A novel dense granule protein, GRA22, is involved in regulating parasite egress in Toxoplasma gondii

Molecular & Biochemical Parasitology 189 (2013) 5–13

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Molecular & Biochemical Parasitology

A novel dense granule protein, GRA22, is involved in regulating parasite egress in Toxoplasma gondii Tadashi Okada a , Dini Marmansari a , Zeng-mei Li a , Altanchimeg Adilbish a , Shishenkov Canko a , Akio Ueno a , Haruhi Shono b , Hidefumi Furuoka b , Makoto Igarashi a,∗ a b

National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido 080-8555, Japan Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan

a r t i c l e

i n f o

Article history: Received 2 October 2012 Received in revised form 8 April 2013 Accepted 15 April 2013 Available online 23 April 2013 Keywords: Toxoplasma gondii Dense granule protein Egress

a b s t r a c t The intracellular protozoan parasite Toxoplasma gondii is capable of invading any nucleated cell and replicates within a parasitophorous vacuole (PV). This microenvironment is modified by secretory proteins from organelles named rhoptries and dense granules. In this report, we identify a novel dense granule protein, which we refer to as GRA22. GRA22 has no significant homology to any other known proteins. GRA22 possesses a signal peptide at the N-terminal end which is responsible for dense granule and PV localization. The RH strain GRA22 contains 12 copies of tandem repeats consisting each of 21 amino acids located between the 42nd and 293rd amino acid residues from a full length of 624 amino acids. On the other hand, ME49 strain GRA22 has 10 copies of tandem repeats. The Neospora caninum GRA22 ortholog completely lacks this repetitive sequence. GRA22 knock out parasites show a similar growth rate as the parental strain. However, the timing of egress is earlier than that of the parental strain. These results suggest that GRA22 is involved in regulating parasite egress in T. gondii. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Toxoplasma gondii is an obligate intracellular apicomplexan parasite and one of the most common human and veterinary pathogens world-wide [1,2]. It is an important opportunistic pathogen of immunocompromised patients such as those with acquired immune deficiency syndrome (AIDS) or of transplant patients [3,4]. T. gondii is usually transmitted through oocystscontaminated food or water and tissue cysts in raw or undercooked meat. Up to a third of the world’s population are chronically infected by T. gondii [5]. It is parasitizing an unexceptionally wide range of warm-blooded animals [6]. T. gondii is capable of invading any nucleated cell and replicates in a parasitophorous vacuole (PV) [7]. This vacuole is formed within the host cells during the entry of T. gondii and is compartmentalized from the host cell cytoplasm by a PV membrane. The dense granule of T. gondii is a vesicular organelle containing secretary proteins, which are called dense granule proteins that participate in the modification of the PV and PV membrane to maintain intracellular parasitism in host cells [8]. Approximately 20 dense granule proteins, granule protein (GRA)

1 – GRA10, GRA12, GRA14, GRA15, protease inhibitors (TgPI-1, TgPI-2), isoforms of nucleotide triphosphate hydrolases (NTPaseI, NTPase-II), cyclophilin (Cy-18), and T. gondii patatin-like protein (TgPL1) [9–14], have been reported. The released proteins are targeted to either the vacuolar space (Cy-18, TgPI-1, TgPI-2, TgPL1, GRA1, 2), the PV membrane (GRA3, 5, 7, 8, 10, 14) or the intravacuolar tubular network (GRA2, 3, 4, 6, 7, 9, 12, 14, NTPase), which is thought to be important for the structure of the PV and for nutrient acquisition by the parasite from the host cell [9]. Therefore, dense granule proteins are believed to be important for the parasite’s growth and proliferation. However, the precise role of dense granule proteins is still unknown. In this study, we identify a novel dense granule protein, GRA22. GRA22 has no significant homology to any other known proteins, and has unique tandem repeat sequences. We generated the GRA22 knock out strain and analyzed its phenotypes. The GRA22 knock out strain shows early egress from host cells. This result suggests that GRA22 is involved in regulating parasite egress in T. gondii. 2. Materials and methods 2.1. Enzymes and chemicals

Abbreviations: PV, parasitophorous vacuole; T. gondii, Toxoplasma gondii; IFA, indirect immunofluorescence assay. ∗ Corresponding author. Tel.: +81 155495642; fax: +81 155495643. E-mail address: [email protected] (M. Igarashi). 0166-6851/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molbiopara.2013.04.005

Restriction endonucleases were purchased from Toyobo Co., Ltd. (Osaka, Japan) and Promega (CA, USA). Other DNA-modifying enzymes were purchased from TaKaRa Shuzo Co., Ltd. (Kyoto,

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T. Okada et al. / Molecular & Biochemical Parasitology 189 (2013) 5–13

Table 1 Primers used in this study. GRA1 promoter 400 bp truncated GRA22 Full length GRA22 GRA22 5 amplification GRA22 3 amplification GRA22 coding region (PCR1) PCR2 PCR3 GRA22N GRA22TR

