Role of Peroxidoxins in Leishmania chagasi Survival

EVIDENCE OF AN ENZYMATIC DEFENSE AGAINST NITROSATIVE STRESS*

Stephen D. Barr and Lashitew GedamuDagger

From the Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada

Received for publication, December 19, 2002, and in revised form, January 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanisms by which Leishmania parasites survive exposure to highly reactive oxygen (ROS) and nitrogen (RNS) species within phagosomes of macrophages are not well known. Recently it has been shown that RNS alone is sufficient and necessary to control Leishmania donovani infection in mice (Murray, H. W., and Nathan, C. F. (1999) J. Exp. Med. 189, 741-746). No enzymatic defense against RNS has been discovered in Leishmania to date. We have previously isolated two peroxidoxins (LcPxn1 and LcPxn2) from Leishmania chagasi and showed that recombinant LcPxn1 protein was capable of detoxifying hydrogen peroxide, hydroperoxide, and hydroxyl radicals (Barr, S. D., and Gedamu, L. (2001) J. Biol. Chem. 276, 34279-34287). In further characterizing the physiological role of peroxidoxins in Leishmania survival, we show here that recombinant LcPxn1 protein can detoxify RNS in addition to ROS, whereas recombinant LcPxn2 protein can only detoxify hydrogen peroxide. LcPxn1 and LcPxn2 are localized to the cytoplasm, and overexpression of LcPxn1 in L. chagasi parasites enhanced survival when exposed to exogenous ROS and RNS and enhanced survival within U937 macrophage cells. Site-directed mutagenesis studies revealed that the conserved Cys-52 residue is essential for detoxifying hydrogen peroxide, t-butyl hydroperoxide, and hydroxyl radicals, whereas the conserved Cys-173 residue is essential for detoxifying t-butyl hydroperoxide and peroxynitrite. This is the first report of an enzymatic defense against RNS in Leishmania.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Leishmania is a protozoan parasite that affects over 12 million people worldwide with an estimated 2 million new cases each year. Depending on the species involved, symptoms range from the self-healing cutaneous form (e.g. Leishmania major) to the fatal visceral form (e.g. Leishmania chagasi). The parasites are transmitted as promastigotes from the gut of its sandfly vector to mammalian host macrophages wherein they transform into amastigotes and proliferate. As a macrophage defense mechanism, nitric oxide (·NO),1 peroxynitrite (ONOO-), hydroxyl radicals (·OH), hydrogen peroxide (H2O2), hydroperoxide (ROOH), and superoxide radicals (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>) are produced in an attempt to destroy the parasites. These reactive nitrogen species (RNS) and oxygen species (ROS) readily react with proteins, DNA, and lipids and have been implicated in a wide variety of cell functions such as signal transduction, redox homeostasis, apoptosis, aging, activation of T lymphocytes, control of blood pressure, tumor progression, protection of eye tissue, and pathogen infection/defense (1-4).

Numerous reports have shown that Leishmania parasites are susceptible to ROS-mediated killing (5-9) and RNS-mediated killing (10-17). It has been shown that RNS alone is both necessary and sufficient to control Leishmania donovani infection in mice (17) and more recently that both ROS and RNS produced by macrophages act together early to control infection by L. chagasi (18) and L. donovani (11, 17). These studies suggest that further characterization of antioxidant molecules within Leishmania and the role that they play in parasite survival in the promastigote and amastigote stages could lead to the development of novel strategies to compromise parasite survival.

Despite the ability of ROS and RNS to control Leishmania infection within macrophages, strains causing cutaneous and visceral leishmaniasis persist long enough within macrophages to produce skin lesions or death. The molecular mechanisms by which Leishmania circumvent the toxic effects of these reactive species is not fully understood. Some Leishmania molecules implicated in antioxidant defense against ROS include intracellular thiols (19), lipophosphoglycan (20, 21), iron superoxide dismutase (22), HSP70 (23), ovothiol A and trypanothione (24), and peroxidoxins (25-28). The mechanisms by which Leishmania withstand the toxic effects of RNS is much less well-defined. Glutathione has recently been implicated in protecting L. major from ·NO-induced cytotoxicity (29). To date, an enzymatic defense against RNS has not been identified in Leishmania.

Peroxidoxins (or peroxiredoxins) are highly conserved enzymes found in all kingdoms ranging from bacteria to humans. 2-Cys peroxidoxin proteins are characterized by two conserved cysteine residues corresponding to approximately positions 47 and 170 and exist in nature predominantly as head-to-tail dimers, although high molecular weight multimers have been reported (25, 30-32). Peroxidoxins were initially characterized as enzymes able to detoxify ROS, namely H2O2 and alkyl hydroperoxides (33), with ·OH recently being added to the substrate list (25). Peroxidoxins have also been implicated in detoxifying RNS in bacteria, yeast, and human cells (34-36).

