臺灣博碩士論文加值系統

腸出血性大腸桿菌O157:H7是致病性大腸桿菌其中之一，會造成出血性腹瀉，且可能倂發溶血性尿毒症候群，約有3~5%致死率。其毒力因子包括了第三型分泌系統，藉由此分泌系統將蛋白質送入腸道細胞，使細胞骨架改變產生高台狀構造，造成A/E病灶，讓細菌能夠緊密黏附到細胞表面。構成此第三型分泌系統所需相關蛋白質之基因，主要位於腸出血性大腸桿菌染色體的LEE 致病島嶼上，l0045為其中一個功能尚未清楚了解的開放性閱讀框架。為了研究L0045於第三型分泌系統扮演之角色，我們建構一個l0045基因剔除突變株，並發現剔除l0045會影響第三類分泌系統蛋白質的分泌以及部分蛋白質的表現。此外，我們同時觀察到一旦過量表現L0045於腸出血性大腸桿菌野生株，會嚴重影響第三類分泌系統蛋白質表現以及分泌，我們以過量表現L0045突變蛋白質之試驗，確認此影響是由過量之野生型L0045所造成。由過量表現L0045於腸出血性大腸桿菌野生株及JM109野生株之實驗，顯示腸出血性大腸桿菌野生株存在(JM109缺乏)特有之L0045調控機制，可感測到L0045表現過多而促使過量之L0045降解至無法以西方墨點法偵測的範圍內，且不影響細菌生長。為了進一步尋找可能之調節機制，我們將L0045過量表現於腸出血性大腸桿菌grlA基因剔除突變株，結果顯示可感測L0045表現量之相關蛋白質會被LEE致病島嶼之正向調控因子GrlA所調控。我們也以過量表現L0045突變蛋白質之試驗，確認偵測L0045表現量之蛋白質並非感測L0045酵素之產物而為L0045蛋白質。總括來說，本篇研究提出了一個腸出血性大腸桿菌如何調控L0045的可能機制，並提供了找尋偵測L0045表現量之感測蛋白質的可能方向。

Enterohemorrhagic E. coli (EHEC), which is an etiological agent for food-borne illnesses, is one of the pathogenic E. coli strains. Infection of EHEC often leads to bloody diarrhea and sometimes to serious diseases of hemolytic-uremic syndrome and acute kidney failure. EHEC infects large intestinal cells and forms typical histological lesions called attaching and effacing lesions (A/E lesion). The known mechanism is that the bacteria employ a type III secretion system to deliver several effector proteins into the infected cells. The effector proteins then trigger the formation of pedestal structure that tightens the attachment of the bacteria to the host cells. Most of the genes involved in the formation of A/E lesion reside in the locus of enterocyte effacement (LEE), a pathogen island. The LEE island contains 41 open reading frames. Some of the genes have been well studied as their roles in the type III secretion system are well understood whereas the others are not. l0045 is among the genes that have not been well characterized. To investigate the role of L0045 in the type III secretion system, an l0045-deletion mutant strain was created. The deletion of l0045 results in the attenuation of the type III secretion and decrease of the expression of representative LEE proteins. However, when L0045 expression is driven by a strong promoter, the intracellular levels of Tir and EspA in EHEC were severely suppressed. Furthermore, under this circumstance, the protein level of L0045 in EHEC was hardly detected. And this was in contrast to an observation that a huge amount of L0045 was seen when similarly performed in K-12 of the JM109 strain, a result suggesting that a network must exist in EHEC to tightly regulate the synthesis of L0045. This regulation could link to GrlA, one of the LEE regulator, since deleting grlA from EHEC increased an expression of L0045.

