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

项目摘要/摘要 革兰氏阴性菌构成了大多数被确认为对抗生素产生紧急威胁的抗生素耐药性生物 疾病控制和预防中心的人类健康。革兰氏阴性菌的一个主要原因 对现代抗生素有抵抗力的是其外膜脂多糖(LPS)的不透性 一层。这种不透性是由于邻近的内毒素分子之间强烈的侧向相互作用造成的 包括类脂A，核心糖，和O抗原。纳米疗法能够克服这一障碍 成功地将抗生素输送到革兰氏阴性菌将对人类健康具有极其重要的意义。它是 我们打算通过利用从描绘机制中获得的知识来开发这种纳米疗法 革兰氏阴性噬菌体通过这种方式将其外部细菌磷脂(BPL)膜与 其宿主的内毒素，以启动感染。嵌入到囊状病毒BPL中的是多聚体蛋白复合体， 被称为尖峰，识别革兰氏阴性宿主并进行融合。这些蛋白质是相似的 到，但不同于，更好地研究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)。这项提案的目的是调查 来自囊状病毒家族三个不同成员的刺激物识别其宿主和 推动膜融合。这项提案的目标是确定峰值的结构，以建立 生物化学和生物物理研究的结构支架(目标1)，确定细胞的机制 识别(目标2)，并确定导致内毒素-BPL融合的生化决定因素(目标3)。这个 该项目的长期目标是利用尖峰将抗生素输送到特定的致病革兰氏菌株。 阴性菌。

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.