We have previously isolated two peroxidoxin genes from L. chagasi that are differentially regulated, where LcPxn1 RNA transcripts are highly abundant in the amastigote stage and LcPxn2 transcripts are highly abundant in the promastigote stage (25). Recombinant LcPxn1 protein was shown to detoxify H2O2, ROOH, and ·OH, but the mechanism of its action and the role that L. chagasi peroxidoxins play in detoxifying RNS and in parasite survival has not been characterized. In this report, we demonstrate that recombinant LcPxn1 protein, but not LcPxn2, can detoxify RNS in addition to ROS and show that LcPxn1 protects L. chagasi parasites from ROS- and RNS-mediated toxicity in vitro and enhances survival within macrophages. Furthermore, we have identified the key catalytic residues of LcPxn1 involved in detoxifying both ROS and RNS, which differ from peroxidoxins isolated from other organisms.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Site-directed Mutagenesis-- LcPxn1 mutants C52A, C173A, and C52A/C173A were generated by site-directed PCR mutagenesis as previously described (37). All peroxidoxin constructs were amplified using PCR, cloned into the pGEX-2T vector (Amersham Biosciences), and confirmed by sequencing. Transformed Escherichia coli DH5alpha cells were grown shaking at 37 °C in Luria-Bertani broth containing 100 µg ml-1 ampicillin for 8 h, after which 0.2 mM isopropyl-1-thio-beta -D-galactoside was added to the culture and shaken overnight. GST fusion proteins were harvested by sonication and passed over a glutathione-agarose resin column as described by manufacturer (Amersham Biosciences). The fusion proteins were cleaved with thrombin overnight at 24 °C and further purified, and protein purity (>95%) was verified on a SDS-PAGE gel. Protein concentrations were determined using the BCA protein assay kit (Pierce Chemical, Rockford, IL).

Peroxide Assays-- Peroxide metabolism was measured as previously described (38). Briefly, the reaction mixture contained 50 mM Tris-HCl (pH 8.0), 0.2 mM dithioerythritol (DTE), 50 µM H2O2 or 50 µM t-butyl hydroperoxide, and 0.125 µg/ml protein (preincubated with 0.2 mM DTE for 30 min at 37 °C). The reaction was stopped with the addition of 1 ml of trichloroacetic acid (10% w/v). 0.2 ml of 10 mM ferrous ammonium sulfate and 0.1 ml of 2.5 M potassium thiocyanate were added, and the peroxide concentrations were determined spectrophotometrically at 480 nm using known amounts of peroxide (1-50 µM) as a standard. All solutions were made fresh immediately before use.

Deoxyribose Degradation Assay for ·OH Scavenging-- The production of ·OH and the ·OH-induced damage of 2-deoxy-D-ribose were measured as previously described (39). A 50-µl 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 DTE, and 0.125 µg/µl protein (proteins were preincubated in 0.2 mM DTE for 30 min at 37 °C). 21 µM ferrous ammonium sulfate was added, and the tubes were incubated at 37 °C for 15 min. 100 µl of thiobarbituric acid (1% w/v) and 100 µl of trichloroacetic acid (2.8% w/v) were then added to the mixture, and the mixture was boiled for 10 min. Fluorescence was measured in a 96-well plate using a SpectraMax Gemini plate reader (Molecular Devices) with six reads per well (excitation = 532 nm; emission = 553 nm). All solutions were made fresh immediately before use.

·OH-induced DNA Nicking Assay-- 3 µM FeCl3, 0.1 mM EDTA, and 10 mM DTE were allowed to react for 10 min at 37 °C to generate ·OH as previously described (40). 0.5 mg/ml protein (preincubated with 0.2 mM DTE for 30 min at 37 °C) was then added to the mixture and incubated at 37 °C for 30 min. 2 µg of pGEM-2 plasmid (Promega) was then added to each tube, and the mixture was incubated at 37 °C for 4 h. The DNA was separated on a 1% agarose gel containing 0.2 µg/ml ethidium bromide at a 100-V constant. All solutions were made fresh immediately before use.

Pyrogallol Red Bleaching Assay for ONOO- Scavenging-- Reagent peroxynitrite was generated from acidified hydrogen peroxide and nitrite using the quenched-flow method (41) and passaged over MnO2 column as previously described (42). The reaction assay was carried out as previously described (43). The reaction mixture contained 100 mM phosphate buffer (pH 7.0), 1 µM DTE, 50 µM Pyrogallol Red (epsilon  = 2.4 × 104 mol-1.liter.cm-1) and 20 µM protein at 25 °C. 20 µM of reagent peroxynitrite was added to the reaction for 5 min after which the absorbance at 542 nm was measured. All solutions were made fresh immediately before use.

ONOO--induced DNA Nicking Assay-- A reaction mixture containing 50 mM sodium phosphate (pH 7.0), 10 mM NaCl, 0.1 mM diethylenetriaminepentaacetic acid, 0.5 µg of intact pGEM-2 plasmid DNA, and 20 µM protein was prepared as previously described (44). 50 µM reagent ONOO- was added to the reaction mixture and incubated at room temperature for 5 min. The DNA was separated on a 1% agarose gel containing 0.2 µg/ml ethidium bromide at a 100-V constant. All solutions were made fresh immediately before use.

·NO Detoxification Assay-- ·NO levels were measured as previously described (45). 100 mM sodium nitroprusside and 0.125 µg/ml protein were incubated in phosphate-buffered saline (PBS), pH 7.4, for 5 min. 2,2'-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was then added to the reaction mixture (5 mM final concentration), and the ·NO-induced oxidation of ABTS to ABTS+ was measured by monitoring the change in absorbance at 420 nm at room temperature. All solutions were made fresh immediately before use.

