由於抗生素的濫用，使多重抗藥性菌株面臨無藥可醫的情形。鮑氏不動桿菌 （Acinetobacter baumannii, AB）就是其中之一。病人若是感染多重抗藥性鮑氏不動桿菌 （multiple drug resistance Acinetobacter baumannii, MDRAB），將面臨無藥可醫的情形。有鑑於此，我們想要找尋新的殺菌劑來殺死多重抗藥性菌株。最近的文獻指出，抗菌胜肽 （antimicrobial peptide）在生物體扮演抵抗部份外來入侵為生物的角色。本研究首先以生物資訊學軟體分析本實驗室分離之鮑氏不動桿菌噬菌體的溶菌酶 （lysin）及 Holin 胺基酸序列，並藉以設計出具有不同特性 （二級結構、帶電性、疏水性及疏水性矩）的六條胜肽來進行殺菌實驗。在殺菌實驗結果顯示當靜電荷為+6、疏水性為-0.99及疏水性矩為0.49的 LysAB2 113-145 G1對於多重抗藥性鮑氏不動桿菌具有最好的殺菌效果，其最低抑菌濃度為8 μM，而最小殺菌濃度為16 μM。在溶血實驗試驗中顯示這六條抗菌胜肽的溶血率隨著胜肽的濃度增加而升高，顯示出這六條抗菌胜肽具有部分細胞毒性。在協同作用試驗中發現當 LysAB2 113-145 G1結合 LysAB2 113-145 G3，或者以 LysAB2 113-145 G1結合HolAB2 18-37 G1不具有協同作用且不為拮抗作用，但所需的抗菌胜肽濃度降低，但此時仍有部分細胞毒性。經掃描式電子顯微鏡 (scanning electron microscope, SEM)觀察到抗菌胜肽能直接破壞鮑氏不動桿菌。在細菌細胞膜通透性試驗中發現，抗菌胜肽可以造成細菌細胞膜通性改變。由以上實驗結果得知，本實驗所設計的胜肽是一種有效對抗細菌的抗菌胜肽，並有潛力作為殺菌劑之潛力。未來我們希望此研究能應用在新型抗菌藥劑之研發上。

The emergence of multidrug-resistant strains of Acinetobacter baumannii (MDRAB) constitutes a serious threat to public health. The increasing problem of antibiotic resistance among pathogenic bacteria requires the development of new antimicrobial agents. This study was designed to test the possibility that antimicrobial peptides could be derived from the genomic sequences of phage lysin and Holin. Base on A. baumannii phage2 lysin and Holin amino acid sequences, we designed and produced six putative peptides (LysAB2 113-145, LysAB2 133-145 G1, LysAB2 113-145 G2, LysAB2 113-145 G3, HolAB2 18-37 and HolAB2 18-37 G1). Synthetic these six peptides displayed potent activity against A. baumannii (minimum inhibitory concentration, MICs ranging from 8-128 μM; minimum bactericidal concentration, MBCs ranging from 8-128 μM) while displaying hemolytic activity against human erythrocytes (HC50＝32 μM). We investigated antimicrobial peptides for potential synergy with each other against clinical strains of multidrug resistance A. baumannii. The bactericidal test results show that these peptides can effectively restrain parts of Gram negative bacteria. Under the Scanning Electron Microscope, it was observed that these peptides can directly destroy A. baumannii. Treating bacteria with antimicrobial peptides clearly enhanced permeation of the bacterial cytoplasmic membrane. The possible relevance of these results showed these peptides may be used to design new potent antimicrobial candidates much needed in face of the ever threatening drug resistance problems.

