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Arch Microbiol (2008) 189:375–384
DOI 10.1007/s00203-007-0328-4
ORIGINAL PAPER
Cloning and expression of trypanothione reductase 
from a New World Leishmania species
Denise Barçante Castro-Pinto · Marcelo Genestra · Gustavo B. Menezes · 
Mariana Waghabi · Antonio Gonçalves · Catarina De Nigris Del Cistia · 
Carlos Mauricio R. Sant’Anna · Leonor L. Leon · Leila Mendonça-Lima 
Received: 13 June 2007 / Revised: 27 September 2007 / Accepted: 9 November 2007 / Published online: 5 December 2007
© Springer-Verlag 2007
Abstract Trypanothione disulWde (T[S]2), an unusual
form of glutathione found in parasitic protozoa, plays a cru-
cial role in the regulation of the intracellular thiol redox
balance and in the defense against oxidative stress. Trypa-
nothione reductase (TR) is central to the thiol metabolism
in all trypanosomatids, including the human pathogens Try-
panosoma cruzi, Trypanosoma brucei and Leishmania.
Here we report the cloning, sequencing and expression of
the TR encoding gene from L. (L.) amazonensis. Multiple
protein sequence alignment of all known trypanosomatid
TRs highlights the high degree of conservation and illus-
trates the phylogenetic relationships. A 3D homology
model for L. amazonensis TR was constructed based on the
previously reported Crithidia fasciculata structure. The
puriWed recombinant TR shows enzyme activity and in vivo
expression of the native enzyme could be detected in infec-
tive promastigotes, both by Western blotting and by immu-
noXuorescence.
Keywords Trypanothione reductase (EC 1.8.1.12) · 
Leishmania (L.) amazonensis · Thiol metabolism · 
Gene cloning · Leishmaniasis
Introduction
A promising route towards development of improved thera-
peutic agents for diseases caused by human pathogens, such
as parasitic protozoa, is the identiWcation of key diVerences
between the metabolism of the host and the parasite, and
the development of inhibitors of parasite-speciWc enzymes
(Amssoms et al. 2002). Trypanothione, an unusual form of
glutathione found in parasitic protozoa of the Kinetoplasti-
dae family, plays a crucial role in regulating the intracellu-
lar thiol redox balance and in the defence against chemical
and oxidative stress (reviewed in Müller et al. 2003). It
contains two molecules of glutathione joined by a poly-
amine linker. Trypanothione reductase (TR; EC 1.8.1.12) is
a NADPH-dependent Xavoprotein oxidoreductase essential
in maintaining the intracellular concentration of reduced
trypanothione (T[SH]2), and plays a central role in the thiol
metabolism of all trypanosomatids (Cunningham and Fair-
lamb 1995; Fairlamb et al. 1985; Fairlamb and Cerami
1985, 1992; Henderson et al. 1987, 1988; Jockers-Sherubi
et al. 1989; Shames et al. 1986, 1988; Taylor et al. 1994). It
is also an essential enzyme in the detoxiWcation process. TR
catalyzes the transfer of electrons from NADPH to its
speciWc substrate via a FAD prosthetic group and a redox
Communicated by Ercko Stackebrandt.
Nucleotide sequence data reported in this paper is available in the 
GenBankTM database under accession number DQ530259.
D. B. Castro-Pinto · M. Genestra · L. L. Leon
Laboratory of Trypanosomatid Biochemistry, 
Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil
G. B. Menezes · M. Waghabi · L. Mendonça-Lima (&)
Laboratory for Functional Genomics and Bioinformatics,
Fiocruz. Av. Brasil 4365 Manguinhos, Rio de Janeiro, 
RJ 21040-900, Brazil
e-mail: lmlima@Wocruz.br
A. Gonçalves
Laboratory for Immunopathology, Oswaldo Cruz Institute, 
FIOCRUZ, Rio de Janeiro, RJ, Brazil
C. De Nigris Del Cistia · C. M. R. Sant’Anna
Department of Chemistry, ICE, UFRRJ, Seropedica, 
Rio de Janeiro, RJ, Brazil
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376 Arch Microbiol (2008) 189:375–384
active cysteine disulWde. The enzyme is a homodimer and
each subunit comprises a NADPH-binding domain, a
FAD-binding domain, and an interface domain; residues
from the FAD-binding domain and the interface domain
form the trypanothione disulWde-binding site (Bond et al.
1999).
Parasites of the genus Leishmania can infect reptiles and
several orders of mammals, showing a worldwide distribu-
tion in tropical and subtropical regions (Cupolillo et al.
