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研究生：	許博淳 Hsu, Po-Chuen
論文名稱：	分析一個大腸桿菌蛋白酵素之功能 Functional study of an Escherichia coli protease
指導教授：	鄧景浩 Teng, Ching-Hao
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	59
中文關鍵詞：	Rep蛋白酵素、受質、ECK1083蛋白質、細胞壁水解酵素
外文關鍵詞：	Rep protease, substrates, ECK1083, peptidoglycan hydrolase
相關次數：	點閱：127 下載：1
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大腸桿菌會造成許多腸道外的感染症,像是泌尿道感染,菌血症,細菌性腦膜炎且具有相當的致死率。由於使用抗生素對抗細菌性感染的手段造成許多具有抗藥性的大腸桿菌有持續性被篩選出現的現象。隨著抗藥性菌株的出現,使得臨床上在抗生素的選擇性受到了限制,進而可能提升病人的死亡率,所以發展出對抗具有抗藥性的大腸桿菌的抗菌手段是急迫被需要的。大腸桿菌蛋白酵素Rep是一個良好的且新穎的抗菌目標蛋白,原因是因為rep基因剔除的大腸桿菌會提升對許多抗生素的感受性,除此之外,我們實驗室先前的研究也發現Rep蛋白酵素的功能也有參與在致病性的大腸桿菌造成高度菌血症的過程中,且無法生長在低滲透壓與高溫的培育環境,如果能夠更加了解Rep這個蛋白酵素在大腸桿菌中扮演的角色與功能,可以有助於我們發展對抗抗藥性大腸桿菌的新穎手法, 在本篇的研究中, 我們利用了E. coli proteome chip assay去找出Rep蛋白酵素的受質, 並想進一步分析Rep蛋白酵素的受質在rep基因剔除的大腸桿菌中扮演的角色。
透過E. coli proteome chip assay發現有100個大腸桿菌的蛋白質可能是Rep蛋白酵素的受質,且其中有13個大腸桿菌的蛋白質在in vitro的情況下會被Rep 蛋白酵素進行切割。而在這13個大腸桿菌蛋白質中,只有ECK1083這個蛋白質似乎是有參與在rep基因剔除的大腸桿菌在低滲透壓以及高溫的環境下有生長缺陷的現象有關, 因為我們發現eck1083和rep 基因同時剔除的大腸桿菌可以生長在低滲透壓以及高溫的培育環境。
由於我們想要進一步想去驗證ECK1083在rep基因剔除的大腸桿菌中所扮演的角色,於是建構了eck1083基因融合了3xFlag序列的大腸桿菌突變株,以便利用anti-Flag抗體偵測ECK1083蛋白質的含量。由於先前的研究證實ECK1083是一個坐落在大腸桿菌inner membrane上的細胞壁水解酵素,所以我們將野生株與rep基因剔除的大腸桿菌培育在低滲透壓與高溫的環境中,並利用西方點墨法分析ECK1083在野生株與rep基因剔除的大腸桿菌突變株中的含量。我們發現rep基因剔除的大腸桿菌突變株的ECK1083含量相較於野生株的大腸桿菌有明顯的上升,且進一步確認了eck1083 gene的轉錄作用在野生株與rep基因突變株中是非常相似的。根據結果推測在rep基因剔除的大腸桿菌中,ECK1083的含量上升是因為沒有Rep蛋白酵素進行調節所造成。而沒有調節的ECK1083可能是造成rep基因剔除的大腸桿菌沒有辦法在低滲透壓與高溫的環境下存活的原因之一。

