Valdiation of Trypanosoma brucei trypanothione synthetase as drug target

Free Radical Biology & Medicine, Vol. 36, No. 10, pp. 1289–1302, 2004Copyright D 2004 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2004.02.008

Original Contribution

VALIDATION OF Trypanosoma brucei TRYPANOTHIONE SYNTHETASE

AS DRUG TARGET

MARCELO A. COMINI,* SERGIO A. GUERRERO,

y SIMON HAILE,z ULRICH MENGE,

§

HEINRICH LUNSDORF,§and LEOPOLD FLOHE*

*Department of Biochemistry, Technical University of Braunschweig, Braunschweig, Germany; yFacultad de Bioquımica yCiencias Biologicas, Universidad Nacional del Litoral, Santa Fe, Argentina; zZentrum fur Molekulare Biologie der UniversitatHeidelberg (ZMBH), Heidelberg, Germany; and §National Research Centre for Biotechnology (GBF), Braunschweig, Germany

(Received 3 December 2003; Revised 29 January 2004; Accepted 2 February 2004)

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Abstract—In trypanosomes, the parasite-specific thiol trypanothione [T(SH)2] fulfills various functions, the best

established being detoxification of H2O2 and organic hydroperoxides and ribonucleotide reduction. Recently, a

trypanothione synthetase (Tb-TryS) gene from Trypanosoma brucei was isolated and the heterologously expressed Tb-

TryS catalyzed the entire synthesis of T(SH)2 from glutathione (GSH) and spermidine in vitro. To confirm the in situ

function of the complex Tb-TryS activities and to evaluate the importance of T(SH)2 metabolism in T. brucei, TryS

suppression by double-stranded RNA interference was performed. Knockdown of TryS led to depletion of both T(SH)2and glutathionylspermidine (Gsp) and accumulation of GSH, while concomitantly impairment of viability and arrest of

proliferation were observed. TryS-downregulated cells displayed a significantly increased sensitivity to H2O2 and tert.-

butyl hydroperoxide. These data verify the hypothesis that in T. brucei, a single enzyme synthesizes the spermidine-

conjugated thiols (Gsp and T(SH)2) and further confirms the significance of trypanothione in the defense against

oxidative stress and the maintenance of viability and proliferation in unstressed parasites. D 2004 Elsevier Inc. All

rights reserved.

Keywords—Trypanothione biosynthesis, Drug target, RNA interference, Oxidative stress, Hydroperoxide detoxifica-

tion, Free radicals

INTRODUCTION

The genus Trypanosoma comprises several important

vector-transmitted pathogens. In Central Africa, T. brucei

brucei and T. congolense are the causative agents of

Nagana disease of cattle, which markedly contributes to

the poor nutritional status of the population in endemic

areas. T. brucei rhodesiense and T. brucei gambiense

cause the two forms of African sleeping sickness, which,

untreated, are lethal in humans. They had been almost

dress correspondence to: Prof. Dr. Med. Leopold Flohe,

A GmbH, Universitatsplatz 2, D-39106 Magdeburg, Germany.

9-331-7480950; E-mail: [email protected].

1289

eradicated in the sixties, but have more recently reached

an all-time maximum of an estimated 500,000 cases

associated with more than 100,000 fatalities per year

(http://www.who.int/emc/diseases/tryp/trypanodis.html).

Any attempts to manage spread of the disease by

vaccination failed, because the African trypanosomes

escape the immune response by their ability to change

the surface antigens in short intervals [1]. Treatments

currently available were developed mostly in the first

half of the past century, and they suffer from poor

efficacy, development of resistance, and substantial tox-

icity. Accordingly, recent search for novel trypanocidal

drugs focuses on inhibition of metabolic pathways that

are of vital importance to the pathogens but are absent in

their hosts.

http:\\www.who.int\emc\diseases\tryp\trypanodis.html

M. A. COMINI et al.1290

Such a unique metabolic pathway, which T. brucei

shares with other trypanosomatids [2] and possibly some

other protozoa [3], is the biosynthesis and use of trypa-

nothione [N1,N8-bis(glutathionyl)spermidine, T(SH)2]. In

trypanosomatids, T(SH)2 in many aspects substitutes for

the redox mediator glutathione (GSH) that is abundant in

mammalian hosts [4]: T(SH)2 mediates the NADPH-

dependent reduction of hydroperoxides by means of a

most complex cascade of oxidoreductases [5], thus

mimicking hydroperoxide detoxification by the glutathi-

one system of mammals [6]; it provides the reduction

equivalents to ribonucleotide reductase via a thioredoxin-

related protein, tryparedoxin (TXN) [7], a pathway

commonly depending on the thioredoxin or glutaredoxin

system [8]; T(SH)2 has further been implicated in the

detoxification of xenobiotics in analogy to the mercap-

turic acid pathway [4] and replaces GSH in the trypano-

somal glyoxalase system (Krauth-Siegel, personal

communication). The biosynthesis of T(SH)2, however,

appears to differ between trypanosomatid genera. For the

insect pathogen Crithidia fasciculata, two distinct

enzymes were reported to catalyze the stepwise ligation

of the two GSH molecules to spermidine [9–11], where-

as from T. cruzi [12] and T. brucei [13,14], trypanothione

synthetases (TryS) that could catalyze both steps of

Fig. 1. Formation and functions of trypanothione. The scheme is deducpathways such as trypanothione [T(SH)2] regeneration by trypanothionhydroperoxides and ribonucleotides, observed cosubstrate functions iglutathione (GSH) or ascorbate, and some redox or nucleophilic reactidocumented by experimental data for T(SH)2. T(SH)2biosynthesis, depeenzyme, trypanothione synthetase (TryS), or may involve two enzyproblems specifically addressed in this article are T(SH)2 biosynthesis

T(SH)2 biosynthesis were cloned and heterologously

expressed (Fig. 1).

