Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80

Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA.
Eukaryotic Cell (Impact Factor: 3.18). 03/2009; 8(4):530-9. DOI: 10.1128/EC.00358-08
Source: PubMed


As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Deltaku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Deltaku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Deltaku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.

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Available from: Vern B Carruthers
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    • "type I RH tachyzoites and their transgenic derivatives were grown in human foreskin fibroblasts (HFFs) as described inRoos et al. (1994). The following lines have been described before: RH::Δku80 (Huynh and Carruthers, 2009) was kindly shared by Vern Carruthers (University of Michigan), TATi::Δku80 (Sheiner et al., 2011) was kindly shared by Boris Striepen (University of Georgia), and TgNuf2-cKD was generated by Farrell and Gubbels (2014). The TgEB1-KO line was established by replacing the endogenous locus with a 1.5-kb PCR product composed of an HXGPRT drug-selectable cassette and the TgEB1-cKD line with a 4.1-kb PCR product composed of DHFR-Tet7Sag4-TgEB1-(CDS)-2xMyc. "
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    ABSTRACT: Toxoplasma gondii replicates asexually by a unique internal budding process characterized by interwoven closed mitosis and cytokinesis. Although it is known that the centrosome coordinates these processes, the spatiotemporal organization of mitosis remains poorly defined. Here we demonstrate that centrosome positioning around the nucleus may signal spindle assembly: spindle microtubules (MTs) are first assembled when the centrosome moves to the basal side, and become extensively acetylated after the duplicated centrosomes reposition to the apical side. We also tracked the spindle MTs using the MT plus-end binding protein TgEB1. Endowed by a C-terminal NLS, TgEB1 resides in the nucleoplasm in interphase and associates with the spindle MTs during mitosis. TgEB1 also associates with the subpellicular MTs at the growing end of daughter buds toward the completion of karyokinesis. Depletion of TgEB1 results in escalated disintegration of kinetochore clustering. Furthermore, we show that TgEB1's MT association in Toxoplasma and in a heterologous system (Xenopus) is based on the same principles. Finally, overexpression of a high MT affinity TgEB1 mutant promotes the formation of overstabilized MT bundles resulting in avulsion of otherwise tightly clustered kinetochores. Overall, we conclude that centrosome position controls spindle activity and that TgEB1 is critical for mitotic integrity.
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    • "All rights reserved. DHFR vector (Bougdour et al., 2013) using the LIC cloning method as described (Huynh et al., 2009). Approximately 20 µg of the resulting vector was linearized within the region of homology with AvrII before transfection. "
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    ABSTRACT: Toxoplasma gondii and Plasmodium species are obligatory intracellular parasites that export proteins into in the infected cells in order to interfere with host-signaling pathways, acquire nutrients or evade host defense mechanisms. With regard to export mechanism, a wealth of information in Plasmodium spp. is available, while the mechanisms operating in T. gondii remain uncertain. The recent discovery of exported proteins in T. gondii, mainly represented by dense granule resident proteins, might explain this discrepancy and offers a unique opportunity to study the export mechanism in T. gondii. Here, we report that GRA16 export is mediated by two protein elements present in its N-terminal region. Because the first element contains a putative PEXEL linear motif (RRLAE), we hypothesized that GRA16 export depended on a maturation process involving protein cleavage. Using both N- and C-terminal epitope tags, we provide evidence for protein proteolysis occurring in the N-terminus of GRA16. We show that TgASP5, the T. gondii homolog of Plasmodium Plasmepsin V, is essential for GRA16 export and is directly responsible for its maturation in a PEXEL-dependent manner. Interestingly, TgASP5 is also involved in GRA24 export, though the GRA24 maturation mechanism is TgASP5-independent. Our data reveal different modus operandi for protein export, in which TgASP5 should play multiple functions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Aug 2015 · Cellular Microbiology
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    • "The parasite strain RH / ku80 - / hxgprt - ( Huynh and Carruthers , 2009 ) ( ATCC : PRA319 ) was used as the parental strain of the trans - genic parasites throughout this study . Parasites were maintained and serially passaged to new host Vero cells as described else - where ( Roos et al . "
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    ABSTRACT: Toxoplasma gondii is the causative pathogen for Toxoplasmosis. Bumped kinase inhibitor 1NM-PP1 inhibits the growth of T. gondii by targeting TgCDPK1. However, we recently reported that resistance to 1NM-PP1 can be acquired via a mutation in T. gondii mitogen-activated protein kinase like 1 (TgMAPKL-1). Further characterization of how this TgMAPKL-1 mutation restores the inhibitory effect of 1NM-PP1 would shed further light on the function of TgMAPKL-1 in the parasite life cycle. Therefore, we made parasite clones with TgMAPKL-1 mutated at the gatekeeper residue Ser 191, which is critical for 1NM-PP1 susceptibility. Host cell lysis of RH/ku80-/HA-TgMAPKL-1S191A was completely inhibited at 250 nM 1NM-PP1, whereas that of RH/ku80-/HA-TgMAPKL-1S191Y was not. By comparing 1NM-PP1-sensitive (RH/ku80-/HA-TgMAPKL-1S191A) and -resistant (RH/ku80-/HA-TgMAPKL-1S191Y) clones, we observed that inhibition of TgMAPKL-1 blocked cell cycle progression after DNA duplication. Morphological analysis revealed that TgMAPKL-1 inhibition caused enlarged parasite cells with many daughter cell scaffolds and imcomplete cytokinesis. We conclude that the mutation in TgMAPKL-1 restored the cell cycle-arresting effect of 1NM-PP1 on T. gondii endodyogeny. Given that endodyogeny is the primary mechanism of cell division for both the tachyzoite and bradyzoite stages of this parasite, TgMAPKL-1 may be a promising target for drug development. Exploration of the signals that regulate TgMAPKL-1 will provide further insights into the unique mode of T. gondii cell division.
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