目錄 I
中文摘要 III
英文摘要 IV
第一章 前言 1
1.1鮑氏不動桿菌 (Acinetobacter baumannii)之特性 1
1.2 噬菌體之特性及應用 1
1.3 溶菌酶及 Holin之特性及應用 3
1.4 抗菌胜肽簡介 7
1.5 抗菌胜肽結構分類 8
1.5.1 雙親性和疏水性α-螺旋胜肽（amphipathic and hydrophobic α-helices） 8
1.5.2 β-褶板抗菌胜肽 10
1.5.3擁有特殊胺基酸組成的抗菌胜肽 11
1.6 抗菌活性機制 12
1.6.1抗菌胜肽目標特異性和選擇毒性的機制 12
1.6.2膜的組成、疏水性作用力和極性 12
1.6.3抗菌胜肽作用機制 14
1.6.4 結構參數 22
1.7 實驗目的 26
第二章 材料與方法 27
2.1 培養基 27
2.2 本實驗所用鮑氏不動桿菌菌株 28
2.3 人工合成抗菌胜肽設計 29
2.4 殺菌試驗 29
2.5 最低抑菌濃度測定 (Minimal Inhibitory Concentration；MIC) 30
2.6 最小殺菌濃度測定 （Minimum bactericidal concentration；MBC） 30
2.7 抗菌胜肽協同作用試驗 30
2.8 紅血球溶血試驗 31
2.9掃描式電子顯微鏡 31
2.10細菌細胞膜之通透性試驗 32
第三章 結果 33
3.1 抗菌胜肽設計和胺基酸序列分析結果 33
3.2 抗菌胜肽殺菌實驗結果 34
3.3 抗菌胜肽協同作用實驗結果 34
3.4紅血球溶血試驗結果 35
3.5 掃描式電子顯微鏡結果 35
3.6 細菌細胞膜之通透性試驗結果 36
第四章 討論 37
第五章 圖表 40
圖一、溶菌酶序列之分析結果 40
圖二、Holin序列跨膜區之分析結果 41
圖三、Holin序列分析結果 42
圖四、抗菌胜肽二級結構分析結果 43
圖五（a）、抗菌胜肽協同作用測試之結果 44
圖五（b）、抗菌胜肽協同作用測試之結果 45
圖六、抗菌胜肽溶血活性測試之結果 46
圖七、抗菌胜肽殺菌試驗之結果圖 47
圖八、抗菌胜肽殺菌於高倍率掃描式電子顯微鏡之結果 48
圖九、細菌細胞膜之通透性試驗結果圖 49
表一、本實驗所合成之抗菌胜肽簡介 50
表二、抗菌胜肽對鮑氏不動桿菌的最低抑菌濃度與最小殺菌濃度之結果 51
表三、抗菌胜肽對不同細菌的最低抑菌濃度與最小殺菌濃度之結果 52
第六章 參考文獻 53

1.Baumann, P., Isolation of Acinetobacter from soil and water. J Bacteriol, 1968. 96(1): p. 39-42.
2.Barbe, V., et al., Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res, 2004. 32(19): p. 5766-79.
3.Pagel, J.E. and P.L. Seyfried, Numerical taxonomy of aquatic Acinetobacter isolates. J Gen Microbiol, 1976. 96(2): p. 220-32.
4.Hsueh, P.R., et al., Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan. Emerg Infect Dis, 2002. 8(8): p. 827-32.
5.Jain, R. and L.H. Danziger, Multidrug-resistant Acinetobacter infections: an emerging challenge to clinicians. Ann Pharmacother, 2004. 38(9): p. 1449-59.
6.Russel, M., N.A. Linderoth, and A. Sali, Filamentous phage assembly: variation on a protein export theme. Gene, 1997. 192(1): p. 23-32.
7.Frye, J.G., et al., Host gene expression changes and DNA amplification during temperate phage induction. J Bacteriol, 2005. 187(4): p. 1485-92.
8.Ziedaite, G., et al., The Holin protein of bacteriophage PRD1 forms a pore for small-molecule and endolysin translocation. J Bacteriol, 2005. 187(15): p. 5397-405.
9.Young, I., I. Wang, and W.D. Roof, Phages will out: strategies of host cell lysis. Trends Microbiol, 2000. 8(3): p. 120-8.
10.López, R., E. García, and P. García, Enzymes for anti-infective therapy: phage lysins. Drug Discovery Today: Therapeutic Strategies, 2004. 1(4): p. 469-474.
11.Rotem, S., et al., Identification of antimicrobial peptide regions derived from genomic sequences of phage lysins. Peptides, 2006. 27(1): p. 18-26.
12.Loessner, M.J., Bacteriophage endolysins--current state of research and applications. Curr Opin Microbiol, 2005. 8(4): p. 480-7.
13.Bernhardt, T.G., et al., Breaking free: "protein antibiotics" and phage lysis. Res Microbiol, 2002. 153(8): p. 493-501.
14.Hermoso, J.A., J.L. Garcia, and P. Garcia, Taking aim on bacterial pathogens: from phage therapy to enzybiotics. Curr Opin Microbiol, 2007. 10(5): p. 461-72.
15.During, K., et al., The non-enzymatic microbicidal activity of lysozymes. FEBS Lett, 1999. 449(2-3): p. 93-100.
16.Smith, D.L., et al., Purification and biochemical characterization of the lambda holin. J Bacteriol, 1998. 180(9): p. 2531-40.
17.Hancock, R.E. and R. Lehrer, Cationic peptides: a new source of antibiotics. Trends Biotechnol, 1998. 16(2): p. 82-8.
18.Steiner, H., et al., Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature, 1981. 292(5820): p. 246-8.
19.Fernandez de Caleya, R., et al., Susceptibility of phytopathogenic bacteria to wheat purothionins in vitro. Appl Microbiol, 1972. 23(5): p. 998-1000.