2000). More than 30 species of Leishmania that infect
mammals have been described, and these can be divided
into two sub-genera: L. (Leishmania) and L. (Viannia)
(Lainson and Shaw 1987). Leishmanias can produce a wide
spectrum of diseases, depending both on the infecting spe-
cies and on the immunological status of the host, ranging
from simple cutaneous and mucocutaneous to diVuse cuta-
neous and visceral manifestations (reviewed by McMahon-
Pratt and Alexander 2004). L. (L.) amazonensis, a New
World Leishmania species, is the only member of the L.
Leishmania sub-genus causing cutaneous leishmaniasis in
the Americas, having also been reported as causing visceral
leishmaniasis in some regions of Brazil (Barral et al. 1991;
Leon et al. 1992).
Genes encoding TR have been described from several
trypanosomatid species, such as the human pathogens
Trypanosoma cruzi (Sullivan and Walsh 1991) and
T. brucei (Aboagye-Kwarteng et al. 1992), the cattle path-
ogen T. congolense (Shames et al. 1988), the insect patho-
gen Crithidia fasciculata (Aboagye-Kwarteng et al.
1992), and the Old World Leishmania species L. (L.)
donovani (two diVerent strains, one from Ethiopia and one
from India—Taylor et al. 1994; Mittal et al. 2005). TR
sequences can also be derived from the L. major, L. bra-
ziliensis and L. infantum genome projects (http://
www.sanger.ac.uk). Here we report the cloning, sequenc-
ing and heterologous expression of the TR gene of Leish-
mania (L.) amazonensis. A 3D homology model for
L. amazonensis TR was constructed based on the previ-
ously reported C. fasciculata TR structure (Kuriyan et al.
1991), and compared to the structure of the T. cruzi
enzyme (Zhang et al. 1996), showing a high conservation
of the disulWde substrate binding site. This structural con-
servation suggests that inhibitors developed for L. ama-
zonensis TR should also eVectively inhibit the T. cruzi
enzyme. PuriWed recombinant L. amazonensis TR shows
enzyme activity equivalent to the activity of the native
enzyme, previously reported by us (Castro-Pinto et al.
2004) and a mouse antiserum developed against rTR
recognizes a 53 kDa protein in the soluble lysate of L.
amazonensis infective promastigotes. This antibody also
shows strong reactivity by immunoXuorescence,
localizing with the Xagellar pocket region and other intra-
cellular structures in L. amazonensis promastigotes.
Materials and methods
Parasites
Leishmania (L.) amazonensis (MHOM/BR/77/LTB0016
strain) infective promastigotes were obtained from
amastigotes isolated from Balb/c mice lesions, as previ-
ously described (Cysne-Finkelstein et al. 1998). Parasites
were inoculated in biphasic medium consisting of NNN
(Novy, McNeal and Nicole), with 15% agar and deWbrin-
ated rabbit blood as the solid phase and Schneider’s
medium, pH 7.2 as liquid phase (Schneider’s Insect
Medium, Sigma Cell Culture, USA), supplemented with
inactivated fetal calf serum (FCS; 10%), 1 mM L-gluta-
mine, 100 U penicillin G and 100 �g/ml streptomycin
(Sigma) and incubated at 26°C.
Cloning and sequencing of the Leishmania amazonensis 
TR gene
Genomic DNA was isolated from promastigote forms,
essentially as described (Ozaki and Traub-Cseko 1984).
The TR coding sequence was obtained by PCR ampliWca-
tion of genomic DNA using primers TR1 (5�-CGGGATCC
ATGGCCCGCGCGTACGACCTCGTGGTGC-3�) and
TR2 (5�-CCCAAGCTTGCTGCTGAGCTTTTCGACGC
C-3�), designed based on the reported TR gene sequence of
L. donovani (Accession number: Z23135). The “PCR
supermix high Wdelity” kit (Invitrogen) was used in all
DNA ampliWcations. PCR conditions were as follows: ini-
tial denaturation at 94°C (4 min.) followed by 5 cycles of
denaturation (94°C, 30 s),annealing (55°C, 30 s) and
extension (72°C, 2 min) and a further 30 cycles with an
annealing temperature of 65°C. The 1,500 bp DNA
fragment ampliWed from L. amazonensis DNA was puriWed
with QIAquick (Qiagen) and cloned in the pCR-Blunt
II-TOPO (Invitrogen) vector.
DNA sequences were obtained from puriWed plasmid
DNA using primers TR1, TR2, and the internal primers
TR5 (5�-CGCACACGGTGGTGGTGCGC-3�) and TR6
(5�-TTCGTGTGGTCGGTGCCGCTGGGC-3�). Sequenc-
ing reactions were performed with the BigDye 3.11 kit
(Applied Biosystems) according to manufacturer’s instruc-
tions, and run on an ABI3730 DNA analyzer. DNA
sequences were assembled locally with the Wisconsin
Package (Genetics Computer Group—GCG, Madison,
Wisconsin).
