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INTRODUCTION |
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
) 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.
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EXPERIMENTAL PROCEDURES |
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 DH5
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-
-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
(
= 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%
-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
-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.
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RESULTS |
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
(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.
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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
(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).
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 -tubulin antibody to
serve as a loading control (lower panel).
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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.
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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.
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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.
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DISCUSSION |
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.