Functional characterisation of the iron superoxide dismutase gene repertoire in Trypanosoma brucei.

Abstract

Superoxide dismutases (SOD) are a family of antioxidant enzymes that function by removing superoxide anions from the cellular environment. Here, we show that the African trypanosome, Trypanosoma brucei, expresses four SOD isoforms, three of which we have validated biochemically as iron dependent, a feature normally associated with prokaryotic SODs. Localisation studies reveal that two of the enzymes are found predominantly in a parasite-specific organelle, the glycosome (TbSODB1 and TbSODB2), while the other two are targeted to the mitochondrion (TbSODA and TbSODC). Functional analysis of the SOD repertoire in bloodstream form parasites was performed using an inducible RNA interference (RNAi) approach. Down-regulation of the glycosomal SOD transcripts corresponded with a significant reduction in the corresponding proteins and a dramatic level of cell death within the population. The importance of one of the mitochondrial enzymes (TbSODA) only became apparent when parasites were exposed to the superoxide-generating agent paraquat following induction of RNAi. These experiments therefore identify essential components of the superoxide metabolising arm of the T. brucei oxidative defence system and validate these enzymes as parasite-specific targets for drug design.

Full text

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Original Contribution
Functional characterisation of the iron superoxide dismutase gene
repertoire in Trypanosoma brucei
Shane R. Wilkinson*, S. Radhika Prathalingam, Martin C. Taylor, Aiyaz Ahmed,
David Horn, John M. Kelly
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
Received 26 April 2005; revised 10 June 2005; accepted 27 June 2005
Available online 18 August 2005
Abstract
Superoxide dismutases (SOD) are a family of antioxidant enzymes that function by removing superoxide anions from the cellular
environment. Here, we show that the African trypanosome, Trypanosoma brucei, expresses four SOD isoforms, three of which we have
validated biochemically as iron dependent, a feature normally associated with prokaryotic SODs. Localisation studies reveal that two of the
enzymes are found predominantly in a parasite-specific organelle, the glycosome (TbSODB1 and TbSODB2), while the other two are
targeted to the mitochondrion (TbSODA and TbSODC). Functional analysis of the SOD repertoire in bloodstream form parasites was
performed using an inducible RNA interference (RNAi) approach. Down-regulation of the glycosomal SOD transcripts corresponded with a
significant reduction in the corresponding proteins and a dramatic level of cell death within the population. The importance of one of the
mitochondrial enzymes (TbSODA) only became apparent when parasites were exposed to the superoxide-generating agent paraquat
following induction of RNAi. These experiments therefore identify essential components of the superoxide metabolising arm of the T. brucei
oxidative defence system and validate these enzymes as parasite-specific targets for drug design.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Trypanosoma brucei; Oxidative defence; Superoxide dismutase; RNA interference; Glycosome; Mitochondrion; Free Radical
Introduction
Protozoan parasites belonging to the family trypanoso-
matidae cause debilitating diseases in many regions of the
developing world. Over 30 million people are infected by
Trypanosoma brucei, Trypanosoma cruzi, or Leishmania,
parasites that respectively cause African sleeping sickness,
American trypanosomiasis, and visceral/cutaneous leishma-
niasis (http://www.who.int). There is no immediate prospect
of a vaccine for any of these diseases and current drug
treatments are problematic due to limited efficacy, toxicity,
and cost. Improvements in chemotherapy are urgently
required[1].Manyexistingdrugregimeshavebeenproposed
to mediate part of their trypanocidal action by inducing
oxidative stress. For agents such as nifurtimox and benzni-
dazole this occurs directly via redox cycling of the drug
leading to the formation of reactive oxygen species (ROS)
[2,3]. With other compounds such as difluoromethylorni-
thine, melarsporol, and pentamidine, the effect is indirect,
with oxidative stress arising from a reduction in the effective
thiol pool [4–6]. Biochemical and molecular analysis of the
mechanisms by which parasites combat oxidative stress may
therefore have implications for improved chemotherapeutic
intervention.
ROS such as the superoxide anion (O2S?), H2O2, and
hydroxyl radical are produced as by-products of aerobic
metabolism. In addition, pathogens can also encounter ROS
as a result of drug turnover and host immune responses.
Trypanosomatids have evolved a number of processes for
combating oxidative stress that are distinct from the systems
0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2005.06.022
Abbreviations: SOD, superoxide dismutase (EC 1.15.1.1); ROS,
reactive oxygen species; O2S?, superoxide anion; BSF, bloodstream form;
PCF, procyclic form; NBT, nitroblue tetrazolium; RNAi, RNA interference;
IPTG, isopropyl-h-D-thiogalactopyranoside; PBS, phosphate-buffered saline.
* Corresponding author. Fax: +44 20 7636 8739.
E-mail address: shane.wilkinson@lshtm.ac.uk (S.R. Wilkinson).
Free Radical Biology & Medicine 40 (2006) 198 – 209
www.elsevier.com/locate/freeradbiomed

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found in other eukaryotes [5,7–9]. The enzyme-mediated
pathways used to eliminate hydroperoxides are now well
established.Thesearedependentontheparasite-specificthiol
trypanothione (N1,N8-bisglutathionylspermidine) and its
ancillary enzyme, trypanothione reductase [5,10]. This sys-
tem, analogous to the glutathione redoxcyclefound in higher
eukaryotes, drives a series of two-step oxidoreductase cas-
cades which result in the reduction of H2O2and a range of
short-chain organic, fatty acid, and phospholipid hydro-
peroxides[11–13].Thesedetoxification pathways havebeen
localised to various ROS-generating sites including the mito-
chondrion, glycosome, endoplasmic reticulum, and cytosol.
