EUKARYOTIC CELL, Nov. 2011, p. 1403–1412 1535-9778/11/$12.00 doi:10.1128/EC.05117-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 10, No. 11
A GCN2-Like Eukaryotic Initiation Factor 2 Kinase Increases the Viability of Extracellular Toxoplasma gondii Parasites䌤† Christian Konrad,1,2 Ronald C. Wek,2 and William J. Sullivan, Jr.1,3* Departments of Pharmacology and Toxicology,1 Biochemistry and Molecular Biology,2 and Microbiology and Immunology,3 Indiana University School of Medicine, Indianapolis, Indiana 46202 Received 15 May 2011/Accepted 2 September 2011
Toxoplasmosis is a significant opportunistic infection caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular pathogen that relies on host cell nutrients for parasite proliferation. Toxoplasma parasites divide until they rupture the host cell, at which point the extracellular parasites must survive until they find a new host cell. Recent studies have indicated that phosphorylation of Toxoplasma eukaryotic translation initiation factor 2-alpha (TgIF2␣) plays a key role in promoting parasite viability during times of extracellular stress. Here we report the cloning and characterization of a TgIF2␣ kinase designated TgIF2K-D that is related to GCN2, a eukaryotic initiation factor 2␣ (eIF2␣) kinase known to respond to nutrient starvation in other organisms. TgIF2K-D is present in the cytosol of both intra- and extracellular Toxoplasma parasites and facilitates translational control through TgIF2␣ phosphorylation in extracellular parasites. We generated a TgIF2K-D knockout parasite and demonstrated that loss of this eIF2␣ kinase leads to a significant fitness defect that stems from an inability of the parasite to adequately adapt to the environment outside host cells. This phenotype is consistent with that reported for our nonphosphorylatable TgIF2␣ mutant (S71A substitution), establishing that TgIF2K-D is the primary eIF2␣ kinase responsible for promoting extracellular viability of Toxoplasma. These studies suggest that eIF2␣ phosphorylation and translational control are an important mechanism by which vulnerable extracellular parasites protect themselves while searching for a new host cell. Additionally, TgIF2␣ is phosphorylated when intracellular parasites are deprived of nutrients, but this can occur independently of TgIF2K-D, indicating that this activity can be mediated by a different TgIF2K. tional control to remain viable during the times it must persist without host cells (18). Toxoplasma can cause congenital birth defects, ocular disease, and life-threatening opportunistic infection (46). Current treatments consist of antifolates, which are problematic due to toxicity issues; therefore, there is an urgent need to develop novel therapies to treat this parasitic infection (6). Phosphorylation of eIF2␣ has recently been shown to be critical during multiple phases of the life cycle of apicomplexan parasites (8, 17, 18, 27, 51). We generated a Toxoplasma mutant that no longer phosphorylates TgIF2␣ by mutating the regulatory serine (Ser71) to alanine (18). The TgIF2␣-S71A mutant suffered a significant fitness defect in vitro and in vivo because the mutants were more susceptible to extracellular exposure. Toxoplasma expresses four putative eIF2␣ kinases designated TgIF2K-A to -D, and the underlying protein kinase mediating this translational control in response to extracellular stress has not yet been identified (18, 27). In this study, we hypothesized that extracellular parasites endure nutrient deprivation through the activity of a GCN2 orthologue. We show that TgIF2K-D is a GCN2-like eIF2␣ kinase in Toxoplasma. Through the generation of TgIF2K-D mutants, TgIF2K-D was shown to enhance the viability of parasites during times when they are deprived of their host cells. Parasites with a knockout of TgIF2K-D are unable to phosphorylate TgIF2␣ and initiate translational control in response to extracellular stress, phenocopying the TgIF2␣-S71A mutant (18). Intracellular parasites also phosphorylate TgIF2␣ during nutrient deprivation, but this can occur independently of TgIF2K-D, suggesting the involvement of another TgIF2K. This study indicates that TgIF2K-D is the eIF2␣ kinase facil-
The ability to rapidly respond to stress is essential for cellular survival. Translational control through the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2␣) is a well conserved mechanism used by cells to repress global protein synthesis during times of nutrient scarcity (14, 48). In a GTP-driven process, eIF2 delivers the initiator tRNA to the translational machinery. When GCN2 phosphorylates eIF2␣ at its regulatory serine residue, this translation initiation factor becomes an inhibitor of its guanine nucleotide exchange factor, eIF2B, which results in reduced levels of the eIF2-GTP-MettRNAi ternary complex that are accessible to the translational machinery. Consequently, general protein synthesis is diminished, which conserves cellular resources and provides time for the cell to reprogram its genome to adapt to the stress. In addition to the GCN2 protein kinase that is activated during nutritional starvation, other eIF2␣ kinases respond to different stress conditions (41, 48). These protein kinases include PERK/PEK, activated by endoplasmic reticulum (ER) stress, and HRI and PKR, which respond to heme depletion and viral infection, respectively. We have previously shown that the obligate intracellular parasite Toxoplasma gondii (phylum Apicomplexa) relies on phosphorylation of eIF2␣ (designated TgIF2␣) and transla-
