小肠结肠炎耶尔森菌AmpC β-内酰胺酶表达调控基因功能研究

发布时间：2018-05-19 16:16

  本文选题：小肠结肠炎耶尔森菌 + AmpC　； 参考：《江苏大学》2017年博士论文

【摘要】：目的:小肠结肠炎耶尔森菌(Yersinia enterocolitica)是一种革兰阴性无芽胞杆菌,属于肠杆菌科耶尔森菌属,是耶尔森菌属中的三大致病菌之一,可通过水和食品传播以胃肠道症状为主的疾病。和其他肠杆菌科细菌一样,Y.enterocolitica染色体携带ampR-ampC基因,表达产生AmpCβ-内酰胺酶,从而对部分青霉素类和头孢类抗生素产生天然耐药。然而,尽管肠杆菌科细菌AmpC β-内酰胺酶表达调控机制研究较早,但目前尚没有报道Y.enterocolitica ampC表达调控机制的文献。因此,本研究通过分子生物学、免疫学及微生物学技术对Y. enterocolitica AmpC β-内酸胺酶表达调控机制进行研究,旨在进一步阐明该菌的耐药机制。方法:1.采用生物信息学技术对ampC上游的启动子区进行预测,利用PCR方法扩增预测序列,将扩增产物与原始质粒pBBRLux连接,构建重组质粒pLUXampC。该质粒可根据ampC基因的启动子强弱而发出相应的生物冷光,可用于指示细菌AmpC β-内酰胺酶的表达量。2.采用BLAST程序寻找可能的Y.enterocoltica AmpD蛋白、低分子量青霉素结合蛋白(Low-Molecular-Mass Penicillin-Binding Proteins)以及β-N-乙酰葡萄糖胺酶(NagZ)编码基因。通过无缝克隆技术将目的基因的上、下游同源臂(约1000bp)连接至自杀质粒敲除载体pDS132,通过无痕敲除技术构建相关目的基因缺失株。3.将ampC启动子活性检测质粒pLUXampC转化到相应的ampD1、ampD2、ampD3、pbp4、pbp5a、pbp5b、pbp7、ampR 以及 nagZ缺失株中。常规培养后,检测细菌的生物发光强度,以判断各缺失株AmpC β-内酰胺酶的表达量。4.将处于对数生长期的细菌超生破碎,离心后取上清液,获得细菌酶粗提物。分别使用头孢硝噻吩和4-nitrophenyl N-acetyl-β-D-glucosaminide作为反应底物,对待测菌株粗提物种的β-内酰胺酶活性和β-N-乙酰葡萄糖胺酶(NagZ)活性进行测定。5.采用微量肉汤稀释法测定各缺失株的抗生素最低抑制浓度(MIC)。结果:1.成功构建基于生物发光技术的ampC启动子活性检测质粒,并命名为pLUXampC。2.根据蛋白质序列的同源性分析得知Y. enterocolitica 105.5R(r)中WP_005156822、WP_005164953 和 WP_013649890 这三个蛋白均具有与 AmpD相同的蛋白功能结构域,将其命名为AmpD1、AmpD2和AmpD3,并构建了七种与之相关的单、双、三基因缺失株。AmpD1、AmpD2和AmpD3均为有功能的AmpD同系物蛋白,同时参与ampC的表达调控。AmpD蛋白缺失会导致细菌β-内酰胺酶活性的增高,三基因缺失株YE△D123则出现了完全去阻遏持续高产AmpC β-内酰胺酶的表型特点。3.构建了15种与低分子量青霉素结合蛋白(LMMPBPs) PBP4、PBP5a、PBP5b和PBP7相关的单、双、三、四基因缺失株。发现以PBP5b为首的LMM PBPs对细菌ampC调控起关键作用。各缺失株的ampC启动子活性出现不同程度的升高,四基因缺失株YEA4A5aA5bA7出现了与YEAD123相似的持续高产AmpC β-内酰胺酶表型。4.为比较上述两AmpC过表达菌株的区别,将YE△D123和YEA4A5aA5bA7的β-N-乙酰葡萄糖胺酶基因(nagZ)敲除,发现该基因的敲除对于YE△D123无影响,而对YEA4A5aA5bA7的影响较大,nagZ基因缺失株YE△4A5aA5b△7△Z的β-内酰胺酶活性降低至野生株相同水平。结论:1.通过构建重组质粒pLUXampC建立了基于生物发光技术的细菌ampC表达水平检测方法。该方法可在不影响细菌活性的情况下对细菌的AmpC β-内酰胺酶表达量进行监测;2.Y.enterocolitica可通过三个AmpD同系物对细菌的AmpC β-内酰胺酶进行调控。其中AmpD1功能较强,单缺失株YE△D1(ampD 缺失)的AmpC酶表达量即出现明显的上升;而AmpD2和AmpD3功能相对较弱,其负调控功能只有在双、三基因缺失株中才能体现,在肠杆菌科细菌中发现多AmpD参与的ampC逐级调控机制;3.Y.enterocolitica的四种低分子量青霉素结合蛋白(LMM PBPs)均参与细菌AmpC β-内酰胺酶的表达调控,LMM PBPs缺失会导致细菌AmpC β-内酰胺酶表达量不同程度的上调。其中以PBP5b的功能最强,单缺失株YE△5b的β-内酰胺酶活性即出现明显上升;而PBP4、PBP5a和PBP7的功能相对较弱,主要与PBP5b相协同而发挥作用:4.Y.enterocolitica具有NagZ依赖/NagZ非依赖两种AmpC β-内酰胺酶调控途径。
[Abstract]:Objective: Yersinia enterocolitica (Yersinia enterocolitica) is a gram-negative bacillus free bacillus, belonging to the genus Kyel Sen, one of the three general germs of the genus Jerson, which can transmit the gastrointestinal symptoms through water and food. The Y.enterocolitica chromosome, like the Enterobacteriaceae, is the same as that of the Enterobacteriaceae. With ampR-ampC gene, AmpC beta lactamase is produced to produce natural resistance to some penicillins and cephalosporins. However, although the regulation mechanism of the expression of AmpC beta lactamase in Enterobacteriaceae is early, there is no literature on the regulatory mechanism of Y.enterocolitica ampC. The regulation mechanism of Y. enterocolitica AmpC beta lactamase expression was studied by subbiology, immunology and microbiology. The aim of this study was to further elucidate the resistance mechanism of the bacteria. Method: 1. using bioinformatics technology to predict the promoter region of the upstream of ampC, amplification of the prediction sequence by PCR method, and the amplification products and the original plasmids PBBRLux connection and construction of recombinant plasmid pLUXampC., the plasmid can produce corresponding biological cold light according to the promoter of ampC gene, and can be used to indicate the expression of AmpC beta lactamase,.2. using BLAST program to find the possible Y.enterocoltica AmpD protein, low molecular Liang Qing mycin binding protein (Low-Molecular-Mass Penicillin-Bi). Nding Proteins) and beta -N- acetylglucosaminase (NagZ) encoding gene. Through seamless cloning technology, the downstream homologous arm (about 1000bp) of the target gene was connected to the suicide plasmid knockout carrier pDS132, and the related target gene deletion.3. was constructed to convert the ampC promoter activity detection plasmid pLUXampC to the corresponding a through the null knockout technique. MpD1, ampD2, ampD3, pbp4, pbp5a, pbp5b, pbp7, ampR, and nagZ missing strains. After routine culture, the bioluminescence intensity of