For the metabolism of O2S?, trypanosomatids express
proteins belonging to the superoxide dismutase (SOD)
family of antioxidant enzymes [14–17]. These metallopro-
teins function by removing excess O2S?to form H2O2and
O2[18]. They have been identified universally in aerobic
organisms [19,20] and, based on the metal cofactor at their
catalytic core, can be subdivided into three major groups:
copper/zinc (Cu/Zn-SOD), manganese (Mn-SOD), and iron
(Fe-SOD). Generally, eukaryotes possess both Cu/Zn-and
Mn-SODs, but lack Fe-SODs [21]. Trypanosomatids differ
from this generalization and are reported to express only
SODs of the Fe isoform [14–17]. This is similar to the
situation observed in the protozoan parasites Plasmodium
falciparum and Entamoeba histolytica, where enzyme-
mediated O2S?metabolism is exclusively Fe-SOD depend-
ent [22–24]. Plants also possess Fe-SODs but this activity is
restricted to the chloroplast and represents only part of the
total SOD repertoire of that organelle [25,26]. The
importance of SODs to trypanosomatids has been demon-
strated by generating cell lines with altered levels of
expression. Overexpression of T. cruzi SODB1 results in
cells that are hypersensitive to the trypanocidal agents
benznidazole and gentian violet [27]. Similar studies in
Leishmania chagasi produced cells with an increased
resistance toward paraquat and nitroprusside [28]. Deletion
of one copy of the L. chagasi SODB1 generated an
increased sensitivity to these agents and a significant
reduction in survival within human macrophages [29].
With the completion of the trypanosomatid genome
sequencing projects it is now possible to investigate the full
SOD complement of these parasites. Here we report the
dissection of the O2S?metabolising arm of T. brucei in terms
of biochemical properties, subcellular localisation, and func-
tional importance.
Materials and methods
Parasites
T. brucei single marker cell line (SMB) BSF that
constitutively express T7 RNA polymerase and the tetracy-
cline repressor protein [30] were grown at 37-C under a 5%
CO2atmosphere in modified Iscove’s medium [31] con-
taining 2 Ag ml?1G418. Transformed SMB cells were
maintained in this growth medium supplemented with 2.5
Ag ml?1hygromycin. T. brucei PCF cells (PCF 29-13) that
constitutively express T7 RNA polymerase and the tetracy-
cline repressor protein [30] were grown at 27-C in SDM-79
medium [32] supplemented with 25 Ag ml?1G418 and 25
Ag ml?1hygromycin. Transformed PCF 29-13 parasites
were maintained in the above medium containing 2.5 Ag
ml?1phleomycin. Tetracycline-free fetal calf serum (Auto-
gen Bioclear) was used in the growth media. DNA and total
RNA were extracted from parasites using the DNeasy
Tissue and RNeasy mini kits (Qiagen), respectively. T.
brucei sequence data were obtained from The Wellcome
Trust Sanger Institute website at http://www.sanger.ac.uk/
Projects/T_brucei. Sequencing of the T. brucei genome was
accomplished as part of the Trypanosoma Genome Network
with support by The Wellcome Trust.
Biochemical properties
Four T. brucei genes that encode SOD were identified
from either the T. brucei genome project or EMBL/
GenBank databases. Derivatives of all four genes were
amplified from parasite genomic DNA (see Table 1 for
primer combinations) and cloned into the expression vector
pTrcHis-C (Invitrogen). DNA was sequenced using a Dye
Terminator cycle kit (Applied Biosystems) and an ABI
Prism 377 sequencer.
Escherichia coli strains transformed with the appropriate
expression plasmid were grown in NZCYM broth (Sigma)
containing ampicillin and protein expression was induced
by the addition of isopropyl-h-D-thiogalactopyranoside
(IPTG). Recombinant His-tagged proteins were affinity-
purified on a Ni-NTA matrix column under native con-
ditions as recommended by the manufacturer (Qiagen). The
cell lysis, column wash, and elution steps were all carried
out in the presence of protease inhibitors (Roche). Fractions
were analysed by SDS-PAGE and protein concentrations
were determined by the BCA protein assay system (Pierce).
Enzyme activity was measured using the cytochrome c
[18], nitroblue tetrazolium (NBT) [33], and SOD-525 [34]
(Bioxytech) assays. These were carried out as follows: For
the cytochrome c reaction, the SOD-mediated inhibition of
ferricytochrome c reduction in the presence of a O2S?-
generating system (xanthine/xanthine oxidase) was deter-
mined by monitoring the change in absorbance at 550 nm.
A 1-ml reaction containing 50 mM potassium phosphate,
pH 7.8, 10 AM ferricytochrome c, 50 AM xanthine, 100
AM EDTA, and SOD was incubated at room temperature
for 5 min. The background rate of ferricytochrome c
reduction was determined and the reaction initiated by the
addition of 0.02 unit xanthine oxidase. Control experi-
ments were carried out where SOD was omitted. Activity
was determined by comparing the control and SOD-
containing reactions. One unit of SOD is defined as the
enzyme activity that inhibits cytochrome c reduction by
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xanthine oxidase by 50%. The basis for the NBT assay is
similar to that described above, except that 75 AM NBT
was used in place of ferricytochrome c and activity was
measured by following the change in absorbance at 560
nm. The SOD-525 assay was carried out as recommended
by the manufacturer (Bioxytech). In this reaction SOD
increases the autooxidation of 5,6,6a,11b-tetrahydro-
3,9,10-trihydroxybeno[c]fluorine to yield a chromophore
with maximum absorbance at 525 nm. Interference due to
mercaptans was controlled by pretreating samples with 1-
methyl-2-vinylpyridinium that directly removes mercaptans
by an alkylation reaction. Activity was measured by
comparison of the autooxidation rates in the presence
and absence of SOD.
