AHL-SdiA抑制大肠埃希菌质粒转移

发布时间：2018-03-14 13:13

  本文选题：铜绿假单胞菌　切入点：群体感应系统　出处：《广州中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:质粒转移是细菌传播耐药基因的重要途径。质粒(plasmid)是的环形DNA,它不仅独立于细菌染色体,而且能自主复制。部分质粒具有转移性,并能携带抗生素耐药基因。广泛宿主质粒能穿梭于细菌间,因此质粒被视为传播耐药的关键媒介。质粒水平转移的主要途径是细菌的接合作用。接合作用(conjugation)是细菌借助性菌毛相互沟通、传受DNA的过程。因此,细菌接合作用被视为传播抗生素耐药基因的重要途径。AHL是细菌群体感应系统的信号分子。群体感应(quorum sensing,QS)是细菌感知群体数量变化,并调整群体行为的调控系统。其中,脂肪酰基高丝氨酸内酯(acyl homoserine lactones,AHL)是革兰阴性菌QS系统的信号分子,它通过结合LuxR类受体蛋白,实现调控基因表达。蛋白SdiA是QS系统的受体蛋白,同时也是重要的转录因子。蛋白SdiA存在于大肠埃希菌中,能感知并结合其他细菌分泌的AHL。蛋白SdiA拥有DNA结合域,它能结合基因的启动子,以调控基因表达。因此,QS系统通过AHL-SdiA调控基因表达。一般认为,质粒转移的调控是由质粒与宿主共同调控的过程,而受体菌参与接合调控则少有报道。本文推测,受体菌能通过QS系统调控质粒转移:受体菌分泌AHL,通过与宿主的蛋白SdiA结合,两者形成复合物,以调控接合相关基因traI。其中,蛋白TraI能结合质粒复制起始点oriT,参与质粒的复制及转移全过程,直接调控细菌接合作用。因此,本文探究AHL-SdiA与基因traI的调控关系。另外,本文发现蛋白SdiA对sRNA的调控作用,展望后续研究AHL-SdiA-sRNA对细菌接合作用的调控机制。方法:本实验采用大肠埃希菌SM10λπ作为质粒供体菌。该菌染色体被整合了质粒转移的调控元件(包含基因traI),并含有转移性质粒pUC24T。质粒pUC24T具庆大霉素(Gm)抗性,作为候选实验的筛选标记。受体菌则选用铜绿假单胞菌PA01。PA01基因IasI、基因rhlI分别调控3-oxo-C12-HSL、C4-HSL的催化合成,后两者为PA01最主要的AHL。实验主要通过构建接合模型、运用荧光定量PCR(qPCR)检测基因表达量以及启动子报告质粒来验证实验假设。构建接合模型。分别将供、受体菌培养至0.5麦氏浊度单位(MCF)。利用尿液分析仪测细菌浓度,并调配菌液浓度至1.0×107CFU/mL。供、受体菌各取100μL,37℃混合培养6h。筛选合子并计数,换算接合频率。接合频率高,则说明接合作用则强。qPCR检测基因表达量。收集待测菌液,并抽提细菌全RNA组。将RNA反转录为cDNA。以cDNA作为模板,利用qPCR检测目的基因相对表达量。构建基因traI启动子报告质粒pQF50-PtraI。以质粒pQF50为载体,该质粒缺少Laz启动子。运用分子克隆技术,将基因traI启动子及其上游317 bp片段,克隆于β-半乳糖苷酶基因上游。当基因traI启动子被激活时,其下游β-半乳糖苷酶基因也被激活。后续通过β-半乳糖苷酶试验(ONPG试验)检测酶活性,便能反映出基因traI启动子的表达状况。酶活性高,则说明基因traI表达上调。通过该实验观察AHL-SdiA对基因traI的调控情况。结果:①SM10λ π-PA01 进行接合反应。敲除PA01基因lasI、基因rhlI,即AHL的缺失能提高SM10λ π接合频率(P0.001):若在此基础上,加入3-oxo-C12-HSL、C4-HSL干预接合反应,接合频率则降低(P0.05)。敲除SM10λ π的基因sdiA,能增加SM10λ π-PA01接合频率(P0.001)。该结果提示,AHL与蛋白SdiA共同抑制SM10λ π接合作用。②SM10λ π-EC600进行接合反应。其中,受体菌EC600为大肠埃希菌,自身不合成AHL。加入AHL干预,接合作用受抑制,SM10λπ-EC600接合频率降低(P0.001);而在敲除基因sdiA后,接合频率又回复至原水平。但是,在不加AHL干预时,在敲除基因sdiA后,SM10λπ-EC600的接合频率相近。该结果说明,AHL抑制接合作用依赖于基因sdiA的表达。至此,实验从表型上阐明了 AHL-SdiA抑制质粒转移。③SM10λ π-PA01进行接合反应。敲除PA01基因lasI、基因rhlI,即AHL的缺失能上调SM10λ π基因traI表达(P0.05)。若在此基础上,加入3-oxo-C12-HSL、C4-HSL干预接合反应,SM10λ π基因traI表达降低(P0.05)。敲除SM10λ π的基因sdiA,SM10λ π基因traI表达上调(P0.05)。该结果从分子上阐明了AHL-SdiA抑制质粒转移。④将含有报告质粒pQF50-PtraI大肠埃希菌BW25113泛进行ONPG试验。加入AHL干预培养后,其β-半乳糖苷酶活性降低(P0.05)。而敲除BW25113基因sdiA或不加入AHL干预培养时,则均不降低酶活性。该结果说明,AHL-SdiA抑制基因traI启动子。⑤本实验通过生物信息学方法,预测并初步验证4条sRNA与基因sdiA调控成正相关性。敲除SM10λ π基因sdiA,能下调RnpB、SibA、SibB、EyeA等sRNA的表达。结论:实验通过SM10λ π-PA01、SM10λ π-EC600接合模型,从表型上阐明AHL-SdiA抑制质粒转移。而后,通过qPCR检测基因traI表达变化及pQF50-PtraI启动子报告质粒实验,从分子水平阐明了 AHL-SdiA抑制基因traI启动子,从而抑制质粒转移。最后,实验发现数条sRNA受SdiA调控,为进一步探讨AHL-SdiA-sRNA抑制质粒转移机制指明了方向。
[Abstract]:Objective: plasmid transfer is an important way to the spread of bacteria resistant gene. The plasmid (plasmid) DNA is a ring, it is not only independent of the bacterial chromosome, and can self replicate. Some plasmids with metastasis, and can carry antibiotic resistance genes. Extensive host plasmid shuttling between bacteria, the plasmid is seen as key media the main way of resistance. Horizontal plasmid transfer is conjugation. Bacterial conjugation (conjugation) is a bacterial pili by means of mutual communication, transmission by the DNA process. Therefore, bacterial conjugation is regarded as an important way to spread the antibiotic resistant