WalKR调控金黄色葡萄球菌生物膜形成机制研究

Biofilm can assist Staphylococcus aureus（S. aureus） in evading the host immune system, and represent a diffusion barrier for antibiotics. Biofilm-associated infections caused by S. aureus often lead to persistent infections that are difficult to cure. A better understanding of the development of biofilm at the molecular level is imperative to generate new treatment strategies for these infections. Our previous research found that the expression of WalKR positively correlated with biofilm formation. With CRISPRi system, we have successfully knocked down walKR gene. S. aureus with WalKR low expression led to a reduced biofilm formation. These data indicated that WalKR play an important role in the process of biofilm formation in S. aureus, but the specific mechanism remains to be elucidated. The applicant aimed to explore the scientific problems as follows: 1. Transcriptome Sequencing will be performed to test the effects of WalKR on the expression of genes involved in biofilm formation. Pull down assay with mass spectrometry will be performed to identified new proteins which directly bind to WalR. EMSA and DNase I footprinting will be used to explore the regulatory mechanisms by which WalR and its interactive proteins regulate the expression of genes associated with biofilm formation directly and/or indirectly. 2. Validate the correlation between WalKR and the ability of biofilm formation in clinical isolates. Evaluate the role of WalKR in biofilm-associated infection diagnosis. 3. Evaluate the role of WalKR inhibition involved in biofilm formation regulation and the anti-infection effect. The results of this study will provide the theoretical basis for the hypothesis that WalKR is a key regulator and therapeutic target in biofilm-associated infection caused by S. aureus.

金葡菌常通过形成生物膜逃避宿主免疫攻击，抵御抗菌药物清除，致感染迁延难愈。研究生物膜形成的调控机制对寻找特异的诊疗靶点具有重要意义。申请人前期研究发现双组分调控系统WalKR表达量与金葡菌生物膜形成呈正相关。利用CRISPRi技术构建WalKR敲低株，发现其生物膜形成明显减少，推测WalKR可调控金葡菌生物膜形成。本研究拟1.通过转录组测序揭示WalKR对生物膜形成功能基因及调控因子转录的影响；Pulldown结合质谱方法发现与WalR相互作用的蛋白；EMSA等方法研究WalR及其互作蛋白与目标基因间的作用关系和调控机制。2.检测临床分离的金葡菌，验证WalKR差异表达和生物膜形成之间的关联，评估其作为金葡菌生物膜相关感染诊断分子标志物的潜能。3.靶向干预WalKR验证其对生物膜形成的调控作用和抗感染效应。研究结果将为建立以WalKR为靶点的金葡菌生物膜相关感染诊断和干预方法提供理论依据。

生物被膜是金黄色葡萄球菌（金葡菌）重要的适应性防御机制之一。生物被膜的形成减少了抗菌药物的渗入，阻碍了机体免疫系统对菌体的清除，使感染进入难以治愈的慢性状态。近年来，金葡菌生物被膜相关感染的发病率逐年上升，给临床诊疗带来极大困难。我们前期利用CRISPRi技术构建了 WalKR敲低菌株，发现敲低WalKR基因，菌株生物被膜形成明显减少，推测WalKR参与调控金葡菌生物被膜的形成，具有成为生物被膜相关感染诊断标志物及治疗靶点的潜在价值。本研究：1.构建了WalKR敲低株和过表达株，检测各菌株生物被膜形成能力，证实WalKR可调控金葡菌生物被膜形成。2.转录组测序分析揭示WalKR可调控金葡菌生物被膜形成相关功能基因的转录，上调isaA、ssaA、clfA、fnbA等基因表达。3.利用凝胶迁移阻滞、DNase I足迹印迹等实验证实WalR可直接结合clfA启动子区域，正调控clfA表达。敲除clfA，WalKR过表达菌株生物被膜形成明显减少。4.pulldown结合质谱方法发现WalR蛋白可与MgrA蛋白相互作用。初步阐明WalKR调控金葡菌生物被膜形成的分子机制。5.检测了117株临床分离的金葡菌生物被膜形成能力及WalKR mRNA表达水平，证实WalKR差异表达和生物被膜形成之间存在关联。6.利用WalKR差异表达的临床分离株构建小鼠植入物感染模型，证实金葡菌生物被膜形成能力与WalKR表达水平呈正相关。7.体外实验证实靶向干预WalKR，可影响金葡菌黏附、自溶和eDNA释放，进而影响金葡菌生物被膜形成。8.使用NCTC 8325野生株和WalKR过表达株，构建小鼠置入物感染模型，使用万古霉素和/或WalR抑制剂walrycin B治疗。结果显示，较之于野生株，WalKR过表达菌株植入物表面形成的生物被膜更厚更致密，使用万古霉素和walrycin B治疗，均可减少植入物表面生物被膜的形成。二者联合治疗，抗生物被膜形成的效果更为明显。.本研究揭示了WalKR 在金葡菌生物被膜形成中的重要作用，阐明WalKR调控金葡菌生物被膜形成的分子机制，为建立基于WalKR为靶点的金葡菌生物被膜相关感染诊断方法和干预策略提供理论依据。