Expression profiles of peroxiredoxin proteins of the rodent

malaria parasite Plasmodium yoeliiq

Shin-ichiro Kawazua,*, Tomoyoshi Nozakib,e, Takafumi Tsuboic,d, Yoko Nakanoa,Kanako Komaki-Yasudaa,f, Nozomu Ikenouea, Motomi Toriic, Shigeyuki Kanoa

aResearch Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, JapanbDepartment of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

cDepartment of Molecular Parasitology, Ehime University School of Medicine, Shigenobu-cho, Ehime 791-0295, JapandCell-Free Science and Technology Research Center, Ehime University, Matsuyama, Ehime 790-8577, Japan

ePrecursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, JapanfDomestic Research Fellow, Japan Society for the Promotion of Science, Japan

Received 14 April 2003; received in revised form 25 June 2003; accepted 2 July 2003

Abstract

Patterns of expression of the 2-Cys and 1-Cys peroxiredoxin (Prx) proteins of the rodent malaria parasite Plasmodium yoelii during its life

cycle were observed by immunofluorescent antibody staining and confocal laser scanning microscopy. 2-Cys Prx was expressed in the

parasite cytoplasm throughout the life cycle, and the thioredoxin (Trx)-peroxidase activity of 2-Cys Prx revealed with the recombinant

protein suggested that the Prx is constitutively expressed and, thus, likely plays a housekeeping role in the parasite’s intracellular redox

control. In contrast, 1-Cys Prx showed stage-specific expression in blood-stage parasites. The limited expression of 1-Cys Prx in the

trophozoite cytoplasm suggests that 1-Cys Prx may be involved in haemoglobin metabolism by the parasite, which generates a prooxidative

haem iron and increases intracellular oxidative stress. The antioxidant activity of 1-Cys Prx was tested for its ability to protect yeast enolase

against inactivation of the mixed-function oxidation system. Differential expression of the two Prx proteins during the erythrocytic and insect

stages suggests the importance of these proteins in protecting parasites against oxidative stress, which is generated by the parasite’s

metabolism and also from the environment.

q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Antioxidant; Insect stage; Malaria; Peroxiredoxin; Plasmodium yoelii

1. Introduction

As Plasmodium spp. (malaria parasites) actively pro-

liferate during asexual development in the erythrocytes of

their vertebrate hosts, the parasites are subjected to the toxic

effects of reactive oxygen species (ROS) (Becker et al.,

1994; Flohe et al., 1999). The significant decrease in

glutathione levels in parasite-infected erythrocytes suggests

that the parasites require efficient antioxidant systems

(Muller et al., 2001). Because Plasmodium spp. in

erythrocytes are susceptible to oxidative stresses (Postma

et al., 1996), their antioxidant defences appear to be

potential targets for malaria chemotherapies (Krauth-Siegel

and Coombs, 1999). Malaria parasites develop sexually in

the digestive tract (midgut) of mosquitoes and then mature

intracellularly in the salivary gland. In these environments,

the parasites are also likely to be under the oxidative stress

(Han et al., 2000; Kanzok et al., 2001).

Proteins that are structurally homologous to the thiol-

specific antioxidant of yeast (Chae et al., 1994) have been

identified in all living organisms from bacteria to human and

are referred to as peroxiredoxins (Prx) (Chae et al., 1999).

A sequence alignment among family members revealed the

existence of two groups of Prx that differ in the number of

conserved cysteine residues. The 2-Cys Prx contains both

the conserved residues (Cys47 and Cys170), whereas

0020-7519/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/S0020-7519(03)00184-X

International Journal for Parasitology 33 (2003) 1455–1461

www.parasitology-online.com

q Nucleotide sequence data reported in this paper are available in the

GenBank, EMBL and DDBJ databases under the accession numbers

AB089300 and AB089301.* Corresponding author. Tel.: þ81-3-3202-7181x2878; fax: þ81-3-

3202-7364.

