臺灣博碩士論文加值系統

腸出血性大腸桿菌O157: H7簡稱EHEC，感染人體後會貼附在腸道上皮細胞表面，造成微絨毛消失並使細胞形成高臺狀結構，引發出血性腹瀉。EHEC主要的毒力因子皆位於染色體上的致病島嶼LEE，其轉譯的蛋白質能構成一特化的第三型分泌系統。第三型分泌系統包括位在細菌內、外膜上的基體以及連接宿主細胞膜的纖維狀構造，經由此構造細菌即可將作用蛋白分泌至宿主細胞質中，造成宿主細胞型態改變，而此纖維狀的中空管道即是由EspA所聚合形成。然而，目前尚無明確機制解釋EspA是如何通過基體並且被分泌出細菌外。在先前的研究中，我們證實位在細菌內膜的L0050和CesA2能夠分別與EspA產生交互作用，並維持EspA在細菌中的穩定性。本論文以細菌雙雜合試驗及蛋白質共同純化證實L0050與CesA2之間亦存在交互作用，且進一步發現EspA、L0050與CesA2三者能形成複合體。另外，利用細菌雙雜合系統進行L0050的蛋白質片段分析，結果顯示N端缺失的L0050失去其與EspA結合的能力，但卻能與第三型分泌系統的ATP水解酶EscN產生交互作用。因此，我們推測L0050與CesA2能夠共同將EspA帶到細菌內膜的位置，且L0050藉由與EspA的結合而能和EscN產生交互作用。此時，EscN水解ATP產生能量將EspA-L0050-CesA2複合體分開，使EspA能夠順利被分泌至細菌外。由此可知，EspA到達細菌外部之前需要受到嚴密的保護及調控，且需要許多LEE蛋白質參與其中。

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic diarrhea. The major virulent factors reside in the locus of enterocyte effacement (LEE) in the bacterial genome. The LEE island encodes a set of genes for forming a specialized machinery called the type III secretion system (TTSS). TTSS secretes effectors, which collectively cause morphological changes of the infected cell, via a long filamentous structure that connects bacteria to the host cell membrane. This filament is composed of polymerized EspA and forms a hollow channel through which secreted protein may pass. A cytoplasmic protein, CesAB, has been identified as the first chaperone of EspA, a protein that posses an aggregation tendency. However, the translocation and assembly mechanism of EspA from bacterial cytoplasm to extracellular space could be more complicated than previously thought. In previous studies, we documented that L0050 and CesA2, proteins located in the inner-membrane fraction, could interact with EspA, respectively. Here, we further demonstrated that L0050 interacts with CesA2 by co-purification. Co-purification also revealed that EspA, L0050, and CesA2 could form a complex. Furthermore, mapping of L0050 by a bacterial two-hybrid system has shown that the N-terminal region of L0050 is needed for interaction with EspA, and the N-terminal region-deleted L0050 remains active to interact with TTS ATPase (EscN) that is thought to energize the secretion process. We therefore proposed a model that L0050 and CesA2 cooperate to escort EspA to the inner membrane of EHEC. Through the binding of EspA, L0050 interacts with EscN, which provides energy for efficient translocation of EspA.

