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研究生(外文):Yu-Ling Chen
論文名稱(外文):Characterization of L0038 Encoded in the Pathogenicity Island of Enterohemorrhagic Escherichia coli O157:H7 and Researching of Protein-Protein Interaction in Type III Secretion System by FLIM/FRET
指導教授(外文):Wan-Jr Syu
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Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a pathogen causes hemorrhagic diarrhea and hemolytic-uremic syndrome, employs the type III secretion system to deliver proteins into host cells. Mechanistically, a pedestal-like structure is formed between the bacterium and the host cell, then a tight bacterial attachment is achieved. These characteristics have been attributed to a pathogenic island on the bacterial chromosome known as the locus for enterocyte effacement(LEE), a cluster of 41 predicted open reading frame(ORFs), of which some have been characterized. Among the uncharacterized ORFs, l0038 encoded a protein of 152 amino acid residue with a predicted pI of 5.5. An l0038–deleted strain created by one-step inactivation of chromosomal gene gave decrease of EspA synthesis and lost most of secrected proteins, suggested that L0038 may act as one of the TTSS components.Unfortunately, there is no way to complement the secretion phenotype of the deleted strain byexogenously expressing L0038, so the hypothesis might need further investigation. In this study, the interaction between L0038 and EscU, and also that between EscU and L0050 were demonstrated by bacterial two-hybrid assay. The sequential interactions between EspA, L0050, EscU and L0038 may imply the decrease of EspA in l0038 mutant may be due to the fail in recruitment of EspA to TTSS machinery. The protein interactions indeed play an essential role in building the TTSS and in secreting the TTS proteins. Accordingly, we apply the technology of FRET/FLIM in in vivo analyzing the interactions between bacterial proteins. Co-expression of both fluorescence-tags fused CesT and Tir in JM109 was used as positive control to set up the FLIM assay in bacteria. Although the possible interference of non-intact fusion protein ruined the analysis result, the shorter life time of donor fluorescence as comparing with that with donor fluorescence protein only demonstrated that there is an in vivo protein-protein interaction between CesT and Tir. Results clearly showed the success in applying FLIM assay in analysis of bacterial samples.
中文摘要 3
Abstract 5
第一章 緒論 7
1. 大腸桿菌概論 7
2. 腸出血性大腸桿菌概論 (Enterohemorrhagic E. coli, EHEC) 8
3. 出血性大腸桿菌之致病機制 8
4. 第三型分泌系統 (type III secretion system; TTSS) 10
5. LEE致病島嶼 (the locus of enterocyte effacement island, LEE island) 11
6. L0038先前之研究 12
7. 目前探討蛋白質交互作用之生物化學研究方法 13
8. 螢光原理與特性 14
9. 螢光共振能量轉移(Förster or fluorescence resonance energy transfer; FRET) 15
10. 螢光壽命呈像顯微術(fluorescence lifetime imaging microscopy; FLIM) 17
11. Tir與其侍衛蛋白質CesT (Tir and the chaperone of Tir, CesT) 18
12. 本文動機與方向 19
第二章 材料與方法 21
1. 細菌菌株 (bacterial strain) 21
2. 質體與引子 (plasmids and primers) 21
3. 抗體 (antibodies) 21
4. 細菌的培養 (bacterial culture) 21
5. 細菌質體萃取與純化 (bacterial plasmid extraction) 22
