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博碩士論文 etd-0602115-172442 詳細資訊
Title page for etd-0602115-172442
論文名稱
Title
抗菌肽抑制幽門桿菌上之機制探討與研發應用
Antimicrobial Peptides: Efficacy and Molecular Mechanism of Action against Multi-Drug Resistant Gastric Pathogen Helicobacter pylori Infection
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
208
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2015-06-30
繳交日期
Date of Submission
2015-07-02
關鍵字
Keywords
Tilapia piscidin、Epinecdin-1、免疫調節、幽門螺桿菌、抗菌肽
Tilapia piscidin, Epinecdin-1, Immunomodulation, Antimicrobial peptides, Helicobacter pylori
統計
Statistics
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中文摘要
抗生素所產生的抗藥性是對未來健康福祉的嚴重障礙。頻繁使用抗生素是病原菌產生抗藥性菌株重要的原因之一。革蘭氏陰性的胃幽門螺桿菌 Helicobacter pylori (H. pylori)多重抗藥性菌株multidrug-resistant (MDR)是造成全球健康問題的主因之一。幽門桿菌的感染是十分普遍的,它與各種胃部疾病,包括胃炎,消化性潰瘍,和胃癌有很大的關係。目前治療幽門桿菌的方案(使用蛋白質幫浦抑制劑輔以三重或四重廣效性抗生素)是最理想的,但帶有高度的失敗率。在這樣發展下去有效的抗生素將面臨不足以對抗的境地,因此有必要發展對幽門桿菌新的抗菌手段。最有潛力之一的選擇就是抗菌胜肽Antimicrobial peptides (AMPs),它是一種存在於脊椎與無脊椎動物之中具有攻防兼備能力的細胞溶解胜肽。它們可以迅速殺死各種微生物並具備額外的活性影響先天免疫以及發炎反應。另外對於細菌對常規抗生素的抗藥性也使得抗菌胜肽有機會成為新一代的抗生素。因此它們分子作用機制是相當吸引人並且需要去闡明的。在這裡我們描述兩種新的抗菌胜肽,名為Tilapia Piscidin4 (TP4) 以及 Epinecidin-1 (Epi-1),這是我們篩選出具有生長抑制以及殺菌效果用來抵抗ATCC 43504﹑51653﹑700392 以及臨床分離具有抗生素抗藥性菌株CI-HC-028 (這些菌株為 CagA+﹑VacA+) 等幽門桿菌。TP4 與Epi-1 展現出最小抑菌濃度lowest minimum inhibitory concentrations (MIC)並顯示與抗生素一起時的協同作用。我們刺激sub-MIC以及體內傳遞抵抗試驗表明TP4 與Epi-1具有獨立對抗這些細菌的效果。這些抗菌胜肽製造孔洞破壞細菌細胞膜,在這裡我們提出一個體外膜滲透機制:在幽門桿菌上TP4會經由膜形成微膠粒,Epi-1則是使膜以馬鞍形的弧度張開。這些TP4 與Epi-1的行動模式預測是藉由螢光探針1-N-phenylnaphthylamine (NPN)洩漏試驗證實,以測量幽門桿菌暴露在TP4 與Epi-1抗菌胜肽時所改變的表面膜電位差來驗證。更進一步則以transmission electron microscopic studies (TEM)直接觀察體外殺菌機制,結果證明是使膜去極化以及破壞胞質成分的組成。TP4 與Epi-1在體內的的影響以及在感染幽門桿菌時選擇性的調節宿主系統免疫反應則是在小鼠模組上驗證。TP4 與Epi-1的免疫調控潛力是經由流式細胞儀篩選cytokines ﹑ T cell marker以及splenic subsets分析幽門桿菌感染後的胃癌相關基因表現。經由胃組織的colonization,幽門桿菌具備高度的Regulatory T (Tregs) subsets以及低Th17/Treg比例,並且同時表現前以及抗發炎的cytokines。TP4 與Epi-1選擇性的抑制Treg subset的數量,而且TP4顯著的恢復了Th17/Treg的比例。TP4 與Epi-1甚至向下調控前以及抗發炎的cytokines以及Foxp3轉錄因子在胃部組織的表現。誘導CD4+-Foxp3 Tregs的損耗是因為幽門桿菌在早期被清除; 此外TP4 與Epi-1處理會經由抑制Th17 subsets幫助胃部恢復正常型態。小鼠與兔子的細胞毒性研究(口服﹑皮膚/真皮和眼睛的刺激測試)證實了TP4 與Epi-1的安全性,高劑量的胜肽並未造成在口服﹑真皮和眼睛的刺激測試產生毒性影響。總而言之,我們的結果顯示TP4 與Epi-1清除幽門桿菌是藉由一獨特的模式,在體外以膜溶解以及在體內強力影響調停幽門桿菌誘導宿主反應。高濃度的TP4 與Epi-1並不會造成小鼠與兔子的異常。因此,這兩種抗菌胜肽可以被視為對抗多重抗藥性幽門桿菌感染具有療效的新型藥劑。
Abstract
The ongoing development of antibiotic-resistant infections is a major obstacle in ensuring future health and wellbeing. The frequent misuse of antibiotics is one of the reasons behind the development of resistance in pathogenic strains of bacteria. Emergence of multidrug-resistant (MDR) strains of the Gram-negative gastric pathogen Helicobacter pylori (H. pylori) is a global health concern. H. pylori infection is highly prevalent, and has a strong association with various gastric diseases, including gastritis, digestive ulcers, and gastric cancer. Currently, treatment of H. pylori infection (involving the use of proton pump inhibitors followed by triple and quadruple therapy with broad-spectrum antibiotics) is suboptimal, with high failure rates. This development is predicted to result in imminent inadequacy of applicable effective antibiotics, necessitating the development of novel antimicrobial strategies against H. pylori. Among the potential alternatives are antimicrobial peptides (AMPs), which are cytolytic peptides that serve in vertebrates and invertebrates for both offensive and defensive purposes. They can rapidly kill a broad range of microbes, and have additional activities that impact on the quality and effectiveness of innate responses and inflammation. Furthermore, the challenge of bacterial resistance to conventional antibiotics and the unique mode of action of AMPs have made such peptides promising candidates for the development of a new class of antibiotics. Accordingly, the molecular basis of their action is of considerable interest and needs to be elucidated. Here, we