Construction of Expression Vectors-- The Leishmania-specific expression vector pX63NEO (kindly provided by Dr. S. M. Beverley, Washington University in St. Louis, MO) was used to express LcPxn1, LcPxn1-C52A, LcPxn1-C173A, and LcPxn1-C52A/C173A in L. chagasi. The coding regions were PCR-amplified using a 5'-primer (5'-ACCAGGGATCCATGTCCTGCGGTGACGCC-3') and a 3'-primer (5'-ACATCGGATCCTTACTTATTGTGATCGACCTTCAGGCC-3') with incorporated BamHI sites (underlined). The pX63NEO vector and the PCR products were digested with BamHI, ligated, and sequenced for the correct orientation. To express LcPxn1 and LcPxn2 as GFP fusions, the coding region of GFP was PCR-amplified using a 5'-primer (5'-GTCGGATCCATGGTGAGCAAGGGCGAGG-3') and a 3'-primer (5'-CCGGAATTCGTACTTGTACAGCTCGTCC-3'). The stop codon of GFP was mutated from TAA to TAC (boldface). The PCR fragment was digested with BamHI and EcoRI and purified. The coding region of LcPxn1 was PCR-amplified using a 5'-primer (5'-GTCGAATTCATGTCCTGCGGTGACGCC-3') and a 3'-primer (5'-ACATCTCTAGATTACTTATTGTGATCGACCTTCAGGCC-3'). The coding region of LcPxn2 was PCR- amplified using a 5'-primer (5'-GTCGAATTCATGTCCTGCGGTGACGCC-3') and a 3'-primer (5'-GTCTCTAGATTACTGTTTGCTGAAGTACC-3'). The PCR products of LcPxn1 and LcPxn2 were each digested with EcoRI and XbaI and purified. PCR products of GFP and LcPxn1 or LcPxn2 were mixed and ligated and cloned into the BamHI and XbaI restriction sites of the pXNEO vector (provided by Dr. S. M. Beverley). All constructs were re-confirmed by sequencing at the University of Calgary DNA Sequencing Laboratory.

Western Blotting-- To detect GFP-LcPxn fusion proteins, 8 × 109 parasites were pelleted by centrifugation and washed once in ice-cold PBS. Parasites were resuspended in 2.5 ml of pre-chilled lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM CaCl2, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 0.5% Triton X-100) and placed on ice for 30 min. Lysate was centrifuged at 1,000 × g for 10 min and then 10,000 × g for a further 10 min at 4 °C. 40 µl of the supernatant was boiled in sample buffer (2% SDS, 60 mM Tris, pH 6.8, 2.5% beta -mercaptoethanol) for 5 min and resolved on a 10% SDS-PAGE gel and transferred to Hybond-P membrane (Amersham Biosciences). Western blotting was performed using anti-GFP (1:2500) antibodies and detected using an enhanced chemiluminescence method (Amersham Biosciences). To compare the overexpression of LcPxn1 and LcPxn1 mutants (Fig. 2), 5 × 106 parasites of each transfectant were washed in PBS, freeze-thawed, and boiled in sample buffer for 10 min before loading onto a 10% SDS-PAGE gel. Samples were subjected to Western blot as described above with anti-LcPxn1 antibodies. Monoclonal antibody (E7) to beta -tubulin was purchased from Developmental Studies Hybridoma Bank, University of Iowa.

[3H]Uracil Incorporation Assay-- The [3H]uracil incorporation assay was performed as previously described (5, 20, 46). Briefly, triplicate samples of 2 × 106 midlog phase promastigotes or stationary phase promastigotes were washed with and resuspended in 100 µl of Krebs-Ringer phosphate-glucose solution, pH 7.4 (0.154 M NaCl, 0.154 M KCl, 0.11 M CaCl2, 0.154 M MgSO4·7H2O, 0.1 M Na2HPO4·2H2O, pH 7.4, 10 mM glucose). Parasites were exposed for 2 h at 26 °C to either reagent 100 µM H2O2, 100 µM t-butyl hydroperoxide (tBOOH), 5 mM sodium nitroprusside, 1 mM ONOO- or the ·OH-generating system. ·OH was generated as previously described (20). The ·OH-generating system contained 1.0 mM xanthine, 2.4 × 10-2 units/ml xanthine oxidase, and 30 µM Fe2+ for 90 min at 26 °C. The parasites were then grown for another 2 h at 24 °C in the presence of 2 µCi of [3H]uracil (Amersham Biosciences) for measurement of RNA synthesis. The parasites were solubilized in 2 ml of solubilization solution (0.1% SDS, 0.1% diethyl pyrocarbonate, 25 mM HEPES, 10 mM EDTA, 100 µg/ml uracil) for 15 min on ice. After 15 min, 200 µl of trichloroacetic acid (100% w/v) was added to the tubes, and then they were placed on ice for 15 min. Samples were then applied to Whatman GF-C glass microfiber filters and washed with 10 ml of ice-cold trichloroacetic acid (10% w/v) and then with 10 ml of ice-cold ethanol (95%) using a vacuum apparatus. Filters were air-dried, placed in 2 ml of scintillation fluid, and monitored for incorporated [3H]uracil for 5 min.

