Title Page
Contents
ABSTRACT 8
Chapter 1. Introduction 9
Chapter 2. Material and Methods 16
2.1. Peptide synthesis 16
2.2. Bacteria strains 16
2.3. Minimum inhibitory concentration (MIC) 16
2.4. Cytotoxicity 16
2.5. Hemolysis 17
2.6. Biofilm inhibition assay 17
2.7. Bacteria outer membrane permeability test 18
2.8. Circular dichroism (CD) analysis 18
2.9. Antimicrobial activity time-course assay 18
2.10. Bacteria morphology imaging 18
2.11. Limulus amebocyte lysate (LAL) assay 19
2.12. BODIPY-TR-cadaverine (BC) displacement assay 19
2.13. Isothermal titration calorimetry (ITC) 19
2.14. Saturation transfer difference (STD)-nuclear magnetic resonance (NMR) 20
2.15. Suppression of LPS-induced Inflammatory cytokines 20
2.16. Suppression of colistin-resistant bacteria-induced inflammatory cytokines 20
2.17. Inhibition of nitric oxide (NO) production by T. ni cecropin in response to various Toll-like receptors (TLRs) 21
2.18. Secreted embryonic alkaline phosphatase (SEAP) assay 21
2.19. Surface plasmon resonan ce (SPR) 21
2.20. Flow cytometry 22
2.21. Data analysis 22
Chapter 3. Results 23
3.1. Antibacterial Activities of T. ni cecropin. 23
3.2. Toxicity of T. ni cecropin to mammalian cells 24
3.3. T. ni cecropin inhibits ColREC biofilm formation 25
3.4. Antibacterial mechanisms of T. ni cecropin against Gram-negative bacteria 26
3.4.1. LPS neutralizing capacity of T. ni cecropin 26
3.4.2. Membrane depolarization ability of T. ni cecropin against E. coli 27
3.4.3. T. ni cecropin induces E. coli cell membrane damage 28
3.4.4. T. ni cecropin directly interacts with LPS 29
3.4.5. Secondary structure of T. ni cecropin 30
3.5. Inhibition of cytokine production in RAW 264.7 cells stimulated by LPS or ColREC 31
3.6. T. ni cecropin selectively targets the TLR4-inflammatory signaling pathway 32
Chapter 4. Discussion 35
Chapter 5. Conclusion 38
Reference 39
Abstract (in Korean) 44
Table 1. Minimal inhibitory concentration of antimicrobial peptides against various bacteria. 23
Figure 1. Structures of (a) polymyxins and (b) Trichoplusia ni cecropin A (T. ni cecropin). Red indicates hydrophilic and blue indicates hydrophobic 10
Figure 2. Chemical structure of lipid A moiety of Escherichia coli 12
Figure 3. Mechanisms of colistin-resistance in Gram-negative bacteria 12
Figure 4. Mechanism of antimicrobial peptide against bacteria membrane 14
Figure 5. Cytotoxicity of peptides against (a) RAW 264.7 cells, (b) L929, and (c) sheep red blood cells (sRBCs). *p 〈 0.05, ** p 〈 0.01, *** p 〈 0.001; and ns, nonsignificant compared... 25
Figure 6. Inhibitory effects of peptides in biofilm assays performed in (a) ColREC 1557 and (b) ColREC 12. Data are presented as the mean ± SEM from triplicate experiments. *p 〈 0.05, ** p... 26
Figure 7. Measurement of interaction between lipopolysaccharide (LPS) and peptides. (a) Limulus amebocyte lysate assay showing the LPS neutralization capacities of peptides. (b)... 27
Figure 8. Fluorescence intensity as measured via peptide-induced membrane disruption. Relative fluorescence intensities in (a) E. coli, (b) ColREC 1557, and (c) ColREC 12 as measured using 1-... 28
Figure 9. (a) Time-dependent killing activity of T. ni cecropin. Field emission-scanning electron microscopy images showing the morphology of E. coli treated with T. ni cecropin. (b)... 29
Figure 10. Reference one-dimensional nuclear magnetic resonance (NMR) spectrum for T. ni cecropin (top) and saturation transfer difference (STD) NMR spectrum obtained through... 30
Figure 11. Secondary structures of T. ni cecropin in membrane mimetic environments as observed via circular dichroism spectroscopy. DPC: dodecylphosphorcholine; SDS: sodium... 30
Figure 12. Anti-inflammatory effect in RAW 264.7 cells induced by stimulants. Graphs show the dose-dependent nitrite and interleukin 6 (IL-6) inhibitory effect of the peptide (a, b) on LPS... 32
Figure 13. Measurement of interaction between Toll-like receptor 4 (TLR4) and peptides. (a) Specific agonist treatment for each TLR receptor shows TLR4 selectivity of T. ni... 34