E-mail address: skawazu@ri.imcj.go.jp (S.-i. Kawazu).

the 1-Cys group lacks the conserved Cys170 residue (Chae

et al., 1999). Several Prx proteins act as the terminal

peroxidase that reduces hydrogen peroxide and organic

hydroperoxides with electrons donated by the thioredoxin

(Trx) system (Chae et al., 1994) or other physiological

reductants (Kang et al., 1998; Fisher et al., 1999).

We and others recently reported the expression of 1-Cys

and 2-Cys Prx proteins in blood-stage Plasmodium

falciparum (Kawazu et al., 2000, 2001; Rahlfs and Becker,

2001; Krnajski et al., 2001). These proteins, which have

thiol-dependent peroxidase activities, are expressed during

the trophozoite stage of the parasites (Kawazu et al., 2000;

Krnajski et al., 2001). The 2-Cys Prx of P. falciparum

(PfTPx-1) was suggested to be the terminal peroxidase of

the Trx system during the blood stage, whereas physio-

logical electron donors for the 1-Cys Prx (Pf1-Cys-Prx) are

not known (reviewed in Rahlfs et al., 2002).

Despite such data accumulated from blood-stage para-

sites, the function of Plasmodium Prx proteins during the

insect stage remains poorly understood. In this report, we

examined the expression patterns of 2-Cys and 1-Cys Prx

proteins of the rodent malaria parasite Plasmodium yoelii

during the blood and insect stages by immunofluorescent

antibody staining and confocal laser scanning microscopy.

The antioxidant activities of P. yoelii Prx were also tested

with recombinant proteins.

2. Materials and methods

2.1. Parasites

Plasmodium yoelii 17XL was maintained by mosquito

transmission in Anopheles stephensi interspersed by a

maximum of two serial passages in Crj:ICR mice purchased

from Charles River, Japan. Blood-stage parasites used for

nucleic acid and protein extractions were prepared from

blood of parasite-infected mice. Blood was passed through a

CF11 column (Whatman) to remove leucocytes, and the

parasite-infected erythrocytes were lysed with phosphate-

buffered saline (PBS) containing 0.05% saponin. The

parasite pellet was washed several times with PBS, snap-

chilled in liquid nitrogen, and stored at 280 8C until use.

The animal experiments in this study were carried out either

at Ehime University School of Medicine or at the

International Medical Center of Japan in compliance with

the Guide for Animal Experimentation.

2.2. Ookinete culture and sporozoite preparation

P. yoelii ookinetes were obtained by in vitro culture

of the parasite-infected blood as described previously (Tsuboi

et al., 1997). For the sporozoite preparation, mosquitoes were

fed on P. yoelii-infected mice. Fourteen days after feeding,

salivary glands of the mosquitoes were dissected and minced

in M199 medium (Gibco BRL) containing 0.2% bovine

serum albumin to release sporozoites. Parasite-infected

blood, ookinetes, and sporozoites were spotted onto Multitest

slides (Flow Laboratories) and fixed with ice-cold acetone for

indirect immunofluorescence assay.

2.3. Cloning and expression of P. yoelii peroxiredoxins

Approximately 400–500 bp of the 50 regions of Prx

cDNAs were obtained from the P. yoelii expressed sequence

tags database. These fragments (GenBank accession

numbers BM166616 and BM162294) were identified as

those sequences that had significant identities to the Pf1-

Cys-Prx and PfTPx-1 cDNAs of P. falciparum (GenBank

accession numbers AB020595 and AB037568, respec-

tively). The 30 regions of the P. yoelii cDNAs were obtained

by 30 rapid amplification of cDNA ends (RACE)–PCR.

Template cDNA was synthesised from blood-stage parasite

mRNA with Ready-To-Go You-Prime First-Strand Beads

and Not I–(dT)18 primer (Amersham Pharmacia Biotech).