20.Hancock, R.E.W., Peptide Antibiotics. Antimicrobial Agents and Chemotherapy, 1999. 43: p. 1317-1323.
21.Hancock, R.E., The role of cationic antimicrobial peptides in innate host defences. Trends in Microbiology., 2000. 8: p. 402-410.
22.Hancock, R.E., Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis, 2001. 1(3): p. 156-64.
23.Epand, R.M. and H.J. Vogel, Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta, 1999. 1462(1-2): p. 11-28.
24.Bulet, P., R. Stocklin, and L. Menin, Anti-microbial peptides: from invertebrates to vertebrates. Immunol Rev, 2004. 198: p. 169-84.
25.Simmaco, M., et al., Temporins, antimicrobial peptides from the European red frog Rana temporaria. Eur J Biochem, 1996. 242(3): p. 788-92.
26.Reddy, K.V., R.D. Yedery, and C. Aranha, Antimicrobial peptides: premises and promises. Int J Antimicrob Agents, 2004. 24(6): p. 536-47.
27.Cornet, B., et al., Refined three-dimensional solution structure of insect defensin A. Structure, 1995. 3(5): p. 435-48.
28.Otvos, L., Jr., The short proline-rich antibacterial peptide family. Cell Mol Life Sci, 2002. 59(7): p. 1138-50.
29.Brewer, D., H. Hunter, and G. Lajoie, NMR studies of the antimicrobial salivary peptides histatin 3 and histatin 5 in aqueous and nonaqueous solutions. Biochem Cell Biol, 1998. 76(2-3): p. 247-56.
30.Tsai, H. and L.A. Bobek, Human salivary histatins: promising anti-fungal therapeutic agents. Crit Rev Oral Biol Med, 1998. 9(4): p. 480-97.
31.Vunnam, S., P. Juvvadi, and R.B. Merrifield, Synthesis and antibacterial action of cecropin and proline-arginine-rich peptides from pig intestine. J Pept Res, 1997. 49(1): p. 59-66.
32.Selsted, M.E., et al., Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem, 1992. 267(7): p. 4292-5.
33.Yeaman, M.R. and N.Y. Yount, Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev, 2003. 55(1): p. 27-55.
34.Tytler, E.M., et al., Molecular basis for prokaryotic specificity of magainin-induced lysis. Biochemistry, 1995. 34(13): p. 4393-401.
35.Koppelman, C.M., et al., Escherichia coli minicell membranes are enriched in cardiolipin. J Bacteriol, 2001. 183(20): p. 6144-7.
36.Tossi, A., L. Sandri, and A. Giangaspero, Amphipathic, alpha-helical antimicrobial peptides. Biopolymers, 2000. 55(1): p. 4-30.
37.Dathe, M., et al., Optimization of the antimicrobial activity of magainin peptides by modification of charge. FEBS Lett, 2001. 501(2-3): p. 146-50.
38.Matsuzaki, K., et al., Modulation of magainin 2-lipid bilayer interactions by peptide charge. Biochemistry, 1997. 36(8): p. 2104-11.
39.Mavri, J. and H.J. Vogel, Ion pair formation of phosphorylated amino acids and lysine and arginine side chains: a theoretical study. Proteins, 1996. 24(4): p. 495-501.
40.Peschel, A., et al., Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J Biol Chem, 1999. 274(13): p. 8405-10.
41.Yang, L., et al., Crystallization of antimicrobial pores in membranes: magainin and protegrin. Biophys J, 2000. 79(4): p. 2002-9.
42.Bello, J., H.R. Bello, and E. Granados, Conformation and aggregation of melittin: dependence on pH and concentration. Biochemistry, 1982. 21(3): p. 461-5.
43.Dathe, M. and T. Wieprecht, Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim Biophys Acta, 1999. 1462(1-2): p. 71-87.
44.Oishi, O., et al., Conformations and orientations of aromatic amino acid residues of tachyplesin I in phospholipid membranes. Biochemistry, 1997. 36(14): p. 4352-9.
45.Toke, O., Antimicrobial peptides: New candidates in the fight against bacterial infections. Biopolymers, 2005. 80(6): p. 717-735.
46.Yang, L., et al., Barrel-stave model or toroidal model? A case study on melittin pores. Biophys J, 2001. 81(3): p. 1475-85.
47.Hara, T., et al., Effects of peptide dimerization on pore formation: Antiparallel disulfide-dimerized magainin 2 analogue. Biopolymers, 2001. 58(4): p. 437-46.
48.Uematsu, N. and K. Matsuzaki, Polar angle as a determinant of amphipathic alpha-helix-lipid interactions: a model peptide study. Biophys J, 2000. 79(4): p. 2075-83.
49.Shai, Y. and Z. Oren, From "carpet" mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides. Peptides, 2001. 22(10): p. 1629-41.