The relevance of the trypanothione system to viability

and virulence of trypanosomatids has been amply cor-

roborated by genetic techniques: (1) a conditioned

knockout of the T(SH)2-regenerating trypanothione re-

ductase (TR) in T. brucei caused increased hydroperox-

ide sensitivity, arrest of proliferation and loss of virulence

[17]; (2) a dominant negative approach leading to re-

duced TR activity in Leishmania donovani impaired

parasite survival in macrophages [18]; (3) suppression

of TXN biosynthesis in T. brucei by RNAi proved to

inhibit growth even without any H2O2 challenge [19]; (4)

knockdown of the TXN-dependent tryparedoxin peroxi-

dase (TXNPx) provoked cell death and an increased

susceptibility to exogenous peroxide [19]. Expectedly,

g-glutamylcysteine synthetase, the key enzyme for GSH

synthesis, also proved to be essential [20]. In view of this

overwhelming evidence, proof of a pivotal role for TryS

may seem superfluous. Validation of TryS as a drug

target was nevertheless considered mandatory for several

reasons: (1) because the genome analysis of T. brucei has

not yet been completed, alternative routes of T(SH)2biosynthesis, e.g., analogous to that of Crithidia, might

still be envisaged; (2) the hypothetical presence of a

ed from recent reviews [4,15,16] and comprises well-establishede reductase (TR) and tryparedoxin (TXN)-mediated reduction ofn enzymatic or nonenzymatic reactions such as regeneration ofons inferred from GSH biochemistry that are not yet adequatelynding on the trypanosomatid genera, can be achieved by a singlemes, glutathionylspermidine synthetase (GspS) and TryS. Thein T. brucei and its relevance to hydroperoxide metabolism.

dsRNA interference of trypanothione synthetase from Trypanosoma brucei 1291

glutathionylspermidine synthetase (GspS), in the absence

of TryS, might lead to an accumulation of glutathionyl-

spermidine (Gsp), a compound whose metabolic poten-

tial has not yet been evaluated in any depth; (3) GSH

accumulating due to deficient TryS might take over vital

T(SH)2 functions, e.g., detoxifying hydroperoxides by

any of the recently discovered glutathione peroxidase-

related proteins [16]; (4) thioredoxin, which is present in

T. brucei [21], might replace the T(SH)2/TXN couple,

e.g., in ribonucleotide reduction. We therefore knocked

down TryS expression in T. brucei by dsRNA and

evaluated the consequences thereof by the following

readouts: phenotypic changes; levels of GSH, Gsp, and

T(SH)2; hydroperoxide sensitivity.

EXPERIMENTAL PROCEDURES

Tb-TryS RNAi plasmid construction

The vectors p2T7.ISG75a (a gift from J. E. Donelson,

University of Iowa, Ames, USA) [22] and p2T7TA-177

(a gift from C. Clayton, ZMBH, Heidelberg, Germany)

[23] were used to construct the Tb-TryS RNAi plasmids.

In both vectors, dsRNAi generation is led by two head-

to-head T7 promoters, each one regulated by a tetracy-

cline (tet) operator (Fig. 2). The plasmid pMCL 347.1

[13] containing the Tb-TryS gene (Accession No.

AY155570) was used as DNA template for PCRs.

Two different regions of the 5V coding sequence from

Tb-TryS were targeted (Fig. 2). To generate the plasmid

p2T7-1, a fragment between 719 and 998 bp of the Tb-

TryS coding sequence was amplified from template

DNA by PCR with the following oligonucleotides:

Fo719H 5V-CCCCCCAAGCTTGAATTCTACAAAA-CATTTGGTAAGGAG-3V (adds a HindIII site) and

(Re998B) 5 V-CCCCCCGGATCCGAATTCTT-

GAGTGTTTTCATCAAAAACAAA-3V (adds a BamHI

site). The plasmid p2T7.ISG75a and the PCR product

were digested with HindIII and BamHI, and ligated with

T4 DNA ligase. For construction of p2T7TA-177-28, a

1.1 kb fragment (nucleotides 795–1884) of the coding

region of Tb-TryS was obtained by PCR from

pMCL347.1 using the following oligonucleotides:

(Fo3) 5V-GTTGTACCTTAACTGCGTCCGTTACGG-TAC-3V and (ReB) 5V-CGCGGGATCCCTACATTT-

GAATACGTACGG-3V (adds a BamHI site). Digestion

of this product with XhoI and BamHI gave a 650-bp

fragment that was inserted into the corresponding sites

of p2T7TA-177. Escherichia coli DH5-a was used as

bacterial host for cloning and plasmid purification. All

insertions were confirmed by restriction analysis and

DNA sequencing. Plasmid DNA used for transfection

was prepared with the HiSpeed Plasmid Midi Kit or

Qiagen Plasmid Giga Kit (Qiagen, Hilden, Germany),

and was linearized with NotI.

