Characterisation of Toxoplasma gondii engineered to express mouse interferon-gamma.
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-gamma) dependent immunity to T. gondii, the parasites were stably transfected with IFN-gamma gene, under control of the GRA1 promoter. Immunofluorescence analyses showed that recombinant mouse IFN-gamma localised to discrete punctuate structures consistent with dense granules and secreted into the vacuolar space. The production of IFN-gamma 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-gamma. Addition of anti-mouse IFN-gamma antibody resulted in growth recovery of the recombinant parasites, suggesting that IFN-gamma, 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, N(G)-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.
Nishikawa
Yoshifumi
Y
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Xuenan
Xuan
X
Makala
Levi
L
Vielemeyer
Ole
O
Joiner
Keith A
KA
Nagasawa
Hideyuki
H
D000818:Animals; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D006790:Host-Parasite Interactions; D007371:Interferon-gamma / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D009569:Nitric Oxide / Q000032:analysis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010274:Parasitology / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014122:Toxoplasma / Q000235:genetics* / Q000276:immunology; D014124:Toxoplasmosis, Animal / Q000276:immunology*; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D019323:omega-N-Methylarginine / Q000494:pharmacology
D012333:RNA, Messenger; D011994:Recombinant Proteins; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse