Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy.

Abstract

The protozoan parasite Toxoplasma gondii infects a wide range of vertebrate hosts and is an important opportunistic pathogen in immunocompronised humans. Although Toxoplasma is amenable to both biochemical and cellular experimental approaches, the molecular basis of its success as an intracellular parasite is poorly understood. To provide a system for molecular genetic analyses, we An alternative strategy for stable transformation is based on rescuing cells from an auxotrophic condition by introducing a metabolic gene that allows growth. The production of auxotrophs and transformation by conversion to prototrophy is widely used in bacteria and yeast and provides a wide range of independent selectable markers. In the case of an obligate intracellular parasite like Toxoplasma, it is not possible to culture cells in strictly defined medium. It has also not been possible to generate auxotrophic mutants because of the difficulty in controlling the host-cell pool of nutrients.To circumvent the difficulty presented by an obligate intracellular existence, we have adapted a strategy that takes advantage of the naturally occurring tryptophan auxotrophy in Toxoplasma. The requirement for tryptophan allows efficient inhibition of Toxoplasma growth in host cells depleted of tryptophan (16). In mammalian tissue culture cells, tryptophan auxotrophy can be complemented by expression of the Escherichia coli trpB gene, which encodes the 13 subunit of tryptophan synthase, an enzyme that catalyzes the production of tryptophan from indole and seine (17). To adapt this strategy to Toxoplasma, we constructed a plasmid containing the trpB gene flanked by Toxoplasma sequences from the single-copy SAG] gene, which encodes the major tachyzoite surface antigen p30 (18). MATERIALS AND METHODSParasite Growth. The RH strain of Toxoplasma gondii was maintained by serial, 2-day passage on monolayers of human foreskin fibroblasts (HFFs) (4-7). Parasites were harvested from freshly lysed monolayers, separated by 3.0-pim membrane filters and washed in Hanks' balanced salt solution (7 5508The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Molecular genetic strategies in Toxoplasma gondii: close in on a successful invader

“…The HGPRT gene of T. gondii converts 6-thioxanthine into an inhibitor of GMP syntase which kills the parasites, whereas mycophenolic acid efficiently kills parasites lacking the enzyme [20]. As an alternative to drug resistances, stable selection can be achieved by complementation of the naturally occurring tryptophan auxotrophy of Toxoplasma by the bacterial tryptophan synthase (trpB) gene [7]. Finally, expression of the components of the site-specific recombinase system cre loxP from bacteriophage P1 to T. gondii allows specific excision of DNA segment and thus an unlimited use of all the All rights reserved.…”

“…In the early 1990s, the establishment of stable transformation and gene replacement for Leishmania species and Trypanosoma brucei has provided a tremendous boost to research on kinetoplastids [14]. Toxoplasma gondii was the first member of the phylum Apicomplexa to be transfected [5], and elaboration of appropriate methods for selection of stable transformants of this obligate intracellular parasite have followed [6][7][8][9]. As expected, overcoming the barrier of transfectability for one member of Apicomplexa led rapidly to the establishment of stable transformation and homologous recombination for others such as the important human pathogen Plasmodium falciparum [10] and the rodent malaria parasite P. berghei [11].…”

Overproduction of the Pro-Apoptotic Molecule, Programmed Cell Death 5, in Toxoplasma gondii Leads to Increased Apoptosis of Host Macrophages

“…TgPDCD5 or 106-122: Electroporation of tachyzoites was performed as described previously [31]. In brief, purified T. gondii PLK tachyzoites were resuspended at 10 7 cells/ml with cytomix buffer (120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 -KH 2 PO 4 , 2 mM EDTA, 5 mM MgCl 2 , 25 mM HEPES [pH 7.6]), supplemented with 2 mM ATP and 5 mM glutathione.…”

Recombinant Attenuated Toxoplasma gondii Expressing the Plasmodium yoelii Circumsporozoite Protein Provides Highly Effective Priming for CD8+ T Cell-Dependent Protective Immunity Against Malaria

“…Electroporation of tachyzoites was performed as described (8). After transfection, tachyzoites were allowed to infect HFF in drug-free culture medium for 18 h to permit the phenotypic expression of the HXGPRT minigene selectable marker, at which time MPA was then added at a final concentration of 25 g/ml in combination with xanthine (50 g/ml), as described (10).…”