Trypanosome growth and transfection

The bloodstream forms (BSFs) of T. brucei strain

427, cell lines 90-13 (a gift from M. Boshart, LMU,

Munich, Germany) [24] and 1313-514 (a gift from C.

Clayton, ZMBH, Heidelberg, Germany) [[25]; Storm et

al., unpublished] were aerobically cultivated at 37jCunder 5% CO2 in HMI-9 medium [26] supplemented

with 10% fetal calf serum containing 2 Ag ml�1 G418

and 5 Ag ml�1 hygromycin or 2.5 Ag ml�1 phleomy-

cin, respectively. These cell lines harbor integrated

genes for T7 RNA polymerase and tet repressor protein

[[24,25]; Storm et al., unpublished]. Transfections were

carried out according to Wirtz et al. [27], but with

3�107 cells and 100 Ag of linearized plasmids. Imme-

diately after electroporation, cells were resuspended in

12 ml HMI-9 medium with 10% FCS, and 0.5 ml of

the cell suspension was transferred per well into a 24-

well culture plate (3�104 to 1�105 survivors per well).

Cell cultures were allowed to recover for 18 h in a

humidified incubator at 37jC and 5% CO2 in absence

of selection agents. For selection, 2 Ag ml�1 G418, 2.5

Ag ml�1 phleomycin, and 5 Ag ml�1 hygromycin were

added to the growth medium (1 ml per well). Stable

lines were obtained 1–2 weeks later. Cell cultures were

diluted before growing beyond 1�106 cells ml�1. Cell

densities were determined using an Improved Neubauer

chamber. Cells transfected with the empty vectors were

used as controls mimicking wild-type cell lines. Stable

cell lines were cryopreserved in HMI-9 medium with

10% glycerol.

Phenotype analysis of stable cell lines

Synthesis of dsRNA was induced by addition of 1 Agml�1 tet. The RNAi cell lines were seeded at 1�105 cellsml�1 and incubated at 37jC and 5% CO2 in the presence

of tet. Every 20–24 h, cell growth was monitored

microscopically and the culture diluted back to 1�105cells ml�1. As controls, uninduced cultures were grown

in parallel. The growth curves were generated by plotting

the product of cell density and total dilution (data

expressed as means F SD) or as relative cell growth

with control cultures set to 100%.

Gene expression analysis

Total RNA was isolated from 1�107 cells with the

RNeasy Mini Kit ((Qiagen) Hilden, Germany) and spec-

trophotometrically quantified. Treatment with 20 U RN-

ase-free DNase I (Boehringer-Mannheim, Mannheim,

Germany) per microgram of RNA for 1 h at 37jC was

performed, followed by inactivation of the enzyme at

65jC for 10 min in presence of 2.5 mM EDTA. A one-

step RT-PCR kit (Qiagen) was used to examine the

relative level of transcription of the Tb-TryS gene with

Fig. 2. Schematic representation of inducible constructs used for Tb-TyS RNAi. (A) Fragment of vector pMCL347.1 containing the Tb-TryS coding sequence (gray box). Positions(nucleotide number in parentheses) of primers and restriction sites used for further subcloning strategies are shown. The asterisk marks the stop codon. Physical positions of primers Fo1 andRe4 employed for RT-PCR (see Experimental Procedures) are also demonstrated. (B) Construct p2T7-1 obtained by cloning a 280-bp PCR product of the Tb-TryS gene into vectorp2T7.ISG75a. The TryS fragment is flanked by two head-to-head T7 promoters. (C) Construct p2T7TA-177.28 derived from a 1.1-kb fragment of Tb-TryS that was digested with XhoI/BamHI and inserted as a 650 bp product between the two T7 promoters of the p2T7TA-177 vector.

M.A.COMINI

eta

l.1292


h-actin as internal control. The following oligonucleo-

tides were used for Tb-TryS: (Fo1) 5V-ATGAC-

GAAGTCGGCACTTGCAGACACTAAA-3V and (Re4)

5V-TTTGTCAAGTCCAGCCAGTCAGTCTTTCGA-3V;primers for a fragment of copy A of the h-actin cDNA

were as described by Stojdl and Clarke [28]. RT-PCR

components and conditions were optimized so that none

of the RNAs analyzed reached a plateau at the end of the

PCR and the two sets of primers used in each reaction did

not compete with each other [29]. The reaction (50 Al)consisted of 40 ng of total RNA, 0.25 and 0.5 AMprimers for Tb-TryS and h-actin, respectively, 1� Q-

Solution and RT-PCR buffer, 400 AM dNTPs, 2 Al RT-PCR Enzyme Mix, and RNase-free water. The standard

thermal cycler conditions comprised a step of 30 min at

50jC (reverse transcription), followed by 15 min at 95jC(simultaneous inactivation of reverse transcriptases and

activation of HotStarTaq DNA polymerase); 30 PCR

cycles at 95jC for 1 min, 50jC for 1 min, and 72jCfor 1 min; and a final extension at 72jC for 10 min. To

rule out genomic DNA contamination, a PCR containing

total RNA as template and both sets of primers was

performed (Taq PCR Master Mix Kit, Qiagen). The PCR

products (20 Al per lane) were separated on a 1.2%

agarose gel and stained for 1 h with SYBR-Gold nucleic

acid stain (Molecular Probes, Leiden, The Netherlands).

Gel images were acquired with an EASY 429K CCD

camera and the band intensities were quantified by

densitometric scanning with E.A.S.Y. Win32 (both, from

Herolab GmbH, Wiesloch, Germany).