Escherichia coli are one of the most common gram-negative bacteria that cause bacteremia and urinary tract infections. Antibiotic treatment is the traditional way to manage the E. coli-caused infections. Due to the rapid emergence of antibiotic-resistant strains, developing novel strategies against such diseases becomes critical. The E. coli protease, Rep, is a potential target for developing the new antimicrobial strategies, because deletion of rep increased the sensitivity to multiple antibiotics, and decreased pathogenic E. coli’s ability to survive in the bloodstream. Also, the E. coli rep mutant exhibit a growth defect under the combined low osmotic and high temperature stresses. Characterizing the function of Rep would facilitate the development of alternative antibacterial strategies. In this study, we utilize E. coli proteome chip assay to screen for the potential Rep substrates.
The E. coli proteome chip assay revealed that 100 E. coli proteins might be the substrate of Rep. Among these 100 possible substrates, 13 of them could be cleaved by the Rep protease in vitro. Further, one of them, ECK1083, was shown to contribute to the growth defect of the E. coli rep mutant under the combined osmotic and high temperature stresses, because the eck1083 deletion in the rep mutant partially rescued the growth defect.
To assess whether ECK1083 accumulates in the rep mutant, we constructed the strains with eck1083-3xFlag at the chromosomal locus of eck1083 to allow the detection of the ECK1083 protein with the anti-Flag antibody. Since ECK1083 has been identified as an inner membrane protein, the membrane fraction from the wild-type and rep mutant strains were isolated under the combined osmotic and high temperature stresses condition. The level of ECK1083 in the membrane fraction was measured by western blot analysis. The level of ECK1083 in the rep mutant was significantly higher than that in the wild-type strain. Also, the mRNA level of eck1083 in the rep mutant was similar to that of the wild-type strain. These results suggested that the lack of Rep-mediated degradation cause the accumulation of ECK1083 in the E. coli rep mutant and that the accumulation may contribute the growth defect under the combined osmotic and high temperature stresses.

Abstract I
中文摘要 III
誌謝 V
Contents IX
The list of figures XII
The list of tables XIII
Abbreviations XIV
Chapter 1 Introduction 1
1-1 Introduction of Escherichia coli 1
1-2 Introduction the importance of cell envelopment in E. coli 1
1-3 Introduction of Rep protease 2
1-3-1 Function of Rep protease 2
1-3-2 Rep contributes to bacterial pathogenesis 3
1-4 Introduction of E. coli proteome chip 3
1-5 Introduction of ECK1083 4
Specific aim 5
Chapter 2 Materials and Methods 7
3-1 Bacterial strain and culture condition 7
3-2 Overexpression and purification of Rep, K455A-Rep and ΔPDZ-K455A-Rep protease	7
3-3 Rep protease assay 8
3-4 Construction of single substrate gene mutant and substrate rep gene double mutant 8
3-4-1 P1 phage preparation 8
3-4-2 P1 phage transduction 9
3-5 Salt-free LB growth assay 9
3-6 Construction of ΔPDZ-pTR163	10
3-7 Construction of pBAD24-eck1083-HA 10
3-8 Construction of pBAD24-lacZ 11
3-9 Construction of pACYC184-eck1083-HA 11
3-10 Construction of strains with eck1083-3XFlag at chromosomal locus 11
3-11 Isolation of the membrane fraction	12
3-12 Western blot analysis 12
3-13 Miller assay 13
3-14 Analysis of eck1083 gene expression level in wild type and Δrep BW25113 E. coli 13
3-14-1 RNA extraction and DNA digestion 13
3-14-2 Reverse transcription polymerase chain reaction (PCR) 14
3-14-3 Real-time PCR 15
3-15 Statistical analysis 15
Chapter 3 Result	16
4-1. Characterization of E. coli Rep, K455A-Rep and ΔPDZ-  K455A-Rep protease activity 16
4-2. Screening for the potential substrates of the Rep protease by the E. coli proteomic chip 16
4-3. Rep protease assay with the E. coli proteins screened out by the E. coli proteomic array assay 17
4-4. Mutation in eck1083 suppresses the rep mutant growth defect under the combined osmotic and heat stresses condition 18
4-5. Overexpression ECK1083 in E. coli K12 cause the growth defect under the combined osmotic and high temperature stresses 19
4-6. The levels of ECK1083 in E. coli strains with and without the expression of the Rep protease 19
4-7. The arabinose promoter activity of pBAD24 is not affected by existence of Rep 20
4-8. Transcription level of eck1083 in the wild-type BW25113 was similar to that in Δrep-BW25113 21
4-9. Deletion of rep increased the level of ECK1083 when E. coli strains were cultured in salt-free LB at 42℃ 21
4-10. Transcriptional level of eck1083 in wild-type BW25113 was similar to that in Δrep-BW25113 when E. coli strains were cultured in salt-free LB at 42℃ 21
4-11. The regulation of D,D endopeptidase Spr in Δeck1083 rep-BW25113 strain was similar to that in Δrep-BW25113 22
Chapter 4 23
Discussion 23
Tables & Figures 26
Reference 56
                                    

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