PARINT-01290; No of Pages 5 Parasitology International xxx (2014) xxx–xxx

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Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei Miho Usui, Hirono Masuda-Suganuma, Shinya Fukumoto, Jose Ma. M. Angeles, Hassan Hakimi, Noboru Inoue, Shin-ichiro Kawazu ⁎ National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido 080-8555, Japan

a r t i c l e

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Available online xxxx Keywords: Liver stage Malaria Peroxiredoxin Plasmodium berghei Thioredoxin peroxidase

a b s t r a c t Phenotypic observation of thioredoxin peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vitro infection system in order to investigate defective liver-stage development in a mouse infection model. Indirect immunofluorescence microscopy assay with anticircumsporozoite protein antibody revealed that in the liver schizont stage, TPx-1 KO parasite cells were significantly smaller than cells of the wild-type parent strain (WT). Indirect immunofluorescence microscopy assay with anti-merozoite surface protein-1 antibody, which was used to evaluate late schizont-stage development, indicated that TPx-1 KO schizont development was similar to WT strain development towards the merozoite-forming stage (mature schizont). However, fewer merozoites were produced in the mature TPx-1 KO schizont than in the mature WT schizont. Taken together, the results suggest that TPx-1 may be involved in merozoite formation during liver schizont development. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Malaria remains a major public health threat worldwide and is responsible for high burdens of mortality and morbidity in diseaseendemic areas [1]. The invasion of malaria parasites into a patient's body begins in the liver where they replicate and generate thousands of progeny. Thus, inactivating the liver-stage parasite offers clear advantages, which include blocking the developmental phase in liver cells before parasites are able to infect erythrocytes and symptoms begin to develop in the patient. At present, the number of drugs that target the liver-stage parasite is limited due to a lack of studies on the liver-stage parasite, particularly on its metabolism during this stage. Primaquine is the only drug to target the liver-stage parasite that is currently available. However, its use is limited due to the increased risk of hemolysis when it is administered to patients with glucose-6phosphate dehydrogenase (G6PD) deficiency [2]. A better understanding of the liver-stage parasite's basic biology is needed in order to produce drugs that are effective against it in this stage and/or to Abbreviations: AOP, antioxidant protein; CSP, circumsporozoite protein; GSH, glutathione; GPx, GSH peroxidase; MSP-1, merozoite surface protein 1; Prx, peroxiredoxin; ROS, reactive oxygen species; SOD, superoxide dismutase; TSA, thiol-specific antioxidant; Trx, thioredoxin; TPx, Trx peroxidase. ⁎ Corresponding author. Tel.: +81 155 495846; fax: +81 155 495643. E-mail addresses: [email protected] (M. Usui), [email protected] (H. Masuda-Suganuma), [email protected] (S. Fukumoto), [email protected] (J.M.M. Angeles), [email protected] (H. Hakimi), [email protected] (N. Inoue), [email protected] (S. Kawazu).

formulate a strategy to inactivate liver-stage parasites with minimal side effects. Since malaria parasites are sensitive to oxidative stress [3], their antioxidant defense mechanisms represent a potential target for new strategies against malaria. A number of enzymatic and nonenzymatic antioxidants possessed by the malaria parasite allow it to maintain low intracellular levels of reactive oxygen species (ROS). Among these are the superoxide dismutases (SODs), which act as the first line defense against ROS. SODs are a ubiquitous family of enzymes that efficiently catalyze the dismutation of superoxide (O2 −) into oxygen and hydrogen peroxide [4]. Hydrogen peroxide (H2O2) is then reduced into water and oxygen to prevent the oxidation of other cellular components. This reaction is catalyzed by a variety of peroxidases including glutathione (GSH)-dependent peroxidases (GPx) and thioredoxin-dependent peroxidases or peroxiredoxins (Prxs). Genuine GPx does not exist in Plasmodium. However, the GSH system is present in Plasmodium falciparum and GSH is synthesized de novo by γ-glutamyl-cysteine synthetase and glutathione synthetase [5]. GPx and Prx obtain their reducing equivalents from two distinct systems, the GSH and the thioredoxin redox systems, respectively [6–9]. Both comprised a cascade of redox-active proteins which transfer reducing equivalent from NADPH to acceptor molecules, in this case is H2O2. Malaria parasites possess two SODs [10], but do not encode catalase or Gpx [11], the two major antioxidant enzymes in other organisms, indicating that their cellular redox homeostasis is critically dependent on Prx. In addition, Prx is known as a multifunctional molecule; it reduces peroxynitrite (ONOO−) and also is involved in a H2O2-mediated signal transduction cascade [12,13].

