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系統識別號 U0026-0306201318111800
論文名稱(中文) eco60-63 基因組在大腸桿菌K1受到血清攻擊時所扮演的角色
論文名稱(英文) The role of the eco60-63 operon in serum killing of Escherichia coli K1
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 101
學期 2
出版年 102
研究生(中文) 陳冠福
研究生(英文) Kuan-Fu Chen
學號 T16991046
學位類別 碩士
語文別 中文
論文頁數 51頁
口試委員 指導教授-鄧景浩
口試委員-何漣漪
口試委員-吳俊忠
中文關鍵字 大腸桿菌K1  菌血症  受鐵調控基因  血清擊殺 
英文關鍵字 Escherichia coli K1  iron-regulated gene  bacteremia  serum-mediated killing 
學科別分類
中文摘要 大腸桿菌K1(Escherichia coli K1)是造成革蘭氏陰性菌新生兒腦膜炎的主要致病菌。大腸桿菌K1主要透過在血液中的散布來造成新生兒腦膜炎。因此,細菌需要在血流中的存活與增殖以進一步造成中樞神經系統的感染。然而現今對大腸桿菌K1在血流中與宿主血液中的免疫系統互動機制還未清楚。eco60-63基因組由4個基因eco60、eco61、eco62和eco63組成。基因組前端帶有Fur-box相似序列。過去研究已知eco60-63基因組與大腸素攝取、泌尿道感染有關,然而對於此基因組在大腸桿菌K1的致病能力所扮演的角色尚未清楚。根據BLAST分析,Eco60的同源蛋白為與攝取血基質有關的PhuW;Eco61的同源蛋白為參與營養物質傳輸的能量傳遞蛋白TonB;Eco62的許多同源蛋白則皆為TonB依賴型受體(TonB-dependent receptor);Eco63則已知能轉譯為功能尚不清楚的TieB蛋白。
本篇研究主要在探討eco60-63基因組如何在大腸桿菌K1的血流生長的角色。結果顯示,在小鼠菌血症感染模型中,eco60-63基因組突變株與大腸桿菌K1野生株相比較,其血流中的生存能力會顯著的上升。另外,在缺鐵環境下eco60-63基因組表達量會較有鐵環境高。這可能是由於血液中為缺鐵環境導致帶有eco60-63基因組菌株遭受血液免疫系統清除。我們也發現當大腸桿菌K1的eco60-63基因組剔除後會提高其在血清中的生存能力,然而在補體蛋白被高溫去活性的血清中,eco60-63基因組的有無不會影響細菌的血清存活能力。這結果說明細菌表達ecoo60-63基因組可能引起較強的補體系統攻擊。另外我們利用eco60-63基因組個別成員的突變株與帶有His-tag的回補株,透過血清生長測試,證實了eco60和eco62為造成大腸桿菌K1在血清中生存能力下降的主因。
英文摘要 Escherichia coli K1 is the most common Gram-negative bacterium causing neonatal meningitis. Neonatal E. coli K1 meningitis usually occurs via hematogenous spread. Thus, E. coli need to survive and multiply in the blood stream so as to invade the central nervous system. However, the interaction between E. coli K1 and the host immune system in the blood stream is still unclear. The eco60-63 operon contains four genes, eco60, eco61, eco62 and eco63. A near-consensus Fur box is found upstream of eco60-63 operon. Previous studies have shown that eco60-63 operon is associated with colicin uptake and urovirulence of E. coli. However, the role of eco60-63 operon in the pathogenesis of E. coli K1 is still unknown. According to the BLAST search, Eco60 is homologous to the heme acquisition related protein, PhuW; Eco61 is homologous to the TonB protein that is involved in energy transduction of the nutrient transport system; Eco62 is homologous to several TonB-dependent receptors; Eco63 has been known to encode the TieB protein, whose function is not identified yet. This study aims to investigate the role of the eco60-63 operon in blood-stream survival of E. coli K1. In the mouse model of bacteremia, we found that deletion of this operon increased survival of the bacteria in the blood stream. The expression of the eco60-63 operon in the iron-depleted medium was higher than that in the iron-containing medium. These results suggested that the iron-restricted condition of the blood stream might induce the eco60-63 operon's expression which, in turn, cause clearance of the invading E. coli by innate immune system in the blood stream. We also found that the eco60-63 operon deletion mutant strain exhibited significantly higher survival than the wild-type strain in normal human serum (NHS), but the wild-type and mutant strains showed similar survival in the heat-inactivated normal human serum (HI-NHS). These findings suggest that the eco60-63 operon might facilitate serum killing mediated by the heat-labile complement system. In addition, we constructed mutants of individual genes in this operon and the corresponding complemented strains. By using the serum survival assays, we demonstrated that eco60 and eco62 are the genes in the eco60-63 operon responsible for decreasing the serum survival of E. coli K1.
論文目次 中文摘要 I
Abstract II
誌謝 IV
目錄 V
表目錄 VIII
圖目錄 VIII
縮寫表 X
一. 緒論 1
1.1 細菌性新生兒腦膜炎與大腸桿菌K1 1
1.2 補體系統 1
1.3 細菌攝鐵系統 2
1.4 Fur蛋白的調控 3
1.5 大腸素 3
1.6 eco60-63基因組 3
1.7 實驗目的 4
二. 材料與方法 5
2.1 大腸桿菌K1之來源、培養、保存 5
2.2 動物實驗 5
2.3 eco60-63基因組及eco60、eco61、eco62、eco63質體建構 6
2.4 限制酶反應 6
2.5 DNA接合作用 (ligation) 6
2.6 電穿孔勝任細胞製備 6
2.7 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 7
2.8 eco60-63基因體及eco60、eco61、eco62、eco63突變株建構 7
2.9 即時聚合酶鏈鎖反應 (real-time-polymerase chain reaction, RT-PCR) 8
2.10 血清生存實驗 (serum survival assay) 8
2.11 生長曲線 (growth curve) 9
2.12 西方點墨法 (western blot) 9
2.13 細菌外膜分離法 9
2.14 統計方法 10
三. 結果 11
3.1 eco60-63基因組 11
3.2 eco60-63基因組對大腸桿菌K1感染小鼠的毒力影響 12
3.3 大腸桿菌K1在缺鐵環境下eco60-63基因組的表達 12
3.4 eco60-63基因組導致大腸桿菌K1在正常人類血清中的生存能力下降 13
3.5 eco60-63基因組在大腸桿菌K1生長早期造成血清中的生存能力下降 15
3.6 eco60-63基因組在補體擊殺路徑的探討 16
3.7 eco60-63基因組個別基因突變株在血清中生長受到的影響 17
3.8 eco60-63基因組個別基因回補株在血清中受到的生長影響 18
3.9 eco60-63基因組個別基因蛋白的表達以及Eco60在細胞膜的位置 18
四. 討論 20
五. 參考文獻 27
Tables 33
Figures 38
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