CobB调控MurA琥珀酰化影响金黄色葡萄球菌中介耐受万古霉素的作用与机制

Abstract：Arising and prevalence of vancomycin-intermediate Staphylococcus aureus (VISA) have brought a great challenge to clinical treatment. Nowadays, study on the mechanisms underlying VISA formation has mainly focused on the protein dysfunction caused by gene mutation. Whether a protein post-translational modification affects VISA formation is not clear. Thickened cell wall is a common feature for VISA strains. Our previous study demonstrated that the deletion of cobB gene resulted in higher protein lysine succinylation levels in S. aureus strain XN108, accompanying the thinner cell wall and lower vancomycin resistance. Using mass spectrometry, we found the major alteration of succinylation occurred in the UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA), which plays an important role in the cell wall biosynthesis. Therefore, we speculated that CobB may affect vancomycin intermediate resistance of S. aureus by controlling the succinylation of MurA, resulting in the enzymatic activity alteration of MurA and the decreased biosynthesis of bacterial cell wall. To verify the hypothesis, this project aims to firstly confirm the regulation role of CobB on protein succinylation of MurA and the resistance of VISA. The mutants with cobB or murA gene deletion will be constructed to evaluate the level of protein succinylation and drug resistance. Then, the influence of succinylation on MurA enzymatic activity will be evaluated in vitro, and the influence of MurA succinylation on the cell wall synthesis of VISA will also be performed in vivo by site-directed mutation strategies. Our study will not only help to understand the new mechanisms of VISA formation, but also provide new strategies for clinical treatment of VISA infections.

万古霉素中介耐药金葡菌(VISA)的不断出现及流行给临床控制金葡菌感染带来了严峻挑战。迄今，VISA耐药机制的研究主要集中在基因突变所致的蛋白质功能变化上，而对蛋白质翻译后修饰能否影响VISA的耐药性知之甚少。细胞壁增厚是VISA的普遍特征。我们前期发现cobB基因敲除后，金葡菌琥珀酰化修饰水平增高，细胞壁变薄，耐药性降低。结合质谱分析结果，我们推测CobB可能通过调控细胞壁合成酶MurA的琥珀酰化，进而影响VISA耐药性。为验证该假设，本项目拟构建cobB和murA基因敲除株，观察琥珀酰化修饰和耐药水平变化，证实CobB调控MurA琥珀酰化影响VISA耐药性的作用；制备MurA琥珀酰化修饰位点突变蛋白，体外评价琥珀酰化修饰对MurA酶活的影响；构建修饰位点突变株，检测细胞壁厚度改变，从翻译后修饰角度揭示CobB影响VISA万古霉素耐药的新机制，为临床控制耐药金葡菌感染提供新的途径。

万古霉素中介耐药金葡菌（VISA）的流行给临床治疗金葡菌感染造成严重困扰。本项目前期发现蛋白质琥珀酰化修饰与金葡菌万古霉素耐药具有显著相关性。为阐明蛋白质翻译后修饰影响VISA耐药的新机制，本项目首先以XN108株为对象，对该菌的琥珀酰化修饰及乙酰化修饰组学进行分析，共计发现799个蛋白中的3260个赖氨酸位点发生琥珀酰化修饰，1710个蛋白的7935个赖氨酸位点发生乙酰化修饰，蛋白功能分析提示修饰蛋白主要富集在中心代谢相关通路。随后，通过构建cobB基因突变株，证实了CobB蛋白参与调控VISA菌的琥珀酰化修饰，同时该蛋白显著影响细菌万古霉素耐药表型及细胞壁表型。为证实上述表型之间的相关性，对XN108-WT及XN108-ΔcobB敲除株的琥珀酰化修饰组进行比较组学分析，发现金葡菌细胞壁合成酶MurA活性中心K22、K69、K93、K191等赖氨酸位点的修饰水平受CobB蛋白调控。因此，利用同源重组技术在XN108及K65等VISA菌株中构建murA敲除株及上述修饰位点突变株，包括单一位点突变及组合位点突变，共计获得16个不同的突变株，利用肉汤稀释法和E-test法检测突变株的万古霉素耐药水平，发现突变株的MIC值均不同程度降低；其中以K65为背景的突变株，其万古霉素耐药水平从4μg/mL下降至1μg/mL左右，从万古霉素中介耐药菌变成万古霉素敏感菌，该结果表明MurA琥珀酰化修饰对金葡菌中介耐受万古霉素具有重要影响，可作为治疗VISA感染的潜在药物设计靶点。本项目为蛋白质翻译后修饰参与调控金葡菌中介耐受万古霉素的新机制提供了充分证据。本项目投稿会议论文摘要3篇，1篇SCI论文正在审稿，参与培养研究生1名。