Elucidation of the Molecular Mechanism of Staphylococcus aureus Response to Cell-Wall Damage

细胞壁是细菌抵御环境变化和抗菌剂的第一道防线。细胞壁具有选择性渗透性、机械强度和柔韧性，是细胞生长所必需的。因此，细胞壁的完整性是许多抗生素的靶标。我研究的首要目标是了解金黄色葡萄球菌对细胞壁破坏的反应。金黄色葡萄球菌的细胞壁由称为肽聚糖的糖肽聚合物和共价连接到肽聚糖的聚阴离子聚合物组成，称为壁磷壁酸。这两种聚合物的完整性对于细菌建立宿主定植、感染和启动免疫反应是必不可少的。基于基因组的研究表明，细菌对细胞壁损伤的反应一般都有信号转导通路，特别是金黄色葡萄球菌，以感知和反应损伤。特别是，一个双组分的信号转导系统(TCS)，VraSR，涉及金黄色葡萄球菌对以肽聚糖生物合成为靶点的抗生素的反应。另一种TCS，GraSR，参与了阳离子抗菌肽对细胞壁电荷的调节。此外，曾经被认为是真核生物特有的Ser/Thr激酶/磷酸酶信号通路，参与了细胞壁生物合成的调节和细胞壁损伤的感知。最近，我们的团队发现金黄色葡萄球菌的Stk1磷酸化了一种新的D-氨基酯酶FmtA，它调节细胞壁的电荷。尽管这些信号转导通路参与了细胞壁损伤反应的大量信息，但细胞内应力传递和细胞壁损伤反应的机制(S)还不完全清楚。此外，这些信号转导通路之间的网络正在被认识到，但还没有完全阐明，其意义也没有被理解。该研究计划旨在：1)确定VraSR、GraSR和Stk1/STP信号转导的分子机制；2)确定它们之间的联网机制；3)确定VraSR、GraSR和Stk1/STP感知细胞壁损伤的分子机制。我的实验室拥有蛋白质化学、分子生物学和微生物学方面的成熟专业知识，可以实现上述目标。目标蛋白将被分离并通过凝胶电泳法鉴定其活性，它们之间的相互作用将通过下拉试验和等温滴定热法(ITC)进行测试。此外，目标基因将被敲除，以研究体内基因功能，其中包括使用电子显微镜和核磁共振。预期在该计划中获得的知识将对参与微生物应激反应、细胞壁生物合成和基因调控的研究团体产生极大的兴趣。

The cell wall is the first line of the bacterial defense against environmental changes and antimicrobial agents. With its selective permeability, mechanic strength and flexibility, the cell wall is essential to the cell growth. As such, the cell wall integrity is the target of a number of antibiotics. The overarching goal of my research is to understand the Staphylococcus aureus response to cell-wall damage. The cell wall in S. aureus consists of a glycopeptide polymer, referred to as peptidoglycan, and a polyanionic polymer covalently attached to peptidoglycan, referred to as wall teichoic acid. The integrity of both polymers is indispensable for bacteria to establish host colonization, infection and initiate an immune response. Genome-based studies on profiling the bacterial response to cell-wall damage have revealed that bacteria in general, and S. aureus in particular, have signal transduction pathways in place to sense and respond to the damage. In particular, a two-component signal transduction system (TCS), VraSR, is involved with the S. aureus response to antibiotics that target peptidoglycan biosynthesis. Another TCS, GraSR, is involved with the modulation of the cell-wall charge in response to cationic antimicrobial peptides. Furthermore, a Ser/Thr kinase/phosphatase signaling pathway, once considered unique to eukaryotes, is implicated in regulation of the cell-wall biosynthesis and sensing cell wall damage. Recently, our group discovered that Stk1 of S. aureus phosphorylates a novel D-amino esterase, FmtA, which modulates the cell-wall charge. Despite the ample information on the involvement of these signal transduction pathways in response to the cell-wall damage, the mechanism(s) of transducing stress intracellularly and mounting the response to cell-wall damage is(are) not fully understood. Moreover, the networking among these signal transducing pathways is being recognized but not fully elucidated and its significance is not understood. The proposed research program aims to: 1) determine the molecular mechanism of signal transduction by VraSR, GraSR and Stk1/Stp; 2) determine the mechanism of networking among them; and 3) determine the molecular mechanism of sensing cell-wall damage by VraSR, GraSR and Stk1/Stp. My laboratory has the established expertise in protein chemistry, molecular biology and microbiology to achieve the above objectives. The target proteins will be isolated and characterized for their activity by gel electrophoresis, and interactions among them will be tested by pull-down assays and isothermal titration calorimetry (ITC). In addition, the target genes will be knocked out to investigate in vivo gene function, using among others Electron Microscopy, and Nuclear Magnetic Resonance. The knowledge anticipated to be gained in this program will be of great interest to the research communities involved with microbial stress response, cell wall biosynthesis and gene regulation.