Molecular Mechanisms Of T.Gondii ROP18 Kinase And Its Inhibition Of The Host NF-κB Pathway

Background:The obligate intracellular parasite Toxoplasma gondii affects a wide range of hosts, such as mammals and birds, and humans as well.Toxoplasma gondii is an important opportunistic human pathogen. In most people, the clinical symptoms of T. gondii infections is like flu symptoms.But fetus and patients with primary or acquired defects in T cell-mediated immunity have severe complications.The innate immunity response not only limits parasite growth but also promotes the development of adaptive immunity, which is required for long term resistance to infection. T. gondii ROP18 kinase is responsible for promoting p65 degradation, thus providing a survival advantage to the infectious agent.Objective:To demonstrate that virulence factor ROP18 of T. gondii is responsible for for promoting p65 degradation by pull-down, yeast-two-hybrid assays, immunofluorescence and wetern blotting assays.Materials:This study identified the interaction between ROP18 and p65 by yeast two-hybrid screening, pull-down assay and immunoprecipitation assays. The plasmids expressing ROP18 and its mutants were constructed. The ROP18 over-expressing transgenic strain was constructed and confirmed by immunofluorescence and western-blotting assays. Immunofluorescence staining indicated ROP18 and p65 were co-localizated in HFF cells. Immunoprecipitation assay indicated that ROP18 promoted p65 degradation.Results:(1) T.gondii virulence factor ROP18 interacts with p65(2) The over-expressing ROP18 transgenic strain is constructed.(3) ROP18 is co-localizated with p65 in HFF cells.(4) ROP18 interacts with p65 and mediates its degradation.(5) ROP18 targets p65 to a proteasome-ubiquitin-dependent degradation pathway.Conclusions:T.gondii kinase ROP18 inhibits the host NF-κB pathway by promoting p65 degradation.