Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog
© The Author(s). 2016
Received: 22 December 2015
Accepted: 30 June 2016
Published: 21 July 2016
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis. Understanding the mechanism of tachyzoite invasion is important for the discovery of new drug targets and may serve as a model for the study of other apicomplexan parasites. We previously showed that Plasmodium falciparum expresses a homolog of human calcium calmodulin-dependent protein kinase (CaMK) that is important for host cell invasion. In this study, to identify novel targets for the treatment of Toxoplasma gondii infection (another apicomplexan parasite), we sought to identify a CaMK-like protein in the T. gondii genome and to characterize its role in the life-cycle of this parasite.
An in vitro kinase assay was performed to assess the phosphorylation activities of a novel CaMK-like protein in T. gondii by using purified proteins with various concentrations of calcium, calmodulin antagonists, or T. gondii glideosome proteins. Indirect immunofluorescence microscopy was performed to detect the localization of this protein kinase by using the antibodies against this protein and organellar maker proteins of T. gondii.
We identified a novel CaMK homolog in T. gondii, T. gondii CaMK-related kinase (TgCaMKrk), which exhibits calmodulin-independent autophosphorylation and substrate phosphorylation activity. However, calmodulin antagonists had no effect on its kinase activity. In T. gondii-infected cells, TgCaMKrk localized to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylated TgGAP45 for phosphorylation in vitro.
Our data improve our understanding of T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis [1, 2]. In humans, ingested T. gondii cysts release asexually reproducing bradyzoites that differentiate into tachyzoites, which propagate the infection by spreading through the body via the blood and lymphatic systems. While the immune system can normally clear a T. gondii infection, immunocompromised individuals, such as those infected with human immunodeficiency virus, have trouble doing so and can develop severe toxoplasmosis . Although drugs to treat toxoplasmosis are available, they are poorly tolerated, have severe side effects, and are ineffective against chronic Toxoplasma infections [4, 5]. Therefore, new drugs are urgently needed. To discover new drug targets, we must first understand the mechanism of tachyzoite invasion. Such knowledge may also benefit the study of other apicomplexan parasites.
Our laboratory and others have demonstrated the importance of parasitic kinases for T. gondii [6–10]; however, no kinase function has been found to be of critical importance in the primary host, felines. For tachyzoite invasion of a host cell, many kinases are called to action, including T. gondii calcium-dependent protein kinase 1 (TgCDPK1) [7, 11–13], T. gondii cyclic GMP-dependent protein kinase , and TgCDPK1_2 . TgCDPK1 also participates in the egress of tachyzoites from infected cells . Additional T. gondii protein kinases are involved in host manipulation, cell cycle regulation, and functions required for growth, stress responses, and the transition from tachyzoite to bradyzoite . Thus, given their level of involvement in many aspects of the parasitic life-cycle, the kinases encoded by the parasite genome are obvious potential drug targets.
The motility of T. gondii tachyzoites is activated by an increase in the cytosolic Ca2+ concentration , which occurs as the parasites egress from the host cells [17, 18]. This increase in Ca2+ concentration causes the parasites to secrete adhesion molecules from its microneme . An actin/myosin-based motor complex, the glideosome , powers parasite motility and is a conserved feature of apicomplexans . The glideosome of T. gondii is a macromolecular complex that includes myosin A, myosin light chain (TgMLC1), T. gondii glideosome-associated protein 50 (TgGAP50), TgGAP45, aldolase 1, and actin 1 (TgACT1) . TgGAP40, TgGAP70, and TgGAP80 are also glideosome components [22, 23]. The glideosome, which is located between the parasite’s plasma membrane and its inner membrane complex, mediates motility, migration, host cell invasion, and egress. In T. gondii and another apicomplexan family member, Plasmodium falciparum, the phosphorylation of glideosome components within the tachyzoite, or merozoite in the case of P. falciparum, occurs during invasion and helps control gliding, invasion, and egress. In T. gondii, the phosphorylation of Ser163 and Ser167 in TgGAP45 marks the final step in glideosome assembly . Structural modeling studies have shown that Ser163 and Ser167 are Ca2+-insensitive and that Ser184/5 and Thr189 are Ca2+-sensitive phosphorylation sites on TgGAP45 . PfCDPK1 localizes to the periphery of merozoites where it acts on myosin A tail domain-interacting protein (MTIP) and PfGAP45 at the inner membrane complex. PfCDPK1 can also phosphorylate MTIP and PfGAP45 in vitro .
