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。众所周知，阳离子amp形成两亲螺旋，与阴离子细胞膜结合，形成孔，并最终溶解细胞。细菌相对无法抵抗这种一般的作用模式，这使得amp及其模拟物成为抗抗生素耐药菌株的有趣候选药物。虽然已经确定AMPs可以降解细菌膜并最终导致裂解，但大多数机制工作都集中在体外合成囊泡的研究或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。这项工作的主要目标是开发新的荧光显微镜检测方法，直接观察AMPs对单个细胞的细菌膜的实时攻击。这些方法将首次能够在注射AMP后的前10秒到前两个小时的同一时间线上放置许多直接可观察到的事件。测量的事件将包括AMP结合密度、鞭毛运动停止、生长停止、外周质渗漏和细胞质渗漏。去除AMP后的延时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革兰氏阴性大肠杆菌和革兰氏阳性枯草芽孢杆菌中各种不同amp攻击膜的机制。这些包括LL-37、cecropin A、magainin-2、蜂毒素、alamethicin、吲哚霉素、bactenecin-5、1-防御素和蛋白素-1。我们将直接比较每种AMP降解细菌细胞膜的表面浓度与先前体外合成脂质双分子层的研究结果。结果将有助于确定从体外研究中收集的所谓的地毯形成和孔形成机制是否与真正的细菌膜有关。本文开发的方法将广泛适用于研究amp和候选药物与各种细菌菌株的相互作用。

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.