The coding sequence of 1-Cys Prx was amplified by PCR

from blood-stage parasite cDNA with primers 50-CGC GGA

TCC ATG GGA TAT CAT TTA GGA-30 and 50-CGC CTC

GAG TCA TAA ATT AAC AAA TCT-30. The coding

sequence of 2-Cys Prx was amplified by PCR with the

primers 50-CGC GGA TCC ATG CCA TCA ATT GTA

GGA-30 and 50-CGC CTC GAG TTA TAA ATT CGA TAA

ATA-30 from the same cDNA. The primers, which

contained a Bam HI or Xho I site (indicated in italics)

adjacent to the initiation or the termination codon,

respectively (indicated by underline), were designed from

the EST-derived 50 end sequences and the 30 end sequences

determined in this study. PCR products were digested with

Bam HI and Xho I and ligated into expression vector pGEX-

6P-1 (Amersham Pharmacia Biotech). Recombinant plas-

mids were transformed into Escherichia coli strain BL21,

and expression of N-terminal glutathione S-transferase

fusion protein was induced with isopropyl-b-D-thiogalacto-

side at 0.3 mM. Proteins were purified with the Glutathione

Sepharose 4B Column Chromatography System (Amersham

Pharmacia Biotech). The glutathione S-transferase-tag of

the fusion protein was cleaved by PreScission protease

(Amersham Pharmacia Biotech) and then removed with the

glutathione S-transferase–glutathione affinity system.

2.4. Western blot analysis

Recombinant (rPyPrx) proteins or extracts prepared from

blood-stage parasites were solubilised in SDS–PAGE

sample buffer (Laemmli, 1970). After separation by

SDS–PAGE, the proteins were transferred electrophore-

tically to polyvinylidene difluoride sheets (Immobilon

Transfer Membranes; Millipore) and reacted with either

antisera (1:200) or antibodies (25 mg/ml) to recombinant

P. falciparum Prx (rPfPrx) proteins. Antisera against rPfPrx

proteins were produced in rabbits (Kawazu et al., 2001).

The IgG fractions of the antisera were prepared with HiTrap

S.-i. Kawazu et al. / International Journal for Parasitology 33 (2003) 1455–14611456

rProteinA affinity columns (Amersham Pharmacia Biotech)

according to the method of Miller and Stone (1978).

Immune complexes were visualised with horseradish

peroxidase-conjugated anti-rabbit IgG antibody (Cappel).

2.5. Antioxidant activity assay

Antioxidant activities of the recombinant proteins were

examined with mixed-function oxidation (MFO) system

(Kim et al., 1998) with slight modifications. Briefly,

inactivation mixtures (50 ml) containing 50 mM HEPES

(pH 7.0), 10 mM dithiothreitol and 3 mM FeCl3 were pre-

incubated with or without rPyPrx protein at 30 8C for 30

min. After pre-incubation, 0.025 U of yeast enolase

(Oriental Yeast) was added, and the inactivation mixture

was incubated on ice for another 30 min. The enolase

activity in the reaction mixture was assayed in 1.0 ml of

assay mixture containing 50 mM Tris–Cl (pH 7.5), 1 mM

MgCl2, and 1 mM 2-phosphoglyceric acid (Sigma Aldrich).

The production of phosphoenolpyruvate was monitored as

the increase in A240 at room temperature for 100 s.

Nicotinamide-adenine dinucleotide phosphate, reduced

(NADPH) oxidation coupled to the reduction of H2O2 by

rPyPrx protein in the presence of the Trx and Trx reductase

(TrxR) system was evaluated as described (Fisher et al.,

1999). NADPH oxidation was monitored as the decrease in

A340 at room temperature in a 0.1-ml reaction mixture

containing 50 mM HEPES (pH 7.0), 250mM NADPH, 10mM

E. coli Trx (Wako Pure Chemical), 3 U E. coli TrxR (Sigma

Aldrich), 250 mM H2O2, and 4.0 mM of rPyPrx protein.