* Corresponding author. Mailing address: Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, MS A-525, Indianapolis, IN 46202. Phone: (317) 2741573. Fax: (317) 274-7714. E-mail:
[email protected]. † Supplemental material for this article may be found at http://ec .asm.org/. 䌤 Published ahead of print on 9 September 2011. 1403
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itating translational control in extracellular parasites, enhancing the survival of Toxoplasma as it searches for a new host cell. MATERIALS AND METHODS Parasite culture. Toxoplasma tachyzoites were maintained in human foreskin fibroblasts (HFFs) in Dulbecco modified Eagle’s medium (DMEM) containing 25 mM glucose and 4 mM glutamine (Invitrogen) and supplemented with 1% heat-inactivated fetal bovine serum (Gibco/Invitrogen) at 37°C and 5% CO2. Cloning of the TgIF2K-D cDNA. Tachyzoite mRNA was used to generate a cDNA library (Omniscript; Qiagen) for the amplification of the TgIF2K-D open reading frame (ORF). This PCR amplification employed primers specific to the TgIF2K-D gene that was annotated in the Toxoplasma database (www.toxodb .org, TgME49_119610). Tachyzoite mRNA was reverse transcribed using the SuperScript one-step reverse transcription-PCR (RT-PCR) kit (Invitrogen) with random and oligo(dT) primers according to the manufacturer’s recommendations. All PCRs were carried out with Phusion DNA polymerase (Finnzymes) using the provided GC buffer. The GeneRacer kit (Invitrogen) was used for the 5⬘ and 3⬘ rapid amplification of cDNA ends (RACE) of the TgIF2K-D gene. Generation of TgIF2K-D knockout parasites. To generate TgIF2K-D knockout parasites (⌬if2k-d), we amplified ⬃1.5-kb DNA fragments upstream and downstream of the start and stop codons carried in the TgIF2K-D locus. Oligonucleotide primers used to amplify the 5⬘ flanking sequence were designated 1 and 2, and the primers used to amplify the 3⬘ flanking sequence were designated 3 and 4 (see Table S1 in the supplemental material). The amplified DNA was inserted into the pDHFR*-TSc3 vector (35), such that these fragments flanked opposing ends of a modified dihydrofolate reductase-thymidylate synthase (DHFR*-TS) minigene, which confers resistance to pyrimethamine. The resulting knockout vector was designated ⌬TgIF2K-D::DHFR*. Fifty micrograms of the ⌬TgIF2K-D::DHFR* knockout vector was linearized with NotI and transfected into RH strain parasites lacking Ku80 (10, 15) as described previously (35). Transfected parasites were grown in HFF cells in the above-defined DMEM supplemented with 1 M pyrimethamine and cloned by limiting dilutions. Individual parasite clones were screened by PCR to confirm the replacement of the TgIF2K-D genomic locus with the DHFR*-TS minigene. To confirm that the correct insertion occurred at the TgIF2K-D locus, primers complementary to the 3⬘ untranslated region (3⬘-UTR) of the DHFR* minigene (primer 10) and upstream of the insertion site (primer 9) were used in a PCR assay with genomic DNAs purified from the candidate knockout parasites. PCR assays using primers 5 and 6, which are complementary to exon III, and primers 7 and 8, which were used to generate the genetic tagging vector (see below), were carried out to verify the absence of the complete TgIF2K-D genomic locus. Loss of TgIF2K-D mRNA expression was verified by RT-PCR using primers complementary to sequences upstream (primer 11) and downstream (primer 12) of the encoded protein kinase domain. As control, a portion of Toxoplasma actin (TgME49_009030) mRNA was amplified by RT-PCR using primers 13 and 14. Genetic tagging of TgIF2K-D. For the expression of TgIF2K-D tagged with hemagglutinin (HA) at its C terminus, a 1.2-kb DNA fragment containing exon XVIII was amplified using Toxoplasma genomic DNA as the template and primers 7 and 8. The amplified DNA segment was then inserted into the vector 3⫻HA-LIC-DHFR-TS using the ligation-independent cloning method (43). LIC-HA3⫻-DHFR-TS is a derivative of pYFP-LIC-DHFR (15) in which the yellow fluorescent protein (YFP)-coding fragment had been replaced with three contiguous HA tags. Fifty micrograms of the TgIF2K-D-HA3⫻ plasmid was linearized with the restriction enzyme AscI and then transfected into RH⌬ku80 parasites. Following limiting dilutions, positive clones were identified using a monoclonal antibody that specifically recognizes the HA tag (Roche). The 1.2-kb DNA fragment containing exon XVIII (amplified with primers 7 and 8) was also ligated into a LIC-HA2⫻-DD-DHFR-TS vector (15) to generate a TgIF2K-D fusion with 2⫻HA and a Shield-regulated destabilization domain (DD) at the C terminus (2⫻DD). Following transfection of this linearized plasmid, individual parasite clones were screened for the stabilization of TgIF2KD2⫻DD in the presence of Shield-1 (500 nM; Clontech) using the anti-HA monoclonal antibody. Parasite motility and invasion assays. Parasites of the designated strain were released from host cells via syringe passage and filter purified as described above. Motility of extracellular tachyzoites was assessed by monitoring deposits of SAG1 antigen as described previously (4). The number of parasites with antigen trails was determined in 10 random microscopic fields in 3 independent experiments. Adhesion and invasion assays were performed using differential red/ green staining for SAG1 as described previously (16). Attached and invaded tachyzoites were determined in 15 random microscopic fields, and data from 6 independent experiments were compiled.