Determination of thiols

Thiols were extracted according to Huynh et al. [20],

with the only modification that the cells were subjected

to three cycles of freezing and thawing in liquid N2 after

resuspension in buffer and monobromobimane solution.

Separation and analytical conditions were as described

previously [10]. HPLC analysis was performed with a

Jasco HPLC system. Derivatized standards [GSH, Gsp

and T(SH)2] were used for calibration.

Peroxide sensitivity assays

Trypanosome cell lines grown for 48 h in the

presence and absence of tet were harvested by centri-

fugation at 2000 rpm for 10 min at 4jC. Conditionedmedium was discarded and cell density was adjusted at

5�105 cells ml�1 by addition of fresh prewarmed

culture medium free of tet. According to the scale of

the experiment, 10 or 250 ml of this cell suspension

was transferred to a 25 or 300 cm2 culture flask and

tested for sensitivity to a continuous H2O2 challenge by

adding glucose oxidase from Aspergillus niger (GOD,

Roche Diagnostics GmbH, Mannheim, Germany) at

three different end concentrations: 0.9, 0.3, and 0.1

mU ml�1. Sensitivity against organic hydroperoxides

was analyzed by addition of tert.-butyl hydroperoxide

(t-bOOH) at end concentrations of 1, 10, and 100 AM.

The cultures were monitored for cell growth or death by

light microscopy (Olympus BX60 and Nikon TMS). As

criteria to distinguish between live and dead cells, both

morphology and motility were considered. Those cells

showing normal trypanosome morphology despite di-

minished motility were considered still alive, whereas

immobile cells with altered morphology were rated as

dead (see 120 min in Fig. 6).

Light microscopy

Morphological changes of the trypanosomes, pro-

voked by Tb-TryS downregulation with or without

H2O2 challenge were documented by phase contrast

microscopy. Routinely, 1–5 � 107 cells cultured under

different conditions were harvested by centrifugation at

1500 rpm for 10 min at room temperature, washed

once with 50 ml TDB (KCl 5 mM, NaCl 80 mM,

MgSO4 1 mM, Na2HPO4 20 mM, NaH2PO4 2 mM,

and glucose 20 mM) pH 7.7, resuspended, and fixed

with 0.5–1 ml 100 mM cacodylate-buffered 2% glu-

taraldehyde (pH 7.4). Phase contrast microscopy was

performed using an Axioplan microscope (Zeiss, Ober-

kochen, Germany). Digital photographs were taken

using a digital video camera (INTAS focus imager,

INTAS, Gottingen, Germany).

Electron microscopy

Prefixed trypanosomes, as described for light mi-

croscopy, were posttreated and embedded in epoxy

resin as described by Vannier-Santos and Lins [30].

Ultrathin sections were analyzed with a CEM 902

energy-filter transmission electron microscope (Zeiss)

and images were recorded with a 1024 � 1024 CCD

camera (Proscan, Scheuring, Germany). Prefixed trypa-

nosomes were adsorbed to poly-L-lysine-coated glass

cover slides and were prepared for scanning electron

microscopy according to the general protocol. Gold-

sputter coated samples were analyzed with a DSM 982

Gemini scanning electron microscope (Zeiss) in a

magnification range from �1000 to �10,000 at 5 kV

acceleration voltage and 7 mm width.

Hydroperoxide determinations

The rate of production of H2O2 by GOD was

determined by using a continuous spectrophotometric

assay at 340 nm, in which H2O2 reduction by excess

glutathione peroxidase (GPx) is coupled to oxidation of

NADPH by glutathione reductase [31]. Assays were

carried out at 25jC in 100 mM Hepes and 0.1 mM


EDTA (pH 7.2) in a final volume of 800 Al. They

contained 0.19 mM NADPH, 10 mM GSH, 25 mM h-D-glucose, 6.25 U ml�1 GR, and 6.25 U ml�1 bovine

GPx. The concentration of t-bOOH was assessed

accordingly.

RESULTS

Selection of controlled TryS RNAi constructs

Phenotypic changes of T. brucei on impaired synthe-

sis of TryS were to be investigated in experimental

settings requiring hours to weeks. It was therefore

deemed advisable to construct cell lines that allowed a

tightly controlled knockdown of TryS gene expression.

To this end, BSFs of T. brucei stably transfected to

produce the tet repressor (BSF 90-13 and BSF 1313-

514) [[24,25]; Storm et al., unpublished] were trans-

fected with plasmids designed for genomic integration

that expressed TryS dsRNA under the control of a tet

operator (p2T7-1 and p2T7TA-177-28; see Fig. 2). In

principle, such constructs should allow the evaluation of

a TryS knockdown by monitoring phenotype changes

on exposure to tet. A particular advantage of the system

is seen in the possibility of using identical noninduced

clones for control cultures.

In a first set of experiments BSF 90-13 cells were

transfected with the plasmid p2T7-1 and selected for

stable transfection as described under Experimental Pro-

cedures. The impact of depressed TryS synthesis on tet

exposure was then evaluated by monitoring the decrease

in proliferation rate as a putative consequence. As is

demonstrated in Fig. 3A, the transfectants did not con-

sistently meet the promises of the system. As expected,

growth of the empty-vector control did not markedly

differ from that of wild-type T. brucei. Also, growth of

transfectants was generally impaired by tet in compliance

with the prediction, but the results varied substantially

between clones. While 2B1 had died completely within

24 h, others survived more than 3 days or even recovered

after 5 days. More importantly, clone 2B1 also died in the

absence of tet after 3 days (arrow in Fig. 3A). Similar

inconsistencies with analogous systems had previously

been observed and interpreted as leaky expression result-

ing from an imbalance between the transfected tet-re-

sponsive genes and the preexisting capacity to produce

the tet repressor [23,32]. In view of the scattered results,

the BSF 90-13/p2T7-1-based clones were not considered

suitable for any in-depth analysis of a TryS knockdown,

although, taken together, they already pointed to a vital

role for TryS.