gene in.AHL is a signal molecule of quorum sensing systems. Quorum sensing (quorum sensing, QS) is the number of perceived changes in bacterial groups, control system and adjust the group behavior. Among them, fatty acyl homoserine lactone (acyl homoserine, lactones, AHL) is a gram negative bacteria The signal molecules of the QS system, using a combination of LuxR receptor like protein, can regulate gene expression. SdiA protein is a receptor protein of the QS system, but also an important transcription factor. SdiA protein in Escherichia coli, and combined with other bacteria can sense the secretion of AHL. protein with SdiA DNA binding domain, it can be combined with gene the promoter to regulate gene expression. Therefore, QS AHL-SdiA system through the regulation of gene expression. It is generally considered that the regulation of plasmid transfer is by plasmid and host common regulatory process, and participate in the regulation of receptor engagement is rarely reported. It is suggested that receptor bacteria can utilize QS system to control plasmid transfer: the receptor bacteria secrete AHL and through the combination with the host protein SdiA, both the complex formation, in order to control the joint related gene traI., TraI protein can bind to plasmid replication starting point oriT in plasmid replication and transfer process, Direct regulation of bacterial conjugation. Therefore, this paper explores the relationship of AHL-SdiA and regulation of traI gene. In addition, this paper found that the regulation effect of SdiA protein on sRNA and prospects for future research AHL-SdiA-sRNA on the regulation mechanism of bacterial conjugation. Methods: this experiment used Escherichia coli SM10 k'rr53 as donor bacteria. The bacteria chromosome is integrated regulatory elements (including the transfer of plasmid traI gene), and contains a transfer plasmid pUC24T. pUC24T with gentamicin (Gm) resistance as a selective marker candidate experiment. The recipient bacterium Pseudomonas aeruginosa PA01.PA01 gene IasI and gene rhlI were regulated by 3-oxo-C12-HSL, catalytic synthesis of C4-HSL, both for AHL. PA01 the main experiment mainly through the construction of joint model, using fluorescence quantitative PCR (qPCR) to detect gene expression and promoter reporter plasmids to verify the hypotheses. The construction joint model. The supply of bacteria to 0.5 receptor Maxwell turbidity unit (MCF). The measured concentration of bacteria by urine analyzer, and the deployment of the concentration of bacteria to 1 x 107CFU/mL. for each receptor strain 100 L, 37 c mixed culture 6h. screening and zygote counting, conversion joint frequency. The high frequency of engagement, engagement effect of strong detection of.QPCR gene expression. The collection to be tested bacteria, and bacterial RNA extraction group. RNA is transcribed into cDNA. using cDNA as template, the relative expression detected by qPCR gene. The gene traI promoter reporter plasmid pQF50-PtraI. with plasmid pQF50, the plasmid Laz promoter. The lack of molecular cloning, gene traI promoter and its upstream 317 bp fragment cloned into upstream beta galactosidase gene. When traI gene