To determine which metal cofactor is required by the
trypanosomal SODs, assays were carried out in the presence
of H2O2, NaN3, and KCN. The resultant inhibition profiles
were then compared to those obtained for commercial Fe-
SOD, Mn-SOD, and Cu/Zn-SOD (Sigma).
Generation of TbSODB antiserum
The His-tagged TbSODB2 protein was excised from a
Coomassie-stained SDS-PAGE gel and macerated in liquid
nitrogen. The resultant material was suspended in Freund’s
complete adjuvant (Sigma), sonicated (6 ? 10 s), and then
inoculated into mice (BALB/c). At 2-week intervals further
inoculations were carried out using gel-purified TbSODB2
suspended in Freund’s incomplete adjuvant (Sigma). After a
total of five inoculations, the mice were bled and the
specificity of the antiserum tested by Western blotting.
RNA interference
Fragments (518 – 748 bp) corresponding to the coding
sequences of TbSODA, TbSODB1, and TbSODC were
amplified from genomic DNA (see Table 1 for primers),
digested with BamHI + XhoI, and cloned into the
corresponding sites of the vector p2T7Ti[35]. In this vector,
the inserted DNA is flanked by two opposing T7 promoters
with each promoter under the control of a tetracycline
operator. Constructs were linearised with NotI and electro-
porated into BSF parasites, and transformants were cloned
as described [36]. Induction of RNA interference (RNAi)
was initiated by adding 1 Ag ml?1tetracycline to the culture.
Detection of the growth inhibition phenotype
BSF trypanosomes, transformed with each of the RNAi
constructs, were seeded at 1 ? 105cells ml?1and incubated
at 37-C in the presence of tetracycline (1 Ag ml?1). Every
24 h parasite growth was monitored microscopically and the
culture diluted back to 1 ? 105cells ml?1. Control cultures
incubated in the absence of tetracycline were grown in
parallel. Under these conditions untreated cell lines grew
with a doubling time of approximately 7–8 h.
Susceptibility experiments
BSF parasites (tetracycline treated and untreated) were
seeded at 1 ? 103ml?1in a 96-well plate in 200 Al growth
medium containing different concentrations of H2O2
(Sigma), benznidazole (Roche), gentian violet (Gurr), nifur-
Table 1
Oligonucleotides used in this study
GenePrimerSequence (5V To 3V)
Protein expression
TbSODA
TbSODA-4
TbSODA-5
TbSODB1-4
TbSODB1-5
TbSODB2-1
TbSODB2-2
TbSODC-1
TbSODC-2
TbSODC-8
TbSODC-9
TbSODA-1
TbSODA-2
TbSODB1-1
TbSODB1-2
TbSODC-4
TbSODC-5
TbSODA-6
TbSODA-7
TbSODB1-6
TbSODB1-7
TbSODB2-3
TbSODB2-4
TbSODC-14
TbSODC-15
ggatccATGGAGCAGCAGCGGAATTG
aagcttTTACTTCAGAGCCTGTTCATA
ggatccTGACCTTCAGCATTCCACCAC
aagcttCTAGCTTTTCAGAGCTGCTC
ggatccCTCAGCATTCCACCACTTCCG
aagcttTTACAAGTCACTATGCGGTGC
ggatccTTCCAACGCTAGAATTTCCGTGG
gaattcTTACCAGAACTTCATCTCGTA
ggatccGGGCCCCCGATTACTACGTAGAG
ggatccTTGGCACAGCGCTGCAAGCATCA
ggatccTTCAGTTCAACTGGAAAGACG
ctcgagATAGTCCAAATTTCCTTGAGG
ggatccCTTCCGTGGGGGTACGATGGA
ctcgagTGCACGTAGGCCGGACGGTCA
ggatccCTTGGGCGTTTCAATTACATGG
ctcgagCCCGCTTCCAGTGCCGTTCCG
aagcttATGAGGTCTGTCATGATGCGTTGC
tctagaCTTCATAGCCTGTTCATACGTCCT
tctagaATGACCTTCAGCATTCCACCACTT
ggatccCTAGCTTTTCAGCAGCTGCTCCTC
tctagaATGGCTTTCAGCATTCCACCACTT
ggatccTTACAAGTCCATATGCGGTGCCC
aagcttATGCGTCGCGTGGCTTCATTC
tctagaAAGGAAGAAAAAGCCTTGTGA
TbSODB1
TbSODB2
TbSODC
RNAi
TbSODA
TbSODB1
TbSODC
Localisation
TbSODA
TbSODB1
TbSODB2
TbSODC
The sequences in lowercase italics correspond to restriction sites incorporated into the primers to facilitate cloning.
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timox (Bayer), or paraquat (Sigma). After incubation at 37-C
for4days,20AlofthevitalstainAlamar blue(BiosourceUK
Ltd) was added to each well and the plates were incubated for
a further 6 h. The fluorescence of each culture was
determined using a Gemini Fluorescent Plate reader (Molec-
ular Devices) at an excitation wavelength of 530 nm,
emission wavelength of 585 nm, and a filter cutoff at 550
nm. The colour change resulting from the reduction of
Alamar blueisproportionaltothenumberoflive cells,which
was established following production of a standard curve.
