Zhao Yan-Yan, Wang Chun, Wang Wei-Xiao, Han Li-Mei, Zhang Caiyun, Yu Jiao-Yang, Chen Wei, Hu Chun-Mei
Department of Tuberculosis, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, 210003, People's Republic of China.
Clinical Research Center, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, 210003, People's Republic of China.
Infect Drug Resist. 2023 Mar 28;16:1801-1812. doi: 10.2147/IDR.S403232. eCollection 2023.
Drug-resistant tuberculosis (TB) is an emerging threat to public health worldwide. Antimicrobial peptide (AMP) is a promising solution to solve the antimicrobial resistance crisis. The apolipoprotein E mimetic peptide COG1410 has been confirmed to simultaneously have neuroprotective, anti-inflammatory, and antibacterial activity. However, whether it is effective to inhibit growth of mycobacteria has not been investigated yet.
The peptide COG1410 was synthesized with conventional solid-phase peptide synthesis and qualified by HPLC and mass spectrometry. Micro-dilution method was used to determine the minimal inhibitory concentration. A time-kill assay was used to determine the bactericidal dynamics of antimicrobial peptide and relative antibiotics. Static biofilm formation was conducted in 24-well plate and the biofilm was separated from planktonic cells and collected. The mechanism of action of COG1410 was explored by TEM observation and ATP leak assay. The localization of COG1410 was observed by confocal laser scan microscopy. The drug-drug interaction was determined by a checkerboard assay.
COG1410 was a potent bactericidal agent against in vitro and within the macrophages with MIC 16 μg/mL, but invalid against and . A time-kill assay showed that COG1410 killed as potent as clarithromycin, but faster than LL-37, another short synthetic cationic peptide. 1× MIC COG1410 almost reduced 90% biofilm formation of . Additionally, COG1410 was able to penetrate the cell membrane of macrophage and inhibit intracellular growth. TEM observation and ATP leak assay found that COG1410 disrupted cell membrane and caused release of cell contents. Confocal fluorescence microscopy showed that FITC-COG1410 aggregated around cell membrane instead of entering the cytoplasm. Although COG1410 had relative high cytotoxicity, it exhibited strong additive interaction with regular anti-TB antibiotics, which reduced the working concentration of COG1410 and expanding safety window. After 30 passages, there was no induced drug resistance for COG1410.
COG1410 was a novel and potent AMP against by disrupting the integrity of cell membrane.
耐多药结核病是全球公共卫生面临的新威胁。抗菌肽是解决抗菌药物耐药危机的一个有前景的办法。载脂蛋白E模拟肽COG1410已被证实同时具有神经保护、抗炎和抗菌活性。然而，其对分枝杆菌生长的抑制作用是否有效尚未得到研究。
采用传统固相肽合成法合成肽COG1410，并通过高效液相色谱和质谱进行鉴定。采用微量稀释法测定最低抑菌浓度。采用时间杀菌试验测定抗菌肽和相关抗生素的杀菌动力学。在24孔板中进行静态生物膜形成实验，将生物膜与浮游细胞分离并收集。通过透射电镜观察和ATP泄漏试验探索COG1410的作用机制。通过共聚焦激光扫描显微镜观察COG1410的定位。采用棋盘法测定药物相互作用。
COG1410是一种对体外及巨噬细胞内的结核分枝杆菌有效的杀菌剂，最低抑菌浓度为16μg/mL，但对其他分枝杆菌无效。时间杀菌试验表明，COG1410杀灭结核分枝杆菌的效果与克拉霉素相当，但比另一种短合成阳离子肽LL-37更快。1×最低抑菌浓度的COG1410几乎可减少结核分枝杆菌90%的生物膜形成。此外，COG1410能够穿透巨噬细胞膜并抑制细胞内结核分枝杆菌的生长。透射电镜观察和ATP泄漏试验发现，COG1410破坏细胞膜并导致细胞内容物释放。共聚焦荧光显微镜显示，异硫氰酸荧光素标记的COG1410聚集在细胞膜周围而未进入细胞质。尽管COG1410具有相对较高的细胞毒性，但它与常规抗结核抗生素表现出强烈的相加作用，这降低了COG1410的工作浓度并扩大了安全窗口。传代30次后，未诱导出COG1410的耐药性。
COG1410是一种新型且有效的抗菌肽，通过破坏细胞膜完整性来对抗结核分枝杆菌。
