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J. Biol. Chem., Vol. 279, Issue 50, 52300-52311, December 10, 2004

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The Expression of Lactate Dehydrogenase Is Important for the Cell Cycle of Toxoplasma gondii^*

Fatme Al-Anouti, Stanislas Tomavo, Stephen Parmley¶, and Sirinart Ananvoranich||

From the Chemistry and Biochemistry Department, University of Windsor, Windsor, Ontario N9B 3P4, Canada, Equipe de Parasitologie Moléculaire, Laboratoire de Chimie Biologique, UGSF, CNRS UMR 8576, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France, and ^¶Department of Immunology and Infectious Diseases, Palo Alto Medical Foundation, Palo Alto, California 94305

Received for publication, August 11, 2004 , and in revised form, September 30, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In Toxoplasma gondii, lactate dehydrogenase is encoded by two independent and developmentally regulated genes LDH1 and LDH2. These genes and their products have been implicated in the control of a metabolic flux during parasite differentiation. To investigate the significance of LDH1 and LDH2 in this process, we generated stable transgenic parasite lines in which the expression of these two expressed isoforms of lactate dehydrogenase was knocked down in a stage-specific manner. These LDH knockdown parasites exhibited variable growth rates in either the tachyzoite or the bradyzoite stage, as compared with the parental parasites. Their differentiation processes were impaired when the parasites were grown under in vitro conditions. In vivo studies in a murine model system revealed that tachyzoites of these parasite lines were unable to form significant numbers of tissue cysts and to establish a chronic infection. Most importantly, all mice that were initially infected with tachyzoites of either of the four LDH knockdown lines survived a subsequent challenge with tachyzoites of the parental parasites (10⁴), a dose that usually causes 100% mortality, suggesting that live vaccination of mice with the LDH knockdown tachyzoites can confer protection against T. gondii. Thus, we conclude that LDH expression is essential for parasite differentiation. The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Toxoplasma gondii is a ubiquitous protozoan parasite that has the ability to infect a variety of warm-blooded vertebrates including humans (1). This parasite has a complex life cycle involving both sexual and asexual multiplication. The sexual cycle occurs exclusively in feline intestinal epithelial cells. In humans and other intermediate hosts, the parasite exists in the following two asexual forms: rapidly dividing tachyzoites and slowly replicating bradyzoites. Initial and acute infection is characterized by the presence of tachyzoites that are normally cleared by the host immune response. In most cases of human toxoplasmosis, infection ultimately becomes chronic when tachyzoites differentiate into bradyzoites. The bradyzoites can remain dormant within tissue cysts protected from the host immune response (2). In patients with immunodeficiencies such as AIDS or other malignancies, bradyzoites that differentiate into tachyzoites after release from the cysts can give rise to a recurrent infection that can prove fatal. Congenital malformations are observed when an acute infection is acquired during pregnancy (3). At present, no effective treatment for chronic toxoplasmosis is available. Therefore, cell culture and animal models for the study of chronic Toxoplasma infection are crucial. The differentiation of tachyzoites to bradyzoites can be induced in vitro, although the genetic regulatory signals that control this differentiation are still unknown (2, 4). Several studies have identified stage-specific proteins including the cyst wall glycoprotein 1 (5, 6), surface antigen SAG4A¹ (7), metabolic enzymes, lactate dehydrogenase (LDH), enolase (ENO) (8–10), and the stress-response protein bradyzoite surface antigen 1 (11). Some of these genes are developmentally regulated and suspected to be crucial for T. gondii differentiation (12, 13).

Lactate dehydrogenase is a glycolytic enzyme that catalyzes the interconversion of pyruvate to lactate. This enzyme plays an indispensable role when glycolysis becomes the only pathway to provide energy under anaerobic conditions (14). LDH is considered as a potential anti-parasitic drug target because its unique structural and kinetic properties differ from those of human host cells (14–16). Among the reported LDH sequences, those of T. gondii and all members of the Plasmodium genus carry a pentapeptide insertion into the enzyme-active site (16). The activity of LDHs is generally inhibited by an excess of pyruvate. However, this property is greatly reduced in the LDHs from Plasmodium and Toxoplasma (15, 16).

In T. gondii, two LDH isoforms are present, LDH1 and LDH2. The genes encoding the isoforms show 64% nucleotide sequence identity, and their gene products share 71% amino acid sequence identity (17). The genes encoding LDH1 and LDH2 isoforms are developmentally regulated. The mRNA of LDH2 is detected in the bradyzoite stage only, whereas the transcript of LDH1 is found in both bradyzoites and tachyzoites. The LDH1 gene product is only expressed in tachyzoites, whereas that of LDH2 is expressed in only bradyzoites. It is speculated that LDH1 is replaced by LDH2 during development from tachyzoites to bradyzoites as a consequence of post-transcriptional induction of LDH2 (8, 17). During differentiation, T. gondii modifies its morphology and energy metabolism to adapt to its surrounding environment. Aerobic respiration and glycolysis are functional in tachyzoites, whereas glycolysis is the only energy source in bradyzoites (15, 18). Therefore, LDH, which is important for anaerobic respiration, might be a promising target for developing drugs for chronic toxoplasmosis.

