Molecular functions of the murine guanylate binding protein (mGBP) 2 in the immune defense against Toxoplasma gondii

干扰素γ（IFN γ）是一种促炎细胞因子，其在感染的遏制和清除中起关键作用。具有遗传缺陷的患者、鼠模型系统以及体外实验已经证明，IFN γ是宿主防御的必需细胞因子，导致有效控制，特别是细胞内病原体。IFN γ受体刺激主要通过Jak/Stat介导的大量转录应答导致细胞活化。鸟苷酸结合蛋白（GBP）属于响应IFN γ刺激而大量表达的GTP酶的主要家族。GBP已被证明对于限制细胞内寄生虫（即，全球分布的刚地弓形虫）的复制至关重要。T.弓形虫的生长限制是由一组GBP募集到寄生虫空泡（PV）的膜上，易位到PV空间，并最终与T.弓形虫采用生物化学和成像（激光扫描显微镜/多参数荧光图像光谱）技术，它可以证明，鼠GBP（mGBP）驻留在至少两个离散的亚细胞水库和攻击寄生虫空泡膜（PVM）作为精心策划的，超分子复合物形成大，密集包装的多聚体，包括多达几千个单体。显著的mGBP富集导致PVM完整性的丧失，随后mGBP 2直接攻击寄生虫的质膜。最近，我们发现了mGBP 2的新的相互作用伙伴，如细胞骨架相关蛋白（Ckap 4）、膜联蛋白A5（AnxA 5）、半乳糖凝集素9（Gal 9）和干扰素刺激基因15（ISG 15），它们可以在T.刚地。在即将到来的资助期内，mGBP 2的PVM和寄生虫相互作用的分子内和分子间先决条件将被解开，Ckap 4，Anxa 5，Gal 9和ISG 15在弓形虫感染中的作用和效应功能将被研究。此外，将鉴定具有抗寄生虫活性的mGBP 2的新型相互作用伴侣。该项目将为GBP效应分子介导的细胞自主免疫提供重要的动态和分子感知，以抵御重要的细胞内病原体。

Interferon gamma (IFNgamma) is a proinflammatory cytokine which plays a crucial role in the containment and clearance of infections. Patients with genetic defects, murine model systems as well as in vitro experiments have demonstrated that IFNgamma is an essential cytokine for host defense leading to effective control, especially of intracellular pathogens. IFNgamma receptor stimulation results in activation of cells, primarily through a Jak/Stat mediated massive transcriptional response. Prominently, Guanylate-binding proteins (GBPs) belong to a major family of GTPases being abundantly expressed in response to IFNgamma stimulation. GBPs have been shown to be crucial for restriction of the replication of intracellular parasites, i.e. the worldwide distributed Toxoplasma gondii. T. gondii growth restriction is mediated by recruitment of a set of GBPs to the membrane of the parasitophorous vacuole (PV), translocation into the PV space, and, ultimately, the association of GBP molecules with the membrane of the T. gondii parasite. Employing biochemical and imaging (Laser Scanning Microscopy / Multiparameter Fluorescence Image Spectroscopy) techniques it could be demonstrated that murine GBPs (mGBPs) reside in at least two discrete subcellular reservoirs and attack the parasitophorous vacuole membrane (PVM) as orchestrated, supramolecular complexes forming large, densely packed multimers comprising up to several thousand monomers. The dramatic mGBP enrichment results in the loss of PVM integrity, followed by a direct assault of mGBP2 upon the plasma membrane of the parasite. Recently, we could identify novel interaction partners of mGBP2, such as Cytoskeleton associated protein (Ckap4), Annexin A5 (AnxA5), Galectin 9 (Gal9), and Interferon Stimulated Gene 15 (ISG15) which can be detected at the PV of T. gondii. In the upcoming funding period, the intra- and intermolecular preconditions of the PVM and parasite interactions of mGBP2 will be unraveled and the roles and effector functions of Ckap4, Anxa5, Gal9, and ISG15 in toxoplasma infection will be investigated. Furthermore, novel interactions partners of mGBP2 with anti-parasitic activities will be identified. This project will provide vital dynamic and molecular perceptions into cell-autonomous immunity mediated by GBP effector molecules in host defense against important intracellular pathogens.