臺灣博碩士論文加值系統

出血性大腸桿菌 (EHEC) 屬於病原性大腸桿菌之一，其感染人類腸內上皮細胞後會破壞腸內微絨毛而產生所謂的 A/E lesion 病灶。一般來說，引發 A/E lesion 病灶的特性已證明和細菌染色體上致病島嶼 LEE有關聯性，該島嶼內共包含有 41 個開放閱讀框架 (open reading frame)，而約可歸為五個主要的操縱子 (operon)。EHEC 致病過程是藉由第三型分泌系統 (TTSS) 將分泌性蛋白質送至宿主細胞，在宿主細胞內引發訊息傳導，造成宿主肌動蛋白聚集與微絨毛消失。 l0032 為 LEE3 operon 中功能未知的開放性閱讀框架。我們以同源性基因重組 (homologous recombination) 刪除 l0032 得到的突變株，發現其體內第三型分泌系統蛋白質的合成並未受到影響。另外，我們也發現轉位器 (translocator) 的分泌量有下降的趨勢，但作用蛋白 (effector) 卻能正常地被分泌。由此可以推論 L0032 在第三型分泌系統中，可能扮演著調控轉位器的分泌的角色。另一方面，我們利用細部分離法 (fractionation) 證實了 L0032 主要位於細菌的內膜部份。l0033 為本論文中所研究的另一個功能未知的開放性閱讀框架。本實驗室先前的研究發現， l0033 突變株的分泌性蛋白質 EspA 的合成量會下降，Tir、EspA、EspD、EspB 的分泌亦受到影響。本論文進一步探討 EspA 受到調控的層次，發現 L0033 可能參與在 EspA 轉錄後調控的過程。我們也觀察到 EspA 在野生株中遠比 l0033 突變株中來得更穩定，推測 L0033 可能扮演著穩定 EspA 的角色。此外，經由鎳離子親和性管柱層析法，我們認為 L0033 和 EspA 之間可能存在著相互作用的關係。到底 L0033 和 EspA 如何作用，仍有待我們進一步的研究。

Enterohemorrhagic Escherichia coli (EHEC) forms typical histological lesions termed attaching and effacing (A/E) lesions on infected large intestine tissue. Genetically, EHEC has a set of virulent genes organized on a locus of enterocyte effacement (LEE) involved in the A/E lesion. The LEE island comprises 41 open reading frames (ORFs), of which most are organized in five operons. l0032 is an uncharacterized ORF of LEE3 operon. When l0032 was deleted from the LEE in EHEC, the synthesis of type III secretion proteins remained as usual. On the other hand, the secretion of the translocators was reduced but Tir could be secreted normally. Thus, L0032 is required for the secretion of translocators. To analyze the localization of L0032 in the bacteria, protein fractionation was carried out and we found that L0032 was mainly located in the inner membrane fraction. We also studied L0033. l0033 had been previously studied and in preliminary results, we found that the synthesis of EspA was significantly reduced, and the type III secretion was completely abolished in a l0033-deletion mutant. We speculated that L0033 may be involved in the post-transcriptional regulation of EspA, particularly focusing on the stabilization of EspA during the secretion process. Our results also suggested that L0033 likely formed complexes with EspA under native condition. Therefore, we assumed that L0033 may act as an auxiliary protein to assist EspA to pass through the channel during the assembly of the type III secretion system.