Over the last few years, effective methods of transfection and targeted disruption of genes has made T. gondii amenable to genetic manipulations (19, 20). In addition to these conventional gene manipulations, we previously reported that the introduction of double-stranded RNA (dsRNA), presumably via RNAi, can lower the expression of T. gondii genes (21, 22). Here we report the first success in knocking down the expression of the essential genes LDH1 and LDH2 in T. gondii. Significantly, we have provided the evidence that the expression of LDH1 and LDH2 is essential for parasite development. The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
T. gondii
All experiments were carried out using human foreskin fibroblasts (HFF) that were grown in Dulbecco's modified Eagle's medium, supplemented with 10% cosmic calf serum (Hyclone, Logan, UT), 5 µg/ml streptomycin, and 5 units/ml penicillin in a 5% CO₂ atmosphere. Generation of LDH knockdown parasites was performed using the HXG-PRT-deficient (HXGPRT) T. gondii, strain PLK (23) (AIDS Research and Reference Reagent Program, National Institutes of Health, catalogue number 2860). Parasites were maintained as tachyzoites using HFF monolayers grown in minimum Eagle's medium supplemented with 1% dialyzed fetal bovine serum (Wisent, Montreal, Quebec, Canada). To differentiate the parasites into bradyzoites, freshly lysed parasites were allowed to invade HFF for 4 h. The media were subsequently replaced with RPMI containing 10 mM HEPES and 5% fetal bovine serum, pH 8.2. The medium was replaced every 2 days to maintain the pH (6). Parasites were harvested immediately after they had lysed out of HFF monolayers. After 4–6 days of bradyzoite differentiation using the alkaline method (7), cysts were freed from cells by scraping and digestion with 170 mM NaCl-pepsin (0.1 mg/ml) to 60 mM HCl for 1 min at 37 °C followed by neutralization with 94 mM Na₂CO₃. Collected parasites were passed through a 27-gauge needle twice and filtered through a 3-µm filter (Nucleopore). Filter-purified parasites were then collected by centrifugation at 15,000 rpm for 10 min and washed twice with Dulbecco's phosphate-buffered saline.

Plasmid Construction and Transformation
Bradyzoite-specific Expression Plasmids—A DNA fragment (993 bp) encompassing the LDH2 promoter region, referred to as L2, was amplified by PCR from genomic DNA isolated from T. gondii PLK strain using the oligonucleotides KpnILDH2(sense) and LDH2(antisense). The fragment was cloned into pBluescript SK+ (Stratagene) at KpnI and a blunted XhoI site. The second L2 fragment was produced by PCR using the oligonucleotides SacILDH2(sense) and XbaILDH2(antisense) and was cloned between SacI and XbaI sites. Portions of LDH1 and LDH2 ORF (nucleotides 1–487, GenBank^TM accession numbers U35118 [GenBank] and U23207 [GenBank] , respectively) were amplified by PCR from plasmids pMAL-C2-LDH1 and pMAL-C2-LDH2 (8) and cloned at the SmaI site. The 1.8-kb HXGPRT minigene was isolated from plasmid pTUB5mycGFPHXGPRT (obtained from Dr. Dominique Soldati, Imperial College London, UK) by SacII digestion and subcloned into the constructed plasmids. The resultant plasmids, which had two L2 fragments arranged in a head-to-head fashion flanking LDH1 and LDH2 cDNA, were named pL2-LDH1-L2 and pL2-LDH2-L2, respectively.

Tachyzoite-specific Expression Plasmids—The plasmid p30/11UP30/11, which contains the ORF of uracil phosphoribosyltransferase (UPRT, 21), was removed by digestion with NsiI and AgeI. The LDH1 and LDH2 ORF portions were then cloned separately by blunt end ligation to replace the UPRT cDNA. The resultant plasmids had two modified SAG1 promoters arranged in a head-to-head fashion flanking either LDH1 or LDH2 coding region. The 1.8-kb HXGPRT minigene was cloned at SacII site to give rise to plasmids p30/11LDH130/11 and p30/11LDH230/11.

Generation of Stable Parasite Lines
The resultant plasmids were individually introduced into PLKHXGPRT parasites by electroporation (20). Stable populations were selected using 25 µg/ml mycophenolic acid and 50 µg/ml xanthine. Electroporation with p30/11LDH130/11 and pL2LDH1L2 gave rise to transgenic parasite lines TL1T and BL1B, which produce dsRNA homologous to LDH1 in the tachyzoite and bradyzoite stage, respectively. Electroporation with p30/11LDH230/11 and pL2LDH2L2 resulted in the generation of parasite lines TL2T and BL2B, which express dsRNA homologous to LDH2 in the tachyzoite and bradyzoite stage, respectively. Four transgenic parasite lines were generated, namely TL1T, TL2T, BL1B, and BL2B. For clarity, T and B refer to the tachyzoite- or the bradyzoite-specific expression, respectively, whereas L1 and L2 refer to the LDH1 and LDH2, respectively.

Southern and Northern Blot Analysis
Genomic DNA was prepared as described previously (21), and RNA extractions were carried out by using Trizol reagent (Invitrogen.). For Southern blot analysis, 10 µg of genomic DNA was digested with the BamHI or PstI restriction enzymes and resolved on 0.7% agarose. Resolved DNA fragments were then transferred onto Hybond-N⁺ nylon membrane (Pall Life Sciences) using 10x SSC as transfer buffer. Hybridization was carried out at 68 °C in the presence of the DIG-labeled heat-denatured probe. The blots were then washed with a solution of 0.5x SSC and 0.01% SDS at 65 °C. Immunological detection was performed using alkaline phosphatase-conjugated anti-DIG Fab fragments and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo[3.3.1.1^3,7]decan}-4-yl)phenyl phosphate chemiluminescent substrate (Roche Applied Science). The signal was finally revealed by the Alpha Imager system (Alpha Innotech) and quantified using the ChemImager version 5.5. For Northern blotting, 8 µg of total RNA were electrophoresed onto 1% agarose, 2.7% formaldehyde gel. The RNA was transferred to Hybond-N⁺ membrane and probed as for Southern blots except that the hybridization was performed at 58 °C and the washes were at 54 °C. Immunological detection was carried out to reveal hybridization signals. The probe was then removed by incubating the blot in a denaturing buffer containing 200 mM NaOH and 0.1% SDS. The blot was then re-hybridized with another probe. For each sample, the signal detected for each target gene expression was normalized by the signals detected for a constitutively expressed gene that was used as an internal standard. Thus, the presented data exhibit no influence from variable sample handling. Dot blots were performed using low amounts of RNA (2 µg), and hybridization was carried out under similar conditions as those of Northern blot (22).