Localisation
The vectors used for localisation were generated as
follows. TbSODA was amplified from parasite genomic
DNA (Table 1 for primers). The resultant DNA fragment was
digested with HindIII/XbaI and cloned into the correspond-
ing sites of the vector pLEW-tagC. The ligation was
performed such that an amino acid epitope (9E10) derived
from the human c-myc protein was added to the carboxyl
terminal of TbSODA. A similar approach was used to tag
TbSODB1 and TbSODB2 (Table 1 for primers), except that
HindIII/XbaI-digested DNA fragments were cloned into the
vector pLEW-tagN such that the c-myc (9E10) epitiope was
added to the amino terminal of each protein. A 306-bp DNA
sequence corresponding to the amino terminal of TbSODC
was amplified from parasite genomic DNA (Table 1 for
primers) and digested with HindIII/XbaI. This was used in a
three way ligation with HindIII/BamHI-digested pLEW100
[30] and a XbaI/BamHI-digested DNA fragment encoding
the eGFP (Clontech). The cloning was carried out such that
the eGFP gene was inserted in-frame at the 3V end of the
TbSODC-derived DNA fragment.
Constructs were linearised with NotI (for the TbSODA,
TbSODB1, and TbSODC-based vectors) or SacII (for the
TbSODB2-containing construct) and then electroporated
into the PCF 29-13 parasites [36]. Transformed cells were
selected in SDM-79 medium containing 25 Ag ml?1G418,
25 Ag ml?1hygromycin, and 2.5 Ag ml?1phleomycin.
Expression of the tagged proteins was initiated by adding 1
Ag ml?1tetracycline to the culture.
Trypanosomes expressing tagged versions of TbSODA
or TbSODC were suspended at 5 ? 106cells ml?1in SDM-
79 medium containing 5 AM MitoTracker Orange (Molec-
ular Probes) and incubated at 27-C for 20 min. Cells were
washed once in phosphate-buffered saline (PBS) and then
incubated at 27-C for 60 min in fresh growth medium in the
absence of MitoTracker. Parasites were pelleted, washed
twice in PBS, and then fixed in 2% paraformaldehyde in
PBS. Aliquots of the cell suspension (1 ? 105cells) were
then air-dried onto microscope slides. To localise TbSODA,
fixed parasites were blocked with 50% horse serum/0.1%
saponin in PBS for 20 min at room temperature and stained
with mouse anti-c-myc (9E10) antibody (diluted 1:200)
(Santa Cruz Biotechnology). After 60 min, the slides were
washed extensively with 0.1% saponin in PBS and then
incubated with Alexa-Fluor 488 goat anti-mouse (1:400)
(Molecular Probes). Trypanosomes expressing tagged
TbSODB1 and TbSODB2 were fixed and blocked as
described above without MitoTracker staining. Cells were
costained with mouse anti-c-myc (9E10) antibody (1:200)
and anti-TbgGAPDH serum (1:400) raised in rabbits and
then incubated with Alexa-Fluor 488 goat anti-mouse and
Alexa-Fluor 546 goat anti-rabbit serum (Molecular Probes)
(both diluted 1:400). For all localisation experiments para-
site DNA was stained with 200 pM DAPI (Sigma) in 50%
glycerol in PBS and slides were viewed using a Zeiss LSM
510 confocal microscope.
Results
Identification of the T. brucei SOD repertoire
The biochemical characterisation of a T. brucei SOD has
previously been reported [17]. To determine the full
repertoire of parasite isoforms we carried out a protein
motif search of the T. brucei GeneDB database using a Fe-
SOD/Mn-SOD consensus sequence (DxWEH[STA][FY]
[FY] from PROSITE: PDOC00083). This identified open-
reading frames with the potential to encode four distinct
SODs. These were designated TbSODA (T. brucei GeneDB
web link Tb05.27M3.490), TbSODB1 (Tb11.01.7550),
TbSODB2 (Tb11.01.6660), and TbSODC (Tb11.01.7480)
with TbSODB1 corresponding to the enzyme analysed by
Kabiri and Steverding [17] (Fig. 1). Northern blot analysis
indicated that all genes are expressed in both the insect and
the bloodstream forms (BSF) of the parasite (see Fig. 5A).
Examination of the four parasite proteins suggested that they
were all related to the Fe group, having 42–54% identity to
bacterial Fe-SODs and a lower identity to Fe-SODs of a
plant origin (33–41%). Significant similarity was also
observed to bacterial Mn-SODs (29–39% identity) reflect-
ing the relatedness of Fe and Mn isoforms. Sequence
analysis of the T. brucei enzymes reveals that they all have
ligands implicated in Fe or Mn binding (His28, His76,
Asp161, His165; numbering relates to the TbSODB1
sequence) (Fig. 1) and a series of conserved residues that
facilitate this interaction (His32, His33, Tyr36, Trp80,
Ser123, Trp125, Trp163, Glu164, His165, Ala166,
Tyr167, Tyr168, Asn173) [20]. Mn-and Fe-SODs can be
readily distinguished from each other despite the consid-
erable similarity at their catalytic core. Four residues
characteristic of Fe-SODs are present in all the T. brucei
SODs (Ala71, Gln72, Phe78, Ala145) (Fig. 1), while the
amino acids that typify Mn-binding enzymes are absent
[20,37]. Additionally these four residues, in conjunction
with amino acids at other sites, play a role in mediating
oligomerisation [20]. Fe-SODs can be divided into those
that function in a homodimeric form and those that are
homotetrameric. Examination of the four parasite SODs
demonstrates that they have residues found only in the
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homodimeric enzymes (Asn68, Ala71, Gln72, Phe78,
Phe121, Ala145, Pro148).
