Abstract Melimine and Mel4 are cationic antimicrobial peptides which can resist biofilm development once bound to biomaterials. The aim of the current study was to determine the mode of action of bound melimine and Mel4 against S. aureus. The peptides were covalently attached to glass using an azidobenzoic acid linker. The amount of attached peptides was confirmed by XPS and amino acid analysis and their covalent attachment by SDS extraction. The release of autolysins after interaction of S. aureus with immobilized peptides was determined in cell free supernatants. The interaction of immobilized peptides with lipoteichoic acid was confirmed by ELISA. Membrane damage by surface bound peptides was assessed using DiSC(3)-5 (membrane potential sensitive), Syto-9 (membrane permeable) and PI (membrane impermeable) dyes with fluorescence microscopy. Release of ATP and nucleic acids (DNA/RNA) was measured in the surrounding fluid. Attachment of the peptides resulted in increased N% for melimine (5.4 ± 1.8%) and for Mel4 (4.8 ± 1.8%). The concentrations of immobilised amino acids were 0.297 nmole for melimine and 0.358 nmole for Mel4. SDS extraction released < 15% of peptides from the glass. The immobilized peptides bound ≥ 4 times more LTA than control surfaces. More autolysins (8 ± 2%; p = 0.026) were released from Mel4 than melimine or control surfaces. Membrane depolarization occurred at 15 min and was associated with a reduction in bacterial viability ≥ 37% for both peptides (p < 0.001). Disruption of the membrane potential resulted in loss of ATP from melimine (0.9 ± 0.4 nM) or Mel4 (0.6 ± 0.3 nM) coated surfaces compared to control (p < 0.001). Melimine coatings yielded 27 ± 11% (p = 0.026) and Mel4 gave 17 ± 12% (p = 0.150) PI stained cells after 4 h. DNA/RNA was released only by melimine coatings (2.1 ± 0.1 times; p = 0.011) compared to process control at 6 h. Both bound peptides resulted in the release of ATP, but only melimine released DNA/RNA while Mel4-coating resulted in the release of autolysins. Since the mode of action of melimine and Mel4 relate to the cell surface, they have potential for the development of infection-resistant implants.

中文翻译：

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表面结合的抗菌肽三聚氰胺和 Mel4 与金黄色葡萄球菌的相互作用

摘要 三聚氰胺和Mel4是阳离子抗菌肽，与生物材料结合后可抵抗生物膜的形成。当前研究的目的是确定结合三聚氰胺和 Mel4 对金黄色葡萄球菌的作用模式。使用叠氮苯甲酸接头将肽共价连接到玻璃上。通过 XPS 和氨基酸分析以及通过 SDS 提取的共价连接来确认连接肽的量。在无细胞上清液中测定金黄色葡萄球菌与固定肽相互作用后自溶素的释放。ELISA 证实了固定肽与脂磷壁酸的相互作用。使用 DiSC(3)-5（膜电位敏感）、Syto-9（膜渗透）和 PI（膜不渗透）染料和荧光显微镜评估表面结合肽对膜的损伤。测量周围液体中 ATP 和核酸 (DNA/RNA) 的释放。肽的附着导致三聚氰胺 (5.4 ± 1.8%) 和 Mel4 (4.8 ± 1.8%) 的 N% 增加。三聚氰胺的固定化氨基酸浓度为 0.297 nmole，Mel4 为 0.358 nmole。SDS 提取从玻璃中释放出 < 15% 的肽。固定化肽结合的 LTA 是对照表面的 ≥ 4 倍。与三聚氰胺或对照表面相比，Mel4 释放出更多的自溶素（8 ± 2%；p = 0.026）。膜去极化发生在 15 分钟，并且与两种肽的细菌活力降低 ≥ 37% 相关（p < 0.001）。与对照相比，膜电位的破坏导致三聚氰胺 (0.9 ± 0.4 nM) 或 Mel4 (0.6 ± 0.3 nM) 涂层表面的 ATP 损失 (p < 0.001)。三聚氰胺涂层产生 27 ± 11% (p = 0.026) 并且 Mel4 在 4 小时后产生 17 ± 12% (p = 0.150) PI 染色细胞。与 6 小时的过程控制相比，仅通过三聚氰胺涂层释放 DNA/RNA（2.1 ± 0.1 倍；p = 0.011）。两种结合的肽都导致 ATP 的释放，但只有三聚氰胺释放 DNA/RNA，而 Mel4 涂层导致自溶素的释放。由于三聚氰胺和 Mel4 的作用方式与细胞表面有关，因此它们具有开发抗感染植入物的潜力。