EUKARYOT. CELL Comparative fitness assay. The comparative fitness assay was carried out as described previously by Joyce et al. (18), with the exception that SYBR greenbased quantitative real-time PCR (qPCR) was performed using primers that specifically delineated between parental ⌬Ku80 parasites, referred to as wild type (WT), and ⌬if2k-d parasites. In brief, equal numbers of filter-purified parental and ⌬if2k-d parasites (5 ⫻ 105) were cocultured in the same flask of HFF host cells. At 72 h postinfection, 105 parasites of the mixed population were isolated from the lysed culture and then transferred to a fresh HFF monolayer for an additional 72 h. This resulted in a total of 6 days of HFF infection by using two serial passages. Genomic DNA (gDNA) from the parasite samples was isolated using the DNeasy kit (Qiagen) and used in qPCR assays. Primers used to distinguish WT from ⌬if2k-d parasites included primers 15 and 16 and primers 17 and 18, as indicated. qPCR measurements were normalized by amplifying the 5⬘-UTR of TgIF2K-D, which is present in both WT and ⌬if2k-d parasites (primers 19 and 20). Twenty-five nanograms of gDNA was used in the qPCR assays, which were performed in triplicate using the 7500 real-time PCR system (Applied Biosystems). Relative quantification software (SDS software, version 1.2.1) was used for the analysis. As a specificity control, SYBR green assays employing gDNA purified from either WT or ⌬if2k-d parasites were carried out to verify the specificity of primers in the qPCR assay (data not shown). Parasite proliferation assays. Toxoplasma recovery from extracellular stress was analyzed using standard doubling and plaque assays (35). Parental ⌬ku80 (WT), ⌬if2k-d, and TgIF2␣-S71A (18) parasites were physically released from host cells by syringe passage and then filter purified to remove host cell debris. A total of 106 parasites were subjected to an extracellular stress assay for 0, 8, or 10 h in culture medium at 37°C and 5% CO2 without host cells prior to infecting HFF host cells, as described previously (18). Parasites were quantitated using a standard counting assay, with counts performed every 8 h postinfection. Parasite counting assays were carried out in triplicate using separate biological samples, and results of a representative experiment are shown. In the plaque assays, 500 WT, ⌬if2k-d, TgIF2␣-S71A, or TgIF2K-D2xDD parasites were used to infect HFF monolayers in 12-well plates following extracellular incubation for up to 10 h, as indicated. The degree of host cell lysis at 7 days postinfection was determined by crystal violet staining of methanol-fixed cells. Measurements of the lysed areas were done using an Alpha Innotech imaging system, and results of a representative experiment of three independent experiments are presented. Analysis of nutrient starvation of intracellular Toxoplasma. To deprive intracellular Toxoplasma of nutrients, we employed a method recently developed by Anthony Sinai (University of Kentucky, unpublished). For these experiments, HFF cells and parasites were maintained in alpha minimal essential medium (␣MEM)–7% fetal bovine serum (FBS)–2 mM glutamine (complete medium [CM]) (Gibco). Tachyzoites (106) were allowed to infect HFF monolayers in T-25 flasks for 24 h, at which point medium and extracellular parasites were removed by washing with prewarmed Hank’s balanced salt solution (HBSS) lacking glucose. Nutrient starvation was induced by incubating infected HFFs in CM diluted to a 6% final concentration as the starvation medium (6% SM) for 8 h; control flasks were incubated in CM. These conditions do not overtly affect the viability of HFF host cells or induce host cell autophagy (Anthony Sinai, personal communication). Following the 8-h incubation in diluted medium, the infected monolayer was washed with ice-cold HBSS and parasites were harvested by syringe passage and filter purified. Analysis of TgIF2␣ phosphorylation was performed by immunoblotting as described previously (44). To monitor the recovery from intracellular nutrient deprivation, 6%-SM was replaced with CM at 4 or 8 h after exposure. The number of parasites per vacuole was counted 24 and 36 h later. Western blotting. Western blot analyses of TgIF2␣ phosphorylation were carried out as previously described (18, 27). To analyze the stabilization of the TgIF2K-D2xDD protein, intracellular parasites were grown for up to 24 h in medium supplemented with 500 nM Shield-1 prior to physical release from the host cells. HA-tagged proteins were detected by Western blot analyses after parasite lysates were resolved on a 3 to 8% Tris-acetate polyacrylamide gradient gel. A rat monoclonal antibody that specifically recognizes the HA tag (Roche) was used as a primary antibody, and an anti-rat IgG antibody conjugated with horseradish peroxidase (GE Healthcare) was used as a secondary antibody. HA-tagged proteins were visualized using a chemiluminescence Western blotting substrate (Pierce). Measurements of protein synthesis. Intracellular parasites were mechanically released from host cells as described above, and 2.5 ⫻ 107/ml tachyzoites were transferred into Toxoplasma culture medium lacking methionine and cysteine. Labeling was initiated by adding [35S]Cys/Met label (ICN) to a final concentration of 200 Ci/ml. After 1 h of incubation in DMEM at 37°C and 5% CO2, samples were immediately put on ice. Parasites were harvested by centrifugation at 4°C, and cell pellets were washed twice with ice-cold phosphate-buffered saline
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(PBS) and then lysed in 100 l RIPA buffer (44). Uptake of the 35S during the 1-h pulse radiolabeling was similar for the WT and mutant parasites. For each sample, equal amounts of proteins were precipitated by adding trichloroacetic acid (TCA) to a final concentration of 10%. After incubation on ice for 30 min, samples were collected by centrifugation at 10,000 ⫻ g for 30 min at 4°C. The TCA precipitates were washed twice with acetone and resuspended in an equal volume of PBS. Incorporation of the radiolabeled amino acids was determined using a scintillation counter. Results of all radiolabeling experiments are presented as averages for three independent samples, with P values and standard errors determined using analysis of variance (ANOVA). Immunofluorescence assays. HFF monolayers were grown on coverslips, infected for 24 h, and then fixed in 3% paraformaldehyde. Immunofluorescence analyses using a rat monoclonal antibody that recognizes the HA tag (Roche) followed by goat anti-rat Alexa Fluor 488 as the secondary antibody (Invitrogen) was performed as previously described (29).