Alignment of trypanosomatid TR protein sequences
Alignment of known trypanosomatid TR protein sequences
was done with the T-CoVee program (at http://www.ch.
embnet.org/software/TCoVee.html; Notredame et al. 2000).
123
Arch Microbiol (2008) 189:375–384 377
A dendrogram showing the phylogenetic relationship
between the various TRs was generated with Mega3.
3D homology modeling of L. amazonensis TR 
and molecular modeling study
The template sequence for the L. amazonensis TR model-
ing, chain A of Crithidia fasciculata TR (PDB code:
1FEC), was found with the Swiss-Model BLAST tool
(Altschul et al. 1990), so as to select the most appropriate
modeling templates for Swiss-Model. The template shares a
high degree of sequence identity (79%) with the target and
its 3D structure has the best resolution (1.7 Å) among the
template sequences with the higher BLAST scores. The 3D
model was constructed with the First Approach Mode of
the Swiss-Model server (Peitsch 1995; Guex and Peitsch
1997; Schwede et al. 2003), which includes model genera-
tion with ProModII and energy minimization with GRO-
MOS96 (Gunsteren et al. 1996). The integrated sequence
alignment tools and structural superposition algorithms of
the Deep View (Swiss-PDB Viewer) 3.7 program (Guex
et al. 1999; Guex and Peitsch 1997) were used to compare
the modeled structure with known experimental TR
structures.
As the homology model was constructed without the
ligands, a subsequent molecular modeling study was per-
formed in order to evaluate the trypanothione binding in
the L. amazonensis TR. For construction of the substrate
binding site model, all amino acid residues and water
molecules with at least one atom located 4.0 Å from the
trypanothione molecule were selected from the T. cruzi
TR crystal structure in complex with trypanothione (PDB
code 1BZL) (Bond et al. 1999). This selected binding
site was composed by 7 water molecules and 23 amino
acid residues from A and B chains of T. cruzi TR
homodimer, which were replaced by the corresponding
amino acid residues from the L. amazonensis TR model
with the Deep View 3.7 program. Hydrogen atoms were
included to both structures with the PC Spartan Pro pro-
gram (Wavefunction, Inc.) and the coordinates of the
substrate molecule and of all hydrogen atoms were pre-
liminary energy minimized with the molecular mechan-
ics approach (MMFF force Weld). Subsequently, these
models were reoptimized with the PM3 semi empirical
approach as implemented in Mopac2002 program
(Fujitsu Ltd). Only the coordinates of the peptide atoms
were held Wxed during energy minimization at the semi
empirical level, which was accomplished with the default
options of the Mopac2002 program. All calculations
were carried out with the PM3 method (Stewart 1989a, b)
using the linear scaling approach, which enables fast
quantum calculations on systems composed of many
hundreds of atoms.
Expression and puriWcation of rTR
For expression of recombinant L. amazonensis TR, the
DNA fragment containing the TR coding sequence was
ampliWed from the pCR-Blunt II-TOPO construct using
primers TR3 (5�-ATGGCCCGCGCGTACGACCTCG-3�)
and TR4 (5�-AAGCTTGCTGCTGAGCTTTTCGAC-3�).
The puriWed DNA fragment was subcloned into the pBAD-
Thio/Topo (Invitrogen) expression vector, the mixture
transformed into E. coli TOP10, following the manufac-
turer’s protocol, and transformants selected on Luria Ber-
tani (LB; Invitrogen) agar (1.5%; Difco) plates containing
100 �g/ml ampicillin (Sigma). Plasmid DNA was puriWed
from cultures grown from a single colony using the Wizard
plasmid miniprep kit (Promega). Recombinant TR was
puriWed from 1 L of induced culture (in LB medium con-
taining 100 �g/ml ampicillin and 0.02% L-arabinose, for
induction of the pBAD promoter). BrieXy, E. coli Top10
cells harboring the expression construct were grown at
37°C with agitation to late log phase (OD600 nm = 0,8), arab-
inose was added to a Wnal concentration of 0.02% and the
culture incubated for a further 2 h. Cells were harvested by
centrifugation at 3,000g for 10 min at 4°C. The wet cell
pellet was ressuspended in 200 ml of cold lysis buVer
(50 mM Tris–Cl pH 7.5/100 mM KCl/5 mM EDTA) and
cells mechanically lysed in a Bead Beater apparatus (Glen
Mills). The lysate was centrifuged at 4,000g for 15 min and
the recombinant protein found in the insoluble, inclusion
body fraction. Inclusion bodies were washed several times
in buVer containing 50 mM Tris–Cl pH 8.5/0.5% Triton X-
100/5 mM EDTA/150 mM NaCl, before solubilization in
8 M urea/50 mM Tris–Cl pH 8. After centrifugation, the
supernatant fraction was adjusted to a Wnal concentration of
6 M urea/50 mM Tris–Cl pH 8.5, 5 mM imidazole/0.3 M
NaCl before chromatography on a Ni2+-charged Chelating
Sepharose Fast Flow (GE Healthcare) column. rTR con-
taining fractions were eluted in 0.2 M imidazole/50 mM
Tris–Cl pH8.5/0.3 M NaCl/6 M urea, pooled and dialyzed
against PBS (0.14 M NaCl/2.7 mM KCl/6.4 mM Na2HPO4/
0.88 mM KH2PO4, pH 7), for renaturation. PuriWed rTR
concentration was determined with the RC-DC kit (Bio-
Rad).
