Agricultural Research Service, National Animal Disease Center, Ruminant Diseases and Immunology Research Unit, USDA, Ames, IA, USA.
Agricultural Research Service, National Animal Disease Center, Food Safety and Enteric Pathogens Research Unit, USDA, Ames, IA, USA.
Sci Rep. 2021 Sep 29;11(1):19276. doi: 10.1038/s41598-021-98860-6.
Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasures to control MDR Salmonella outbreaks. In this study, antimicrobial activity of cationic α-helical bovine NK-lysin-derived antimicrobial peptides was evaluated against MDR Salmonella outbreak isolates. NK2A and NK2B strongly inhibited MDR Salmonella growth while NK1 and NK2C showed minimum-to-no growth inhibition. Scrambled-NK2A, which is devoid of α-helicity but has the same net positive charge as NK2A, also failed to inhibit bacterial growth. Incubation of negatively charged MDR Salmonella with NK2A showed increased Zeta potential, indicating bacterial-peptide electrostatic attraction. Confocal and transmission electron microscopy studies revealed NK2A-mediated damage to MDR Salmonella membranes. LPS inhibited NK2A-mediated growth suppression in a dose-dependent response, suggesting irreversible NK2A-LPS binding. LPS-NK2A binding and bacterial membrane disruption was also confirmed via electron microscopy using gold nanoparticle-NK2A conjugates. Finally, NK2A-loaded polyanhydride nanoparticles showed sustained peptide delivery and anti-bacterial activity. Together, these findings indicate that NK2A α-helicity and positive charge are prerequisites for antimicrobial activity and that MDR Salmonella killing is mediated by direct interaction of NK2A with LPS and the inner membrane, leading to bacterial membrane permeabilization. With further optimization using nano-carriers, NK2A has the potential to become a potent anti-MDR Salmonella agent.
多药耐药（MDR）沙门氏菌对公共健康构成威胁。非抗生素疗法可以作为控制 MDR 沙门氏菌爆发的重要对策。在这项研究中，评估了阳离子α-螺旋牛 NK 细胞素衍生的抗菌肽对 MDR 沙门氏菌爆发分离株的抗菌活性。NK2A 和 NK2B 强烈抑制 MDR 沙门氏菌的生长，而 NK1 和 NK2C 则表现出最小至无生长抑制作用。缺乏α-螺旋但具有与 NK2A 相同净正电荷的 scrambled-NK2A 也不能抑制细菌生长。带负电荷的 MDR 沙门氏菌与 NK2A 孵育显示出增加的 Zeta 电位，表明细菌-肽静电吸引。共聚焦和透射电子显微镜研究表明 NK2A 介导的 MDR 沙门氏菌膜损伤。LPS 以剂量依赖性方式抑制 NK2A 介导的生长抑制，表明 NK2A-LPS 结合不可逆。通过使用金纳米粒子-NK2A 缀合物的电子显微镜，还证实了 LPS-NK2A 结合和细菌膜破坏。最后，载有 NK2A 的聚酸酐纳米颗粒显示出持续的肽递药和抗菌活性。总之，这些发现表明 NK2A 的α-螺旋和正电荷是抗菌活性的前提条件，并且 MDR 沙门氏菌的杀伤是由 NK2A 与 LPS 和内膜的直接相互作用介导的，导致细菌膜通透性增加。通过使用纳米载体进一步优化，NK2A 有可能成为一种有效的抗 MDR 沙门氏菌剂。