A second approach (Fig. 3B) yielded data that are

more consistent. It made use of the strain BSF 1313-

514, which was designed for more reliable tet repressor

expression by introduction of a double tet repressor

gene [[25]; Storm et al., unpublished; C. Clayton,

personal communication] and a p2T7TA-177-based

vector (p2T7TA-177-28), which targets a different site

for genomic integration [23]. All p2T7TA-177-28-trans-

fected BSF 1313-514 cells, if not exposed to tet, grew

as fast as empty-vector-transfected ones, but stopped

growing 72–120 h after induction. Three of these

clones that behaved most similarly (2A2a, 2C1a,

4C1a) were selected for further analysis. As is evident

from Fig. 4A, which shows the absolute cell densities

reached at given times, the cultures had not even died

completely after 120 h despite persistent induction by

tet and resumed normal growth at about 200 h. This

escape from the consequences of p2T7TA-177-28

transfection is evidently caused by a reversion to

wild-type TryS expression, as demonstrated by RT-

PCR (Fig. 4B). TryS mRNA was markedly reduced

24 h after induction and further decreased to marginal

levels over the following 48 h but had returned to

almost pre-induction levels 10 days after induction,

whereas in the uninduced cultures, the levels of TryS

transcript remained constant (Fig. 4B).

The ‘‘second-generation’’ transfectants thus clearly

reveal that depressed TryS expression results in sub-

stantial impairment of viability and spontaneous prolif-

eration, which had been chosen as a putative selection

criterion for a consistent and regulated response to

specific dsRNA synthesis. The results, however, also

show that even with the improved vector/host combi-

nations, a persistent gene knockdown is hard to

achieve in trypanosomatids [17]. The time window of

depressed TryS expression, though, could be rated

satisfactory for the evaluation of its metabolic and

functional consequences.

Metabolic consequences of TryS knockdown

Without induction, the transfectants contained GSH,

Gsp, and T(SH)2 in concentrations similar to those

reported for wild-type T. brucei trypomastigotes by

Ariyanayagam et al. [33]. Induction of TryS expression

by tet led to a substantial loss of both Gsp and T(SH)2(Table 1). The effect of TryS knockdown on Gsp and

T(SH)2 levels was already fully established 24 h after

induction and did not change significantly over the next

2 days. GSH content was slightly depressed during the

first 2 days after induction and had increased signifi-

cantly after 3 days, which may be interpreted as resulting

simply from accumulation due to impaired consumption

by TryS or from a compensatory response to impaired

T(SH)2 synthesis. These findings prove that TryS in T.

brucei is responsible for both steps of T(SH)2 synthesis.

Also, lack of Gsp accumulation in the induced cultures

further sheds doubt on the hypothetical existence of a

distinct GspS in T. brucei.

Fig. 3. Cell growth analysis of TryS RNAi knockdown in individual T. brucei cell lines. Proliferation of tet-induced (filled symbols) andnoninduced cell lines (j) was monitored daily for 6 days. Cell densities were averaged from duplicate cell counts. Data are expressed aspercentages of growth of the pertinent noninduced clone set at 100% (j). (A) Proliferation of five individual clones (2B1 ., 2B4 z,2C3 n, 2C1 x, 2B2 E) of BSF 90-13 cells transfected with the p2T7.ISG75a-derived Tb-TryS RNAi vector p2T7-1. The growth curveof cells transfected with the empty vector p2T7.ISG75a (w) is shown as an additional control. The arrow marks the time point when alsothe noninduced clone 2B1 died. (B) Proliferation of five representative BSF 1313-514 clones (4A2a ., 4A4az, 2C1an, 4C1a x, 2A2aE) transfected with the p2T7TA-177-derived TryS RNAi knockdown vector p2T7TA-177-28. A clone transfected with the empty vectorp2T7TA-177 (w) is included as an additional control.


Hydroperoxide sensitivity

TryS RNAi transfectants were exposed to a steady

flux of H2O2 by incubation with glucose and GOD to

mimic the oxidant attack of phagocytes (Figs. 5A–5C).

Cells were pre-induced for 48 h with tet to guarantee a

substantial and sustained decrease in T(SH)2 (Table 1)

without any significant impairment of viability and

proliferation (Fig. 4A). They compared with uninduced

ones as controls. Only at the highest GOD concentration

was cell count markedly affected after 4 h in the

uninduced control cultures. In contrast, the cell count

had significantly decreased at the latest after 60 min in all

induced samples.

