MraZ通过影响毒力相关基因的表达增强金黄色葡萄球菌毒力的研究

Staphylococcus aureus can causing a variety of diseases ranging from benign, supercial skin infection to life-threatening infections. Many virulence factors are involved in S. aureus infections. In our previous study, mraZ has been knocked out in S. aureus MW2 (MW2), named MW2 ΔmraZ. Compared with MW2 wild type strain, the diameters of hemolysis ring on blood agar plate and the abscess size in mice caused by MW2 ΔmraZ were significantly smaller and the survival rate of greater wax moth infected by MW2 ΔmraZ increased significantly. The RNAs of MW2 ΔmraZ and MW2 wild type strain were sequenced. The differential expression genes between MW2 ΔmraZ and MW2 wild type strain (≥2 folds) were 324 including 7 virulence-associated genes. Therefore, we speculate that MraZ encoded by mraZ can enhance virulence of Staphylococcus aureus via regulating the expression of virulence-associated genes. To corroborate our speculation, we will construct complementary strain of MW2 ΔmraZ with plasmid containing mraZ and further investigate the role of MraZ in the course of S. aureus infection by mice infection model, and in adhension and invasion of S. aureus to epithelial cells and immune evasion of S. aureus from host. Based on the data of RNA sequencing obtained from previous study, the virulence-associated candidate genes regulated by MraZ will be investigated and the regulation mechanism of MraZ on the expression of virulence-associated candidate genes will also investigated. The mechanism of mraZ expression will be further studied. The findings of the present study will be helpful for finding MraZ as an important potential target for antivirulence strategy for treating S. aureus infections.

金黄色葡萄球菌可引起从浅表的皮肤感染到威胁生命的严重感染，由多种毒力因子参与。我们前期研究利用同源重组技术敲除了金黄色葡萄球菌MW2（MW2）的mraZ基因，发现与MW2野生株相比，mraZ敲除株在血平板上的溶血环明显缩小，感染大蜡螟后生存率明显增加，皮下感染小鼠后的脓肿面积显著缩小；转录组分析发现，与MW2野生株相比，mraZ敲除株差异表达的基因有324个（≥2倍），包括7个毒力相关基因。我们推测MraZ可能通过影响毒力相关基因的表达增强金黄色葡萄球菌的毒力。为了验证该推测，本项目拟通过构建mraZ阴性回复突变株，利用小鼠感染模型研究MraZ是否可以增加金黄色葡萄球菌感染的严重程度；研究MraZ是否可以增强金黄色葡萄球菌粘附、侵袭和免疫逃逸的能力；挖掘前期转录组数据研究受MraZ调控的相关毒力基因和调控机制以及mraZ基因表达调控机制。为治疗金黄色葡萄球菌感染的抗毒力策略提供更多的靶标。

近年来，多重耐药的耐甲氧西林金黄色葡萄球菌日益流行，这对感染性疾病的抗生素治疗提出了巨大挑战。针对毒力因子的抗毒力策略是一种有希望的治疗金黄色葡萄球菌感染的新方法。本项目构建金黄色葡萄球菌的mraZ的阴性突变菌株和回复菌株证实mraZ基因对金黄色葡萄球菌毒力的影响以及探索MraZ影响毒力的机制。比较野生株、阴性突变株和回复株的表型，发现mraZ不影响金黄色葡萄球菌的生长速度以及色素的产生。mraZ缺失突变菌株在大蜡螟模型以及小鼠皮肤感染模型中的毒力显著降低，对人嗜中性粒细胞和全血的抗性也显著降低，表明MraZ对毒力和免疫逃避的重要贡献支持了金黄色葡萄球菌感染的致病性。RNA-Seq和定量RT-qPCR显示MraZ是一种多功能的调节因子，它参与多种生物学过程。缺失mraZ可导致与毒力相关的sarA、agr、clfA、hly/hla、hld和sec4基因的表达下调,且MraZ可能通过与sarA基因的启动子序列结合直接调控sarA基因的转录从而影响金黄色葡萄球菌的转录。通过该研究我们对金黄色葡萄球菌的毒力机制有了进一步的了解，为治疗金黄色葡萄球菌的感染的抗毒力策略提供了一个靶标。