Polymyxins are increasingly used as the last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, efforts to address the resistance in superbugs are compromised by a poor understanding of the bactericidal modes because high-resolution detection of the cell structure is still lacking. By performing molecular dynamics simulations at a coarse-grained level, here we show that polymyxin B (PmB) disrupts Gram-negative bacterial membranes by altering lipid homeostasis and asymmetry. We found that the binding of PmBs onto the asymmetric outer membrane (OM) loosens the packing of lipopolysaccharides (LPS) and induces unbalanced bending torque between the inner and outer leaflets, which in turn triggers phospholipids to flip from the inner leaflet to the outer leaflet to compensate for the stress deformation. Meanwhile, some LPSs may be detained on the inner membrane (IM). Then, the lipid-scrambled OM undergoes phase separation. Defects are created at the boundaries between LPS-rich domains and phospholipid-rich domains, which consequently facilitate the uptake of PmB across the OM. Finally, PmBs target LPSs detained on the IM and similarly perturb the IM. This lipid Scramble, membrane phase Separation, and peptide Translocation model depicts a novel mechanism by which polymyxins kill bacteria and sheds light on developing a new generation of polymyxins or antibiotic adjuvants with improved killing activities and higher therapeutic indices.

多粘菌素越来越多地用作治疗多重耐药革兰氏阴性菌引起的感染的最后一线治疗选择。然而，由于仍然缺乏细胞结构的高分辨率检测，对杀菌模式的了解不足，导致解决超级细菌耐药性的努力受到影响。通过在粗粒度水平上进行分子动力学模拟，我们发现多粘菌素 B (PmB) 通过改变脂质稳态和不对称性来破坏革兰氏阴性细菌膜。我们发现，PmB 与不对称外膜 (OM) 的结合松开了脂多糖 (LPS) 的堆积，并引起内叶和外叶之间不平衡的弯曲扭矩，进而触发磷脂从内叶翻转到外叶以补偿应力变形。同时，一些LPS可能被滞留在内膜（IM）上。然后，脂质混杂的 OM 发生相分离。在富含 LPS 的结构域和富含磷脂的结构域之间的边界处产生缺陷，从而促进 PmB 穿过 OM 的摄取。最后，PmB 以 IM 上滞留的 LPS 为目标，并类似地扰乱 IM。这种脂质争夺、膜相分离和肽易位模型描述了多粘菌素杀死细菌的新机制，并为开发具有改进的杀灭活性和更高治疗指数的新一代多粘菌素或抗生素佐剂提供了线索。