双组份系统PhoBR对鱼源无乳链球菌致病性调控机制的研究

发布时间：2018-04-15 18:05

  本文选题：无乳链球菌 + 双组份系统PhoBR　； 参考：《广东海洋大学》2017年博士论文

【摘要】：无乳链球菌是养殖鱼类链球菌病的重要病原之一,可引起鱼类游动异样、败血症、脑膜炎和突眼等症状,给鱼类养殖业带来严重的经济损失。目前无乳链球菌的研究多集中在人类领域,针对鱼源无乳链球菌致病机理了解甚少。细菌的双组分信号转导系统(two-component signal transduction system,TCS)能够感应外界环境变化,并将信号传递到细胞内,引起细菌作出相应应答调控,其在细菌适应宿主体内微环境变化的调控方面发挥重要作用。前人研究表明,phoB基因作为双组分系统PhoBR的反应调控因子(Response regulator),既参与细菌无机磷浓度调节,又负责调控生物膜形成以及多种毒力因子/蛋白表达。而关于无乳链球菌PhoB的相关功能研究在国内外尚属空白。因此,本研究以分离于卵形鲳湽的无乳链球菌TOS01强毒株为野生株,利用同源重组技术,构建无乳链球菌phoB基因缺失突变株SAΔphoB,并通过转录组和蛋白组学研究无乳链球菌PhoB对生物膜形成和毒力相关基因的调控,利用lac Z报告基因和细菌单杂交方法验证其对溶血素的调控功能,阐明PhoB对毒力基因的调控作用。主要研究结果和结论如下:1.海水养殖卵形鲳湽链球菌的分离鉴定2012-2014年,从北海、湛江和深圳患病卵形鲳湽中分离到9株链球菌TOS01-TOS09,其中TOS01和TOS06为共感染菌株。结合生理生化方法和分子生物学技术鉴定TOS01-05为无乳链球菌、TOS06-08为海豚链球菌和TOS09为停乳链球菌停乳亚种。半致死量LD50测定结果显示,无乳链球菌TOS01的LD50(6.38×104 CFU)较海豚链球菌TOS06(1.47×107 CFU)和停乳链球菌停乳亚种TOS09(2.57×106 CFU)高,并且所有菌株对头孢拉定、头孢噻肟和红霉素极度敏感。2.无乳链球菌TOS01 phoB基因缺失株的构建及其生物学特性研究以无乳链球菌TOS01强毒株为野生株,扩增phoB上下游同源臂及红霉素基因,采用分段酶切连接方法将其连接到温敏型自杀质粒pSET4s载体上,利用同源重组技术,成功构建了无乳链球菌phoB基因缺失突变株SAΔphoB。同时利用大肠杆菌-革兰氏阳性穿梭质粒pDL276构建了互补株CΔphoB。分析突变株SAΔphoB生物学特性显示,SAΔphoB能够稳定遗传,但生长速率明显降低,链条由原来的两个或短链状变为几十个长链状排列,表面缺少野生态的凹凸不平胞外基质而变得较为光滑,静止期生物膜厚度显著高于TOS01和CΔphoB,溶血活性、细胞的侵入、黏附以及抗吞噬能力则低于TOS01和CΔphoB;使用卵形鲳湽为模型比较了三种菌株的毒力,显示phoB缺失后导致细菌毒力明显下降;以低浓度突变株SAΔphoB免疫卵形鲳湽获得高达93.1%相对免疫保护率,表明其可作为候选疫苗保护卵形鲳湽免受无乳链球菌危害。3.PhoB对生物膜形成和毒力基因调控比较野生株TOS01和突变株SAΔphoB转录组和蛋白图谱,分别有521个基因和60个蛋白发生了差异表达,涉及磷酸转运系统、应激反应、碳水化合物、脂肪酸和氨基酸代谢、ABC转运系统以及毒力和生物膜形成方面,其中33个与毒力相关的基因和蛋白在限磷条件下受PhoB的调控表达,主要涉及无乳链球菌黏附、定植和入侵;12个与细胞生物膜相关,编码荚膜多糖的5个基因表达水平则下调,表明无乳链球菌的PhoB参与调控细菌生物膜形成和毒力基因表达。4.PhoB调控溶血素相关基因的功能验证通过构建lacZ报告基因载体和细菌单杂交方法检测了无乳链球菌PhoB对cyl、hemolysin III、hemolysin A和ciaR基因启动子区的结合作用。结果显示,PhoB可直接与hemolysin A和ciaR基因启动子区的结合,但未与cyl和hemolysin III基因启动子区的相结合,表明PhoB对其调控是间接的。通过构建18bp DNA随机片段文库法利用细菌单杂交系统发现PhoB结合的保守序列为TTGGAGAA(G/T)。
[Abstract]:Streptococcus agalactiae is an important pathogen of farmed fish streptococcus disease, can cause the fish swimming again, sepsis, meningitis and proptosis, and caused serious economic losses to aquaculture. The current study of Streptococcus agalactiae and more concentrated in the field of human beings, the source of fish Streptococcus agalactiae pathogenesis is poorly understood. Two component signal transduction bacteria (two-component signal transduction system system, TCS) can induce the changes of the external environment, and send the signal into cells, causing bacteria to make corresponding response regulation, play an important role in the regulation to adapt to changes in the microenvironment in the host bacteria. Previous studies showed that phoB gene as a two-component system PhoBR response control factor (Response regulator), is involved in the bacterial inorganic phosphorus concentration regulation, and is responsible for the regulation of biofilm formation and virulence factor expression / protein. Study on the function of PhoB of Streptococcus agalactiae is still blank at home and abroad. Therefore, in this study, isolated from Trachinotus have Streptococcus agalactiae TOS01 strain for wild-type strain by homologous recombination technology, construction of Streptococcus agalactiae phoB gene deletion mutant SA Delta phoB, and through the regulation of transcriptomics and proteomics study on related genes of Streptococcus lactis PhoB on biofilm formation and virulence, verify its regulation on hemolysin function by lac Z reporter gene and the bacterial one hybrid method, clarify the roles of PhoB on regulation of virulence genes. The main results and conclusions are as follows: 1. seawater cultured Trachinotus have Streptococcus isolation and identification of 2012-2014 years, from Beihai isolated, 9 strains of Streptococcus