骨架蛋白IQGAP1在分枝杆菌PDIM脂质介导感染及肉芽肿形成的作用与机制研究

The PDIM lipid is the specific constituent of pathogenic mycobacterial species, which can mediate granuloma formation although the detailed mechanism is incomplete until now. We have successfully identified the IQGAP1 protein with macrophage proteomics, and western validation found that the PDIM could induce the elevated expression of IQGAP1. Then we used a morpholino (MO) to inhibit the iqgap1 gene in zebrafish embryos and performed blood infection with mycobacterium marinum and found decreased survival in the animals. Finally, we detected positive signals of IQGAP1 in the organs of mice and monkeys after infection with mycobacterium tuberculosis H37Rv strain, especially more positively expressed in the granuloma, however the significance and mechanism of IQGAP1 during this process in unclear. In this project we will focus on the role of IQGAP1 in infection and granuloma formation after challenge by mycobacterium. First, we will silence the iqgap1 gene expression in macrophages with its specific siRNA and do the infection with mycobacterium marinum, to compare IQGAP1 protein level and its-related cytokine profiles mediated by PDIM lipid. Then the iqgap1 gene specific MO will be designed and synthesized and injected at the one-cell stage zebrafish embryo. After blood infection with mycobacterium, we will monitor the migration and recruitment of immune cells, including neutrophils and macrophages, with confocal microscopy. Moreover, we will mutate the iqgap1 gene in zebrafish with Cas9 system and investigate the role of IQGAP1 in granuloma formation. Finally, we will do microarray screening to get the candidate genes which are critical to granuloma formation, and validate its role during the formation of granuloma with different techniques. To summarize, the results of this research not only describe the role of IQGAP1 in the mycobacterial infection, but also clarify the significance and molecular mechanism of IQGAP1 protein in the PDIM-mediated granuloma formation.

PDIM脂质是致病性分枝杆菌特有的细胞壁组分，能介导肉芽肿形成，但机制尚不完善。IQGAP1在多种细菌侵染宿主及感染环节扮演重要角色。我们在PDIM脂质感染的巨噬细胞蛋白组学中筛选并鉴定到IQGAP1；MO抑制斑马鱼iqgap1基因后，感染动物死亡率增加；在结核病小鼠和猴子等组织也检测到IQGAP1高表达，但机制有待深入。本项目通过siRNA沉默iqgap1基因，研究PDIM对巨噬细胞IQGAP1及其相关通路分子的调控效应；MO抑制iqgap1表达，confocal实时动态观察感染后免疫细胞的迁移与募集；Cas9突变iqgap1基因对PDIM介导肉芽肿形成的影响，microarray筛选肉芽肿形成相关基因，并进一步验证关键基因的功能。研究成果既能揭示IQGAP1在分枝杆菌感染中的作用，也能阐明IQGAP1对PDIM脂质介导肉芽肿形成的意义与机制，为研究分枝杆菌细胞壁脂质功能提供新思路。

结核分枝杆菌作为一种典型的胞内寄生菌，它既不含内毒素，也不产生侵袭性酶类和外毒素，其致病主要与细胞壁中高含量的脂质成分密切相关。脂质成分约占细胞壁干重的60%以上，其高含量与细菌毒力密切相关，如phthiocerol dimycocerosates （PDIM）成分。其中PDIM是所有致病性分枝杆菌细胞壁特有脂质成分，能介导细菌的免疫逃逸和肉芽肿形成，但机制尚不完善。.前期工作通过比较野生型（wide-type, WT）海分枝杆菌与PDIM缺失突变株（∆PDIM）感染巨噬细胞的蛋白组学差异，筛选到IQGAP1分子。IQGAP1是一类具有重要调控效应的骨架蛋白，进化上高度保守，在宿主—病原体互作过程中发挥重要作用，但在分枝杆菌感染及肉芽肿形成中的作用未见报道。本研究中，通过体外Raw264.7巨噬细胞感染实验和小鼠感染实验，发现IQGAP1在WT_Mm感染过程中表达上调，并且这种上调效应主要与细菌毒力因子如PDIM等相关； IQGAP1可抑制MKK3以降低p38MAPK磷酸化水平，最终影响NF-κBp65活性抑制宿主早期免疫反应。上调的IQGAP1可促进VEGF表达有助于细菌存活。抑制IQGAP1有助于缓解分枝杆菌诱导的组织病理损伤。.本研究全面系统研究了骨架蛋白IQGAP1在分枝杆菌感染中的作用，首次揭示了IQGAP1通过MKK3调控p38MAPK信号通路影响炎症介质和诱导VEGF表达促进分枝杆菌的免疫逃逸，阐明IQGAP1对肉芽肿形成的作用及骨架蛋白的小分子抑制剂对分枝杆菌感染的干预效应。研究结果既能明晰IQGAP1在分枝杆菌致病中的重要作用，也能为IQGAP1作为抗结核治疗新靶标的研究提供详实的实验数据。