本文選題：小腸結(jié)腸炎耶爾森菌 + AmpC�。� 參考：《江蘇大學(xué)》2017年博士論文

【摘要】：目的:小腸結(jié)腸炎耶爾森菌(Yersinia enterocolitica)是一種革蘭陰性無芽胞桿菌,屬于腸桿菌科耶爾森菌屬,是耶爾森菌屬中的三大致病菌之一,可通過水和食品傳播以胃腸道癥狀為主的疾病。和其他腸桿菌科細(xì)菌一樣,Y.enterocolitica染色體攜帶ampR-ampC基因,表達(dá)產(chǎn)生AmpCβ-內(nèi)酰胺酶,從而對部分青霉素類和頭孢類抗生素產(chǎn)生天然耐藥。然而,盡管腸桿菌科細(xì)菌AmpC β-內(nèi)酰胺酶表達(dá)調(diào)控機(jī)制研究較早,但目前尚沒有報道Y.enterocolitica ampC表達(dá)調(diào)控機(jī)制的文獻(xiàn)。因此,本研究通過分子生物學(xué)、免疫學(xué)及微生物學(xué)技術(shù)對Y. enterocolitica AmpC β-內(nèi)酸胺酶表達(dá)調(diào)控機(jī)制進(jìn)行研究,旨在進(jìn)一步闡明該菌的耐藥機(jī)制。方法:1.采用生物信息學(xué)技術(shù)對ampC上游的啟動子區(qū)進(jìn)行預(yù)測,利用PCR方法擴(kuò)增預(yù)測序列,將擴(kuò)增產(chǎn)物與原始質(zhì)粒pBBRLux連接,構(gòu)建重組質(zhì)粒pLUXampC。該質(zhì)�？筛鶕�(jù)ampC基因的啟動子強(qiáng)弱而發(fā)出相應(yīng)的生物冷光,可用于指示細(xì)菌AmpC β-內(nèi)酰胺酶的表達(dá)量。2.采用BLAST程序?qū)ふ铱赡艿腨.enterocoltica AmpD蛋白、低分子量青霉素結(jié)合蛋白(Low-Molecular-Mass Penicillin-Binding Proteins)以及β-N-乙酰葡萄糖胺酶(NagZ)編碼基因。通過無縫克隆技術(shù)將目的基因的上、下游同源臂(約1000bp)連接至自殺質(zhì)粒敲除載體pDS132,通過無痕敲除技術(shù)構(gòu)建相關(guān)目的基因缺失株。3.將ampC啟動子活性檢測質(zhì)粒pLUXampC轉(zhuǎn)化到相應(yīng)的ampD1、ampD2、ampD3、pbp4、pbp5a、pbp5b、pbp7、ampR 以及 nagZ缺失株中。常規(guī)培養(yǎng)后,檢測細(xì)菌的生物發(fā)光強(qiáng)度,以判斷各缺失株AmpC β-內(nèi)酰胺酶的表達(dá)量。4.將處于對數(shù)生長期的細(xì)菌超生破碎,離心后取上清液,獲得細(xì)菌酶粗提物。分別使用頭孢硝噻吩和4-nitrophenyl N-acetyl-β-D-glucosaminide作為反應(yīng)底物,對待測菌株粗提物種的β-內(nèi)酰胺酶活性和β-N-乙酰葡萄糖胺酶(NagZ)活性進(jìn)行測定。5.采用微量肉湯稀釋法測定各缺失株的抗生素最低抑制濃度(MIC)。結(jié)果:1.成功構(gòu)建基于生物發(fā)光技術(shù)的ampC啟動子活性檢測質(zhì)粒,并命名為pLUXampC。2.根據(jù)蛋白質(zhì)序列的同源性分析得知Y. enterocolitica 105.5R(r)中WP_005156822、WP_005164953 和 WP_013649890 這三個蛋白均具有與 AmpD相同的蛋白功能結(jié)構(gòu)域,將其命名為AmpD1、AmpD2和AmpD3,并構(gòu)建了七種與之相關(guān)的單、雙、三基因缺失株。AmpD1、AmpD2和AmpD3均為有功能的AmpD同系物蛋白,同時參與ampC的表達(dá)調(diào)控。AmpD蛋白缺失會導(dǎo)致細(xì)菌β-內(nèi)酰胺酶活性的增高,三基因缺失株YE△D123則出現(xiàn)了完全去阻遏持續(xù)高產(chǎn)AmpC β-內(nèi)酰胺酶的表型特點。3.構(gòu)建了15種與低分子量青霉素結(jié)合蛋白(LMMPBPs) PBP4、PBP5a、PBP5b和PBP7相關(guān)的單、雙、三、四基因缺失株。發(fā)現(xiàn)以PBP5b為首的LMM PBPs對細(xì)菌ampC調(diào)控起關(guān)鍵作用。各缺失株的ampC啟動子活性出現(xiàn)不同程度的升高,四基因缺失株YEA4A5aA5bA7出現(xiàn)了與YEAD123相似的持續(xù)高產(chǎn)AmpC β-內(nèi)酰胺酶表型。4.為比較上述兩AmpC過表達(dá)菌株的區(qū)別,將YE△D123和YEA4A5aA5bA7的β-N-乙酰葡萄糖胺酶基因(nagZ)敲除,發(fā)現(xiàn)該基因的敲除對于YE△D123無影響,而對YEA4A5aA5bA7的影響較大,nagZ基因缺失株YE△4A5aA5b△7△Z的β-內(nèi)酰胺酶活性降低至野生株相同水平。結(jié)論:1.通過構(gòu)建重組質(zhì)粒pLUXampC建立了基于生物發(fā)光技術(shù)的細(xì)菌ampC表達(dá)水平檢測方法。該方法可在不影響細(xì)菌活性的情況下對細(xì)菌的AmpC β-內(nèi)酰胺酶表達(dá)量進(jìn)行監(jiān)測;2.Y.enterocolitica可通過三個AmpD同系物對細(xì)菌的AmpC β-內(nèi)酰胺酶進(jìn)行調(diào)控。其中AmpD1功能較強(qiáng),單缺失株YE△D1(ampD 缺失)的AmpC酶表達(dá)量即出現(xiàn)明顯的上升;而AmpD2和AmpD3功能相對較弱,其負(fù)調(diào)控功能只有在雙、三基因缺失株中才能體現(xiàn),在腸桿菌科細(xì)菌中發(fā)現(xiàn)多AmpD參與的ampC逐級調(diào)控機(jī)制;3.Y.enterocolitica的四種低分子量青霉素結(jié)合蛋白(LMM PBPs)均參與細(xì)菌AmpC β-內(nèi)酰胺酶的表達(dá)調(diào)控,LMM PBPs缺失會導(dǎo)致細(xì)菌AmpC β-內(nèi)酰胺酶表達(dá)量不同程度的上調(diào)。其中以PBP5b的功能最強(qiáng),單缺失株YE△5b的β-內(nèi)酰胺酶活性即出現(xiàn)明顯上升;而PBP4、PBP5a和PBP7的功能相對較弱,主要與PBP5b相協(xié)同而發(fā)揮作用:4.Y.enterocolitica具有NagZ依賴/NagZ非依賴兩種AmpC β-內(nèi)酰胺酶調(diào)控途徑。
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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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本文編號：1910764