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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7719完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂 | |
| dc.contributor.author | Yi-Rou Lu | en |
| dc.contributor.author | 呂怡柔 | zh_TW |
| dc.date.accessioned | 2021-05-19T17:51:13Z | - |
| dc.date.available | 2022-09-08 | |
| dc.date.available | 2021-05-19T17:51:13Z | - |
| dc.date.copyright | 2017-09-08 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-14 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7719 | - |
| dc.description.abstract | 細菌會利用不同的致病因子(virulence factor)來抵禦或逃脫宿主的免疫攻擊，並提升在宿主環境中生存或造成威脅的能力，而細菌分泌蛋白質的過程為一種與宿主互動的重要策略。已知革蘭氏陰性菌演化出六型分泌系統(type I to type VI secretion system, T1SS to T6SS)，而第六型分泌系統是最近期所發現，會透過組裝精密的Hcp-VgrG-PAAR突刺裝置，將作用蛋白質(effector protein)運輸至目標細胞或環境中。據研究指出高於25%的革蘭氏陰性菌帶有構成T6SS核心構造之13個保守基因，而本篇研究對象克雷伯氏肺炎桿菌NTUH-K2044也被發現有T6SS locus-I和locus-III的存在，故想深入探討其T6SS是否具有功能，且會受何種未知機制所調控。
本研究結果顯示，T6SS相關基因(hcp、vgrG1、vgrG2、icmF1和icmF2)廣泛分布於不同來源的克雷伯氏肺炎桿菌臨床菌株，其中以分離自社區型化膿性肝膿瘍(PLA)菌株同時帶有此五個基因之比率為最高（32/42, 76.19%），但隨機挑選的47株hcp+vgrG1+vgrG2+icmF1+icmF2+菌株，包括野生型NTUH-K2044，並未在西方墨點法偵測到Hcp蛋白質的表現，故認為克雷伯氏肺炎桿菌之T6SS在一般實驗室培養條件下可能受細菌內未知機制所壓抑。接續利用即時定量聚合酶鏈鎖反應分析發現H-NS蛋白質會在轉錄層次上負向調控hcp、vgrG1和vgrG2 mRNA表現量，且以西方墨點法在hns基因剔除株中可偵測到Hcp蛋白質的表現。另外，野生型NTUH-K2044在與真核細胞(HL-60, Caco-2 cells)作用後，其hns mRNA表現量雖有顯著降低，但並無提升T6SS相關基因的表現，顯示T6SS在NTUH-K2044與真核細胞作用之下會受到更嚴謹且未知的機制所調控。接著利用凝膠阻滯分析法確認H-NS可直接與T6SS locus-I上之啟動子序列或是基因編碼序列相互結合，藉此抑制T6SS相關基因的表現。最後，在與Caco-2細胞貼附實驗中，發現T6SS相關基因會影響NTUH-K2044對腸道細胞的貼附能力，而其中詳細的分子機制為何仍需更深入的研究。 | zh_TW |
| dc.description.abstract | Bacteria can use different virulence factors to resist or escape host immune response, even to enhance survival ability in host environment. Currently, Gram-negative bacteria have been known to evolve six types of secretion system (T1SS to T6SS), wherein the type six secretion system (T6SS) was discovered recently, which can transport effector proteins to target cells or environment through assembling a delicate Hcp-VgrG-PAAR needle-like structure. Several studies have indicated that up to 25% of Gram-negative bacteria genome includes 13 conserved genes, which encoded the core components of T6SS. In fact, two contiguous putative T6SS loci (locus-I, locus-III) have been found in the Klebsiella pneumoniae NTUH-K2044 genome in a previous study. Therefore, the aim of this study is to investigate whether the NTUH-K2044 have functional T6SS or not, and to identify the unknown regulation mechanisms of its T6SS.
