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

干扰素是一种促炎细胞因子，在控制和清除感染方面起着至关重要的作用。有遗传缺陷的患者、小鼠模型系统以及体外实验都证明，IFNGamma是宿主防御所必需的细胞因子，可以有效地控制，特别是对细胞内病原体的控制。IFNGamma受体刺激导致细胞激活，主要是通过Jak/Stat介导的大规模转录反应。尤其值得一提的是，鸟苷结合蛋白(GBP)属于一个主要的GTP酶家族，在对IFN-γ刺激的反应中大量表达。GBP已被证明对于限制细胞内寄生虫的复制是至关重要的，即分布在世界各地的弓形虫。弓形虫的生长限制是通过将一组GBP募集到寄生虫液泡(PV)的膜上，转位到PV空间，最终GBP分子与弓形虫寄生虫膜的结合来调节的。利用生化和成像(激光扫描显微镜/多参数荧光图像光谱)技术，可以证明小鼠GBPs(MGBPs)存在于至少两个离散的亚细胞储存库中，并以有序的超分子复合体的形式攻击寄生液泡膜(PVM)，形成包含多达数千个单体的大而密集的多聚体。MGBP的戏剧性浓缩导致PVM完整性的丧失，随后mGBP2直接攻击寄生虫的质膜。最近，我们发现了新的mGBP2相互作用伙伴，如细胞骨架相关蛋白(Ckap4)、膜联蛋白A5(AnxA5)、Galectin 9(Gal9)和干扰素刺激基因15(ISG15)，这些都可以在弓形虫的PV中检测到。在即将到来的资助期间，mGBP2的PVM和寄生虫相互作用的分子内和分子间的前提条件将被揭开，Ckap4、Anxa5、Gal9和ISG15在弓形虫感染中的作用和效应功能将被研究。此外，mGBP2与抗寄生虫活性的新的相互作用伙伴将被确定。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，以防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.