Abstract i
中文摘要 ii
目錄 iii
第一章 緒論 1
1.大腸桿菌概論 1
2.腸出血性大腸桿菌概論 (Enterohemorrhagic E. coli, EHEC) 2
3.出血性大腸桿菌之致病機制 2
3.1 類志賀氏毒素 (Shiga-like toxin) 2
3.2 溶血素 (Hemolysin) 3
3.3 A/E病灶 (A/E lesion) 3
4.LEE致病島嶼 (the locus of enterocyte effacement island; LEE island) 4
5.第三型分泌系統 (type III secretion system; TTSS) 5
6.第三型分泌系統的侍衛蛋白 (chaperones of type III secretion system) 6
7.EspA與其侍衛蛋白 (EspA and EspA chaperones) 7
8.L0050先前研究 8
9.EscN先前研究 8
10.本文動機及方向 9
第二章 材料與方法 10
1.細菌菌株 (Bacterial strain) 10
2.質體與抗體 (Plasmids and primers) 10
3.抗體 (Antibodies) 10
4.細菌的培養 (Bacterial culture) 10
5.細菌質體萃取與純化 (Bacterial plasmid extraction and purification) 10
6.聚合酶連鎖反應 (Polymerase chain reaction, PCR) 11
7.瓊脂凝膠製作及電泳 (Agarose gel electrophoresis) 11
8.質體構築 (Plasmid construction) 12
8.1 限制酶切割 (Restriction enzyme digestion) 12
8.2 DNA接合反應 (DNA ligation) 12
9.勝任細胞製備 (Competent cell preparation) 12
10.細菌轉型 (Transformation) 13
11.蛋白質之過量表現 (Protein over-expression) 13
12.SDS聚丙烯醯氯凝膠電泳 (SDS-PAGE) 13
13.西方墨點法 (Western blotting) 14
14.鎳離子管柱共純化 (Nickel-column co-purification) 15
15.細菌雙雜合系統 (Bacterial two-hybrid system) 16
16.β-半乳糖苷酶活性測試 (β-galactosidase activity assay; Miller assay) 16
17.生物資訊學工具 (Bioinformatics tools) 17
第三章 結果 18
1.利用細菌雙雜合系統分析L0050與CesA2之交互作用 18
2.EspA、L0050及CesA2三者形成複合體 19
2.1 以帶有Hisx6的CesA2進行鎳離子親和性管柱純化 19
2.2 以帶有Hisx6的L0050進行鎳離子親和性管柱純化 20
3.L0050生物資訊分析 21
4.構築l0050缺失突變片段之表現載體 21
5.利用細菌雙雜合系統定義L0050與EspA結合之區域 21
6.利用細菌雙雜合系統分析L0050與EscN之交互作用 22
7.利用細菌雙雜合系統定義L0050與EscN結合之區域 23
8.L0050的氨基端及羧基端分別與EspA及EscN結合 23
第四章 討論 25
參考文獻 29
附錄 35

1. Nataro JP, Kaper JB (1998) Diarrheagenic Escherichia coli. Clin Microbiol Rev 11: 142-201.
2. Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, et al. (1983) Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 308: 681-685.
3. Riley LW, Junio LN, Libaek LB, Schoolnik GK (1987) Plasmid-encoded expression of lipopolysaccharide O-antigenic polysaccharide in enteropathogenic Escherichia coli. Infect Immun 55: 2052-2056.
4. Wells JG, Davis BR, Wachsmuth IK, Riley LW, Remis RS, et al. (1983) Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J Clin Microbiol 18: 512-520.
5. Orskov F, Orskov I, Villar JA (1987) Cattle as reservoir of verotoxin-producing Escherichia coli O157:H7. Lancet 2: 276.
6. Schmidt H, Karch H, 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.
7. Schmidt H, Beutin L, Karch H (1995) Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect Immun 63: 1055-1061.
8. Moon HW, Whipp SC, Argenzio RA, Levine MM, Giannella RA (1983) Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun 41: 1340-1351.
9. Knutton S, Baldwin T, Williams PH, McNeish AS (1989) Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun 57: 1290-1298.
10. Rosenshine I, Ruschkowski S, Stein M, Reinscheid DJ, Mills SD, et al. (1996) A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation. EMBO J 15: 2613-2624.
11. Donnenberg MS, Kaper JB, Finlay BB (1997) Interactions between enteropathogenic Escherichia coli and host epithelial cells. Trends Microbiol 5: 109-114.
12. DeVinney R, Gauthier A, Abe A, Finlay BB (1999) Enteropathogenic Escherichia coli: a pathogen that inserts its own receptor into host cells. Cell Mol Life Sci 55: 961-976.
13. Campellone KG, Robbins D, Leong JM (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.
14. de Grado M, Rosenberger CM, Gauthier A, Vallance BA, Finlay BB (2001) Enteropathogenic Escherichia coli infection induces expression of the early growth response factor by activating mitogen-activated protein kinase cascades in epithelial cells. Infect Immun 69: 6217-6224.
15. Chiu HJ, Lin WS, Syu WJ (2003) Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7. Arch Microbiol 180: 218-226.
16. Clarke SC, Haigh RD, Freestone PP, Williams PH (2003) Virulence of enteropathogenic Escherichia coli, a global pathogen. Clin Microbiol Rev 16: 365-378.
17. Frankel G, Phillips AD, Rosenshine I, Dougan G, Kaper JB, et al. (1998) Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol Microbiol 30: 911-921.
18. Perna NT, Mayhew GF, Posfai G, Elliott S, Donnenberg MS, et al. (1998) Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect Immun 66: 3810-3817.
19. Elliott SJ, Wainwright LA, McDaniel TK, Jarvis KG, Deng YK, et al. (1998) The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 28: 1-4.
