双组分信号转导系统VraSR调控表皮葡萄球菌生物膜形成的分子机制研究

The pathogenesis of Staphylococcus epidermidis depends mostly on its ability to form biofilm on the implanted medical biomaterial surface, and the bacteria within biofilm usually increase drug resistance and cause persistent infection, which brings huge problems for clinical treatment without effective antibiotics against biofilm. The two-component regulatory system (TCS) is capable of rapidly sensing environmental stimuli and controlling multi-biological properties in bacteria. TCS-VraSR (vancomycin-resistant associated Sensor and Regulator) induces an increase of drug resistance through modulating the transcription of genes involved in synthesis of cell wall in S. aureus. Similarly, there exists TCS-VraSR in S. epidermidis. Previous study found that S. epidermidis VraSR shares about 90% homologous identity with that of S. aureus, and deletion of VraSR resulted in decreased biofilm formation, increased dead cells in biofilm, and improved susceptibility to antibiotics in S. epidermidis. These preliminary results indicated that S. epidermidis VraSR, being not identical with that in S. aureus, play an important role in the pathogenesis. Then further study on phenotype changes will be performed after construction of VraSR complementary strain and over-express strain. Microarray will be used to analyze expression level of genes involved in phenotype change, and the suspicious ones regulated by VraSR will be confirmed by Chromatin Immunoprecipitation (ChIP) and Electrophoretic Mobility Shift Assay (EMSA). It lays a theoretical foundation for the control of biofilm infection in S. epidermidis.

表皮葡萄球菌主要在植入性医疗材料表面形成生物膜而引起持续性感染及耐药性增加，目前尚无控制细菌生物膜感染的有效药物。双组分信号转导系统（TCS）能感应环境中压力刺激因子的变化，调控细菌的多种生物学功能。金黄色葡萄球菌TCS-VraSR通过调控与细胞壁合成相关基因的转录而介导耐药性的产生，表皮葡萄球菌中也存在类似TCS。本课题前期研究发现表皮葡萄球菌VraSR与金黄色葡萄球菌类似物的同源性约90%，而敲除VraSR后导致表皮葡萄球菌生物膜形成能力降低，生物膜内的死细菌数显著增多，对抗生素的敏感性增强，提示VraSR在表皮葡萄球菌致病性中发挥重要作用，且其作用机制与金黄色葡萄球菌不尽相同。后续将构建VraSR互补株及过表达株，分析与VraSR突变相关的生物学表型，利用基因芯片技术筛选出可能受VraSR调控的靶基因，并用染色质免疫共沉及凝胶阻滞试验确证，为控制表皮葡萄球菌生物膜感染奠定基础。

表皮葡萄球菌主要在介入性生物材料表面形成生物被膜而致病，是引起医院内感染的重要病原菌之一。万古霉素耐药相关双组分系统VraSR与金黄色葡萄球菌的药物敏感性及毒力相关，敲除VraSR后可恢复万古霉素耐药金黄色葡萄球菌的药物敏感性。表皮葡萄球菌的生物学特点及致病性不同于金黄色葡萄球菌，目前尚不清楚VraSR在表皮葡萄球菌中的生物学作用。. 本项目以万古霉素中介敏感的表皮葡萄球菌临床分离株S.epidermidis strain 1457（SE1457）为研究对象，通过同源重组构建vraSR敲除突变株（∆vraSR）来研究VraSR在表皮葡萄球菌生物膜形成及药物敏感性中的作用。首次发现表皮葡萄球菌VraSR能选择性感应作用于细胞壁的压力因子（如氨苄青霉素，万古霉素），而对抑制蛋白质合成的抗生素（氯霉素）、热、氧化压力、高渗透压、低氧等压力因子无明显反应。与野生株SE1457相比，∆vraSR突变株体内和体外的生物膜形成能力显著降低，生物膜内的死细菌数增多，对抑制细胞壁合成的抗生素和SDS的敏感性增加，电镜显示∆vraSR细胞壁表面粗糙、部分断裂。与生物膜形成降低相对应，∆vraSR细胞间多糖粘附素（PIA）合成降低100倍。RNA-Seq提示∆vraSR有73个基因差异表达，下调基因主要涉及生物膜形成、细菌程序性细胞死亡、糖代谢、磷酸戊糖途径和三羧酸循环等。qRT-PCR进一步验证，icaA和lrgAB下调，同时icaR和cidA上调，而耐药相关基因pbp2, serp1412, murAA等无显著变化。凝胶阻滞试验进一步发现，磷酸化的VraR能与vraSR、ica操作子的启动子区结合。上述结果提示，表皮葡萄球菌VraSR存在自我调节机制，通过ica依赖途径直接调控生物膜的形成，通过CidA-LrgAB途径影响细菌存活，可能通过代谢途径间接调控表皮葡萄球菌的药物敏感性。