Macrophage Infection Assay-- Infection of U937 cells from human origin (American Type Cell Collection, Rockville, MD) was carried out as previously described (47). U937 cells were seeded at a concentration of 2.5 × 105 cells/cm2 in eight-chamber slides and differentiated into adherent macrophages by treatment with 7.5 ng of phorbol myristate acetate (Sigma) per milliliter of RPMI 1640 with 10% fetal calf serum, 2 mM glutamine, and 50 µg/ml gentamicin (Invitrogen) (RPMI) under 5% CO2 at 37 °C for 72 h. Non-adherent cells were washed three to five times with warm RPMI media followed by incubation with L. chagasi parasites at a parasite to U937 cell ratio of 10:1 for 6 h. Non-engulfed parasites were washed away three to five times with warm RPMI and incubated in fresh RPMI media. The level of infection in 200 U937 cells was determined at 12, 24, and 48 h by optical microscopy following Diff Quick staining of cell preparations (48). Values are expressed as the average number of parasites per infected U937 cell.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombinant LcPxn1 Protein Can Detoxify RNS, Whereas LcPxn2 Cannot-- Assays containing reagent ONOO- or the ·NO donor sodium nitroprusside and target molecules susceptible to RNS-induced oxidation were used to test whether recombinant LcPxn1 protein can detoxify RNS. Pyrogallol Red (PR) has been previously shown to be bleached by ONOO- but not by decomposed ONOO-, nitrite, or nitrate (49). When added to the reaction mixture, recombinant LcPxn1 and the ONOO- scavenger Trolox significantly (p < 0.001) protected PR from bleaching compared with boiled LcPxn1, GST, and BSA controls (Fig. 1A). DNA has also been shown to be a target of ONOO-, which converts the supercoiled DNA into a slower migrating nicked DNA (50). Consistent with the results observed above, LcPxn1 protein and Trolox were able to protect supercoiled DNA from ONOO--induced nicking, whereas boiled LcPxn1, GST, and BSA were unable to provide protection (Fig. 1A, inset). Known scavengers of H2O2, ·OH, ·NO, and O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (catalase, mannitol, PTIO, and superoxide dismutase, respectively) did not significantly protect the PR from bleaching or the supercoiled DNA from nicking (data not shown). Recombinant LcPxn2 protein did not significantly protect PR (Fig. 1A) or the supercoiled DNA from nicking (Fig. 1A, inset) compared with boiled LcPxn2, GST, or BSA controls.


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Fig. 1.   Recombinant LcPxn1 protein assays. Recombinant LcPxn1 protein and the various LcPxn1 protein mutants were tested for their ability to detoxify various RNS and ROS. A, activity was assessed by the ability of the LcPxn1 proteins to protect 50 µM Pyrogallol Red from ONOO--induced bleaching and to protect supercoiled pGEM-2 plasmid (s) from ONOO- attack into the slower migrating nicked band (n) (A, inset); B, activity toward protecting 5 mM ABTS from ·NO-induced oxidation into the highly absorbing green ABTS+ complex. C, activity expressed as nanomoles/min/µg of recombinant protein toward 100 µM H2O2 and 100 µM t-butyl hydroperoxide (D); E, activity toward protecting 0.8 mM 2-deoxy-D-ribose from ·OH-induced damage and the ability to protect supercoiled (s) pGEM-2 DNA from ·OH-induced nicking (n) (E, inset). The average ± S.E. of at least four independent experiments are shown. Insets were representative of three independent experiments.

A colorimetric assay for measuring ·NO produced from sodium nitroprusside was used to test the ability of recombinant LcPxn1 protein to protect ABTS from ·NO-induced oxidation into the strongly absorbing green ABTS+ complex (45). When added to the reaction mixture, LcPxn1 protein and the ·NO scavenger PTIO significantly (p < 0.0001) protected ABTS from oxidation compared with boiled LcPxn1 and GST controls (Fig. 1B). There was no significant difference in protection between LcPxn2 protein and boiled LcPxn2 or GST controls. As further controls, scavengers of H2O2, ·OH, ONOO-, and O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (catalase, mannitol, Trolox, and superoxide dismutase, respectively) did not significantly protect ABTS from oxidation (data not shown). Taken together, the above data suggest that recombinant LcPxn1 protein is capable of detoxifying RNS whereas LcPxn2 does not appear to detoxify RNS.

Amino Acid Residues Involved in RNS- and ROS-detoxifying Activity-- The two cysteine residues corresponding to Cys-47 and Cys-170 in all 2-Cys peroxidoxins are highly conserved among all organisms. The amino terminus Cys-47 has been implicated as the catalytic residue in the detoxification of H2O2 and alkyl hydroperoxides (51, 52) and peroxynitrite (36). To identify the catalytic residues in Leishmania LcPxn1, we performed site-directed mutagenesis of the corresponding conserved Cys-52 and Cys-173 residues and constructed three recombinant LcPxn1 protein mutants containing Cys to Ala mutations (LcPxn1-C52A, LcPxn1-C173A, and LcPxn1- C52A/C173A).