目次 i
表次 iv
圖次 v
Abstract 1
中文摘要 2
第一章 緒論 3
1. 大腸桿菌概論 3
2. 腸出血性大腸桿菌 (Enterohemorrhagic E. coli；EHEC) 4
3. 腸出血性大腸桿菌的毒力因子 4
3.1 類志賀氏毒素 (Shiga-like toxin；SLT) 4
3.2 溶血素 (hemolysin) 5
3.3 LEE致病島嶼 (locus of enterocyte effacement, LEE island) 5
4. 第三型分泌系統 (type III secretion system, TTSS) 6
5. LEE致病島嶼的架構 7
6. 本文研究動機及目的 10
第二章 材料及方法 11
1. 細菌菌株 (bacterial strain) 11
2. 質體與引子 (plasmids and primers) 11
3. 抗體 (antibodies) 11
4. 細菌的培養 (bacterial culture) 11
5. 細菌之質體萃取與純化 (plasmid extraction) 11
6. 瓊脂凝膠製作及電泳 (agarose gel electrophoresis) 12
7. 聚合酶連鎖反應 (polymerase chain reaction; PCR) 12
8. 限制酶之切割 (restriction enzyme digestion) 12
9. DNA接合反應 (DNA ligation) 13
10. 勝任細胞之製備 (competent cells preparation) 13
10.1 一般實驗菌株勝任細胞之製備 13
10.2 出血性大腸桿菌勝任細胞之製備 13
11. 細胞轉型 (transformation) 14
11.1熱刺激法 (heat-shock) 14
11.2電穿孔法 (electroporation) 14
12. 基因剔除試驗 (gene knockout assay) 15
13. 蛋白質之過量表現 (protein over-expression) 16
14. SDS聚丙烯醯胺凝膠電泳 (SDS-PAGE) 16
15. 西方墨點法 (Western blot) 17
16. 出血性大腸桿菌蛋白質之分層試驗 (fractionation) 18
16.1 膜區間質蛋白質之純化 (periplasm) 18
16.2 細胞質蛋白質之純化 (cytoplasm) 18
16.3 內膜蛋白質之純化 (inner membrane) 19
16.4 外膜蛋白質之純化 (outer membrane) 19
17. 出血性大腸桿菌之蛋白質合成測試 (bacterial lysate) 19
18. 出血性大腸桿菌分泌性蛋白質抽取 (secreted-protein) 20
19. 細菌生長曲線之測定 (bacterial growth curve) 20
20. 生物資訊學工具 (bio-informatics tools) 20
第三章 結果 22
1. L0045的生物資訊分析 22
2. L0045之特性 22
3. L0045的分布 23
4. L0045突變株的分析 23
5. 過量表現L0045對LEE致病島嶼蛋白質表現及分泌之影響 24
6. L0045過量表現於JM109及腸出血性大腸桿菌野生株 25
7. L0045過量表現於grlA剔除之腸出血性大腸桿菌突變株 26
8. 過量表現缺少訊息序列之突變L0045於腸出血性大腸桿菌野生株 27
9. 過量表現失去酵素活性之突變L0045於腸出血性大腸桿菌野生株 28
10. L0045突變型之蛋白質分層試驗 28
第四章 討論 30
1. L0045及lytic transglycosylase 30
2. L0045對於第三型分泌系統之影響 31
3. 過量表現L0045對LEE致病島嶼之影響 32
4. L0045之調控機制 32
5. 由突變之L0045探討其分布 35
6. 未來展望 35
第五章 參考文獻 37
圖表 43

1. Bentley, R. and R. Meganathan, Biosynthesis of vitamin K (menaquinone) in bacteria. Microbiology and Molecular Biology Reviews, 1982. 46(3): p. 241-280.
2. Reid, G., J. Howard, and B.S. Gan, Can bacterial interference prevent infection? Trends in Microbiology, 2001. 9(9): p. 424-428.
3. Boyce, T.G., D.L. Swerdlow, and P.M. Griffin, Escherichia coli O157: H7 and the hemolytic-uremic syndrome. New England Journal of Medicine, 1995. 333(6): p. 364-368.
4. Moon, H.W., et al., Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infection and Immunity, 1983. 41(3): p. 1340-1351.
5. Roe, A., D. Hoey, and D. Gally, Regulation, secretion and activity of type III-secreted proteins of enterohaemorrhagic Escherichia coli O157. Biochemical Society Transactions, 2003. 31: p. 98-103.
