金黄色葡萄球菌AgrA/C双组分信号转导模型的构建与功能评价

Construction and evaluation of AgrA/C two component signal transduction model of Staphylococcus aureus

【背景】抗生素的无序使用加剧了耐药性金黄色葡萄球菌超级菌株的出现,由其引发的感染已成为最难解决的感染性疾患。在生物体系外构建AgrA/C双组分系统的跨膜信号转导过程,对解决金黄色葡萄球菌的耐药性问题和发现新型抗菌药物具有重要的研究意义。【目的】人工模拟构建金黄色葡萄球菌AgrA/C双组分信号转导模型,为生物体外研究金黄色葡萄球菌双组分信号转导的机制及以其为靶点的药物筛选提供新途径。【方法】在大肠杆菌宿主细胞中大量表达AgrA和Agr C蛋白,利用亲和层析和分子筛凝胶层析对其进行分离纯化,利用非放射性凝胶阻滞实验(EMSA)检测AgrA蛋白活性,并检测Agr C激酶活性;进而利用脂质体介导法在体外组装AgrA/C双组分信号转导模型,应用EMSA方法进行评价。【结果】分离纯化得到AgrA和Agr C蛋白,二者纯度均达到90%以上,均具有活性。在生物体系外构建了金黄色葡萄球菌AgrA/C双组分信号转导模型,该系统可增强AgrA对DNA的延滞作用,具有信号传递功能。【结论】初步构建AgrA/C双组分信号转导模型,该模型具有信号传递能力,有望作为针对金黄色葡萄球菌开发新型抗菌药物的筛选平台。

[Background] The abuse of antibiotics accelerated the emergence of antibiotic-resistant of Staphylococcus aureus, and the infections caused by these superbugs have become one of the most challenging tasks in clinic. Reconstruction of the transmembrane signal transduction process of AgrA/C pathway in vitro will be very helpful in solving the problem of antibiotic resistance in S. aureus and developing new antibiotics against these bacteria. [Objective] To realize in vitro construction of an AgrA/C two component signal transduction model for investigating mechanism of two component signal transduction in S. aureus and drug screening. [Methods] AgrA and Agr C proteins were expressed in Escherichia coli C43（DE3） and purified by affinity and size exclusion chromatography, and then their biological activities were analyzed. The AgrA/C two component signal transduction model was constructed in vitro using detergent-mediated method and the validity of the model was verified through the electrophoretic mobility shift assay（EMSA）. [Results] The purity of AgrA and Agr C was over 90%. AgrA protein can bind to the target DNA and Agr C has the kinase activity. The AgrA/C two component signal transduction model was constructed in vitro. The model could enhance the delay of DNA mobility shift by AgrA, indicating the artificial simulation model can transfer signal. [Conclusion] We designed and constructed the AgrA/C two component signal transduction model in vitro successfully. This model system is expected to be a screening platform for new antibacterial drugs targeting S. aureus.