promoter is activated, downstream of the beta galactosidase gene has also been activated. Follow through beta galactose glucoside enzyme test Test (ONPG test) to detect enzyme activity, can reflect the expression status of traI gene promoter. High enzyme activity, the expression of traI gene. Through the experimental observation on regulation of AHL-SdiA gene of traI. Results: 1. SM10 k'rr53 -PA01 conjugation reaction. PA01 gene knockout lasI gene rhlI. The loss of AHL can improve SM10 k'rr53 joint frequency (P0.001): 3-oxo-C12-HSL based on C4-HSL, intervention of conjugation reaction decreased, joint frequency (P0.05). SdiA gene knock in SM10 lambda PI, lambda PI -PA01 SM10 can increase the joint frequency (P0.001). The results suggest that the effect of AHL and protein SdiA inhibits SM10 k'rr53 bonding. The SM10 k'rr53 -EC600 conjugation reaction. Among them, EC600 receptor bacteria were Escherichia coli, their synthesis of AHL. into AHL intervention, joint action by inhibiting SM10, lambda PI -EC600 joint (P0.001); reduce the frequency in sdiA gene knockout, Joint frequency and return to the original level. However, in the absence of AHL intervention, in the knockout of sdiA gene, SM10 k'rr53 -EC600 joint. The results show that the similar frequency, AHL suppressed the expression of conjugation depends on the gene of sdiA. Thus, experiment illustrates the inhibition of AHL-SdiA plasmid transfer from the phenotype of the SM10. K'rr53 -PA01 conjugation reaction. PA01 gene knockout lasI gene, rhlI, that is the absence of AHL could up regulate the expression of SM10 lambda PI gene traI (P0.05) 3-oxo-C12-HSL. Based on C4-HSL, joint intervention reaction, reduce the expression of SM10 k'rr53 gene traI (P0.05). SdiA gene knock in SM10 lambda PI expression of SM10 gene, lambda PI traI (P0.05). The results demonstrated that AHL-SdiA inhibited plasmid transfer from the molecule. The report will contain plasmid pQF50-PtraI of Escherichia coli BW25113 pan ONPG test with AHL after intervention training, reduce the glycosidase activity of - galactosidase (P0.05). Knockdown of BW25113 sdiA or without AHL intervention training, which does not reduce the enzyme activity. The results showed that AHL-SdiA inhibited traI gene promoter. In this experiment by using bioinformatics methods, prediction and preliminary validation of 4 sRNA and sdiA gene regulation into a positive correlation. The knockout of SM10 gene can k'rr53 sdiA the down-regulation of RnpB, SibA, SibB, EyeA and sRNA expression. Conclusion: through the experiment of SM10 lambda PI -PA01, PI lambda SM10 -EC600 joint model, clarify the inhibition of AHL-SdiA plasmid transfer from the phenotype. Then, changes and pQF50-PtraI promoter report to plasmid traI gene qPCR expression was detected by experiments, from the molecular level to clarify the inhibition of AHL-SdiA gene the traI promoter, thereby inhibiting plasmid transfer. Finally, the experiment found that the number of sRNA regulated by SdiA, pointed out the direction for further study of AHL-SdiA-sRNA inhibiting plasmid transfer mechanism.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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