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 journalArticle

36 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
Externally publishedYes

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Keywords

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

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

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