http://dx.doi.org/10.1016/j.parint.2014.09.013 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Usui M, et al, Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei, Parasitology International (2014), http://dx.doi.org/10.1016/j.parint.2014.09.013

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M. Usui et al. / Parasitology International xxx (2014) xxx–xxx

The Prxs are a family of proteins that are structurally homologous to the thiol-specific antioxidant (TSA) of yeast [14]. Prxs have been identified in all living organisms from bacteria to humans [15,16]. There are three subtypes of Prxs, namely 1-Cys Prx, typical 2-Cys Prx and atypical 2-Cys Prx. 1-Cys and 2-Cys Prxs are distinguished by a number of conserved cysteine residues in their catalytic sites. Although the proposed cellular function and electron donor for 1-Cys Prx are not fully understood [16,17], 2-Cys Prx has been found to act as a terminal peroxidase that reduces hydrogen peroxide and organic hydroperoxides using electrons donated by the thioredoxin (Trx) system [16,14]. During this catalytic action, typical 2-Cys Prx forms a homodimer through an intersub-unit disulfide bond that is reduced by an electron donated by Trx. Atypical 2-Cys Prx forms a monomer with an intramolecular disulfide bond that is also reduced by Trx during the catalytic action. Several findings have been reported on the cellular functions of 2-Cys Prx in mammals, including its involvement in the modulation of cytokine-induced H2O2 levels, which have been shown to mediate signaling cascades leading to cell proliferation, differentiation and apoptosis [16,18]. Plasmodium species possess six peroxidases localized in the cytoplasm, mitochondrion and apicoplast and nucleus [19] (Table 1). These include 1-Cys Prx, two typical 2-Cys Prxs, 1-Cys antioxidant protein (AOP), GSH peroxidase-like thioredoxin peroxidase (TPxGl) [19] and nuclear Prx (nPrx), which was previously known as merozoite capping protein-1 (MCP-1) [20]. The 1-Cys Prx [21] and one of the 2-Cys Prxs (thioredoxin peroxidase-1; TPx-1) [22,23] are expressed in cytosol, whereas the other 2-Cys Prx (TPx-2) is expressed in the mitochondrion [23,24]. The AOP has a signal that targets apicoplast [25]. TPxGl is suggested to be localized in cytosol and apicoplast [26,27]. nPrx was recently found in the nucleus of the parasite [20]. The phenotype of typical 2-Cys Prx (TPx-1) gene-disrupted parasite population (TPx-1 KO) has previously been studied in P. berghei [28]. In a mouse infection model, the parasite population showed defective growth in the liver stage. Thus, in the present study, to further investigate the phenotypes that have thus far been found in mouse infection experiments, we observed the phenotype of the TPx-1 KO population during liver-stage development using an in vitro infection system with HepG2 cells.

2. Materials and methods 2.1. Parasites The P. berghei ANKA strain was obtained from the Armed Forces Research Institute of Medical Sciences, Thailand. The TPx-1 KO population (referred in our previous study as Prx KO) with a targeted disruption of pbtpx-1 (PlasmoDB, PBANKA_130280) was established by double-crossover homologous recombination [29].

2.2. Infection of mosquitoes Six-week-old ICR mice (Clea Japan) were infected with P. berghei by intraperitoneal (i.p.) injection of parasites that had been stored as frozen stock at −80 °C. The parasitemia of the animals was monitored daily by light microscopic observation of Giemsa-stained thin blood smears. Anopheles stephensi mosquitoes were maintained on 10% sugar solution at 27 °C and 80% relative humidity under a 12 h light/ dark cycle. A. stephensi mosquitoes were fed on mice for 2 h at room temperature (RT), or 19 °C, when the number of microgametocytes that could exflagellate in vitro had reached 20–30 per 1 × 105 erythrocytes [30]. The parasite-infected mosquitoes (100–200 mosquitoes in each group) were maintained at 19 °C with 10% sugar solution. The animal experiments in this study were carried out in compliance with the Guide for Animal Experimentation at Obihiro University of Agriculture and Veterinary Medicine (Permission number: 23–43). 2.3. Maintenance of hepatoma cells and sporozoite infections HepG2 cells, which are usually used as host cells in the P. berghei liver-stage infection model system [31,32], were maintained in Eagle's minimum essential medium (MEM) (Sigma Aldrich Japan Co., Tokyo, Japan) supplemented with 10% heat inactivated-fetal bovine serum (HI-FBS), 1% MEM nonessential amino acid (Nacalai Tesque Inc., Kyoto, Japan) and 1% penicillin/streptomycin (Invitrogen Japan, Tokyo, Japan). The cells were constantly subcultured until use by trypsinization and kept at 37 °C in a 5% CO2 cell incubator. HepG2 cells (5 × 104 per well) were maintained in 8-chamber plastic Lab-Tek slides (Nalge Nunc International, Cergy Pontoise, France). The salivary glands of parasite-infected mosquitoes were excised and sporozoites were released by gentle triturating of the organ. HepG2 cells were inoculated with sporozoites (1 × 104 per well) and incubated for 3 h. After washing, the infected cells were cultured at 37 °C in a 5% CO2 cell incubator. 2.4. Indirect immunofluorescence microscopy assay The infected HepG2 cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries Ltd., Osaka, Japan) for 15 min and permeabilized with 0.1% polyoxyethylene (10) octylphenyl ether (equivalent to Triton®-X 100) (Wako Pure Chemical Industries) for 15 min. The cells were incubated for 30 min at room temperature with phosphate-buffered saline containing 5% skim milk (Wako Pure Chemical Industries) for blocking. The samples were stained with antibody against circumsporozoite protein (CSP) of P. berghei at 1:100 dilution. Monoclonal antibody against CSP (MRA-100) [33] was supplied by the Malaria Research and Reference Reagent Resource Center (MR4/ATCC, Manassas, VA, USA). Alexa-Fluor®488 conjugated goat anti-mouse IgG (Invitrogen Japan; 1:1000 dilution) was used as the secondary antibody. Hoechst-33342 (Dojindo, Kumamoto, Japan;

Table 1 Peroxidases of Plasmodium parasites. Gene name

Abbr.

PlasmoDB IDa

Localization

Citation

Thioredoxin peroxidase-1

TPx-1

Cytoplasm

[22]

Thioredoxin peroxidase-2

TPx-2

Mitochondrion

[23]

1-Cys peroxiredoxin

1-Cys Prx

Cytoplasm

[21]

1-Cys antioxidant protein GSH peroxidase-like thioredoxin peroxidase

AOP TPxGl

Apicoplast Apicoplast, Cytoplasm

[25] [26,27]

nuclear Prx

nPrx

PF3D7_1438900 PBANKA_130280 PF3D7_1215000 PBANKA_143080 PF3D7_0802200 PBANKA_122800 PF3D7_0729200 PF3D7_1212000 PBANKA_061050 PF3D7_1027300

Nuclear

[20]

a

Upper ID is for Plasmodium falciparum 3D7 strain and lower ID is for P. berghei ANKA strain.