6. 聚合酶連鎖反應 (polymerase chain reaction; PCR) 22
7. 瓊脂凝膠製作及電泳 (agarose gel electrophoresis) 23
8. 質體構築 (plasmid construction) 23
9. 勝任細胞製備 (Competent cell preparation) 24
10. 細菌轉型 (Transformation) 25
11. 蛋白質之過量表現 (Protein expression) 25
12. SDS聚丙烯醯胺凝膠電泳 (SDS-PAGE) 26
13. 西方墨點法 (Western blotting) 26
14. 鎳離子管柱純化(nickel ion-column purification) 27
15. 三氯醋酸沉澱 (TCA precipitation) 28
16. 細菌雙雜合試驗 (bacterial two-hybrid system) 29
17. β-半乳糖苷酶活性測試 (β-galactosidase activity assay; Miller Assay) 29
18. 染色體基因一步失活法(One-step inactivation of chromosomal gene) 30
19. 螢光壽命顯微術(FLIM)之架構 31
第三章 結果 33
1. l0038基因刪除突變株之構築及篩選 33
2. l0038基因刪除突變株對第三型分泌蛋白質合成與分泌之影響 33
3. 利用細菌雙雜合系統,證明L0038與EscU以及EscU與L0050之交互作用 34
4. 構築各表現螢光蛋白質之質體 35
5. 以西方點墨法確認各融合蛋白質最佳表現量之時間點 36
6. 以螢光顯微鏡觀察各螢光蛋白質最佳表現量之時間點 36
7. 透過Ni2+親合性管柱純化確認CesT與Tir之螢光融合蛋白之間仍具有交互作用 37
8. 螢光壽命呈像顯微術(FLIM)接收的訊號 37
9. 實驗樣本之供體螢光(Tir-GFP)壽命降低 38
10. 實驗組與對照組螢光壽命的比較 38
第四章 討論 40
參考文獻 46
附錄 63
1. Molecular epidemiologic investigation and analysis of toxin of enterohemorrhagic E. coli O157:H7 from cattle in southern Taiwan. Lin, K.-H. 2009b.
2. http://www.who.int/mediacentre/factsheets/fs125/en/.
3. Albertazzi,L.F., Arosio,D.F., Marchetti,L.F., Ricci,F.F., and Beltram,F. (2009). Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair. Photochem. Photobiol. 2009 Jan-Feb;85, 287-297.
4. Boute,N., Jockers,R., and Issad,T. (2002). The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol. Sci. 23, 351-354.
5. Campellone,K.G., Robbins,D., and Leong,J.M. (2004). EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nck-independent actin assembly. Dev. Cell 7, 217-228.
6. Creasey., Delahay,R., Delahal., Daniell,S., Daniell S.J., Frankel,G., and Frankel,G. (2003). Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli. Microbiology. 2003 Aug;149, 2093-2106.
7. Deng,W., Puente,J.L., Gruenheid,S., Li,Y., Vallance,B.A., Vazquez,A., Barba,J., Ibarra,J.A., O'Donnell,P., Metalnikov,P., Ashman,K., Lee,S., Goode,D., Pawson,T., and Finlay,B.B. (2004). Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc. Natl. Acad. Sci. U. S. A 101, 3597-3602.
8. Donnenberg,M.S., Kaper,J.B., and Finlay,B.B. (1997). Interactions between enteropathogenic Escherichia coli and host epithelial cells. Trends Microbiol. 5, 109-114.
9. Donnenberg,M.S., Yu,J., and Kaper,J.B. (1993). A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J. Bacteriol. 175, 4670-4680.
10. Ekins,R.P. and Chu,F.W. (1991). Multianalyte microspot immunoassay--microanalytical "compact disk" of the future. Clin. Chem. 37, 1955-1967.11. Elliott,S.J., Hutcheson,S.W., Dubois,M.S., Mellies,J.L., Wainwright,L.A., Batchelor,M., Frankel,G., Knutton,S., and Kaper,J.B. (1999). Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol. Microbiol. 33, 1176-1189.
12. Elliott,S.J., Krejany,E.O., Mellies,J.L., Robins-Browne,R.M., Sasakawa,C., and Kaper,J.B. (2001). EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri. Infect. Immun. 69, 4027-4033.
13. Elliott,S.J., Sperandio,V., Giron,J.A., Shin,S., Mellies,J.L., Wainwright,L., Hutcheson,S.W., McDaniel,T.K., and Kaper,J.B. (2000). The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 68, 6115-6126.
