VraSR在低水平万古霉素耐药金黄色葡萄球菌致病中的作用及机制研究

The adaptive change and regulation mechanism of bacteria under antibiotics are an important scientific issue. Upon treatment with vancomycin, Staphylococcus aureus can develop to low level vancomycin resistant strains (including hVISA and VISA) , and display reduced virulence and increased biofilm formation commonly, but the mechanisms are still not so clear. In our previous study, we found that there was a correlation between vraSR (vancomycin resistance-associated sensor regulator) and genes associated with virulence and biofilm formation and their regulatory gene (accessory gene regulator, agr) at transcriptional level in a series of laboratory-induced vancomycin-resistant strains. Furthermore, VraR can bind to the agr promoter region. These results indicated that VraSR may regulate the expression of genes relevant to virulence and biofilm formation. In this study, (1) We will use vraSR knock-out mutations, complemented mutations, vraSR over-expression strains and induced resistant strains to investigate the effects of VraSR on phenotype, genic mutation, genetic transcription, and the pathogenicity of hVISA/VISA. (2) We will use EMSA and DNase I footprinting to explore the regulatory mechanisms by which VraR regulates the expression of genes associated with virulence and biofilm formation directly and/or indirectly and feedback regulation mechanism by which Agr regulates vraSR. The results of this study will enrich the regulatory network of S.aureus, and provide a theoretical basis for the diagnosis and intervention strategies targeting the VraSR against hVISA/VISA infection.

细菌在抗菌药物作用下适应性改变和调控机制是重要的科学问题。金葡菌在万古霉素作用下可发展为低水平万古霉素耐药金葡菌（包括hVISA和VISA），并常伴有毒力减低、生物膜形成增加，其机制尚不清楚。我们前期使用系列万古霉素诱导耐药菌株研究发现，金葡菌耐药二元调控子vraSR与毒力、生物膜相关基因及其调控基因agr在转录水平存在相关性，且VraR可结合agr启动子，证明VraSR对毒力和生物膜也具有调控作用。本课题将：（1）使用vraSR敲除、回补和过表达株及万古霉素诱导耐药株，深入研究VraSR表达对金葡菌生物表型、基因突变、基因转录及致病力的影响。（2）采用凝胶迁移阻滞、DNase I足迹等方法研究VraR对毒力和生物膜相关基因直接和/或间接调控机制，及Agr对vraSR反馈调控机制。研究结果将丰富金葡菌基因调控网络，为基于VraSR为靶点的hVISA/VISA感染诊断和干预策略提供理论依据。

细菌在抗菌药物作用下适应性改变和调控机制是重要的科学问题。金葡菌在万古霉素作用可诱导为低水平万古霉素耐药，常伴有生物膜形成增加、毒力减低。我们在前期研究中发现，金葡菌万古霉素耐药二元调控子VraSR通过结合agr对毒力和生物膜也有调控作用。本课题旨在揭示VraSR在低水平万古霉素耐药金葡菌致病中的作用和机制。主要研究内容和结果如下：1. 体外试验证明VraSR表达能够增加金葡菌对万古霉素耐药，降低细菌色素形成，减少溶血素等毒力基因表达、增强生物膜形成、抵抗吞噬细胞的吞噬和杀菌，抑制细胞炎症反应、抑制巨噬细胞凋亡和坏死，抑制巨噬细胞自噬流。2. 动物试验表明，无论是皮下接种或静脉注射建模，小鼠均未发生致死性全身感染，而是表现为局部脓肿。表明VraSR表达使金葡菌致病力减弱，但抵御宿主细胞清除能力增强，细菌从侵袭性感染转变为局部持续性感染。3.我们证明VraSR可以与rot基因启动子直接结合，增强rot表达。此外，我们还证明AgrA可以负调控vraSR。研究结果表明，在金葡菌耐药基因和毒力基因之间存在复杂的相互调控网络。本研究阐明了VraSR的表达除调控金葡菌对万古霉素耐药以外，还具有调控细菌毒力、生物膜和致病力等多种作用，丰富了金葡菌的基因调控网络，为基于 VraSR 为靶点的 hVISA/VISA 感染诊断和干预策略提供理论依据。.在该项目的资助下，我们发表了标注项目资助号的标注论文6篇，其中4篇为中文核心，2篇为SCI收录期刊，另有两篇SCI论文正在撰写中。