CcpA通过crt操纵子增强金黄色葡萄球菌毒力的作用与机制研究

金葡菌的重要生物学特性之一便是能产生具有抗氧化杀伤能力的金黄色色素，该色素也是细菌重要的毒力因子。研究表明，金葡菌色素合成由crt操纵子控制，该操纵子可受SigB调控，但是否存在其他调控途径尚未见报道。我们前期发现，敲除能量代谢调控基因ccpA可使金葡菌色素合成显著下降，敲除菌毒力减弱，但SigB表达无变化；报告系统检测证实CcpA对crt启动子具有调控作用，信息学分析提示crt启动子区存在CcpA结合位点，故推测CcpA可通过调控crt操纵子增强金葡菌毒力。为此，本项目拟构建单/双目标基因敲除及回补菌株，再通过细胞抗氧化杀伤及小鼠感染实验探讨CcpA上调细菌色素合成，促进金葡菌毒力的作用；进一步采用EMSA、DNase I足迹法和蛋白-DNA相互作用等实验阐明CcpA调控crt操纵子的分子机制。研究结果不仅能加深我们对金葡菌色素合成调控机理的理解，亦可为临床抗金葡菌感染治疗提供新的靶标。

One of the most important biological characteristics of Staphylococcus aureus is the production of golden carotenoid pigment staphyloxanthin (STX), an important virulence factor for S. aureus owing to its antioxidant activity. Previous studies have proven that the synthesis of STX is fully controlled by the crt operon, which activity can be regulated by the staphylococcal alterative sigma factor SigB. However, whether there exist other regulatory pathways has not been reported yet. We previously found that deletion of the catabolite control protein A gene (ccpA) in S. aureus strain USA300 resulted in significantly reduced of the STX production, as well as the virulence of the ccpA mutant compared with that of the wild-type strain, but the expression of SigB was unchanged as determined by Western blot. The LacZ reporter assay revealed that CcpA had a regulatory effect on the crt promoter, and the bioinformatics analysis suggested that there is a putative CcpA binding site in the crt promoter regions. Therefore, we speculated that CcpA can enhance the virulence of S. aureus through regulation of the crt operon activity. In order to test this hypothesis, the single/double genes (ccpA and crt) deletion mutants will be constructed, and the antioxidant experiment of phagocytes as well as the S. aureus-infected mouse models will further be performed to explore the effects of CcpA-controlled STX on the promotion of bacterial virulence in vitro and in vivo. Then, the molecular mechanism underlying CcpA regulation of the crt operon will be elucidated through the electrophoretic mobility shift assay (EMSA), DNA foot-printing experiment and protein-DNA interaction experiment. The research results will not only deepen our understanding of the regulatory pathway of the golden staphyloxanthin biosynthesis, but also suggest new targets for the establishment of novel therapeutic strategies against S. aureus infections.