2.6. Indirect immunofluorescence microscopy

Acetone-fixed parasites on Multitest slides prepared as

described above were blocked with PBS containing 5% non-

fat dry milk for 30 min at 37 8C. Slides were then incubated

simultaneously with the rabbit antisera to rPfPrx proteins

(1:100) and mouse monoclonal antibody to Pys 25 (1:200,

Tsuboi et al., 1997), which was used for identification of

gametocytes (Kimura et al., 1999), for 60 min at 37 8C and

rinsed with PBS. After incubation with fluorescein isothio-

cyanate (FITC)-conjugated anti-rabbit IgG antibody (1:50,

Cappel) and Alexa Fluor 546-conjugated anti-mouse IgG

antibody (1:50, Molecular Probes) for 30 min at 37 8C, slides

were rinsed with PBS. 40,6-Diamidino-2-phenylindole

(DAPI) staining was done after the second antibody reaction

to visualise the parasite nuclei. The slides were mounted

under cover glasses in Prolong Antifade Kit (Molecular

Probes) and observed with a laser scanning microscopes

(ECLIPSE C1, Nikon and Model LSM510, Carl Zeiss).

2.7. Quantitative real time reverse transcription

PCR (RT–PCR)

Total RNA (approximately 300 ng) isolated from the

blood-stage parasites with TRIzol Reagent (Gibco BRL)

was reverse transcribed with Ready-To-Go You-Prime

First-Strand Beads and pd(N)6 random hexamers (Amer-

sham Pharmacia Biotech). The polymerase chain reaction

was performed with approximately 100 pg of template

cDNA and the sequences-specific oligonucleotide primers

on an ABI PRISM 7700 Sequence Detection System

(Applied Biosystems). Continuous fluorescence observation

of amplifying DNA was done with SYBR Green PCR

Master Mix Kit (Applied Biosystem). After cycling, PCR

products were checked by electrophoresis on agarose gels to

confirm the specificity of each amplification. To compare

the relative amounts of PCR products, the fluorescence

intensity was recorded by cycles for each amplification and

analysed with the ABI Prism Sequence Detection System

software Version 1.7 (Applied Biosystems).

3. Results

3.1. Cloning, sequencing, and expression of P. yoelii

Prx genes

The coding sequences of P. yoelii 2-Cys Prx (GenBank

accession no. AB089301) and 1-Cys Prx (GenBank

accession no. AB089300) were 83.1 and 76.8%, respec-

tively, identical at the amino acid level to those of the

corresponding P. falciparum Prx proteins. These genes were

identified on the MALPY00116 (2-Cys Prx) and

MALPY01293 (1-Cys Prx) contigs, respectively, in the

P. yoelii genome database of the Plasmodium Genome

Consortium PlasmoDB (PlasomDB; http://PlasmoDB.org).

The database search revealed that these genes did not

contain introns. The deduced amino acid sequences of

P. yoelii Prx proteins do not contain putative mitochondrial

or nuclear localisation signals (Park et al., 2000).

Recombinant P. yoelii Prx (rPyPrx) proteins were

produced to test if rabbit anti-sera raised against recombi-

nant P. falciparum Prx (rPfPrx) proteins recognise the

homologous proteins in P. yoelii. The rPyPrx proteins had

molecular masses of 22 kDa (2-Cys Prx) and 25 kDa (1-Cys

Prx) on 12.5% SDS–PAGE gels, which are consistent with

the sizes predicted from the amino acid sequences (Fig. 1A).

On Western blots, each anti-rPfPrx serum specifically

recognised the appropriate rPyPrx (Fig. 1A). These antisera

also specifically recognised native proteins with molecular

masses of 22 kDa (2-Cys Prx) and 25 kDa (1-Cys Prx) in the

blood-stage parasites of P. yoelii on Western blots (Fig. 1B).

An additional band with a molecular mass of approximately

40 kDa was detected on Western blots of 2-Cys Prx (Fig. 1B,

lane 2) and is the dimeric form of the protein (Chae et al.,

1994; Kawazu et al., 2001).

3.2. Antioxidant activities of the rPyPrx proteins

The antioxidant activity of rPyPrx protein was evaluated

with the MFO system. This assay is based on a system that

S.-i. Kawazu et al. / International Journal for Parasitology 33 (2003) 1455–1461 1457

generates hydroxyl radicals to damage an enzyme (enolase).