20. Hacker J, Kaper JB (2000) Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol 54: 641-679.
21. Cornelis GR (2002) Yersinia type III secretion: send in the effectors. J Cell Biol 158: 401-408.
22. Galan JE (2001) Salmonella interactions with host cells: type III secretion at work. Annu Rev Cell Dev Biol 17: 53-86.
23. Nguyen L, Paulsen IT, Tchieu J, Hueck CJ, Saier MH, Jr. (2000) Phylogenetic analyses of the constituents of Type III protein secretion systems. J Mol Microbiol Biotechnol 2: 125-144.
24. Gophna U, Ron EZ, Graur D (2003) Bacterial type III secretion systems are ancient and evolved by multiple horizontal-transfer events. Gene 312: 151-163.
25. Cornelis GR (2006) The type III secretion injectisome. Nat Rev Microbiol 4: 811-825.
26. Marlovits TC, Kubori T, Lara-Tejero M, Thomas D, Unger VM, et al. (2006) Assembly of the inner rod determines needle length in the type III secretion injectisome. Nature 441: 637-640.
27. Wilson RK, Shaw RK, Daniell S, Knutton S, 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.
28. Ide T, Laarmann S, Greune L, Schillers H, Oberleithner H, et al. (2001) Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli. Cell Microbiol 3: 669-679.
29. Arnold R, Jehl A, Rattei T (2010) Targeting effectors: the molecular recognition of Type III secreted proteins. Microbes Infect 12: 346-358.
30. Sory MP, Cornelis GR (1994) Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol 14: 583-594.
31. Anderson DM, Schneewind O (1997) A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 278: 1140-1143.
32. Cheng LW, Anderson DM, Schneewind O (1997) Two independent type III secretion mechanisms for YopE in Yersinia enterocolitica. Mol Microbiol 24: 757-765.
33. Arnold R, Brandmaier S, Kleine F, Tischler P, Heinz E, et al. (2009) Sequence-based prediction of type III secreted proteins. PLoS Pathog 5: e1000376.
34. Lower M, Schneider G (2009) Prediction of type III secretion signals in genomes of gram-negative bacteria. PLoS One 4: e5917.
35. Samudrala R, Heffron F, McDermott JE (2009) Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems. PLoS Pathog 5: e1000375.
36. Akeda Y, Galan JE (2005) Chaperone release and unfolding of substrates in type III secretion. Nature 437: 911-915.
37. Page AL, Parsot C (2002) Chaperones of the type III secretion pathway: jacks of all trades. Mol Microbiol 46: 1-11.
38. Gauthier A, Finlay BB (2003) Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli. J Bacteriol 185: 6747-6755.
39. Neves BC, Mundy R, Petrovska L, Dougan G, Knutton S, et al. (2003) CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD. Infect Immun 71: 2130-2141.
40. Wilharm G, Dittmann S, Schmid A, Heesemann J (2007) On the role of specific chaperones, the specific ATPase, and the proton motive force in type III secretion. Int J Med Microbiol 297: 27-36.
41. Yip CK, Finlay BB, Strynadka NC (2005) Structural characterization of a type III secretion system filament protein in complex with its chaperone. Nat Struct Mol Biol 12: 75-81.
42. Phan J, Austin BP, Waugh DS (2005) Crystal structure of the Yersinia type III secretion protein YscE. Protein Sci 14: 2759-2763.
43. Wainwright LA, Kaper JB (1998) EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli. Mol Microbiol 27: 1247-1260.
44. Elliott SJ, O'Connell CB, Koutsouris A, Brinkley C, Donnenberg MS, et al. (2002) A gene from the locus of enterocyte effacement that is required for enteropathogenic Escherichia coli to increase tight-junction permeability encodes a chaperone for EspF. Infect Immun 70: 2271-2277.
45. Abe A, de Grado M, Pfuetzner RA, Sanchez-Sanmartin C, Devinney R, et al. (1999) Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion. Mol Microbiol 33: 1162-1175.
46. Elliott SJ, Hutcheson SW, Dubois MS, Mellies JL, Wainwright LA, et al. (1999) Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol Microbiol 33: 1176-1189.
47. Creasey EA, Delahay RM, Bishop AA, Shaw RK, Kenny B, et al. (2003) CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map. Mol Microbiol 47: 209-221.
48. Creasey EA, Friedberg D, Shaw RK, Umanski T, Knutton S, et al. (2003) CesAB is an enteropathogenic Escherichia coli chaperone for the type-III translocator proteins EspA and EspB. Microbiology 149: 3639-3647.
