Dynamics of Antimicrobial Peptide Interactions with Bacterial Membranes

描述(申请人提供)：在植物、动物和人类中已经发现了各种各样的自然表达的抗菌肽(AMP)。AMP以其抑制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形成两亲性螺旋，结合到阴离子细胞膜上，形成毛孔，最终溶解细胞。细菌相对无法抵抗这种一般的作用模式，这使得AMP和它们的模拟物成为对抗抗生素耐药菌株的有趣的候选药物。虽然AMPs降解细菌膜并最终导致溶解是公认的，但大多数的机制工作要么集中在体外合成囊泡的研究上，要么集中在AMPS对细菌的长期整体效应上。AMP攻击细菌膜的详细机制还不是很清楚。这项工作的主要目标是开发新的荧光显微镜分析方法，直接实时观察AMPS对单个细胞的攻击。这些方法将首次实现在注射AMP后的前10秒到前2小时的相同、统一的时间线上放置许多直接可观察的事件。测量的事件包括AMP结合密度、鞭毛运动停止、生长停止、周质渗漏和细胞质渗漏。移除AMP后的时间推移成像将揭示哪些发作症状是可逆的，哪些是不可逆转的，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的AMP攻击革兰氏阴性大肠杆菌和革兰氏阳性枯草芽孢杆菌的膜的机制。这些药物包括LL-37、抗菌素A、麦维菌素-2、蜂毒素、阿拉米星、吲哚青霉素、杆菌素-5、1-防御素和前列环素-1。我们将直接将每个AMP降解细菌细胞膜的表面浓度与先前在人工合成脂质双层上的体外研究结果进行比较。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形成和孔形成机制是否与真实的细菌膜相关。这里开发的方法将广泛适用于AMP和候选药物与各种细菌菌株相互作用的研究。 与公共卫生相关：细菌对药物的抗药性越来越强。抗菌肽是整个动物界对抗病原体的第一线防御措施，但我们并不真正了解它们是如何杀死细菌细胞的。这项工作将开发新的成像方法，使人们能够以比以往任何时候更好的空间和时间分辨率直接观察抗菌剂杀死细菌的机制。

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.