DIG-labeled DNA probes specific to UPRT and ROP1 were produced by PCR as described previously (22). For the detection of ENO2 mRNA, primers 5ENOx and 3ENOx were used for the synthesis of an ENO2-specific probe. The oligonucleotides LDH1–5'-UTR and LDH2– 5'-UTR specific to LDH1 and LDH2 mRNA were synthesized (Sigma Genosys) and subjected to tailing reactions using terminal transferase. The reactions were performed at 37 °C for 1 h in a 20-µl volume containing 100 pmol of the oligonucleotide, 25 mM Tris-HCl, pH 6.6, 0.2 M potassium cacodylate, 5 mM CoCl₂, 50 µM DIG-dUTP, 0.5 mM dATP, and 25 units of terminal deoxynucleotidyltransferase (Roche Applied Science).

RT-PCR
Reverse transcription (RT) reactions were carried out at 37 °C for 60 min using 2 µg of DNase-treated total RNA as initial template, 10 pmol of oligonucleotide primer in a total volume of 20 µl containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl₂, 0.5 mM of each dNTP, 50 units of RNase inhibitor, and 200 units of Moloney murine leukemia virus-reverse transcriptase. For the detection of LDH1 antisense RNA, the oligonucleotide LDH-ORF(sense) was used as the RT primer. In parallel, for the detection of LDH2 antisense RNA, the oligonucleotide LDH2forward was utilized as the RT primer. Eight µl of the RT reaction mixtures were used for the PCR amplifications. The PCR was performed in a volume of 100 µl containing 75 mM Tris-HCl, pH 8.8, 50 mM KCl, 2 mM MgCl₂, 50 µM dNTPs, 10 pmol of primers (LDH-ORF(sense) and 3LDH-ORF(antisense) for RT-PCR of LDH1 antisense RNA or LDH2forward and LDH2reverse for RT-PCR of LDH2 antisense RNA), and 2 units of TaqDNA polymerase. To rule out the presence of genomic DNA contamination in the extracted RNA samples, control experiments were conducted by directly subjecting RNA preparations to PCR. The sequences of all primers are listed in Table I.

dsRNA Transfection and Measurement of T. gondii Growth
Parasites were freshly harvested from infected HFF cells as described above and resuspended in the cytomix buffer (120 mM KCl, 0.15 mM CaCl₂, 10 mM K₂HPO₄, KH₂PO₄, pH 7.6, 25 mM HEPES, pH 7.6, 2 mM EDTA, 5 mM MgCl₂) freshly supplemented with 2 mM ATP and 5 mM glutathione. The tested dsRNA was added to the resuspended parasites in 4-mm electroporation cuvettes, and electroporation was carried out using the BTX Electro cell Manipulator (25 megohms, 25 microfarads, and 1.8 keV). After a 20-min incubation period at room temperature, the parasites were inoculated in triplicate into confluent HFF cells grown in 24-well plates at 10⁴ parasites per well and incubated at 37 °C. The extracellular parasites were removed by replacing the medium 4 h post-infection. Parasite growth was subsequently measured by the [³H]uracil or [³H]hypoxanthine incorporation assay (20).

Immunofluorescence Assay
Parasites were inoculated onto HFF monolayers grown on glass slides. Cultures containing tachyzoites were then allowed to grow for 2–3 days, whereas intracellular bradyzoites were cultured for 4 days before analysis. All experiments were set up in duplicate and repeated at least twice. Cells were fixed with 3% paraformaldehyde in PBS for 10 min and then permeabilized with 0.2% Triton X-100 in PBS for 15 min. Cells were then blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. Slides were then incubated for 1 h with either the rabbit LDH1 or LDH2 antiserum (diluted 1:500). For cyst staining, Dolichos biflorus agglutinin conjugated to FITC (diluted 1:300, Sigma) was added for 1 h. After three washes with PBS, cells were incubated for 1 h with goat anti-rabbit IgG conjugated to rhodamine (diluted 1:5000, Molecular Probes). Staining of the nuclei was carried out by incubation in the presence of 4',6-diamidino-2-phenylindole (Sigma) for 1 h and three washings with PBS (13). After drying, slides were overlaid with fluoromount and examined with a Leica DMIRB microscope. All images were taken with a cooled Q-Imaging CCD camera using the Improvision Openlab software.

Enzymatic Assays
Tachyzoites and bradyzoites (10⁷ parasites) were harvested and lysed in 1 ml of a solution containing 50 mM HEPES, pH 7.4, containing 20% glycerol, 0.25% Triton X-100, and 0.5 mM phenylmethylsulfonyl fluoride protease inhibitor. Cell-free lysates were obtained by centrifugation for 10 min at 14,000 rpm and kept at–80 °C until use. Protein concentrations were determined using the Bio-Rad protein assay kit with bovine serum albumin (Promega) as standard. LDH enzymatic assays were carried out at 37 °C in a final volume of 1 ml. Reactions were initiated by the addition of 1.6 mM pyruvate (Sigma) to a mixture containing 100 mM Tris, pH 7.4, 200 mM NaCl, 0.2 mg/ml NADH (Sigma), and 30 µg of protein extract. All assays were monitored at 340 nm for the change in absorbance related to the change in the concentration of NADH (18). To confirm the specificity of the reactions, control assays that lacked protein extracts were carried out.