report our recent analyses of two novel AMPs, called Tilapia Piscidin 4 (TP4) and Epinecidin-1 (Epi-1), which we screened for growth inhibitory and/or bactericidal effects against H. pylori strains ATCC 43504, 51653, and 700392, and clinical isolate-antibiotic resistant strains CI-HC-028 (these strains are CagA+, VacA+). TP4 and Epi-1 displayed low minimum inhibitory concentrations (MIC) and synergistic activity with conventional antibiotics. Our simulated sub-MIC and in vivo passaging resistant assay showed that TP4 and Epi-1 efficacy is independent of resistance in bacteria. These AMPs create pores that cause bacterial membrane disruption, and here, we propose in vitro mechanisms of membrane permeabilization: TP4 acts via Membrane micelle formation, while Epi-1 acts via Saddle splay curvature generation. These modes of action of TP4 and Epi-1 were demonstrated using hydrophobic fluorescent probe 1-Nphenylnaphthylamine (NPN) leakage assay, and detection of changes in surface charge by measuring the zeta potential of H. pylori upon exposure to TP4 or Epi-1. Furthermore, transmission electron microscopic studies (TEM) were performed to evidence the in vitro direct killing mechanism underlying the membrane depolarization and disruption of periplasmic constituents. The in vivo efficacy of TP4 and Epi-1, as well as selective modulation of H. pylori-biased host immune responses, were demonstrated in a mouse model. The immunomodulatory potentials of TP4 and Epi-1 were analyzed in the host during H. pylori infection based on gastric gene expression, and analysis of cytokines, cell markers, and splenic subsets by flow cytometry. During colonization of gastric tissue, H. pylori maintains high regulatory T (Tregs) subsets and a low Th17/Treg ratio, and express both pro- and anti-inflammatory cytokines. TP4 and Epi-1 selectively suppressed the Treg subset population, and TP4 significantly restored the Th17/Treg ratio. Furthermore, TP4 and Epi-1 down regulated gene expression for both pro- and antiinflammatory cytokines and Foxp3 transcription factor in gastric tissue. The induced depletion of CD4+-Foxp3 Tregs results in early clearance of H. pylori density; moreover, TP4 and Epi-1 treatment helped restore normal gastric morphology by moderate suppression of Th17 subsets. Mouse and rabbit toxicity studies (oral, skin/dermal, and eye irritation tests) were performed to evaluate the safety of TP4 and Epi-1; crucially, high doses of peptides did not exert toxic effects in oral, dermal, or eye irritation models. Collectively, our results indicate that TP4 and Epi-1 clear H. pylori colonization through unique modes of membrane lysis in vitro, and strong in vivo efficacy and modulation of H. pylori-induced host responses. High concentrations of TP4 and Epi-1 did not induce abnormalities in mice or rabbits. Therefore, both peptides may be considered promising and novel agents for the monotherapeutic treatment of multidrug resistant H. pylori infections.
目次 Table of Contents
CONTENTS
I. Abstract Chinese………..……………………………………………………………..…vi-vii English…………………………….…………………………………….……...viii-x
II. Abbreviations ……………………………………………………………..…..xiv-xv
CHAPTER I
A. HELICOBACTER PYLORI INFECTION AND ITS TREATMENT
1. Introduction.……………………………………………………………………..…1
1.1. Helicobacter pylori discovery and morphology……………………………….2-5
1.2. Virulence factors ……………………………………………………….….......6-7
1.2.1. Flagellae ………………………………………………………….………8
1.2.2. Adhesion proteins....………………………..…………………….….........9
1.2.3. VacA………………………………………………………………….10-11
1.2.4. CagA....…………………………………………………………….....12-13
1.2.5. Urease B ...…………………………………………………………...14-16
1.2.6. Lipopolysaccharides (LPS) ……………………………….……….…17-19
1.3. H. pylori and Host Immune Responses...…………………………………...20-22
1.4. Clinical aspects of H. pylori-associated diseases………………...……..…...23-25
1.5. Antibiotic therapy and its failure ……………………………………….…...26-28