We previously characterized P. falciparum protein kinase 2 (PfPK2), which is a unique homolog of human Ca2+ calmodulin-dependent protein kinase (CaMK) . PfPK2 phosphorylates its substrate in a Ca2+- and calmodulin-dependent manner. In the present study, we identified a homolog of PfPK2 in T. gondii, T. gondii CaMK-related kinase (TgCaMKrk) (ToxoDB ID: TGME49_315190; GenBank accession number: AB699221), which exhibits autophosphorylation and histone phosphorylation activity. However, calmodulin antagonists had no effect on its kinase activity. We further show that TgCaMKrk is expressed in T. gondii-infected cells and localizes to the apical ends of extracellular and intracellular tachyzoites, and that it specifically targets TgGAP45 for phosphorylation in vitro.
Target cells and parasites
Tachyzoites of T. gondii RH strain were used in this study. The parasite was maintained in monolayers of Vero cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 7.5 % fetal calf serum (FCS), 2 mM l-glutamine, 20 mM HEPES (pH 7.5), streptomycin, and penicillin.
Sequence analysis and identification of the TgCaMKrk open reading frame (ORF)
T. gondii RH strain mRNA was isolated from infected Vero cells by using TRIZOL (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A cDNA library of T. gondii RH strain was amplified by RT-PCR using parasite mRNA as the template and SuperScrip III Reverse Transcriptase with oligo dT primer (Invitrogen, Carlsbad, CA, USA). The TgCaMKrk ORF was identified by sequence analysis of the T. gondii cDNA library by using an Applied Biosystems 3130 Genetic Analyzer.
The list of primers used to generate the expression vectors
Wheat germ cell-free protein synthesis system
Protein expression using a wheat germ cell-free protein synthesis system (CellFree Sciences) was achieved as described previously [27, 28]. Briefly, at the transcription step, 2 μg of pEU-GST-GFP or pEU-GST-TgCaMKrk was mixed with 18 μl of transcription mixture (transcription buffer with 2.5 mM NTP mix, 1 U/μl RNase inhibitor, and 1 U/μl SP6 RNA polymerase; CellFree Sciences) and incubated at 37 °C for 6 h. Each mRNA generated was then mixed with 10.8 μl of WEPRO1240G (CellFree Sciences) and 40 ng/μl creatine kinase (Roche, Mannheim, Germany), transferred to the bottom of the SUB-AMIX solution (CellFree Sciences) to form a bilayer, and incubated at 16 °C for 20 h. To determine whether the resultant protein was bound to Ca2+ or calmodulin upon translation, DMSO (negative control), the calmodulin antagonist W-7 (0.66 mM; BIOMAL, Plymouth Meeting, PA, USA), or calmidazolium (0.05 mM; Sigma-Aldrich, St. Louis, MO, USA) was added to the SUB-AMIX solution.
Purification of recombinant proteins
Wheat germ extracts were mixed with 10 μl of a 50 % slurry of glutathione-Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 16 h. The beads were then washed three times with phosphate-buffered saline (PBS). Purified protein captured on the beads was separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after boiling and was then either silver stained or immunoblotted with anti-α-GST antibodies (Sigma-Aldrich). Purification of the recombinant proteins expressed in Escherichia coli XL-1 blue transformed with plasmids harboring MBP-TgGAP45, MBP-TgACT1, MBP-TgMLC1, or MBP-TgGAP50 was performed as described elsewhere .
