Interference of Toxoplasma gondii with IFN-γ-regulated gene expression of its host cell

Toxoplasma gondii is an obligate intracellular parasite that is able to persist within its host for the host s life span. The parasite has evolved multiple strategies in order to establish and maintain this persistence. One such strategy is the inhibition of IFN-γ-induced MHC class II expression via interference with IFN-γ-mediated signalling. In this study, it could be shown that viable T. gondii inhibit IFN-γ-induced MHC class II expression in the monocyte/macrophage cell line RAW 264.7. More importantly, analysis of the effect of T. gondii on a number of other IFN-γ-responsive genes revealed that the parasite interferes with mRNA levels of most but not all of the genes analysed so far. Parasite requirements for inhibition of IFN-γ-induced MHC class II up-regulation were analysed and the mechanisms involved on the host cell level were then further elucidated. It is shown that invasion of the host cell but not intracellular replication is required to enable the parasite to interfere with IFN-γ-induced MHC class II expression in murine macrophages. Furthermore, a T. gondii lysate was found to mimic the effect of viable parasites on MHC class II expression, suggesting that a parasite-derived factor rather than the process of host cell invasion itself is involved. However, analyses of the underlying cell-biological mechanisms revealed that viable parasites and T. gondii lysate differentially interfere with IFN-γ-mediated MHC class II expression. Thus, T. gondii exert diverse effects on those processes leading to MHC class II expression. The molecule within the parasite lysate mediating the inhibition of MHC class II is partially sensitive to pronase treatment indicating the involvement of a protein. Furthermore, it appears to be released by extracellular parasites, since T. gondii separated from the host cells by a semipermeable membrane also inhibit MHC class II expression of non-infected macrophages. However, different preparations of T. gondii-derived excretory/secretory (ES) antigens from the three specific ES organelles of the parasite did not interfere with IFN-γ-regulated MHC class II expression. Analyses of the mechanisms of the T. gondii-mediated abrogation of MHC class II expression on the host cell level revealed that viable parasite and the parasite lysate differentially interfere with the IFN-γ-induced signalling. Viable T. gondii and the parasite lysate do not abrogate the IFN-γ-induced nuclear translocation of Stat1γ, but both interfere with the expression of the class II transactivator (CIITA). However, only viable parasites inhibit the transcript levels of interferon regulatory factor 1 (IRF-1). In addition, both parasite lysate and viable T. gondii abrogate the IFN-γ-induced transcriptional activity of the CIITA promoter IV while only viable T. gondii are also able to interfere with a minimal GAS-containing promoter as determined by luciferase reporter assays. This suggests the importance of the GAS-sequence and Stat1γ for interference of viable parasites with the transcriptional activation of IFN-γ-responsive genes. Furthermore, DNA-binding studies by EMSA revealed, that T. gondii lysate does not alter the DNA-binding activity of Stat1γ to the GAS-sequence. In contrast, viable parasites clearly diminish the formation of the IFN-γ-induced GAS-Stat1γ-Stat1γ complex but instead induce the formation of a modified GAS-binding complex of lower electromobility, which was found to contain Stat1γ. Precipitation with oligonucleotides containing the GAS-sequence, 2D-gelelectrophoresis and MALDI-Tof analyses revealed, that T. gondii interferes with the association of γ-actin to GAS-containing DNA, which is independent of stimulation with IFN-γ and of Stat1γ-binding to GAS. Furthermore, viable parasites abrogate the acetylation of histone 4 on the CIITA promoter IV, further indicating that T. gondii interferes with the assembly of the basal transcription machinery. In conclusion, T. gondii alter the IFN-γ-induced chromatin remodelling at the CIITA promoter IV and diminish the recruitment of γ-actin to the basal tra nscriptional machinery. This may then abrogate IFN-γ-induced expression of MHC class II genes and possibly other IFN-γ-triggered immune responses facilitating intracellular survival of the parasite and its ability to establish persistent infections.