SDS-PAGE and Western Blot Analysis
Approximately 10⁷ tachyzoites and bradyzoites were collected and boiled in SDS loading buffer (6.25 mM Tris-HCl, pH 6.8, 2% SDS, 10% sucrose, 0.05% bromphenol blue, and 1 mM 2-mercaptoethanol) for 10 min. The samples were resolved on a 9% SDS-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane by electro-blotting (Bio-Rad) for 2 h at 80 mV. The blots were then incubated in a solution containing the polyclonal antibody specific to LDH1 or LDH2 for 1 h at room temperature. The secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase) was subsequently used. After washing with 20 mM Tris-HCl and 0.3% Tween 20, chemiluminescent detection was performed using the chemi-glow detection kit (Alpha Innotech). Control samples were LDH1 and LDH2 fusion proteins prepared from bacteria harboring plasmids pMAL-C2-LDH1 and pMAL-C2-LDH2 (8). The bacterial cultures were induced with 1 mM isopropyl 1-thio--D-galactopyranoside for 4 h and collected. Crude protein extracts containing LDH1 and LDH2 fusion proteins were obtained after sonication in a buffer containing 100 mM Tris-HCl, pH 7.6, and were run along with parasite protein lysates on 9% SDS-polyacrylamide gels.

Experimental Infections in Mice
Purified tachyzoites from the parental PLKHXGPRT described above, TL1T, TL2T, BL1B, and BL2B, were inoculated into groups of five female 8–9-week-old BALB/c mice at 10³ or 10⁴ parasites per mouse and monitored until death or survival for 1 or 2 months. To check that surviving mice were infected and the immune response had developed in the infected mice, the serum of each mouse was tested against the total extract antigens prepared from the parental parasites using Western blots. To assess whether the primary infection of mice with TL1T, TL2T, BL1B, and BL2B tachyzoites conferred protection against the parental parasites, infected mice that survived after 30 days post-inoculation were challenged with the parental parasites (10⁴ tachyzoites per mouse) and then monitored for death or survival for 30 days.

In vivo cyst formation was determined by harvesting mouse brain at 8 weeks after infection. Cysts were purified using Percoll gradients. After resuspension of the pellets in PBS in a final volume of 100–200 µl, the purified cysts were counted using inverted phase microscopy.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Transient Silencing of the LDH Expression—We first verified whether the expression of LDH1 and LDH2 can be down-regulated following the introduction of dsRNA. Transient down-regulation assays as reported previously (21, 22) were used. Briefly, equal amounts of tested dsRNAs (4 µg) specific to LDH1, LDH2, ROP1, GFP, or UPRT were individually introduced into filter-purified parasites by electroporation. 24 h following electroporation, the total RNAs were isolated, and Northern blot analysis was used in the assessment of the down-regulation of the target genes. A control electroporation (mock) was performed using the buffer lacking dsRNA. When the steady state levels of the constitutively expressed and non-target gene enolase 2 (ENO2) were analyzed by Northern blot, single bands were revealed (Fig. 1A, upper panel). When the ENO2 probe was removed and the blot was hybridized to the probe specific to LDH1, transcripts of 1.8 kb were revealed from all samples. The sample isolated from LDH1 dsRNA electroporation exhibited relatively less abundance, as compared with other samples (Fig. 1A, 2nd panel). When the probe specific to LDH2 was used on the same blot following the LDH1 probe removal, no signal was detected in all samples tested (Fig. 1A, 3rd panel), confirming that LDH2 was not expressed at this growth stage. Again, the LDH2 probe was removed prior to the next hybridization. The probe specific to ROP1 revealed transcripts of 2.1 kb in all samples tested, and the hybridization signal was lower in the sample isolated from ROP1 dsRNA electroporation (Fig. 1A, 4th panel). Similarly, the UPRT-specific probe showed a 2.2-kb band corresponding to UPRT transcripts, with the signal of the sample isolated from the UPRT dsRNA electroporation having less intensity (Fig. 1A, bottom panel). Hence, we demonstrated that this transient down-regulation exhibited high specificity toward target genes.

View larger version (34K): [in this window] [in a new window]   FIG. 1. Transient silencing by dsRNAs. A, Northern blot analysis. The dsRNA specific to LDH1, LDH2, ROP1, UPRT, and GFP were individually electroporated into tachyzoites. Total RNA samples extracted from the parasites electroporated with the homologous dsRNA and the mock (buffer) were subjected to Northern blot analysis. Chemiluminescent detection using the DIG-labeled probes (indicated for each panel) was used to reveal the steady state levels of the target transcripts as described under "Experimental Procedures." B, inhibition of parasite growth. The in vitro synthesized dsRNAs homologous to LDH1 and LDH2 were individually electroporated into purified parasites. Four µg of each RNA species were tested, and the [3H]uracil and [3H]hypoxanthine assimilation assays were conducted 24 h following electroporation. The dsRNA homologous to nontarget ROP1 was used as a control. Mock electroporations were performed using the electroporation buffer with no RNA species. C, dose dependence exhibited by dsRNA homologous to LDH1. Different concentrations of dsRNA homologous to LDH1 (10, 4, 0.4, and 0.04 µg) were tested for their abilities to lower uracil and hypoxanthine incorporation.

We reported previously (21) that transient down-regulation upon the introduction of dsRNA is dose-dependent. Hence, it is interesting to know if the effect of LDH1 down-regulation could be enhanced by increasing the amount of dsRNA. When 10 µg of LDH1 dsRNA were electroporated into purified tachyzoites, no enhancement of the down-regulation was observed (Fig. 1C). When the concentrations of LDH1 dsRNA were lowered (as low as 0.04 µg), the nucleotide incorporation was similar to that of the mock electroporation, thus indicating that the threshold of LDH1 knockdown is reached when 4 µg of dsRNA was used (Fig. 1C).