Please cite this article as: Usui M, et al, Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei, Parasitology International (2014), http://dx.doi.org/10.1016/j.parint.2014.09.013

M. Usui et al. / Parasitology International xxx (2014) xxx–xxx

1:500 dilution) staining was performed with the secondary antibody reaction to visualize the parasite nuclei. The slides were mounted with Prolong® Gold antifade reagent (Invitrogen Japan) and observed under a confocal laser-scanning microscope (TCSSP5, Leica Microsystem, Wetzlar, Germany). At 40 h and 50 h post-infection, the area of the liver-stage parasite was measured using TSCCP5 software (Leica Microsystem) to evaluate its size. Anti-CSP antibody is used to determine the P. berghei parasite diameter [34]. At 50 and 60 h postinfection (cytomere stage to merozoite-forming stage), the samples were stained with antibodies against merozoite surface protein-1 (MSP-1) of P. berghei (provided by Dr. A. Holder, National Institute for Medical Research, London, UK) at 1:500 dilution. At 50 h postinfection, each of the developmental stages towards merozoite formation was counted. At 60 h post-infection, the number of merozoites in each schizont was evaluated. MSP-1 expression was used as marker for development and merozoite formation in mature liver-stage parasites [35]. 3. Result and discussion 3.1. TPx-1 KO was smaller than WT in the liver schizont and cytomere stages In the observation with the anti-CSP antibody, the mean numbers of parasite-infected cells in each chamber for the WT and TPx-1 KO populations were 293 and 300 (P = 0.90) (Fig. 1A). This result supported our previous finding from a mouse infection experiment which showed that TPx-1 KO sporozoites were as infective to hepatocytes as the WT population [28]. The area of TPx-1 KO parasites was, however, significantly smaller than that of WT parasites at the schizont and cytomere stages (40 h and 50 h post-infection, respectively). The mean area values calculated for TPx-1 KO were approximately 3/4 of WT at the schizont stage (40 h) and 1/2 of WT at the cytomere stage (50 h) (Fig. 1B). The mean area values calculated for WT and TPx-1 KO at the

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trophozoite stage (17 h post-infection) [36], were 16.53 μm2 and 14.95 μm2, respectively (n = 15, P = 0.79) (Fig. 1C). These results suggested that TPx-1 KO parasites were smaller than WT parasites in the schizont and cytomere stages. 3.2. Development of TPx-1 KO liver schizont is comparable to WT To observe liver schizont development in detail, mouse monoclonal antibody against P. berghei MSP-1 was used. It served as a marker for development and merozoite formation in the liver-stage parasites [35]. MSP-1 is primarily located on the rim of the developing schizont. Afterwards, its expression widens within the parasite and the final location is in the cell membrane of the merozoite (Fig. 2A). The staining patterns of MSP-1 in TPx-1 KO and WT schizonts were therefore evaluated at 50 h post-infection according to three criteria: no staining of MSP-1 (a), rim staining of MSP-1 (b), and merozoite staining of MSP-1 (c) (Fig. 2B). In WT, the percentages of the (a), (b), and (c) staining patterns were 25.40%, 42.45% and 32.15%, respectively. In TPx-1 KO, the percentages of the (a), (b), and (c) staining patterns were 21.65%, 42.95% and 35.40% respectively. No statistically significant difference was found between TPx-1 KO and WT in the percentage of any of these staining patterns. This result indicated that the development of the TPx-1 KO liver schizont could be comparable to WT towards the merozoiteforming stage (mature schizont). 3.3. TPx-1 KO liver schizont produces fewer merozoites than the WT We next evaluated merozoite formation in TPx-1 KO liver schizonts. To assess the number of merozoites formed in each mature schizont, the sizes of merozoites in TPx-1 KO were compared with WT at 60 h postinfection. Merozoite numbers were assessed in this way because of the technical difficulty in counting their number in a threedimensional schizont image under confocal microscopy. The result

Fig. 1. The infection rate and area of parasites in the wild-type parent strain (WT) and the pbtpx-1 disrupted population (TPx-1 KO). (A) The number of parasite-infected cells per chamber was counted at 40 h (schizont stage) post-infection in WT and TPx-1 KO liver-stage parasites. The averages of three independent experiments are shown. (B) The area of the parasite was measured for WT and TPx-1 KO at 40 h and 50 h (cytomere stage) using TCSSP5 software (Leica Microsystem). The data of three independent experiments (n = 15) are shown. (C) The area was measured at 17 h (trophozoite stage) (n = 15). The bar in the center indicates mean and error bars represent standard deviation. The nonparametric Mann–Whitney U-test was performed to compare the values of WT and TPx-1 KO parasites. Asterisks (*) indicate statistical significance. *: P b 0.001; **: P b 0.0001. P b 0.05 was considered statistically significant.