49. Su MS, Kao HC, Lin CN, Syu WJ (2008) Gene l0017 encodes a second chaperone for EspA of enterohaemorrhagic Escherichia coli O157 : H7. Microbiology 154: 1094-1103.
50. Daniell SJ, Kocsis E, Morris E, Knutton S, Booy FP, et al. (2003) 3D structure of EspA filaments from enteropathogenic Escherichia coli. Mol Microbiol 49: 301-308.
51. Delahay RM, Knutton S, Shaw RK, Hartland EL, Pallen MJ, et al. (1999) The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli. J Biol Chem 274: 35969-35974.
52. Deng W, Puente JL, Gruenheid S, Li Y, Vallance BA, et al. (2004) Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci U S A 101: 3597-3602.
53. Ku CP, Lio JC, Wang SH, Lin CN, Syu WJ (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.
54. Pallen MJ, Beatson SA, Bailey CM (2005) Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion. BMC Microbiol 5: 9.
55. Jackson MW, Plano GV (2000) Interactions between type III secretion apparatus components from Yersinia pestis detected using the yeast two-hybrid system. FEMS Microbiol Lett 186: 85-90.
56. Hueck CJ (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62: 379-433.
57. Dreyfus G, Williams AW, Kawagishi I, Macnab RM (1993) Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens. J Bacteriol 175: 3131-3138.
58. Eichelberg K, Ginocchio CC, Galan JE (1994) Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J Bacteriol 176: 4501-4510.
59. Fan F, Macnab RM (1996) Enzymatic characterization of FliI. An ATPase involved in flagellar assembly in Salmonella typhimurium. J Biol Chem 271: 31981-31988.
60. Woestyn S, Allaoui A, Wattiau P, Cornelis GR (1994) YscN, the putative energizer of the Yersinia Yop secretion machinery. J Bacteriol 176: 1561-1569.
61. Auvray F, Ozin AJ, Claret L, Hughes C (2002) Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH. J Mol Biol 318: 941-950.
62. Pozidis C, Chalkiadaki A, Gomez-Serrano A, Stahlberg H, Brown I, et al. (2003) Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization. J Biol Chem 278: 25816-25824.
63. Akeda Y, Galan JE (2004) Genetic analysis of the Salmonella enterica type III secretion-associated ATPase InvC defines discrete functional domains. J Bacteriol 186: 2402-2412.
64. Jouihri N, Sory MP, Page AL, Gounon P, Parsot C, et al. (2003) MxiK and MxiN interact with the Spa47 ATPase and are required for transit of the needle components MxiH and MxiI, but not of Ipa proteins, through the type III secretion apparatus of Shigella flexneri. Mol Microbiol 49: 755-767.
65. Minamino T, Gonzalez-Pedrajo B, Kihara M, Namba K, Macnab RM (2003) The ATPase FliI can interact with the type III flagellar protein export apparatus in the absence of its regulator, FliH. J Bacteriol 185: 3983-3988.
66. Minamino T, MacNab RM (2000) Interactions among components of the Salmonella flagellar export apparatus and its substrates. Mol Microbiol 35: 1052-1064.
67. Silva-Herzog E, Dreyfus G (1999) Interaction of FliI, a component of the flagellar export apparatus, with flagellin and hook protein. Biochim Biophys Acta 1431: 374-383.
68. Zarivach R, Vuckovic M, Deng W, Finlay BB, Strynadka NC (2007) Structural analysis of a prototypical ATPase from the type III secretion system. Nat Struct Mol Biol 14: 131-137.
69. Blaylock B, Riordan KE, Missiakas DM, Schneewind O (2006) Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator, YscL. J Bacteriol 188: 3525-3534.
70. Andrade A, Pardo JP, Espinosa N, Perez-Hernandez G, Gonzalez-Pedrajo B (2007) Enzymatic characterization of the enteropathogenic Escherichia coli type III secretion ATPase EscN. Arch Biochem Biophys 468: 121-127.
71. Quinaud M, Chabert J, Faudry E, Neumann E, Lemaire D, et al. (2005) The PscE-PscF-PscG complex controls type III secretion needle biogenesis in Pseudomonas aeruginosa. J Biol Chem 280: 36293-36300.
72. Sun P, Tropea JE, Austin BP, Cherry S, Waugh DS (2008) Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG. J Mol Biol 377: 819-830.