In vitro kinase assay
Purified GST-GFP, GST-TgCaMKrk, or GST-TgCaMKrkKA captured on glutathione-Sepharose beads was rinsed twice with washing buffer (50 mM Tris-HCl, pH 9.0, and 2 mM DTT). Kinase assay reactions were performed with the purified GST fusion proteins at 37 °C for 30 min in 50 μl of kinase buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 50 mM MgCl2, 0.1 % Nonidet P-40, 1 mM DTT, 5 μM ATP, and 10 μM CaCl2) containing 5 μCi of [γ-32P]ATP. After incubation, the samples were washed three times with TNE buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM EDTA), and the phosphorylated proteins were separated by 10 % SDS-PAGE. The gels were stained with Coomassie Brilliant Blue (CBB), dried, and exposed to X-ray film [27, 28].
Rabbit anti-GRA6 polyclonal antibodies were kindly sent to us by Dr. L.D. Sibley (Washington University School of Medicine, St. Louis, MO, USA); rabbit anti-M2AP (microneme protein 2-associated protein) polyclonal antibodies were kindly provided by Dr. V. Carruthers (John Hopkins University, Baltimore, MD, USA); rabbit anti-ROP1 polyclonal antibodies were a gift from Dr. J. Dubremetz (University of Montpellier, Montpellier, France); and rabbit anti-TgGAP45 polyclonal antibodies were a gift from Dr. D. Soldati (University of Geneva, Switzerland).
Production of anti-TgCaMKrk antibodies
Mouse antiserum against TgCaMKrk was prepared by immunizing BALB/c mice with purified GST-TgCaMKrk expressed in a wheat germ cell-free system, purified on glutathione-Sepharose beads, extensively washed with buffer C, and eluted with elution buffer (10 mM glutathione and 500 mM Tris-HCl, pH 8.0). For the first immunization, a mixture of the eluted supernatants and Freund’s complete adjuvant (Rockland, Gilbertsville, PA, USA) was injected intraperitoneally into the mice. After 2 weeks, a mixture of the eluted supernatants and Freund’s incomplete adjuvant (Rockland) was injected three times, at 2-week intervals. One week after the final injection, mouse antiserum against TgCaMKrk was collected.
Immunoblotting and IFA
T. gondii RH strain was propagated and lysed by passing the cells though a #27 syringe and was filtered by using a 5-μm filter. The lysate was subjected to immunoblotting as described elsewhere . For indirect immunofluorescence microscopy (IFA), purified extracellular tachyzoites were fixed on a 14-well slide with 40 % formaldehyde and were permeabilized with 0.1 % Triton X-100. The slide was incubated with antibodies against TgCaMKrk and GRA6, TgGAP45, ROP-1, or M2AP, respectively, for 60 min at 37 °C and then rinsed three times with PBS. After incubation with AlexaFluor 488-conjugated goat anti-mouse IgG (H + L) and AlexaFluor 546-conjugated goat anti-rabbit IgG (H + L) (Invitrogen) for 60 min at room temperature, the slide was rinsed three times with PBS. Intracellular tachyzoites were prepared by infecting Vero cells cultured in an eight-well chamber slide for 48 h; the slide was then fixed and stained as described above.
Identification of TgCaMKrk
We previously characterized PfPK2, a unique CaMK encoded by the P. falciparum genome . Calmodulin antagonists inhibited PfPK2 kinase activity in vitro and markedly decreased ring-stage parasitemia in invasion assays . CaMK plays important roles in Ca2+ signaling during various cellular events, including carbohydrate metabolism, transcription, spermatogenesis, transcription, neuronal memory, and mitochondrial biogenesis . Therefore, we speculated that such a gene likely exists in another apicomplexan parasite, T. gondii. In this study, we sequenced and identified an ORF (9,591 bp) of a novel T. gondii gene that included a sequence homologous to the catalytic domain of PfPK2. These sequence analysis data were consistent with transcriptional data from the RNA-seq of T. gondii ME49 strain performed by Drs. J. Yamagishi and X. Xuan (Obihiro University of Agriculture and Veterinary Medicine; personal communication). The putative catalytic domain of TgCaMKrk shares 57 % identity and 88 % similarity with that of PfPK2; however, it has no homologous regulatory (autoinhibitory and calmodulin-binding) domain and a long nonconserved region (Fig. 1a). We named this gene T. gondii CaMK-related kinase (TgCaMKrk) (GenBank accession number: AB699221).