Please cite this article as: Usui M, et al, Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei, Parasitology International (2014), http://dx.doi.org/10.1016/j.parint.2014.09.013

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Fig 2. Quantitative evaluation of liver merozoite formation. (A) The developmental stages of parasites in the wild-type parent strain (WT) at 50–55 h post-infection. The stage labeled with “merozoite staining of MSP-1” was found at 55 h post-infection. MSP-1 is stained with Alexa-Fluor®488 (green). Nuclei are stained with Hoechst-33342 (blue). Scale bar indicates 10 μm. (B) The developmental stages of parasites in WT and the pbtpx-1 disrupted population (TPx-1 KO) by counting the parasites at 50 h post-infection with different staining patterns according to the criteria indicated by the representative images (upper panel): no MSP-1 staining (a), rim staining of MSP-1 (b), merozoite staining of MSP-1 (c). MSP-1 is stained with AlexaFluor®488 (green). Scale bar indicates 10 μm. The percentage of each staining pattern in WT and TPx-1 KO parasites is shown in lower panel. The average of two independent experiments (n = 70) is shown. Error bars represent standard deviation. Mann–Whitney U-test was performed to compare the values of WT and TPx-1 KO parasites. P b 0.05 was considered statistically significant.

showed that the mean merozoite size in TPx-1 KO was 1.40 μm2, while that of WT was 1.36 μm2 (n = 50, P = 0.35) (Fig. 3). Taken together with the finding that the liver schizont and cytomere stages were smaller in size in the TPx-1 KO parasite than in WT, it is suggested that the

TPx-1 KO liver schizont produces fewer merozoites than the WT liver schizont. It has been reported that the size of the Plasmodium yoelii liver schizont (stages found in liver cells after 50 h of sporozoite infection) correlates with the number of merozoites formed within the mature schizont [37]. 3.4. Conclusion

Fig. 3. Evaluation of merozoite size in the wild-type parent strain (WT) and the pbtpx-1 disrupted population (TPx-1 KO) liver schizonts. Liver merozoite area was measured using TSCCP5 software (Leica Microsystem) at 60 h post-infection (n = 50). The bar in the center indicates mean and error bars represent standard deviation. Mann–Whitney U-test was performed to compare the values of WT and TPx-1 KO parasites. P b 0.05 was considered statistically significant.

In the mouse infection model, liver-stage development was found to be defective in the TPx-1 KO population [28]. In order to further investigate defective liver-stage development in a mouse infection model, an in vitro infection system was utilized in this study to allow us to observe the sample easily and use the same cell group for diachronic observation. The findings of the present study, together with previous results, suggested that the developmental disorders in the liver stage of TPx-1 KO took place in or after the liver schizont stage. The mean schizont area values calculated for TPx-1 KO are the data obtained from two-dimensional observation, and the size difference between WT and KO when observed three-dimensionally tends to be greater. Thus, we thought our in vitro results may support our previous in vivo observations. The intracellular ROS condition may be more severe in the schizont stage than in earlier stages due to ATP production during karyokinesis, which is extensive in this stage. The highest expression level of TPx-2, the mitochondrion Prx, has also been observed in WT at the liver schizont stage [36]. The development of TPx-1 KO was comparable to WT during the liver schizont to cytomere stage. However, the mature schizont produced fewer merozoites in TPx-1 KO than in WT. In the mosquito stage of the TPx-1 KO population, the number of oocysts formed in the midgut was comparable to WT; however, the TPx-1 KO

Please cite this article as: Usui M, et al, Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei, Parasitology International (2014), http://dx.doi.org/10.1016/j.parint.2014.09.013