LDH Knockdown Parasite Lines—To investigate the physiological roles of LDH1 and LDH2 in parasite development, we primarily aimed to lower LDH1 expression in a tachyzoite-specific manner and to lower LDH2 expression in a bradyzoite-specific manner. The two transgenic parasite lines generated for this purpose were TL1T and BL2B, respectively. However, to gain a better insight into the knockdown effect and the LDH expression pattern, we generated two additional transgenic parasite lines, BL1B and TL2T. In BL1B, LDH1 expression was knocked down in the bradyzoite conditions, whereas in TL2T, LDH2 expression was knocked down in the tachyzoite conditions. The genomic arrangement of these transgenic parasite lines was investigated by using Southern blot analyses. Fig. 2A shows a Southern blot analysis using the LDH1 probe. A 12.2-kb DNA band was detected in the sample isolated from the parental parasites and digested with BamHI (Fig. 2A, lane 1). This single band corresponds to the digested genomic locus of the LDH1 gene. Additional bands were detected in the BamHI-digested DNA samples isolated from the transgenic parasite lines (Fig. 2A, lanes 2–5). Similar results were obtained when the PstI-digested DNA samples were used for the analysis. A single 3-kb band was revealed for the sample isolated from the parental parasites (Fig. 2A, lane 6), whereas additional bands were detected for the samples isolated from the transgenic parasite lines (Fig. 2A, lanes 7–10). The LDH1 probe was removed, and the blot was hybridized with the LDH2 probe. A 9.2-kb band of the BamHI-digested LDH2 genomic locus was detected in the sample isolated from the parental parasites (Fig. 2B, lane 1), indicating no cross-hybridization of the LDH2 probe to LDH1 fragment. In addition to this band, the digested samples obtained from the transgenic parasite lines showed extra bands (Fig. 2B, lanes 2–5). Likewise, the PstI-digested samples isolated from the parental parasites showed three different bands of 1.3, 1.8, and 2.2 kb in size (Fig. 2B, lane 6). The samples isolated from the transgenic parasite lines and digested with the same restriction endonuclease showed additional random bands (Fig. 2B, lanes 7–10). The digestion patterns revealed by the LDH1 and LDH2 individual probes indicated that the transgenes, expressing dsRNA, were integrated into the genomes of the parasite lines. An additional hybridization with an HXGPRT-specific probe using the same blot also confirmed the integration (data not shown).

View larger version (35K): [in this window] [in a new window]   FIG. 2. Stable parasite lines. A and B, Southern blot analysis. Genomic DNA was isolated from the parental, TL1T, TL2T, BL1B, and BL2B parasites. DNA was digested with either BamHI or PstI and subjected to Southern analysis using a DIG-labeled LDH1 cDNA fragment (A) or LDH2 cDNA fragment (B). C, expression of dsRNAs. An image of an agarose gel resolving the RT-PCR products obtained using RNA samples extracted from the knockdown parasites. The expected size of the RT-PCR product was 487 bp (as indicated) when using the primer pair LDH-ORF(sense) and 3LDH-ORF(antisense) or the primer pair LDH2forward and LDH2reverse. Lane 1 contains DNA markers of 1000, 750, 500, and 250 bp, respectively. The RT-PCR products amplified from the RNA samples isolated from the parental, TL1T, and BL1B tachyzoites are shown in lanes 2, 4, and 6, respectively. The RT-PCR products amplified from the RNA samples isolated from the parental, TL1T, and BL1B bradyzoites are shown in lanes 3, 5, and 7, respectively. The oligonucleotide primer LDH-ORF(sense) was used during the RT step for the samples in lanes 2–7. The RT-PCR products amplified from RNA samples isolated from parental, TL2T, and BL2B tachyzoites are in lanes 8, 10, and 12, respectively. The RT-PCR products amplified from RNA samples isolated from the parental, TL2T, and BL2B bradyzoites are in lanes 9, 11, and 13, respectively. The oligonucleotide primer LDH2 forward was used during the RT step for the samples in lanes 8–13.

The steady state levels of the LDH1 and LDH2 mRNAs in the knockdown parasite lines were then assessed. The dot blot analyses were performed by using LDH1 and LDH2 gene-specific probes (Fig. 3A). The ROP1-specific probe was used to allow a normalization of signals obtained from LDH1- and LDH2-specific probes. For comparison, the ratios of LDH1/ROP1 and LDH2/ROP1 were calculated for each sample. It was observed that the ratio between LDH1/ROP1 remained constant (1:1) for the tachyzoites (T) and bradyzoites (B) of the parental, TL2T, and BL2B parasites and for the TL1T bradyzoites and the BL1B tachyzoites. It was as expected because LDH1 mRNA is expressed constitutively in tachyzoites and bradyzoites, although the LDH1 product is formed only in tachyzoites (17). Reduced LDH1/ROP1 ratios of 1.0:0.26 and 1.0:0.29 were observed for the TL1T tachyzoites and the BL1B bradyzoites, respectively, indicating gene- and stage-specific knockdown of LDH1 expression (Fig. 3A). When the signals from the LDH2-specifc probe and ROP1 probe were measured and calculated, we detected negligible levels of LDH2 mRNA in the tachyzoites of all parasites including the parental, TL1T, BL1B, TL2T, and BL2B parasites (Fig. 3A, bottom panel). The calculated ratios of LDH2/ROP1 at 1:1 for all tested bradyzoites were obtained, except the BL2B-bradyzoites, for which the ratio of LDH2/ROP1 was 1.0:0.60, indicating that the expression of LDH2 was lowered only in the bradyzoite of BL2B parasite line (Fig. 3A).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Phenotypical studies of LDH knockdown parasites. A, LDH1 and LDH2 transcription profiles. Total RNA was extracted from the tachyzoites (T) and bradyzoites (B) of all four parasite lines along with the parental (P) and subjected to dot-blot analysis. DIG-labeled probes specific to LDH1 and LDH2 5'-UTR were used to assess the steady state levels of the LDH1 and LDH2 target transcripts. A probe specific to ROP1 was used to control for the amount of RNA per sample. B, Western blot analysis. Images of Western blots of tachyzoite protein extracts probed with the LDH1 antiserum (upper panel) and bradyzoite protein extracts probed with the LDH2 antiserum (lower panel). LDH1 and LDH2 fusion proteins were included as controls.