In the absence of rPyPrx protein, the hydroxyl radicals

inactivate enolase (Fig. 2A). In the present study, addition of

the recombinant proteins to the system prevented an

inactivation of enolase in a concentration-dependent

manner, indicating that PyPrx proteins have antioxidant

activities (Fig. 2A). The Trx-linked peroxidase activity of

rPyPrx was tested by monitoring oxidation of NADPH in

the presence of the E. coli Trx/TrxR system (Fig. 2B). After

addition of H2O2, the assay with rPy 2-Cys Prx showed a

marked decrease in A340, which was attributed to oxidation

of NADPH. This result suggests that the rPy 2-Cys Prx is a

Trx peroxidase. In contrast, NADPH oxidation in the assay

with rPy 1-Cys Prx was negligible.

3.3. Expression of the P. yoelii Prx proteins

during the blood and insect stages

Immunofluorescence microscopy of P. yoelii blood-stage

parasites revealed that 2-Cys Prx was expressed in

the parasite cytoplasm during the early to late trophozoite,

schizont, and gametocyte stages (Fig. 3C,D). There was no

specific fluorescence in the nuclei or plasma membranes of

parasites. Simultaneous application of the mouse

monoclonal antibody to Pys25 (Tsuboi et al., 1997), a

molecule expressed in the cytoplasm of P. yoelii gameto-

cytes, with rabbit anti-Prx serum differentiated gametocytes

from trophozoites and schizonts on merged images of Alexa

Fluor 546 (red) and FITC (green) staining (Fig. 3D). In

contrast, substantial 1-Cys Prx staining was detected only in

a small number of trophozoites in the cytoplasm (Fig. 3G,

H). Expression of 1-Cys Prx in schizonts was very weak

(Fig. 3H) in comparison with that in parasites of the same

stage stained with anti-2-Cys Prx serum (Fig. 3D). 1-Cys

Prx was not detected in gametocytes (Fig. 3H).

Immunostaining of insect-stage parasites with anti-serum

against 2-Cys Prx revealed that 2-Cys Prx was expressed

throughout the cytoplasm of ookinetes and sporozoites

(Fig. 3I–L). 2-Cys Prx was also detected in the cytoplasm of

zygotes (data not shown). In contrast, 1-Cys Prx was not

detected during the zygote, ookinete, and sporozoite stages

(data not shown). Table 1 summarises the patterns of

expression of these Prx proteins during each developmental

stage of P. yoelii as determined by the immunofluorescent

antibody staining. Immunostaining with the pre-immune

sera detected no fluorescence for Prx proteins in blood-stage

(Fig. 3A,B,E,F) or insect-stage parasites (data not shown).

3.4. Expression 1-Cys and 2-Cys Prx mRNAs in blood-stage

P. yoelii

The lower immunofluorescent staining of 1-Cys Prx

observed in the parasites during the blood and insect stages,

except for trophozoites, might be due to a lower affinity of

the anti-serum used for 1-Cys Prx protein. To eliminate this

possibility, levels of 1-Cys Prx and 2-Cys Prx mRNAs in

blood-stage P. yoelii parasites were compared with the

quantitative real time RT–PCR method. We verified that

the level of 1-Cys Prx mRNA was lower than that of 2-Cys

Prx; the ratio of the absolute quantities of these two mRNA

species was 1:40 (Table 2).

4. Discussion

Here, we have described molecular cloning, biochemical

characterisation, and expression patterns of the 2-Cys and

1-Cys Prx proteins of the rodent malaria parasite P. yoelii.