The sensitivity of the transfectants to organic hydro-

peroxides was investigated with a bolus (1–100 AM) of t-

Fig. 4. Cell growth and Tb-TryS gene expression analysis of selectedRNAi cell lines. Cell growth of induced and noninduced T. brucei celllines was performed in 25 cm2 cell culture flasks and monitored dailyover a period of 9 days. Cell densities were averaged from cultures ofeach clone counted twice. (A) The growth pattern of three stable Tb-TryS RNAi cell lines (2A2a, 2C1a, 4C1a) harboring the constructp2T7TA-177-28 is presented in absolute cell counts (z). Pertinentnoninduced cultures (j) and empty vector (p2T7TA-177 transfectedcells) in the presence (.) and absence (o) of tet served as controls.*Significant difference, tet-induced transfectans versus uninducedcultures p < .01 (n=3 for three different clones, two tailed Student’st test). (B) Representative gene expression analysis of induced Tb-TrySRNAi (Tet +) and noninduced (Tet�) cell line 2A2a. Primer pairs Fo1/Re4 (Fig. 1A) and Foh/Reh were used for one-step RT-PCR amplifi-cation of 700- and 300-bp fragments from the TryS and h-actintranscripts, respectively.

Table 1. Thiol Content in Tb-TryS RNAi Cell Linesa

Tetracycline Thiol (nmol/108cells)

induction

GSH Gsp T(SH)2

Baseline � 1.69 F 0.20b 0.22 F 0.03b 0.30 F 0.07b

� 1.5 F 0.10c 0.21 F 0.06c 0.42 F 0.20c

24 h � 1.46 F 0.66 0.24 F 0.08 0.35 F 0.05+ 1.07 F 0.33d 0.07 F 0.04e 0.05 F 0.01e

48 h � 1.77 F 0.29 0.23 F 0.07 0.33 F 0.03+ 1.06 F 0.48d 0.06 F 0.02e 0.02 F 0.01e

72 h � 1.84 F 0.10 0.18 F 0.07 0.22 F 0.05+ 2.46 F 0.28e 0.09 F 0.07e 0.03 F 0.02e

a Determinations were done with tet-induced and uninduced cultures

of cell lines 2A2a and 2C1a.b The values represent means F SD for the uninduced T. brucei cell

lines (n = 6) before H2O2 challenge.c Values for T. brucei bloodstream trypomastigote reported by

Ariyanayagam et al. [33].d,e Values with a significance level of dp < .05 or p < .01, two-

tailed Student’s t test between induced (n = 2) and pooled uninduced

controls (n = 6).


bOOH as model compound. t-bOOH at the concentrations

chosen proved to be toxic to the T. brucei cultures

irrespective of the TryS knockdown (Figs. 5D–5F). Nu-

merically the induced cells were more affected through-

out. However, due to progressing damage in controls, a

significant difference between induced and uninduced

cells was usually no longer detectable at later time points.

The morphological phenomena that were associated

with H2O2-mediated cell damage were investigated by

light and electron microscopy. Some characteristic fea-

tures of the H2O2-exposed trypanosomes are compiled in

Figs. 6 and 7. After 30 min, most cells are still mobile but

a considerable proportion appear stumpy, with a flagel-

lum that looks retracted and is sometimes detached from

the undulating membrane (30 min in Fig. 6). After 60

min, the whole cell body appears less homogeneous by

phase-contrast microscopy, and scanning electron mi-

croscopy reveals characteristic wrinkles on the entire

surface and stalked blebs usually pulling out near the

flagellar pocket (60 min in Fig. 6). At later time points,

rounded cell bodies with a thin, stretched-out flagellum

predominate. At this stage mobility, if any, is restricted to

rotation due to sporadic beats of the flagellum. Scanning

microscopy shows the cell surface is covered with deep

clefts and holes (120 min in Fig. 6).

Transmission electron microscopy provided comple-

mentary information on the cellular ultrastructure, which

confirmed and, in part, explained the phenomena ob-

served by phase-contrast and scanning electron micros-

copy. The cells subjected to H2O2 exposure for 30 min

(Fig. 7B) are hard to distinguish from unchallenged cells

(Fig. 7A). However, in challenged parasites the endo-

plasmatic reticulum more often appears inflated, the

mitochondrion could be described as slightly swollen,

the membrane of the flagellum is less tightly packed

around the microtubules and the paraflagellar rod (Fig.

7B, inset 2), and the kinetoplast material appears less

dense (Fig. 7B, inset 1). After a 60-min challenge, the

loss of volume control of the entire cell and its organelles

becomes more obvious (Fig. 7C). While the corset of

subpellicular microtubules still keeps the cell in a rea-

sonable shape (Fig. 7C, inset 2), the cell body is rounded

up, the endoplasmatic reticulum is widened throughout,

and the flagellar membrane looks damaged and loosely

surrounds the axonema (Fig. 7C, inset 4). Large vesicles

with a double membrane, sometimes including rudimen-

tary kinetoplast material, clearly reveal an advanced state

of mitochondrial swelling (Fig. 7C, inset 3), and, most

**

Fig. 5. Hydroperoxide sensitivity of Tb-TryS RNAi cell lines. Peroxide challenge assays were performed in T. brucei cultures preinducedover 48 h (black bars) and noninduced controls (empty bars). Cell growth/death was monitored by light microscopy over a period of 4 h.Mean cell density was determined from duplicate cell counts. The data are presented as means and SD of the averaged cell densities ofeach of the clones tested (2A2a, 2C1a, 4A1a). The asterisks indicate significant differences versus uninduced controls: p < .05, **p <.01 (n= 3 for three different clones; two tailed Student’s t test). The black rhombi represent significant differences of xp < .05, xxp <.01 (n= 3 for three different clones, two tailed Student’s t test) of hydroperoxide-challenged controls against t0 (A–C) H2O2 sensitivity:5� 105 cells ml� 1 were exposed to three different GOD concentrations [0.9 (A), 0.3 (B), and 0.1 (C) mU ml� 1 yielding initial H2O2

fluxes of 170, 60, and 20 pmol min� 1 ml� 1, respectively, under the experimental conditions]. (D–F) Organic peroxide sensitivity:5� 105 cells ml� 1 were exposed to three tert.-butyl hydroperoxide concentrations: 100 (D), 10 (E), and 1 (F) AM.


characteristically, the nuclear DNA is always seen con-

densed, leaving most of the nucleus a huge empty vesicle

(Fig. 7C, inset 1). At 120 min of H2O2 challenge, little

else than assemblies of swollen vesicles and vacuoles

surrounded by a ruptured pelliculum is left (Fig. 7D).