5 -ttttggatcccgaaggctgtagtactgg-3 5 -ttgaattcatggcatttcaaggagatgac-3 5 -ttgaattcatgtggtttttgcctcgcctggac-3 5 -tttttctagatgcccgtttcctccctggctacagcctc-3 5 -ttttatcgataagcgacacttgcgcgacacgcaca-3 5 -cacattggcatcattgaa-3 5 -cctcttcaaatgtgaatgctc-3 5 -atatgaccgtctggcaaacatggct-3 5 -ttgaattcatgtggtttttgcctcgcctggac-3 5 -ttttggccgatgcggcccccaggcaggaagatggcag-3

5 -ttttgctagccttgcttgatttcttcaa-3 5 -ttttgcggccgcgtgcccgggtttgaataaagagg-3 5 -ttttgcggccgcgtgcccgggtttgaataaagagg-3 5 -ttttggatcctaactacttcaccgcgtttggttcac-3 5 -ttttgggcccaggaagccgaagcggcggttcagaacg-3 5 -gtgcccgggtttgaataaagagg-3 5 -aatcaaccgaattcatttgggaga-3 5 -gaactggtgtgtttgaatggc-3 5 -ttttgtcgactgagcgtccaaacctgcatc-3 5 -ttttggccgcatcggccagtacgaaaccctcgtcctc-3

Japan). All reagents used were commercially available and of analytical grade.

by SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/) and (http://www.cbs.dtu.dk/services/SignalP/) algorithm.

2.2. Animals

2.5. Generation of recombinant GRA22 protein

Eight weeks old female Balb/c mice used in our experiments were purchased from CLEA, Japan. The mice were used for polyclonal anti-GRA22 antisera production and for the parasite virulence assay. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Obihiro University of Agriculture and Veterinary Medicine.

The pGEX-5X-1 plasmids with truncated GRA22 cDNA was transformed into E. coli strain BL21 (DE3) pLysS-competent cells and grown in LB medium supplemented with 50 ␮g/ml ampicillin with vigorous shaking at 37 ◦ C up to an optical density of 0.6 measured at 600 nm. Then, the GST-fused protein was induced with isopropyl-␤-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM with mild shaking at 25 ◦ C overnight. The cells were centrifuged and the bacterial pellet was resuspended in pre-chilled STE buffer (150 mM NaCl, 50 mM Tris–HCl [pH 9.5] and 1 mM EDTA [pH 8.0]), then stored at −20 ◦ C. After thawing, the cells were disrupted by sonication on ice, and 20% (w/v) Triton X-100 in 1× PBS was added to the samples to form a final concentration of 1% (w/v) Triton X-100. Cell debris was removed by centrifugation (7000 × g, 20 min), and the resulting supernatant was recovered to a new tube. Recombinant GRA22 protein in the soluble fraction was affinity purified by glutathione-Sepharose beads according to the manufacturer’s protocols (GE Healthcare, Buckinghamshire, UK). Beads were washed twice with 1× PBS containing 1% (w/v) Triton X-100 and once with 1× PBS, and then bound proteins were eluted with elution buffer (200 mM NaCl, 20 mM reduced glutathione, 100 mM Tris–HCl [pH 9.5] and 5 mM EDTA [pH 8.0]). The eluted fractions were dialyzed against 1× PBS and the amount of recombinant protein was calculated using the Coomassie protein assay reagent kit according to the manufacturer’s protocols (Thermo Scientific, MA, USA).

2.3. Parasites The highly virulent type I RH strain with hypoxanthinexanthine-guanine phosphoribosyl transferase (HXGPRT) knockout (RHhxgprt) [15] and the weakly virulent type II ME49 strain of T. gondii were used. T. gondii tachyzoites were maintained in our laboratory through serial passage in Vero or human foreskin fibroblast (HFF) cells grown in modified Eagle’s medium (Sigma–Aldrich, Dorset, UK) supplemented with 5% fetal calf serum (FCS). 2.4. GRA22 cDNA cloning and sequencing analysis T. gondii GRA22 cDNA containing truncated or the entire coding region was RT-PCR amplified from RH strain total RNA as a template using primers described in Table 1. The amplified truncated cDNA was double digested with EcoRI and NotI and subcloned into identical restriction sites of bacterial expression plasmid with GST-tag, pGEX-5X-1 (GE Healthcare, Buckinghamshire, UK). For constructing expression vector in T. gondii, DNA fragments of the GRA1 promoter (600 bp) amplified using primers in Table 1 was replaced with CMV promoter of pcDNA6-V5-His (Invitrogen), which we named pGRA. The amplified full length cDNA was double digested with EcoRI and NotI and subcloned into identical restriction sites of pGRA. Plasmids were transformed into Escherichia coli DH5␣competent cells. Individual colonies were grown at 37 ◦ C overnight with rotary shaking in 10 ml of Luria–Bertani (LB) medium with 50 ␮g/ml of ampicillin. Plasmid DNA was extracted using the plasmid purification kit (QIAGEN, Venlo, Netherlands). After plasmid preparation, 5 ␮l (out of 30 ␮l) of each sample was treated with the restriction enzymes, EcoRI and NotI, to check for the presence of insert DNA. Only plasmids containing the expected insert size were used for the subsequent nucleotide sequencing analysis. Cycle sequencing reactions were carried out using a BigDye Terminator Cycle Sequencing kit Ver. 3.1 according to the manufacturer’s protocol (Applied Biosystems, CA, USA), and each sample was analyzed using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, CA, USA). The nucleotide sequence of the plasmid was compared with that of the Toxoplasma genome sequence in ToxoDB (http://toxodb.org/toxo/, gene ID; TGGT1 125960). The hydrophobicity of the deduced amino acid sequence was analyzed