50.Christensen, B., et al., Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proc Natl Acad Sci U S A, 1988. 85(14): p. 5072-6.
51.Brogden, K.A., Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol, 2005. 3(3): p. 238-50.
52.Mangoni, M.L., et al., Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli. Biochem J, 2004. 380(Pt 3): p. 859-65.
53.Lehrer, R.I., et al., Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J Clin Invest, 1989. 84(2): p. 553-61.
54.Gennaro, R. and M. Zanetti, Structural features and biological activities of the cathelicidin-derived antimicrobial peptides. Biopolymers, 2000. 55(1): p. 31-49.
55.Park, C.B., et al., Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of buforin II. Proc Natl Acad Sci U S A, 2000. 97(15): p. 8245-50.
56.Futaki, S., et al., Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem, 2001. 276(8): p. 5836-40.
57.Casteels, P., et al., Functional and chemical characterization of Hymenoptaecin, an antibacterial polypeptide that is infection-inducible in the honeybee (Apis mellifera). J Biol Chem, 1993. 268(10): p. 7044-54.
58.Boman, H.G., B. Agerberth, and A. Boman, Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infect Immun, 1993. 61(7): p. 2978-84.
59.Patrzykat, A., et al., Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli. Antimicrob Agents Chemother, 2002. 46(3): p. 605-14.
60.Falla, T.J., D.N. Karunaratne, and R.E. Hancock, Mode of action of the antimicrobial peptide indolicidin. J Biol Chem, 1996. 271(32): p. 19298-303.
61.Dathe, M., et al., Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides. FEBS Lett, 1997. 403(2): p. 208-12.
62.Bessalle, R., et al., Augmentation of the antibacterial activity of magainin by positive-charge chain extension. Antimicrob Agents Chemother, 1992. 36(2): p. 313-7.
63.Eisenberg, D., A problem for the theory of biological structure. Nature, 1982. 295(5845): p. 99-100.
64.Finer-Moore, J. and R.M. Stroud, Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc Natl Acad Sci U S A, 1984. 81(1): p. 155-9.
65.Eisenberg, D., Three-dimensional structure of membrane and surface proteins. Annu Rev Biochem, 1984. 53: p. 595-623.
66.Sitaram, N. and R. Nagaraj, Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity. Biochim Biophys Acta, 1999. 1462(1-2): p. 29-54.
67.Wilkins, M.R., et al., Protein identification and analysis tools in the ExPASy server. Methods Mol Biol, 1999. 112: p. 531-52.
68.Rice, P., I. Longden, and A. Bleasby, EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet, 2000. 16(6): p. 276-7.
69.Cole, C., J.D. Barber, and G.J. Barton, The Jpred 3 secondary structure prediction server. Nucleic Acids Res, 2008. 36(Web Server issue): p. W197-201.
70.McGuffin, L.J., K. Bryson, and D.T. Jones, The PSIPRED protein structure prediction server. Bioinformatics, 2000. 16(4): p. 404-5.
71.Tossi A, S.L., Giangaspero A, New consensus hydrophobicity scale extended to non-proteinogenic mino acids. Peptides, 2002.
72.Nilsson, J., B. Persson, and G. von Heijne, Consensus predictions of membrane protein topology. FEBS Lett, 2000. 486(3): p. 267-9.
73.Conlon, J.M., et al., Potent and rapid bactericidal action of alyteserin-1c and its [E4K] analog against multidrug-resistant strains of Acinetobacter baumannii. Peptides, 2010. 31(10): p. 1806-10.
74.Elemam, A., J. Rahimian, and M. Doymaz, In vitro evaluation of antibiotic synergy for polymyxin B-resistant carbapenemase-producing Klebsiella pneumoniae. J Clin Microbiol, 2010. 48(10): p. 3558-62.
75.Nomura, K., et al., Induction of morphological changes in model lipid membranes and the mechanism of membrane disruption by a large scorpion-derived pore-forming peptide. Biophys J, 2005. 89(6): p. 4067-80.
76.Haro, A., et al., Reconstitution of holin activity with a synthetic peptide containing the 1-32 sequence region of EJh, the EJ-1 phage holin. J Biol Chem, 2003. 278(6): p. 3929-36.
77.Becker, S.C., J. Foster-Frey, and D.M. Donovan, The phage K lytic enzyme LysK and lysostaphin act synergistically to kill MRSA. FEMS Microbiol Lett, 2008. 287(2): p. 185-91.
78.Matsuzaki, K., et al., Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria. Biochim Biophys Acta, 1997. 1327(1): p. 119-30.
79.Henk, W.G., et al., The morphological effects of two antimicrobial peptides, hecate-1 and melittin, on Escherichia coli. Scanning Microsc, 1995. 9(2): p. 501-7.