臺灣博碩士論文加值系統

腸出血性大腸桿菌利用第三型分泌系統將作用蛋白與毒性因子送入細胞中，改變細胞骨架形成高臺狀隆起以緊緊地與宿主細胞相連，並造成人類腸內上皮細胞微絨毛之脫落稀疏化，稱之為沾黏/剝落病灶。造成此一病理現象之蛋白主要座落於細菌染色體上之致病島嶼 ；此致島嶼包含 41 個開放性閱讀框架，約可歸為五個主要的操縱子：LEE 1~LEE5 (Tir)。
第三型分泌系統主要由膜內基座與膜外針狀結構所構成，而 EspA 為構成此一膜外針狀結構的主要蛋白。 EspA 被報導具有強烈自我聚集之傾向，因此在構築第三型分泌系統時，需要有侍衛子 : CesAB (L0052), CesA2 (L0017), 和 EscL (L0050) 之協助，確保針狀結構之順利合成。
本文主要探討位於第一個操縱子上，功能尚未明瞭之蛋白 L0053。 利用細胞細部分離法得知 L0053 位於細菌的內膜與細胞質中，另外，L0053 與 EspA之內膜侍衛子 CesA2 具有交互作用之能力，更甚者，EHEC L0053 突變株與 CesA2 突變株一樣可以造成第三型分泌系統生理失能現象：細胞中可見 EspA的量急劇減少，但對EspB無顯著之影響。無疑地 ，L0053 為第三型分泌系統中不可或缺之一員。
為更進一步探討 L0053 於第三型分泌系統中扮演的角色。首先，本文利用細菌雙雜合系統得知 L0053 結合上 CesA2 N’第19~36 胺基酸進行交互作用；但卻無法與 EspA 另一個侍衛子 CesAB 進行交互作用，此線索更加促使本文進一步瞭解 L0053 與 CesA2 交互作用對於第三型分泌系統和其膜外針狀結構主要蛋白 EspA 的重要性。本文證實 L0053 失能的突變株除嚴重影響 EspA 的表現外，當細菌生長至一定濃度時，L0053 失能的突變株相較於野生株， EspA 的蛋白穩定度大幅度降低，此現象與 CesA2 失能的突變株相同。本文亦利用生物資訊比對 L0053 與其同源蛋白相似程度，發現 EHEC L0053 雖與 YscE 與 PscE 具有一定程度之序列相似度；但本文觀察 EHEC L0053 與 EHEC CesA2 並不會互相調控彼此之表現與穩定度，且 L0053 突變株並不會影響 CesA2 之細胞座落 ，因此推測 EHEC L0053 除藉由 CesA2 調控影響 EspA 外，尚有其他路徑調控 EspA 與第三型分泌系統。L0053 的完整功能仍待進一步釐清。

Enterohemorrhagic Escherichia coli (EHEC) utilizes Type III Secretion System (TTSS) to deliver virulent effectors, a result leading to cytoskeleton rearrangement and pedestal structure formation. This pedestal structure holds EHEC tightly to host cells and causes human epithelial cells becoming loosened and sparse, a pathogenesis named attaching and effacing lesion (A/E lesion). The effector proteins are encoded by genes clustered in the locus of enterocyte effacement (LEE) island in bacterial chromosome. The LEE island contains 41 open reading frames (ORFs) that could be divided into five major operons, LEE1 to LEE5.
TTSS is structurally comprised of a membrane basal body and an external needle complex. The needle structure is organized mainly by EspA that possesses a biochemical tendency of self-polymerization. Thus, in the formation of TTSS, EspA needs chaperones or binding proteins for stabilization before the needle structures are assembled. Currently known chaperones are: CesAB (L0052), CesA2 (L0017), and EscL (L0050).
Here, we focus on a less characterized protein, L0053, that also causes poor EspA expression when its cognate gene is deleted. L0053 is the fourth ORF in LEE1. By protein fractionation, L0053 is found in the inner membrane and cytosol fractions. Experimentally demonstrated is that L0053 has the ability to interact with one of EspA chaperones, CesA2. Moreover, EHEC strain with l0053 deleted (L53) shows that EspA dramatically diminishes while another secretion protein, EspB, has no apparent effect. However, a consequence is that Tir, EspB, and EspA all disappear from bacteria supernatant.
BacterioMatchTM Two-Hybrid-System was used to map how L0053 binds to CesA2 and the N-terminal 19~36 amino acids of CesA2 were concluded to be critical. No binding of L0053 has been observed with any other EspA chaperone. Intriguingly, by the same approach, the same N-terminal region of CesA2 was found critical for the binding of EspA. However, L0053 and CesA2 have no ability to modulate the stability and localization of the expressed proteins to each other. And over-expression CesA2 in EHEC L53 strain couldn’t restore the physiological ability of EspA. Thus, we conclude that EHEC L0053 may serve as a co-chaperone for CesA2 so that CesA2 could execute its interaction with EspA at a right timing after EspA is released from ribosome while moving forwards to the assembly during de novo synthesis.

