臺灣博碩士論文加值系統

大多數的一般的大腸桿菌對人來說是無害的，但是腸出血性大腸桿菌 (EHEC)對人的健康卻有嚴重的危害。被這種出血性的大腸桿菌感染會有許多病徵，例如胃部痙攣、腹瀉、發燒、嘔吐以及溶血性尿毒症，而其中最嚴重的是溶血性尿毒症 (HUS)，有此症狀之後可能會導致腎功能失調，若更嚴重甚至會造成腎衰竭，其死亡率非常高。EHEC的感染在全世界的衛生健康議題上也是一種不可忽視的威脅，尤其是對於開發中國家或較落後的地區：印度、非洲或是中東地區。出血性大腸桿菌與一般的大腸桿菌最大的不同在於：前者有志賀毒素 (shiga-like toxin)，是感染後造成細胞死亡的主因之一。第三型分泌系統 (Type III secretion system)是EHEC中另一個主要的致病機轉，EHEC會利用由LEE 致病基因群 (locus of enterocyte effacement pathogenicity island)上所表現的Tir與Esp這些功能性蛋白，藉由此系統分泌並且能夠造成宿主細胞產生attaching and effacing lesion (A/E lesion)。除了上述兩種感染系統之外，EHEC還具有溶血素 (enterohemolysin)可以造成人類紅血球破裂而導致溶血現象。先前比較了EHEC與非致病的大腸桿菌菌種K-12的全基因體序列差異，挑選了一個只出現在EHEC中，並且被生物資訊軟體推測為脂肪酸合成相關的基因，我們將它命名為 “fasyn”。在這段特殊的基因簇內，包含了許多基因，但只有四個基因z4863, z4864, z4865, 以及 z4866被軟體推測跟已知的脂肪酸合成基因有一定的相似度。我們的實驗室建立了這四種基因以及整個fasyn的基因剔除菌株，並且已經證明了∆fasyn的確會造成第三型分泌系統中的蛋白質表現量降低。在本篇論文中，我們試圖了解這些基因所轉譯的蛋白質功能，藉由細菌雙雜合系統 (bacterial two hybrid system)來檢視可能的蛋白質反應。利用即時逆轉錄聚合酶鏈式反應 (qRT-PCR)來測試許多不同的調控基因，以了解他們的RNA表現量是否與先前的RNA sequencing結果一致。此外，利用最小抑制濃度（MIC）之測試來檢驗fasyn對常用抗生素的效果。最後，我們分析了野生型菌株與fasyn剃除菌株的脂質組成，發現有三種脂質有明顯的差異，然而其脂質的差異是否可以直接連結到fasyn存在與否所導致仍有待釐清。

Most Escherichia coli (E. coli) strains are harmless to humankind, but some strains like enterohaemorrhagic Escherichia coli (EHEC) are hazardous to human health. EHEC can cause diseases with several different symptoms including stomach muscle spasms, diarrhea, fever, vomiting, and hemolytic uremic syndrome (HUS). The most severe presentation is the HUS because it could cause dysfunction and failure of kidney, which have an extremely high opportunity of causing death. Therefore, EHEC infection is one of important public health threats in the world, including developed countries and the stragglier areas, like India, Africa, or middle-east countries. The most different factor between EHEC and commensally seen E. coli is that EHEC secretes Shiga-like toxin, and this is the main factor causing cell death. Type-3 secretion system is another pathogenic determinant of EHEC. EHEC injects effector proteins such as Tir and Esp proteins encoded from the locus of enterocyte effacement (LEE) pathogenicity island into the host cell through this secretion system and causes attaching and effacing (A/E) lesion. Besides these two infection systems, EHEC also secretes enterohemolysin that can cause human erythrocyte burst that leads to hemolysis. Previously, comparing the differences of E. coli genome between EHEC and K-12 strain revealed one of gene clusters unique in EHEC, of which function was annotated to associate with fatty acid synthesis. We name these gene cluster “fasyn.” The fasyn cluster contains several genes, but only four consecutive genes (z4863 to z4866) have certain degrees of identity to the known fatty acid synthesis genes. We have generated knockout strains of individual genes and of the entire fasyn. We have also demonstrated that Δfasyn decreases the secretion of type-3 secreted proteins. In this thesis, we aimed at better understanding the functions of proteins encoded by these genes. By bacterial two-hybrid system, possible protein-protein interactions among these proteins were examined. By using RT-qPCR, we examine numerous regulatory genes to see whether their RNA levels match those from the RNA sequencing results. Moreover, by using minimal inhibitory concentration(MIC) test, we examined the effect of fasyn on commonly used antibiotics. Lastly, we analyzed the lipid composition differences between the wild type strain and the fasyn deletion strain. Three major lipids were readily found distributed differently between the two strains, which are isogenic. However, whether the lipid differences could be linked directly to the fasyn remains to be clarified.

Abstract----i
中文摘要----iii
Content----v
1. Introduction----1
2. Material and method----6
2.1 Bacterial strains and cultures----6
2.2 Plasmid construction----6
2.3 Bacterial two hybrid system----6
2.4 Type III secretion assay----7
2.5 SDS-PAGE electrophoresis----8
2.6 Western blotting assay----9
2.7 Bacterial RNA extraction----10
2.8 cDNA preparation and qRT-PCR----11
2.9 Minimal inhibitory concentration (MIC) test----11
2.10 Cell fractionation and lipid extraction and----12
2.11 LC/MS/MS----13
3. Results----14
3.1 Effect of knockout fasyn cluster on RNA expression----14
3.2 The effect of fasyn deletion on antibiotic minimal inhibitory concentration (MIC)----15
3.3 Over-expressed, non-lee encoded regulators in ∆fasyn----16
3.4 Examination of additional regulators affected in ∆fasyn by RT-qPCR----17
3.5 The ability of protein-protein interaction between genes encoded in fasyn cluster----18
3.6 The comparison of total lipid composition between wild type and ∆fasyn strain----18
4. Discussions----20
5. Table----27
5.1 Table 1----27
5.2 Table 2----28
5.3 Table 3----30
5.4 Table 4----32
5.5 Table 5----33
5.6 Table 6----34
6. Figures----36
6.1 Figure 1----36
6.2 Figure 2----38
6.3 Figure 3----40
6.4 Figure 4----41
6.5 Figure 5----43
7. References----44
8. Supplementary figure----49

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