Interestingly, intact axonemata can still be identified by

their characteristic architecture of nine peripheral dou-

blets of microtubules plus two central single tubules (Fig.

Fig. 6. Light and scanning electron micrographs of GOD-treated Tb-TryS RNAi cells. Peroxide challenge was performed on T. bruceicell line 2A2a preinduced 48 h with tet. GOD was added at a final concentration of 0.3 mU ml� 1. Samples were taken at the timesindicated and processed for light microscopy (left) and SEM (right).


Fig. 7. Transmission electron micrographs of ultrathin sections from GOD-treated Tb-TryS RNAi cells. Peroxide challenge wasperformed on tet-induced (48 h) and noninduced T. brucei cell line 2A2a. GOD was added at a concentration of 0.3 mU ml� 1. Insetsshow further details of the respective cultures. (A) Section of a control trypanosome (uninduced, unchallenged) showing normal cellmorphology. (B–D) Sections of tet-induced trypanosomes challenged with H2O2 for 30, 60, and 120 min, respectively. For detaileddescriptions of the morphological changes, see text. A, axonema; ER, endoplasmatic reticulum; G, Golgi apparatus; K, kinetoplast; M,mitochondrion; N, nucleus; P, paraflagellar rod. Arrowheads indicate subpellicular microtubules. A detailed description of trypanosomemorphology can be obtained from [34–36].


7D, inset 1), although they are no longer surrounded by

any membrane and likely represent the thin stretched-out

‘‘flagellum’’ that characterizes dying trypanosomes in

light microscopy.

Qualitatively the morphological investigations did not

disclose any differences in the response to H2O2 in tet-

induced and uninduced cells. However, the onset of

morphological alterations was delayed in the uninduced

cells by at least 2 h at high H2O2 exposure, which

complies with the survival data shown in Fig. 5A. On

medium challenge (Fig. 5B), as used for most of the

morphological studies (Figs. 6 and 7), the uninduced

cells survived with minor morphological changes for

18 h, whereas the tet-induced cells had completely

disappeared. Interestingly, similar though minor morpho-

logical alterations were observed in unchallenged tet-


induced cells in parallel with the impaired survival (Fig.

4A) starting at Day 2 of incubation (not shown). This

observation might indicate that the T(SH)2-deficient cells

cannot even cope with the oxidative challenge resulting

from their own metabolism.

DISCUSSION

A novel host/vector combination allowed well-con-

trolled suppression of TryS expression in T. brucei by

dsRNA. Evidently, the expression of tet repressor and the

tet-regulated RNAi gene was better balanced than in

previously used systems [22,24,32], with the favorable

outcome that the synthesis of T(SH)2 was inhibited only

when the cultures were exposed to tet, which by itself

does not affect growth or viability of wild-type T. brucei.

Accordingly, the clones stably transfected to form TryS

dsRNA fragments showed unchanged TryS mRNA lev-

els and behaved like wild-type T. brucei unless exposed

to tet. By means of this system essentially three messages

were obtained: (1) In T. brucei TryS is essential for the

entire synthesis of T(SH)2. (2) A decline in T(SH)2 levels

to about 15% of normal leads to proliferation arrest and,

if sustained for days, impairs viability. (3) Sensitivity to

both H2O2 and organic hydroperoxides is significantly

enhanced at depressed T(SH)2 levels.

The last finding was not particularly surprising, as

conditioned knockouts of trypanothione reductase simi-

larly increased hydroperoxide sensitivity [17]. Taken

together, the results underscore the importance of the

trypanothione-mediated hydroperoxide detoxification

system in T. brucei which comprises regeneration of

T(SH)2 by TR at the expense of NADPH, reduction of

TXN by T(SH)2, reduction of the peroxiredoxin-type

TXN peroxidase (TXNPx) by reduced TXN, and ulti-

mately hydroperoxide reduction by TXNPx, as was first

demonstrated for C. fasciculata [5] and has meanwhile

been confirmed to operate in most trypanosomatids

[2,4,15,37–39]. The relevance of this system to antioxi-

dant defense is further corroborated by RNAi assays to

knock down TXN and TXNPx in T. brucei [19]. Interest-

ingly, we found that incomplete suppression of TryS, as

demonstrated by still detectable TryS mRNA and about

15% of normal T(SH)2 levels, did have such a dramatic

effect on antioxidant capacity. The data would be in line

with kinetic analyses [40–42] indicating that the reduction

of TXN by T(SH)2 might be the bottleneck of the entire

system and would be strongly affected by a decline in

T(SH)2, as is observed here. Our results further demon-

strate that GSH, even if accumulated, as in sustained TryS

knockdown (see Table 1), cannot substitute for T(SH)2 in

trypanosomal antioxidant defense. This finding and the

observation that GPx-I from T. brucei does not play a

critical role in detoxification of hydroperoxides [19] rule

out any relevant role for the glutathione peroxidase-related

proteins of T. brucei in antioxidant defense that would be

independent of T(SH)2. In T. brucei and T. cruzi, these

proteins have, in fact, been shown to display a weak

glutathione peroxidase activity in vitro but to be more

efficiently reduced by TXN [43,44] and thus also depend

on T(SH)2.