When compared with each other, the T. brucei SODs
were shown to have 36–90% identity. Alignment of the
TbSODB1 and TbSODB2 sequences revealed that these two
proteins have a high degree of similarity (90% identity),
with the only significant differences at their carboxyl
termini (Fig. 1). The genes presumably arose through
duplication. Analysis of the carboxyl-terminal region
revealed that TbSODB2 contains a SKL-type tripeptide
(SDL) that may function to target the protein to the
glycosome, a parasite-specific organelle related to the
peroxisome of other eukaryotes [38]; this sequence (SDL)
has been shown to act as a glycosomal targeting signal in
other trypanosomatid proteins [29]. Although TbSODB1
has no clearly defined localisation signal, PSORT analysis
did indicate that it may also be found in this organelle. Other
potential targeting signals were also observed in TbSODA
and TbSODC. Both proteins have amino terminal exten-
sions of 35 and 97 residues, respectively. Examination of
these regions suggested that both may function in mito-
chondrial targeting (prediction by iPSORT) (Fig. 1). Several
mitochondrially targeted SODs have been reported from a
variety of organisms, generally belonging to the Mn-SOD
subgroup [39,40].
Biochemical characterisation of T. brucei SOD repertoire
To determine the activity of the four putative SODs, each
gene was cloned into the vector pTrcHis-C and expressed in
E.coli BL21+ (for TbSODB1, TbSODB2, and TbSODC) or
TOP10 (for TbSODA). In this system, the parasite enzymes
are expressed as recombinant proteins tagged at their amino
terminal with a histidine-rich sequence and an epitope
detectable with the anti-Xpress monoclonal antibody (Invi-
trogen). For TbSODA and TbSODC, the expressed re-
combinant proteins had been designed to lack the amino
terminal extensions (Fig. 1). After induction with IPTG,
bands of approx 25 kDa were detected in the soluble
fraction of lysates by Western blot analysis. These proteins
could be readily purified by one round of affinity
chromatography on a nickel-nitrilotriacetic acid column
(Fig. 2A).
The SOD activity of each trypanosomal isoform was then
investigated using the cytochrome c assay [18]. For
TbSODA, TbSODB1, and TbSODB2, activity was readily
detected (Fig. 2B; Table 2). No activity was detected with
TbSODC. Two further versions of this gene were cloned
into the expression construct containing larger portions of
the amino terminal extension. Although recombinant
proteins were readily purified, these also lacked SOD
activity. To confirm that TbSODA, TbSODB1, and
TbSODB2 are Fe-SODs, we examined the activity of each
enzyme in the presence of the inhibitors H2O2, NaN3, and
KCN. The resultant inhibition profiles were then compared
against commercial Fe-, Mn-, and Cu/Zn-SODs (Sigma)
assayed in the same way (Table 3). In these experiments Cu/
Zn-SOD was shown to be cyanide-sensitive but insensitive
to both peroxide and azide, while Fe-SOD was inhibited by
peroxide and azide, but unaffected by cyanide treatment. No
significant inhibition of Mn-SOD activity was observed
with any of the agents. Therefore, analysis of the T. brucei
Fig. 1. Comparison of T. brucei superoxide dismutases. Four SOD sequences identified from the T. brucei genome project and EMBL/GenBank databases were
aligned using CLUSTALW with Fe-and Mn-SOD sequences from E. coli (Accession Nos. P09157 and P00448, respectively). Amino acids that are common
with the TbSODB1 sequence are highlighted in grey. Residues in red represent ligands that bind the metal cofactor and are conserved between Fe-SODs and
Mn-SODs, while residues in yellow are characteristic of Fe-SODs [20,37]. The putative targeting sequences identified in TbSODA and TbSODC (both in blue)
and glycosomal tripeptide in TbSODB2 (in green) are noted. The underlined sequences correspond to the 5V primers used to produce the E. coli expression
vectors (Materials and methods).
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SOD activity indicates that all three enzymes display the
distinctive inhibition profiles of the Fe isoform (Table 3).
Localisation of SOD isoforms in T. brucei
The subcellular location of the trypanosomal SODs was
examined by expressing c-myc (9E10) or enhanced green
fluorescent protein (eGFP)-tagged versions of each enzyme
in procyclic form (PCF) parasites. DNA sequences for
TbSODA, TbSODB1, and TbSODB2 were amplified and
ligated in-frame with the 9E10 epitope from the human c-
myc protein using the vectors pLEW-tagC and pLEW-tagN.
For TbSODA this resulted in a protein tagged at its carboxyl
terminus, while TbSODB1 and TbSODB2 were tagged at
their amino termini. The localisation constructs were
electroporated into T. brucei PCF29-13 and recombinant
parasites selected. To induce expression of the tagged
protein, cells were incubated in the presence of tetracycline
for 48 h and lysates examined by Western blotting using a
monoclonal antibody against the c-myc (9E10) epitope (Fig.
3A). Extracts derived from parasites expressing tagged SOD
all contained a single band of the expected size. Attempts to
tag the carboxyl terminal of TbSODC with c-myc (9E10)
were also carried out and although genetically modified
parasites were obtained, the expressed level of recombinant
protein was very low after induction with tetracycline. To
circumvent this problem a 306-bp DNA fragment contain-
ing the amino terminal leader sequence of TbSODC (Fig. 1)
was amplified and cloned in-frame and upstream of the
eGFP gene. The construct was introduced into T. brucei and
transformants were selected as described above. Addition-
ally, a construct containing a nontagged version of eGFP
was made and analysed in parallel.
