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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 廖淑貞 | |
dc.contributor.author | Yi-Lin Tsai | en |
dc.contributor.author | 蔡易霖 | zh_TW |
dc.date.accessioned | 2021-06-08T03:18:18Z | - |
dc.date.copyright | 2017-03-01 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2017-01-01 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21061 | - |
dc.description.abstract | 前言:奇異變形桿菌(Proteus mirabilis)是經常導致泌尿道感染的病原菌,在長期使用尿導管及糖尿病的患者,容易造成伺機性感染。為了解高糖環境易感染的原因,我們研究與糖相關的cAMP receptor protein(Crp)。Crp不只調控碳水化合物代謝,也會影響致病及壓力抵抗的調控。到目前為止,Crp不僅在P. mirabilis尚未被研究外,也無文獻探討Crp與糖尿病泌尿道感染間的關聯。目的:探討尿道病原菌奇異變形桿菌cAMP受體蛋白質所扮演的角色。結果:觀察Crp活化態與glucose相關性,以plac-gfpuv reporter assay得知Crp活化態受到glucose調控。為了解Crp調控網路,構築crp突變株及互補株以reporter assay及real-time PCR研究與ptsG(glucose permease)、cyaA(adenylyl cyclase)、hfq(RNA chaperone)及本身crp基因調控,發現Crp-cAMP自我調控及正調控ptsG,負調控cyaA及hfq外,Hfq也會正調控crp。研究Crp-cAMP與糖尿病泌尿道感染的關聯,以streptozotocin構築糖尿病鼠後,進行泌尿道定植發現crp突變較容易定植腎臟,不易定植膀胱外,糖尿病鼠也較容易定植,顯示Crp可能在糖尿病鼠的腎臟定植扮演角色。為探討crp突變易腎臟定植,以腎臟表皮細胞做貼附實驗,發現crp突變較易貼附於腎臟表皮細胞,以transcriptome分析發現與腎臟貼附相關的pmp纖毛表現會於crp突變株及10% glucose預處理之野生株中上升,以reporter assay、real-time PCR及DNase I footprinting來研究Crp-cAMP與pmpA的調控,發現Crp-cAMP負調控pmpA。為了解Pmp在腎臟定植的角色,構築pmpA突變株及pmpA/crp雙突變株,發現PmpA的缺失降低腎表皮細胞的貼附與定植能力。此外,E. coli中Crp-cAMP透過RpoS影響壓力抵抗,在P. mirabilis的crp突變株及10% glucose預處理之野生株會增加對於pH 3、30 mM H2O2及THP-1巨噬細胞內存活能力,推測可能與RpoS有關。另外也發現Crp-cAMP參與調控移動性,以transcriptome、real-time PCR及reporter assay分析發現Crp-cAMP會正調控flhDC及glnA,萃取flagellin及TEM觀察後發現crp突變顯著的減少鞭毛抗原的合成。結論:Crp-cAMP調控許多毒性因子,如移動性、鞭毛抗原、抗酸或H2O2、巨噬細胞內存活、透過Pmp影響腎臟表皮細胞貼附和腎臟定植。因此Crp對於P. mirabilis在泌尿道感染上扮演著重要的角色,且Pmp纖毛標的具有作為設計抗感染藥物的潛力。 | zh_TW |
dc.description.abstract | Introduction: Proteus mirabilis is a common human pathogen causing recurrent or persistent urinary tract infections (UTIs), especially in patients with indwelling catheters. The underlying mechanisms for P. mirabilis to establish UTIs are still not fully elucidated. The ability of P. mirabilis to colonize within the host urinary tract is closely related to fimbria-mediated adhesion. Previously, we found a conserved cAMP receptor protein, Crp, is involved in the carbon metabolism of P. mirabilis. Many studies have shown that the Crp is also associated with virulence factor production in pathogenic bacteria. Specific aims: we investigated the roles of Crp in uropathogenic Proteus mirabilis. Results: In this work, we first demonstrated that alteration of Crp activity was in response to glucose. Then we investigate the correlation of Crp with major glucose PTS transporter gene ptsG, adenylate cyclase gene cyaA and Hfq by XylE reporter assay and real-time RT-PCR. The results indicate that the Crp-cAMP increased ptsG and its own gene mRNA level, and decreased cyaA and hfq mRNA level. In addition, we found the loss of crp increased kidney colonization and survival in macrophages. Transcriptome analysis revealed Crp-regulated genes. The candidate genes were validated by real-time PCR and reporter assay. We found that Crp could regulate multiple virulence factors, including Pmp fimbriae, flagellin, stress (acid or H2O2) tolerance, and modulation of host immune responses, which may contribute to colonization in mice. Conclusion: For the first time, we found upregulation of Pmp fimbriae in crp mutant is critical to the kidney colonization. In this regard, it is tempting to suggest that Pmp fimbriae may serve as the target for design of novel drugs for diabetic Proteus infection. