分泌蛋白Efb在金黄色葡萄球菌感染中抑制巨噬细胞NF-κB信号通路机制研究

The secretory proteins play an important role in negative regulating macrophage immune response during the infection of Staphylococcus aureus. Our previous studies have preliminarily confirmed that Efb could interact with TRAF3 and inhibit the activation of NF-κB pathway in macrophages. The degradation of TRAF3 through K48-linked ubiquitination can activate NF-κB pathway. Therefore, we speculated that negatively regulating NF-κB pathway of macrophages through inhibiting the degradation of TRAF3 by Efb might be an immune evasion stratage of staphylococcus aureus. Therefore, We use Efb as research objects in this project. Firstly, the negative regulation of NF-κB pathway by Efb and the interaction of endogenous Efb and TRAF3 will be investigated by infecting mouse peritoneal macrophages with WT、ΔEfb and ΔEfb+Flag-Efb strains. Secondly, the specific mechanism of Efb and TRAF3 will be investigated by overexpression the mutants or domains of Efb, TRAF3 and related proteins. Finally, mouse bacteremia models or other models will be used to verify the interaction of Efb and TRAF3 during the infection. At the molecular, cellular and animal levels, elucidating the mechanism of negative regulation of NF-κB pathway by Efb will be of great significance in understanding the pathogenesis of Staphylococcus aureus infection diseases.

金黄色葡萄球菌通过分泌蛋白抑制巨噬细胞免疫反应是其感染宿主的重要策略。前期实验显示Efb可显著抑制巨噬细胞NF-κB通路激活，但机制尚不清楚。初步研究显示，Efb可与TRAF3发生互作并抑制其降解。TRAF3 K48位泛素化降解可导致NF-κB通路激活。我们推测Efb抑制TRAF3 K48位泛素化降解进而抑制NF-κB通路激活可能是金葡菌调控巨噬细胞免疫反应的新机制。本项目拟以Efb为研究对象，通过野生菌株、Efb敲除株及回补株感染小鼠腹腔巨噬细胞验证Efb对NF-κB通路抑制作用，并探讨内源性Efb和TRAF3互作关系；然后通过相关蛋白及其点突变、结构域过表达实验等，进一步明确Efb和TRAF3的互作机制；最后利用小鼠菌血症等模型验证Efb和TRAF3互作在疾病发生过程中的效应。以期在分子、细胞、动物水平上阐明Efb抑制巨噬细胞NF-κB通路的机制，为深入理解金葡菌致病机制提供理论支撑。

许多致病微生物通过分泌效应分子劫持宿主免疫细胞内关键调控分子以促进发病。然而，金黄色葡萄球菌分泌的效应分子在宿主细胞内作用机制尚不清楚。在这项研究里，我们揭示了金黄色葡萄球菌将细胞外纤维蛋白原结合蛋白（Efb）在巨噬细胞中抑制宿主免疫反应的核心机制。该机制如下：RING finger蛋白114（一种宿主E3连接酶）介导Efb第71位赖氨酸的K27泛素化，这有助于肿瘤坏死因子受体相关因子（TRAF3）向Efb募集，导致炎症信号级联的抑制。此外，Efb K71R突变体失去抑制炎症的能力，并表现出致病性降低。因此，我们的研究发现了1种金黄色葡萄球菌抑制宿主防御的机制，这极有可能是一个开发有效的抗金黄色葡萄菌免疫调节剂的靶点。