To characterize the phenotype of the LDH knockdown parasite lines, we examined the multiplication rates of tachyzoites and bradyzoites. Because T. gondii divides by a unique binary division, the number of parasites per vacuole reflects the division rate. The parental, BL1B, TL2T, and BL2B tachyzoites multiplied at similar rates, of which 8 and 64 parasites/vacuole were obtained for most vacuoles, at times 48 and 96 h after infection (Fig. 4, A and B, respectively). TL1T tachyzoites grew more slowly. Values of 2 and 8 parasites/vacuoles at 48 and 96 h post-infection were scored. Under bradyzoite growth conditions, 32 parasites/vacuole were counted in most vacuoles of the parental parasites (Fig. 4C). It was detected that 8 parasites/vacuole for BL2B and TL1T bradyzoites and 26 parasites/vacuole for TL2T and BL1B bradyzoites were formed.

View larger version (20K): [in this window] [in a new window]   FIG. 4. Parasite growth. The percentage distribution of vacuole size (number of parasites/vacuole) was determined at 48 (A) and 96 h (B) after infection with the parental and parasite lines grown under tachyzoite culture conditions, and at 96 h under bradyzoite differentiation conditions (C). The number of parasites per vacuole was counted for 100 vacuoles in two different experiments. , parental (P); , TL1T; , BL2B; , BL1B; •, TL2T.

View larger version (68K): [in this window] [in a new window]   FIG. 5. Differentiation of stable parasite lines. A, the parental and knockdown parasite lines were grown in HFF monolayers under either tachyzoite or bradyzoite conditions. Nuclei were stained with 4',6-diamidino-2-phenylindole (blue). The tachyzoites of the parental parasites (images 1 and 2) and TL1T line (images 3 and 4) were subjected to immunofluorescence assays using the LDH1 antiserum and a rhodamine conjugate (red) carried out as described under "Experimental Procedures." FITC-labeled D. biflorus agglutinin in conjunction with the LDH2 antiserum was used to stain the cyst wall of bradyzoites (green) and LDH2 (red) (images 5–7) and BL2B-bradyzoites (images 8–10). Bars on the micrographs are 10 (for images 3 and 4) and 20 µm (images 1 and 2 and 5–10). B, normalized fluorescence values from LDH1 staining for the parasite lines under tachyzoite culture conditions. C, normalized fluorescence values from LDH2 staining when the parasites were induced to differentiate in vitro into bradyzoites.

The Knockdown Expression of LDH Attenuates the Parasite Virulence in Mice—To explore the effect of LDH1 and LDH2 knockdown on parasite growth in vivo, TL1T, BL1B, TL2T, and BL2B parasites were inoculated into female BALB/c mice at doses up to 10³ tachyzoites. After 4–5 days of infection, all mice infected with the parental parasite and LDH knockdown parasites showed the same characteristic symptoms of disease. However, the groups of mice infected with the four knockdown parasite lines recovered faster than those infected with the parental parasite. At 14 days post-inoculation, infection with TL1T, BL1B, and BL2B tachyzoites was not lethal to the mice, whereas the infection with TL2T tachyzoites and the parental parasites caused 20 and 60% mortality, respectively (Fig. 6A). When an independent experiment using a dose of 10⁴ tachyzoites per mouse was performed, 40 and 100% mortality rates were observed at day 11 in mice infected with TL2T tachyzoites and the parental parasite, respectively. In this case, 100% survival was again obtained in the groups of mice infected with TL1T, BL1B, or BL2B (Fig. 6B), suggesting that LDH knockdown attenuated T. gondii virulence in mice. In order to ensure that the surviving mice had truly been infected, serum was collected 30 days post-inoculation and was tested in Western blot. A pool of the pre-immune sera from these same mice was also tested and failed to detect T. gondii antigens as expected. Numerous antigens of molecular masses ranging from 14 to 90 kDa were recognized by sera from mice surviving infection with TL1T, BL1B, TL2T, and BL2B tachyzoites (Fig. 6C). Similar antigenic profiles were observed when the sera from the two mice surviving infection with 10³ tachyzoites of the parental parasites (Fig. 6A) were tested (Fig. 6C, panel P, lanes 1 and 2). Except for an antigen with an apparent molecular mass of 43 kDa, which was not observed in four out of five mice infected with TL1T (Fig. 6C, panel TL1T, lanes 1–4), and one of the parental parasite (lane 1), numerous antigens of the same molecular masses were detected in all sera tested. To determine whether the surviving mice had developed protective immunity 30 days post-infection, mice initially infected with TL1T, BL1B, TL2T, and BL2B were challenged with 10⁴ parental parasite tachyzoites. As shown in Fig. 6B (arrows), all mice infected with the LDH knockdown parasites (TL1T, BL1B, TL2T, and BL2B) survived a subsequent challenge with 10⁴ tachyzoites of the parental parasite, a dose that usually causes 100% mortality, suggesting that live vaccination of mice with the LDH knockdown tachyzoites can confer protection against T. gondii.

