【作者】 张丹凤；

【导师】 彭宣宪；

【作者基本信息】 厦门大学 ， 生化与分子生物学， 2007， 博士

【摘要】 采用蛋白质组学和基因组学研究方法,对革兰氏阴性病原菌大肠杆菌K-12的外膜蛋白进行了较系统的研究,重点筛选和鉴定了与消毒剂耐受和抗生素耐药相关的蛋白质组,并对这些差异外膜蛋白进行验证和一系列的功能研究,有助于了解细菌对消毒剂耐受和对抗生素耐药的机制,这对于寻找新药物靶点和研制新的抗菌药物或疫苗具有重要的意义。通过对多种消毒剂的蛋白质组学进行分析,成功地在大肠杆菌K-12中分别鉴定出与异丙醇(7种)、乙醇(7种)和苯酚(5种)耐受相关的外膜蛋白,并发现异丙醇与乙醇耐受相关外膜蛋白一致,均为Tsx、OmpC、OmpF、FadL、OmpT、OmpA和LamB,其中FadL、OmpT、OmpA、LamB4种外膜蛋白是三者所共有的。在对这些结果进行Western blotting证实的基础上,采用基因敲除菌株、补救菌株和高表达菌株进一步进行消毒剂耐受试验,证明了LamB、OmpC和OmpA是大肠杆菌K-12对醇类消毒剂耐受相关最重要的外膜蛋白:LamB、OmpA和OmpT是大肠杆菌K-12对苯酚耐受相关的重要外膜蛋白,尤其以OmpA和OmpT为著。进一步实验显示,大肠杆菌K-12对这三种消毒剂的耐受还受EnvZ/OmpR双成分调节系统的调节。这些结果均显示外膜蛋白在革兰氏阴性细菌对消毒剂耐受中起到重要作用,其特征与消毒剂种类有关。通过亚蛋白质组学技术,研究大肠杆菌K-12对四环素和卡那霉素耐药相关的功能外膜蛋白。结果发现,在四环素耐药菌株中共有11个差异点,其中有8个点为外膜蛋白,属于6种外膜蛋白,分别为LamB、FirnD、TolC、OmpC、Tsx和OmpW;在卡那霉素耐药菌株的11个差异蛋白点中,有9个点为外膜蛋白,共为7种外膜蛋白,它们是OmpA、TolC、FadL、OstA、Tsx、OmpW和MipA。经比较发现,TolC、Tsx和OmpW为2种抗生素耐药菌株共同变化的外膜蛋白,Western blotting结果进一步证实了2-DE结果的可靠性。在此基础上,对这些外膜蛋白进行了较深入的功能分析。结果发现,在大肠杆菌K-12对四环素耐药过程中,LamB、TolC和OmpC这3个外膜蛋白发挥重要作用;在大肠杆菌K-12对卡那霉素耐药过程中,MipA和TolC是特别重要的外膜蛋白,其中关于MipA是细菌耐药相关外膜蛋白的报道在国内外尚属首次。采用功能基因组学的方法,构建了两株分别含有TolC长、短片段胞外结构域突变的突变株△tolC-pET-28a-tolC1和△tolC-pET-28a-tolC2,并通过耐药实验证明了TolC除了具有药物外排功能之外,还能感知外界环境药物浓度的新功能,而且这两个胞外区域对该功能的作用不一样。更多还原

【Abstract】 Using proteomics and genomics technologies,we invesigated the outer membrane proteins of Gram-negative bacterium E.coli K-12 in response to disinfectant-tolerance and drug-resistance.This investigation was performed with the use of proteomic methodologies for altered proteome,Western blotting for verification of the altered proteins,and gene-defieient and protein-overexpressed approch for protein function. These studies would help us to understand disinfectant-tolerance and drug-resistance mechanisms of bacteria,find new drug targets,and develop new anti-bacteria agents or vaccine.Seven,seven and five altered outer membrane proteins were characterized in isopropanol-tolerant,ethanol-tolerant and phenol-tolerant E.coli K-12 strains,respectively, in which altered outer membrane proteins in both of isopropanol-tolerance and ethanol-tolerant strains were identical(Tsx,OmpC,OmpF,FadL,OmpT,OmpA and LamB).Out of the seven proteins,altered FadL,OmpT,OmpA,LamB changed were also characterized in phenol-tolerant strain.Following the verification of 2-DE by Western blotting,function of the altered proteins was investigated by gene knockout,gene complementation and gene overexpression experiments.LamB,OmpC and OmpA were determined as the most crucial outer membrane proteins in response to alcohols-tolerance, and LamB,OmpA and OmpT to phenol-tolerance,especially OmpA and OmpT.Further investigation indicated that bacterial tolerance to the threee disinfectants was regulated by EnvZ/OmpR two component signal transduction system.These results showed that outer membrane proteins of Gram-negative bacteria played important role in disinfectant tolerance,in which the characteristic feature of the altered outer membrane proteins was correlated to disinfectant types.Through the sub-proteomics to study functional outer membrane proteins relevant to E.coli K-12 resistant to tetracycline and kanamycin,we found 11 altered spots in tetracycline-resistant strain and 8 of them were identified as outer membrane proteins which belonged to 6 types of outer membrane proteins(LamB,FimD,TolC,OmpC,Tsx and OmpW);11 altered spots in E.coli K-12 resistant to kanamycin and 9 of them were identified as outer membrane proteins which belonged to 7 types of outer membrane proteins(OmpA,TolC,FadL,OstA,Tsx,OmpW and MipA).Furthermore,TolC,Tsx and OmpW were commonly changed outer membrane proteins in these two drug-resistant strains.The reliability of 2-DE was confirmed by Western blotting.On the basis of these results,further functional analysis of these outer membrane proteins showed that LamB,TolC and OmpC were critical in the process of tetracycline-resistance,and MipA and TolC were especially important to kanamycin-resistance.Importantly,MipA was first reported here to be an outer membrane protein in response to bacterial drug-resistance.Moreover,we constructed two mutants,ΔtolC-pET-28a-tolC1 andΔtolC-pET-28a-tolC2,which respectively contained mutated TolC long and short extracellular domains for investigating ability of extracellular domains in response to an antibiotic.Our results indicated that TolC could sense the concentration of environmental drugs,which is a novel finding.更多还原