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研究生： 凃逸凡
Tu, I-Fan
論文名稱： 幽門桿菌Lon蛋白酶減緩抗生素咪唑尼達活化
Lon protease decreases activation of the antibiotic metronidazole in Helicobacter pylori
指導教授： 詹鴻霖
Chan, Hong-Lin
吳世雄
Wu, Shih-Hsiung
口試委員: 林念璁
Lin, Nien-Tsung
蘇士哲
Sue, Shih-Che
李岳倫
Lee, Yueh-Luen
高茂傑
Kao, Mou-Chieh
學位類別： 博士
Doctor
系所名稱： 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年： 2014
畢業學年度： 102
語文別： 英文
論文頁數： 94
中文關鍵詞： 幽門桿菌 、Lon蛋白酶 、咪唑尼達
外文關鍵詞： H. pylori, Lon protease, metronidazole
相關次數： 點閱：4  下載：0 
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    • 原核生物內的Lon蛋白酶參與水解結構折疊有誤的蛋白質，同時能藉由控制其下游受質數量多寡來調控各種生物生裡反應。幽門桿菌的Lon蛋白酶(HpLon)至今角色未明，只知道在細菌生長過程中會持續表現。在本研究中發現磷酸複合體(polyphosphate)會增強HpLon蛋白酶及短肽鍊酶水解活性，同時磷酸複合體也會影響HpLon與雙股DNA結合能力。為了瞭解HpLon在幽門桿菌中扮演的生物角色，本研究採用trapping approach搭配蛋白質體學方法辨識何種蛋白會與HpLon交互作用。實驗結果顯示大部分會與HpLon結合的蛋白質大多參與咪唑尼達(metronidazole)活化有關，特別是RdxA蛋白為幽門桿菌菌體於活化咪唑尼達專一性最高的酵素。咪唑尼達常用於治療幽門桿菌感染的第一線藥物之一。實驗中將HpLon基因從幽門桿菌中剃除，發現該突變株對於咪唑尼達敏感度增加，顯示HpLon與咪唑尼達活化有關。HpLon藉由降低RdxA之NADPH氧化活性及咪唑尼達還原活性來減緩咪唑尼達活化速率，這樣的調控方式是藉由HpLon上的AAA+ module (ATPases Associated with diverse Activities)結合NADPH再作用於RdxA，即便幽門桿菌菌體內的HpLon失去蛋白酶活性，也不會影響幽門桿菌對咪唑尼達的敏感度。本研究為第一篇針對幽門桿菌HpLon蛋白做功能性探討，同時釐清HpLon如何從蛋白質層面調控RdxA活性，藉以影響咪唑尼達在菌體內的活化。

    The prokaryotic ATP-dependent Lon protease participates in the turnover of misfolded proteins and mediates the activity of regulatory proteins in diverse cellular processes. The lon gene of Helicobacter pylori strains is constitutively expressed during growth. The biological role of Lon protease in H. pylori (HpLon) is unclear so far. In this study, we found that polyphosphate (polyP) enhanced the protease and peptidase activity of HpLon. PolyP also competed with double strand DNA (dsDNA) for HpLon binding. In order to elucidate the function and physiological role of HpLon in H. pylori, a trapping approach was used to identify putative Lon binding partners in the bacterium. Many of HpLon-interacting partners were found to be associated with the activation of metronidazole (Mtz), which is widely used in first-line therapy against H. pylori infections. In addition, we constructed a lon-deficient mutant more susceptible to Mtz than the wild-type form. A proteomic analysis identified RdxA, which encodes oxygen-insensitive NADPH nitroreductase, a major Mtz-activating enzyme in H. pylori. RdxA is a physiological interacting partner of HpLon and is not cleaved by the protease. We found that the AAA+ module (ATPases Associated with diverse Activities) in HpLon causes a decrease in both NADPH oxidase and Mtz reductase activity in RdxA. This result explains why the protease-defective lon mutant (lonS741A mutant) exhibits the same level of Mtz susceptibility as wild-type H. pylori. Our results are consistent with a model in which HpLon recruits the ADP moiety of NADPH through the AAA+ module to interact with RdxA and thereby further decreases its NADPH-oxidizing and Mtz-reducing activities. This is the first study to figure out how HpLon regulates RdxA activity and participate the process of Mtz activation in H. pylori.

