兰州大学机构库 >第二临床医学院
双组分系统RstA/RstB激活外排泵并调节荧光假单胞菌多重耐药性
Alternative TitleThe RstA/RstB two-component system activates efflux pumps and influences multi-drug resistance in Pseudomonas fluorescens
李谛音
Thesis Advisor王志平
2019-03-15
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Degree Discipline外科学
Keyword荧光假单胞菌 双组分系统 多重耐药性
Abstract【目的】荧光假单胞菌2P24中双组分系统RstA/RstB的功能鉴定及其对微生物多重耐药性影响的分子机制探讨。【方法】 利用共适应性分析预测双组份系统RstA/RstB的潜在功能及与其它蛋白的相关关系;采用同源重组技术构建rstA、rstBsensor、rstBkinase、emhABC、cmeAB、mfs等基因缺失菌株及D52A、D52E单点突变菌株;采用倍比稀释法检测多种抗生素对各菌株的最小抑制浓度;通过qRT-PCR和β-半乳糖苷酶实验检测emhABC与cmeAB基因的转录、表达水平;表达纯化Mbp-RstA、Mbp-D52A、Mbp-D52E及Mbp标签蛋白,分别通过凝胶阻滞实验与DNaseI足迹实验检测分析各蛋白与靶基因启动子区域的结合活性与结合位点;利用高能磷酸化试剂与交联试剂完成相应蛋白的体外磷酸化与体外交联;利用非标定量比较蛋白质组学分析方法及生物信息学分析探究RstA可能参与的生物学过程。【结果】经Cofitness分析发现RstA/RstB双组分系统与细菌外排泵EmhABC及CmeAB存在较强的共适应性。作为荧光假单胞菌2p24中一组尚未被鉴定的双组分系统,我们首次发现RstA/RstB与微生物多重耐药性密切相关,并且可以直接调控激活外排泵EmhABC及CmeAB的表达,该调控依赖于RstB激酶结构域的催化活性和RstA第52位天冬氨酸的磷酸化,而被磷酸化后的RstA很可能通过形成二聚体完成功能激活。在随后的蛋白质组学结果中,我们发现该双组分系统参与多种细菌生理代谢过程,尤其是对多种胁迫信号的响应,如抗热损伤、缺氧生存、次级代谢产物生成、生物膜形成及有害物质排出等,是一个以抗胁迫为主要功能的全局性转录因子。【结论】RstA/RstB双组份系统是荧光假单胞菌2P24一组重要的感受-应答调控系统,通过复杂机制调控多个外排泵的表达进而影响微生物多重耐药性,同时很可能在多个生理过程中扮演重要角色,作为一个全局性调控因子对微生物在复杂环境中的适应与生存至关重要。本研究为假单胞菌属多重耐药性的研究提供了新的线索,并为新型抗生素或抗生素佐剂的研发提供理论基础。
Other Abstract【Objective】This work is to identify the function of a two-component system RstA/RstB and investigate the molecular mechanism of RstA/RstB on multidrug resistance in Pseudomonas fluorescens strain 2P24.【Methods】Cofitness analysis was performed to explore possible functions of the two-component system RstA/RstB and its correlation with other proteins. Mutant strains,including genes deletion of rstA, rstBsensor, rstBkinase, emhABC, cmeAB, mfs and singel point mutations of D52A, D52E, were constructed by homologous recombination and minimum inhibitory concentration values of several antibiotics were detected among this strains. Quantitative real-time PCR assay and β-galactosidase assay were performed to detect the transcriptional levels of target genes, emhABC and cmeAB. Protein expression and purification in vitro included Mbp-RstA, Mbp-D52A, Mbp-D52E and Mbp-tag. Electrophoretic mobility shift assay(EMSA)and DNase I footprinting assay were employed to access the interaction activities and binding domains between RstA and target promotors. In vitro phosphorylation and protein cross-linking were performed by using high-energy phosphorylation reagents and cross-linking reagents. Exploring the biological processes in which RstA may be involved by using label-free quantitative proteomic analysis and bioinformatics analysis. 【Results】As results, RstA/RstB has similar fitness pattern with efflux pumps EmhABC and CmeAB. As a two-component system of Pseudomonas fluorescens 2p24, RstA/RstB has not yet been identified and we first confirmed that RstA/RstB can modulate the efflux pumps’ expression and further modify multiple-antibiotic resistance. We identified that phosphorylation on site D52 of RstA induced the dimerization and gain of function. Also, the catalytic activity of the RstB kinase domain is necessary. Intriguingly, we found out that RstA possibly functioning as a global regulator involved in many biological processes, especially responding to many kinds of stresses, such as heat damage resistance, anaerobic survival, secondary metabolites production, biofilm formation and harmful substances efflux.【Conclusion】The RstA/RstB two-component system is an important sensory-response regulation system of Pseudomonas fluorescens 2P24, which regulates the expression of multiple efflux pumps through complex mechanisms and affects the multidrug resistance. As a global regulator, RstA plays an important role in many physiology processes, which is critical for the adaptation and survival of bacteria in complex environments. This study provides new clues for the study of multi-drug resistance of Pseudomonas and provides a theoretical basis for the development of new antibiotics or antibiotic adjuvants.
Pages67
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/338659
Collection第二临床医学院
Affiliation第二临床医学院
First Author AffilicationSecond Clinical School
Recommended Citation
GB/T 7714
李谛音. 双组分系统RstA/RstB激活外排泵并调节荧光假单胞菌多重耐药性[D]. 兰州. 兰州大学,2019.
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