rTR activity assays
rTR activity was assayed spectrophotometrically by mea-
suring NADPH consumption at 340 nm, as previously
described (Cunnigham et al. 1995; Girault et al. 2001). The
reaction mixture (0.1 ml) containing rTR (3.5–14 �g/ml) in
40 mM Hepes (pH 7.5)/1 mM EDTA, was pre-incubated
with NADPH (150 �mol/l) for 5 min at 27°C prior to initi-
ating the enzyme reaction by addition of the substrate
(50 �M T[S]2; Bachem) (Krauth-Siegel et al. 1987). Three
123
378 Arch Microbiol (2008) 189:375–384
independent experiments were performed and the data ana-
lyzed statistically by one-way ANOVA and Student’s t test
(P < 0.05).
Production of L. amazonensis rTR antiserum
PuriWed rTR was used to immunize Balb/c mice for antise-
rum production. A measure of 50 �g of the puriWed protein
emulsiWed in Freund’s incomplete adjuvant were used to
immunize 6 Balb/c mice (6–7 weeks old) by intraperitoneal
injection. Two identical booster injections were given at
15-day intervals.
Western blot analysis
The L. amazonensis infective promastigotes, obtained as
described above, were harvested by centrifugation at 500g
for 10 min and washed twice with PBS. The Wnal pellet was
ressuspended in 40 mM HEPES/1 mM EDTA, lysed in a
Dounce homogenizer, centrifuged at 12,500g for 15 min
and the supernatant (soluble lysate) collected (Castro-Pinto
et al. 2004). Total protein concentration was measured with
the RC-DC kit (BioRad). Approximately 100 �g of proteins
in the soluble lysate fraction were resolved by SDS-PAGE
on 12% gels in a mini-Protean 3 system (BioRad). Proteins
were electro-transferred to Hybond-C membrane (GE
Healthcare) with a Trans-Blot apparatus (BioRad). Primary
mice polyclonal sera anti-rTR was used in a 1:500 dilution,
and bound antibodies were revealed using an alkaline-phos-
phatase-coupled goat anti-mouse IgG antibody (Dako
Cytomation; 1:1,000 dilution in 5% low-fat milk/TBS).
Blots were developed by incubation in 100 mM Tris–Cl pH
9.5/100 mM NaCl/5 mM MgCl2 containing NBT (300 �g/
ml; BioRad) and BCIP (150 �g/ml; BioRad).ImmunoXuorescence:
Promastigote forms of L. amazonensis in late log phase
(1 £ 107 promastigotes/ml) were Wxed in 4% paraformalde-
hyde for 10 min in ice, washed twice with PBS and adhered
to poly-L-lysine covered slides (2 £ 106 parasites/slide).
Cells were treated with 0.25% Triton X-100 for 5 min at
4°C and blocked with 1% bovine serum albumin (fraction
V, Sigma) in 0.01% Triton X-100/PBS for 1 h at room tem-
perature. Parasites were incubated with the anti-TR poly-
clonal antibody (1:100) or with control polyclonal antibody
(1:100), in blocking solution for 16 h at 4°C. Slides were
washed three times in PBS and incubated for 1 h at room
temperature with secondary antibody (FITC-conjugated
anti-mouse IgG diluted 1:200 in blocking solution—
Sigma). Nuclear and kinetoplast (mitochondrial) DNA was
stained with 10 �M DAPI (Sigma). Over 100 cells were
analysed in a Nikon EC-600 epiXuorescence microscope.