Purification of the TgCaMKrk kinase domain by using a wheat germ cell-free protein synthesis system
Protein kinase activity of TgCaMKrk
It is possible that the kinase activity we detected in these experiments was caused by a contaminating kinase(s) that physically associates with GST-TgCaMKrk, or that it was non-specifically selected for by the glutathione-Sepharose beads. To exclude these possibilities, we constructed a mutant that lacked intrinsic kinase activity but retained its overall structure. We used site-directed mutagenesis to replace Lys218 of TgCaMKrk with an Ala residue to generate the GST fusion protein GST-TgCaMKrkKA (see Fig. 1b). Lys218 was chosen because it is conserved in subdomain II of known protein kinases and is required for kinase activity , and because mutation of the corresponding lysine in PfPK2 resulted in a loss of kinase activity . The autophosphorylation activity of GST-TgCaMKrkKA was tested as before using purified protein and [γ-32P]ATP. The wild-type fusion protein was labeled by autophosphorylation (Fig. 3b, Lane 2), whereas the mutant was not (Fig. 3b, Lane 3). These results indicate that the substitution of Lys218 abolished the kinase activity of TgCaMKrk and that contaminating kinases were not a factor in our kinase assay.
Neither Ca2+ nor calmodulin are required for the protein kinase activity of TgCaMKrk
We also tested the effect of calmodulin antagonists on the phosphorylation activity of TgCaMKrk, because we had previously demonstrated their inhibition of PfPK2 activity in vitro and of erythrocyte invasion by P. falciparum . The translation mixture (i.e. WEPRO1240G) used in the wheat germ cell-free system contained wheat calmodulin, so we eliminated the wheat calmodulin at the translation step by incubating the translation mixture with the calmodulin antagonist W-7 or calmidazolium. After translation and purification, TgCaMKrk activity was assessed by scintillation counting. Both autophosphorylation and histone phosphorylation were slightly reduced after the addition of W-7 (Fig. 4b and c), while the activity of autophosphorylation of PfPK2 was greatly reduced after the addition of W-7. Thus, neither Ca2+ nor calmodulin is required for the kinase activity of TgCaMKrk. These results are consistent with the fact that the putative amino acid sequence of TgCaMKrk has a conserved catalytic domain but lacks the regulatory domain, which comprises the autoinhibitory and calmodulin-binding domains in CaMK-like proteins (Fig. 1a) .
TgCaMKrk is expressed at the periphery of T. gondii tachyzoites
PfPK2 is expressed in the membrane of the parasite . Our previous IFA data showed that PfPK2 is produced by merozoites during erythrocyte invasion . To determine the localization of the TgCaMKrk protein in T. gondii, rabbit anti-GRA6, -GAP45, -ROP1 and -M2AP antibodies were employed to show the dense granules, glideosome, rhoptry, and apical ends. As shown in Fig 5b, in the released tachyzoite, the TgCaMKrk signal did not completely co-localize with any of the markers we used, but it was located more toward the apical end of the M2AP signal and closest to the GAP45 signal (Fig. 5b, 2nd panel). In the growing tachyzoites in the infected cells, the TgCaMKrk signal localized to the apical ends and again did not completely co-localize with any of the marker signals (Fig. 5b, 5th–8th panels). Pre-immune mouse serum did not stain any parasites (data not shown). The TgCaMKrk signal at the more apical end of M2AP suggests an apical end location for TgCaMKrk; however, further study is required to determine the precise localization of this protein.
