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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳垣崇 | |
| dc.contributor.author | Shih-Jung Wu | en |
| dc.contributor.author | 鄔式絨 | zh_TW |
| dc.date.accessioned | 2021-06-14T17:17:18Z | - |
| dc.date.available | 2013-09-11 | |
| dc.date.copyright | 2008-09-11 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-25 | |
| dc.identifier.citation | 1. Boix, E., and Nogues, M. V. (2007) Mol Biosyst 3, 317-335
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41105 | - |
| dc.description.abstract | 抗菌胜肽廣泛存在於植物、昆蟲、兩棲類和哺乳類，在先天性免疫之防禦機制上扮演重要的角色。在本論文中，我們合成一段含正電荷之胜肽-(R3S1)3，對綠膿桿菌有高度專一性的抗菌能力。我們發現綠膿桿菌之外膜蛋白OprI和OprF可和此胜肽結合，若加入過量之重組蛋白rOprI、rOprF、anti-OprI或anti-OprF專一性抗體，則可抑制胜肽之抗菌活性，因此推測外膜蛋白OprI和OprF可以調控綠膿桿菌對抗菌胜肽(R3S1)3之感受性。另外，以螢光和電子顯微鏡觀察顯示，綠膿桿菌在經過胜肽處理之後，細菌細胞膜之通透性顯著增強，外膜上會凸起產生水泡，內外膜分離，細胞質內物質有高度濃縮現象，最後會排出菌體外，而使細胞形成空洞狀。由免疫組織染色法得知，(R3S1)3穿透細菌細胞膜，累積在細胞質內高度濃縮區域，由於(R3S1)3可以和去氧核醣核酸(DNA)或核醣核酸(RNA)結合，因此我們推測此胜肽可與細胞內核酸結合，進而影響細菌之生理功能。
此胜肽(R3S1)3 除了具有殺菌特性外，也能穿透真核細胞HaCaT和HeLa之細胞膜及核膜，並聚集在細胞核某特定區域，但不造成細胞毒性，因此它可能為一具細胞穿透性之胜肽(Cell-penetrating peptide)。本論文有關(R3S1)3透過外膜蛋白OprI及OprF進入細菌，破壞及干擾菌體結構及成份之機制，提供了發展抗菌藥物之重要線索。 | zh_TW |
| dc.description.abstract | Short cationic antimicrobial peptides are widely present in living organisms for innate defense against invading microorganisms. They are generally amphipathic, small and cationic with at least two positive residues. We demonstrated a synthetic cationic peptide, (R3S1)3, possessing antimicrobial activity, especially against Pseudomonas aeruginosa. Our results showed that the outer membrane proteins of P. aeruginosa, OprI and OprF, interact with the peptide. The antimicrobial activity of (R3S1)3 were repressed by excess amounts of rOprI, rOprF, anti-OprI or anti-OprF. The bacterial membrane became permeable, the chromatin condensed in cytosol and blebs formed on outer membrane of bacteria after (R3S1)3 treatment analyzed by TEM. In addition, the (R3S1)3 translocated across the cytoplasmic membrane, localized in the cytosol of P. aeruginosa and bound to intracellular target, like nucleic acids, analyzed by immunohistochemistry. The (R3S1)3 exerted not only antimicrobial activity but also penetrated eukaryotic cell and nuclear membranes. These action mechanism of antimicrobial peptide (R3S1)3 against P. aeruginosa through the bacterial outer membrane proteins, OprI/OprF, provide important clues for development of new antimicrobial agents. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T17:17:18Z (GMT). No. of bitstreams: 1 ntu-97-R95445115-1.pdf: 8844425 bytes, checksum: 18b7e4c3b4426d10f6c334e80294fbd0 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | Index
口試委員會審定書…………………………………...……………..……..I 誌謝……………………………………………………...…………...……II 中文摘要……………...............………………………………………….III ABSTRACT…………….................……………………………………..IV ABBREVIATIONS……………...………………………………………..V CAPTER 1 INTRODUCTION……………...……………………...…….1 1.1 Antimicrobial peptides/proteins.….....………………………….1 1.1.1 General concept………………...………..…………………1 1.1.2 Nonribosomally synthesized peptides………….....………..2 1.1.3 Ribosomally synthesized peptides……………...…………..2 1.1.4 Mechanism of action…………………..…...………………3 1.2 Outer membrane proteins……………………...………..............5 1.2.1 Outer membrane proteins of Pseudomonas aeruginosa........5 1.2.2 Outer membrane protein F (OprF)………………....