In studying the amino acid residues involved in detoxifying ROS, we found that LcPxn1-C52A protein failed to detoxify H2O2, t-butyl hydroperoxide (tBOOH), and ·OH (p < 0.001 in each case) compared with wild-type LcPxn1 protein (Fig. 1, C, D, and E, respectively). In addition, LcPxn1-C52A protein failed to protect supercoiled DNA from ·OH-induced nicking (Fig. 1E, inset). Because there was no significant ROS-detoxifying activity by LcPxn1-C52A compared with boiled LcPxn1 or GST, Cys-52 appears to be essential for detoxifying ROS, which is consistent with previous findings with other peroxidoxins. There was no significant difference observed in the ability of LcPxn1-C173A to detoxify H2O2 (Fig. 1C) or ·OH (Fig. 1E) compared with wild-type LcPxn1 and LcPxn1-C173A protected supercoiled DNA from ·OH-induced nicking (Fig. 1E, inset). In contrast, LcPxn1-C173A did not demonstrate significant tBOOH-detoxifying activity compared with boiled LcPxn1 and GST (Fig. 1D). Taken together, these results suggest that the Cys-173 residue is not essential for detoxifying H2O2 or ·OH but is essential in detoxifying tBOOH. LcPxn1-C52A/C173A protein did not demonstrate any significant activity against H2O2, tBOOH, or ·OH compared with boiled LcPxn1 and GST and did not prevent supercoiled DNA from ·OH-induced nicking. LcPxn2 protein was found to detoxify only H2O2. It did not demonstrate significant activity toward tBOOH or ·OH (p > 0.05), nor did it protect DNA from ·OH-induced nicking compared with controls (Fig. 1, C-E).

In studying the amino acid residues involved in detoxifying RNS, we found that LcPxn1-C52A protein exhibited similar levels of activity in detoxifying ONOO- and protecting supercoiled DNA from ONOO--induced nicking compared with wild-type LcPxn1 protein (Fig. 1A), suggesting that Cys-52 is not essential for detoxifying ONOO-. LcPxn1-C173A protein did not demonstrate a significant difference in detoxifying ONOO- compared with boiled LcPxn1 or GST (p < 0.005) and could not protect DNA from nicking (Fig. 1A), suggesting that Cys-173 is the catalytic cysteine residue and is essential for detoxifying ONOO-. Both LcPxn1-C52A/C173A and LcPxn2 proteins did not detoxify ONOO- and could not prevent ONOO--induced nicking of DNA. Interestingly, wild-type LcPxn1, LcPxn1-C52A, LcPxn1-C173A, and LcPxn1-C52A/C173A proteins were all found to significantly (p < 0.002) detoxify ·NO compared with boiled LcPxn1, LcPxn2, and GST controls (Fig. 1B). Remarkably, LcPxn1-C52A/C173A protein provided significantly (p < 0.0001) more protection from ·NO-induced oxidation compared with wild-type LcPxn1 (Fig. 1B). Collectively, these results suggest that the Cys-52 residue is essential for detoxifying H2O2, tBOOH, and ·OH, Cys-173 is essential for detoxifying tBOOH and ONOO-, and neither Cys-52 nor Cys-173 are essential for detoxifying ·NO (summarized in Table I).


                              
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Table I
Summary of the antioxidant profiles of recombinant LcPxn1 and LcPxn1 mutants and LcPxn2 proteins

Overexpression of LcPxn1 Protein in L. chagasi Enhances Survival against ROS and RNS-- To test whether LcPxn1 protein can protect L. chagasi parasites from exposure to an environment enriched in ROS and RNS, we overexpressed LcPxn1 protein in the parasites to see if they exhibited enhanced survival. Parasites were transfected with the pX expression vector containing the various constructs of LcPxn1 and selected at 800 µg/ml G418. Southern blot verified the presence of the vectors (data not shown), and Western blot and densitometry analysis showed that each of the early log and stationary phase transfectants had more than a 1.8-fold increase in the level of LcPxn1 protein compared with the control transfectant which contained the pX vector alone (Fig. 2).


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Fig. 2.   Western blot analysis of L. chagasi overexpressers. Lysates from early log phase or stationary phase parasites transfected with pX, pX-LcPxn1, pX-LcPxn1-C52A, pX-LcPxn1-C173A, or pX-LcPxn1-C52A/C173A expression vectors were separated on a 10% SDS-PAGE gel and subjected to Western blotting using LcPxn1 antisera (upper panel). The same blots were stripped and incubated with monoclonal beta -tubulin antibody to serve as a loading control (lower panel).

Both early log phase and stationary phase parasites overexpressing LcPxn1 protein exhibited a significant (p < 0.01 in each case) enhanced survival upon exposure to H2O2, tBOOH, ·OH, ONOO-, and ·NO compared with control parasites containing the expression vector alone (Fig. 3, A-E). Consistent with previous findings (5), we found that the control (pX) stationary phase parasites were significantly (p < 0.01) more resistant to H2O2 toxicity than early log phase parasites (Fig. 3A). We also found that the control (pX) stationary phase parasites were significantly more resistant to tBOOH (p < 0.001) (Fig. 3B) and ONOO- (p < 0.03) (Fig. 3D).


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Fig. 3.   Parasite protection assays. Early log phase (black bars) or stationary phase (open bars) L. chagasi parasites overexpressing LcPxn1 or the various mutant LcPxn1 proteins were re-suspended in 100 µl of Krebs-Ringer phosphate-glucose solution, pH 7.4, and exposed to 100 µM H2O2 (A), 100 µM t-butyl hydroperoxide (B), ·OH-generating system (C), 1 mM ONOO- (D), or 5 mM nitroprusside (E) for 2 h as described under "Experimental Procedures." Parasite viability was assessed by determining the percentage of [3H]uracil incorporation into parasite RNA. The average ± S.E. values of at least three independent experiments are shown.