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:18:18Z (GMT). No. of bitstreams: 1 ntu-105-D01424003-1.pdf: 2923967 bytes, checksum: 53cdfa649a2a10b7bb47bfe658fa8807 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 中文摘要 III Abstract IV 目錄 V 圖目錄 VIII 表目錄 VIII 第一章 文獻回顧 1 第一節 奇異變形桿菌簡介 1 1.1 奇異變形桿菌毒性因子(Virulence factors) 1 1.1.1 鞭毛(Flagella) 2 1.1.2 纖毛(Fimbriae) 2 1.1.3 溶血酶(Haemolysin) 2 1.1.4 尿素酶(Urease) 2 1.1.5 生物膜(Biofilm) 3 1.1.6 胺基酸分解酶(Deaminase) 3 1.1.7 IgA蛋白酶(IgA-degrading protease) 3 1.2 奇異變形桿菌表面移行能力(Swarming motility) 3 第二節 cAMP receptor protein簡介 5 2.1 Crp介紹(cAMP receptor protein) 5 2.2 Crp的活化及調控網路(Regulatory network of Crp) 5 第三節 研究目的 7 第二章 材料與方法 8 第一節 實驗設計 8 第二節 實驗流程 9 第三節 實驗方法 10 3.1 實驗菌株與質體(Bacterial strains and plasmids) 10 3.2 生物資訊分析(Bioinformatic analysis) 10 3.2.1 生物資訊網站(Website) 10 3.2.2 生物資訊軟體(Software) 11 3.3 構築質體與突變株(Construction of plasmids and P. mirabilis mutants) 12 3.3.1 構築突變株(Construction of mutant strain) 12 3.3.2 基因體DNA萃取(Genomic DNA extraction) 12 3.3.3 聚合酶連鎖反應(Polymerase chain reation,PCR) 12 3.3.4 限制酶酶切(Restriction enzyme digestion) 13 3.3.5 DNA純化(DNA purification) 13 3.3.6 質體DNA萃取(Plasmid DNA extraction) 14 3.3.7 DNA黏合作用(DNA ligation) 14 3.3.8 構築過度表達株(Construction of overexpression strain) 15 3.3.9 質體DNA轉型作用(Transformation) 15 3.3.10 細菌接合生殖(Conjugation) 16 3.3.11 南方墨點法(Southern blot analysis) 17 3.3.12 核酸定序與序列分析(Sequencing) 18 3.4 基因表達(Gene expression) 18 3.4.1 Promoter活性分析(Reporter assay) 18 3.4.2 RNA萃取(RNA extraction) 18 3.4.3 RNA反轉錄(Reverse transcription) 19 3.4.4 同步定量PCR(Real-time PCR) 19 3.4.5 反轉錄PCR(RT-PCR) 20 3.4.6 電泳遷移率變動分析(EMSA) 20 3.4.7 DNase I足印法(DNase I footprinting) 21 3.5 蛋白質萃取(Protein extraction) 21 3.5.1 鞭毛萃取(Flagellin extraction) 21 3.5.2 蛋白質濃度測定(Bio-Rad protein assay) 22 3.5.3 不連續膠體電泳(SDS-PAGE) 22 3.6 抗藥性及壓力耐受性(Antimicrobial resistant and stress tolerance) 22 3.6.1 生長曲線(Growth curve) 22 3.6.2 最小抑制濃度(Minimum inhibitory concentration,MIC) 23 3.6.3 氧化壓力抵抗(Oxydative stress tolerance) 23 3.6.4 耐酸性(Acid resistance) 23 3.7 移動性及電子顯微鏡觀察(Motility assays and TEM) 24 3.7.1 表面移行(Swarming) 24 3.7.2 泳動(Swimming) 24 3.7.3 電子顯微鏡(TEM) 24 3.8 細胞及動物實驗(Cell and animal experiments) 24 3.8.1 巨噬細胞殺菌試驗(Macrophage infection assay) 24 3.8.2 上皮細胞貼附試驗(Epithelial cell adhesion assay) 25 3.8.3 動物實驗(Animal experiments) 25 第三章 結果 26 第一節 P. mirabilis cAMP receptor protein之特性 26 第二節 P. mirabilis Crp對於泌尿道定植之影響 28 第三節 P. mirabilis Crp影響腎臟與膀胱上皮細胞貼附能力及調控纖毛表現 29 第四節 P. mirabilis crp突變增加巨噬細胞THP-1內存活能力 31 第五節 P. mirabilis crp突變增加抗H2O2及酸能力 31 第六節 P. mirabilis crp突變增加rpoS mRNA表現量 31 第七節 P. mirabilis Crp調控flhDC影響移動性與鞭毛濃度 32 第四章 討論 33 第五章 結論 37 第六章 參考文獻 52 第七章 個人簡歷 62 | |
dc.language.iso | zh-TW | |
dc.title | 探討尿道病原菌奇異變形桿菌cAMP受體蛋白質所扮演的角色 | zh_TW |
dc.title | The roles of cAMP receptor protein in uropathogenic Proteus mirabilis | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 鄧麗珍,賈景山,許萬枝,楊翠青 | |
dc.subject.keyword | 奇異變形桿菌,泌尿道感染,糖尿病,cAMP受體蛋白質, | zh_TW |
dc.subject.keyword | Proteus mirabilis,Urinary Tract Infection,Diabetes mellitus,cAMP receptor protein, | en |
dc.relation.page | 66 | |
dc.identifier.doi | 10.6342/NTU201603867 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2017-01-03 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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