Dynamics of Antimicrobial Peptide Interactions with Bacterial Membranes

抗菌肽与细菌膜相互作用的动力学

• 批准号: 8986794
• 负责人: James C. Weisshaar
• 金额: $ 29.41万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8986794
• 关键词: Animals; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Benchmarking; Binding; Biochemical; Biological Assay; Carpet; Cell Death; Cell membrane; Cells; Charge; Communities; Cytoplasm; Defensins; Dependence; Diffuse; Diffusion; Drug resistance; Dyes; Epithelial Cells; Escherichia coli; Event; Exhibits; Extracellular Matrix; Fluorescence Microscopy; Gastrointestinal tract structure; Goals; Gram-Positive Bacteria; Grant; Growth; Health; Hospitals; Host Defense; Human; Human body; Hybrids; Immune response; Immune system; In Vitro; Individual; Infection; Invaded; Label; Learning; Length; Life; Light; Lipid Bilayers; Lipids; Lipopolysaccharides; Lung; Measurement; Measures; Membrane; Methods; Microbe; Microbial Biofilms; Modeling; Moths; Multi-Drug Resistance; Oxidative Stress; Penetration; Peptides; Phase; Plants; Property; Pseudomonas aeruginosa; Resolution; Route; Septate; Site; Staphylococcus aureus; Structure; Study models; Surface; Testing; Therapeutic Agents; Time; Tissues; Vesicle; Work; antimicrobial; antimicrobial peptide; bactericide; base; biophysical analysis; cathelicidin; cecropin; cecropin A; cell growth; commensal microbes; copolymer; design; drug candidate; imaging modality; insight; interest; killings; macrophage; member; mutant; natural antimicrobial; neutrophil; novel; oxidative damage; pathogen; pathogenic bacteria; periplasm; resistant strain; synthetic drug; synthetic peptide; temporal measurement

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.

描述（由申请人提供）：已在植物、动物和人类中发现了多种天然表达的抗微生物肽（AMP）。 AMP 因其具有阻止革兰氏阴性和革兰氏阳性细菌生长的一般能力而引人注目。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。细菌相对无力抵抗这种一般作用模式，使得 AMP 及其模拟物成为对抗抗生素耐药菌株的有趣候选药物。迄今为止，几乎所有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。 AMP 对细菌膜攻击的详细机制尚不清楚。 这项工作开发了新型荧光显微镜检测方法，以便实时直接观察 AMP 对单个细胞细菌膜的攻击。在 α-螺旋肽攻击大肠杆菌时，我们观察到以下事件：与外膜 (OM) 结合并在其内扩散、跨 OM 易位、OM 透化为周质 GFP、细胞突然收缩和细胞生长停止、细胞质膜 (CM) 透化为染料 Sytox Green，以及细胞膜内氧化损伤的开始 细胞质。我们计划将我们的工作从 α-螺旋 AMP 扩展到防御素，另一类重要的人类 AMP。除了对模式物种大肠杆菌和枯草芽孢杆菌的研究之外，我们还将观察 AMP 对铜绿假单胞菌和金黄色葡萄球菌非致病菌株的影响。我们将启动针对模型生物膜中细菌的 AMP 活性研究。我们的结果将测试先前关于合成脂质双层的研究与真实细菌生长停止机制的相关性。 我们开发新的方法来杀死耐药病原体至关重要。通过解剖 通过了解天然 AMP 杀死细菌的步骤，我们将为 AMP 的合成模拟物提供新的设计标准，这可能在临床上有用。这里开发的新方法将广泛适用于天然 AMP、旨在模拟 AMPS 的合成共聚物和合成药物的机理研究。