Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, People's Republic of China; Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, People's Republic of China; Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Eur J Med Chem. 2018 Feb 10;145:263-272. doi: 10.1016/j.ejmech.2017.12.066. Epub 2017 Dec 29.
Salmonellae, gram-negative bacteria, are facultative intracellular pathogens that cause a number of diseases in animals and humans. The poor penetration ability of antimicrobial agents limits their use in the treatment of intracellular bacterial infections. In this study, the cell-penetrating peptides (CPPs) bLFcin and Tat were separately conjugated to the antimicrobial peptide N2, and the antibacterial activity and pharmacodynamics of the CPPs-N2 conjugates were first evaluated against Salmonellae typhimurium in vitro and in macrophage cells. The cytotoxicity, cellular uptake and mechanism of cellular internalization of the CPPs-N2 conjugates were also examined in RAW264.7 cells. Similar to N2, CPPs-N2 have two reverse β-sheets and three loops. The minimal inhibitory concentration (MIC) of CPPs-N2 was approximately 2 μM, which was higher than that of N2 (0.8 μM). The dose-time curves and cytotoxicity assay showed that both peptide conjugates were more effective than N2 alone at concentrations ranging from 0.25 to 1 × MIC, and they exhibited low cytotoxicity (9.78%-13.54%) at 100 μM. After 0.5 h incubation, the cell internalization ratio of B6N2 and T11N2 exceeded 28.3% and 93.5%, respectively, which was higher than that of N2. The uptake of B6N2 and T11N2 was reduced by low temperature (82.1%-91.7%), chlorpromazine (35.7%-75.1%), and amiloride (26.0%-52.1%), indicating that macropinocytosis and clathrin-mediated endocytosis may be involved. Approximately 98.85% and 91.35% of bacteria were killed within 3 h by T11N2 and B6N2, respectively, which was higher than the percentage killed by N2 (69.74%). Compared with the bactericidal activity of N2 alone, the bactericidal activity of T11N2 and B6N2 was increased by 53.7%-99.6% and 85.3-85.8%, respectively. Both CPPs-N2 conjugates may be excellent candidates for novel antimicrobial agents to treat infectious diseases caused by intracellular pathogens.
沙门氏菌是一种兼性细胞内病原体，革兰氏阴性细菌，可导致动物和人类的多种疾病。抗菌药物的穿透能力差限制了它们在治疗细胞内细菌感染中的应用。在这项研究中，细胞穿透肽（CPP）bLFcin 和 Tat 分别与抗菌肽 N2 缀合，首先评估 CPP-N2 缀合物对体外和巨噬细胞中的鼠伤寒沙门氏菌的抗菌活性和药效。还在 RAW264.7 细胞中检查了 CPP-N2 缀合物的细胞毒性、细胞摄取和细胞内化机制。与 N2 相似，CPP-N2 具有两个反向 β-折叠和三个环。CPP-N2 的最小抑菌浓度（MIC）约为 2 μM，高于 N2（0.8 μM）。剂量-时间曲线和细胞毒性测定表明，两种肽缀合物在 0.25 至 1×MIC 的浓度范围内均比 N2 单独作用更有效，并且在 100 μM 时表现出低细胞毒性（9.78%-13.54%）。孵育 0.5 小时后，B6N2 和 T11N2 的细胞内化率分别超过 28.3%和 93.5%，高于 N2。B6N2 和 T11N2 的摄取被低温（82.1%-91.7%）、氯丙嗪（35.7%-75.1%）和氨甲蝶呤（26.0%-52.1%）降低，表明可能涉及巨胞饮作用和网格蛋白介导的内吞作用。在 3 小时内，T11N2 和 B6N2 分别杀死了约 98.85%和 91.35%的细菌，高于 N2 杀死的百分比（69.74%）。与 N2 单独的杀菌活性相比，T11N2 和 B6N2 的杀菌活性分别提高了 53.7%-99.6%和 85.3-85.8%。CPP-N2 缀合物都可能是治疗细胞内病原体引起的感染性疾病的新型抗菌药物的优秀候选物。
