Unveiling the mechanism of bactericidal activity of a cecropin A-fused endolysin LNT103
- 주제(키워드) Gram-negative bacteria , endolysin , lipopolysaccharide , membrane permeability
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 윤현진
- 발행년도 2023
- 학위수여년월 2023. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032689
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Bacteriophages encode endolysins at the end of their lytic cycle to degrade the peptidoglycan layer of the host bacterium, leading to release of progeny phages. In virtue of the bacteriolytic activity, endolysins have been explored as an alternative antibacterial agent. However, the outer membrane present in Gram-negative bacteria obstructs the access of exogenous endolysins to the peptidoglycan lying beneath the outermost membranous structure. In order to overcome the restriction of intrinsic endolysins against Gram-negative bacteria, an endolysin encoded by Pseudomonas aeruginosa phage PBPA90 was engineered by substituting the 15 amino acids (mtPA90) and further by fusing the antimicrobial peptide cecropin A to its N-terminus (LNT103). Cecropin A of LNT103 improved the interaction with lipopolysaccharides (LPS) and accelerated the disruption of both bacterial membranes including the outer membrane and the inner membrane. LNT103 did not compromise the integrity of LPS structure. However, an LPS mutant strain with an altered lipid A structure was more susceptible to both endolysins of mtPA90 and LNT103, suggesting that the integrity of lipid A is important to obstruct endolysin penetration into bacterial membrane. This study revealed that an endolysin fused with cecropin A could not only degrade its intrinsic target peptidoglycan layers but also destabilize both bacterial membranes, leading to faster inactivation of Gram-negative bacteria.
more목차
1. Introduction 1
2. Materials and Methods 3
2.1 Bacterial strains and growth conditions 3
2.2 Expression and purification of endolysins 5
2.3 Structural modeling 5
2.4 Bacterial viability assay 5
2.5 Turbidity reduction assay 6
2.6 LPS extraction and analysis 6
2.7. Interaction with LPS 7
2.8. SYTO9/propidium iodide (PI) staining 7
2.9. β-lactamase and β-galactosidase assay 8
2.10. Electron microscopy analysis 8
2.11. Statistical analysis 9
3. Results 10
3.1 Cecropin A fusion facilitates the interaction with bacterial LPS 10
3.2 Lipid A of LPS impedes the penetration of endolysins across the outer membrane 14
3.3 Cecropin A fusion increased the permeability across the inner and outer membranes 23
4. Discussion 28
5. Conclusion 30
6. Reference 31
