Dynamics of Antimicrobial Peptide Interactions with Bacterial Membranes

描述（由申请人提供）：在植物、动物和人类中发现了多种天然表达的抗菌肽（AMP）。AMP显著地具有阻止革兰氏阴性和革兰氏阳性细菌生长的一般能力。阳离子AMP与带负电荷的细菌细胞膜结合，并明显降低其屏障功能，最终导致生长停止和细胞死亡。细菌抵抗这种一般作用模式的相对不能力使得AMP及其模拟物成为针对耐药性菌株的有趣的候选药物。迄今为止，几乎所有的机制研究都集中在体外合成脂质囊泡或AMP对细菌的长期、大量影响上。AMP攻击细菌膜的详细机制还不清楚。 这项工作开发了新的荧光显微镜检测，以直接观察攻击的抗菌肽对细菌膜的个别细胞在真实的时间。α-螺旋肽对E.在大肠杆菌中，我们观察到这样的事件：与外膜（OM）结合并在外膜（OM）内扩散，穿过OM的易位，OM对周质GFP的透化，细胞突然收缩和细胞生长的停止，细胞质膜（CM）对染料Sytox绿色的透化，以及细胞质内氧化损伤的开始。我们计划将我们的工作从α-螺旋AMP扩展到防御素，这是另一类重要的人类AMP。除了对模式种E. coli和B.枯草杆菌，我们将观察AMP对非致病性铜绿假单胞菌和S.金黄色。我们将开始研究AMP对生活在模型生物膜中的细菌的活性。我们的研究结果将测试相关的合成脂质双层生长停止机制在真实的细菌的先前研究。 至关重要的是，我们必须开发新的手段来杀死耐药病原体。通过解剖 天然抗菌肽杀死细菌的步骤，我们将提供新的设计标准，合成模拟抗菌肽，可能证明临床上有用的。本文所开发的新方法将广泛应用于天然AMPS、合成共聚物和合成药物的机理研究。

DESCRIPTION (provided by applicant): A wide variety of naturally expressed anti-microbial peptides (AMPs) have been discovered in plants, animals, and humans. AMPs are remarkably for their general ability to halt growth of both Gram negative and Gram positive bacteria. Cationic AMPs bind to negatively charged bacterial cell membranes and evidently degrade their barrier function, eventually leading to the halting of growth and cell death. The relative inabilit of bacteria to resist this general mode of action makes AMPs and their mimics interesting drug candidates against antibiotic-resistant strains. Almost all mechanistic studies thus far have focused either on synthetic lipid vesicles in vitro or on long-time, bulk effects of AMPs on bacteria. The detailed mechanisms of AMP attack on bacterial membranes are not well understood. This work develops novel fluorescence microscopy assays in order to directly observe the attack of AMPs on bacterial membranes for individual cells in real time. On attack of α-helical peptides on E. coli, we observe such events as: binding to and diffusion within the outer membrane (OM), translocation across the OM, permeabilization of the OM to periplasmic GFP, abrupt cell shrinkage and the halting of cell growth, permeabilization of the cytoplasmic membrane (CM) to the dye Sytox Green, and the onset of oxidative damage within the cytoplasm. We plan to extend our work from α-helical AMPs to defensins, the other important class of human AMPs. In addition to studies of the model species E. coli and B. subtilis, we will observe AMP effects on non-pathogenic strains of P. aeruginosa and S. aureus. We will initiate studies of AMP activity against bacteria living in model biofilms. Our results will test the relevance of previous studies on synthetic lipid bilayers to growth-halting mechanisms in real bacteria. It is critical that we develop new means to kill drug-resistant pathogens. By dissecting the steps by which natural AMPs kill bacteria, we will provide new design criteria for synthetic mimics of AMPs that may prove clinically useful. The novel methods developed here will be widely applicable to mechanistic studies of natural AMPs, synthetic copolymers designed to mimic AMPS, and synthetic drugs.