Mechanism of membrane fusion involving the Gram-negative bacteria outer membrane

项目总结/摘要 革兰氏阴性菌构成了大多数抗生素耐药微生物，被认为是对人类健康的紧迫威胁。 美国疾病控制和预防中心（Center for Disease Control and Prevention）。革兰氏阴性菌 对现代抗生素具有抗性的原因是它们的外膜脂多糖（LPS）的不可渗透性 层.这种不可渗透的性质是由于相邻LPS分子之间的强横向相互作用， 包括脂质A、核心糖和O-抗原。纳米治疗能够克服这一障碍， 成功地将抗生素递送到革兰氏阴性菌中对于人类健康将是极其重要。是 我们打算通过使用从描述机制中获得的知识来开发这种纳米治疗剂， 革兰氏阴性噬菌体将其外部细菌磷脂（BPL）膜与 启动感染。包埋在囊病毒BPL中的是多聚体蛋白复合物， 称为刺突，其识别革兰氏阴性宿主并进行融合。这些蛋白质类似于 与更好地研究的负责膜融合的真核蛋白（例如HIV env，West 尼罗河病毒糖蛋白E和疱疹病毒科gB蛋白）。这项建议的目的是调查 来自囊病毒家族三个不同成员的刺突识别其宿主的机制， 驱动膜融合。本提案的目标是确定尖峰的结构，以建立一个 生物化学和生物物理研究的结构支架（目的1），确定细胞的机制， 识别（目的2），并确定负责LPS-BPL融合的生化决定簇（目的3）。的 该项目的长期目标是使用长钉将抗生素输送到致病性革兰氏菌的特定菌株， 阴性细菌。

PROJECT SUMMARY/ABSTRACT Gram-negative bacteria constitute the majority of antibiotic resistant organisms identified as urgent threats to human health by the Center for Disease Control and Prevention. A major reason why Gram-negative bacteria are resistant to modern antibiotics is the impermeable nature of their outer membrane lipopolysaccharide (LPS) layer. This impermeable nature is due to the strong lateral interaction between neighboring LPS molecules that includes lipid A, the core saccharide, and the O-antigen. Nanotherapeutics capable of overcoming this barrier to successfully deliver antibiotics to Gram-negative bacteria will be of immense importance for human health. It is our intent to develop such nanotherapeutics by using the knowledge attained from delineating the mechanism by which a Gram-negative bacteriophage fuses its external bacterial phospholipid (BPL) membrane with the LPS of its host for initiating infection. Embedded into the Cystovirus BPL are multimeric protein complexes, referred to as spikes, that recognize the Gram-negative host and perform fusion. These proteins are analogous to, but different from, the better studied eukaryotic proteins responsible for membrane fusion (e.g. HIV env, West Nile Virus glycoprotein E, and Herpesviridae gB protein). The intent of this proposal is to investigate the mechanism by which spikes from three different members of the Cystovirus family recognize their hosts and drive membrane fusion. The goals of this proposal are to determine the structure of the spikes for establishing a structural scaffold for biochemical and biophysical studies (Aim 1), determine the mechanism of cellular recognition (Aim 2), and determine the biochemical determinants responsible for LPS- BPL fusion (Aim 3). The long-term goal of this project is to use the spikes for delivery of antibiotics to specific strains of pathogenic Gram- negative bacteria.