苯唑西林通过SarA促进金黄色葡萄球菌膜泡释放的作用与机制研究

膜泡是细菌分泌的膜状结构，可携带蛋白质等成分，在疫苗或疫苗载体研发中具有广阔应用前景。膜泡分泌包括生成和释放两个阶段，相关调控机制不清，探索促进膜泡分泌的途径和机制是其开发利用的前提。我们前期发现，亚抑菌浓度苯唑西林作用下，金葡菌转录因子SarA和自溶素Sle1表达上调，膜泡产量显著提高，敲除sarA则膜泡产量降低；信息学分析发现sle1基因调控区存在SarA的结合位点，故推测苯唑西林通过SarA上调Sle1表达，降低细胞壁交联度，促进金葡菌的膜泡释放。为此，本项目拟构建目标基因敲除和回补金葡菌株，检测肽聚糖交联度和膜泡产量变化；利用基因报告系统、EMSA等实验，阐明苯唑西林通过SarA促进金葡菌膜泡释放的作用机制；再利用蛋白质组学比较抗生素处理前后细菌膜泡组分的差异，观察抗生素刺激制备的膜泡疫苗对细胞和动物的保护作用。结果对理解金葡菌膜泡释放机理有重要意义，也可为高效制备膜泡提供新途径。

Bacterial membrane vesicles (MVs) are spherical nanoparticles secreted by bacteria during their normal growth. MVs that incorporated with various bacterial proteins have emerged as attractive and effective vaccines or delivery systems in recent years. The secretion of bacterial MVs is divided into two steps, production and release. However, the mechanisms underlying MV production and release are not clear. To explore the strategy and mechanism for promotion of high-level production of MVs is the key for MV applications. In our previous study, we showed that sub-inhibitory concentrations of oxacillin remarkably enhanced the production of MVs by Staphylococcus aureus strains. RT-qPCR detection and Western blot analysis revealed that the expressions of regulator SarA and autolysin Sle1 genes were upregulated. Deletion of sarA decreased the production of MVs in S. aureus. Bioinformatic analysis revealed a putative binding site of SarA exits in the promoter regions of sle1 gene. Therefore, we suppose that sub-MIC oxacillin can trigger the SarA-dependent upregulation of autolysin Sle1, which promotes the release of S. aureus MVs through the alteration of cell wall crosslinking. To text this hypothesis, the sarA and sle1 deletion S. aureus mutants and corresponding complement strains will be constructed to verify the role of SarA-controlled Sle1 in the release of bacterial MVs. Next, the gene reporter assay, site-directed mutation assay, electrophoretic mobility shift assay, and DNA footprinting experiment will be performed to elucidate the molecular mechanism by which SarA regulates sle1 expression. Lastly, the alterations in the MV components secreted by S. aureus in presence and absence of oxacillin will be compared using proteomic technology. The protective roles of oxacillin-stimulated MV vaccine against dengue virus challenge will also be evaluated in culture cells as well as animal models. The results of present project will not only be important for understanding the release of bacterial MVs, but also provide a new way in the preparation of high-level S. aureus MVs before the translation of bacterial vesicles into real-life applications.