In support of our findings that Cys-52 is essential in detoxifying H2O2, tBOOH, and ·OH and that Cys-173 is essential in detoxifying tBOOH and ONOO-, parasites overexpressing LcPxn1-C52A did not exhibit an enhanced survival upon exposure to H2O2, tBOOH, or ·OH but did exhibit enhanced survival upon exposure to ONOO- compared with pX control parasites (Fig. 3, A-D). LcPxn1-C173A exhibited an enhanced survival upon exposure to H2O2 and ·OH but not upon exposure to tBOOH or ONOO- (Fig. 3, A-D), which is consistent with our observations with recombinant LcPxn1-C173A protein (Fig. 1). Contrary to our findings with recombinant LcPxn1 proteins, parasites overexpressing LcPxn1-C52A and LcPxn1-C173A did not exhibit enhanced survival upon exposure to ·NO (Fig. 3E). Parasites overexpressing LcPxn1-C52A/C173A did not exhibit enhanced survival upon exposure to any of the ROS or RNS.

Cellular Localization of LcPxn1 and LcPxn2 Proteins-- To further define the functions of LcPxn1 and LcPxn2 in parasite survival, we studied the cellular localization of these proteins within L. chagasi. The amino acid sequence of LcPxn1 does not appear to contain a typical organellar-targeting signal sequence, which suggests that it may be localized to the cytoplasm. The last three amino acids at the carboxyl terminus of LcPxn2 are SKQ and conspicuously resembles the glycosomal targeting signal sequence SKL. Although mutational analysis of the SKL glycosomal targeting signal in Trypanosoma brucei showed that this signal is highly degenerate, mutation of the signal to SKQ was not sufficient to target proteins to the glycosome but rather remained cytosolic (53).

We created GFP-LcPxn1 and GFP-LcPxn2 fusion protein gene constructs and overexpressed them in L. chagasi parasites. Parasites were selected at 50 µg/ml G418, and Western blot analysis of each parasite extract with anti-GFP revealed the presence of a fusion protein of ~48 kDa suggesting that both the GFP-LcPxn1 and GFP-LcPxn2 fusion proteins were intact (Fig. 4A). No bands corresponding to the GFP protein alone (27 kDa) were observed in either of the extracts isolated from parasites expressing the fusion proteins. Fluorescence microscopy showed that both the GFP-LcPxn1 and GFP-LcPxn2 fusion proteins are localized throughout the parasite, including the flagella, similar to the fluorescence pattern observed with the control parasites expressing the GFP protein alone (Fig. 4, B-D). The fluorescence patterns observed for both LcPxn1 and LcPxn2 are distinct from the pattern of glycosomal localization (data not shown). These results suggest that both LcPxn1 and LcPxn2 are localized to the cytoplasm.


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Fig. 4.   LcPxn1 and LcPxn2 cellular localization. A, lysates from wild-type L. chagasi cells transfected with pX-GFP, pX-GFP-LcPxn1, and pX-GFP-LcPxn2 were separated on a 10% SDS-PAGE gel and subjected to Western blot analysis using anti-GFP antibodies. A band of ~48 kDa was observed in lanes 2 and 3, which corresponds to the sizes of LcPxn1 (21 kDa) and LcPxn2 (22 kDa) fusion proteins with GFP (27 kDa). GFP fluorescence patterns of L. chagasi parasites expressing GFP alone (B), LcPxn1-GFP (C), or LcPxn2-GFP (D) are shown.

Overexpression of LcPxn1 Protein in L. chagasi Enhances Intracellular Survival within Macrophages-- During the initial stages of infection with a foreign pathogen, an oxidative burst occurs in human macrophages (9, 18, 54, 55), including U937 cells (56-58) in which ROS is produced in response to phagocytosis. Human macrophages, including U937 cells, have also been shown to produce RNS once infection is established (18, 59, 60).

We have previously shown that the level of LcPxn1 mRNA expression increases significantly toward the amastigote phase compared with early log phase parasites (25). To gain insight into the role that LcPxn1 plays in intracellular survival within macrophages, we tested the ability of L. chagasi parasites overexpressing LcPxn1 to survive within the human macrophage cell line U937. The average number of amastigotes overexpressing wild-type LcPxn1 per infected macrophage at 12, 24, and 48 h post infection was 8.3 (±0.44), 12.1 (±0.35), and 14.2 (±0.65), respectively (Fig. 5). These numbers are significantly (p < 0.05) greater than pX control parasites at the corresponding time intervals with 5.9 (±0.1), 8.5 (±0.76), and 9.4 (±0.4), respectively (Fig. 5). At 12 h post infection, parasites overexpressing LcPxn1-C173A had a significantly (p < 0.05) higher average number of amastigotes per infected macrophage of 7.6 (±0.31) compared with 5.9 (±0.10) for the pX control parasites. At 24 and 48 h post infection, there was no significant difference in the average numbers between parasites overexpressing LcPxn1-C173A and the pX control parasites. Parasites overexpressing LcPxn1-C52A or LcPxn1-C52A/C173A did not exhibit a significant increase in parasite load compared with the pX control parasites at any time interval (Fig. 5). No significant difference in the percentage of infected U937 cells was observed at any time interval (data not shown).