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population produced fewer sporozoites in the oocysts [28]. TPx-1 may protect the parasite from oxidative stress in the developmental stages when the parasites multiply exponentially, such as in the oocyst stage in the midgut of mosquitoes and the schizont stage in the liver of mammalian hosts. On the other hand, multiple functions of TPx-1 other than peroxidase have also been reported, including the functions of molecular chaperone and cell signaling sensor protein [38]. The molecular chaperone activity of TPx-1 in P. falciparum has recently been suggested [39]. The mechanisms by which TPx-1 is able to support sporozoite formation in oocysts and merozoite formation in liver schizonts are of interest and comprehensive profiling of gene expression in the oocyst and the liver schizont by RNA sequencing may yield useful information. The liver stage is the least studied stage in Plasmodium development. This is mainly due to its relative inaccessibility and low abundance of parasites in this stage which hinder detailed cellular and molecular studies [40]. For anti-malarial intervention, however, this stage of Plasmodium infection bears enormous potential. Our results suggested that the loss of TPx-1 affected the liver-stage development of the malaria parasite. The in vitro experiments with HepG2 cell-infection model may allow us to use a pro-electron reagent and the antioxidizer which are not suitable in in vivo experiments. Further study to elucidate the role of TPx-1 in the liver stage will provide greater insight into the contribution of this antioxidant protein in this stage and may yield novel antimalarial drugs and vaccination strategies. Acknowledgments We are very grateful to Dr. V. Nussenzweig of the New York University School of Medicine (New York, USA) for providing Hybridoma 3D11 producing antibodies against CSP of P. berghei, to the Malaria Research and Reference Reagent Resource Center (MR4/ATCC) and to Dr. A. Holder of the National Institute for Medical Research (London, UK) for the kind gift of the antibodies against MSP-1 of P. berghei. We are also grateful to Dr. T. Ishino of Ehime University in Japan and Dr. S.M. Kanzok of Loyola University, Chicago for their kind advice regarding the liver-stage experiments. This work was supported by a Grantin-Aid for Japan Society for the Promotion of Science (JSPS) Fellows (255843) and Scientific Research (23390098) from the JSPS. References [1] WHO. Summary and key points. World Malaria Report 2012. Geneva: WHO; 2012 IX–XIII. [2] Baird JK, Lacy MD, Basri H, Barcus MJ, Maguire JD, Bangs MJ, et al. Randomized, parallel placebo-controlled trial of primaquine for malaria prophylaxis in Papua, Indonesia. Clin Infect Dis 2001;33:1990–7. [3] Müller S, Gilberger TW, Krnajski Z, Lüersen K, Meierjohann S, Walter RD. Thioredoxin and glutathione system of malaria parasite Plasmodium falciparum. Protoplasma 2001;21:43–9. [4] Fridovich I. Superoxide radical and superoxide dismutases. Annu Rev Biochem 1995; 64:97–112. [5] Jortzik E, Becker K. Thioredoxin and glutathione systems in Plasmodium falciparum. Int J Med Microbiol 2012;302(4–5):187–94. [6] Arnér ES, Holmgren A. Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem 2000;267(20):6102–9. [7] Becker K, Gromer S, Schirmer RH, Müller S. Thioredoxin reductase as a pathophysiological factor and drug target. Eur J Biochem 2000;267(20):6118–25. [8] Yodoi J, Masutani H, Nakamura H. Redox regulation by the human thioredoxin system. Biofactors 2001;15(2-4):107–11. [9] Filomeni G, Rotilio G, Ciriolo MR. Cell signalling and the glutathione redox system. Biochem Pharmacol 2002;64(5–6):1057–64. [10] Sienkiewicz N, Daher W, Dive D, Wrenger C, Viscogliosi E, Wintjens R, et al. Identification of a mitochondrial superoxide dismutase with an unusual targeting sequence in Plasmodium falciparum. Mol Biochem Parasitol 2004;137:121–32.

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Please cite this article as: Usui M, et al, Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei, Parasitology International (2014), http://dx.doi.org/10.1016/j.parint.2014.09.013

Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei.

Phenotypic observation of thioredoxin peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vi...
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