Li Bing, Yang Na, Wang Xiumin, Hao Ya, Mao Ruoyu, Li Zhanzhan, Wang Zhenlong, Teng Da, Wang Jianhua
Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
Key Laboratory for Feed Biotechnology, Ministry of Agriculture and Rural Affairs, Beijing, China.
Front Microbiol. 2020 Jun 5;11:1057. doi: 10.3389/fmicb.2020.01057. eCollection 2020.
Insect defensins are promising candidates for the development of potent antimicrobials against antibiotic-resistant (). An insect defensin, DLP4, isolated from the hemolymph of larvae, showed low antimicrobial activity against Gram-positive (G) pathogens and high cytotoxicity, which limited its effective therapeutic application. To obtain more potent and low cytotoxicity molecules, a series of peptides was designed based on the DLP4 template by changing the conservative site, secondary structure, charge, or hydrophobicity. Among them, a variant designated as ID13 exhibited strong antibacterial activity at low MIC values of 4-8 μg/mL to G pathogens (: 4 μg/mL; : 8 μg/mL; : 4 μg/mL; : 4 μg/mL), which were lower than those of DLP4 (: 16 μg/mL; : 64 μg/mL; : 32 μg/mL; : 16 μg/mL), and cytotoxicity of ID13 (71.4% viability) was less than that of DLP4 (63.8% viability). ID13 could penetrate and destroy the cell membrane of CVCC 546, resulting in an increase in potassium ion leakage; it bound to genomic DNA (gDNA) and led to the change of gDNA conformation. After treatment with ID13, perforated, wrinkled, and collapsed CVCC 546 cells were observed in electron microscopy. Additionally, ID13 killed over 99.99% of within 1 h, 2 × MIC of ID13 induced a post-antibiotic effect (PAE) of 12.78 ± 0.28 h, and 10 mg/kg ID13 caused a 1.8 log (CFU/g) (CFU: colony-forming units) reduction of in infected mouse thigh muscles and a downregulation of TNF-α, IL-6, and IL-10 levels, which were superior to those of DLP4 or vancomycin. These findings indicate that ID13 may be a promising peptide antimicrobial agent for therapeutic application.
昆虫防御素是开发针对抗生素耐药菌的强效抗菌剂的有潜力的候选物。从幼虫血淋巴中分离出的一种昆虫防御素DLP4，对革兰氏阳性（G+）病原体显示出低抗菌活性且细胞毒性高，这限制了其有效的治疗应用。为了获得更有效且细胞毒性低的分子，基于DLP4模板通过改变保守位点、二级结构、电荷或疏水性设计了一系列肽。其中，一种名为ID13的变体对G+病原体在4 - 8μg/mL的低最低抑菌浓度（MIC）值下表现出强抗菌活性（金黄色葡萄球菌：4μg/mL；枯草芽孢杆菌：8μg/mL；表皮葡萄球菌：4μg/mL；溶血葡萄球菌：4μg/mL），这些值低于DLP4的（金黄色葡萄球菌：16μg/mL；枯草芽孢杆菌：64μg/mL；表皮葡萄球菌：32μg/mL；溶血葡萄球菌：16μg/mL），并且ID13的细胞毒性（存活率71.4%）小于DLP4的（存活率63.8%）。ID13可以穿透并破坏CVCC 546的细胞膜，导致钾离子泄漏增加；它与基因组DNA（gDNA）结合并导致gDNA构象改变。用ID13处理后，在电子显微镜下观察到CVCC 546细胞出现穿孔、起皱和塌陷。此外，ID13在1小时内杀死了超过99.99%的金黄色葡萄球菌，2倍MIC的ID13诱导了12.78±0.28小时的抗生素后效应（PAE），并且10mg/kg的ID13使感染小鼠大腿肌肉中的金黄色葡萄球菌减少了1.8对数（CFU/g）（CFU：菌落形成单位），并下调了肿瘤坏死因子-α、白细胞介素-6和白细胞介素-10的水平，这些均优于DLP4或万古霉素。这些发现表明ID13可能是一种有前景的用于治疗应用的肽类抗菌剂。
