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研究生(外文):Yi-Ting Chen
論文名稱(外文):Lon Protease-Dependent Proteolysis of EmrR, a Transcriptional Repressor of Multidrug Efflux Pump in Escherichia coli K-12
指導教授(外文):Shih-Hsiung Wu
口試委員(外文):Po-Huang LiangKuo-Feng HuaHsiao-Ching Lin
外文關鍵詞:Lon proteasevirulencequinolone antibiotic resistanceMDR efflux pump
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隨著抗生素的濫用導致致病菌演化成具有抗藥性的結果下,細菌感染造成的致死率逐年上升。近年來新興的抗致病因子概念開始發展,這項概念是藉由抑制致病菌中對宿主有害的致病因子,使這些致病菌在與體內的共生菌競爭結果下而被淘汰。Lon蛋白質水解酶屬於AAA+ 蛋白質家族,負責參與蛋白質的品質管理以及降解與生理調控和致病力相關的調節因子。然而,針對Lon蛋白酶的降解規則目前尚未研究透徹,這增加了鑑定Lon蛋白酶受質的困難。
在本篇研究中,我們以大腸桿菌K-12菌株為模型,發現EmrR蛋白和IhfB蛋白是Lon蛋白酶的受質。EmrR為多重抗生素輸出幫浦的調控蛋白,藉由研究大腸桿菌不同的生長階段,發現大腸桿菌在穩定期時EmrR蛋白會被Lon蛋白酶降解,同時在報導因子分析試驗 (reporter assay) 中我們也得到了相同結論。然而,利用不同大腸桿菌突變菌株,發現細菌對萘啶酸 (nalidixic acid) 的抗性與Lon蛋白酶的參與關係相當複雜,我們推論這個現象可能由許多不同因素參與,無法藉由單一蛋白的研究來解釋。除此之外,降解萘啶酸所引發的EmrR蛋白似乎並非單由Lon蛋白酶所主導。因此,我們認為Lon蛋白酶所主導的降解可能是由於在不同生長階段中DNA拓譜的改變而導致的結果。在蛋白質序列比對以及分子嵌合模擬中,我們假設EmrR蛋白的降解辨識序列 (degron) 可能位在N端或C端。另外,利用胞外蛋白質水解分析法以及圓二色光譜,我們認為Lon蛋白酶必須經由連接分子的協助來辨認EmrR蛋白,經由報導因子分析試驗後,發現DnaK蛋白有可能就是這個連接分子,這個結果提出一個伴護蛋白協助的蛋白降解現象。最後,我們也發現EmrR蛋白的表現有助於大腸桿菌感染真核細胞,這項新發現表示EmrR蛋白可望當作抑菌治療中的標的蛋白之一。

With an alarming rise of antibiotic resistance in pathogenic bacteria, the number of casualties due to bacterial infection expediently increases. An emerging concept of anti-bacterial virulence has been developed. By compromising harmful effects of the virulent factors, the bacteria were disarmed and soon die out due to natural selection when competing for the resource with the “good” bacteria inhabited in our body. Lon protease, belonging to AAA+ (ATPase associated with a variety of cellular activities) superfamily, participates in the protein quality control and the degradation of several regulatory proteins crucial for well-regulated growth as well as virulence. So far, no degradation rule has been reported, and thus makes substrate screening difficult.
In this study, we used Escherichia coli K-12 as model and identified EmrR and IhfB as the substrates of Lon. EmrR is a negative regulator of the multidrug resistance (MDR) efflux pump EmrAB in E. coli. By growth phase-dependent data sampling, we observed the degradation of EmrR was growth phase-dependent, which was in good agreement with emrRAB-based reporter assay. By gene deletion studies, we found that Lon-mediated nalidixic acid resistance was highly complicated since this phenomenon is comprised of more than one factor at a time, which cannot be well-explained by single protein participation model. However, the addition of nalidixic acid seemed irrelevant to Lon-dependent EmrR degradation. Together with growth-phase degradation and NA addition results, the stability of EmrR might be originated from the different DNA topology during the different growth phase. Along with protein alignment and docking calculation, we hypothesized the degron (degradation signal) of EmrR might be situated at the N- or C-terminus. Through circular dichroism and in vitro degradation assay, we believed the recognition of EmrR by Lon should be adaptor-mediated. By deletion and reporter assays, we found DnaK might serve as the critical adaptor for this degradation event to occur. To summarize, we observed a unique “chaperone-assisted proteolysis” in Lon-dependent EmrR proteolysis. These findings provided the importance of the “cross-talk” between chaperones and proteases for the properly targeted degradation. Also, the newly identified function of cell invasion in EmrR offers a direction for future drug development.

中文摘要 i
Abstract ii
List of Contents iii
List of Figures v
List of Tables vi
1. Introduction 1
1.1 AAA+ superfamily and the AAA+ protease 1
1.2 Lon protease 4
1.3 Multiple-drug resistance 6
1.4 Multidrug efflux pumps 8
1.5 The local repressor EmrR 12
1.6 Aims and background of this study 15
2. Materials and Methods 16
2.1 Chemicals, antibodies, and reagents 16
2.2 Plasmids and cloning 17
2.3 Construction of in-frame gene deletion mutants by optimized high-throughput knockout method 24
2.4 In vivo degradation assay 28
2.5 Endogenous EmrR degradation 28
2.6 Western blotting 29
2.7 Protein expression and purification 29
2.8 EMSA analysis of EmrR and the DNA fragment of emrRAB promoter 31
2.9 Fluorescence reporter assay 31
2.10 Susceptibility of nalidixic acid 32
2.11 Native PAGE 33
2.12 Induction of EmrR by nalidixic acid 33
2.13 In vitro proteolysis assay 34
2.14 Circular Dichroism 34
2.15 Invasion assay 35
3. Results and Discussions 36
3.1 In vivo validation of ten potential substrates 36
3.2 The abundance of EmrR was affected by Lon-dependent degradation in stationary phase 40
3.3 Lon protease modulated the emrRAB promoter strength 42
3.4 Highly complicated regulatory network of nalidixic acid resistance in E.coli 45
3.5 Protein alignment reveals the N- or C-terminals might serve as the degron of EmrR for Lon-dependent proteolysis 48
3.6 Molecular simulation of nalidixic acid-EmrR complex reveals possible mechanism for nalidixic acid-induced DNA disassociation 50
3.7 The stability of EmrR was not “solely” Lon-dependent upon nalidixic acid treatment 53
3.8 Lon degrades EmrR through an adaptor-mediated proteolysis 56
3.9 The strength of emrRAB promoter was mitigated in dnaK deletion mutant 60
3.10 Overexpression of EmrR leads to more efficient invasion to eukaryotic cells 62
4. Reference 64

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