Indolicidin為一有潛力的抗生藥物，其具有相當廣泛的抗生活性，對抗細菌、真菌、病毒皆有不錯的能力，且是一段具有不錯經濟效益的短鏈胜肽。然而，Indolicidin對人類紅血球的溶血活性卻限制其進一步的發展。雖然，擁有低溶血血性的IL之類似物(ILK7、ILF89、ILK7F89)已經被設計，但它們的行為機制仍然不是非常清楚。本研究主要是探討其生物活性與膜擾亂之間的關係。我們藉由螢光異位向性實驗研究膜擾亂的情形，並以恆溫滴定微卡計研究胜肽與膜之間的熱變化。而我們使用兩種仿生物細胞膜的微脂粒，仿細菌細胞膜(POPG/POPC=1, PG/PC-SUVs)與仿人類紅血球細胞膜(純 POPC, PC-SUVs)。而我們進一步探討IL及其類似物對抗革蘭氏陽性菌(staphylococcus epidermidis)的活性，結果發現IL的類似物皆擁有較IL更強或相當的抗菌活性。另一方面，在POPC微脂粒的作用中，胜肽對膜的擾亂與膜擾亂焓和其溶血活性沒有很強烈的關係，所以對於溶血活性主要仍是與其胜肽吸附量有關。因此，對於抗菌活性胜膜的擾亂程度確實是一很重要的影響因素。

Indolicidin (IL) is a cationic antibacterial peptide with broad-spectrum against many pathogens; therefore, it is a potential peptide antibiotic. However, the hemolytic activity of IL limits its clinical application. Although the IL-analogues (ILK7, ILF89 and ILK7F89) with lower hemolysis have been designed, the action mechanisms of them were still under debating. In this study, we tried to examine the relationships between bioactivity and membrane perturbation. The membrane perturbation was investigated through the help of fluorescence anisotropic measurement. The membrane association enthalpy and membrane perturbation enthalpy were examined by isothermal titration calorimeter (ITC) and peptide-membrane adsorption isotherm. Two types of small unilamellar vesicles (SUVs) were used to mimic the bacterial-like membrane (POPG/POPC=1) and erythrocyte-like membrane (pure POPC). The antibacterial activity of IL and its analogues against Gram-positive bacteria (staphylococcus epidermidis) were also examined. The results revealed that IL-analogues with enhancing or similar antibacterial activity than that of IL peptide.
The interaction of peptide and PG/PC-SUVs revealed that the peptide with high antibacterial activity owns strongly membrane perturbation and highly membrane perturbation enthalpy. On the contrary, as peptide and PC-SUVs interaction, the membrane perturbation of peptide action and membrane perturbation enthalpy are not strongly correlated with its hemolytic activity. Particularly, the hemolysis is related to the amounts of peptide adsorption. Consequently, the membrane perturbation suffered by peptide is an important implication on antibacterial activity.

1. Diamond, R.E.W.H.a.G., The role of cationic antimicrobial peptides in innate host defences. TRENDS IN MICROBIOLOGY, 2000. 8(9): p. 402-410.
2. Yechiel Shai , Z.O., From “carpet” mechanism to de-novo designed diastereomeric cellselective antimicrobial peptidesPeptides, 2001. 22(2001): p. 1629–1641.
3. Shai, Y., Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by K-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochimica et Biophysica Acta. 1462(1999): p. 55-70.
4. Revital Halevy , A.R., Sofiya Kolusheva , Robert E.W. Hancock , Raz Jelinek, Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay. Peptides, 2003. 24(2003): p. 1753–1761.
5. Rozek, A., Friedrich, C. L., and Hancock, R. E. W., Structure of the Bovine Antimicrobial Peptide Indolicidin Bound to Dodecylphosphocholine and Sodium Dodecyl Sulfate Micelles. Biochemistry, 2000. 39(51): p. 15765-15774.
6. Schluesener HJ, R.S., Melms A, Jung S., Leukocytic antimicrobial peptides kill autoimmune T cells. Journal of neuroimmunology, 1993. 47.
7. Giacometti, A., Cirioni, O., Greganti, G. et al, In Vitro Activities of Membrane-Active Peptides against Gram-Positive and Gram-Negative Aerobic Bacteria. Antimicrob. Agents Chemother, 1998. 42(12): p. 3320-3324.
8. Friedrich, C.L., Moyles, D., Beveridge, T. J. et al, Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria,” Antimicrob. Agents Chemother, 2000. 44(8): p. 2086-2092.
9. Falla, T.J., and Hancock, R. E., Improved activity of a synthetic indolicidin analog Antimicrob. Agents Chemother, 1997. 41(4): p. 771-775.
10. Subbalakshmi, C., Krishnakumari, V., Sitaram, N. et al, Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes. J. Biosci, 1998. 23: p. 9-13.
11. Lee, D.G., Kim, H. K., Kim, S. A. et al, Fungicidal effect of indolicidin and its interaction with phospholipid membranes. Biochemical and Biophysical Research Communications, 2003. 305(2): p. 305-310.
12. Stephen B. Aley, M.Z., Michel Hetsko, Michel E. Selsted, and Frances D. Gillin,, Killing of Giardia lamblia by Cryptdins and Cationic Neutrophil Peptides. Infection and Immunity, 1994 62(12): p. 5397-5403.
13. Robinson, W.E., McDougall, B., Tran, D. et al., Anti-HIV-1 activity of indolicidin, an antimicrobial peptide from neutrophils. J Leukoc Biol, 1998. 63( 1): p. 94-100.