Content

Abstract (Chinese) ...…………………………………………… 4 Abstract …………………………………………………………….5 1. Introduction ……………………………………………………6 1-1. Escherichia coli (E. coli) ………………………………6 1-2. Diarrheagenic E. coli ...……………………………….7
1-2-1. Enterotoxigenic E. coli (ETEC) ………………………7
1-2-2. Enteroaggregative E. coli (EAEC) ……………………8
1-2-3. Diffusely adherent E. coli (DAEC)…………………...8 1-2-4. Enteroinvasive E. coli (EIEC)………………………8
1-2-5. Enteropathogenic E. coli (EPEC) ……………………………….9 1-2-6. Enterohemorrhagic E. coli (EHEC) …………………………...9 1-3. Enterohemorrhagic E. coli (EHEC) and O157:H7 ……………..10 1-4. Virulent Factors of EHEC ………………………………………..12 1-4-1. Shiga Toxin (Stx) ………………………………..………………12 1-4-2. Enterohemolysin ………………………………………………..13 1-4-3. Adhersin ………………………………………………………...14 1-4-4. Type III Secretion System and Effector Molecules ………..…14 1-5. Genetic Content of EHEC O157:H7……………………………..14 1-6. The Locus of Enterocyte Effacement (LEE) ……………………15 1-7. Type III Secretion System (TTSS) ……………………………….17 1-8. l0032 ...……………………………………………………………..21 1-9. l0033 …….…………………………………………………………21 1-10. Rationales and Aims …….………………………………….……21 2. Materials and Methods ...…………………………………………...22 2-1. Bacterial Strain and Culture conditions ………………………...22 2-2. Oligonucleotide Primers ………………………………………….23 2-3. Antibodies …………………………………………………………23 2-4. Molecular Cloning ………………………………………………..23 2-5. Polymerase Chain Reaction (PCR) ……………………………...23 2-6. Preparation of Competent Cells …………………………………24 2-6-1. Competent Cell used for Heat-shock Transformation ……….24 2-6-2. Competent Cell used for Electroporation Transformation ….24 2-7. Transformation ………………………………………………...…25 2-7-1. Heat-shock Method …………………………………………….25 2-7-2. Electroporation …………………………………………………25 2-8. Plasmid …………………………………………………………….26 2-9. Agarose Gel Electrophoresis ……………………………………..26 2-10. Gene Knockout …………………………………………………..27 2-11. Protein Expression and Purification …………………………...28 2-12. Sodium Dodecyl Sulfate-Polyarylamide Gel Electrophoresis
(SDS-PAGE) …………………………………………….………...29 2-13. Western Blotting ………………………………………………...30 2-14. Polyclonal Antibody Preparation ………………………………31 2-15. Preparation of Secreted protein ………………………………...32 2-16. Preparation of Intracellular Protein …………………………...32 2-17. Fractionation of Bacteria Protein ………………………………33 2-18. Analysis of EspA Stability ………………………………………34 2-19. Affinity Purification with Ni2+ column …………………………35 2-20. Bioinformatics Analysis …………………………………………35 3. Results ………………………………………………………………..37 3-1. L0032 ………………………………………………………………37 3-1-1. Physical Properties of L0032 …………………………………..37 3-1-2. Construction of l0032-deletion Mutant and its Genotype ……37 3-1-3. Specificity of L0032 Polyclonal Antibody ……………………..38 3-1-4. Phenotype of l0032-deletion Mutant …………………………..38 3-1-5. L0032 is Located on the Inner Membrane in EHEC ………...39 3-2. L0033 ………………………………………………………………40 3-2-1. Sequence Analysis of L0033 ……………………………………40 3-2-2. Expression of L0033 in E. coli K12 Strains and in EHEC …...41 3-2-3. Absence of L0033 also Affects the Expression of EspA
From Plasmids …………………………………………………43 3-2-4. Stability of Exogenous EspA in l0033-deletion Mutant ……...43 3-2-5. L0033 can associate with EspA under Native Condition …….44 4. Discussion ……………………………………………………………46 4-1. L0032 is Required for the Secretion of Translocators ………….46 4-2. The Regulation of L0033 …………………………………………47 4-3. L0033 is implicated in the Post-transcriptional Regulation of
EspA ……………………………………………………………….48 4-4. The Relationship between EspA and L0033 …………………….50 5. Reference …………………………………………………………….53 6. Tables ………………………………………………………………...61 7. Figures ……………………………………………………………….65

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