SignalP

2.6. Generation of polyclonal anti-GRA22 antisera The polyclonal anti-GRA22 antisera were produced in Balb/c mice. Mice were immunized as described previously [16]. In brief, mice were immunized subcutaneously at three different inoculation sites with 10 ␮g of recombinant GRA22 protein emulsified with an equal volume of Freund’s complete adjuvant. Two weeks later, mice were immunized with the same dose of antigen emulsified with Freund’s incomplete adjuvant. On day 28, mice were immunized with one more dose of antigen with Freund’s incomplete adjuvant. Mice were sacrificed 7 days later under isoflurane anesthesia and blood for serum preparation was collected. All efforts were made to minimize suffering. The reactivity of the sera collected was tested using western blot and indirect immunofluorescence assay (IFA). 2.7. Western blot analysis T. gondii tachyzoite lysates were dissolved in SDS-PAGE sample buffer (62.5 mM Tris–HCl [pH 6.8], 2% (w/v) SDS, 140 mM 2mercaptoethanol, 10% (w/v) glycerol and 0.02% (w/v) bromophenol blue), heated at 95 ◦ C for 5 min and separated on a 6 or 10% polyacrylamide gel. All separated proteins were electrically transferred

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onto a polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore, MA, USA) using a Western blot apparatus (HorizeBlot Type AE-6677, ATTO Bioscience & Biotechnology, Tokyo, Japan). The separated proteins were probed with primary antibodies at 1:500 in 1× PBS containing 1% (w/v) skimmed milk (PBS-M). Bound antibodies were detected using anti immunoglobulin G (IgG) conjugated with horseradish peroxidase from sheep (GE Healthcare, Buckinghamshire, UK) diluted at 1:1000 in PBS-M. Peroxidase activity was revealed by using enhanced chemiluminescence (ECL) as described previously [17]. Molecular mass standards (SeeBlue plus2 Pre-stained standard – Life Technologies, CA, USA) were used. 2.8. Indirect immunofluorescence assay (IFA) The indirect immunofluorescence assay (IFA) was performed as described previously [18]. Parasites were inoculated onto glass coverslips with confluent monolayers of HFF cells and incubated in a 37 ◦ C CO2 incubator. After washing with PBS three times, the coverslips were fixed and permeabilized with 4% formaldehyde–0.2% Triton X-100 in PBS, pH 7.0, for 15 min. After washing with PBS three times, samples were blocked for 10 min in blocking solution (3% bovine serum albumin (BSA) fraction V in PBS, pH 7.0). Samples were then incubated for 1 h with polyclonal mouse anti-GRA22 antisera, polyclonal rabbit anti-GRA1 antisera, or polyclonal rabbit anti-SAG1 antisera diluted 1:500 in blocking solution. After washing three times with PBS, samples were incubated for 1 h with Alexa fluor 488 conjugated goat anti-mouse or Alexa fluor 594 conjugated goat anti-rabbit antibody diluted 1:1000 in blocking solution. After washing three times with PBS, samples were examined using a Leica TCS NT Confocal Laser Scanning Microscope (Leica Microsystems, Wetzlar, Germany). 2.9. Deletion of the gene encoding GRA22 For generation of a GRA22 deletion plasmid, upstream (3 kbp) and downstream (2.5 kbp) flanking regions of the GRA22 locus were amplified from T. gondii strain RH genomic DNA using primers in Table 1 and subcloned into pBluescript SK II vector. The HXGPRT expression cassette was subcloned between them. The GFP expression cassette was subcloned before the upstream DNA fragment for negative selection. One hundred ␮g of the final construct, pGRA22::HXGPRT, was transfected by electroporation into T. gondii strain RHhxgprt. For selection of transformants, the transfected parasites were grown in medium containing 25 ␮g/ml mycophenolic acid and 50 ␮g/ml xanthine. After 15 days of selection, the parasites were cloned by limiting dilution. GFP-negative parasites were isolated and the absence of the GRA22 gene was screened by PCR for the GRA22 coding region (Table 1, PCR1). Homologous recombination was examined by PCR using primers of the GRA22 flanking region (upstream 3.1 kbp, downstream 2.6 kbp) and inside of the HXGPRT expression cassette (Table 1, PCR2 and PCR3). Gene disruption was confirmed by IFA and western blot analysis using anti-GRA22 polyclonal antisera. 2.10. Analysis of the parasite growth kinetics Parasites (3.0 × 105 parasites per 100 mm tissue culture dish) were inoculated onto glass coverslips with confluent monolayers of HFF cells or Vero cells, and incubated in a 37 ◦ C CO2 incubator. The parasites were fixed and permeabilized (4% formaldehyde–0.2% Triton X-100 in PBS, pH 7.0, for 15 min) at indicated time points. Then the parasites were stained using anti-SAG1 polyclonal antisera (1:1000) and the number of parasites per PV were counted. Each time point represents the mean value of three experiments with a standard deviation. Data values were statistically analyzed using

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Student’s t-test. P-values < 0.05 were considered to be statistically significant.