14. Vanesa, C.A.M., and Viviana, C., Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus. International journal of antimicrobial agents, 2004. 23( 4): p. 382-389.
15. Falla, T.J., Karunaratne, D. N., and Hancock, R. E. W, Mode of Action of the Antimicrobial Peptide Indolicidin. J. Biol. Chem, 1996. 271(32): p. 19298-19303.
16. Manhong Wu, E.M., Roland Benz, Robert E. W. Hancock, Mechanism of Interaction of Different Classes of Cationic Antimicrobial Peptides with Planar Bilayers and with the Cytoplasmic Membrane of Escherichia coli. Biochemistry, 1999. 38(22): p. 7235-7242.
17. Yang Sung-Tae, S.S.Y., K.-S. H. et al., Design of perfectly symmetric Trp-rich peptides with potent and broad-spectrum antimicrobial activities. International journal of antimicrobial agents, 2006. 27(4): p. 325-330.
18. Zhang, L., Rozek, A., and Hancock, R. E. W., Interaction of Cationic Antimicrobial Peptides with Model Membranes. J. Biol. Chem, 2001. 276(38): p. 35714-35722.
19. Yau, W.-M., Wimley, W. C., Gawrisch, K. et al., The Preference of Tryptophan for Membrane Interfaces. Biochemistry, 1998. 37(42): p. 14713-14718.
20. Schibli, D.J., Epand, R. F., Vogel, H. J. et al., Tryptophan-rich antimicrobial peptides: comparative properties and membrane interactions. Biochemistry and Cell Biology, 2002. 80: p. 667-677.
21. Zhao, H., Mattila, J.-P., Holopainen, J. M. et al., Comparison of the Membrane Association of Two Antimicrobial Peptides, Magainin 2 and Indolicidin. Biophysical Journal, 2001. 81(5): p. 2979-2991.
22. Chilukuri Subbalakshmi, N.S., Mechanism of antimicrobial action of indolicidin. FEMS Microbiology Letters, 1998. 160(1): p. 91-96.
23. Hsu, C.-H., Chen, C., Jou, M.-L. et al., Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA. Nucl. Acids Res, 2005. 33(13): p. 4053-4064.
24. Shaw, J.E., Alattia, J.-R., Verity, J. E. et al., Mechanisms of antimicrobial peptide action: Studies of indolicidin assembly at model membrane interfaces by in situ atomic force microscopy. Journal of Structural Biology, 2006. 154(1): p. 42-58.
25. Hsu, J.C.Y., and Yip, C. M., Molecular Dynamics Simulations of Indolicidin Association with Model Lipid Bilayers. Biophys. J, 2007 92(12): p. 100-102.
26. Ahmad, I., Perkins, W. R., Lupan, D. M. et al., Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity. Biochimica et Biophysica Acta (BBA)- Biomembranes, 1995. 1237(2): p. 109-114.
27. Subbalakshmi, C., Krishnakumari, V., Nagaraj, R. et al., Requirements forantibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin. FEBS Letters, 1996. 395(1): p. 48-52.
28. Subbalakshmi, C., Bikshapathy, E., Sitaram, N. et al., Antibacterial and Hemolytic Activities of Single Tryptophan Analogs of Indolicidin. Biochemical and Biophysical Research Communications, 2000. 274(3): p. 714-716.
29. Papo, N., and Shai, Y., Exploring Peptide Membrane Interaction Using Surface Plasmon Resonance: Differentiation between Pore Formation versus Membrane Disruption by Lytic Peptides. Biochemistry, 2003. 42(2): p. 458-466.
30. Mechler, A., Praporski, S., Atmuri, K. et al., Specific and Selective Peptide-Membrane Interactions Revealed Using Quartz Crystal Microbalance. Biophysical Journal, 2007. 93(11): p. 3907-3916.
31. Adam A. Strömstedt , L.R., Artur Schmidtchen , Martin Malmsten Interaction between amphiphilic peptides and phospholipid membranes. Current Opinion in Colloid & Interface Science, 2010(12).
32. Ana Lu´ cia C.F. Souto, E.F.P., Clo´ vis R. Nakaie, Shirley Schreier,, Fluorescence and circular dichroism study of the interaction between indolicidin, a tryptophan-rich antimicrobial peptide, and model membranes. Progr Colloid Polym Sci, 2004. 128.
33. Royer, C.A., Probing Protein Folding and Conformational Transitions with Fluorescence. Chemical Reviews, 2006. 106(5): p. 1769-1784.
34. Caputo, G.A., and London, E., Using a Novel Dual Fluorescence Quenching Assay for Measurement of Tryptophan Depth within Lipid Bilayers To Determine Hydrophobic α-Helix Locations within Membranes. Biochemistry, 2003. 42(11): p. 3265-3274.
35. L. Miccoli. Szczepaniak, D., Savonni~re, .Muller, C. Carr6, and a.M. Donner, Interaction of a Phosphatidylcholine Derivative of 1,6- Diphenyl-l,3,5-hexatriene (DPH) with Intact Living Cells: Steady-State Fluorescence Polarization and Phase Fluorometry Studies Journal of Fluorescence, 1993. 3(4).
36. M. Mun˜oz , N.R.I.H.V.G.C.M.M.A.B., Fluorescence analysis of the interaction of two peptide sequences of hepatitis GB virus C with liposomes. Talanta, 2003. 60.
37. Seelig, J., Titration calorimetry of lipid–peptide interactions. Biochimica et Biophysica Acta, 1997. 1331.