Images were captured with Image-Pro and processed with
Adobe Photoshop. Data presented in the Wgure are repre-
sentative of at least 90% of the parasitic population.
Results
The coding sequence for L. amazonensis TR was obtained
through PCR ampliWcation from genomic DNA, cloned and
sequenced. The sequenced fragment corresponding to L.
amazonensis DNA (after removal of sequences correspond-
ing to the oligonucleotides used in ampliWcation) contained
an open reading frame of 1428 nucleotides, leading to a
deduced amino acid sequence of 476 residues (of the 491
amino acids for the complete expected protein), sharing
89% identity to L. donovani TR.
The alignment of the L. amazonensis TR deduced
amino acid sequence and all trypanosomatid TRs reported
to date (including data from genome projects available at
http://www.sanger.ac.uk) is shown in Fig. 1. TR sequences
show high conservation, particularly around the active site
and co-factor binding sites. A dendrogram derived from
this alignment (Fig. 2) evidences the phylogenetic rela-
tionships among TRs from various trypanosomatid. TR
sequences from New World Leishmanias (L. amazonensis
and L. braziliensis) form a separate group from Old World
species, as expected. Corroborating previous molecular
phylogenetic studies (Croan et al. 1997), our data show
that the New World Leishmania species (L. amazonensis
and L. braziliensis) are in distinct groups, although L. ama-
zonensis is only distantly related to the other members of
the L. Leishmania sub-genus (L. donovani, L. infantum and
L. major).
In order to analyse the probable L. amazonensis TR
structure, we used a homology-base approach, leading to a
3D model. A least squares Wt of L. amazonensis TR on C.
fasciculata TR provided a very low root-mean-square
deviation (RMSD) for C� atoms, 0.07 Å, as expected by
the high degree of sequence identity. The Ramachandran
plot (Ramachandran and Sasisekharan 1968) depicted in
Fig. 3 shows only three non-glycine residues outside the
most favoured regions. The most notable outlier, Phe45,
corresponds to a residue in the C. fasciculata TR that is
also an outlier, Tyr44. The strained conformation of
Phe45 is probably a consequence of the simultaneous
interaction of its peptidic NH group with the peptidic CO
group of Ala46 (N–O = 3.84 Å) and of its peptidic CO
group with the peptidic NH group of Gly41 (O–
N = 3.00 Å). The inter-species sequence identity can be
even greater in speciWc binding sites (as highlighted in
Fig. 1). The L. amazonensis TR modeled structure is also
very similar to TR structures from other species. Compar-
ison of our model to the TR structure from another human
123
Arch Microbiol (2008) 189:375–384 379
pathogen, T. cruzi, shows a RMS deviation of 0.65 Å for
C� atoms. Analysis of both structures revealed elevated
structural similarity in speciWc sites of the enzymes, such
as the trypanothione binding site (Fig. 4).
The cloned L. amazonensis TR coding region was
subcloned into an E. coli expression vector leading to
high-level expression of a 69.2 kDa recombinant fusion
protein (1 mg of puriWed rTR per liter of induced cul-
ture; Fig. 5). Activity of puriWed rTR was measured in
relation to NADPH consumption (Fig. 6). A concentra-
tion of 50 �M trypanothione (T[S]2) was used, as
reported in the literature (Girault et al. 2001). The puri-
Wed rTR shows enzyme activity similar to the native par-
asite enzyme previously reported by us (Castro-Pinto
et al. 2004). In a parallel assay, based on DTNB-medi-
ated regeneration of T(SH)2 over a pH range of 3–10,
rTR was most active in pH 7.5 (data not shown), in
accordance with the pH proWle for native TR obtained in
the absence of DTNB (Castro-Pinto et al. 2004; Hamil-
ton et al. 2006).
Fig. 1 Alignment of known TR 
protein sequences. TR 
sequences are from: Leishmania 
donovani strain MOHM/ET/67/
HU3 (DON; P39050) and strain 
MHOM/IN/83/Dd8 (Dd8; 
AJ415162), L. infantum (INF; 
Sanger genome project, contig 
1163), L. major Friendlin (MAJ; 
CAJ01955), L. amazonensis 
(AMA; this work), L. brazilien-
sis (BRA; our unpublished data 
and sequences 
brazil527h08.p1k, 
brazil345e11.p1k for N-and 
C-terminal fragments, respec-
tively, from the Sanger genome 
project), Crithidia fasciculata 
(FAS; P39040), Trypanosoma 
cruzi (CRU; P28593), 
T. congolense (CON; P13110) 
and T. brucei (BRU; P39051). 
Shaded residues indicate the 
FAD and NADPH binding sites 
and the active site, as described 
for L. donovani TR (Taylor et al. 
