Kumar S Dinesh, Shin Song Yub
Department of Biomedical Science, Graduate School, Chosun University, Gwangju, 61452, Republic of Korea.
Department of Biomedical Science, Graduate School, Chosun University, Gwangju, 61452, Republic of Korea; Department of Cellular & Molecular Medicine, School of Medicine, Chosun University, Gwangju, 61452, Republic of Korea.
Eur J Med Chem. 2020 Oct 15;204:112580. doi: 10.1016/j.ejmech.2020.112580. Epub 2020 Jul 16.
Antimicrobial peptides (AMPs) have gained increasing attention to combat antibiotic-resistant pathogens. dCATH (duck cathelicidin) is a 20-residue avian cathelicidin with potent bactericidal activity. However, its therapeutic application is limited due to high mammalian cell cytotoxicity. To develop therapeutically useful AMPs with enhanced antimicrobial and cell-selective property, we designed a series of 12-meric (dodeca) short amphiphilic peptides based on dCATH. Among these, Trp and Lys-rich dCATH 12-4 and dCATH 12-5 exhibited higher selectivity towards bacterial cells than erythrocytes and macrophages. Additionally, these AMPs significantly reduced NO and TNF-α secretion in LPS-stimulated macrophage cells, suggesting their anti-inflammatory properties. Various fluorophore-based studies and confocal microscopic observations demonstrated that dCATH 12-4 and dCATH 12-5 could penetrate the bacterial cell membrane and accumulate in the cytoplasm, without disrupting membrane integrity. Results from the microscopic examination and gel-retardation DNA binding assay suggested that both the designed AMPs could bind with bacterial DNA, subsequently leading to cell death via arrest of DNA synthesis. Fluorescence spectroscopy and flow cytometry analysis revealed that the designed AMPs induced strong binding to LPS oligomers which resulted in dissociation of LPS aggregates, thereby preventing LPS from binding to the carrier protein lipopolysaccharide-binding protein (LBP) or alternatively to CD14 receptors of macrophage cells. Additionally, both dCATH 12-4 and dCATH 12-5 demonstrated synergistic actions with various conventional antibiotics against antibiotic resistant pathogens, thus indicating their ability as promising adjuncts to combination therapy. In summary, these findings contribute to the design of short AMPs with bactericidal and immunomodulatory properties for combating bacterial infection and sepsis.
抗菌肽（AMPs）在对抗抗生素耐药病原体方面越来越受到关注。dCATH（鸭cathelicidin）是一种由20个氨基酸残基组成的禽类cathelicidin，具有强大的杀菌活性。然而，由于其对哺乳动物细胞的高细胞毒性，其治疗应用受到限制。为了开发具有增强抗菌和细胞选择性的治疗性有用的AMPs，我们基于dCATH设计了一系列12肽（十二肽）短两亲性肽。其中，富含色氨酸和赖氨酸的dCATH 12-4和dCATH 12-5对细菌细胞的选择性高于红细胞和巨噬细胞。此外，这些AMPs显著降低了脂多糖刺激的巨噬细胞中一氧化氮和肿瘤坏死因子-α的分泌，表明它们具有抗炎特性。各种基于荧光团的研究和共聚焦显微镜观察表明，dCATH 12-4和dCATH 12-5可以穿透细菌细胞膜并积聚在细胞质中，而不会破坏膜的完整性。显微镜检查和凝胶阻滞DNA结合试验的结果表明，这两种设计的AMPs都可以与细菌DNA结合，随后通过阻止DNA合成导致细胞死亡。荧光光谱和流式细胞术分析表明，设计的AMPs与脂多糖寡聚物有强烈结合，导致脂多糖聚集体解离，从而防止脂多糖与载体蛋白脂多糖结合蛋白（LBP）或巨噬细胞的CD14受体结合。此外，dCATH 12-4和dCATH 12-5都与各种传统抗生素对耐药病原体表现出协同作用，因此表明它们作为联合治疗的有前途的辅助药物的能力。总之，这些发现有助于设计具有杀菌和免疫调节特性的短AMPs，以对抗细菌感染和败血症。