TOS01-TOS09 in Shenzhen and Zhanjiang have Trachinotus, including TOS01 and TOS06 co infection strains. Combined with physiological and biochemical methods and molecular biological identification technology TOS01-05 Streptococcus agalactiae, TOS06-08 Streptococcus iniae and TOS09 Streptococcus dysgalactiae. Semi lethal dose LD50 assay results showed that Streptococcus agalactiae TOS01 LD50 (6.38 x 104 CFU) with Streptococcus iniae TOS06 (1.47 * 107 CFU) and Streptococcus dysgalactiae (2.57 TOS09 * 106 CFU). And all the strains of cefradine, construction of cefotaxime and erythromycin sensitive.2. of Streptococcus agalactiae TOS01 strain with phoB gene deletion and Study on the biological characteristics of the Streptococcus agalactiae TOS01 strain for wild strains, phoB amplification of the upstream and downstream homologous arm and erythromycin gene, using segmented enzyme connection method to connect it to the temperature sensitive type Dutch act plasmid pSET4s, by homologous recombination technology, the successful construction of Streptococcus agalactiae phoB gene deletion mutant SA Delta phoB. and Escherichia coli - gram positive shuttle plasmid pDL276 to construct C Delta phoB. complementation strain Analysis of the mutant SA phoB biological characteristics showed that SA phoB could be inherited, but the growth rate decreased significantly, the chain from the original two or short chain into dozens of long chains, lack of surface uneven wild ecological extracellular matrix and is relatively smooth, quiescent biofilm thickness was significantly higher than that of TOS01 and C Delta phoB, hemolytic activity, cell invasion, adhesion and anti phagocytic ability is lower than that of TOS01 and C phoB; use of Trachinotus have compared the virulence of three strains as a model, that the absence of phoB resulted in decreased bacterial virulence; with low concentration of mutant SA phoB have immune Trachinotus get up to 93.1% the relative rate of immune protection, that can be used as a candidate vaccine against Streptococcus agalactiae Trachinotus have harm.3.PhoB on biofilm formation and virulence genes of wild strain TOS01 and mutant SA phoB transcription and protein atlas group, There were 521 genes and 60 differentially expressed proteins, involved in phosphate transport system, stress response, carbohydrate, amino acid and fatty acid metabolism, ABC transporter and virulence and biofilm formation, 33 of them associated with virulence gene and protein in phosphorus limited condition under the regulation of PhoB expression, mainly related to Streptococcus agalactiae adhesion, colonization and invasion; 12 with the cell membrane, reduced the expression level of 5 genes encoding capsular polysaccharide, showed that Streptococcus agalactiae PhoB is involved in the regulation of bacterial biofilm formation and virulence gene expression regulation of.4.PhoB gene hemolysin related functions are verified by the lacZ reporter gene vector was constructed and bacterial one hybrid detection of Streptococcus agalactiae PhoB on CYL, hemolysin III, hemolysin A and ciaR gene promoter binding promoter region. The results showed that PhoB can directly with hemolysin A and ciaR The binding of promoter region, but not combined with the promoter region of CYL and hemolysin III, indicates that PhoB is indirectly regulated by it. By constructing 18bp DNA random fragment library, we found that the conserved sequence of PhoB binding was TTGGAGAA (G/T) by using bacterial single hybrid system.

【学位授予单位】：广东海洋大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S941.4
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本文编号：1755197