    Table of content Abstract / 1 致謝 / 3 Table of content / 4 Abbreviations / 8 Chapter 1. Introduction 1.1 Helicobacter / 9 1.1.1 Overview / 9 1.1.2 The bacteriology of H. pylori / 9 1.1.3 The pathogenesis of H. pylori / 10 1.2 The mechanism of antibiotic action and antibiotic resistant in H. pylori 1.2.1 Overview of antimicrobial agents against H. pylori / 11 1.2.2 The introduction of Metronidazole (Mtz) and Mtz resistance mechanism among the pathogens / 13 1.2.3 Mtz resistance mechanism of H. pylori / 14 1.2.4 The introduction of RdxA in H. pylori / 15 1.2.5 The introduction of Nitazoxanide (NTZ) and mode of action / 16 1.3 The treatment against H. pylori infection 1.3.1 The introduction of current therapy for H. pylori infection / 17 1.3.2 Clinical effect of antimicrobial resistance / 18 1.4 The introduction of Lon protease 1.4.1 The biochemistry and structure studies in Lon protease / 18 1.4.2 Biological role of ATP-dependent Lon protease / 21 1.5 The current studies of polypohosphate (polyP) in H. pylori / 22 1.6 The current studies in H. pylori Lon (HpLon) / 23 1.7 The aim of this work / 23 Chapter 2. Materials and Methods 2.1 Bacterial strains and growth conditions / 25 2.2 Allelic exchange mutagenesis and complementation in H. pylori / 25 2.3 Expression and purification of RdxA and HpLon / 26 2.4 ATPase activity assay / 27 2.5 Protease activity assay / 27 2.6 Peptidase assay / 28 2.7 Gel mobility shift assays (GMSA) / 28 2.8 Circular-dichroism spectroscopy (CD) / 28 2.9 Intrinsic tryptophan fluorescence measurements / 29 2.10 HpLon trapping proteins identification / 29 2.11 Antibiotic disk diffusion assay and E test susceptibility assay / 30 2.12 Alkaline gel DNA analysis / 30 2.13 NADPH oxidase detection / 31 2.14 Measurement of Mtz reduction / 31 Chapter 3. Result 3.1 The biophysical and biochemistry of HpLon determination / 33 3.2 HpLon trapping proteins are involved in the Mtz-induced metabolic changes in H. pylori / 35 3.3 The antibiotic susceptibility of H. pylori / 36 3.4 RdxA is not a substrate of HpLon protease / 37 3.5 The AAA+ module of HpLon modulates RdxA oxidase and reductase activities / 37 3.6 NADPH abates the ATPase activity in HpLon / 38 Chapter 4. Discussion 4.1 The PolyP effect on Mtz-susceptibility of H. pylori / 40 4.2 Comparison of the potential role of EcLon and HpLon for the development of multi-drug resistant bacteria / 41 4.3 The effect of HpLon on Mtz susceptibility in H. pylori 4.3.1 HpLon affects NADPH oxidation and Mtz reduction of RdxA through AAA+ module / 42 4.3.2 The binding partner of HpLon identification represents the relationship between HpLon and Mtz / 44 4.3.3 Comparison of two nitroreductase in H. pylori, RdxA and FrxA, for the contribution of Mtz activation / 45 4.4 The proposed mechanism of HpLon on the development of Mtz resistance in H. pylori / 46 Chapter 5. Reference / 48 Chapter 6. Table Table 1. Bacterial strains and plasmids in this study/ 64 Table 2. Trapping proteins were identified by proteomics analysis / 66 Table 3. Antibiotics susceptibility in H. pylori reference strain 26695 and its derivative lon mutant /68 Table 4. Comparison the Mtz susceptibility in wildtype and derivative lon mutant among H. pylori strains / 69 Table 5. HpLon affects RdxA activity, including NADPH oxidase and Mtz reductase / 70 Chapter 7. Figure Fig.1 Multiple alignment of LonA protease sequences among bacteria and the identification of functional domains in HpLon / 71 Fig. 2 Enzymatic activity determination of HpLon / 73 Fig. 3 Protease and peptidase activity of HpLon determination in presence of polyP / 74 Fig. 4 The effect of HpLon or HpLon_α and polyP interaction on DNA binding by Gel mobility shift assays (GMSA) analysis / 75 Fig. 5 The effect of polyP on the HpLon structure by CD and intrinsic Trp fluorescence spectrum / 76 Fig. 6 HpLon detection in H. pylori derivative mutants by western blot / 77 Fig. 7 H. pylori susceptibility to metronidazole / 78 Fig. 8 Mtz induces DNA fragmentation in H. pylori / 79 Fig. 9 HpLon protease activity is not necessary for RdxA and Mtz activation / 80 Fig. 10 Pull-down assay of HpLon and RdxA complex after overexpression in E. coli / 82 Fig. 11 NADPH competes with ATP for HpLon binding / 84 Fig. 12 NADH oxidation by RdxA in presence of polyP / 85 Fig. 13 The disk diffusion assay of Nitazoxanide (NTZ, A) and Nitrofurantoin (B) to detect the susceptibility of H. pylori 26695 and its lon mutant / 86 Fig. 14 Proposed model for HpLon effect on Mtz activation in H. pylori / 87 Chapter 8. Appendix Supporting fig. 1 Structure-based studying in RdxA and FrxA / 88 Supporting fig. 2 The crystal structure of the AAA+ module (PDB: 1D2N) / 90 Supporting table 1. The summary of transcriptional factors and regulators in H. pylori / 91 Supporting table 2. The mechanism of clinical antibiotics for H. pylori treatment / 92 Supporting table 3. Recommended first-line therapies for Helicobacter pylori infection in 2011 to 2012 / 93 Supporting table 4. The enzymatic activity comparison of RdxA and FrxA / 94

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