6. O'Brien, A.D. and R.K. Holmes, Shiga and Shiga-like toxins. Microbiology and Molecular Biology Reviews, 1987. 51(2): p. 206-220.
7. O'Brien, A.D. and G.D. LaVeck, Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli. Infection and Immunity, 1983. 40(2): p. 675-683.
8. Strockbine, N.A., et al., Two toxin-converting phages from Escherichia coli O157: H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infection and Immunity, 1986. 53(1): p. 135-140.
9. Tesh, V.L. and A.D. O'Brien, The pathogenic mechanisms of Shiga toxin and the Shiga-like toxins. Molecular Microbiology, 1991. 5(8): p. 1817-1822.
10. Schmidt, H., L. Beutin, and H. Karch, Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157: H7 strain EDL 933. Infection and Immunity, 1995. 63(3): p. 1055-1061.
11. Schmidt, H., H. Karch, and L. Beutin, The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coliα-hemolysin family. FEMS Microbiology Letters, 1994. 117(2): p. 189-196.
12. Donnenberg, M.S., J.B. Kaper, and B.B. Finlay, Interactions between enteropathogenic Escherichia coli and host epithelial cells. Trends in Microbiology, 1997. 5(3): p. 109-114.
13. Frankel, G., et al., Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Molecular Microbiology, 1998. 30(5): p. 911-921.
14. Kenny, B., et al., Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell, 1997. 91(4): p. 511-520.
15. Sansonetti, P.J., et al., Enterohemorrhagic Escherichia coli O157: H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated. Infection and Immunity, 1999. 67(5): p. 2389-2398.
16. Gophna, U., E.Z. Ron, and D. Graur, Bacterial type III secretion systems are ancient and evolved by multiple horizontal-transfer events. Gene, 2003. 312: p. 151-163.
17. Donnenberg, M.S., L.C. Lai, and K.A. Taylor, The locus of enterocyte effacement pathogenicity island of enteropathogenic Escherichia coli encodes secretion functions and remnants of transposons at its extreme right end. Gene, 1997. 184(1): p. 107-114.
18. Elliott, S.J., et al., The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Molecular Microbiology, 1998. 28(1): p. 1-4.
19. Hueck, C.J., Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiology and Molecular Biology Reviews, 1998. 62(2): p. 379-433.
20. Cornelis, G.R., The type III secretion injectisome. Nature Reviews Microbiology, 2006. 4(11): p. 811-825.
21. Moraes, T.F., T. Spreter, and N.C.J. Strynadka, Piecing together the Type III injectisome of bacterial pathogens. Current Opinion in Structural Biology, 2008. 18(2): p. 258-266.
22. Garmendia, J., G. Frankel, and V.F. Crepin, Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. 2005, Am Soc Microbiol. p. 2573-2585.
23. Galan, J.E. and H. Wolf-Watz, Protein delivery into eukaryotic cells by type III secretion machines. Nature, 2006. 444: p. 567-573.
24. Creasey, E.A., et al., Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli. 2003, Soc General Microbiol. p. 2093-2106.
25. Koster, M., et al., The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocolitica forms a ring-shaped multimeric complex. Molecular Microbiology, 1997. 26(04): p. 789-797.
26. Wilson, R.K., et al., Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli. Cellular Microbiology, 2001. 3(11): p. 753-762.
27. Ide, T., et al., Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli. Cellular Microbiology, 2001. 3(10): p. 669-679.
28. Iizumi, Y., et al., The enteropathogenic E. coli effector EspB facilitates microvillus effacing and antiphagocytosis by inhibiting myosin function. Cell Host & Microbe, 2007. 2(6): p. 383-392.
29. Su, M.S.W., et al., Gene l0017 encodes a second chaperone for EspA of enterohaemorrhagic Escherichia coli O157: H7. Microbiology, 2008. 154(4): p. 1094.
30. Gauthier, A. and B.B. Finlay, Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli. Journal of Bacteriology, 2003. 185(23): p. 6747-6755.