RESULTS Characterization of the GCN2-like kinase TgIF2K-D. The predicted gene TgME49_119610 (ToxoDB.org) was previously designated TgIF2K-D and is suggested to encode an orthologue of GCN2 (27), the eIF2␣ kinase that is well-documented as a responder to nutrient starvation stress in other species (14, 48). We used RT-PCR to identify and characterize the fulllength TgIF2K-D cDNA. Our analysis revealed a predicted TgIF2K-D product consisting of 2,729 amino acid residues (GenBank accession number JF827031), which modifies the predicted sequence for TgME49_119610 due to a discrepancy at the exon 3/intron 3 boundary. The predicted start codon for the TgIF2K-D ORF matches the consensus sequence for translation initiation in Toxoplasma (39) and is preceded by an in-frame stop codon. RACE analyses indicated a 5⬘ untranslated region (5⬘-UTR) of 2,151 bp, which is consistent with the transcriptional start site (TSS) derived from the Full-parasites database (49) and chromatin immunoprecipitation-on chip (ChIP-Chip) data available in the ToxoDB, and a 3⬘-UTR of ⬃1,000 bp (see Fig. S1 in the supplemental material). The 5⬘-UTR was further validated by RT-PCR using primers flanking the TSS (see Fig. S2 in the supplemental material). An alignment between TgIF2K-D and the eIF2␣ kinases from multiple species was compiled using BLAST and CLUSTALW (see Fig. S3 in the supplemental material). TgIF2K-D (residues 1,318 to 1,630) has the central features characteristic of eIF2␣ kinases, including an insert between subdomains IV and V (Fig. 1; see Fig. S3 in the supplemental material). As judged by BLAST analyses, this portion of TgIF2K-D is most closely related to putative eIF2␣ kinases from the parasites Plasmodium falciparum (AAN37036; 4e⫺14) and Trypanosoma brucei (XP_828792.1; 6e⫺10) (25), followed by characterized GCN2 orthologues from Arabidopsis thaliana (CAD30860; 6e⫺32) (52), Drosophila melanogaster (AAC13490; 8e⫺27) (28), Schizosaccharomyces pombe (AAU11313; 2e⫺25) (50), and Saccharomyces cerevisiae (AAA34636; 1e⫺22) (47). Another hallmark feature of GCN2 is an RWD domain, which is present between residues ⬃800 and ⬃1000 of TgIF2K-D, with a significance of 4e⫺6 as determined by the motif search program Pfam (9, 26) (see Fig. S4 in the supplemental material). The RWD in GCN2 from S. cerevisiae was reported to directly bind to the activator protein GCN1 (14, 26), and residue changes in GCN2 that blocked this binding, or abolition of the GCN2/GCN1 association by GCN1 binding with another RWD-containing protein, YIH1, blocked
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FIG. 1. Domain structure of TgIF2K-D. TgIF2K-D contains a protein kinase domain (black boxes) with an insert (I) characteristic of eIF2␣ kinases and signature regulatory regions, including the RWD domain (dark gray) and a proposed HisRS-related region (light gray). The conserved C-terminal homology (C-term) domain is denoted with a stippled box. The numbers below the diagram demarcate the amino acid residues for each of the domains of TgIF2K-D. A diagram of mouse GCN2 (MmGCN2) is shown for comparison.
GCN2 phosphorylation of eIF2␣ in yeast depleted of amino acids (13, 14, 36, 37). Toxoplasma also has a predicted GCN1 orthologue (TGME49_031480) and a YIH1-related protein (TGME49_112350), supporting the idea that this network functions to regulate a GCN2-related eIF2␣ kinase in this parasite. The sequences of the histidyl-tRNA synthetase (HisRS) domain, which stimulates eIF2␣ kinase activity by binding to uncharged tRNAs accumulating during nutrient deprivation (14), appears to be less well conserved in the protozoan GCN2like kinases. Analysis of the sequences flanking the C-terminal end of the protein kinase domain (residues 1750 to 2360) identified the PRGGRVY2299 sequence as the closest match to the histidine B sequence (AAGGRYD), which is characteristic for the HisRS-related domains (42). This weaker conservation of the HisRS-related sequences is a feature shared with other GCN2-related protein kinases from apicomplexans, including P. falciparum (8). TgIF2K-D also lacks the pseudokinase domain found in mammalian and yeast GCN2s, which is thought to contribute to the eIF2␣ kinase activity (33). The C terminus of GCN2 is important for dimerization and ribosome association (14, 48), and this region in TgIF2K-D (residues 2436 to 2499) is rich in hydrophobic and basic residues, which are suggested to contribute to these regulatory processes in this eIF2␣ kinase. Interestingly, this region shares sequence identity with GCN2-like kinases encoded in the apicomplexans Neospora caninum (NCLIV_010550, 3e⫺30), Cryptosporidium muris (CMU_027700; 0.011), Plasmodium falciparum (PF14_ 0264; 9e⫺08), Plasmodium berghei (PBANKA_101620, 4.8e⫺08), Plasmodium knowlesi (PKH_113740, 1.1e⫺07), and Plasmodium vivax (PVX_085120; 2e⫺07) (see Fig. S5 in the supplemental material). We designated this conserved region the C-terminal homology (CTH) region (Fig. 1). Based on the presence of sequences related to the eIF2 kinases juxtaposed to the signature RWD domain, a putative histidine B-like sequence, and a C terminus rich in hydrophobic and basic residues, TgIF2K-D is suggested to be a parasite orthologue of GCN2 (Fig. 1). We therefore hypothesize that
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FIG. 2. Size and localization of TgIF2K-D. (A) TgIF2K-D3⫻HA protein was detected by Western blotting by probing parasite lysates with anti-HA antibody. Densitometry values for the slowest-migrating protein (upper arrow) are listed below the anti-HA blot. The fastermigrating TgIF2K-D3⫻HA variants (lower arrow) diminished when parasites were subjected to extracellular stress for 4 or 8 h. Samples were normalized in the immunoblot analysis using antibody specific for Toxoplasma tubulin. (B) Western blot of TgIF2K-D3⫻HA parasites with antibodies specific for total TgIF2␣ or phosphorylated TgIF2␣ (TgIF2␣⬃P) during 0, 4, or 8 h of extracellular stress. (C) Immunofluorescence analysis using a rat monoclonal HA antibody and an anti-rat Alexa 488 conjugate (green) was performed to show localization of TgIF2K-D3⫻HA protein in intra- and extracellular parasites. Nuclear DNA was costained with 4,6-diamidino-2-phenylindole (DAPI) (blue). TgIF2K-D3⫻HA does not colocalize with nuclear DNA, indicating a cytoplasmic localization in the parasite.