Content

中文摘要.................................................................................................................................i
Abstract..................................................................................................................................ii
Content..................................................................................................................................iii
I. Introduction.....................................................................................................1
I-1. Enterobateriaceae.................................................................................................................1
I-2. Escherichia coli (E. coli).....................................................................................................1
I-3. Pathogenic E. coli................................................................................................................2
I-4. Enterohemorrhagic E.coli; EHEC.......................................................................................2
I-5. The Locus of Enterocyte Effacement Island; LEE Island...................................................3
I-6. Type III Secretion System; TTSS........................................................................................4
I-7. Chaperones of Type III Secretion System...........................................................................5
I-8. EspA and EspA Chaperones................................................................................................5
II. Aims and Rationales......................................................................................7
III. Materials and Methods................................................................................8
III-1. Bacteria Strains.................................................................................................................8
III-2. Bacteria Culture and Growth Conditions..........................................................................8
III-3. Primers for Molecular Cloning.........................................................................................8
III-4. Plasmids used in this study...............................................................................................8
III-5. Plasmid Construction........................................................................................................8
III-5-1. Restriction enzyme digestion.........................................................................................8
III-5-2. DNA ligation..................................................................................................................9
III-6. Bacterial Plasmid Extraction and Purification..................................................................9
III-7. Polymerase Chain Reaction, PCR.....................................................................................9
III-8. Agarose Gel Electrophoresis...........................................................................................10
III-9. Competent Cell preparation............................................................................................10
III-9-1. Competent Cell Used for Heat-shock Transformation................................................10
III-9-2. Competent Cell Used for Electroporation Transformation..........................................10
III-10. Transformation..............................................................................................................11
III-10-1. Heat-shock method....................................................................................................11
III-10-2. Electroporation..........................................................................................................11
III-11. SDS-PAGE....................................................................................................................11
III-12. Western Blotting...........................................................................................................12
III-13. The Antibodies Used in this Study................................................................................12
III-14. Protein Over-expression................................................................................................12
III-15. Nickel-column Protein Purification..............................................................................13
III-16. Bacterial Two-hybrid System........................................................................................13
III-17. β-galactosidase Activity Assay; Miller Assay............................................................14
III-18. Protein Stability Assay..................................................................................................15
III-19. Bioinformatics tools......................................................................................................15
IV. Results..........................................................................................................16
IV-1. Interaction between L0053 and CesA2 seen with BacterioMatchTM Two-Hybrid
System............................................................................................................................16
IV-2. Bio-informatics and secondary structure prediction for L0053......................................16
IV-3. Mapping L0053 and CesA2 interaction region with partial deletion constructs of L0053
under BacterioMatchTM Two-Hybrid System...............................................................17
IV-4. Mapping L0053 and CesA2 interaction region with partial-deleted L0017 (CesA2)
constructs under BacterioMatchTM Two-Hybrid System.............................................17
IV-5. Properties associated with the deletion of l0053 in EHEC.............................................17
IV-5-1. Genetic organization of the l0053-deletion mutant.....................................................17
IV-5-2. Phenotype of l0053-deletion mutant on representative TTSS proteins.......................17
IV-6. L0053 located in Inner Membrane and Cytosol of EHEC..............................................18
IV-7. L0053 modulating EspA via CesA2................................................................................18
IV-7-1. Effect of deleting l0053 on ectopically expressed EspA.............................................18
IV-7-2. Reporter assay of L0053 regulation on EspA..............................................................18
IV-7-3. L0053 influences EspA protein stability......................................................................19
IV-8. Physiologically meaning of CesA2 and L0053 interaction ability.................................20 IV-8-1. L0053 does not influence ectopically expressed CesA2 nor does CesA2 influence
plasmid-encoded L0053.............................................................................................20
IV-8-2. CesA2 protein dynamic in bacteria..............................................................................20
IV-9. Comparison of the EspA level in L0053 versus in L0017..............................................20
V. Discussion......................................................................................................21


VI. Tables...........................................................................................................25
Table 1......................................................................................................................................25
Table 2......................................................................................................................................25
Table 3......................................................................................................................................26
Table 4......................................................................................................................................28
Table 5......................................................................................................................................29
VII. Figures........................................................................................................30
Figure 1....................................................................................................................................30
Figure 2....................................................................................................................................31
Figure 3....................................................................................................................................32
Figure 4....................................................................................................................................33
Figure 5....................................................................................................................................34
Figure 6....................................................................................................................................35
Figure 7....................................................................................................................................36
Figure 8....................................................................................................................................37
Figure 9....................................................................................................................................38
Figure 10..................................................................................................................................39
Figure 11..................................................................................................................................40
Figure 12..................................................................................................................................41
Figure 13..................................................................................................................................42
Figure 14..................................................................................................................................43
Figure 15..................................................................................................................................44
Figure 16..................................................................................................................................45
Figure 17..................................................................................................................................46
Figure 18..................................................................................................................................47
Figure 19..................................................................................................................................48
Figure 20..................................................................................................................................49
VIII. Reference..................................................................................................51
IX. Appendix.....................................................................................................56
Appendix 1...............................................................................................................................56
Appendix 2...............................................................................................................................57
Appendix 3...............................................................................................................................58
Appendix 4...............................................................................................................................59
Appendix 5...............................................................................................................................60
Appendix 6...............................................................................................................................61
Appendix 7...............................................................................................................................62
Appendix 8...............................................................................................................................63
Appendix 9...............................................................................................................................64
Appendix 10.............................................................................................................................65
Appendix 11.............................................................................................................................66

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