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自然表达的抗微生物肽（amp）。抗菌肽具有显著的抑制革兰氏阴性和革兰氏阳性细菌生长的能力。阳离子amp与带负电荷的细菌细胞膜结合，明显降低其屏障功能，最终导致生长停止和细胞死亡。细菌相对不能抵抗这种一般的作用模式，使得amp及其模拟物成为抗抗生素耐药菌株的有趣候选药物。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AMPs对细菌的长期整体效应上。AMP攻击细菌膜的详细机制尚不清楚。这项工作开发了新的荧光显微镜分析，以便直接观察AMPs对单个细胞的细菌膜的攻击。当α-螺旋肽攻击大肠杆菌时，我们观察到以下事件：与外膜（OM）的结合和扩散，跨OM的易位，OM对质周GFP的渗透，细胞突然收缩和细胞生长停止，细胞质膜（CM）对Sytox Green染料的渗透，以及细胞质内氧化损伤的发生。我们计划将我们的工作从α-螺旋AMPs扩展到防御素，这是另一类重要的人类AMPs。除了研究模式种大肠杆菌和枯草芽孢杆菌外，我们还将观察AMP对非致病性铜绿假单胞菌和金黄色葡萄球菌的作用。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之前关于合成脂质双分子层与真实细菌生长停止机制的研究的相关性。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

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.