1994). Asterisks indicate 
identity, (:) and (.) indicate 
conservative and semi-
conservative substitutions, 
respectively. Residues in the 
L. amazonensis sequence 
corresponding to the primers 
used in ampliWcation are 
underlined. Sanger genome 
project sequences retrieved at 
http://www.sanger.ac.uk. 
Alignment generated with 
T-CoVee (Notredame et al. 
2000) at http://
www.ch.embnet.org/software/
TCoVee.html 
123
380 Arch Microbiol (2008) 189:375–384
To further assess TR expression in vivo, a polyclonal
murine serum was produced against the puriWed L. amazon-
ensis rTR. This serum was used in a Western blot assay and
recognizes a protein of the expected size in the soluble
lysate of infective promastigotes (Fig. 7, panels a and b).
Since this serum was produced against a thioredoxin fusion
protein it could be argued that it might be able to recognize
a similar molecule (thioredoxin or tryparedoxin) in Leish-
mania. We, therefore, included as control a murine serum
obtained against a non-related thioredoxin-fusion protein of
mycobacterial origin (expressed in the same E. coli vector).
No bands are detected in the control experiment (Fig. 7,
panel c).
To address the question of trypanothione reductase
localization in L. amazonensis, we performed immunoXuo-
rescence experiments with infective promastigotes. Both
the anti-rTR murine polyclonal serum (Fig. 8a and c) and
the control serum against the unrelated thioredoxin-fusion
protein (Fig. 8b and d) were used. The anti-rTR serum
shows strong immunoreactivity localizing with the region
of the Xagellar pocket of the parasite, and also with another
possible intracellular structure at the posterior end of the
cell which we are unable, at this point, to precisely identify.
The control antiserum shows no signiWcant staining, as
already expected from the Western blot experiments.
Fig. 2 Phylogenetic relationships between trypanosomatid TRs. Den-
drogram showing the phylogenetic relationships between trypanoso-
matid TRs generated with the program Mega3, based on the alignment
in Fig. 1, using the neighbor-joining method with bootstrap option
(percentages of bootstrap support for each node are indicated in the Wg-
ure). Species of the (L) L. (Leishmania) and (V) L. (Viannia) subgen-
era indicated to the left, and old and new world species (old and new,
respectively) indicated to the right
 don
 Dd8
 infant
 major
 amaz
 brazfasc
 cruzi
 cong
 brucei71
100
99
100
99
100
51
0.05
L. donovani (Eth)
L. donovani (In)
L. infantum
L. major
L. amazonensis
L. braziliensis
C. fasciculata
T. cruzi 
T. congolense
T. brucei
O
ld
we
N
L
V
Fig. 3 Ramachandran plot for the L. amazonensis TR model
Fig. 4 Superposition between the energy minimized (PM3 method)
trypanothione binding sites of T. cruzi TR and L. amazonensis TR.
Color code: C (T. cruzi TR): green, C (L. amazonensis TR): gray, C
(trypanothione in T. cruzi TR): cyan, C (trypanothione in L. amazon-
ensis TR): pink, N: blue, O: red, S: yellow. Hydrogen atoms were omit-
ted to improve clarity (please refer to color Wgure in online version)
Fig. 5 PuriWed recombinant L. 
amazonensis TR. A measure of 
1 �g of puriWed recombinant 
protein was resolved on a 12% 
SDS-PAGE gel, silver stained. 
The predicted molecular mass of 
rTR is 69.2 kDa (53 kDa relative 
to L. amazonensis TR plus the 
13 kDa N-terminal E. coli thio-
redoxin fusion and the C-termi-
nal fusions, including a 6-
histidine tag, encoded by the 
expression vector). Size markers 
indicated to the right, in kDa
92.0
52.2
35.7
28.9
123
Arch Microbiol (2008) 189:375–384 381
Discussion
Trypanothione plays a central role in maintaining the thiol-
redox balance in trypanosomatid parasitic protozoa, in a
pathway not shared by the human host. Enzymes involved
in the biosynthesis and utilization of T(S)2 are, therefore,
potential targets for the development of new anti-trypano-
somatid chemotherapies. Trypanothione reductase (TR) is
the equivalent to the human glutathione reductase (GR); it
has been described in a number of trypanosomatid species
such as the human pathogens T. cruzi (Krauth-Siegel et al.
