Yang Lei, Sun Yawei, Xu Yanzhao, Hang Bolin, Wang Lei, Zhen Ke, Hu Bing, Chen Yanan, Xia Xiaojing, Hu Jianhe
College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang, China.
Front Microbiol. 2020 Feb 7;11:174. doi: 10.3389/fmicb.2020.00174. eCollection 2020.
The increasing rates of resistance to traditional anti- agents have made the treatment of invasive salmonellosis more problematic, which necessitates the search for new antimicrobial compounds. In this study, the action mode of BSN-37, a novel antibacterial peptide (AMP) from bovine spleen neutrophils, was investigated against serovar Typhimurium ( Typhimurium). Minimum inhibitory concentrations (MICs) and time-kill kinetics of BSN-37 were determined. The cell membrane changes of Typhimurium CVCC541 (ST) treated with BSN-37 were investigated by testing the fluorescence intensity of membrane probes and the release of cytoplasmic β-galactosidase activity. Likewise, cell morphological and ultrastructural changes were also observed using scanning and transmission electron microscopes. Furthermore, the cytotoxicity of BSN-37 was detected by a CCK-8 kit and real-time cell assay. The proliferation inhibition of BSN-37 against intracellular Typhimurium was performed in Madin-Darby canine kidney (MDCK) cells. The results demonstrated that BSN-37 exhibited strong antibacterial activity against ST (MICs, 16.67 μg/ml), which was not remarkably affected by the serum salts at a physiological concentration. However, the presence of CaCl led to an increase in MIC of BSN-37 by about 4-fold compared to that of ST. BSN-37 at the concentration of 100 μg/ml could completely kill ST after co-incubation for 6 h. Likewise, BSN-37 at different concentrations (50, 100, and 200 μg/ml) could increase the outer membrane permeability of ST but not impair its inner membrane integrity. Moreover, no broken and ruptured cells were found in the figures of scanning and transmission electron microscopes. These results demonstrate that BSN-37 exerts its antibacterial activity against Typhimurium by a non-lytic mode of action. Importantly, BSN-37 had no toxicity to the tested eukaryotic cells, even at a concentration of 800 μg/ml. BSN-37 could significantly inhibit the proliferation of intracellular Typhimurium.
对传统抗菌药物的耐药率不断上升，使得侵袭性沙门氏菌病的治疗变得更加棘手，这就需要寻找新的抗菌化合物。在本研究中，对一种来自牛脾脏中性粒细胞的新型抗菌肽（AMP）BSN-37针对鼠伤寒血清型（鼠伤寒）的作用模式进行了研究。测定了BSN-37的最低抑菌浓度（MIC）和时间杀菌动力学。通过检测膜探针的荧光强度和细胞质β-半乳糖苷酶活性的释放，研究了用BSN-37处理的鼠伤寒CVCC541（ST）的细胞膜变化。同样，使用扫描电子显微镜和透射电子显微镜也观察了细胞形态和超微结构的变化。此外，通过CCK-8试剂盒和实时细胞检测法检测了BSN-37的细胞毒性。在Madin-Darby犬肾（MDCK）细胞中进行了BSN-37对细胞内鼠伤寒的增殖抑制实验。结果表明，BSN-37对ST表现出较强的抗菌活性（MIC为16.67μg/ml），生理浓度的血清盐对其影响不显著。然而，与ST相比，CaCl的存在导致BSN-37的MIC增加了约4倍。100μg/ml浓度的BSN-37与ST共孵育6小时后可完全杀灭ST。同样，不同浓度（50、100和200μg/ml）的BSN-37可增加ST的外膜通透性，但不损害其内膜完整性。此外，在扫描电子显微镜和透射电子显微镜图像中未发现细胞破裂和穿孔。这些结果表明，BSN-37通过非溶菌作用模式对鼠伤寒发挥抗菌活性。重要的是，即使在浓度为800μg/ml时，BSN-37对受试真核细胞也没有毒性。BSN-37可显著抑制细胞内鼠伤寒的增殖。