TgCaMKrk specifically phosphorylates the glideosome protein TgGAP45 in vitro
This is the first report to describe the identification and characterization of a CaMK-like protein in T. gondii, TgCaMKrk. GST-TgCaMKrk containing the kinase domain was successfully expressed and purified by using a wheat germ cell-free protein synthesis system. TgCaMKrk was shown to have both autophosphorylation and substrate phosphorylation activity, as well as a conserved catalytic lysine (Lys218). The catalytic domain of TgCaMKrk shares 88 % similarity with that of PfPK2; however, TgCaMKrk has no regulatory domain. The regulatory domain of PfPK2 consists of autoinhibitory and calmodulin-binding domains. The lack of such a domain in TgCaMKrk is reflected by the fact that calmodulin antagonists (W-7 and calmidazolium) did not inhibit TgCaMKrk autophosphorylation or the histone phosphorylation activity of TgCaMKrk. Some regulation of TgCaMKrk must occur, however, because continuous phosphorylation by this protein would presumably affect the parasite’s life-cycle. A BLAST search via ToxoDB (http://toxodb.org/toxo/) using the putative amino acid sequence of TgCaMKrk did not reveal a domain homologous to the regulatory domain of PfPK2. Therefore, it is likely that another parasite protein regulates the kinase function of TgCaMKrk.
Our IFA data showed that TgCaMKrk localizes to the apical ends of extracellular tachyzoites. The localization patterns of TgCaMKrk in extracellular tachyzoites were more posterior to those of GAP45. Antibodies against TgGAP45 stained the inner membrane complex, but not the plasma membrane . Previous fractionation analysis of P. falciparum indicated that PfPK2 is also expressed in the parasite’s inner membrane . These findings suggest that TgCaMKrk could be secreted as it is associated with apical organelles, and it could have activity against host proteins for pathogenesis, and localize with a specific organelle in the inner membrane complex in tachyzoites.
TgCaMKrk specifically phosphorylated TgGAP45 in the glideosome in vitro. The residues of TgGAP45 phosphorylated by TgCaMKrk were most likely Ser163 and Ser167. Mutation of these residues to Glu prevented association of the MyoA-MLC1-GAP45 complex with GAP50, but did not have any obvious effects on motility or host cell invasion, suggesting that Ser163 and Ser167 phosphorylation controls the final step in assembly of the myosin XIV motor complex .
TgGAP70 localized to the apical cap of extracellular tachyzoites, whereas TgGAP45 localized to the periphery . When we examined the homology between TgGAP45 and TgGAP70, we found that the similarity was approximately 73 % (Genetyx). However, the critical serine residues corresponding to Ser163 and Ser167 of TgGAP45 are not conserved in TgGAP70. Further analysis of TgCaMKrk in extracellular tachyzoites is needed to clarify its substrates.
T. gondii kinases play important and diverse roles in the parasite’s life-cycle . Inhibitor studies suggest that kinases are crucial regulators of parasite invasion. Therefore, such proteins represent good potential drug targets for blocking parasite-specific activity. On the basis of the data presented here, we suggest that TgCaMKrk should be explored as a drug target, because it localizes to the apical ends of T. gondii tachyzoites and could be associated with the glideosome. Future studies should focus on the regulatory mechanism for this protein kinase and its overall impact on the life-cycle of T. gondii.
In this study, we identified TgCaMKrk, which exhibits autophosphorylation and histone phosphorylation activity. TgCaMKrk is expressed in T. gondii-infected cells and localizes to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylates TgGAP45 in vitro. Our data further our understanding of T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.
This study was supported by grants-in-aid for Young Scientists, Exploratory Research, and Scientific Research on Innovative Areas (3308 and 3407) from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan; by the Bio-oriented Technology Research Advancement Institution (BRAIN); by the Program to Disseminate Tenure Tracking System and the Adaptable & Seamless Technology Transfer Program through Target-driven R&D (A-STEP) from the Japan Science and Technology Agency (JST); by the Ito Foundation; and by the Promotion for Young Research Talent and Network from Northern Advancement Center for Science & Technology (NOASTEC).
Availability of data and material
The complete coding sequence of the Toxoplasma gondii mRNA for the CaMK-related kinase was registered in GenBank (accession number: AB699221). The plasmids and antibody produced in the study are available with the suitable process.
KK designed the experiments. KK, TS, HT, RT, HG, AI performed the experiments. TH and HA offered useful advice. KK wrote and edited the manuscript and supervised the study. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All animal work was conducted in accordance with the guidelines of the University of Tokyo (Permit number, P08-183).
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