TgIF2K-D plays a critical role during nutrient deprivation experienced by extracellular Toxoplasma. TgIF2K-D is expressed in intra- and extracellular parasites. Using RH⌬ku80 parasites engineered to have greater frequencies of homologous recombination (10, 15), we endogenously tagged TgIF2K-D with three HA epitopes (3⫻HA) at the C terminus. Western blot analyses of total protein lysate using anti-HA antibody identified three clustered protein bands with a molecular mass similar to the deduced 289 kDa for TgIF2K-D (Fig. 2A). These proteins were not present in the untagged parental parasites referred to as wild-type (WT). We also observed that upon extracellular incubation for up to 8 h, a condition that induces high levels of TgIF2␣ phosphorylation (18) (Fig. 2B), the levels of the faster-migrating TgIF2KD3⫻HA variants diminished while those of the slower-migrating protein increased slightly (Fig. 2A). The difference between these variants of TgIF2K-D may be attributable to a posttranslational modification(s), such as protein phosphorylation, or to alternative mRNA splicing, which may contribute to TgIF2K-D activation by stress. While we did not detect alternative mRNA splice products during our analysis of the TgIF2K-D cDNAs, alternative mRNA splicing was reported in
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earlier studies of mammalian GCN2, although its biological significance is not yet understood (42). To identify the cellular location of TgIF2K-D, we also carried out immunofluorescence microscopy. The HA-tagged TgIF2K-D localized to the parasite cytosol in both intra- and extracellular parasites (Fig. 2C). A cytosolic localization is consistent with reports on GCN2 in other species (14, 48). TgIF2K-D facilitates TgIF2␣ phosphorylation and translational control in extracellular parasites. Extracellular stress is a potent inducer of TgIF2␣ phosphorylation, and loss of translational control in the TgIF2␣-S71A mutant reduced parasite viability (18). To address whether TgIF2K-D is required to manage extracellular stress, we generated knockdown and knockout parasite clones in the RH⌬ku80 background. The knockdown of TgIF2K-D involved an in-frame fusion of two HA tags and a 12-kDa destabilization domain (DD) at the C terminus of the endogenous TgIF2K-D in the RH⌬ku80 strain (see Fig. S6 in the supplemental material). The parasite clone, designated TgIF2K-D2xDD, allowed tunable expression of the TgIF2K-D protein. In the absence of the stabilizing ligand Shield-1, DD-tagged proteins are rapidly degraded (1, 2); TgIF2K-D2xDD parasites cultured without Shield-1 had no detectable levels of TgIF2K-D protein as assayed by Western blot analysis (see Fig. S6 in the supplemental material). The knockout of TgIF2K-D eliminated the entire genomic locus through homologous recombination and allelic replacement with a modified dihydrofolate reductase-thymidylate synthase (DHFR-TS) minigene, which confers resistance to pyrimethamine (Fig. 3A) (5). ⌬if2k-d was verified by PCR analyses of genomic DNA purified from pyrimethamine-resistant clones (Fig. 3B). In addition, total RNAs from the parental strain and a ⌬if2k-d knockout clone were isolated for RT-PCR analysis of the TgIF2K-D transcript. While TgIF2K-D mRNA was amplified from parental parasites, the corresponding transcript was not detected in ⌬if2k-d parasites (Fig. 3C). This parasite clone represents the first knockout of an eIF2␣ kinase in Toxoplasma. Next we addressed whether TgIF2K-D is required for induced TgIF2␣ phosphorylation when the parasite is outside the host cell. As observed previously (18), parental WT parasites showed TgIF2␣ phosphorylation after 8 h of incubation in the extracellular environment (Fig. 4A). In comparison, there was minimal TgIF2␣ phosphorylation in the TgIF2K-D2xDD knockdown or ⌬if2k-d knockout parasites following extracellular exposure (Fig. 4A). To test the specificity of TgIF2K-D in responding to extracellular stress, we subjected WT and ⌬if2k-d parasites to the calcium ionophore A23187, a known inducer of ER stress and TgIF2␣ phosphorylation (27). As shown in Fig. 4B, the ⌬if2k-d parasites were not defective for TgIF2␣ phosphorylation in response to ER stress. These results support the model that each TgIF2␣ kinase in Toxoplasma recognizes distinct stress arrangements and TgIF2K-D is central for inducing TgIF2␣ phosphorylation when parasites are outside the host cell. Under stress conditions, eIF2␣ phosphorylation represses general translation as part of the cellular stress response (41). To compare translational control in WT versus ⌬if2k-d parasites, we measured the incorporation of radiolabeled Cys/Met in parasites subjected to extracellular stress for 1 and 8 h. As expected, protein synthesis was repressed by greater than 90%
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FIG. 3. Generation of a TgIF2K-D knockout. (A) The TgIF2K-D genomic locus, depicted with 18 exons, was replaced by a minigene conferring resistance to pyrimethamine (DHFR*) using homologous recombination in ⌬ku80 RH strain parasites. The numbered arrows indicate the positions of primers used to screen genomic DNA from transfected pyrimethamine-resistant clones and parental (WT) parasites. Primer sequences are listed in Table S1 in the supplemental material. (B) Genomic PCR assays used gDNA harvested from WT or ⌬if2k-d parasites and the indicated primers to validate replacement of the TgIF2K-D genomic locus. (C) The absence of TgIF2K-D mRNA in the ⌬if2k-d parasites was confirmed by RT-PCR analysis using primers upstream and downstream of the encoded protein kinase domain (primers 11 and 12). Toxoplasma actin mRNA was amplified as a positive control (primers 13 and 14). A no-template control (Ø) was included in all PCRs.
in WT parasites subjected to 8 h of extracellular stress; however, in the ⌬if2k-d and TgIF2␣-S71A mutant parasites, protein synthesis was diminished by only about 40% (Fig. 4C). We conclude that TgIF2K-D is likely to be the primary eIF2␣ kinase that mediates translational control in response to extracellular stress.
