CobB通过GlnA琥珀酰化 负调控金黄色葡萄球菌万古霉素中介耐药性的作用与机制

The increasing prevalence of vancomycin-intermediate Staphylococcus aureus (VISA) has become a global public health concern. Currently, major studies on the mechanisms of VISA formation are focus on the mutations in certain genes, and studies from the perspective of post-translation modification are limited. Succinylation is a novel protein modification involving glycolysis, protein metabolism and other biological processes. In our previous studies, we found the succinylation levels of total proteins in VISA were significantly decreased than those in proteins in vancomycin-susceptible S. aureus (VSSA) . Further studies showed that the succinylation level of VISA strain XN108 increased in the cobB deletion mutant, accompanying by the thinner cell wall and decreased vancomycin resistance level. The mass spectrometry revealed that the most changed succinylation occurred in the glutamine synthase (GlnA), which catalyzes the biosynthesis of glutamine contributing to the cell wall synthesis in S. aureus. Therefore, we speculate that CobB affects VISA resistance through decreasing the succinylation of GlnA. To test this hypothesis, both cobB and glnA gene deletion and complemetant strains will be constructed, and the influence of certain genes on the succinylation in VISA will be evaluated. Then, the succinylation sites on GlnA will be analyzed and site-directed mutations will be performed. The enzyme activities and vancomycin resistance levels will also be determined to clarify the mechanism of CobB regulating GlnA succinylation. The study is expected to provide new ideas on VISA resistance and laid the foundation for the prevention and control of VISA infections.

万古霉素中介耐药金葡菌VISA的不断流行，已成为全球性公共卫生问题。目前对VISA形成机制的研究主要集中在基因突变上，从翻译后修饰角度开展研究极少。琥珀酰化是一种新的蛋白质修饰，涉及糖酵解，蛋白质代谢等多种生物学过程。课题组前期发现VISA菌总蛋白的琥珀酰化水平较敏感菌显著降低；敲除cobB基因的VISA菌之琥珀酰化水平显著升高，同时细胞壁变薄，万古霉素耐药水平降低；质谱鉴定发现谷氨酰胺合成酶GlnA琥珀酰化改变最明显，而谷氨酰胺是细胞壁合成的重要原料。由此推测CobB可能通过抑制GlnA琥珀酰化影响VISA耐药性。故本项目将构建cobB及glnA的敲除株及回补株，检测琥珀酰化对VISA耐药性的影响；分析GlnA琥珀酰化位点并进行定点突变，评价突变体酶活性及对细菌耐药水平影响，阐明CobB调控GlnA琥珀酰化的机制。为理解琥珀酰化修饰对VISA耐药性的作用，防控VISA菌感染奠定基础。

万古霉素中介耐药金葡菌VISA的不断流行，已成为全球性公共卫生问题。目前对VISA形成机制的研究主要集中在基因突变上，从蛋白质饭后修饰的角度开展研究较少。赖氨酸琥珀酰化是一种新近发现的蛋白质翻译后修饰，影响糖酵解，蛋白质代谢等多种生物学过程。但琥珀酰化与金葡菌耐药的研究罕见。本研究通过同源序列比对，在金葡菌中发现了和大肠杆菌cobB基因的同源序列，利用同源重组技术对XN108中的cobB基因进行敲除，Wersten blot检测发现cobB基因敲除后，金葡菌的琥珀酰化修饰程度上升。同时对XN108-WT和XN108-ΔcobB的生物学表型进行检测，发现CobB对VISA菌株的致病性影响不显著，但显著影响细菌的万古霉素耐药性。为探索其具体机制，利用非标记定量（LFQ）修饰组学检测技术结合LC-MS/MS检测了两种细菌的琥珀酰化组，发现83个蛋白上的98个位点发生了琥珀酰化上调，只有2个蛋白上的3个位点发生了琥珀酰化下调。值得注意的是质谱发现多个与细胞壁合成相关的蛋白如谷氨酰胺合成酶GlnA、谷氨酰胺果糖-6-磷酸转氨酶GlmS、以及UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶MurA等直接参与细胞壁合成的酶发生琥珀酰化修饰。以GlnA，MurA为例构建模拟琥珀酰化突变菌株。结果表明过表达PXR-glnA(K236EK299E)质粒导致其万古霉素耐药MIC下降为6-8μg /ml，采用定点突变技术构建了MurA琥珀酰化修饰突变株，结果表明XN108-MurA（K69E）由原来的12μg/ml降为6μg/ml。综上所述，本研究在VISA菌株XN108中发现CobB具有去琥珀酰化功能，进一步研究发现CobB可以通过影响细胞壁关键蛋白MurA的琥珀酰化，影响金葡菌的万古霉素耐药性。研究结果表明蛋白质琥珀酰化修饰可以影响金葡菌的万古霉素耐药性，为阐明VISA的形成机理提供了一种全新的角度。