B. POST ANTIBIOTIC ERA: ANTIMICROBIAL PEPTIDES
2. Background ……………………………………………………………………......29
2.1. AMPS: A diverse collection of biomolecules …………..…………………..30-32
2.2. AAMPs: Anionic antimicrobial peptides …………………….………..…....32-36
2.3. CAMPs: Cationic antimicrobial peptides ……….…………....………….........37
2.3.1. Anti-bacterial peptides……………………………………………..…37-38
2.3.2. Anti-fungal peptides ……………………………………………………..39
2.3.3. Anti-parasite peptides ……………………………………………..........40
2.4. Anti-viral peptides ……………………………………………...….………..40-41
2.5. Tools for delivery of antimicrobial peptides ……………………………....42-43
2.6. Commercialization and clinical development of antimicrobial peptides.….44-45
2.7. Specific aims of the present investigation ……………………………….…… 46

CHAPTER II
CATIONIC ANTIMICROBIAL PEPTIDE: TILAPIA PISCIDIN 4 (TP4)
3. Introduction.…………………………………………...…………….…………..47-51
3.1. Materials and Method.. ……………………………………..……………….52-61
3.2. Results.……………………………………………………..………………..62-75
3.3. Figures and tables ……………………..…………………..……………….76-90



CHAPTER III
CATIONIC ANTIMICROBIAL PEPTIDE: EPINECIDIN-1 (EPI-1)
4. Introduction …………………………………………...…………….………….91-93
4.1. Materials and Methods...…………..……………………………….………94-101
4.2. Results.………………………………………………………………...….102-109
4.3. Figures and tables..………………………………………………………..110-121

CHAPTER IV
DISCUSSION AND FUTURE PROSPECTS
5. Discussion….………………………………………………………………….122-129
6. Conclusion………………………………………………………………….…130-131
7. Future Prospects ....……………………………………………………………132-133
8. References………...………….………………………………………………..134-189

APPENDICES
1. List of Primers ...…………………………………………………………..190-191
2. Curriculum Vitae.………………………………………………………….192-193
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