Parasites lacking TgIF2K-D exhibit a fitness defect. Previously we reported that TgIF2␣-S71A mutants are outcompeted by wild-type parasites when placed in a “head-to-head” competition assay, as the mutant struggles to cope with the extracellular environment experienced while finding a new host cell (18). Given that the ⌬if2k-d mutant failed to phosphorylate
FIG. 4. TgIF2K-D phosphorylates TgIF2␣ and represses protein synthesis in response to extracellular stress. (A) Wild-type (WT), TgIF2KD2xDD (DD), and ⌬if2k-d parasites were exposed for 0 or 8 h to the extracellular environment. TgIF2␣ phosphorylation was analyzed by separating cell lysates via denaturing SDS-PAGE, followed by Western blotting using antibodies to total TgIF2␣ or phosphorylated TgIF2␣ (TgIF2␣⬃P). (B) WT and ⌬if2k-d tachyzoites were treated with 5 M calcium ionophore A23187 for 30 min and then analyzed for TgIF2␣⬃P by immunoblotting. (C) WT, TgIF2␣-S71A, and ⌬if2k-d parasites were physically released from host cells and incubated for 1 or 8 h in DMEM culture medium. One hour prior to harvesting, the parasites were incubated in the presence of [35S]Cys/Met. Lysates were prepared, and equal amounts of protein were precipitated with TCA. Levels of incorporation of radiolabeled amino acids were determined via scintillation counting. Three experiments were performed, and incorporation of the radiolabel is represented as a percentage of that measured for parasites subject to 1 h of stress. Error bars indicate the standard error, and significance was determined using a two-tailed Student’s t test, with P ⬍ 0.05, as indicated by the asterisks.
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FIG. 5. TgIF2K-D contributes to the fitness of Toxoplasma tachyzoites. (A) Schematic of the “head-to-head” fitness assay. (B) Map of primers (arrows) used to distinguish between wild-type (WT) and ⌬if2k-d parasites. Relative levels of WT and ⌬if2k-d parasites were determined using a SYBR green assay with primers 26 and 27 or primers 28 and 29, as indicated. Samples were normalized for the amplification of a DNA fragment carrying the 5⬘-UTR, which is conserved between WT and ⌬if2k-d parasites (primers 24 and 25). Error bars indicate standard errors, and significance was determined using a two-tailed Student’s t test, with P ⬍ 0.01, as indicated by the asterisk.
TgIF2␣ in response to extracellular stress (Fig. 4A), we tested whether the ⌬if2k-d parasites would be outcompeted by parental wild-type parasites using the head-to-head fitness assay. Equal numbers of WT and ⌬if2k-d parasites were premixed and transferred into the same culture flask containing a confluent monolayer of HFF cells (Fig. 5A). Samples were taken prior to infection and after day 6 for genomic DNA isolation. The relative amounts of WT and ⌬if2k-d parasites were determined using a SYBR green-based quantitative PCR assay and primers specific for WT or ⌬if2k-d parasites. Primers that amplify DNAs from both strains were used to ensure normalization between the samples (Fig. 5B). WT parasites outgrew the mutant parasites by day 6 (Fig. 5C), establishing that parasites lacking TgIF2K-D exhibit reduced fitness in the parasite lytic cycle. TgIF2K-D promotes the viability of extracellular tachyzoites. We evaluated whether the reduced fitness seen in the ⌬if2k-d parasites involved impaired motility or host cell invasion using gliding assays and a red/green adhesion and invasion assay. As reported for the TgIF2␣-S71A mutant, ⌬if2k-d parasites ex-
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hibited no deficiencies in motility or the ability to attach and invade host cells (see Fig. S7 and S8 in the supplemental material). As mentioned above, parasites deficient for TgIF2K-D suffer a loss in viability due to an inability to respond appropriately to the extracellular stress experienced while outside host cells. To further address the role of TgIF2K-D in the extracellular stress response, the WT and different mutant parasites (TgIF2␣S71A, ⌬if2k-d, and TgIF2K-D2xDD without Shield) were incubated outside host cells in DMEM for between 0 and 10 h prior to being applied to a fresh host cell monolayer. After 7 days, the infected host cells were fixed and stained to determine the degree of host cell lysis. With increased periods of extracellular stress, ⌬if2k-d parasites showed sharply reduced infection and lysis of host cells that was similar to that measured for the TgIF2␣-S71A mutants (Fig. 6A). This defect was more pronounced in the ⌬if2k-d parasites than in the TgIF2K-D2xDD knockdown parasites, suggesting that there are residual levels of functional TgIF2K-D despite the absence of Shield. To further characterize the role of translational control in the resistance to extracellular stress, we also analyzed the doubling rate of the ⌬if2k-d parasites. ⌬if2k-d parasites proliferated at a rate similar to that of the WT when allowed to infect a new host cell monolayer immediately upon release from their initial host cells (Fig. 6B, 0-h extracellular stress). However, consistent with the plaque assay, extracellular stress led to a significant reduction in the proliferation of ⌬if2k-d parasites. WT parasites subjected to extracellular stress for 10 h grew to an average of ⬃17 parasites/vacuole, but ⌬if2k-d parasites grew only to ⬃10 parasites/vacuole (Fig. 6B). This reduction in doubling time was also observed when the TgIF2␣-S71A mutants were subjected to extracellular stress prior to infection