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。已知阳离子AMP形成两亲性螺旋，其结合到阴离子细胞膜，形成孔，并最终裂解细胞。细菌抵抗这种一般作用模式的相对无能使得AMP及其模拟物成为对抗耐药性菌株的有趣候选药物。虽然它是公认的AMP降解细菌膜，并最终导致裂解，大多数的机械工作集中在体外合成囊泡的研究或AMP对细菌的长期，批量的影响。AMP攻击细菌膜的详细机制还不清楚。 这项工作的主要目标是开发新的荧光显微镜检测，直接观察攻击的抗菌肽对细菌膜在真实的时间，为个别细胞。这些方法将首次能够将许多直接可观察到的事件放置在同一个统一的时间线上，涵盖注射AMP后的前10秒到前两个小时。测量的事件将包括AMP结合密度、鞭毛运动的停止、生长的停止、周质的泄漏和细胞质的泄漏。移除AMP后的延时成像将揭示哪些症状是可逆的，哪些是不可逆的，以及细胞恢复生长的时间尺度。这些新方法将用于探索性研究各种不同AMP攻击革兰氏阴性大肠杆菌和革兰氏阴性大肠杆菌细胞膜的机制。大肠杆菌和革兰氏阳性枯草芽孢杆菌。这些包括LL-37、天蚕抗菌肽A、爪蟾抗菌肽-2、蜂毒肽、丙甲菌素、吲哚菌素、bactenecin-5、1-防御素和保护素-1。我们将直接比较每个AMP降解细菌细胞膜的表面浓度与以前的研究结果在体外合成脂质双层。这些结果将有助于确定从体外研究中收集到的所谓的地毯状形成和孔形成机制是否与真实的细菌膜相关。 本文所建立的方法将广泛应用于抗菌肽和候选药物与多种细菌菌株相互作用的研究。 公共卫生相关性：细菌对药物的耐药性越来越强。抗菌肽是整个动物王国对抗病原体的前线防御，但我们并不真正了解它们如何杀死细菌细胞。这项工作将开发新的成像方法，使直接观察抗菌剂的杀菌机制具有比以往更好的空间和时间分辨率。

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 are known to form amphipathic helices that bind to anionic cell membranes, form pores, and eventually lyse the cell. The relative inability of bacteria to resist this general mode of action makes AMPs and their mimics interesting drug candidates against antibiotic-resistant strains. While it is well established that AMPs degrade bacterial membranes and eventually lead to lysis, most of the mechanistic work has focused either on studies of synthetic vesicles in vitro or on long-time, bulk effects of AMPs on bacteria. The detailed mechanism of AMP attack on bacterial membranes is not well understood. The primary goal of this work is to develop novel fluorescence microscopy assays that directly observe the attack of AMPs on bacterial membranes in real time, for individual cells. For the first time, these methods will enable placement of a number of directly observable events on the same, unified time line covering the first 10 seconds to the first two hours after injection of the AMP. Measured events will include AMP binding density, halting of flagellar motion, cessation of growth, leakage of the periplasm, and leakage of the cytoplasm. Time lapse imaging after removal of the AMP will reveal which symptoms of the attack are reversible and which are not, as well as the time scale on which cells recover growth. The new methods will be used in an exploratory study of the mechanisms by which a variety of different AMPs attack membranes in both the Gram negative E. coli and the Gram positive Bacillus subtilis. These include LL-37, cecropin A, magainin-2, melittin, alamethicin, indolicidin, bactenecin-5, 1-defensin, and protegrin-1. We will directly compare the surface concentrations at which each AMP degrades bacterial cell membranes with results of previous studies in vitro on synthetic lipid bilayers. The results will help determine whether purported carpet- forming and pore-forming mechanisms gleaned from in vitro studies are relevant to real bacterial membranes. The methods developed here will be widely applicable to studies of both AMPs and drug candidates interacting with a variety of bacterial strains. PUBLIC HEALTH RELEVANCE: Bacteria are increasingly resistant to drugs. Antimicrobial peptides are the front-line defense against pathogens throughout the animal kingdom, but we do not really understand how they work to kill bacterial cells. This work will develop novel imaging methods that will enable direct observation of the mechanisms of bacterial killing by antimicrobials with better spatial and time resolution than ever before.