Data_Sheet_1_Antibiofilm property and multiple action of peptide PEW300 against Pseudomonas aeruginosa.docx

Pseudomonas aeruginosa (P. aeruginosa), an opportunistic pathogen, is often associated with difficulties in treating hospital-acquired infections. Biofilms formed by P. aeruginosa significantly improve its resistance to antimicrobial agents, thereby, posing a great challenge to the combat of P. aeruginosa infection. Antimicrobial peptides (AMPs) have recently emerged as promising antibiofilm agents and increasingly attracting the attention of scientists worldwide. However, current knowledge of their antibiofilm behavior is limited and their underlying mechanism remains unclear. In this study, a novel AMP, named PEW300, with three-point mutations (E9H, D17K, and T33A) from Cecropin A was used to investigate its antibiofilm property and antibiofilm pathway against P. aeruginosa. PEW300 displayed strong antibacterial and antibiofilm activity against P. aeruginosa with no significant hemolysis or cytotoxicity to mouse erythrocyte and human embryonic kidney 293 cells. Besides, the antibiofilm pathway results showed that PEW300 preferentially dispersed the mature biofilm, leading to the biofilm-encapsulated bacteria exposure and death. Meanwhile, we also found that the extracellular DNA was a critical target of PEW300 against the mature biofilm of P. aeruginosa. In addition, multiple actions of PEW300 including destroying the cell membrane integrity, inducing high levels of intracellular reactive oxygen species, and interacting with genomic DNA were adopted to exert its antibacterial activity. Moreover, PEW300 could dramatically reduce the virulence of P. aeruginosa. Taken together, PEW300 might be served as a promising antibiofilm candidate to combat P. aeruginosa biofilms.

铜绿假单胞菌（Pseudomonas aeruginosa，简称P. aeruginosa）是一种机会致病菌，常与医院获得性感染的治疗难题紧密相关。由铜绿假单胞菌形成的生物被膜（Biofilms）可显著增强其对抗菌剂的耐药性，进而为该菌感染的防控带来极大挑战。抗菌肽（Antimicrobial peptides, AMPs）近年来作为极具潜力的抗生物被膜制剂，日益受到全球科研工作者的关注。然而，目前学界对其抗生物被膜行为的认知仍较为有限，其潜在作用机制尚未阐明。本研究以源自天蚕素A（Cecropin A）、携带E9H、D17K及T33A三点突变的新型抗菌肽PEW300为研究对象，探究其对铜绿假单胞菌的抗生物被膜活性及相关作用通路。实验结果表明，PEW300对铜绿假单胞菌表现出强劲的抗菌与抗生物被膜活性，且对小鼠红细胞与人胚肾293细胞（human embryonic kidney 293 cells）无显著溶血反应与细胞毒性。此外，抗生物被膜通路分析结果显示，PEW300可优先解离成熟生物被膜，使被膜包裹的细菌暴露并被杀灭。与此同时，本研究还发现胞外DNA是PEW300靶向作用于铜绿假单胞菌成熟生物被膜的关键靶点。除此之外，PEW300可通过多种途径发挥抗菌活性：破坏细胞膜完整性、诱导高水平胞内活性氧，以及与基因组DNA结合。此外，PEW300还可显著降低铜绿假单胞菌的毒力。综上，PEW300有望成为对抗铜绿假单胞菌生物被膜的极具应用前景的候选制剂。