2.11. Measurement of PV size Parasites (3.0 × 105 parasites per 100 mm tissue culture dish) were inoculated onto glass coverslips with confluent monolayers of HFF cells, and incubated in a 37 ◦ C CO2 incubator for 48 h. The parasites were fixed and permeabilized (4% formaldehyde–0.2% Triton X-100 in PBS, pH 7.0) for 15 min, then they were stained using antiSAG1 polyclonal antisera (1:1000) and PV size were investigated using a Leica TCS NT Confocal Laser Scanning Microscope (Leica Microsystems, Wetzlar, Germany). Data values were statistically analyzed using Student’s t-test. P-values < 0.05 were considered to be statistically significant.

2.12. Observation of parasite plaque Parasites (1.0 × 104 parasites per 100 mm tissue culture dish) were inoculated onto glass coverslips with confluent monolayers of HFF cells, and incubated in a 37 ◦ C CO2 incubator for 5 days. The parasites were fixed and permeabilized (4% formaldehyde–0.2% Triton X-100 in PBS, pH 7.0) for 15 min, then they were stained using anti-SAG1 polyclonal antisera (1:1000) and the plaque were observed.

2.13. Sandwich enzyme linked immunosorbent assay (ELISA) Parasites (1 parasite per well, 96-well plate) were inoculated with HFF cells and incubated in a 37 ◦ C CO2 incubator for 10 days. The MIC10 antigen in culture supernatant was measured using sandwich ELISA as described previously [16]. Data values were statistically analyzed using Student’s t-test. P-values < 0.05 were considered to be statistically significant.

2.14. GRA22 complementation assay One hundred ␮g of pGRA containing full length cDNA and 10 ␮g of plasmid containing the GFP and DHFR-TS expression cassette were co-transfected by electroporation into the GRA22 knock out strain. For selection of transgenic parasites, the transfected parasites were grown in medium containing 1 ␮g/ml pyrimethamine. After 15 days of selection, the transgenic parasites were cloned by limiting dilution. GRA22 expression was confirmed by western blot analysis using anti-GRA22 polyclonal antisera. Growth kinetics analysis was performed as mentioned above (see Section 2.10). Each time point represents the mean value of three experiments with a standard deviation. Data values were statistically analyzed using Student’s t-test. P-values < 0.05 were considered to be statistically significant.

2.15. Construction of deletion mutants, generation of transgenic parasites, localization and complementation assay For constructing the deletion mutant, the N-terminal half of GRA22 (GRA22N) cDNA was amplified using primers in Table 1, double digested with EcoRI and SalI and subcloned into EcoRI and XhoI restriction sites of pGRA. Tandem repeat deletion (GRA22TR) was made by PCR amplification of pGRA containing full length GRA22 cDNA as a template using the primers described in Table 1, digested with SfiI and self-ligated. Generation of transgenic parasites, IFA and growth kinetics analysis were performed as mentioned above.

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Fig. 1. Structure, molecular weight, and localization of GRA22. (A) Schematic diagram of GRA22. The RH strain GRA22 protein consists of 624 amino acids. The N-terminal SP means signal peptide sequence. The RH strain GRA22 has 12 unique tandem repeat sequences consisting each of 21 amino acids (K E K/N A/V S V A F/S Q G D D A R V L T S G/D E/K G/E) located between the 42nd and 293rd amino acid residues. The ME49 strain GRA22 lacks two copies of this repetitive sequence, and the Neospora caninum GRA22 ortholog completely lost this region. (B) Western blot analysis of the GRA22 protein. Anti-GRA22 polyclonal antisera detected a band at 85 and 80 kDa in T. gondii strains RH and ME49, respectively. (C) Localization of GRA22 protein in RH strain tachyzoite. Anti-GRA1 polyclonal antisera were used as the dense granule protein marker. Cells were fixed with formalin (upper panels) or cold methanol (lower panels), respectively. White scale bars are 5 ␮m.

2.16. Parasite egress assay Parasites (2.0 × 105 parasites per 100 mm tissue culture dish) were inoculated onto glass coverslips with confluent monolayers of HFF cells, and incubated in a 37 ◦ C CO2 incubator for 20 h. The medium was washed off twice with PBS and replaced with Hanks balanced salts solution (Life Technologies, CA, USA) containing 1 mM MgCl2 , 1 mM CaCl2 , 10 mM NaHCO3 , and 20 mM HEPES (pH 7.6). Then parasites were treated with A23187 (100 nM, Sigma–Aldrich, Dorset, UK) for 0, 2, 5, and 10 min. The solution was washed off twice with PBS and the parasites were fixed and permeabilized (4% formaldehyde–0.2% Triton X-100 in PBS, pH 7.0) for 15 min. Then the parasites were stained using anti-SAG1 polyclonal antisera (1:1000) and lysed vacuoles were counted. Each time point represents the mean value of three experiments with a standard deviation. Data values were statistically analyzed using Student’s t-test. P-values < 0.05 were considered to be statistically significant. 2.17. Parasite virulence assay The parasite virulence assay was performed as follows. Purified parasites (1.0 × 102 parasites) were injected intraperitoneally into 8-weeks old female Balb/c mice (parental strain: n = 15, gra22 strain: n = 14). The mice survival rate was measured every day. 2.18. Transmission electron microscopy Parasites were grown for 24 h in Vero cells, detached by a cell scraper, and spun down at 200 × g for 5 min. Cells were washed once in phosphate buffer (100 mM, pH 7.2) then fixed in 2% paraformaldehyde–2.5% glutaraldehyde (Nissin EM, Tokyo, Japan) in phosphate buffer for 1 h at 24 ◦ C. Cells were washed in phosphate buffer and post fixed in 1% osmium tetroxide (Nissin EM, Tokyo,