T. brucei cells induced to express the tagged SODs were
fixed, permeabilised, and then treated as described. For both
TbSODA and TbSODC-eGFP a lattice-like structure spread
throughout the cell was observed. This was reminiscent of the
pattern reported for proteins localised to the large-single
mitochondrion of the parasite [41]. To confirm this, cells were
costained with MitoTracker (a mitochondrial-specific dye)
(Fig. 3C). When the images were superimposed there was a
pattern of colocalisation (yellow staining), indicating the
presence of TbSODA and TbSODC at the same sites as
MitoTracker throughout the mitochondrion. When cells
Fig. 2. Biochemical properties of TbSODs. (A) Ni-NTA-purified TbSODs (indicated by the arrows) were resolved on a 12% SDS-PAGE gel and visualised by
Coomassie staining. Markers show molecular mass in kDa. (B) SOD activity was assayed by following the O2S?-mediated reduction of ferricytochrome c [18]
in the presence of the T. brucei enzymes and compared against reactions where SOD was omitted (control). Each assay was performed in triplicate and
representative experiments are shown. No SOD activity was observed with the TbSODC recombinant protein.
Table 2
Specific activities of T. brucei SODs
Protein Specific activity (U mg?1) T
standard deviation
TbSODA
TbSODB1
TbSODB2
800 T 108
2144 T 132
2304 T 48
Enzyme activities of the purified, recombinant T. brucei SODs were
determined using the cytochrome c assay [18]. Specific activity is measured
as SOD units per milligram of protein and the values shown are the means
from three experiments T standard deviation.
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expressing an untagged version of eGFP were examined,
fluorescence was detected throughout the whole cell (Fig. 3B).
Staining of T. brucei expressing tagged TbSODB1 and
TbSODB2 gave a punctuate pattern characteristic of
proteins found in the glycosome [42,43]. Costaining experi-
ments using the c-myc (9E10) monoclonal antibody (green),
an antibody that recognises glycosomal GAPDH (red) and
DAPI (blue), showed that both of these SODs colocalise
with the glycosomal marker (Fig. 3C). Both TbSODB1 and
TbSODB2 may also be present at other sites; a faint
background fluorescence was observed throughout the main
body of the cell but absent from the nucleus. Additional
costaining using endoplasmic reticulum and mitochondrion
markers demonstrated that TbSODB1 was not targeted to
these organelles.
Investigating the functional significance of T. brucei SODs
using RNAi
To determine whether any of the enzymes were essential
for viability of BSF parasites an RNAi-based approach was
employed. DNA fragments (518–748 bp) corresponding to
the central regions of three SODs (TbSODA, TbSODB1, and
TbSODC) were generated using PCR. These were cloned
into p2T7Ti[35], the sequence of the inserted DNA was
confirmed and the RNAi constructs were used to transform
BSF T. brucei. Parasite clones were selected and integration
into the genome was checked by Southern hybridisation.
The effect of inducing RNAi was first examined by
following the cumulative cell density of tetracycline-treated
BSF T. brucei compared to that of untreated cultures (Fig. 4).
In the absence of tetracycline, all recombinant cell lines were
found to grow at approximately the same rate as the parental
cells. Addition of tetracycline to recombinant parasites
harbouring the TbSODA or TbSODC RNAi constructs did
notresultinamajoralterationinthegrowthrate(Figs.4Aand
C), even though the endogenous mRNA was shown to be
significantly reduced (Fig. 5A). In contrast, induction of
double-stranded RNA corresponding to TbSODB had a
dramatic effect (Fig. 4B). Within 48 h a significant reduction
in the growth rate of these cells could be observed coupled
with a reduction of both TbSODB1 and TbSODB2 endog-
enous mRNA (Fig. 5A). In the next 24 h, most of the cells in
the population died. This was repeated with a second clone,
which showed a similar rapid decline in growth rate. Three to
4 days after exposure to tetracycline, the cumulative cell
density of treated parasites was less than 1% that of the
noninduced cultures. The TbSODB1 and TbSODB2 genes
have high identity including four stretches between 50 and
114 bp where the sequence is completelyconserved. It can be
inferred that each of the corresponding transcripts should be
equally susceptible to RNAi-mediated knockdown [44].
Comparison of parasite extracts derived from tetracycline-
treatedcellsharbouringtheTbSODBRNAiconstruct against
untreated controls demonstrated that the level of TbSODB
protein was significantly reduced (Fig. 5B).
An elevated level of SODB1 in T. cruzi has been shown to
affect the sensitivity toward benznidazole and gentian violet
[27]. Here we examined whether down-regulation of
TbSODs affects the resistance of T. brucei toward various
trypanocidal agents. Induced and noninduced BSF cells
harbouring eitherthe TbSODA or TbSODC RNAiconstructs
were grown in the presence of nifurtimox, benznidazole,
H2O2, gentian violet, or paraquat (methyl viologen) and the
concentration that inhibited parasite growth by 50% (IC50)
was determined. With most of these agents no significant
difference was observed in the IC50 value between the
induced and noninduced recombinant lines when compared
with controls. However, cells where the TbSODA transcript
wasdown-regulatedexhibitedanincreasedsensitivitytoward
the O2S?inducer paraquat (Fig. 6). Tetracycline treatment
resulted in cells that were 5-fold more sensitive than the
controls. Even in the absence of tetracycline these recombi-
nant parasites were 2-fold more sensitive. Analysis of a
second TbSODA RNAi clone gave a similar hypersensitivity
pattern in both tetracycline treated and untreated cultures.
This suggests that in this context there is sufficient ‘‘leaky’’
expression of double-stranded RNA to mediate an RNAi
effect in the absence of tetracycline. It has previously been
reported that this inducible system is not always completely
repressed in the absence of tetracycline [30, 45]. Similar
growth inhibition experiments using cells transformed with
the TbSODB construct could not be performed due to the
rapidity of cell death when RNAi was induced.
