Effect of Proline to Peptoid Residue Substitution on the Structure, Cell Selectivity and Mechanism of Antibacterial Action of Antimicrobial Peptide Tritrpticin-amide
- Author(s)
- 주만용
- Issued Date
- 2006
- Abstract
- 본 연구에서는 proline을 포함하는 β-turn 항균 펩타이드에 있어서 proline의 peptoid residue로의 치환이 세균특이성 및 항균작용기작에 미치는 영향을 조사하기 위하여, 항균 펩타이드 tritrpticin-amide (TP; VRRFPWWWPFLRR-NH2) 및 TP의 5, 9번째에 있는 2개의 proline잔기를 Ala-peptiod residue (Nala), Leu-peptiod residue (Nleu), Phe-peptiod residue (Nphe), Lys-peptiod residue (Nlys)로 각각 치환시킨 펩타이드를 합성하였다.
TP의 proline을 Nala, Nleu 또는 Nphe로 치환시킨 펩타이드(TPa, TPl 및 TPf)는 거의 박테리아의 항균활성이 유지되었으나 포유동물세포(mammalian cells)에 있어서의 강한 세포독성(cytotoxicity)을 나타내었다. 반면에 proline의 Nlys로의 치환 (TPk)은 항균활성을 증가시켰으며, 포유동물세포에 대한 세포독성을 현저히 감소시켰다.
트립토판 형광실험 (Tryptophan fluorescence experiment)을 통하여 항균 펩타이드 TPk의 박테리아 세포특이성은 negatively charged phospholipid에 대한 우선적 상호작용과 관련성이 있다는 사실을 알았다. 흥미롭게도, TPk는 Staphylococcus aureus에 대한 membrane deploarization 활성 및 negatively charged vesicle내에 포획된 fluorescent dye의 leakage 활성에 있어서 매우 덜 효과적이였다. 더구나, confocal laser-scanning microscopy를 통하여 항균 펩타이드 TPk는 Escherichia coli 및Staphylococcus aureus의 세포막을 통과하여 세포질내에 축적되지만, TP 및 TPf는 세포막을 통과하지 않고 세포막 표면이나 외부에 결합된다는 사실을 알았다.
Gel retardation assay를 통하여 항균 펩타이드 TPk는 TP 및 TPf보다 매우 강한 DNA 결합능력을 보였다. 이들 결과로부터TPk의 살균작용기작은 세균세포막을 통과한 후, 세포내 표적 (intracellular target)의 저해에 의한 것임을 시사하였다. 또한 TP에 있어서 proline을 lysine으로 치환시킨 펩타이드 TPK는 Staphylococcus aureus의 cytoplasmic membrane을 효과적으로 depolarization시킨다는 사실을 알았다. TPK는 박테리아 세포막을 모방하는 리포좀 (bacterial membrane-mimicking liposome)으로부터 빠르고, 효과적인 dye leakage를 유도하였다. 이 사실은 항균 펩타이드 TPk의 박테리아 세포막에 효과적인 통과(penetration)는 Lys peptoid residue의 치환에 의한 positive charge의 증가 및 펩타이드의 backbone change에 관련된 이중효과 때문이라는 사실을 시사하였다.
이상의 연구 결과로부터 tritrpticin과 같이 proline을 포함하는 β-turn구조 항균 펩타이드에 있어서 proline잔기의 Lys peptoid residue (Nlys)로의 치환은 세포내 표적 작용기작 (intracellular target mechanism)을 나타내는 새로운 박테리아세포 특이성 항균 펩타이드의 설계를 위한 유용한 수단임을 알았다.|To investigate the effect of proline → peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing β-turn antimicrobial peptides, tritrpticin-amide (TP, VRRFPWWWPFLRR-NH2) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 were replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue) or Nlys (Lys peptoid residue) were synthesized, respectively. Peptides with Pro → Nleu (TPl) or Pro → Nphe (TPf) substitution retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro → Nlys (TPk) substitution increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the cell selectivity of TPk was closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at causing the leakage of a fluorescent dye entrapped within negatively charged vesicles and depolarizing of the membrane potential of Staphylococcus aureus and Escherichia coli spheroplasts. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is maybe due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes. TPK effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro → Nlys substitution in Pro-containing β-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.
- Alternative Title
- 항균펩타이드 tritrpticin-amide의 구조, 세포특이성 및 항균작용기작에 있어서 proline의 peptoid잔기로의 치환이 미치는 영향
- Alternative Author(s)
- Zhu, Wan Long
- Affiliation
- 조선대학교 대학원
- Department
- 일반대학원 생물신소재학과
- Advisor
- 신송엽
- Awarded Date
- 2007-02
- Table Of Contents
- CONTENTS = Ⅰ
LIST OF TABLES = Ⅳ
LIST OF FIGURES = Ⅴ
ABSTRACT = 1
국문 초록 = 54
I. INTRODUCTION = 3
II. MATERIALS AND METHODS = 6
II-1. Materials = 6
II-2. Peptide Synthesis and purification = 6
II-3. Microorganisms = 7
II-4. Antimicrobial activity assay = 7
II-5. Hemolytic activity assay = 8
II-6. Cytotoxicity against mammalian cells = 9
II-7. Circular dichroism (CD) spectroscopy = 9
II-8. Preparation of small unilamellar vesicles (SUVs) = 10
II-9. Tryptophan fluorescence blue shift = 10
II-10. Tryptophan fluorescence quenching by acrylamide = 11
II-11. Preparation of calcein-loaded LUVs and leakage assay = 11
II-12. Membrane depolarization assay with bacteria = 12
II-13. Confocal laser-scanning microscope in bacterial strains = 14
II-14. DNA binding assay = 14
II-15. Peptide internalization and visualization in mammalian cells by confocal laser-scanning microscope = 15
Ⅲ. RESULTS = 16
Ⅲ-1. Peptide design = 16
Ⅲ-2. Antimicrobial activities = 16
Ⅲ-3. Hemolytic activities = 17
Ⅲ-4. Cytotoxicity against mammalian cells = 17
Ⅲ-5. CD spectroscopy = 18
Ⅲ-6. Tryptophan fluorescence blue shift = 18
Ⅲ-7. Tryptophan fluorescence quenching studies = 19
Ⅲ-8. Peptide-induced dye leakage from negatively charged LUVs = 20
Ⅲ-9. Membrane potential depolarization = 20
Ⅲ-10. Confocal laser-scanning microscopy in bacterial strains = 21
Ⅲ-11. DNA binding activity = 22
Ⅲ-12. Confocal laser-scanning microscopy in HeLa cells = 22
Ⅳ. DISCUSSION = 38
Ⅴ. REFERENCES = 44
Ⅵ. ACKNOWLEDGMENT = 52
Ⅶ. ABBREVIATIONS = 53
- Degree
- Master
- Publisher
- 조선대학교 대학원
- Citation
- 주만용. (2006). Effect of Proline to Peptoid Residue Substitution on the Structure, Cell Selectivity and Mechanism of Antibacterial Action of Antimicrobial Peptide Tritrpticin-amide.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/6471
http://chosun.dcollection.net/common/orgView/200000233842
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