Japan) for 1 h. The cells were then rinsed extensively in dH2 O prior to en bloc staining with 1% aqueous uranyl acetate for 1 h. Following several rinses in dH2 O, samples were dehydrated in a graded series of ethanol and embedded in Quetol-812 resin (Nissin EM, Tokyo, Japan). Sections of 70–80 nm were cut, stained with uranyl acetate and lead citrate, and viewed on a Hitachi 7700 transmission electron microscope. 3. Results 3.1. Identification of the novel dense granule protein, GRA22 We isolated TGGT1 125960 cDNA encoding a protein of 624 amino acids, with a deduced molecular weight of 67.7 kDa and a calculated pI of 5.0. The encoded protein had a predicted signal peptide sequence at the N-terminal end and 12 unique tandem repeat sequences consisting of each 21 amino acids (K E K/N A/V S V A F/S Q G D D A R V L T S G/D E/K G/E) located between the 42nd and 293rd amino acid residues (Fig. 1A). A search of ToxoDB identified an ortholog in ME49 strain (TGME49 015220) and also in Neospora caninum (NCLIV 052190). Interestingly, TGME49 015220 lacked two copies of this repetitive sequence and NCLIV 052190 completely lacked this region. To examine the expression and subcellular localization, polyclonal antisera against recombinant TGGT1 125960 protein were raised and used for western blot analysis. The result showed that 85 and 80 kDa bands were detected in RH and ME49 strain total cell lysates, respectively (Fig. 1B). To identify subcellular localization, TGGT1 125960 antiserum was used for IFA where strong signals in the dense granules and PV which overlapped with GRA1 staining were found (Fig. 1C). This result indicated that TGGT1 125960encoded protein was a dense granule protein. Therefore, we named TGGT1 125960 as GRA22.

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3.2. Targeted disruption of the GRA22 gene To understand the function of GRA22, we disrupted the GRA22 gene by homologous recombination in RHhxgprt strain parasites (Fig. 2A). The GRA22 knock out plasmid (pGRA22::HXGPRT) was transfected into RHhxgprt strain parasites. The transfected parasites were selected with mycophenolic acid and xanthine, and cloned by limiting dilution. The GFP-negative parasites were then screened by PCR for the existence of the GRA22 coding region (Fig. 2A, PCR1). PCR analysis showed that the parental strain (Fig. 2B, lane wt) had an amplified 670 bp band, which was absent in a candidate of the GRA22 disruption parasite (Fig. 2B, lane KO). To confirm the successful homologous recombination, we performed PCR analysis using primers which annealed the GRA22 flanking region and HXGPRT expression cassette (Fig. 2A, PCR2 and PCR3). These primers were designed to amplify the DNA fragment only in knockout parasites. PCR analysis showed that the candidate GRA22 disruption parasite (Fig. 2C, lane KO) had an amplified band. On the other hand, the signals were absent in the parental strain (Fig. 2C, lane wt). Finally, we confirmed GRA22 disruption by western blot analysis (Fig. 2E) and IFA (Fig. 2F) using anti-GRA22 polyclonal antisera. GRA22 protein was detected in the parental strain (Fig. 2E, lane wt) but was undetected in the candidate GRA22 disruption parasite (Fig. 2E, lane KO). Similarly, the candidate strain showed the lack of GRA22 expression by IFA (Fig. 2F). These data indicate the successful generation of the GRA22 knockout (gra22) strain. 3.3. The GRA22 knockout strain showed early egress from host cells To examine whether deletion of GRA22 affects the parasite’s multiplication, we analyzed growth kinetics of the gra22 and parental strains. After infection to host cells, parasites were fixed at indicated time points and stained using anti-SAG1 polyclonal antisera. Then we counted parasite numbers per PV. The gra22 strain showed a similar growth rate as the parental strain until 24 h. This result suggests that deletion of GRA22 did not affect the parasite’s growth. However, an increase of the average parasite number of gra22 strain in PV was suppressed relative to the parental strain after 24 h (Fig. 3A). We considered that this result presumed two possibilities. One possibility was that the growth of gra22 strain had slowed down after 24 h. Another possibility was that the timing of egress in gra22 strain was earlier than in the parental strain, and that the egressed parasite re-infected surrounding host cells. To distinguish these possibilities, first we compared the growth rate by measuring PV size infection after 48 h (Fig. 3B). Small PVs, which seemed to be secondary infections, were excluded and the average size of large PVs were measured. The average PV sizes of the gra22 strain were the same as the parental strain. This data indicates that the growth of the gra22 and parental strain were similar after 24 h. Next, we calculated the ratio of PVs which contained only one parasite (Fig. 3C), and examined the ratio of parasite egress at each individual time point (Fig. 3D). The ratio of PVs which contained only one parasite in gra22 strain was elevated from 24 h onward, whereas, the ratio in the parental strain remained low (Fig. 3C). The ratio of parasite egress in the gra22 strain was significantly higher than that in the parental strain (Fig. 3D). To further confirm this phenotype, we observed parasite plaque size and parasite density infection after 5 days (Fig. 3E). The plaque size of gra22 and the parental strain was not significantly different. However, the parasite density in plaque formed by the gra22 strain seemed to be lower than that of the parental strain. To confirm this result, we measured the amount of MIC10 antigen in culture supernatant released from a plaque by sandwich ELISA (Fig. 3F),

