Characterisation of Toxoplasma gondii engineered to express mouse interferon-gamma

Yoshifumi Nishikawa, Xuan Xuenan, Levi Makala, Ole Vielemeyer, Keith A. Joiner, Hideyuki Nagasawa

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

Recent studies have shown the feasibility of using Toxoplasma gondii as an expression system for heterologous protein. For better understanding of the mechanism of interferon-gamma (IFN-γ) dependent immunity to T. gondii, the parasites were stably transfected with IFN-γ gene, under control of the GRA1 promoter. Immunofluorescence analyses showed that recombinant mouse IFN-γ localised to discrete punctuate structures consistent with dense granules and secreted into the vacuolar space. The production of IFN-γ was detectable in both extracellular parasites and the parasite-infected cells. Growth of the recombinant parasites was inhibited in the mouse macrophage cell line (J774A.1 cells), but not in monkey kidney adherent fibroblasts (Vero cells), demonstrating the species-specificity of IFN-γ. Addition of anti-mouse IFN-γ antibody resulted in growth recovery of the recombinant parasites, suggesting that IFN-γ, secreted from the parasitised host cells across the parasitophorous vacuole membrane, acted in a paracrine manner. Reverse transcription (RT)-PCR analysis revealed significant expression of inducible nitric oxide synthase mRNA and high levels of nitric oxide production in recombinant parasite-infected J774A.1 cells. A competitive inhibitor of the L-arginine-dependent effector pathway, NG-monomethyl-L-arginine, inhibited the reduction of recombinant parasite growth in J774A.1 cells. Taken together, our data suggest that the T. gondii expression system may provide a new tool for cytokine gene expression and that parasites engineered to express a cytokine gene may be rationally designed for use in studies on immune responses to T. gondii.

Original languageEnglish (US)
Pages (from-to)1525-1535
Number of pages11
JournalInternational Journal for Parasitology
Volume33
Issue number13
DOIs
StatePublished - Nov 2003

Keywords

  • Expression system
  • Interferon-gamma
  • Nitric oxide
  • Toxoplasma gondii

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

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