14. Fields S., Song and Song,O. (1989). A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul;%20;340, 245-246.
15. Gavin,A.C., Bosche,M., Krause,R., Grandi,P., Marzioch,M., Bauer,A., Schultz,J., Rick,J.M., Michon,A.M., Cruciat,C.M., Remor,M., Hofert,C., Schelder,M., Brajenovic,M., Ruffner,H., Merino,A., Klein,K., Hudak,M., Dickson,D., Rudi,T., Gnau,V., Bauch,A., Bastuck,S., Huhse,B., Leutwein,C., Heurtier,M.A., Copley,R.R., Edelmann,A., Querfurth,E., Rybin,V., Drewes,G., Raida,M., Bouwmeester,T., Bork,P., Seraphin,B., Kuster,B., Neubauer,G., and Superti-Furga,G. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141-147.
16. Gophna,U., Ron,E.Z., and Graur,D. (2003). Bacterial type III secretion systems are ancient and evolved by multiple horizontal-transfer events. Gene 312, 151-163.
17. Hu,J.C. (2001). Model systems: Studying molecular recognition using bacterial n-hybrid systems. Trends Microbiol. 9, 219-222.
18. Huang,L.H. and Syu,W.J. (2008). GrlA of enterohemorrhagic Escherichia coli O157:H7 activates LEE1 by binding to the promoter region. J. Microbiol. Immunol. Infect. 41, 9-16.
19. Jarvis,K.G., Giron,J.A., Jerse,A.E., McDaniel,T.K., Donnenberg,M.S., and Kaper,J.B. (1995). Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc. Natl. Acad. Sci. U. S. A 92, 7996-8000.
20. Jerse,A.E., Yu,J., Tall,B.D., and Kaper,J.B. (1990). A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. U. S. A 87, 7839-7843.
21. Kenny,B., Abe,A., Stein,M., and Finlay,B.B. (1997). Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract. Infect. Immun. 65, 2606-2612.
22. Kenny,B. and Finlay,B.B. (1995). Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells. Proc. Natl. Acad. Sci. U. S. A 92, 7991-7995.
23. Kenny,B. and Jepson,M. (2000). Targeting of an enteropathogenic Escherichia coli (EPEC) effector protein to host mitochondria. Cell Microbiol. 2, 579-590.
24. Kenny,B., Lai,L.C., Finlay,B.B., and Donnenberg,M.S. (1996). EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells. Mol. Microbiol. 20, 313-323.
25. Klein,J.R., Fahlen,T.F., and Jones,B.D. (2000). Transcriptional organization and function of invasion genes within Salmonella enterica serovar Typhimurium pathogenicity island 1, including the prgH, prgI, prgJ, prgK, orgA, orgB, and orgC genes. Infect. Immun. 68, 3368-3376.
26. Ku,C.P., Lio,J.C., Wang,S.H., Lin,C.N., and Syu,W.J. (2009). Identification of a third EspA-binding protein that forms part of the type III secretion system of enterohemorrhagic Escherichia coli. J. Biol. Chem. 284, 1686-1693.
27. Lai,L.C., Wainwright,L.A., Stone,K.D., and Donnenberg,M.S. (1997). A third secreted protein that is encoded by the enteropathogenic Escherichia coli pathogenicity island is required for transduction of signals and for attaching and effacing activities in host cells. Infect. Immun. 65, 2211-2217.
28. Lio,J.C. and Syu,W.J. (2004). Identification of a negative regulator for the pathogenicity island of enterohemorrhagic Escherichia coli O157:H7. J. Biomed. Sci. 11, 855-863.
29. MacBeath,G. (2002). Protein microarrays and proteomics. Nat. Genet. 32 Suppl, 526-532.
30. McDaniel,T.K., Jarvis,K.G., Donnenberg,M.S., and Kaper,J.B. (1995). A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc. Natl. Acad. Sci. U. S. A 92, 1664-1668.
