This corresponding to uncropped images of S3 Fig.

Antimicrobial resistance represents a critical global public health challenge, leading to increased mortality and morbidity due to the ineffectiveness of current antibiotics against bacterial infections. Antimicrobial peptides (AMPs) offer a promising alternative for treating bacterial infections because of their broad-spectrum activity, biocompatibility, and rapid bactericidal action. Recent studies have demonstrated that Ib-M peptides exhibit bactericidal activity against pathogenic Escherichia coli clinical isolates. The objective of this study was to evaluate the mechanisms by which Ib-M peptides destabilize and disrupts E. coli membranes. We showed by dilutions assays that Ib-M peptides had a minimum inhibitory concentration (MIC) of 12.5 µM against E. coli. Electron microscopy studies confirmed that Ib-M peptides were directly implicated in E. coli membrane disruption, altered bacterial shape and subsequent disintegration. To understand bacterial membrane interaction with Ib-M peptides at the molecular level, we evaluated structure-function relationships using circular dichroism spectroscopy and in silico simulations. These studies demonstrated the strong amphipathic, hydrophobic and cationic properties of Ib-M peptides. At sublethal concentrations, these peptides interacted with bacterial lipopolysaccharides (LPS), leading to outer and inner membrane permeation and cytoplasmic membrane depolarization. This effect was transient at sublethal Ib-M peptides concentrations, as evidenced by the recovery of bacterial growth in lag phase kinetics. At higher concentrations, there was high depolarization of cytoplasmic membrane, disruption of outer membrane and inner membranes and irreversible bacterial lysis. When mammalian cells were exposed Ib-M1 peptide cytotoxic effect was only reached when MIC was increased 10-fold. In conclusion, Ib-M peptides inhibited E. coli growth by disrupting bacterial membranes via interactions with LPS and increased membrane permeation yet, they have low cytotoxicity on mammalian cells. This study highlights the mechanisms of action on Ib-M peptides as antimicrobials and paves the way for further research on the clinical use of these peptides as antimicrobial agents against multidrug resistant bacterial infections.

抗菌药物耐药性（Antimicrobial resistance）是一项至关重要的全球公共卫生挑战，当前抗生素无法有效对抗细菌感染，进而导致死亡率与发病率攀升。抗菌肽（Antimicrobial Peptides, AMPs）凭借其广谱抗菌活性、生物相容性以及快速杀菌作用，成为治疗细菌感染的极具前景的替代方案。近期研究表明，Ib-M肽对致病性大肠埃希菌（Escherichia coli，E. coli）临床分离株具有杀菌活性。本研究旨在探究Ib-M肽破坏大肠埃希菌细胞膜的作用机制。我们通过稀释实验证实，Ib-M肽对大肠埃希菌的最低抑菌浓度（minimum inhibitory concentration, MIC）为12.5 μM。电镜研究证实，Ib-M肽可直接介导大肠埃希菌细胞膜破坏，改变细菌形态并最终导致其裂解。为从分子层面解析Ib-M肽与细菌细胞膜的相互作用，我们借助圆二色光谱法（circular dichroism spectroscopy）与in silico模拟开展了结构-功能关系研究。上述研究证实，Ib-M肽具备显著的两亲性、疏水性与阳离子特性。在亚致死浓度下，此类肽可与细菌脂多糖（Lipopolysaccharides, LPS）结合，引发外膜与内膜通透性增加以及细胞质膜去极化。亚致死浓度的Ib-M肽所引发的该效应具有可逆性，细菌生长动力学滞后阶段的恢复即可证实这一点。当浓度升高至一定水平时，细胞质膜会发生高度去极化，外膜与内膜遭到破坏，最终导致细菌不可逆裂解。当哺乳动物细胞暴露于Ib-M1肽时，仅当浓度提升至MIC的10倍时才会观察到细胞毒性效应。综上，Ib-M肽通过与脂多糖结合、增加细胞膜通透性来破坏细菌细胞膜，从而抑制大肠埃希菌的生长，且对哺乳动物细胞的细胞毒性较低。本研究阐明了Ib-M肽作为抗菌剂的作用机制，为后续将此类肽开发为对抗多重耐药细菌感染的临床抗菌制剂奠定了研究基础。