The morphological phenomena resulting from TryS

knockdown, in particular membrane damage associated

with loss of permeability control, would also comply

with the assumption that impaired hydroperoxide metab-

olism is the most prominent result of lowered T(SH)2levels. In this context it may be recalled that glutathione

peroxidase, which is the mammalian equivalent of the

trypanosomal trypanothione peroxidase system, had once

been rediscovered as ‘‘contraction factor II,’’ which

means a factor preventing ’’high-amplitude swelling’’

of mitochondria [45], now commonly referred to as

‘‘permeability pore transition’’ [46]. The contraction

factor activity could later be attributed to the ability of

glutathione peroxidase to prevent peroxidation of unsat-

urated lipids in mitochondrial membranes [47,48]. Be-

cause protection against lipid peroxidation by broad

spectrum peroxidases such as glutathione and trypare-

doxin peroxidases has become a widely accepted con-

cept, one might argue that even the obvious membrane

damage that is seen in later phases of TryS knockdown

cultures without any artificial peroxide challenge is due

to compromised defense against endogenously produced

hydroperoxides, a fact that has been shown to occur in T.

cruzi at least [49–51]. Such endogenous production of

H2O2 would occur predominantly in mitochondria and

would have to be balanced by the mitochondrial iso-

enzymes of TXNPx [37–39] that equally depend on the

trypanothione system [52]. It appears, however, hazard-

ous to overemphasize the qualitative similarities of

H2O2-induced and spontaneously developing morpho-

logical criteria of cell death. Almost identical images of

progressing cell disintegration have been reported for

trypanosomes dying from exposure to antimicrobial

peptides such as human defensins and cathelicidins

[53] or dibutyltin chloride and analogs [54]. In both

cases, the trypanocidal mechanism appears not to have

been analyzed in depth, but to implicate oxidative stress

would be mere speculation. In fact, the multiple hypo-

thetical or established functions of T(SH)2 in trypano-

somes compiled in Fig. 1 provide various explanations

for the impaired proliferation and viability of the unchal-

lenged TryS knockdown cultures. Certainly, the arrest of

proliferation is most likely due to limited ribonucleotide

reduction, which is required to ensure adequate DNA

synthesis. This assumption is corroborated by the finding

that thioredoxin is a poor substitute for tryparedoxin in

ribonucleotide reduction [21] and that silencing of the


thioredoxin gene, in contrast to TXN knockdown [19],

did not result in any obvious phenotype [55].

Irrespective of the uncertainties about the mechanisms

by which TryS knockdown affects proliferation, viability,

and, as may be inferred from the analogous TR knockout,

virulence, our data unambiguously demonstrate a pivotal

role for TryS in T. brucei. As a drug target TryS deserves

particular interest within the trypanothione system: (1) In

T. brucei, at least, TryS appears to be the only enzyme

that catalyzes glutathionylation of spermidine and Gsp

and thereby fuels the T(SH)2-mediated antioxidant de-

fense and other indispensable T(SH)2 functions. (2) An

incomplete knockdown of TryS by RNAi yielded phe-

notypic changes similar to those that required a more

than 90% knockout of TR. (3) In contrast to TXN and

TXNPx, TryS is a protein of low abundance and thus

should be more easily inhibited. (4) TryS, in contrast to

all other components of the trypanothione system, does

not have any close relatives within vertebrates; it is a

rather unique protein that, apart from some motifs

reminiscent of ATP binding sites, does not have any

significant sequence similarity to any known mammalian

protein [13]. With these characteristics, TryS qualifies

not only as a validated but as a most attractive target for

the design of trypanocidal drugs.

Acknowledgments—We especially thank M. Boshart, J. E. Donelson,and C. Clayton for kindly providing plasmids, strains, and/orprotocols. L. Krauth-Siegel and C. Clayton are also acknowledgedfor sharing unpublished results. L. Storm and C. Hartmann areacknowledged for constructing plasmids 1313 and 514, respectively.We thank H. Budde for providing technical instructions to handletrypanosomes. Sample preparation for electron microscopy by E. Barthis gratefully acknowledged. This work was supported by the BMBFgrant ARG 02/006.

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ABBREVIATIONS

BSF—blood stream form

dsRNA—double-stranded ribonucleic acid

GOD—glucose oxidase

GPx—glutathione peroxidase

GR—glutathione reductase

GSH—glutathione

Gsp—glutathionylspermidine

GspS—glutathionylspermidine synthetase

PCR—polymerase chain reaction

RNAi—ribonucleic acid interference

RT-PCR—reverse transcription polymerase chain

reaction

t-bOOH— tert.-butyl hydroperoxide

tet— tetracycline

TryS— trypanothione synthetase

T(SH)2— trypanothione

TXN—tryparedoxin

TXNPx—tryparedoxin peroxidase

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Valdiation of Trypanosoma brucei trypanothione synthetase as drug target