Our results showed that five of the T6SS genes (hcp, vgrG1, vgrG2, icmF1, icmF2) that were highly related to the assembly and function of T6SS were widespread in various clinical isolates of K. pneumoniae, and strains isolated from community-acquired pyogenic liver abscess (PLA) have a higher prevalence (32/42, 76.19%). The Hcp protein was detected neither in randomly selected 47 hcp+vgrG1+ vgrG2+icmF1+icmF2+ strains nor in the wild-type NTUH-K2044. Thus, we assumed that T6SS was repressed via unknown mechanisms in general in vitro culture conditions. Next, we found that transcription levels of hcp, vgrG1 and vgrG2 were suppressed by H-NS via real-time PCR analysis. The Hcp protein expression was significantly increased in the hns deletion mutant strain. The hns transcription level of the NTUH-K2044 was significantly decreased after interacting with both HL-60 and Caco-2 cells, however, the expression of T6SS-related genes did not increased, revealing that a more stringent and unknown mechanisms behind when interacting with eukaryotic cells. In addition, the gel retardation assay confirmed that H-NS could bind to the promoter sequences or coding region of T6SS locus-I, indicating the suppression of transcription of T6SS-related genes is through direct binding. At last, we found that T6SS-related genes were involved in the NTUH-K2044 adhesion to Caco-2 intestinal cells from adherence assays, while the detailed molecular mechanisms still need further study. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:51:13Z (GMT). No. of bitstreams: 1 ntu-106-R04445116-1.pdf: 2802663 bytes, checksum: 543185f8eed0076bd88760e1b7d66a91 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員審定書 I
致謝 II 中文摘要 III Abstract V 目錄 VII 表目錄 X 圖目錄 XI 第一章、 緒論 1 1.1 克雷伯氏肺炎桿菌(Klebsiella pneumoniae)與其臨床重要性 1 1.2 革蘭氏陰性細菌獨有之分泌系統(Secretion System) 2 1.3 第六型分泌系統(Type VI Secretion System, T6SS) 3 1.4 細菌對第六型分泌系統之調控機制 6 1.4.1 鐵離子調控機制 6 1.4.2 H-NS蛋白質的調控機制 6 1.4.3 轉錄後調控機制：The Gac/Rsm pathway 7 1.4.4 群體感應(Quorum sensing)調控機制 7 1.4.5 轉譯後調控機制：Threonine phosphorylation pathway 8 1.5 H-NS (Histone-like nucleoid structuring protein)蛋白質 8 1.6 研究動機 9 第二章、 材料方法 10 2.1 材料 10 2.1.1 克雷伯氏肺炎桿菌之臨床菌株(clinical strains) 10 2.1.2 其他菌株與質體(plasmids) 10 2.1.3 培養基(media) 11 2.1.4 抗生素(antibiotics) 11 2.1.5 引子(primers) 12 2.1.6 HL-60細胞株培養與分化 12 2.1.7 Caco-2細胞株培養 12 2.2 方法 13 2.2.1 聚合酶鏈鎖反應(Polymerase chain reaction；PCR) 13 2.2.2 聚丙烯醯胺膠體電泳(Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis；SDS-PAGE)與蛋白質染色(Coomassie blue staining) 14 2.2.3 西方墨點法(Western Blot) 17 2.2.4 萃取克雷伯氏肺炎桿菌之RNA 19 2.2.5 反轉錄定量聚合酶鏈鎖反應(Quantitative real-time reverse-transcription PCR；RT-qPCR) 20 2.2.6 克雷伯氏肺炎桿菌與真核細胞株交互作用模式 22 2.2.7 Caco-2細胞貼附試驗(Cell adherence assay) 23 2.2.8 NTUH-K2044 之T6SS locus-I之kp1_2392(impB) ~ kp1_2400(vgrG1)操縱子(operon)位置分析 24 2.2.9 表現及純化克雷伯氏肺炎桿菌之H-NS蛋白質 24 2.2.10 凝膠阻滯分析(Gel retardation assay) 27 第三章、 結果 29 3.1 研究策略 29 3.2 克雷伯氏肺炎桿菌臨床菌株之T6SS相關基因盛行率 29 3.3 克雷伯氏肺炎桿菌臨床菌株之Hcp蛋白質的表現 30 3.4 NTUH-K2044對T6SS的調控機制 30 3.4.1 尋找NTUH-K2044之H-NS蛋白質 31 3.4.2 NTUH-K2044之H-NS對T6SS相關基因或蛋白質表現的影響 31 3.4.3 NTUH-K2044與真核細胞株交互作用對T6SS相關基因表現的影響 32 3.5 NTUH-K2044 之T6SS locus-I之kp1_2392(impB) ~ kp1_2400 (vgrG1)操縱子位置分析 32 3.6 凝膠阻滯分析(Gel retardation assay)實驗 33 3.7 野生型NTUH-K2044及其T6SS基因剔除株與Caco-2細胞之貼附實驗(Cell adherence assay) 34 第四章、 總結與討論 36 第五章、 參考文獻 57 附錄一、鐵離子濃度對NTUH-K2044之vgrG1及vgrG2 mRNA表現量的影響 64 附錄二、suhB基因剔除株(△kp1_0314和△kp1_1580)之Hcp蛋白質表現 65 | |
| dc.language.iso | zh-TW | |
| dc.title | 克雷伯氏肺炎桿菌第六型分泌系統之調控 | zh_TW |
| dc.title | Regulation of type 6 secretion system (T6SS) in Klebsiella pneumoniae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 謝世良,張永祺(yungchiychang@ntu.edu.tw),潘怡均 | |
| dc.subject.keyword | 克雷伯氏肺炎桿菌,第六型分泌系統,H-NS蛋白質,凝膠阻滯分析法, | zh_TW |
| dc.subject.keyword | Klebsiella pneumoniae,Type six secretion system,H-NS,Gel retardation assay, | en |
| dc.relation.page | 65 | |
| dc.identifier.doi | 10.6342/NTU201702520 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2017-08-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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