………6 1.2.3 Outer membrane protein I (OprI)…………..……...……….7 1.3 Specific aims………………………………………………........7 CAPTER 2 MATERIALS AND METHODS…………………………....8 2.1 Peptides……………………….……………………………...…8 2.2 Microbes and growth conditions..................................................8 2.3 Antimicrobial activity assay.…………………………………....8 2.4 Fluorescence microscopy…………………………………….…8 2.5 Membrane permeability assay…………………………………..9 2.6 Inhibition of bactericidal activities of AMPs………………...…9 2.7 Binding properties of (R3S1)3 to P. aeruginosa…………..……10 2.8 Isolation of bacterial membrane fraction………………..….…10 2.9 Pull-down assay…......................................................................11 2.10 Extraction of genomic DNA from P. aeruginosa………….......11 2.11 Plasmid construction……………………………………...…...11 2.12 Purification of rOprI and rOprF……………….………...….....12 2.13 Preparation of polyclonal antibodies……………..……………13 2.14 Slot blotting…………………………………………...….……13 2.15 CD analysis of rOprI………………………..………...……….14 2.16 Analysis of OprI polymerization…………….………………...14 2.17 Analysis of OprF gene in P. aeruginosa strains……….………15 2.18 Transmission electron microscopy (TEM)…………....……….15 2.19 Confocal laser microscopy…………………………...………..15 2.20 Nucleic acids binding………………………………….………16 2.21 Hemolytic assay……………………………………...………..16 CAPTER 3 RESULTS……………………………………………...........18 3.1 Antimicrobial activity of (R3S1)3………………...………….…18 3.2 Effect of (R3S1)3 on bacterial membrane permeability……..….18 3.3 Factors affecting bactericidal activity and bacterial binding ability of (R3S1)3…………………………………....………….19 3.3.1 Effects of LPS and divalent cations on the bactericidal activity of (R3S1)3………………………....………………19 3.3.2 Effects of LPS and divalent ion on the binding ability of (R3S1)3 to P. aeruginosa……………..…………………… 19 3.4 Identification of (R3S1)3-binding protein of P. aeruginosa……20 3.5 Expression and preparation of recombinant OprI……………..20 3.6 Role of OprI………..………………………………………….21 3.6.1 Specific interaction between (R3S1)3 and native OprI…….21 3.6.2 Specific interaction between rOprI and AMPs………...….21 3.6.3 Antimicrobial activity of (R3S1)3 repressed by rOprI or anti-OprI antibody…………………………………...……22 3.7 Other properties of OprI…………...………...………………...22 3.8 Properties of another outer membrane protein OprF...………...23 3.8.1 Expression and preparation of recombinant OprF from P. aeruginosa………………………………………...………23 3.8.2 Interaction of rOprF with (R3S1)3, rOprI and hRNase7...…23 3.8.3 Repression of antimicrobial activity of (R3S1)3……….......24 3.8.4 Analysis of OprF gene in P. aeruginosa……………….…24 3.8.5 Role of OprF in the susceptibility and permeability of bacteria to (R3S1)3………………………………...……….24 3.9 Morphological changes of bacteria and internalization of (R3S1)3………………………………………………………....25 3.9.1 Morphological change after (R3S1)3 treatments……...……25 3.9.2 Internalization of (R3S1)3 into bacterial cytosol……...……25 3.10 Intracellular targets of (R3S1)3………….……………………...26 3.11 Hemolytic activity of (R3S1)3…………...……………….…….26 3.12 Internalization of (R3S1)3 into mammalians cell lines….....…...26 CAPTER 4 DISCUSSION………………………………...…………….28 REFERENCES……………….….…………………………………...….32 FIGURES………………………………………………………..……….36 Fig.1 Antimicrobial activity of (R3S1)3 against various microbes….