of the HFF cells. Collectively, these studies establish that TgIF2K-D is critical for promoting survival of extracellular tachyzoites through translational control mediated by the phosphorylation of TgIF2␣. TgIF2␣ is phosphorylated in starved intracellular parasites in the absence of TgIF2K-D. In addition to nutrient deprivation experienced while outside a host cell, parasites can be deprived of host nutrients while intracellular as well. Toxoplasma is auxotrophic for several key metabolites and amino acids, including tryptophan. Tryptophan starvation has been shown to be a mechanism of gamma interferon (IFN-␥)-mediated parasite growth inhibition (31). We tested whether intracellular parasites phosphorylated TgIF2␣ in response to nutrient starvation and whether TgIF2K-D mediated this activity. We subjected infected HFFs to starvation medium for 8 h, which is composed of complete medium diluted to a 6% final concentration in HBSS (6% SM). TgIF2␣ phosphorylation was detected in parasites incubated in 6% SM for 8 h, indicating that translational control is initiated in response to nutrient deprivation in intracellular parasites (Fig. 7A). The ⌬if2k-d mutants were still able to phosphorylate TgIF2␣ in response to nutrient deprivation in intracellular parasites (Fig. 7A), suggesting that a different TgIF2K is activated in starved intracellular parasites. To further characterize the role of TgIF2␣ phosphorylation in intracellular parasites when nutrients become limiting, we analyzed the recovery of wild-type, ⌬if2k-d, and TgIF2␣-S71A mutants following exposure to 6% SM using a standard dou-
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FIG. 6. TgIF2K-D protects Toxoplasma against extracellular stress. (A) Five hundred wild-type (WT), TgIF2K-D2xDD (DD), ⌬if2k-d, or TgIF2␣-S71A (S71A) parasites physically released from host cells were incubated extracellularly in DMEM culture medium for the designated times before being allowed to infect HFF monolayers in 12-well plates. At day 7 postinfection, infected host cells were fixed and stained with crystal violet, and the degree of host cell lysis was determined using an Alpha Innotech imager. Three independent experiments were carried out, each in triplicate, and results of a representative experiment are shown as the percentage of the values for corresponding nonstressed parasite strain. (B) Proliferation of wild-type (WT), ⌬if2k-d, and TgIF2␣-S71A (S71A) parasites following extracellular incubation was determined by counting the number of tachyzoites in 50 random vacuoles at the designated time points postinfection. Three independent experiments were carried out, and the average number of parasites per vacuole for one representative experiment is shown. Error bars, representing standard errors, and significance were determined using a two-tailed Student’s t test (P ⬍ 0.05).
bling assay. Wild-type and ⌬if2k-d parasites exhibited no difference in their ability to recover following incubation for 4 or 8 h in 6%-SM (Fig. 7B). In contrast, TgIF2␣-S71A parasites exhibited a greater defect in recovering from the intracellular nutrient starvation (Fig. 7B). These data demonstrate that phosphorylation of TgIF2␣ promotes the viability of intracellular tachyzoites that experience nutrient deprivation but that this response can be mediated by a TgIF2K other than TgIF2K-D. DISCUSSION In this study, we generated and characterized the first eIF2␣ kinase knockout in the obligate intracellular parasite Toxoplasma. The TgIF2K-D knockout showed reduced TgIF2␣ phosphorylation and translational control in response to extracellular stress, along with reduced viability when outside the
host cell (Fig. 4 and 6). This phenotype was also observed for the TgIF2␣-S71A mutant, supporting the idea that induced TgIF2K-D phosphorylation of TgIF2␣ is central for Toxoplasma to persist in the extracellular environment (Fig. 6) (18). Intracellular tachyzoites proliferate within a parasitophorous vacuole membrane that operates as a molecular sieve and regulates the acquisition of nutrients from the host cell (38, 40). Upon exit from their host cell, the tachyzoites must find a new host cell in order to survive and replicate. The extracellular environment is likely to be reduced in essential nutrients that are available to the parasite, and/or the tachyzoites may not be equipped with the uptake mechanisms needed to acquire them. Our data suggest that TgIF2␣ phosphorylation serves to protect the parasite during this period of vulnerability. Reductions in global translation would allow the tachyzoites to conserve energy and nutrients and may also
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FIG. 7. Induction of TgIF2␣ phosphorylation in starved intracellular Toxoplasma. (A) Intracellular wild-type (WT) or ⌬if2k-d parasites were incubated in CM or 6% SM for 8 h. TgIF2␣ phosphorylation was analyzed by Western blotting using antibodies to total TgIF2␣ or phosphorylated TgIF2␣ (TgIF2␣⬃P). (B) Proliferation of intracellular wild-type (WT), ⌬if2k-d, and TgIF2␣-S71A (S71A) parasites following 4 or 8 h of incubation in 6% SM was determined by counting the number of tachyzoites in 50 random vacuoles at the designated time points. Three independent experiments were carried out, and the average number of parasites per vacuole for one representative experiment is shown. Error bars represent standard errors, and significance was determined using a two-tailed Student’s t test (P ⬍ 0.01).