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which is correlated with a parasite number [16]. First, we compared the expression level of MIC10 in parental and gra22 strain by Western blot analysis. The level of MIC10 in gra22 strain was same as parental strain (data not shown) and not affected by GRA22 deletion. Then we performed a sandwich ELISA. The result showed that the amount of MIC10 released from a plaque of gra22 strain was significant lower than that from parental strain (Fig. 3F). These data indicated that the parasite density in plaque formed by the gra22 strain was lower than that of the parental strain and the gra22 strain egressed before parasite number had fully increased. Taken together, the gra22 strain had an early egress phenotype, and growth rate was similar to that of the parental strain. To confirm the GRA22 knockout phenotypes, we performed complementation analysis of the gra22 strain. The GRA22 expression plasmid was transfected in the gra22 strain, and generated transgenic parasites. GRA22 expression was confirmed by western blot analysis using anti-GRA22 antisera (Fig. 3G). Then we analyzed the growth kinetics of those parasites at indicated time points (Fig. 3A and C). The growth kinetics of GRA22-complemented gra22 strains were similar to that of the parental strain. These data suggest that GRA22 was involved in regulating parasite egress.

3.4. Determination of the GRA22 functional region GRA22 has unique tandem repeat sequences in the N-terminal half region. To determine the GRA22 functional domain, we constructed two deletion mutants of GRA22 (Fig. 4A), one was the N-terminal half which contained unique tandem repeat sequences (GRA22N) while the other was the deletion of unique tandem repeat sequences (GRA22TR), and generated transgenic parasites in a GRA22 knockout background. Expression was confirmed by western blot analysis using anti-GRA22 antisera (Fig. 4B). Then we analyzed the localization of deletion mutants (GRA22N and GRA22TR) and the growth kinetics of those parasites at indicated time points (Fig. 4C–E). The localization of these mutant proteins was similar to that of endogenous GRA22. The growth kinetics of the GRA22N transgenic strain did not result in the recovery of the early egress phenotype, and the percentage of PVs which contained only one parasite was higher than the gra22 strain. In contrast, the growth kinetics of the GRA22TR transgenic strain was partially recovered. These results suggest that the GRA22 C-terminal region contributes to the function of GRA22.

3.5. Calcium ionophore-induced parasite egress assay Intracellular calcium signaling was associated with parasite egress. So we performed a calcium ionophore-induced parasite egress assay for parental, gra22, and GRA22-complemented gra22 strain (Fig. 5). The timing of egress in the gra22 strain was significantly faster than that in the parental strain. On the other hand, GRA22-complemented gra22 strains showed similarity to the parental strain.

3.6. GRA22 was not essential for virulence To investigate whether this early egress phenotype affected parasite virulence, we intraperitoneally injected parental or gra22 strain into mice (Fig. 6A). However, the survival rate was not significantly difference between mice infected with these two strains. This data suggests that GRA22 was not essential for virulence in vivo.

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Fig. 2. Targeted disruption of the GRA22 gene. (A) Schematic diagram of the GRA22 gene knock out strategy. Using homologous recombination, the GRA22 coding region was replaced with the selectable marker HXGPRT. PCR1, PCR2 and PCR3 were the primers used to confirm GRA22 deletion. (B) PCR analysis of the parental and gra22 parasite. No DNA amplification of the coding region (PCR1) was observed in the gra22 strain. (C) PCR analysis of the parental and gra22 parasite. Amplified DNA bands between the GRA22 flanking region and HXGPRT expression cassette (PCR2 and PCR3) were observed in the gra22 strain. (D) PCR analysis of the parental and gra22 parasite. The actin coding region was used as a PCR control. (E) Western blot analysis of parental and gra22 parasite lysates. The GRA22 protein was not present in gra22 lysates. Actin was used as the loading control (lower panel). (F) Immunostaining of GRA22 protein in parental and gra22 parasite tachyzoite. Anti-SAG1 polyclonal antisera were used for counter staining. GRA22 was not visible in the gra22 strain.