Constitutive expression of the 2-Cys Prx protein in the

erythrocytic and insect stages suggests the importance of the

protein in the parasite antioxidant defence. It has been

suggested that P. falciparum has a functional 2-Cys

Prx/Trx/Trx reductase (TrxR) cycle that is involved in

detoxification of intracellular ROS (reviewed in Rahlfs et al.,

2002). Although there is presently no evidence for an active

Trx cycle in P. yoelii, the expression of 2-Cys Prx during all

the erythrocytic stages strongly suggests that the parasite

uses this protein as an antioxidant enzyme to cope with

enhanced oxidative stress in parasite-infected erythrocytes

(Becker et al., 1994; Postma et al., 1996). Malaria parasites

Fig. 1. Western blot analyses of recombinant Plasmodium yoelii

peroxiredoxin (rPyPrx) proteins. (A) Purified rPyPrx proteins were

subjected to 12.5% SDS–PAGE under reducing conditions. Lane 1, rPy

2-Cys Prx protein (500 ng); lane 2, rPy 1-Cys Prx protein (500 ng). The

proteins were transferred to Immobilon membranes and reacted with rabbit

anti-rPf 2-Cys Prx serum (2-Cys Prx) or anti-rPf 1-Cys Prx serum (1-Cys

Prx). (B) Extracts prepared from blood-stage parasites were subjected to

12.5% SDS–PAGE under reducing conditions. Thirty-eight micrograms of

extract was separated by electrophoresis (lane 1). Proteins were transferred

to Immobilon membranes and reacted with rabbit anti-rPf 2-Cys Prx IgG

(lane 2) or anti-rPf 1-Cys Prx IgG (lane 3). SDS–PAGE gels were stained

with Coomassie Brilliant Blue. Molecular weight markers in kDa are

indicated on the left.

S.-i. Kawazu et al. / International Journal for Parasitology 33 (2003) 1455–14611458

live in oxygen-rich environments both in vertebrate ery-

throcytes and in the tissues/organs of the mosquito vector.

The lumen of the mosquito midgut, which is where gametes

are fertilised to form zygotes, is filled with host blood

components. This space is likely to be an oxygen-rich

environment. The ookinete, which develops from the zygote,

penetrates epithelial cells to form an oocyst on

the haemolymph side of the midgut. During this process,

the parasite is exposed to nitric oxide (NO) from the host cell

(Han et al., 2000). Reactions between NO and O2 or O2-

generate biologically reactive nitrogen oxide species, which

react with and modulate critical intracellular molecules

(Wink et al., 2000). The sporozoite, which remains within

cells of the host’s salivary glands, is also likely exposed to

host ROS (Kanzok et al., 2001). Expression of 2-Cys Prx by

the sporozoite may benefit the parasite in the insect vector

and also when the parasites are inoculated into the vertebrate

host. It has been reported that the mosquito Anopheles

albimanus, which transmits malaria parasites, possesses a

salivary haem peroxidase that is released into the skin of the

vertebrate host for vasodilation during probing and blood

feeding (Ribeiro and Nussenzveig, 1993). When inoculated

into the vertebrate host, the sporozoite may require a certain

level of antioxidant defences to forestall saliva-derived haem

peroxidase activity. The 2-Cys Prx/Trx/TrxR system

together with other antioxidant systems, such as the

glutathione system (Muller et al., 2001) and glutaredoxin

system (Rahlfs et al., 2001) may be utilised by the parasites to

protect against oxidative stresses. Further efforts to clarify

such functions of 2-Cys Prx may offer new insights into the

biology of malaria parasites and facilitate development of

potential strategies to block the life cycle.

P. yoelii 1-Cys Prx was expressed during the late

trophozoite stage. This stage-specific expression is consistent

with that reported for the Prx protein of P. falciparum

(Kawazu et al., 2000). Trophozoites of malaria parasites

Fig. 2. Antioxidant activity of recombinant Plasmodium yoelii peroxiredoxin (PyPrx) proteins. (A) rPyPrx protein-dependent protection of yeast enolase from

inactivation was examined in the mixed-function oxidation (MFO) system. Enolase activity was measured as the initial rate (0–100 s) of production of

phosphoenolpyruvate under the conditions described in Section 2. Results are expressed as the recovery (percentage) of the rate with various concentrations of

recombinant protein relative to that recorded without the MFO system (–). Data are means ^ S.D. of three similar experiments. (B) Nicotinamide-adenine

dinucleotide phosphate, reduced (NADPH) oxidation coupled by rPy 2-Cys Prx (solid line) or rPy 1-Cys Prx (dotted line) to reduction of H2O2 in the presence

of the Escherichia coli Trx and TrxR system. NADPH oxidation was monitored with an Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech) as

the decrease in A340 after addition of H2O2 to the system. Data are representative of three similar experiments.