induce preferential translation of key proteins required for extracellular survival (e.g., membrane transporters or a new array of metabolic enzymes). Such preferential translation of transcripts, such as ATF4 in mammals and GCN4 in S. cerevisiae, during eIF2␣ phosphorylation is central for ameliorating nutrient stress (14, 41, 45, 48). The ability of the parasite to overcome extracellular stress is suggested to be important for pathogenesis, as demonstrated by our prior report that the TgIF2␣-S71A mutant has reduced virulence when inoculated into mice (18). Toxoplasma strains differing in virulence are also suggested to differ in their ability to initiate translational control; hypervirulent strains are able to phosphorylate TgIF2␣ faster and more robustly than hypovirulent strains during extracellular stress (18). Mechanisms by which Toxoplasma copes with the extracellular environment. The mechanisms employed by tachyzoites to overcome the dramatic changes in their extracellular environment are poorly understood but have recently emerged as a new area of intensive research. Microarray analyses have revealed significant changes in the transcriptome between intra- and extracellular tachyzoites (12, 21). Generally, intracellular parasites favor expression of genes involved in metabolism and DNA replication, while Toxoplasma cells in the extracellular environment activate genes focused on invasion, motility, and signal transduction. Coincident with the reprogramming of the transcriptome, extracellular parasites form a novel plant-like vacuole/vacuolar compartment (PLV/VAC). The PLV/VAC may protect parasites from osmotic or ionic stresses encountered outside host cells or mediate the proteolytic maturation of proproteins targeted to micronemes, a cellular compartment important for the parasite invasion into host cells (11, 24, 30). Several studies have also shown that extracellular parasites undergo a metabolic shift from oxidative phosphorylation to glycolysis in order to generate the ATP required for gliding motility and invasion (22, 32). Collectively, these studies suggest that tachyzoites undergo extensive changes in their morphology, metabolism, and transcriptome when transitioning to the extracellular environment. Translational control through TgIF2␣ phosphorylation pro-
vides an additional mechanism that can modulate Toxoplasma gene expression that is designed to facilitate extracellular survival. In support of this model, our data showed that parasites lacking the GCN2-like TgIF2K-D are significantly impaired in their ability to survive outside host cells. In addition to TgIF2K-D, Toxoplasma is suggested to express three other eIF2␣ kinases that are each proposed to respond to unique stress arrangements or environmental cues. TgIF2K-A resides in the parasite endoplasmic reticulum and is suggested to function analogously to mammalian PEK/PERK (27, 44). TgIF2K-B is a parasite-specific eIF2␣ kinase likely to respond to a cytosolic stress (27). Finally, TgIF2K-C is another GCN2like protein kinase present in the Toxoplasma genome (27). However, this putative eIF2␣ kinase appears to lack an RWD that was reported to be essential for GCN2 activity in the yeast model system (13, 19, 20). We do not yet understand the functional significance of two related GCN2 eIF2␣ kinases in Toxoplasma, although this study demonstrates that deletion of TgIF2K-D alone is sufficient to disrupt the translational control required for the parasite to cope with the extracellular environment. It is tempting to speculate that TgIF2K-C is activated to phosphorylate TgIF2␣ during nutrient deprivation experienced by intracellular parasites. Our data show that while TgIF2␣ is phosphorylated in starved intracellular parasites, TgIF2K-D is dispensable for this response. It will be important to identify the TgIF2K involved in phosphorylating TgIF2␣ under intracellular starvation conditions, since TgIF2␣-S71A is deficient in recovering from this stress. Collectively, our data further highlight the eIF2␣ kinase stress response pathway as a potential therapeutic target. GCN2-like protein kinases in parasites. The tandem arrangement of GCN2-related eIF2␣ kinases is also found in the related parasite Plasmodium falciparum. Conservation of multiple GCN2-related protein kinases may indicate that each phosphorylates eIF2␣ in response to distinct stress conditions. The P. falciparum PF14_0264 product is most closely related to TgIF2K-D and contains an RWD domain, while PfeIK1 appears to lack an RWD domain and has recently been reported to respond to amino acid starvation during the intraerythrocyte ring stage (8). This observation suggests that the RWD/GCN1
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regulatory network may not be essential for invoking translational control during periods of certain nutritional deficiencies. GCN2-like protein kinases lacking the RWD domain are not restricted to Apicomplexa. Three GCN2-related kinases (IFKA through -C) have been described in Dictyostelium, but only IFKC possesses an RWD domain (34). Dictyostelium is capable of developing a fruiting body, a process that is induced upon nutrient starvation. Although they are involved in regulating Dictyostelium development, neither IFKA nor IFKB appears to represent the initial sensor for this stress, supporting the idea that different GCN2 isoforms sense distinct stress conditions (3, 7). The role of IFKC in this process has not yet been studied. In the case of mammalian GCN2, different mRNA isoforms have been identified, leading to the expression of one GCN2 variant lacking the RWD domain (42). The reason for the absence of this domain is still enigmatic but is likely to affect the regulation of eIF2␣ kinase activity. How the different versions of GCN2 protein kinases interplay and respond to stress will be an interesting topic for future investigation. Regulation of translation through TgIF2K-independent mechanisms. Our radiolabeling experiments revealed that ⌬if2k-d and TgIF2␣-S71A mutant parasites subjected to extracellular stress reduce protein synthesis by about 40% (Fig. 4C). While this is much different from the 90% reduction observed in wild-type parasites, the data suggest that other mechanisms of translation regulation occur in addition to TgIF2␣ phosphorylation. The reduction in protein synthesis in parasites incapable of phosphorylating TgIF2␣ could be due to diminished amino acids required to sustain translation. Furthermore, mammalian target of rapamycin complex 1 (mTORC1) is another key player in translation regulation, which is activated and positively regulates translation when nutrients are available (23). A putative orthologue of mTOR is encoded in the Toxoplasma genome (TgTOR, TGME49_116440), suggesting that the mTORC1 pathway of translation control is likely to be conserved in Toxoplasma, but it has not been characterized to date. Overall, the similar phenotypes observed for ⌬if2k-d and TgIF2-S71A mutant parasites suggest that translational control via TgIF2␣ phosphorylation is critical during this stress response.
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ACKNOWLEDGMENTS We thank Bradley R. Joyce (Indiana University School of Medicine) and Anthony Sinai (University of Kentucky) for helpful discussions during the course of this study. We thank David Sibley (Washington University) for providing antitubulin antisera, John Boothroyd (Stanford University, Palo Alto, CA) for supplying rabbit anti-SAG1, and Vernon Carruthers (University of Michigan Medical School) for providing RH⌬ku80 parasites and LIC vectors. Support for this research was provided by the National Institutes of Health (R21 grant AI084031 to R.C.W. and W.J.S.), R01 GM049164 (R.C.W.), and R01 AI077502 (W.J.S.). REFERENCES 1. Banaszynski, L. A., L. C. Chen, L. A. Maynard-Smith, A. G. Ooi, and T. J. Wandless. 2006. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126:995–1004. 2. Banaszynski, L. A., and T. J. Wandless. 2006. Conditional control of protein function. Chem. Biol. 13:11–21. 3. Bowman, R. L., Y. Xiong, J. H. Kirsten, and C. K. Singleton. 2011. eIF2␣ kinases Control chalone production in Dictyostelium discoideum. Eukaryot. Cell 10:494–501. 4. Dobrowolski, J. M., and L. D. Sibley. 1996. Toxoplasma invasion of mam-
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