3.7. Electron microscopic analysis of the GRA22 knockout strain In general, dense granule proteins participate in the modification of PV and the PV membrane [8]. The secreted dense granule proteins target the vacuolar space or are associated with the PV membrane or decorate the intravacuolar network of the membraneous structure within the PV [19]. Dense granule protein knockout parasites often do not display any ultrastructural defect in the PV or the PV membrane. So we performed electron microscopic analysis, focused on the PV membrane region and intravacuolar network of parental and gra22 strain. However,

no gross (Fig. 6B).

changes

were

observed

in

the

gra22

strain

4. Discussion In this study, we identified a novel dense granule protein, GRA22, and performed disruption of the GRA22 gene to understand the function of GRA22. GRA22 (TGGT1 125960) was originally isolated by yeast two-hybrid screening using one cyclin motif containing cDNA as bait. However, physiological and functional

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Fig. 3. Early egress phenotype of the gra22 strain. (A) The graph shows the average number of parasites per PV at the indicated time points. The closed triangle with a line shows the parental strain (parent), the closed square with a line shows the gra22 strain (gra22), and the closed circle with a dashed line shows the GRA22-expressed gra22 strain (gra22 +GRA22). Each time point represents the mean value of three experiments with a standard deviation. *P < 0.05. (B) The graph shows average sizes of large PVs infection after 48 h. Small PVs which seemed to be secondary infections were excluded. The white bar shows the parental strain while the black bar shows the gra22 strain. The graph shows the PV major axis (left) and the PV minor axis (right). Each point represents the mean value with a standard deviation. *P < 0.05. (C) The graph shows the percentage of PV which contains one parasite. The closed triangle with a line shows the parental strain (parent), the closed square with a line shows the gra22 strain (gra22), and the closed circle with a dashed line shows the GRA22-expressed gra22 strain (gra22 +GRA22). Each time point represents the mean value of three experiments with a standard deviation. *P < 0.05. (D) The graph shows the percentage of parasite egress. The white bar shows the parental strain, and the black bar shows the gra22 strain. Each time point represents the mean value of three experiments with a standard deviation. *P < 0.05. (E) Parasite plaque was stained with anti-SAG1 antibody. The photos show the parental strain (left) and the gra22 strain (right). Differences in parasite density are visible. (F) A sandwich ELISA result shows that the amount of MIC10 released from a plaque of the gra22 strain is significant lower than that of the parental strain. The vertical axis shows relative optical density (OD) at 415 nm. Negative control represents no parasite plaque in well. Each column represents the mean value with a standard deviation. *P < 0.05. (G) Western blot analysis of parental (RH), gra22 (none) and GRA22-expressed gra22 (+GRA22) parasite lysates. The GRA22 protein is not present in gra22 lysates. Actin was used as the loading control (lower panel).

interactions between them remain unclear and will be the subject of a future study. TGGT1 125960 is deposited in ToxoDB and 1st methionine is predicted to be a translation initiation codon. However, our investigation using transgenic parasites suggested 3rd methionine as a translational initiation codon (Figs. S1 and S2). Therefore, we conclude that GRA22 from RH strain encodes 624 amino acids (Fig. 1A). The RH strain GRA22-deduced molecular weight was 67.7 kDa. However, our western blot detected the RH strain GRA22 band at 85 kDa (Fig. 1B). The expression of GRA22 cDNA in the gra22

parasite demonstrated a band that also migrated to 85 kDa (Figs. 3G and S2). This result indicates that the identified cDNA contained the entire coding region. The difference between GRA22 protein migration and predicted GRA22 molecular mass may be due to a feature in the protein sequence and/or post translational modification. GRA22 has unique tandem repeat sequences consisting each of 21 amino acids. Similarly, some toxoplasma rhoptry proteins such as rhoptry protein 1 (ROP1) [20] or toxolysin-1 (TLN1) [21] have unique tandem repeat sequences and their protein showed slower migration in SDS-PAGE. Frequently, the unbalanced

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Fig. 4. Complementation analysis of the GRA22 deletion mutant. (A) Schematic diagram of the deletion mutant of GRA22 (GRA22N and GRA22TR). GRA22N consists of the N-terminal half of GRA22 containing a tandem repeat sequence. GRA22TR has a deletion of the tandem repeat sequence GRA22. (B) Western blot analysis of the deletion mutant strains. Deletion mutant of GRA22 protein, GRA22N and GRA22TR, were detected at 54 kDa and 46 kDA, respectively. Actin was used as loading control (lower panel). (C) Localization of deletion mutant proteins in GRA22 knockout tachyzoite. GFP was used as transfection marker. White scale bars are 5 ␮m. (D) The graph shows the average number of parasites per PV at the indicated time points. The closed triangle with a dashed line shows the parental strain (parent), the closed square with a dashed line shows the gra22 strain (gra22), the closed rhombus with a line shows the GRA22N-expressed gra22 strain (gra22 +GRA22N), and the closed circle with a line shows the GRA22TR-expressed gra22 strain (gra22 +GRA22TR). Each time point represents the mean value of three experiments with a standard deviation. *P < 0.05. (E) The graph shows the percentage of PVs which contain one parasite. The closed triangle with a dashed line shows the parental strain (parent), the closed square with a dashed line shows the gra22 strain (gra22), the closed rhombus with a line shows the GRA22N-expressed gra22 strain (gra22 +GRA22N), and the closed circle with a line shows the GRA22TR-expressed gra22 strain (gra22 +GRA22TR). Each time point represents the mean value of three experiments with a standard deviation. *P < 0.05.