Discussion
O2S?can cause biological damage directly, or indirectly,
by the formation of secondary products through interaction
Table 3
Inhibition profiles of T. brucei SODs
TreatmentFe-SODMn-SOD Cu/Zn-SOD TbSODA TbSODB1TbSODB2 TbSODC
Untreated
+2 mM H2O2
+20 M NaN3
+5 mM KCN
0*,#,$
85*,$
34*,$
0#
0*,#,$
14*,$
12*,$
0#
0*,#,$
9*,$
1*,$
76#
0*,$
62$
44$
0#
0*,#
98*
37*
0#
0*,#
nd
nd
nd
nd
100*
38*
0#
The activities of the purified, recombinant T. brucei SODs were determined using either the cytochrome c (#), SOD-525 (*) or nitroblue tetrazolium ($) assays
in the presence of various inhibitors (H2O2; NaN3; KCN) (Materials and methods). The data are presented as percentage inhibition when compared to an
untreated control. Reactions with commercial Fe-SOD, Mn-SOD, and Cu/Zn-SOD (all Sigma) were performed in parallel as control. All experiments were
carried out in triplicate. Nd, no activity detected.
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with reactive oxygen and nitrogen species or transition
metals. In most organisms, SODs form the major enzyme-
mediated defence against this oxidant. These enzymes can
be split into three classes. Those belonging to two of the
groups, the Fe-and Mn-SODs, are commonly found in
prokaryotes or organelles of a bacterial origin, while the
third class of enzyme, the Cu/Zn-SODs, is the major activity
present in higher eukaryotes [21,39,40,46,47]. The Fe-and
Mn-SODs have a high degree of similarity and are distinct
from the Cu/Zn-SODs. This has led to the proposal that Fe-
and Mn-SODs may have evolved from a common ancestral
protein while Cu/Zn-SODs have a different evolutionary
origin [48].
Previous studies on O2S?metabolism in trypanosoma-
tids have resulted in the partial characterisation of Fe-SODs
from several species [14–16,27–29]. To gain an insight into
an entire SOD repertoire we used conserved motifs to search
the now complete T. brucei genome sequence. Two
conserved domains found in Cu/Zn-SODs failed to detect
any homologous parasite sequences, whereas the Fe/Mn-
SOD motif identified four putative T. brucei enzymes,
including one previously analysed [17]. Closer analysis of
Fig. 3. Localisation of TbSODs in T. brucei procyclic cells. (A). Specificity of the c-myc (9E10) antiserum was examined by probing a blot containing cell
lystates from T. brucei procyclic cells containing the TbSODA, TbSODB1, and TbSODB2 localisation vectors. Extracts from cultures grown for 48 h in the
absence (–) or presence (+) of tetracycline were compared. Protein from 1 ? 107cells was loaded in each track and equal loading verified by Coomassie
staining (not shown). (B). Transformed T. brucei expressing untagged eGFP. The two blue spots correspond to the nuclear and mitochondrial genomes of a
trypanosome not expressing eGFP. (C). Panel 1: transformed, fixed T. brucei procyclic cells stained with DAPI (blue). Panel 2: location of the c-myc (9E10) or
eGFP tagged TbSOD (green). Panel 3: parasites stained with Mitotracker (MT) or anti-gGAPDH as indicated (red). Cells were examined by confocal
microscopy (Materials and methods). The pattern of colocalisation on a merged image is shown in panel 4 (yellow).
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the four parasite sequences suggested that all proteins
contain residues characteristic of the Fe isoform (Fig. 1).
Biochemical studies confirmed that three of the four T.
brucei proteins could metabolise O2S?and that this activity
could be inhibited by H2O2and NaN3, but not by cyanide, an
inhibition profile that signifies an Fe-SOD (Fig. 2B; Table 3).
We were unable to detect activity with TbSODC recombi-
nant proteins, despite using a number of different truncated
versions and various bacterial expression systems. This
enzyme contains all of the diagnostic residues of a Fe-SOD
and the lack of activity could be related to the long amino-
terminal extension (Fig. 1). This facilitates mitochondrial
targeting (Fig. 3; see below), but may also perform roles
essential for enzyme activity that are difficult to replicate in
the context of a bacterial expression system.
In most organisms, the SOD repertoire exhibits a high
degree of compartmentalisation. For example, E. coli have a
cytosolic Mn-SOD and an Fe-SOD [49,50] and a periplas-
mic Cu/Zn-SOD [51], while mammals have distinct Cu/Zn-
SOD isoforms at different subcellular locations including
the cytosol, peroxisome, nucleus, and mitochondria, as well
as a mitochondrial Mn-SOD [39,40,46,47]. The requirement
for SOD in different locations reflects the restricted ability
of O2S?to readily cross biological membranes [52]. This
emphasizes the importance of removing this oxidant at, or
near the site of formation, before it can generate other toxic
molecules, such as H2O2, that may access other cellular
compartments.
Here we demonstrated that two of the Fe-SOD isoforms
(TbSODB1 and TbSODB2) are targeted predominantly to
the glycosome with some evidence that TbSODB1 may
additionally be found in the cytosol (Fig. 3C). Glycosomes
are single membrane-bound organelles related to the
peroxisome and are the site of h-oxidation of fatty acids,
ether lipid biosynthesis, purine salvage, and vitamin C
biosynthesis ([53–55], S.R. Wilkinson and J.M. Kelly,
unpublished). Uniquely, they are also the compartment
where glycolysis occurs, a process that is cytosolic in other
eukaryotes. The importance of glycolysis to T. brucei BSF
cells has been well documented [43]. During this life cycle
stage, the parasite is solely reliant on substrate level
phosphorylation for the generation of chemical energy.
