Department of Allied Health Sciences, Faculty of Science, Universiti Tunku Abdul Rahman, Kampar 31900, Perak, Malaysia.
Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Kampar 31900, Perak, Malaysia.
Biomolecules. 2023 Mar 2;13(3):466. doi: 10.3390/biom13030466.
Antibacterial peptides (ABPs) have been proposed as potential candidates for alternative antibacterial agents due to the extensive dissemination of antibiotic resistance. However, ABP isolation from natural resources can be tedious without consistent yield. Moreover, many natural ABPs are not developed for clinical application due to potential toxicity to mammalian cells. Therefore, the objective of this study was to develop a potent ABP with minimal toxicity via phage display selection followed by computer-assisted modification. Briefly, a 12-mer phage-displayed peptide library was used to isolate peptides that bound to the cell surface of with high affinity. The affinity-selected peptide with the highest selection frequency was modified to PAM-5 (KWKWRPLKRKLVLRM) with enhanced antibacterial features by using an online peptide database. Using in vitro microbroth dilution assay, PAM-5 was shown to be active against a panel of Gram-negative bacteria and selected Gram-positive bacteria. Interestingly, the peptide was stable in human plasma by exhibiting a similar bactericidal effect via ex vivo assay. Scanning electron microscopy and SYTOX Green uptake assay revealed that PAM-5 was able to cause membrane disruption and permeabilization of the bacteria. Additionally, the peptide was also able to bind to bacterial DNA as demonstrated by gel retardation assay. In the time-kill assay, PAM-5 was shown to kill the bacteria rapidly in 10 min. More importantly, PAM-5 was non-cytotoxic to Vero cells and non-haemolytic to human erythrocytes at all concentrations tested for the antibacterial assays. Thus, this study showed that the combination of phage display screening and computer-assisted modification could be used to develop potent novel ABPs, and PAM-5 derived from these approaches is worth to be further elucidated for its potential clinical use.
抗菌肽 (ABPs) 由于抗生素耐药性的广泛传播，被认为是替代抗菌药物的潜在候选药物。然而，从天然资源中分离 ABP 可能很繁琐，而且产量也不一致。此外，由于许多天然 ABPs 对哺乳动物细胞可能具有毒性，因此不适合临床应用。因此，本研究的目的是通过噬菌体展示选择和计算机辅助修饰来开发一种毒性最小的有效 ABP。简而言之，使用 12 肽噬菌体展示肽库从 细胞表面高亲和力结合的肽中分离出肽。通过使用在线肽数据库，对选择频率最高的亲和肽进行修饰，得到具有增强抗菌特性的 PAM-5 (KWKWRPLKRKLVLRM)。通过体外微量肉汤稀释试验，发现 PAM-5 对一系列革兰氏阴性菌和选定的革兰氏阳性菌均具有活性。有趣的是，通过体外试验，该肽在人血浆中稳定，表现出相似的杀菌效果。扫描电子显微镜和 SYTOX Green 摄取试验表明，PAM-5 能够导致细菌的膜破裂和通透性增加。此外，凝胶阻滞试验表明，该肽还能够与细菌 DNA 结合。在时间杀伤试验中，PAM-5 在 10 分钟内迅速杀死细菌。更重要的是，在进行抗菌试验的所有测试浓度下，PAM-5 对 Vero 细胞均无细胞毒性，对人红细胞也无溶血作用。因此，本研究表明，噬菌体展示筛选和计算机辅助修饰的组合可用于开发有效的新型 ABPs，并且源自这些方法的 PAM-5 值得进一步研究其在临床上的应用潜力。
