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研究生： 陳怡彣
Yi-Wen Chen
論文名稱： 利用大腸桿菌蛋白質體晶片找出參與第一型線毛表現之細菌蛋白質
Identification of bacterial factors involved in type 1 fimbria expression using an Escherichia coli K12 proteome chip
指導教授： 陳健生
Chien-Sheng Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 生醫理工學院 - 系統生物與生物資訊研究所
Graduate Institute of Systems Biology and Bioinformatics
論文出版年： 2014
畢業學年度： 102
語文別： 英文
論文頁數： 64
中文關鍵詞： 蛋白質體晶片 、第一型線毛 、分子生物 、去氧核醣核酸-蛋白質交互作用
外文關鍵詞： proteome microarray, type 1 fimbria, Molecular biology, DNA-protein interaction
相關次數： 點閱：15  下載：0 
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    • 第一型線毛為一表現在大腸桿菌上的絲狀結構之重要附著因子。因為細菌在感染的第一步便是附著於宿主細胞，所以第一型線毛是致病型大腸桿菌的重要危害因子。第一型線毛的表現是受到一相變化的調控，它使得每一株細菌可在線毛表現相 (phase-ON) 與線毛不表現相 (phase-OFF) 中轉換。而此相變化是受到 314 bp 的 fimS DNA 片段翻轉所調控，此一片段包含了可驅動組成第一型線毛所需要基因表現的啟動子。因此，可與 fimS 結合的細菌蛋白質很有可能參與了調控第一型線毛的表現。為了找出新的第一型線毛調控因子，我們利用大腸桿菌 K12 蛋白質體晶片去篩選可與一包含 fimS 和其相鄰區域的 DNA 片段 (共 602 bp) 交互作用的細菌因子。依此實驗結果我們找出了 Spr 蛋白質並用電泳膠遲緩分析法確認此蛋白質可與 fimS 結合。將大腸桿菌 K1 小兒腦膜炎菌株 RS218 剔除 spr 基因後發現，其生長在瓊脂平板上表現第一型線毛的菌落比例顯著地增加了。此外，我們發現到 Spr 也干預了 fimS 與 FimB 和 FimE 的交互作用，FimB 和 FimE 為一種專一性重組酶，為已知可調節 fimS 翻轉的蛋白質。這些結果顯示 Spr 蛋白質透過直接與 fimS 交互作用來影響第一型線毛的表現，促進了我們對於調控第一型線毛的了解。

    Type 1 fimbriae are filamentous structures on Escherichia coli. These structures are important adherence factors. Because binding to the host cells is the first step of infection, type 1 fimbria is an important virulence factor of pathogenic E. coli. Expression of type 1 fimbria is regulated by a phase variation in which each individual bacterium can alternate between fimbriated (phase-ON) and nonfimbriated (phase-OFF) states. The phase variation is regulated by the flipping of the 314-bp fimS fragment which contains the promoter driving the expression of the genes required for the synthesis of type 1 fimbria. Thus, the bacterial proteins able to interact with fimS are likely to be involved in regulating the expression of type 1 fimbria. To identify novel type 1 fimbria-regulating factors, we utilized an E. coli K12 proteome chip to screen for the bacterial factors able to interact with a 602-bp DNA fragment containing fimS and its adjacent regions. The Spr protein was identified by the proteome chip-based screening and further confirmed to be able to interact with fimS by electrophoretic mobility shift assay. Deletion of spr in the neonatal meningitis E. coli strain RS218 significantly increased the ratio of the bacterial colonies that contained the type 1 fimbria phase-ON cells on agar plates. In addition, Spr interfered with the interactions of fimS with the site-specific recombinases, FimB and FimE, which are responsible for mediating the flipping of fimS. These results suggest that Spr is involved in the regulation of type 1 fimbria expression through direct interaction with the invertible element fimS. These findings facilitate our understanding of the regulation of type 1 fimbria.

    中文摘要 i ABSTRACT ii ABBREVIATIONS iv ACKNOWLEDGMENTS v List of Figures viii List of Tables x I. INTRODUCTION 1 II. EXPERIMENTAL PROCEDURES 6 II.1 Proteome microarray construction 6 II.2 Synthesis of the fimS-related DNA fragments 6 II.3 The E. coli proteome chip assays 7 II.4 Bioinformatics analysis of the chip assays 8 II.5 Electrophoretic mobility shift assay (EMSA) 8 II.6 Invertible element orientation assays 9 II.7 Construction and complementation of the spr deletion mutant of E. coli K1 RS218 10 II.8 The fimS-binding competition assays 11 II.9 Immunofluorescence labeling of type 1 fimbriae on E. coli 12 III. RESULTS 13 III.1 Screening for bacterial factors able to interact with the fimS-containing DNA fragment by E. coli proteome chip assays. 13 III.2 Confirmation of the interactions between the bacterial proteins and the fimS-plus fragment 18 III.3 Spr protein mainly interacts with fimS 23 III.4 The fimS orientation of wild-type, spr deletion mutant, and spr deletion mutant transformed with spr 26 III.5 The fimS orientation reflects the state of type 1 fimbria expression in Δspr-RS218 33 III.6 Spr blocks the binding of fimS with the bacterial factors that have been known to be able to interact with this DNA fragment 36 IV. DISCUSSION 39 V. REFERENCES 46

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