Many of the biochemical processes in glycosomes and
peroxisomes generate H2O2and O2S?[56–58]. To maintain
organelle integrity, organisms have evolved a series of
enzymatic defence strategies specifically located at these
sites; peroxisomes have catalase, glutathione peroxidases,
peroxiredoxins, and Cu/Zn-SODs [46,59,60], while glyco-
somes contain a glutathione peroxidase and two Fe-SODs
[12]. As glycosomal functions are essential to BSF T. brucei
[43], the need to remove damaging H2O2and O2S?is likely
to be crucial [56,61]. This was confirmed by RNAi-
mediated down-regulation of the TbSODB transcripts and
proteins, a process that resulted in rapid cell death (Fig. 4).
We were not able to functionally distinguish between these
SODs using RNAi because of the level of sequence identity.
Localisation studies on the remaining two SODs
(TbSODA and TbSODC) demonstrated that both are
mitochondrial (Fig. 3C). BSF T. brucei possess only a
rudimentary mitochondrion and many functions associated
with this organelle are greatly reduced or absent at this life-
cycle stage, including the Krebs and respiratory cycles [62–
64]. When TbSODA and TbSODC were down-regulated by
RNAi, there was no obvious affect on cell growth under
normal growth conditions (Fig. 4), similar to the observation
made when the T. brucei mitochondrial peroxidase path-
ways were analysed in this manner [35]. However, the
functional importance of one of the mitochondrial SODs
became apparent when cells undergoing RNAi were
Fig. 4. Growth of RNAi cell lines. The growth of induced (+ tet) and
noninduced (– tet) BSF T. brucei was monitored daily over a 6-day period
(Materials and methods). For all the transformed lines, cell counts were
performed on three independent cultures from which the mean cell density
was determined. The data are expressed as cumulative cell density ml–1.
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exposed to the O2S?generating agent paraquat. In this
instance, down-regulation of TbSODA correlated with
increased sensitivity, whereas down-regulation of TbSODC
did not (Fig. 6). This strongly suggests that paraquat
accesses the parasite mitochondrion and undergoes redox
cycling to generate O2S?. Why RNAi-mediated down-
regulation of the two mitochondrial SODs gave distinct
paraquat sensitivity profiles is open for debate. It may be
that the enzymes are found at different submitochondrial
sites or that TbSODA (or an unidentified O2S?metabolising
system) compensates for TbSODC deficiency, while the
reverse situation does not occur.
Similar experiments using nifurtimox, benznidazole, and
gentian violet were also carried out. In all cases these
trypanocidal agent failed to produce an altered sensitivity,
arguing that the main killing activity of these compounds is
not by the production of O2S?in the mitochondrion. This
may be due to inaccessibility or to lack of an appropriate
redox cycling activity at this site. In vitro studies have
shown that the mammalian flavoprotein dihydrolipoamide
dehydrogenase mediates the redox cycling of nitrofuran
drugs, including nifurtimox, leading to the generation of
O2S?[65]. This has led to the proposal that the mitochon-
drial dihydrolipoamide dehydrogenase in trypanosomes
may be responsible for nifurtimox activation within the
parasite [66]. We have now shown that, whereas down-
regulation of TbSODA results in increased sensitivity
toward paraquat, a known O2S?inducing agent, nifurtimox
sensitivity remains unaltered. This implies either that
nifurtimox does not undergo redox cycling in this organelle,
or that, if so, the O2S?level generated is nontoxic.
The reliance on the Fe-SOD isoform for O2S?metabolism
is a common feature of protozoan parasites including
Plasmodium and Entamoeba species [22–24]. Why these
Fig. 6. Down-regulation of TbSODA enhances sensitivity toward paraquat.
The BSF parental (SMB) and RNAi-TbSODA (SODA) cell lines were
grown for 5 days in the presence of tetracycline (1 Ag ml–1) (+), seeded
at 1 ? 103ml–1and then exposed to various concentrations of paraquat
(1–60 AM). After 4 days at 37-C, Alamar blue was added to each culture
and used to determine cell density (Materials and methods). Untreated
parasites (–) were analysed in parallel. The concentration of paraquat that
inhibited parasite growth by 50% (IC50) was calculated. The values shown
are the means from three experiments T standard deviation.
Fig. 5. Down-regulation of gene expression by RNAi. (A). Blots containing 15 Ag of T. brucei total RNA from PCF (P) and tetracycline-induced (+) and
noninduced (–) BSF cell lines were hybridised with radiolabeled probes as indicated. Arrows correspond to the endogenous SOD transcripts. Loading was
judged by ethidium bromide staining of rRNA. (B). Down-regulation of TbSODB was examined by analysing a Western blot containing 15 Ag cell lysate from
tetracycline-induced (+) and noninduced (–) BSF T. brucei cultures with a polyclonal antiserum against TbSODB. A cross-reactive epitope was used as
loading control. Band sizes are in kDa.
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pathogenic organisms have evolved and maintained Fe-
SODs at a variety of subcellular locations is open to
speculation. However, the absence of Fe-SOD activity in
humans, coupled with the essential role played by glyco-
somal Fe-SODs in T. brucei demonstrates that components
of the O2S?metabolising arm of this parasite may now be
regarded as potential targets for chemotherapy.
Acknowledgments
We thank Fred Opperdoes (Universite ´ Catholique de
Louvain, Brussels) for the anti-gGAPDH antibody. T. brucei
sequence data were obtained from The Wellcome Trust
Sanger Institute website at http://www.sanger.ac.uk/Projects/
T_brucei/. Sequencing of the T. brucei genome was
accomplished as part of the Trypanosoma Genome Network
with support by The Wellcome Trust. We acknowledge The
Wellcome Trust for their financial support.
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