臺灣博碩士論文加值系統

鐵是一種豐富的金屬元素並且對於大多數的生物體而言是重要營養素，細菌通常分泌嗜鐵素(siderophore)小分子螯合外界的鐵進行利用。致病菌的攝鐵能力在致病機轉中是重要的因子。因此，致病菌的攝鐵系統通常被認為是一種毒力因子。毒力質體對於致病性大腸桿菌的毒力與致病具有重要性。在新生兒腦膜炎病人分離出來的腸道外致病性大腸桿菌RS218中內含一個質體pRS218。藉由基因體資訊及蛋白質序列搜尋比對，我們發現有3組可能的攝鐵系統位於pRS218。

這3組基因中， fetMP基因組為二價鐵離子運輸系統。FetM為二價鐵離子通透酶，而FetP則具有三價鐵離子還原酶活性，可以促進FetM的運輸功能。eco42-43基因組為一個潛在的血紅素轉運系統，可能利用血紅素所攜帶的鐵離子作為鐵源。此基因組轉譯出的Eco42蛋白可能為第二類螯合酶，能夠螯合金屬離子，而Eco43蛋白則含有保守的外膜血紅素受體蛋白質基序FRAP / NPNL和一個保守的組氨酸殘基。eco60-63基因組已被預測參與攝鐵，實驗顯示這個基因組可能可以被Fur攝鐵調控蛋白所調控。此外Eco62蛋白演化樹親源分析顯示Eco62可能的功能為嗜鐵素受體/血紅素受體。

將菌株RS218分別剔除單套、雙套與三套基因組發現在含鐵及缺鐵條件培養下出現攝鐵系統功能有互補的機制。即突變株所喪失的功能可能藉由細菌中其他攝鐵基因所補償，因此並沒有造成生長缺陷。為避免此機制的影響，我們以非致病性K12大腸桿菌MG1655建構攝鐵能力缺失菌株，並利用這樣的菌株來分析pRS218上的這3組攝鐵相關基因的攝鐵功能。在建造攝鐵能力缺失的MG1655菌株時，我們曾對MG1655的多個攝鐵基因進行剔除，並發現只要對嗜鐵素生合成基因進行剔除就能使MG1655在在缺鐵環境中出現生長缺失。接續利用嗜鐵素生合成基因剔除菌株(IP10)來研究fetMP、eco42-43、eco60-63基因組的攝鐵功能。

我們將這些單套或多套可能攝鐵基因剔除的pRS218經接合生殖送入IP10，然後對於這些接合菌株的攝鐵能力進行分析。結果顯示，這3組基因中僅fetMP基因組可以在缺鐵環境補救攝鐵缺失菌株IP10的生長缺陷。我們也發現此質體會對IP10造成生長負擔。另外，fetMP基因組補救攝鐵缺失菌株的生長缺陷不受到酸鹼值影響。這些結果顯示fetMP基因組可能比eco42-43、eco60-63基因組在攝鐵功能上扮演比較重要的角色。

雖然fetMP、eco42-43、eco60-63基因組對於細菌的生理以及致病力所扮演的角色仍須要進一步的研究，但結果顯示pRS218質體可能增加RS218的攝鐵能力而幫助此菌在缺鐵環境中生長，進而增加細菌的致病力。

Escherichia coli. E. coli K1 RS218, an extraintestinal pathogenic E. coli (ExPEC) strain isolated from a neonate with meningitis patient, containing a plasmid named pRS218. Based on the genomic information and protein blast results, we identified 3 potential iron acquisition systems located on the pRS218. Among the 3 operons of pRS218, the fetMP operon encodes a ferrous iron transport system. The eco42-43 operon encodes a potential heme transport system, which may utilize heme-iron as an iron source. The eco60-63 operon has been predicted to be involved in iron acquisition. We constructed an iron-uptake defected MG1655 E. coli K12 strain and utilize the strain to investigate the iron-uptake function of these operons carried by pRS218. The growth results suggest that the fetMP operon may play a more significant role than the eco42-43 and eco60-63 operons in terms of iron uptake. Overall, our results suggest that the pRS218 plasmid may contribute to the iron-uptake ability of RS218, to facilitate bacterial growth in iron-restricted condition when in host as well as to increase the virulence of the bacterial pathogen.

key word: Escherichia coli, iron-uptake gene

中文摘要I
Extended AbstractII
誌謝IV
目錄V
表目錄VI
圖目錄VI
一、緒論1
1.1 鐵1
1.2 格蘭氏陰性菌的攝鐵系統1
1.2.1 金屬離子運輸系統 (Metal transport system)1
1.2.2 鐵離子複合體運輸系統 (Iron complex transport system)2
1.3 攝鐵系統為毒力因子之一3
1.4 細菌性新生兒腦膜炎3
1.5 腸道外致病型大腸桿菌的親緣性3
1.6 毒性相關質體在大腸桿菌致病中的角色4
1.7 研究目的4
二、材料與方法6
2.1 大腸桿菌之來源、培養、保存6
2.2 電穿孔勝任細胞之製備6
2.3 聚合酶鏈鎖反應(Polymerase chain reaction，PCR)7
2.4 fetMP，eco43，fepA，entA基因剔除菌株之建構7
2.5 P1噬菌體之製備與轉染7
2.6 大腸桿菌與eco60-63 上游Fur結合序列之質體建構8
2.7 限制酶反應9
2.8 Fur titration assay (FURTA)9
2.9 接合生殖(Conjugation)9
2.10 細菌生長曲線9
2.11 血清生存實驗(Serum survival assay)10
三、結果11
3.1 NMEC RS218的攝鐵系統 11
3.2 pRS218中的攝鐵系統11
3.2.1 fetMP (eco10-11)11
3.2.2 eco42-43 12
3.2.3 eco60-63 13
3.3 fetMP、eco43以及eco60-63基因剔除菌株在缺鐵環境下並無生長差異13
3.4 在非致病大腸桿菌K12菌株MG1655中建立攝鐵基因測試系統14
3.5 接合菌株在缺鐵條件下的生長15
3.6 接合菌株在缺鐵的環境中不同酸鹼值並且的生長15
3.7 fetMP、eco42-43基因組與細菌的血清生存能力16
四、討論17
五、參考文獻45

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