31. Mellies, J.L., et al., The Per regulon of enteropathogenic Escherichia coli: identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler). Molecular Microbiology, 1999. 33(2): p. 296-306.
32. Francis, M.S., H. Wolf-Watz, and A. Forsberg, Regulation of type III secretion systems. Current Opinion in Microbiology, 2002. 5(2): p. 166-172.
33. DiRita, V.J., et al., 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. Infection and Immunity, 2000. 68(11): p. 6115-6126.
34. Russell, R.M., et al., QseA and GrlR/GrlA Regulation of the Locus of Enterocyte Effacement Genes in Enterohemorrhagic Escherichia coli? Journal of Bacteriology, 2007. 189(14): p. 5387-5392.
35. Lio, J.C.W. and W.J. Syu, Identification of a negative regulator for the pathogenicity island of enterohemorrhagicEscherichia coli O157: H7. Journal of Biomedical Science, 2004. 11(6): p. 855-863.
36. Deng, W., et al., Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proceedings of the National Academy of Sciences, 2004. 101(10): p. 3597-3602.
37. Huang, L. and W.J. Syu, GrlA of enterohemorrhagic Escherichia coli O 157: H 7 activates LEE 1 by binding to the promoter region. Journal of Microbiology, Immunology and Infection, 2008. 41(1): p. 9-16.
38. Barba, J., et al., A positive regulatory loop controls expression of the locus of enterocyte effacement-encoded regulators Ler and GrlA. Journal of Bacteriology, 2005. 187(23): p. 7918-7930.
39. Datsenko, K.A. and B.L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. 2000, National Acad Sciences. p. 6640-6645.
40. Combet, C., et al., Geno3D: automatic comparative molecular modelling of protein. 2002, Oxford Univ Press. p. 213-214.
41. Pallen, M.J., S.A. Beatson, and C.M. Bailey, Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion. BMC Microbiology, 2005. 5(1): p. 9.
42. Vollmer, W. and U. Bertsche, Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. BBA-Biomembranes, 2008. 1778(9): p. 1714-1734.
43. Holtje, J.V., Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiology and Molecular Biology Reviews, 1998. 62(1): p. 181-203.
44. Suvorov, M., et al., Lytic Transglycosylase MltB of Escherichia coli and Its Role in Recycling of Peptidoglycan Strands of Bacterial Cell Wall. J. Am. Chem. Soc, 2008. 130(36): p. 11878-11879.
45. Park, J.T. and T. Uehara, How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)? Microbiology and Molecular Biology Reviews, 2008. 72(2): p. 211-227.
46. Scheurwater, E., C.W. Reid, and A.J. Clarke, Lytic transglycosylases: bacterial space-making autolysins. International Journal of Biochemistry and Cell Biology, 2008. 40(4): p. 586-591.
47. Vollmer, W., et al., Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiology Reviews, 2008. 32(2): p. 259-286.
48. Koraimann, G., Lytic transglycosylases in macromolecular transport systems of Gram-negative bacteria. Cellular and Molecular Life Sciences (CMLS), 2003. 60(11): p. 2371-2388.
49. Zahrl, D., et al., Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems. 2005, Soc General Microbiol. p. 3455-3467.
50. Allaoui, A., et al., Characterization of the Shigella flexneri ipgD and ipgF genes, which are located in the proximal part of the mxi locus. Infection and Immunity, 1993. 61(5): p. 1707-1714.
51. Galan, J.E. and A. Collmer, Type III secretion machines: bacterial devices for protein delivery into host cells. Science, 1999. 284(5418): p. 1322.
52. Oh, H.S., et al., Pseudomonas syringae Lytic Transglycosylases Coregulated with the Type III Secretion System Contribute to the Translocation of Effector Proteins into Plant Cells? Journal of Bacteriology, 2007. 189(22): p. 8277-8289.
53. Cherepanov, P.P. and W. Wackernagel, Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene, 1995. 158(1): p. 9-14.