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Unveiling the mechanism of bactericidal activity of a cecropin A-fused endolysin LNT113
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 윤현진 | - |
| dc.contributor.author | 조정익 | - |
| dc.date.accessioned | 2025-01-25T01:36:08Z | - |
| dc.date.available | 2025-01-25T01:36:08Z | - |
| dc.date.issued | 2023-08 | - |
| dc.identifier.other | 32865 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/24661 | - |
| dc.description | 학위논문(석사)--분자과학기술학과,2023. 8 | - |
| dc.description.tableofcontents | 1. Introduction 1 <br>2. Materials and Methods 4 <br> 2.1. Bacterial strains and growth conditions 4 <br> 2.2. Expression and purification of endolysins 6 <br> 2.3. Bacterial viability and turbidity reduction assay 6 <br> 2.4. Confocal microscopy 6 <br> 2.5. Limulus amoebocyte lysate (LAL) assay 7 <br> 2.6. LPS extraction and analysis 7 <br> 2.7. SYTO 9/propidium iodide (PI) staining 8 <br> 2.8. Membrane permeability test using β-lactamase and β-galactosidase assay 8 <br> 2.9 Western blot analysis 9 <br> 2.10. Statistical analysis 9 <br>3. Results 10 <br> 3.1. Cecropin A fusion increases the interaction with bacterial LPS 10 <br> 3.2. Integrity of core oligosaccharide and lipid A is critical for LNT113 activity 13 <br> 3.3. Cecropin A fusion increased the permeability across the IM 19 <br>4. Discussion 27 <br>5. Conclusion 29 <br>6. Reference 30 | - |
| dc.language.iso | kor | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Unveiling the mechanism of bactericidal activity of a cecropin A-fused endolysin LNT113 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 대학원 | - |
| dc.contributor.alternativeName | Jeongik Cho | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2023-08 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | T000000032865 | - |
| dc.identifier.url | https://dcoll.ajou.ac.kr/dcollection/common/orgView/000000032865 | - |
| dc.subject.keyword | Endolysin | - |
| dc.subject.keyword | Gram-negative bacteria | - |
| dc.subject.keyword | cecropin A | - |
| dc.description.alternativeAbstract | Endolysins are lytic enzymes produced by bacteriophages at the end of their lytic cycle and degrade the peptidoglycan layer of the bacterial cell wall. Thus, they have been extensively explored as a promising antibacterial agent to replace or supplement current antibiotics. Gram-negative bacteria, however, are prone to resist exogenous endolysins owing to their protective outer membrane. We previously engineered endolysin EC340, encoded by the Escherichia coli phage PBEC131, by substituting its seven amino acids and fusing an antimicrobial peptide cecropin A at its N-terminus. The engineered endolysin LNT113 exerted superior activity to its intrinsic form. This study investigated how cecropin A fusion facilitated the bactericidal activity of LNT113 toward Gram-negative bacteria. Cecropin A of LNT113 markedly increased the interaction with lipopolysaccharides, while the E. coli defective in the core oligosaccharide was less susceptible to endolysins, implicating the interaction between the core oligosaccharide and endolysins. In fact, E. coli with compromised lipid A construction was more vulnerable to LNT113 treatment, suggesting that the integrity of the lipid A layer was important to resist the internalization of LNT113 across the outer membrane. Cecropin A fusion further accelerated the inner membrane destabilization, thereby enabling LNT113 to deconstruct it promptly. Owing to the increased membrane permeability, LNT113 could inactivate some Gram-positive bacteria as well. This study demonstrates that cecropin A fusion is a feasible method to improve the membrane permeability of endolysins in both Gram-negative and Gram-positive bacteria. | - |
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- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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