INHIBITION OF STAT TRANSCRIPTION BY TOXOPLASMA

弓形虫对 STAT 转录的抑制

• 批准号: 9244190
• 负责人: L. David Sibley
• 金额: $ 22.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-05 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9244190
• 关键词: Acute; Adult; Animals; Antibiotic Therapy; Binding; Binding Proteins; Biochemical Genetics; Biological Assay; CRISPR/Cas technology; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Chronic; Complement; Complex; Cyst; Cytokine Receptors; Development; Dimerization; Event; Family; Gene Deletion; Gene Expression; Gene Expression Profiling; Genes; Genetic Transcription; Guanosine Triphosphate Phosphohydrolases; Hela Cells; Hematopoietic; Human; Immune; Immune Response Genes; Immune response; Immune system; Immunity; Immunocompromised Host; Individual; Infection; Infection Control; Interferon Type II; Interferons; Janus kinase; Knowledge; Lead; Life; MHC Class II Genes; Mammalian Cell; Mediating; Methods; Molecular; Mus; NOS2A gene; Natural Immunity; NuRD complex; Nuclear; Nuclear Translocation; Nucleosomes; Parasites; Pathway interactions; Peptide Signal Sequences; Phenotype; Phosphorylation; Proteins; Recruitment Activity; Reporter; Repression; Risk; Rodent; Role; STAT protein; STAT1 gene; Signal Transduction; Technology; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transcription Coactivator; Up-Regulation; Vacuole; Virulence Factors; Zoonoses; antimicrobial; dimer; guanylate; immune activation; improved; inhibitor/antagonist; killings; prevent; promoter; response; therapy design; trafficking; trait; transcription factor; transmission process

Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infection in humans. Although acute infections in healthy adults are normally resolved without serious consequences, infection of immunocompromised individuals can lead to serious complications. Additionally, because chronic infections are not cleared by the immune system or by normal antibiotic treatment, individuals remain chronically infected for life. The presence of semi-dormant tissue cysts that can reactivate predisposes infected individuals to the risk of complications in the event of waning immunity. One of the key features of T. gondii that enables it to persist in the host is the ability to block immune responses, and thereby prevent clearance of the parasite. Among the pathways that are perturbed by T. gondii infection are the signal transducer and activator of transcription (STAT) family of transcription factors. Control of T. gondii infection relies on activation of STAT1 by IFN-γ, which is essential to upregulate antimicrobial pathways in both hematopoietic and non-hematopoietic cells. However, when cells are infected by T. gondii before encountering IFN-γ, the parasite is able to block STAT1-mediated transcription resulting in reduced control of infection. Global analysis of gene expression reveals that the majority of IFN-γ-activated genes are blocked by prior infection with T. gondii. The block in STAT1 signaling occurs in both rodent and human cells, although the molecular basis for this trait has not been previously identified. In preliminary studies we have identified a T. gondii protein that binds to phosphorylated STAT1 dimers and prevents activation of downstream genes. This effector, known as T. gondii Inhibitor of STAT Transcription (TgIST) blocks IFN-γ-induced gene expression mediated by STAT1. TgIST is both necessary and sufficient for blocking STAT1- mediated transcription in mouse and human cells. TgIST binds to a host repressor complex that alters chromatin structure. The proposed studies will define the role of host chromatin modifiers in modulating STAT1 transcriptional complexes using gene disruption by CRISPR/Cas9 combined with various reporter assays. Additionally, we will implement a variety of cellular, biochemical, and genetic methods to define the domains necessary for export and trafficking of TgIST to the host cell nucleus, where it is active.

弓形虫是一种广泛传播的动物寄生虫，可引起人畜共患感染 尽管健康成年人的急性感染通常可以在不发生感染的情况下得到解决。 后果严重，免疫功能低下者感染可导致严重后果 此外，由于慢性感染不能被免疫系统清除，因此会出现并发症。 系统或通过正常的抗生素治疗，个体终生仍处于慢性感染状态。 半休眠组织囊肿的存在可以重新激活，容易被感染 个人在免疫力下降时出现并发症的风险的关键之一。 弓形虫能够在宿主体内持续存在的特征是能够阻断免疫 反应，从而阻止寄生虫的清除。 受弓形虫感染干扰的是信号转导子和转录激活子 (STAT) 转录因子家族对弓形虫感染的控制依赖于激活 STAT1 通过 IFN-γ，这对于上调两种细菌的​​抗菌途径至关重要 造血细胞和非造血细胞 然而，当细胞被 T. 在遇到 IFN-γ 之前，弓形虫能够阻断 STAT1 介导的 转录导致感染控制的降低。 揭示了大多数 IFN-γ 激活基因被先前感染 T. STAT1 信号传导的阻断发生在啮齿动物和人类细胞中，尽管 这种性状的分子基础此前尚未确定。 在初步研究中，我们已经鉴定出一种弓形虫蛋白，该蛋白可与磷酸化的 STAT1 二聚体并阻止下游基因的激活，称为 T。 弓形虫 STAT 转录抑制剂 (TgIST) 阻断 IFN-γ 诱导的基因表达 由 STAT1 介导的 TgIST 对于阻断 STAT1- 来说是必要且充分的。 TgIST 在小鼠和人类细胞中介导的转录与宿主阻遏蛋白结合。 拟议的研究将确定宿主的作用。 使用基因调节 STAT1 转录复合物的染色质修饰剂 此外，我们将通过 CRISPR/Cas9 结合各种报告基因检测进行破坏。 实施各种细胞、生化和遗传方法来定义领域 TgIST 输出和运输至宿主细胞核（TgIST 在细胞核中具有活性）所必需的。