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Fig. 5.   Survival of L. chagasi parasites within U937 cells. U937 cells were incubated with stationary phase parasites transfected with pX (meshed bars), pX-LcPxn1 (black bars), pX-LcPxn1-C52A (slashed bars), pX-LcPxn1-C173A (open bars), or pX-LcPxn1-C52A/C173A (checkered bars) for 6 h as described under "Experimental Procedures." Non-engulfed parasites were washed away, and the infected U937 cells were incubated for 12, 24, and 48 h after which the infected macrophages were stained with Diff Quick and examined using optical microscopy to determine the level of infection which was expressed as the average number of amastigotes per infected macrophage. The average ± S.E. of four independent experiments are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous reports have shown that both ROS and RNS contribute to the early control of Leishmania infection and that RNS alone is necessary and sufficient to control Leishmania infection (11, 17, 18). Clearly, the possession of an antioxidant defense system against ROS and more importantly RNS would provide intracellular pathogens like Leishmania a selective advantage for survival. In further characterizing the role of peroxidoxins in L. chagasi survival, we have shown that recombinant LcPxn1 protein can detoxify RNS in addition to ROS, whereas recombinant LcPxn2 protein appears to play a more limited role by only being able to detoxify H2O2. Overexpressing LcPxn1 within L. chagasi parasites significantly enhanced parasite survival within macrophage cells and upon exposure to exogenously added ROS and RNS. In addition, we have implicated the Cys-52 residue as being essential in detoxifying H2O2, tBOOH, and ·OH and Cys-173 as being essential for detoxifying tBOOH and ONOO-.

Consistent with previous studies on peroxidoxins from other organisms, the Cys-52 residue of LcPxn1 is essential for detoxifying peroxides and is consistent with the proposed mechanism of action for 2-Cys peroxidoxins, which involves the attack of the amino terminus cysteine residue by peroxide to form a sulfenic acid residue intermediate. This intermediate can then react with the adjacent Cys-173 residue and/or a diffusible thiol such as dithioerythritol in vitro or trypanothione in vivo in Leishmania to form a disulphide bond (52, 61). The mechanism of action for the detoxification of ·OH has not been previously described. Similar to the mechanism of action for detoxifying H2O2, our studies revealed that the Cys-52 residue is also essential for detoxifying ·OH, which fits with a mechanism of action where a ·OH can abstract a hydrogen from the Cys-52 R-SH group to form a thiyl radical (R-S·). This radical can subsequently be attacked by another ·OH to form a sulfenic acid residue intermediate and react with the adjacent Cys-173 residue and/or a diffusible thiol. It is debatable whether ·OH can be scavenged in vivo, because ·OH reacts very quickly with almost every type of molecule in living cells, and as such a high concentration of peroxidoxins would have to be present to compete with biological targets. However, peroxidoxins have been shown to be highly abundant in cells such as yeast (0.7% of the total soluble protein), and peroxidoxins are the most abundant protein in erythrocytes after hemoglobin (62, 63). Peroxidoxins have also been shown to be able to protect biological targets such as DNA from attack by ·OH (25, 40). Furthermore, we have shown here that LcPxn1 is cytoplasmic, which significantly increases its chance of coming into contact with ·OH (Fig. 4), and that its overexpression can protect parasites from an exogenous source of ·OH (Fig. 3C).

Numerous studies have identified alkyl hydroperoxides as substrates for peroxidoxins, implicating peroxidoxins as very important enzymes in reducing phospholipid hydroperoxides that can arise from oxidation and thereby protect cells from plasma membrane damage. Leishmania parasites lack typical glutathione peroxidases, which are well-known protectors of lipid peroxides in eukaryotes. We previously demonstrated that recombinant LcPxn1 protein can detoxify alkyl hydroperoxides and have extended these studies to show that LcPxn1 can also protect L. chagasi from tBOOH (Figs. 1D and 3B). Interestingly, we found that both the conserved Cys-52 and Cys-173 residues are essential for detoxifying tBOOH, which suggests an alternative mechanism for the detoxification of ROOH compared with the detoxification of H2O2 and ·OH. Recent studies have emphasized the importance of the microenvironment surrounding the active site residues of peroxidoxins (52, 64). It is therefore possible that the Cys-173 residue of LcPxn1 may be essential for coordinating the active site into a more favorable environment for donating a proton to the poor and much more bulky RO- leaving group. The lack of an available proton donor could cause the sulfenic acid intermediate (R-SOH) that forms on the catalytic cysteine to be further oxidized into R-SOOH, which has been found to lead to reduced activity (65). Remarkably, we could not detect alkyl hydroperoxidase activity with recombinant LcPxn2 protein, which is 89% identical to LcPxn1 (Fig. 1D). There has been a report (26) of a peroxidoxin from L. major (Lmf30 TryP) that is highly homologous to LcPxn2 and is also incapable of significantly detoxifying alkyl hydroperoxides. Crystallographic studies with the peroxidoxin AhpC from Salmonella typhimurium has revealed that structural conformations and the mobility of key residues present in loop structures encompassing the active site cysteines is very important for activity of the protein (64). The main difference between LcPxn1 and LcPxn2 is the presence of a 9-amino acid extension at the carboxyl terminus of LcPxn2. It is possible that this extension alters the microenvironment surrounding the active site into one that is not favorable for activity or is more prone to inactivation as described above. Characterization of the crystal structures of LcPxn1 and LcPxn2 will provide more insight into this mechanism.

