College of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 34134, Korea.
National Marine Biodiversity Institute of Korea (MABIK), 75, Jangsan-ro 101beon-gil, Janghang-eup, Seochun-gun, Chungchungnam-do 33662, Korea.
Mar Drugs. 2020 Jan 15;18(1):56. doi: 10.3390/md18010056.
The rapid emergence of multidrug-resistant pathogens makes an urgent need for discovering novel antimicrobial agents as alternatives to conventional antibiotics. Towards this end, we designed and synthesized a synthetic peptide of 23 amino acids (AAs) (GWLIRGAIHAGKAIHGLIHRRRH) from a defense protein 3 cDNA sequence of Octopus minor. The sequence of the peptide, which was named Octominin, had characteristic features of known antimicrobial peptides (AMPs) such as a positive charge (+5), high hydrophobic residue ratio (43%), and 1.86 kcal/mol of Boman index. Octominin was predicted to have an alpha-helix secondary structure. The synthesized Octominin was 2625.2 Da with 92.5% purity. The peptide showed a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 50 and 200 μg/mL, respectively, against Candida albicans. Field emission scanning electron microscopy observation confirmed that Octominin caused ultrastructural cell wall deformities in . In addition, propidium iodide penetrated the Octominin-treated cells, further demonstrating loss of cell membrane integrity that caused cell death at both MIC and MFC. Octominin treatment increased the production of intracellular reactive oxygen species and decreased cell viability in a concentration dependent manner. Cytotoxicity assays revealed no significant influence of Octominin on the viability of human embryonic kidney 293T cell line, with over 95% live cells in the Octominin-treated group observed up to 100 µg/mL. Moreover, we confirmed the antifungal action of Octominin using a zebrafish experimental infection model. Overall, our results demonstrate the Octominin is a lead compound for further studies, which exerts its effects by inducing cell wall damage, causing loss of cell membrane integrity, and elevating oxidative stress.
耐药性病原体的迅速出现迫切需要发现新的抗菌剂作为传统抗生素的替代品。为此，我们从章鱼的防御蛋白 3 cDNA 序列中设计并合成了一种由 23 个氨基酸 (AA) 组成的合成肽 (GWLIRGAIHAGKAIHGLIHRRRH)。该肽的序列被命名为 Octominin，具有已知抗菌肽 (AMP) 的特征，如正电荷 (+5)、高疏水性残基比例 (43%) 和 1.86 kcal/mol 的 Boman 指数。Octominin 预测具有α-螺旋二级结构。合成的 Octominin 分子量为 2625.2 Da，纯度为 92.5%。该肽对白色念珠菌的最小抑菌浓度 (MIC) 和最小杀菌浓度 (MFC) 分别为 50 和 200 μg/mL。场发射扫描电子显微镜观察证实，Octominin 导致 细胞的超微结构细胞壁变形。此外，碘化丙啶穿透 Octominin 处理的 细胞，进一步证明细胞膜完整性丧失导致细胞在 MIC 和 MFC 时死亡。Octominin 处理以浓度依赖的方式增加细胞内活性氧的产生并降低细胞活力。细胞毒性测定显示，Octominin 对人胚肾 293T 细胞系的活力没有显著影响，在 Octominin 处理组中观察到超过 95%的活细胞，直至 100 μg/mL。此外，我们使用斑马鱼实验感染模型证实了 Octominin 的抗真菌作用。总体而言，我们的结果表明 Octominin 是一种先导化合物，通过诱导细胞壁损伤、破坏细胞膜完整性和增加氧化应激来发挥作用。