View larger version (49K): [in this window] [in a new window]   FIG. 6. Experimental infections in mice. A, the relative time-to-death of mice infected with TL1T, BL1B, TL2T, BL2B, and parental (P) parasites were compared. Female BALB/c mice (groups of five) were inoculated with 103 tachyzoites, and mortality was monitored over 30 days. B, comparative mortality of TL1T, BL1B, TL2T, BL2B, and parental parasites. Female BALB/c mice (groups of five) were inoculated with 104 tachyzoites, and the mortality rate was followed until 30 days post-infection. C, Western blots showing that all surviving mice displayed immune response by producing antibody against numerous Toxoplasma antigens. A pool of pre-immune sera of each tested group was in lanes 1–5 (control). Serum was taken from each infected and surviving mouse 30 days post-infection for TL1T, BL1B, TL2T, BL2B, and parental (P), as indicated at the top of the blot. Protein markers are shown on the left. D, in vivo cyst development of TL1T, BL1B, TL2T, BL2B, and parental parasites. The cysts were isolated from the brains of the living mice analyzed above. Average number of cysts per animal ± S.E. is shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Differentiation and encystation of T. gondii involves the coordinated expression of several genes. A broad spectrum of induction and suppression of gene expression could be observed as developmental dynamics. Despite the amenability of T. gondii to genetic manipulation, silencing the expression of essential genes has proven difficult. Our previous studies (21, 22) have indicated that the introduction of dsRNA is likely to invoke an RNAi mechanism and can be used as a powerful genetic tool for lowering the steady state level of gene expression. In this study, we demonstrated that a similar approach can be used successfully for lowering LDH1 and LDH2 expression. Such effort was reported unattainable via conventional genetic manipulations (6). The ability to lower LDH expression has allowed us to investigate their significance in parasite development.

We initially demonstrated that the transient knockdown of LDH expression gave rise to an altered growth pattern for tachyzoites, as shown by the nucleotide incorporation assays (Fig. 1). The inhibition of LDH1 gene expression in tachyzoites clearly decreased viability and proliferation. As expected, the LDH2 dsRNA electroporation did not affect tachyzoite viability because LDH2 is not expressed at this stage. This finding indicated that the introduction of dsRNA, without the presence of the target LDH2 mRNA, did not interfere with the cell viability. The hpRNAs which targeted a unique sequence on either LDH1 or LDH2 mRNA did not invoke an effective RNAi and did not cause the lowering of LDH expression. However, this finding was not indicative of an ineffectiveness of hpRNAs. We chose only a single site, which might not be accessible to the RISC complex. If other sites had been chosen, the knockdown might have occurred. We have attempted to transiently down-regulate LDH2 expression in the bradyzoite stage by electroporation. However, no conclusive data were obtained. It is likely that the introduction of dsRNA directly into bradyzoites by electroporation was not as efficient as that for tachyzoites.

Four transgenic parasite lines were generated and used for the investigation of the physiological roles and significance of LDH1 and LDH2 in the parasite development. TL1T and BL2B parasite lines were generated to monitor the down-regulation effect of LDH1 and LDH2 in a stage-specific manner, following the pattern of the expressed isoforms. The BL1B parasite line was used for investigating the outcome of lowering untranslated LDH1 mRNA in bradyzoites. TL2T, on the other hand, was a control parasite line for the dsRNA expression in the absence of the target mRNA. Prior to the phenotypical studies, we characterized these parasite lines, in comparison to their parental line, T. gondii, strain PLKHXGPRT. We showed that these parasite lines expressed the LDH1 and LDH2 dsRNA, as designed. Detectable levels of LDH1 dsRNA were expressed in TL1T tachyzoites and BL1B bradyzoites. Similarly, detectable levels of LDH2 dsRNA were detected in TL2T tachyzoites and BL2B bradyzoites. As expected, the steady state levels of target mRNAs were lowered according to the dsRNA expression and RNAi silencing effect. The silencing effect was monitored by dot-blot analysis and Western blot analysis (Fig. 3).

TL1T Parasite Line—TL1T tachyzoites exhibited impaired replication in the HFF monolayer. The doubling time of TL1T tachyzoites was twice that of the parental tachyzoites. It was recently reported that upon inhibiting LDH1 enzymatic activity by gossypol, the tachyzoite growth was reduced in cultured fibroblasts (14, 15). Therefore, the TL1T phenotype is in concordance with these previous enzymatic and inhibition studies. Inhibiting LDH1 enzymatic activity gave rise to slower replication and growth. The finding that the TL1T parasites were unable to kill the mice with a dose that causes 100% mortality in the parental strain and were unable to develop a significant number of cysts in vivo indicated that the host immunity could clear these TL1T parasites more effectively and rapidly after an infection than wild-type parasites. More importantly, the infection with TL1T conferred protection against a new challenge with the parental strain at a dose that kills 100% of infected mice.

BL2B Parasite Line—It is not surprising that BL2B tachyzoites exhibited relatively normal growth similar to the parental parasites (Fig. 4, A and B). As expected, BL2B bradyzoites multiplied significantly slower than those of the parental parasites (Fig. 4B). The percentage survival of the pepsin-released parasites of the BL2B parasite line was low (49%), as compared with the parental parasite (89%). BL2B parasites were unable to develop a significant number of cysts in vitro (Figs. 4C and 5A) and in vivo (Fig. 6D), indicating that the BL2B parasites may be defective in the differentiation process. The fact that few mature cysts (only 2–5 cysts in the whole brain extract) were detected suggests that differentiation is likely completed by this parasite line but at very low efficiency. It was reported that an LDH activity, especially that of LDH2, could be required to acidify the cytosol and keep the parasite in its bradyzoite stage (9, 18). The levels of expressed LDH2 and the steady state LDH2 mRNA were lowered in BL2B, as compared with those of the parental parasites (Fig. 3, B and A). Thus, it is possible that normal functioning of the parasite requires sufficient levels of LDH2. The down-regulation of LDH2 expression in BL2B bradyzoites might be due to a combination of both RNAi and antisense effect. It is thus possible that lowered LDH2 expression might have lead to (i) an insufficient energy supply and (ii) suboptimal conditions required for the bradyzoite development and maintenance. In a murine model system, low efficiency in bradyzoite formation would have left some parasites in the tachyzoites stage to be cleared by the immune system. As observed for this parasite line, BL2B parasites could cause typical symptoms of disease, but they are not lethal. Because only a few cysts were obtained, we were unable to determine by electron microscopy whether they display a normal morphology. The fact that BL2B infection could confer protection against re-infection by T. gondii indicates that the BL2B infection allows a protective immunity to develop.

BL1B Parasite Line—The expression of LDH1 dsRNA in a bradyzoite-specific manner gave rise to the lowered LDH1 mRNA (Fig. 3A), indicative of the RNAi-knockdown of LDH1 expression in the bradyzoite stage. BL1B exhibited a phenotype almost identical to that of the parental parasites. The BL1B tachyzoites and bradyzoites grew at a similar rate as those of the parental parasites. When Dolichos staining was used as the differentiation marker, BL1B was found to differentiate in vitro, similar to that of the parental parasites. Dolichos staining is specific to the cyst wall formation that takes place very early in the differentiation process (2, 4). However, parasites released from the BL1B cysts exhibited less viability (only 68% viability), as compared with the parental parasite (89% viability), suggesting an impairment in cyst development. In addition, the BL1B parasite line also failed to develop a significant number of cysts in the brains of mice. These findings suggest that the maintenance of LDH1 mRNA level may be associated with the differentiation process, perhaps through maintenance of a micro-environmental balance. When we lowered LDH1 mRNA in the bradyzoite stage, we might have offset the level of LDH1 mRNA essential for (i) an optimal interaction with other cellular components important for the parasite differentiation, and/or (ii) parasite readiness to convert to tachyzoites. This notion requires further investigation. Approaches such as those of proteomics and microarray analysis would give better insight into the differences between the BL1B and parental bradyzoites.

TL2T Parasite Line—TL2T tachyzoites produced LDH2 dsRNA in the absence of the target mRNA (Fig. 2C). This parasite line has a similar growth pattern as the parental parasite line, indicating that the presence of dsRNA does not cause any harmful effect to the parasite growth (Fig. 4). The priming of the RNAi system was not effective in lowering the LDH2 expression, as seen by the absence of change in the level of LDH2 mRNA in the TL2T bradyzoites as compared with other three transgenic parasite lines (Fig. 3A). Nonetheless, the LDH2 protein expression levels in the TL2T were lower than that in the parental line (Fig. 3B). The fact that the steady state level of LDH2 mRNA was unchanged, whereas expression of LDH2 isoform was lowered, suggests that the down-regulation of LDH2 expression in TL2T might not be due to the RNAi down-regulation but instead because of an antisense effect. With an antisense effect, translation of the target mRNA is blocked, without significant degradation of the message. This may explain why the mice infected with TL2T yielded fewer cysts in mice as compared with the mice infected with the parental strain. It should be noted that the number of cysts obtained in mice infected with TL2T was slightly higher than those generated by TL1T, BL1B, and BL2B infection. The translational suppression by this putative antisense effect seems to be sufficient to attenuate the virulence of the parasite infection but is not as efficient as that induced by mRNA degradation.

It is important to further investigate how LDH2 dsRNA expression, particularly in the TL2T parasite line, initiates an antisense effect, not an RNAi effect. It has been reported that post-transcriptional regulation via antisense and RNAi share a common pathway, as those observed for the miRNA and siRNA, respectively (28). The degree of complementarity between the siRNAs or miRNAs and the mRNA target is the sole determinant of their function in mRNA degradation and/or translational repression (29). siRNAs recognize mRNA targets by precise complementary base pair interactions leading to subsequent degradation of the target via an RNAi mechanism. On the other hand, miRNAs inhibit the translation of mRNAs via imprecise base pair interactions (30). Both pathways share similar protein partners, RNase III and Argonaute family members (31). It has been recently reported that T. gondii has RNAi-related genes, Dicer and Argonaute family members (32). We have obtained the cDNA encoding putative Dicer and Argonaute and are currently characterizing these genes. Thus, it is highly likely that miRNA as well as siRNA pathways exist in T. gondii.

Because all LDH knockdown parasites are virulence-attenuated in the tested animal model, we question whether the transforming elements and their products (i.e. constructed plasmids and expressed dsRNAs) could be virulence attenuating factors. In order to answer this question, we could further investigate whether other parasite lines expressing dsRNA unrelated to LDH1 and LDH2 are virulence-attenuated. However, this possibility is less likely because a parasite line expressing dsRNA homologous to UPRT exhibits normal growth and differentiation (21).

In conclusion, we have shown that the expression of LDH1 and LDH2 is essential for parasite development and growth. The generation of LDH knockdown parasites that are defective in their abilities to form cysts in vivo verifies the great potential of LDH as a target for designing future drugs that could block tissue cyst formation. The knockdown parasites might also serve as a useful basis for the development of vaccine strategies, especially for livestock.

	FOOTNOTES

 
^* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to S. A.), the Ministry of Colleges and Universities of Ontario (to F. A.), and the CNRS (to S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 519-253-3000 (ext. 3550); Fax: 519-973-7098; E-mail: anans{at}uwindsor.ca.

¹ The abbreviations used are: SAG4A, surface antigen 4A; DIG, digoxigenin; dsRNA, double-stranded RNA; ENO, enolase; FITC, fluorescein-isothiocyanate; GFP, green fluorescent protein; HFF, human foreskin fibroblasts; hpRNA, hairpin RNA; LDH, lactate dehydrogenase; nt, nucleotide; ORF, open reading frame; RNAi, RNA interference; ROP1, rhoptry protein 1; RT, reverse transcription; UPRT, uracil phosphoribosyltransferase; UTR, untranslated region; PBS, phosphate-buffered saline.

	ACKNOWLEDGMENTS

 
We thank Drs. James Gauld and Antonin Gauthier for critical reading of the manuscript, Dr. Kristin Hager for help with the fluorescence microscopy, Marlène Mortuaire for technical assistance, and Gabrielle Oria for iconography. The T. gondii host strain P(LK)HXGPRT was obtained from Dr. David Roos through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health.

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