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) bLFcin₆ and Tat₁₁ 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.

沙门氏菌是革兰氏阴性细菌，是兼性的细胞内病原体，可引起多种动物和人类疾病。抗菌剂渗透性差，限制了它们在细胞内细菌感染治疗中的应用。在这项研究中，将细胞穿透肽（CPPs）bLFcin ₆和Tat ₁₁分别与抗菌肽N2偶联，并首先在体外评估了CPPs-N2偶联物对鼠伤寒沙门氏菌的抗菌活性和药效。和巨噬细胞。还在RAW264.7细胞中检查了CPPs-N2缀合物的细胞毒性，细胞摄取和细胞内在化的机理。与N2相似，CPPs-N2具有两个反向β-折叠和三个环。CPPs-N2的最低抑菌浓度（MIC）约为2μM，高于N2的最低抑菌浓度（0.8μM）。剂量-时间曲线和细胞毒性试验表明，两种肽结合物在0.25至1×MIC的浓度下均比单独的N2更有效，并且在100μM时显示出低的细胞毒性（9.78％–13.54％）。温育0.5小时后，B6N2和T11N2的细胞内在化率分别超过28.3％和93.5％，高于N2。低温（82.1％–91.7％），氯丙嗪（35.7％–75.1％）和阿米洛利（26.0％–52.1％）降低了B6N2和T11N2的吸收，提示可能参与了巨胞饮作用和网格蛋白介导的内吞作用。T11N2和B6N2在3小时内分别杀死了约98.85％和91.35％的细菌，高于N2所杀死的百分比（69.74％）。与单独的N2相比，T11N2和B6N2的杀菌活性分别提高了53.7％–99.6％和85.3–85.8％。两种CPPs-N2偶联物都可能是新型抗微生物剂的极佳候选者，以治疗由细胞内病原体引起的传染病。T11N2和B6N2的杀菌活性分别提高了53.7％–99.6％和85.3–85.8％。两种CPPs-N2偶联物都可能是新型抗微生物剂的极佳候选者，以治疗由细胞内病原体引起的传染病。T11N2和B6N2的杀菌活性分别提高了53.7％–99.6％和85.3–85.8％。两种CPPs-N2偶联物都可能是新型抗微生物剂的极佳候选者，以治疗由细胞内病原体引起的传染病。