S.-i. Kawazu et al. / International Journal for Parasitology 33 (2003) 1455–1461 1459

Fig. 3. Expression of the Plasmodium yoelii Prx proteins during the blood and insect stages. (A,C,E,G) Differential interference contrast. (I,K) Phase contrast

images. (B,D) Triple staining of the parasite cells for 2-Cys Prx (green), Pys25 (red) and nucleus (blue). (F,H) Triple staining of the parasite cells for 1-Cys Prx

(green), Pys25 (red) and nucleus (blue). (J,L) Double staining of the parasite cells for 2-Cys Prx (green) and nucleus (blue). The specimens were stained with

pre-immune (B,F) and anti-Prx rabbit sera (D,H,J,L). The pre-immune sera were used in the same dilution (1:100) to the antisera. The specimens in B, D, F, H, J

and L are identical fields to those in A, C, E, G, I and K, respectively. Letters with arrows in A, C, E, G, I and K indicate early to late trophozoite (T), schizont

(S), gametocyte (G), ookinete (retort) (Ok) and sporozoite (Sp), respectively. Bars indicate 10 mm.

Table 1

Expression of peroxiredoxin proteins in Plasmodium yoelii

2-Cys Prx 1-Cys Prx

Blood stage

Trophozoite þ þ

Schizont þ ^

Gametocyte þ 2

Insect stage

Zygote þ 2

Ookinete þ 2

Sporozoite þ 2

Prx, peroxiredoxin.

Table 2

Expression of peroxiredoxin mRNAs in Plasmodium yoelii blood-stage

parasites

1026 pmol/mla Mean ^ S.D.

Expt. 1 Expt. 2 Expt. 3

2-Cys Prx 120 110 100 110 ^ 10

1-Cys Prx 3 2.7 2 2.6 ^ 0.5

Prx, peroxiredoxin.a pmol of mRNA species/ml of cDNA.

S.-i. Kawazu et al. / International Journal for Parasitology 33 (2003) 1455–14611460

digest the host haemoglobin to release amino acids (Olliaro

and Goldberg, 1995). This process produces large quantities

of prooxidative haem in the food vacuole and results in

generation of ROS in the parasite cytoplasm (Ginsburg et al.,

1999). Thus malaria parasites must produce an effective

antioxidant to cope with such oxidative burdens. Malaria

parasites are known to be highly susceptible to the toxic effect

of the auto-producible haem, and this is the basis of the anti-

malarial action of chloroquine (Ginsburg et al., 1999). Further

studies to clarify the roles of Prx in the malaria parasite may

provide information that allowsutilisation of thisproteinas an

alternative target for anti-malaria chemotherapies.

In summary, the results obtained in this study suggest

that malaria parasites express and utilise 2-Cys and 1-Cys

Prx proteins during both the erythrocytic and insect stages to

protect against oxidative stresses.

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific

Research (C) (13670260 to S.K. 15590378 to T.N., and

14570215 to T.T.) from the Japan Society for the Promotion

of Science (JSPS), Grants-in-Aid for Scientific Research

(14370084 to M.T.) and Scientific Research on Priority

Areas (C) (14021137, 15019127 to S.K., 11147230,

14021134, 15019120 to T.N., and 14021082, 15019072 to

T.T.) from the Ministry of Education, Culture, Sports,

Science and Technology (MEXT) of Japan, and a grant for

Precursory Research for Embryonic Science and Technol-

ogy, Japan Science and Technology Corporation (to T.N.)

and a Grant for International Health Cooperation Research

(15C-5) from the Ministry of Health, Labour and Welfare,

Japan. Sequence data for P. yoelii chromosomes were

obtained from the Plasmodium Genome Consortium

PlasmoDB (PlasomDB; http://PlasmoDB.org).

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