and DNA with damaging results. Peroxidoxin has been shown to enzymatically remove H2O2 and ONOO⫺. We propose that 䡠OH can be added to the list of substrates that can be removed enzymatically by peroxidoxins.

LcPxn3 have evolved to protect the promastigotes from oxidative stress that may result as metabolic changes occur in the parasites in response to starvation and/or overcrowding. Induction of catalase activity in response to caloric restriction has been previously reported (35). As the parasites enter the macrophage, it is postulated that they are exposed to a substantially higher oxidative environment compared with the gut of the sandfly. The putative cytosolically localized LcPxn1 (data not shown) may have evolved to provide a more enhanced and general defense against endogenous and exogenous sources of prooxidants. The potentially different subcellular localization of each of the three peroxidoxins further supports differential functions for these proteins. It will be interesting to see if the possession of three differentially expressed peroxidoxins, particularly LcPxn1, is a factor contributing to the difference between the self-healing cutaneous form and the fatal visceral form of the disease. The pattern of differential expression of peroxidoxin genes observed in L. chagasi and L. major may provide some key insights into the mechanism of gene regulation in Trypanosomatids. Elements within the 3⬘-UTR (and sometimes the intergenic region and 5⬘-UTRs) of many Trypanosomatid genes have been shown to be involved in gene regulation (36 –39). Sequence comparisons of all three L. chagasi peroxidoxins and the one isolated from L. major reveal highly conserved 5⬘-UTRs and coding regions and very different 3⬘-UTRs. It is interesting to note that between the three peroxidoxin genes of L. chagasi and the L. major peroxidoxin, there are distinct patterns of gene expression. The LcPxn1 gene increases in expression toward the amastigote stage, LcPxn2 and LcPxn3 decrease (where LcPxn3 is much lower in abundance), and the L. major peroxidoxin appears to be constitutively expressed. It will be interesting to study the 3⬘-UTRs of all of these genes to see if there are unique sequence elements responsible for the different patterns of gene expression. H2O2 is an uncharged molecule that can diffuse through plasma membranes and oxidize lipids and proteins and inhibit membrane transport processes (40, 41). It has been reported that the H2O2 concentration within the phagosome can reach levels as high as 100 mM (42). H2O2 can also lead to the production of high levels of toxic 䡠OH molecules (Reactions 5 and 8). Therefore, it is evident that an intracellular pathogen

must possess a strong defense against H2O2 and 䡠OH if it is to survive. Such a strong defense in L. chagasi has not been discovered. All peroxidoxins isolated to date have been shown to detoxify H2O2, and we have demonstrated that recombinant LcPxn1 protein also detoxifies H2O2 in vitro. We found it very interesting that LcPxn1 RNA increases substantially toward the amastigote stage, a stage found exclusively within the macrophage in an environment where H2O2 predominates. Knowing that one of the functions of LcPxn1 is in H2O2 detoxification, an increase in the abundance of LcPxn1 transcripts suggests that the parasites may be stockpiling LcPxn1 protein for when they encounter the high concentration of H2O2 found within the phagolysosome. Examples of organisms preparing in advance for the possibility of environmental oxidative stress have been reported (43– 45). H2O2 can also impose toxicity indirectly because it is a precursor for the formation of 䡠OH (Reactions 5, 7, and 8). 䡠OH can initiate lipid peroxidation via hydrogen abstraction. Membrane integrity is essential for L. chagasi to survive; therefore, possessing an enzyme capable of repairing lipid peroxides would be a valuable asset. Glutathione peroxidases are the major enzymes responsible for detoxifying lipid peroxides in many other organisms, but no glutathione peroxidases have been discovered in Leishmania to date. Most isolated peroxidoxins have been shown to detoxify synthetic alkyl hydroperoxides such as t-butyl and cumene hydroperoxides (6), and we have demonstrated that LcPxn1 is no exception. We thus propose that LcPxn1 may be a key player in the removal of lipid peroxides in L. chagasi. Curiously, the peroxidoxin from L. major does not significantly detoxify t-butyl or cumene hydroperoxide (20). The coding region of LcPxn1 is 83% identical to the coding region of the L. major peroxidoxin with the major difference being in the carboxyl terminus. It is possible that the nineamino acid extension of the L. major peroxidoxin and LcPxn2 and LcPxn3 prevents access of the hydroperoxides to the active site. We have not performed a biochemical characterization of the LcPxn2 or LcPxn3 proteins, but it will be interesting to see if they too are unable to detoxify hydroperoxides. The reducing mechanism that maintains peroxidoxins in the active reduced form in vivo is unknown in Leishmania; however, it is speculated that a trypanothione-dependent system may be involved (20, 46). It has been reported that the rate of dithiothreitol-

Differentially Expressed Peroxidoxin Genes from L. chagasi dependent reduction of H2O2 by glutathione peroxidase is 10% that of the rate if glutathione was used as the reducing agent instead (1). Considering the similarity between the glutathionedependent and trypanothione-dependent reducing systems, it is possible that our rates of peroxide-detoxification are underestimated if in fact a trypanothione-dependent reducing system is involved. Because of its extremely high reduction potential, it has been widely believed that 䡠OH is much too reactive to be detoxified by any type of enzyme. We present convincing evidence that recombinant LcPxn1 can enzymatically detoxify 䡠OH. This finding is significant, because this enzymatic activity has never before been clearly demonstrated with any other proteins, and if such activity is conserved within the parasites under in vivo conditions, it may help to explain how L. chagasi can withstand the 䡠OH-enriched environment of the phagolysosome. We found it remarkable that even in an environment of excess H2O2, recombinant LcPxn1 was still able to detoxify 䡠OH and protect DNA from nicking. Proposed mechanisms of H2O2 detoxification in 2-Cys peroxidoxins state that upon reaction with H2O2 and the catalytic Cys52, a sulfenic acid (-SOH) intermediate forms before undergoing a nucleophilic attack with the Cys173 residue on the opposing subunit (30). It is possible that because of its high reduction potential, 䡠OH can outcompete H2O2 for binding to the Cys52 residue to form a sulfenic acid intermediate before being detoxified. However, one cannot rule out the possibility of a second unknown active site. We are currently dissecting the mechanism by which recombinant LcPxn1 detoxifies peroxides and 䡠OH by site-directed mutagenesis and molecular modeling, which could lead to dominant negative and drug design studies. The above findings represent the first clear report for a protein providing an enzymatic defense against hydroxyl radicals. Taken together with the recent finding that bacterial peroxidoxins can detoxify peroxynitrite (7), peroxidoxins appear to play an important role as the last line of defense against oxidative stress (Fig. 7). As such, peroxidoxins from pathogens such as Leishmania could be potential targets for drug and vaccine studies. This paper reports for the first time in Trypanosomatids the differential expression of multiple peroxidoxins. Such contrasting patterns of expression will be a very useful marker in mining the sequence of the 3⬘-UTRs of each of these genes for sequence elements that may be involved in gene regulation. The expression and activities of peroxidoxins described in this paper lead us to believe that by possessing such proteins, L. chagasi has evolved an enhanced defense strategy for survival within macrophages. We are further characterizing the organization of the peroxidoxin gene cluster in L. chagasi and L. donovani in order to identify the flanking sequences of the cluster, which will aid in the generation of knockouts. Our ultimate goal is to define the functional role that these peroxidoxins play in Leishmania survival and pathogenesis. Acknowledgment—We thank Dr. Vasanthakrishna Mundodi for guidance in screening the genomic and cDNA libraries.

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