Bacterial and yeast peroxidoxins have been previously shown to protect cells from ·NO- and ONOO--mediated toxicity (34-36). Our findings also show that LcPxn1 can detoxify ·NO and ONOO- and protect Leishmania from RNS- mediated toxicity, however, the mechanism by which this occurs in Leishmania differs from the proposed mechanism by which bacterial peroxidoxins detoxify ONOO-. The conserved amino terminus cysteine (Cys-46) residue of the AhpC peroxidoxin from S. typhimurium was found to be essential for activity (36), whereas we demonstrate that the carboxyl terminus cysteine (Cys-173) residue of LcPxn1 is essential for activity (Fig. 1A and 3D). This finding will be important for future drug design studies with Leishmania peroxidoxins that target the ability of peroxidoxins to detoxify RNS. A possible mechanism of action is one where the Cys-173 residue can attack the O-O bond in ONOO- resulting in the transfer of one oxygen atom to the Cys-173 and the release of nitrite; alternatively, ONOO- can oxidize the R-SH group of Cys-173 converting it into R-S· and releasing ·NO2 and -OH. Although peroxidoxins have been shown to protect bacteria, yeast, and human cells from the toxic effects mediated by ONOO- and ·NO (34, 35), no evidence has been presented of a recombinant peroxidoxin protein capable of detoxifying ·NO directly. We found that overexpression of LcPxn1 protein in parasites also protected them from both ONOO-- and ·NO-mediated toxicity, but we also found evidence that recombinant LcPxn1 protein can detoxify ·NO (Fig. 1B). Interestingly, site-directed mutagenesis of LcPxn1 protein revealed that neither of the conserved cysteine residues (Cys-52 or Cys-173) were essential for detoxifying ·NO and the mutation of both cysteines led to increased activity. This result suggests that LcPxn1 possesses a different mechanism for detoxifying ·NO, which we are currently investigating. Oddly, the overexpression of wild-type LcPxn1 but not the mutant proteins within the parasites provided significant protection to the parasites when exposed to ·NO (Fig. 3E). It is possible that formation of heterogeneous multimers between mutant and wild-type LcPxn1 monomers within the parasites led to an inhibitory effect.

Of significant interest is our finding that overexpression of LcPxn1 in Leishmania parasites enhanced survival within macrophages (Fig. 5). Despite the findings that ROS and RNS act together early to control Leishmania infection and that RNS alone is sufficient to control infection, L. chagasi persists long enough to establish a potentially fatal infection. Our data show that L. chagasi parasites possess a protective enzymatic defense against ROS and most notably against RNS. Interestingly, overexpression of the Cys-173 mutant in the parasites, which we found to be active only toward H2O2 and ·OH but not RNS, enhanced survival early in infection (12 h post infection) when ROS are produced in abundance during phagocytosis. Later when infection is established (24-48 h post infection) and when inducible nitric-oxide synthase mRNA and protein are expressed, the same parasites overexpressing Cys-173 failed to exhibit enhanced survival. The Cys-52 mutant, which is active only toward RNS, did not provide enhanced survival early in infection and failed to enhance survival at later stages of infection. These findings support previous findings that ROS are important early in controlling Leishmania infection and that RNS alone appear to be sufficient to control infection. Taken together, our results strongly suggest that LcPxn1 enhances parasite survival in culture and within macrophages by providing a last line of defense against the most biologically important ROS and RNS. Because LcPxn1 is predominantly expressed in the amastigote stage, it could provide much needed protection from the ROS and RNS that are produced within the macrophages that are free to diffuse across lipid membranes into the parasites. LcPxn2 is predominantly expressed in the early log phase of the parasites, and our finding that LcPxn2 appears to be able to only detoxify H2O2 suggests that LcPxn2 plays a more limited function in parasite survival in the promastigote stage in the gut of the sandfly vector, which may not be as enriched in ROS and RNS as within the phagosome of the macrophages of the host.

The discovery of an enzymatic defense against RNS in addition to ROS in L. chagasi is very exciting, because it identifies a factor present within the parasites that can detoxify molecules produced by the host that have been previously shown to control Leishmania infection. It will be interesting to see if a homolog to LcPxn1 is present in Leishmania strains that cause the self-healing cutaneous form of leishmaniasis. So far, peroxidoxins isolated from L. major (26) and Leishmania infantum (27, 66) have not been shown to possess the ability to detoxify RNS. It is possible that LcPxn2 is an evolutionary precursor to LcPxn1 that through evolution underwent a deletion in the carboxyl terminus. This deletion could have altered the microenvironment of the active site surrounding the conserved cysteine residues resulting in the acquisition of higher functions such as being able to detoxify phospholipid hydroperoxides, ·OH, ONOO-, and ·NO, which ultimately led to a selective advantage for L. chagasi survival. It will be interesting to see whether the acquisition of the higher functions of LcPxn1 contribute to the pathogenicity of L. chagasi. Our findings provide a better understanding of the mechanisms that Leishmania utilize for intracellular survival and the role that peroxidoxins play in Leishmania survival. We are currently using homologous recombination and antisense technology and mouse infection models to gain a better understanding of the role that peroxidoxins play in the pathogenesis of leishmaniasis.

    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 403-220-5556; Fax: 403-289-9311; E-mail: lgedamu@ucalgary.ca.

Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212990200

    ABBREVIATIONS

The abbreviations used are: ·NO, nitric oxide; ONOO-, peroxynitrite; ·OH, hydroxyl radicals; ROOH, hydroperoxide; tBOOH, tert-butyl hydroperoxide; RNS, reactive nitrogen species; ROS, reactive oxygen species; GST, glutathione S-transferase; GFP, green fluorescent protein; DTE, dithioerythritol; PR, Pyrogallol Red; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ABTS, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide.

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ABSTRACT
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RESULTS
DISCUSSION
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