Toxoplasma gondiistabilises tetrameric complexes of tyrosine-phosphorylated signal transducer and activator of transcription-1 and leads to its sustained and promiscuous DNA binding

Abstract

Toxoplasma gondii is an obligate intracellular parasite that infects up to 30% of humans worldwide. It can lead to severe diseases particularly in individuals with immature or defective immune responses. Control of T. gondii relies on the IFN-γ-induced signal transducer and activator of transcription-1 (STAT1) pathway. T. gondii, however, largely inactivates STAT1-mediated gene transcription by T. gondii inhibitor of STAT1-dependent transcription (TgIST), a parasite effector protein binding to STAT1. Here, we have analysed requirements of STAT1 to bind TgIST and characterised downstream effects on STAT1 signalling. TgIST bound to STAT1 dimers but more efficiently assembled with STAT1 tetramers, which are essential for effective IFN-γ responsiveness. Such binding was abrogated in N-terminal, but not C-terminal deletion mutants of STAT1. Furthermore, TgIST did not bind to the STAT1 substitution mutant that cannot form STAT1 tetramers, resulting in a complete unresponsiveness of parasite-infected STAT1 -expressing cells to IFN-γ. Remarkably, binding of TgIST considerably increased the affinity of the aberrant STAT1 tetramers for DNA consensus sequence binding motifs and even enabled binding to nonconsensus sequences. Consistent with the increased DNA binding, STAT1 from parasite-infected cells remained phosphorylated at Tyr and Ser and was retained within the nucleus in a DNA-bound state. The sustained and promiscuous binding activity particularly of STAT1 tetramers to unspecific DNA sites lacking a consensus STAT1-binding motif is an as yet unrecognised mechanism contributing to the defective IFN-γ-mediated signalling in T. gondii-infected cells.

Epigenetic Control of IFN-γ Host Responses During Infection With Toxoplasma gondii

“…Wild-type cells did not show any significant luciferase activity ( Figure 1G ). In unstimulated luc -transfected cells, infection with T. gondii slightly increased reporter activity ( Figures 1C–F ), consistent with increased binding of STAT1 to DNA in infected cells, and possibly depending on ROP16 as reported for type I parasites ( 20 , 28 , 29 ). Together, these results directly establish a critical impact of native host chromatin on the ability of T. gondii to counteract IFN-γ-dependent activation of both primary and secondary response genes.…”

“…Our results instead suggest that the Mi-2/NuRD complex can only be recruited by TgIST to GAS promoters ( 26 , 27 ) within a native chromatin context and/or that a repressive chromatin environment is crucial for inhibition of IFN-γ-triggered gene expression. The sole increased and sustained binding of STAT1 complexes from T. gondii -infected cells to naked DNA as described by us and others in vitro ( 20 , 26 , 28 , 52 ) appears however to not suffice for repression of gene expression, as we expect such altered binding also occurring at GAS promoters within plasmid DNA.…”

“…The T. gondii effector TgIST, after translocation into the host cell, recruits Mi-2/NuRD to STAT1-responsive promoters ( 26 , 27 ) and represses IFN-γ responses of its host cell ( 17 , 19 , 20 , 26 , 27 ). It also increases binding of STAT1 to naked DNA oligonucleotides in vitro and to native genomic DNA in infected cells, thereby diminishing recycling and reactivation of STAT1 ( 28 , 29 ). Here, we have directly determined whether a native chromatin environment is required for T. gondii to inhibit STAT1-dependent gene transcription in infected monocytic cells.…”

“…T. gondii infection increases binding of activated STAT1 to host chromatin ( 28 , 29 ) and to IFN-γ-responsive native promoters ( 26 , 27 , 29 ), and it impairs histone modifications at promoters of secondary response genes ( 20 , 26 ). T. gondii however also potentiates binding of aberrant STAT1 complexes to naked DNA ( 20 , 26 , 28 , 52 ) raising questions about the role of the host chromatin for repression of IFN-γ-mediated gene expression. Using luciferase reporters, we here provide direct experimental evidence that native chromatin is indispensable for T. gondii to inhibit IFN-γ-regulated gene expression.…”

“…Recently, the GRA protein TgIST was shown translocating into the host cell in an ASP5-dependent manner, entering the host cell nucleus and repressing IFN-γ-regulated gene transcription ( 26 , 27 ). TgIST binds to activated STAT1 complexes ( 20 , 26 , 28 ) and recruits the Mi-2/NuRD chromatin repressor complex to STAT1-responsive promoters ( 26 , 27 ). This is associated with impaired chromatin remodeling at distinct IFN-γ-responsive promoters ( 20 , 26 ), sequestration of STAT1 at GAS and non-GAS promoters ( 27 – 29 ) and impaired nuclear export and recycling of STAT1 ( 28 , 29 ).…”