…36 Fig.2 Membrane permeability of P. aeruginosa after treatments of (R3S1)3, polymyxin B and indolicidin………….………………37 Fig.3 Effects of LPS and divalent cations on antimicrobial activities of various AMPs……………………………………….………….38 Fig.4 The binding ability of (R3S1)3 to bacteria was inhibited by LPS and magnesium…………………………………………………39 Fig.5 Identification of (R3S1)3-binding protein from P. aeruginosa…..40 Fig.6 Analysis of recombinant OprI expressed in E. coli……..………41 Fig.7 Specific interaction of rOprI with various AMPs………………42 Fig.8 Role of OprI in the susceptibility of bacteria to (R3S1)3………..43 Fig.9 Structure of rOprI obtained by computer modeling and CD analysis………………………………………………………....44 Fig.10 rOprI exists in polymers analyzed by gel filtration chromatography………………………..……………………….45 Fig.11 Expression and purification of recombinant OprF from E. coli.46 Fig.12 Specific interaction of rOprF with (R3S1)3-His, hRNase7 and rOprI……………………………………………….......……….47 Fig.13 Role of OprF in the susceptibility of bacteria to (R3S1)3…...…48 Fig.14 Analysis of P. aeruginosa OprF gene in wild-type (H103) and OprF-deficient strain (H636) by PCR……….............................49 Fig.15 Role of OprF in the susceptibility and permeability of bacteria to (R3S1)3 in wild-type (H103) and OprF-deficient strain (H636)..50 Fig.16 Morphological changes and immunohistochemistry of P. aeruginosa after (R3S1)3 treatments……………………………52 Fig.17 Internalization of Alexa fluor 488-(R3S1)3 analyzed by Confocal laser microscopy………………………………………………..53 Fig.18 Gel retardation analysis of the binding of (R3S1)3 to genomic DNA, plasmid DNA and 16S-23S rRNA…………...………….54 Fig.19 Hemolytic activity of (R3S1)3………………………….………55 Fig.20 Internalization of Alexa fluor 488- (R3S1)3 into mammalian cell lines………………………………………………..…...………56 TABLES……………….…………………………………..……………..57 Table.1 Peptides used in this study……………………………….…..57 Table.2 Microbes and culture conditions…………………..…………58 Table.3 Primer sequences used in this study………………………….59 APPENDIX……………………………………………...……………….60 Appendix.1 Pseudomonas aeruginosa genome map…………..……..60 | |
| dc.language.iso | en | |
| dc.subject | 抗菌機制 | zh_TW |
| dc.subject | 細胞穿透胜肽 | zh_TW |
| dc.subject | 外膜蛋白OprI | zh_TW |
| dc.subject | 外膜蛋白OprF | zh_TW |
| dc.subject | 膜通透性 | zh_TW |
| dc.subject | 抗菌胜肽 | zh_TW |
| dc.subject | membrane permeability | en |
| dc.subject | cell-penetrating peptide | en |
| dc.subject | OprF | en |
| dc.subject | OprI | en |
| dc.subject | antimicrobial mechanism | en |
| dc.subject | outer membrane proteins | en |
| dc.subject | antimicrobial peptides | en |
| dc.title | 外膜蛋白OprI/OprF調控綠膿桿菌對陽離子胜肽(R3S1)3之感受性 | zh_TW |
| dc.title | Outer membrane protein, OprI/OprF, modulates the susceptibility of Pseudomonas aeruginosa to cationic peptide, (R3S1)3. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 廖有地 | |
| dc.contributor.oralexamcommittee | 楊瑞彬,王錦堂 | |
| dc.subject.keyword | 抗菌胜肽,外膜蛋白OprI,外膜蛋白OprF,膜通透性,細胞穿透胜肽,抗菌機制, | zh_TW |
| dc.subject.keyword | antimicrobial peptides,outer membrane proteins,OprI,OprF,cell-penetrating peptide,membrane permeability,antimicrobial mechanism, | en |
| dc.relation.page | 60 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-07-27 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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