TY - JOUR
T1 - Characterisation of Toxoplasma gondii engineered to express mouse interferon-gamma
AU - Nishikawa, Yoshifumi
AU - Xuenan, Xuan
AU - Makala, Levi
AU - Vielemeyer, Ole
AU - Joiner, Keith A.
AU - Nagasawa, Hideyuki
N1 - Funding Information:
We thank Dr. J.C. Boothroyd (Stanford University) and Dr. D.S. Roos (University of Pennsylvania) for supplying DNA constructs used in developing recombinant T. gondii. We thank Dr. K.A. Joiner (Yale University) for supplying mAb to GRA2. We appreciate the helpful inputs of Dr. F.G. Claveria of the Biology Department, De La Salle University-Manila, Philippines during her sabbatical stay at the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan. The first author is supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.
PY - 2003/11
Y1 - 2003/11
N2 - 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.
AB - 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.
KW - Expression system
KW - Interferon-gamma
KW - Nitric oxide
KW - Toxoplasma gondii
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U2 - 10.1016/S0020-7519(03)00204-2
DO - 10.1016/S0020-7519(03)00204-2
M3 - Article
C2 - 14572515
AN - SCOPUS:0142248507
SN - 0020-7519
VL - 33
SP - 1525
EP - 1535
JO - International Journal for Parasitology
JF - International Journal for Parasitology
IS - 13
ER -