1987) and T. brucei (Henderson et al. 1987; Aboagye-
Kwarteng et al. 1992), the cattle pathogen T. congolense
(Shames et al. 1988), the insect pathogen C. fasciculata
(Henderson et al. 1987; Aboagye-Kwarteng et al. 1992),
and the Old World Leishmania species L. donovani (Cun-
nigham and Fairlamb 1995). TR sequences can also be
derived from genome projects (L. major, L. infantum and L.
braziliensis.) In a previous work, we described TR activity
in Leishmania amazonensis promastigotes and axenic- and
lesion-derived amastigotes, demonstrating its association
with parasite infectivity. In another work, we evaluated
several tri-peptides (glutathione analogs), and deWned its
eVects on parasite growth, macrophage infection and
enzyme activity. Analysis of parasite TR activity reinforces
the idea that the trypanothione/TR system can be a good
target for experimental chemotherapy of leishmaniasis
(Castro-Pinto et al. 2004, 2007). Here, we report the
sequence of the L. amazonensis TR coding gene. Align-
ment of its amino acid sequence to those of other trypano-
somatid TRs shows a high conservation, particularly
around the active site and co-factor binding sites (Fig. 1),
and a dendrogram derived from this alignment (Fig. 2) evi-
dences the phylogenetic relationships among the various
trypanosomatid TRs. These results suggest that TR
sequences could be useful molecular markers for the study
of Leishmania phylogeny, contributing data to the still
unresolved question of the geographical origin of the genus
(Momen and Cupolillo 2000; Kerr et al. 2000).
The characteristics of the C. fasciculata TR template
used for the construction of the L. amazonensis TR homol-
ogy-based model, i.e. high level of identity (79%) between
primary sequences in conjunction with the high resolution
of the crystallographic structure, point out the reliability of
the structure model of the L. amazonensis enzyme. It is
interesting to compare the L. amazonensis TR model to the
TR structure from another human pathogen, T. cruzi, which
was co-crystallized with the substrate T(S)2 (Bond et al.
1999). There are two substrate molecules co-crystallized in
the T. cruzi TR homodimer, which are interacting with resi-
dues from both chains (Zhang et al. 1996). These regions
are the most interesting for the development of selective TR
inhibitors because of diVerences between human (glutathi-
one) and parasite (trypanothione) substrate structures. Resi-
dues selected with a distance restraint of 6.0 Å from these
substrate molecules are not exactly the same and the two
selected sites were considered for a comparison, revealing
that just one residue, Lys402, is diVerent between both par-
asite-selected sites. In fact, the presence of a lysine residue
Fig. 6 Activity of L. amazonensis rTR. Activity of the L. amazonensis
enzyme was assayed measuring NADPH consumption using diVerent
concentrations of the puriWed recombinant protein (3.5, 7.0 and 14 �g,
pH 7.5). T(S)2 concentration used for all assays was 50 �M
NADPH consumption X recombinant TR concentration
0
0,005
0,01
0,015
0,02
0,025
Control/NADPH
Protein concentration (µg/mL) 
N
A
D
PH
 (3
40
nm
)
3,5 7 14
Fig. 7 Detection of L. amazonensis TR in the soluble lysate fraction
of infective promastigotes. A measure of 100 �g of proteins were re-
solved by SDS-PAGE (coomassie-R250 stained gel shown in a), elec-
tro-transferred and reacted against the anti-rTR polyclonal antibody
(b) or a polyclonal mouse serum raised against an unrelated, thiore-
doxin-fusion recombinant protein of mycobacterial origin (c) as nega-
tive control. Size markers indicated to the left, in kDa
123
382 Arch Microbiol (2008) 189:375–384
at this position is exclusive of T. cruzi TR; in the remaining
known TR sequences, the corresponding amino acid resi-
due is an asparagine.
The side chain amino group of Lys402 is interacting
weakly with the CysI peptidic carbonyl group (N–O =
3.4 Å) in the T. cruzi TR, but the same interaction is appar-
ently not possible in L. amazonensis TR, because the aspar-
agine side chain is shorter than the lysine side chain. In
order to account for the eVect of this amino acid substitu-
tion on the trypanothione binding, we performed an energy
minimization procedure at the semi empirical PM3 level of
the trypanothione active site from L. amazonensis and T.