31. Miyawaki,A. and Tsien,R.Y. (2000). Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472-500.
32. Moon,H.W., Whipp,S.C., Argenzio,R.A., Levine,M.M., and Giannella,R.A. (1983). Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect. Immun. 41, 1340-1351.
33. Neves,B.C., Mundy,R., Petrovska,L., Dougan,G., Knutton,S., and Frankel,G. (2003). CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD. Infect. Immun. 71, 2130-2141.
34. O'Brien,A.D., Lively,T.A., Chang,T.W., and Gorbach,S.L. (1983). Purification of Shigella dysenteriae 1 (Shiga)-like toxin from Escherichia coli O157:H7 strain associated with haemorrhagic colitis. Lancet 2, 573.
35. Pollok,B.A. and Heim,R. (1999). Using GFP in FRET-based applications. Trends Cell Biol. 9, 57-60.
36. Prasher,D.C., Eckenrode,V.K., Ward,W.W., Prendergast,F.G., and Cormier,M.J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229-233.
37. Puig,O., Caspary,F., Rigaut,G., Rutz,B., Bouveret,E., Bragado-Nilsson,E., Wilm,M., and Seraphin,B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218-229.
38. Rabinowitz,R.P., Lai,L.C., Jarvis,K., McDaniel,T.K., Kaper,J.B., Stone,K.D., and Donnenberg,M.S. (1996). Attaching and effacing of host cells by enteropathogenic Escherichia coli in the absence of detectable tyrosine kinase mediated signal transduction. Microb. Pathog. 21, 157-171.
39. Riordan,K.E. and Schneewind,O. (2008). YscU cleavage and the assembly of Yersinia type III secretion machine complexes. Mol. Microbiol. 68, 1485-1501.
40. Rosenshine,I., Ruschkowski,S., Stein,M., Reinscheid,D.J., Mills,S.D., and Finlay,B.B. (1996). A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation. EMBO J. 15, 2613-2624.
41. Schmidt,H., Karch,H., and Beutin,L. (1994). The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coli alpha-hemolysin family. FEMS Microbiol. Lett. 117, 189-196.
42. Sperandio,V., Mellies,J.L., Delahay,R.M., Frankel,G., Crawford,J.A., Nguyen,W., and Kaper,J.B. (2000). Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler. Mol. Microbiol. 38, 781-793.
43. Su,M.S., Kao,H.C., Lin,C.N., and Syu,W.J. (2008). Gene l0017 encodes a second chaperone for EspA of enterohaemorrhagic Escherichia coli O157 : H7. Microbiology 154, 1094-1103.
44. Sukhan,A.F., Kubori,T.F., and Galan,J.E. (2003). Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ. J. Bacteriol. 2003 Jun;185, 3480-3483.
45. tsenko KA,F.A.U. and Wanner,B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 2000 Jun 6;97, 6640-6645.
46. Wainwright,L.A. and Kaper,J.B. (1998). EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli. Mol. Microbiol. 27, 1247-1260.
47. Wallrabe,H., Elangovan,M., Burchard,A., Periasamy,A., and Barroso,M. (2003). Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes. Biophys. J. 85, 559-571.
48. Wilson,R.K., Shaw,R.K., Daniell,S., Knutton,S., and Frankel,G. (2001). Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli. Cell Microbiol. 3, 753-762.
49. Wood,S.E., Jin,J., and Lloyd,S.A. (2008). YscP and YscU switch the substrate specificity of the Yersinia type III secretion system by regulating export of the inner rod protein YscI. J. Bacteriol. 190, 4252-4262.
50. Zhang,J., Campbell,R.E., Ting,A.Y., and Tsien,R.Y. (2002). Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906-918.
51. Zhu,H., Bilgin,M., Bangham,R., Hall,D., Casamayor,A., Bertone,P., Lan,N., Jansen,R., Bidlingmaier,S., Houfek,T., Mitchell,T., Miller,P., Dean,R.A., Gerstein,M., and Snyder,M. (2001). Global analysis of protein activities using proteome chips. Science 293, 2101-2105.
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