bacteria was detected to judge that the expression of AmpC beta lactamase in the missing strains will be in the logarithmic growth period of bacteria, and then take the supernatant to obtain the crude extract of the bacterial enzyme. Nitrophenyl N-acetyl- beta -D-glucosaminide was used as a reaction substrate to measure the beta lactamase activity and the activity of beta -N- acetylglucosaminase (NagZ) in the crude extracts of the strains, and.5. was used to determine the minimum inhibitory concentration (MIC) of the antibiotics in the missing strains by microdilution method (MIC). Results: 1. successfully constructed ampC based on bioluminescence technology. The promoter activity detection plasmid, named pLUXampC.2., was named according to the homology analysis of protein sequence, that the three proteins of WP_005156822, WP_005164953 and WP_013649890 in Y. enterocolitica 105.5R (R) all have the same protein functional domains as AmpD, which are named AmpD1, AmpD2 and AmpD3, and seven are related to them. The single, double and three gene deletion strains.AmpD1, AmpD2 and AmpD3 were all functional AmpD homologues, while participating in the expression of ampC, the deletion of.AmpD protein could lead to the increase of bacterial beta lactamase activity. The three gene deletion strain YE Delta D123 appeared the phenotypic characteristics of the complete depressor and persistent high yield of AmpC beta lactamase,.3. constructed 15 kinds of.3. A single, double, three, four gene deletion strain related to the low molecular weight penicillin binding protein (LMMPBPs) PBP4, PBP5a, PBP5b and PBP7. It was found that LMM PBPs, headed by PBP5b, played a key role in the regulation of bacterial ampC. The ampC promoter activity of the missing strains increased in varying degrees, and the YEA4A5aA5bA7 of the four gene deletion strain appeared to be similar to YEAD123. The continuous high yield AmpC beta lactamase phenotype.4. is the difference between the above two AmpC overexpressed strains and the YE Delta D123 and YEA4A5aA5bA7 beta -N- acetylglucosaminase gene (nagZ) knockout. It is found that the knockout of the gene has no effect on YE Delta D123, but it has great influence on YEA4A5aA5bA7, and the nagZ gene deletion strain delta delta 7 delta beta lactam. The enzyme activity was reduced to the same level as the wild plant. Conclusion: 1. by the construction of recombinant plasmid pLUXampC, the detection method of bacterial ampC expression level based on bioluminescence technology was established. This method can monitor the expression of AmpC beta lactamase in bacteria without affecting the bacterial activity; 2.Y.enterocolitica can pass through three AmpD homologous lines. AmpC beta lactamase was regulated by the bacteria, in which the AmpD1 function was stronger and the AmpC enzyme expression of the single deletion strain YE Delta D1 (ampD deletion) increased obviously; while the function of AmpD2 and AmpD3 was relatively weak, the negative regulatory function was only in the double, three gene deletion strain, and the ampC was found in the Enterobacteriaceae bacteria in ampC. The four low molecular weight penicillin binding protein (LMM PBPs) of 3.Y.enterocolitica participates in the regulation of the expression of the bacterial AmpC beta lactamase, and the deletion of LMM PBPs will lead to the up regulation of the expression of the bacterial AmpC beta lactamase in different degrees. The function of PBP5b is the strongest, and the activity of the beta lactamase activity of the single deletion strain YE delta 5b is that of the beta lactamase. The function of PBP4, PBP5a and PBP7 is relatively weak, and the function of PBP7 is relatively weak, and it plays an important role in coordination with PBP5b: 4.Y.enterocolitica has NagZ dependent /NagZ without dependence on two AmpC beta lactamases.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R378

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相关期刊论文 前1条

1 ZHOU Yan Yan;ZHANG Hong Zhi;LIANG Wei Li;ZHANG Li Juan;ZHU Jun;KAN Biao;;Plasticity of Regulation of Mannitol Phosphotransferase System Operon by CRP-cAMP Complex in Vibrio cholerae[J];Biomedical and Environmental Sciences;2013年10期

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