Activation of NF-κB signaling by dense granule protein GRA15 of a newly discovered type 1 Toxoplasma gondii strain

Abstract

Background: It has been reported that the NF-κB pathway, an important component of host defense system against pathogens infections, can be modulated by GRA15 of type 2 Toxoplasma gondii strains. A newly reported Toxoplasma strain (T.gHB1) belonging to type 1 showed polymorphic phenotypes comparable to the classic type 1 parasite, such as virulence determinant mechanisms. Therefore, it is worth investigating whether the novel type 1 Toxoplasma strain (T.gHB1) has an effect on host NF-κB signaling pathway.Methods: The effects of T.gHB1 on host NF-κB pathway was investigated in HEK293T cells. The GRA15 gene product was analysed by bioinformatics method and its effect on NF-κB was examined by detection of the levels of phosphorylation and nuclear translocation of p65 by Western blotting (WB) and indirect fluorescence assay (IFA). Functional domain of GRA15 was mapped by using the luciferase reporter assay and its effects on p65 nuclear translocation and IκBα phosphorylation were examined by IFA and WB, respectively. Results: We demonstrated that the NF-κB pathway signaling can be activated by the newly identified type 1 T.gHB1 strain of Toxoplasma, while other known type 1 RH parasites exerted an inhibitory effect. T.gHB1 GRA15 possesses only one transmembrane region with an extended C terminal region, which is distinct from other known GRA15 from type 2 parasites. T.gHB1 GRA15 could clearly induce IκBα phosphorylation and p65 nuclear translocation. Dual luciferase assays in HEK293T cells revealed a requirement for 194-518 aa of T.gHB1 GRA15 to effectively activate NF-κB. Conclusions: The overall results indicated that the newly discovered type 1 isolate T.gHB1 and its GRA15 gene product can activate NF-κB through a classical pathway in mammalian cells. These results provide new sights for our understanding of the interaction between Toxoplasma parasites and its host.