parent ΔGRA22 ΔGRA22 +GRA22

% of lysed vacuoles

100

*

80

*

60 40 20 0 0

2

4

6

8

10

minutes Fig. 5. Ca2+ ionophore-induced parasite egress assay of gra22. The graph shows the percentage of lysed vacuoles. The closed triangle with a line shows the parental strain (parent), the closed square with a line shows the gra22 strain (gra22), and the closed circle with a dashed line shows the GRA22-expressed gra22 strain (gra22 +GRA22). Each time point represents the mean value of three experiments with a standard deviation. *P < 0.05.

presence of some amino acids delays the electrophoretic mobility of proteins in SDS-PAGE [22,23]. In this study, we constructed an expression plasmid of two deletion mutants of GRA22 (GRA22N and GRA22TR) (Fig. 4A), and generated transgenic parasites in a GRA22 knockout background. The deduced molecular weight of these GRA22 deletion mutants (GRA22N and GRA22TR) was about 38 and 46 kDa, respectively. Our western blot analysis showed the GRA22N band was detected at 54 kDa, whose migration was slower than the predicted size (Fig. 4B). On the other hand, the GRA22TR band was detected at 46 kDa as expected (Fig. 4B). Moreover, GST fused recombinant protein containing 8 tandem repeats produced in E. coli showed slower migration at 56 kDa than the predicted size (46 kDa) by SDS-PAGE analysis (Fig. S3). Our result suggested that the slower migration of the GRA22 protein was caused by tandem repeat sequences. Highly virulent type I strains such as RH and GT1 strain, and avirulent type III strains such as VEG strain, GRA22 possess 12 tandem repeat sequences. On the other hand, low virulent type II strains such as ME49 strain, GRA22 lack two copies of this repetitive sequence (Fig. 1A). In fact, Western blot analysis showed that GRA22 of ME49 was detected as an 80 kDa band, which was slightly smaller than that of the RH strain (Fig. 1B). Considering the possible involvement of GRA22 in parasite virulence, we performed the intraperitoneal injection of the parental or gra22 strain into

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Fig. 6. GRA22 deletion did not affect parasite virulence and ultrastructure. (A) The graph shows the survival rate of parental or gra22 strain-infected mice. The closed circle with a dashed line shows the parental strain-infected mice (n = 15) and the closed square with a line shows the gra22 strain-infected mice (n = 14). The survival rate was not significantly different between mice infected with these two strains. (B) Electron microscopic analysis of parental (left) and gra22 strain (right). P, PVM, and IVN means parasite, PV membrane, and intravacuolar tubular network, respectively. No gross changes were observed between parental and gra22 strains.

Balb/c mice (Fig. 6A). Their survival rates were not significantly different, so we concluded that GRA22 was not essential for virulence. However, type I RH strain has extremely high virulence. The different phenotype might be seen in a lower virulence strain or in the bradyzoite stage. The gra22 strain showed earlier egress than the parental strain, and GRA22 expression complemented this phenotype (Fig. 3A and C). These results suggest that GRA22 was involved in regulating T. gondii egress. The mechanism of this early egress phenotype is still not clear at the molecular level. Some previous studies reported that elevated Ca2+ was associated with parasite egress from the host cells [24]. T. gondii deficient in Perforin-like protein 1, a calcium-dependent secreted microneme protein, loses the ability to permeabilize the PV membrane and host cell membrane during egress [25]. So we performed a Ca2+ ionophore-induced parasite egress assay of parental, gra22, and GRA22-complemented gra22 strain (Fig. 5). Our results show that the timing of egress in the gra22 strain was significantly faster than that in the parental and GRA22-complemented gra22 strain. The early egress phenotype of the gra22 strain might be associated with Ca2+ sensitivity. In this study, we identified a novel dense granule protein, GRA22, and generated and characterized the gra22 strain. We demonstrated that the gra22 strain showed early egress. However, the regulatory mechanisms of the parasite egress, and/or the function of GRA22 remain unknown. A further study to assess the function of GRA22 in T. gondii is thus needed. Acknowledgements This work was supported in part by Grants-in-Aid for Regional R&D Proposal-Based Program from the Northern Advancement Center for Science & Technology of Hokkaido, Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molbiopara. 2013.04.005. References [1] Black MW, Boothroyd JC. Lytic cycle of Toxoplasma gondii. Microbiology and Molecular Biology Reviews 2000;64:607–23. [2] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. International Journal for Parasitology 2000;30:1217–58. [3] Luft BJ, Remington JS. Toxoplasmic encephalitis in AIDS. Clinical Infectious Diseases 1992;15:211–22. [4] Wong SY, Remington JS. Biology of Toxoplasma gondii. AIDS 1993;7:299–316. [5] Montoya JD, Liesenfeld O. Toxoplasmosis. Lancet 2004;363:1965–76.

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