發(fā)布時間：2018-04-15 18:05

  本文選題：無乳鏈球菌 + 雙組份系統(tǒng)PhoBR�。� 參考：《廣東海洋大學(xué)》2017年博士論文

【摘要】：無乳鏈球菌是養(yǎng)殖魚類鏈球菌病的重要病原之一,可引起魚類游動異樣、敗血癥、腦膜炎和突眼等癥狀,給魚類養(yǎng)殖業(yè)帶來嚴(yán)重的經(jīng)濟損失。目前無乳鏈球菌的研究多集中在人類領(lǐng)域,針對魚源無乳鏈球菌致病機理了解甚少。細(xì)菌的雙組分信號轉(zhuǎn)導(dǎo)系統(tǒng)(two-component signal transduction system,TCS)能夠感應(yīng)外界環(huán)境變化,并將信號傳遞到細(xì)胞內(nèi),引起細(xì)菌作出相應(yīng)應(yīng)答調(diào)控,其在細(xì)菌適應(yīng)宿主體內(nèi)微環(huán)境變化的調(diào)控方面發(fā)揮重要作用。前人研究表明,phoB基因作為雙組分系統(tǒng)PhoBR的反應(yīng)調(diào)控因子(Response regulator),既參與細(xì)菌無機磷濃度調(diào)節(jié),又負(fù)責(zé)調(diào)控生物膜形成以及多種毒力因子/蛋白表達。而關(guān)于無乳鏈球菌PhoB的相關(guān)功能研究在國內(nèi)外尚屬空白。因此,本研究以分離于卵形鯧湽的無乳鏈球菌TOS01強毒株為野生株,利用同源重組技術(shù),構(gòu)建無乳鏈球菌phoB基因缺失突變株SAΔphoB,并通過轉(zhuǎn)錄組和蛋白組學(xué)研究無乳鏈球菌PhoB對生物膜形成和毒力相關(guān)基因的調(diào)控,利用lac Z報告基因和細(xì)菌單雜交方法驗證其對溶血素的調(diào)控功能,闡明PhoB對毒力基因的調(diào)控作用。主要研究結(jié)果和結(jié)論如下:1.海水養(yǎng)殖卵形鯧湽鏈球菌的分離鑒定2012-2014年,從北海、湛江和深圳患病卵形鯧湽中分離到9株鏈球菌TOS01-TOS09,其中TOS01和TOS06為共感染菌株。結(jié)合生理生化方法和分子生物學(xué)技術(shù)鑒定TOS01-05為無乳鏈球菌、TOS06-08為海豚鏈球菌和TOS09為停乳鏈球菌停乳亞種。半致死量LD50測定結(jié)果顯示,無乳鏈球菌TOS01的LD50(6.38×104 CFU)較海豚鏈球菌TOS06(1.47×107 CFU)和停乳鏈球菌停乳亞種TOS09(2.57×106 CFU)高,并且所有菌株對頭孢拉定、頭孢噻肟和紅霉素極度敏感。2.無乳鏈球菌TOS01 phoB基因缺失株的構(gòu)建及其生物學(xué)特性研究以無乳鏈球菌TOS01強毒株為野生株,擴增phoB上下游同源臂及紅霉素基因,采用分段酶切連接方法將其連接到溫敏型自殺質(zhì)粒pSET4s載體上,利用同源重組技術(shù),成功構(gòu)建了無乳鏈球菌phoB基因缺失突變株SAΔphoB。同時利用大腸桿菌-革蘭氏陽性穿梭質(zhì)粒pDL276構(gòu)建了互補株CΔphoB。分析突變株SAΔphoB生物學(xué)特性顯示,SAΔphoB能夠穩(wěn)定遺傳,但生長速率明顯降低,鏈條由原來的兩個或短鏈狀變?yōu)閹资畟�長鏈狀排列,表面缺少野生態(tài)的凹凸不平胞外基質(zhì)而變得較為光滑,靜止期生物膜厚度顯著高于TOS01和CΔphoB,溶血活性、細(xì)胞的侵入、黏附以及抗吞噬能力則低于TOS01和CΔphoB;使用卵形鯧湽為模型比較了三種菌株的毒力,顯示phoB缺失后導(dǎo)致細(xì)菌毒力明顯下降;以低濃度突變株SAΔphoB免疫卵形鯧湽獲得高達93.1%相對免疫保護率,表明其可作為候選疫苗保護卵形鯧湽免受無乳鏈球菌危害。3.PhoB對生物膜形成和毒力基因調(diào)控比較野生株TOS01和突變株SAΔphoB轉(zhuǎn)錄組和蛋白圖譜,分別有521個基因和60個蛋白發(fā)生了差異表達,涉及磷酸轉(zhuǎn)運系統(tǒng)、應(yīng)激反應(yīng)、碳水化合物、脂肪酸和氨基酸代謝、ABC轉(zhuǎn)運系統(tǒng)以及毒力和生物膜形成方面,其中33個與毒力相關(guān)的基因和蛋白在限磷條件下受PhoB的調(diào)控表達,主要涉及無乳鏈球菌黏附、定植和入侵;12個與細(xì)胞生物膜相關(guān),編碼莢膜多糖的5個基因表達水平則下調(diào),表明無乳鏈球菌的PhoB參與調(diào)控細(xì)菌生物膜形成和毒力基因表達。4.PhoB調(diào)控溶血素相關(guān)基因的功能驗證通過構(gòu)建lacZ報告基因載體和細(xì)菌單雜交方法檢測了無乳鏈球菌PhoB對cyl、hemolysin III、hemolysin A和ciaR基因啟動子區(qū)的結(jié)合作用。結(jié)果顯示,PhoB可直接與hemolysin A和ciaR基因啟動子區(qū)的結(jié)合,但未與cyl和hemolysin III基因啟動子區(qū)的相結(jié)合,表明PhoB對其調(diào)控是間接的。通過構(gòu)建18bp DNA隨機片段文庫法利用細(xì)菌單雜交系統(tǒng)發(fā)現(xiàn)PhoB結(jié)合的保守序列為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.

【學(xué)位授予單位】：廣東海洋大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S941.4
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本文編號：1755197