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, Oct 07, 2015
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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.
    Cellular Microbiology 08/2015; DOI:10.1111/cmi.12498 · 4.92 Impact Factor
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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.
    International Journal for Parasitology: Drugs and Drug Resistance 12/2014; 13(1). DOI:10.1016/j.ijpddr.2014.12.001 · 3.29 Impact Factor
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    • "T. gondii tool box has continuingly been expanded to include a tetracyclineinducible gene expression system (Meissner et al., 2001; van Poppel et al., 2006; Jammallo et al., 2011), a site-specific cre/loxP-based recombination system (Heaslip et al., 2010), and a ligand-induced protein stability system (Herm-Götz et al., 2007). Moreover, homologous recombination events could be enhanced with the use of a permissive parasite strain (Huynh and Carruthers, 2009) and well-designed "
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    ABSTRACT: MicroRNAs (miRNAs) are crucial genetic effectors partaking in numerous mechanisms of gene regulation in eukaryotic organisms. Recent discoveries of miRNA in Toxoplasma gondii, an intracellular obligate parasite of the phylum Apicomplexa, suggested possible roles of T. gondii miRNAs (Tg-miRNAs) in the post-transcriptional gene regulation and in the cell biology of the parasite. To gain a better understanding of the involvement of Tg-miRNAs in regulating the parasite gene expression, a dual luciferase reporter system was used in the examination and evaluation of the effects of endogenous Tg-miRNAs, their mimics and inhibitors. A Renilla luciferase (Rnluc) transcript was engineered to carry independent binding sites of two abundant species, namely Tg-miR-60a and Tg-miR-4a, so that the expression of Rnluc was silenced in a sequence specific manner by Tg-miR-60a and Tg-miR-4a. Notably, Tg-miR-60a, but not Tg-miR-4a, caused the levels of Rnluc transcripts to decrease. These findings strongly suggested that T. gondii employs the Tg-miRNA species-specific mode of silencing actions: transcript degradation by Tg-miR-60a, and translational suppression by Tg-miR-4a. Herein we developed a genetic system that exploits and directs the most abundant Tg-miR-60a for loss-of-function analyses in T. gondii. As a proof of principle, we showed that when the binding sites for Tg-miR-60a were introduced into the parasite transcripts via homologous recombination at the locus of (i) DEAD-box RNA helicase (TgHoDI), or (ii) lactate dehydrogenase isoform 1 (TgLDH1), the expression levels of the selected genes can be altered. It was thus proven that inherit Tg-miR-60a could be directed and used to assist in the loss-of-function analyses. Copyright © 2014. Published by Elsevier B.V.
    Journal of Microbiological Methods 12/2014; 108. DOI:10.1016/j.mimet.2014.11.014 · 2.03 Impact Factor
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