cruzi TRs, both containing the substrate molecule. In the
energy-minimized structure resulting from the replacement
of Lys402 by the asparagine residue (Fig. 4), the CysI
peptidic carbonyl group is in fact too far away to form a
Fig. 8 ImmunoXuorescent detection of TR in L. amazonensis
infective promastigotes. L. donovani infective promastigotes were
incubated with the murine anti-rTR serum (a and c) and with a murine
control serum produced against an unrelated thioredoxin-fusion
recombinant protein (b and d). Anti-rTR reactivity was revealed by
FITC-coupled secondary antibody (green); nuclear and kinetoplast
DNA were labeled with DAPI (blue). The region of the Xagellar pocket
(FP), the nucleus (N) and kinetoplast (K) are indicated in the magniW-
cation shown in panel c. The bar represents 5 �m
123
Arch Microbiol (2008) 189:375–384 383
hydrogen bond with the asparagine side chain (N–
O = 6.6 Å). Torsions in the �-glutamyl and cysteinyl back-
bone, however, result in the formation of a hydrogen bond
between the �-GluI peptidic amino group and the CysI pep-
tidic carbonyl group (N–O = 3.3 Å). Because of trypanothi-
one backbone torsions, the spermidine moiety does not
occupy the same position in the minimized L. amazonensis
and T. cruzi TR structures, but this does not inXuence sig-
niWcantly the orientation of the �-GluII–CysII–GlyII frag-
ment, which is very similar in the enzyme from both
species, as is the position of the disulWde bridge (Fig. 4).
The evaluation of activity of rTR showed that this mole-
cule has enzymatic activity after renaturation (Fig. 6), in
agreement with data concerning the native enzyme (Castro-
Pinto et al. 2004). Although the recombinant TR molecule
used in thisstudy carries an N-terminal E. coli thioredoxin
fusion, it has been reported in the literature that thioredoxin
does not function as a substrate for trypanothione reductase
(Reckenfelderbäumer et al. 2000; Schmidt and Krauth-Sie-
gel 2002).
In vivo native expression of the cloned L. amazonensis tr
gene was conWrmed by both western blotting and immuno-
Xuorescence studies. The polyclonal serum obtained
against the puriWed rTR recognizes a band of approxi-
mately 53 kDa in Western blots of soluble lysates of infec-
tive promastigote forms (Fig. 7), compatible with its
expected molecular weight. The speciWc detection of two
bands in the immunoblot could suggest that proteolytic pro-
cessing of TR occurs in L. amazonensis. ImmunoXuores-
cence analysis evidences immunoreactivity of the anti-rTR
serum localizing strongly with the region of the Xagellar
pocket and other intracellular structure(s) in the posterior
end of L. amazonensis promastigotes. The Xagellar pocket
of trypanosomatids is a deep invagination of the plasma
membrane, located at the anterior end of the cell, and its
membrane, representing up to 3% of the cell surface, is the
only known site for endocytosis, secretion of proteins and
addition of integral membrane proteins to the cell surface
(reviewed by Landfear and Ignatushchenko 2001). Since
TR is an essential enzyme in the detoxiWcation processes in
Leishmania, its strategic presence in the Xagellar pocket
could provide this organism with a rapid response at the
point of entry of toxic compounds. Work is underway in
our laboratory to deWne more precisely the nature and
dynamics of these intracellular compartments co-localizing
with TR.
This is the Wrst report on the sequence and expression of
TR from a New World Leishmania species. Despite the fact
that the three-dimensional structures of TRs from two
diVerent trypanosomatids, C. fasciculata and T. cruzi, have
been known for several years, and that high-throughput
screening of inhibitors has been an on-going eVort, eVective
inhibitors of TR suitable to enter clinical phase are still
elusive (Muller et al. 2003). The high sequence and struc-
tural conservation found between the L. amazonensis and T.
cruzi TR disulWde binding sites suggests that inhibitors of
the L. amazonensis enzyme should also eVectively inhibit
TRs from other pathogenic trypanosomatids, making this a
useful model for the screening of new anti-trypanosomatid
drugs. Studies are underway in order to identify new com-
pounds capable of inhibiting L. amazonensis TR.
Acknowledgments We thank Dr. Marcelo Comini (Technical Uni-
versity of Braunschweig/Germany) for donation of trypanothione and
Dr. Elisa Cupolillo for helpful discussions. All DNA sequencing was
performed at the PDTIS-Fiocruz Genomic Platform. D.C-P is a recip-
ient of a scholarship from the Brazilian “Conselho Nacional de Desen-
volvimento CientiWco e Tecnologico - CNPq”. This project received
Wnancial support from CNPq, Faperj and PDTIS/Fiocruz.
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123
	Cloning and expression of trypanothione reductase from a New World Leishmania species
	Abstract
	Introduction
	Materials and methods
	Parasites
	Cloning and sequencing of the Leishmania amazonensis TR gene
	Alignment of trypanosomatid TR protein sequences
	3D homology modeling of L. amazonensis TR and molecular modeling study
	Expression and puriWcation of rTR
	rTR activity assays
	Production of L. amazonensis rTR antiserum
	Western blot analysis
	ImmunoXuorescence:
	Results
	Discussion
	References