Structural and functional determinants of decision-making in bacteriophage host recognition

噬菌体宿主识别决策的结构和功能决定因素

• 批准号: 10451607
• 负责人: Petr G Leiman
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-10 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451607
• 关键词: Antibiotic Therapy; Bacteria; Bacteriophages; Binding; Binding Proteins; Biological Models; C-terminal; Capsid; Cell Surface Receptors; Cell physiology; Cell surface; Cells; Computers; Cryo-electron tomography; Cryoelectron Microscopy; Cytoplasm; DNA; Data; Decision Making; Dengue; Detection; Diagnostic; Energy-Generating Resources; Escherichia coli; Event; Fiber; Fluorescence; Foundations; Goals; Health; Human; Infection; Kinetics; Knock-out; Knowledge; Lipopolysaccharides; Measures; Medical; Membrane; Membrane Proteins; Modeling; Molecular Conformation; Motion; N-terminal; Nature; O Antigens; Organelles; Pathway interactions; Phase; Play; Polysaccharides; Process; Protein Conformation; Proteins; Protocols documentation; Regulation; Resistance; Resolution; Role; Rotation; Shigella dysenteriae; Side; Signal Transduction; Structure; Surface; System; Tail; Therapeutic; VWA7 gene; Vertebral column; Virion; Virus; Virus Activation; West Nile virus; Work; X-Ray Crystallography; ZIKA; antibiotic resistant infections; antimicrobial; contrast imaging; fluorescence imaging; human pathogen; improved; instrument; mass spectrometric imaging; microcalorimetry; mutant; particle; public health relevance; rational design; receptor; receptor binding; small molecule; sugar; tool

Abstract Bacterial viruses (bacteriophages) recognize their host cells with the help of specialized Receptor-Binding Proteins (RBPs) that emanate from the ‘tail’, a host attachment organelle of the phage. RBPs are either long and slender fibers devoid of enzymatic activity or shorter and stockier tailspikes that can digest or modify polysaccharide molecules that extend from or cover the surface of a bacterial cell. Upon host attachment, the tail creates a conduit between the phage capsid and the host cell cytoplasm allowing phage DNA and proteins to be delivered into the cell. Several aspects of this process, especially those that concern the transition from the initial recognition event to irreversible attachment, remain poorly understood. As components of the phage particle, tailspike RBPs are required for both the initial recognition and irreversible attachment. However, isolated tailspike RBPs destroy the cell surface receptor and make the cell resistant to the phage carrying those RBPs. Furthermore, tail fiber RBPs bind to the host cell weakly, but this binding triggers a conformational change in the particle committing it to irreversible attachment. Our goal is to describe this transition and the associated structural transformation of the virus particle for bacteriophage G7C, a virus that infects Escherichia coli and Shigella dysenteriae. G7C has a short tail, 24 tailspike RBPs of two different types, and contains several large proteins inside the capsid. In Aim 1, we will examine the role of different domains of the two tailspike RBPs in host cell recognition and attachment. We will measure the energy of binding of G7C RBPs to their O-antigen substrates. We will also establish the number of RBPs per particle required for infection. We will develop a protocol for fluorescence/phase contrast imaging and computer processing of attachment of the phage to the host cell in a single cell and ensemble modes. In Aim 2, we will examine the structural transformation of the phage particle upon irreversible attachment with the help of cryo-electron microscopy, cryo-electron tomography, and X-ray crystallography. In Aim 3, we will identify the outer membrane receptor for G7C that causes opening of the tail channel and DNA release and examine the structure of G7C bound to that receptor. In summary, the overarching goal of this proposal is to quantitively describe how bacteriophages commit themselves to irreversible attachment, what kind of factors are involved, what is the source of energy that activates the particle for irreversible attachment. The results of the proposed work will lay a foundation for quantitative description of attachment in other phages and, possibly, in other viruses.

抽象的 细菌病毒（噬菌体）借助专门的受体结合来识别宿主细胞 从“尾巴”（噬菌体的宿主附着细胞器）发出的蛋白质 (RBP)。 RBP 要么很长，要么 缺乏酶活性的细长纤维或可以消化或修饰的更短、更粗的尾刺 从细菌细胞表面延伸或覆盖细菌细胞表面的多糖分子。主机附着后， 尾部在噬菌体衣壳和宿主细胞质之间形成管道，允许噬菌体 DNA 和蛋白质 被输送到细胞内。这个过程的几个方面，特别是那些涉及从 对不可逆依恋的最初认识事件仍然知之甚少。作为噬菌体的组成部分 颗粒、尾尖 RBP 是初始识别和不可逆附着所必需的。然而，孤立 尾刺 RBP 会破坏细胞表面受体，并使细胞对携带这些 RBP 的噬菌体产生抵抗。 此外，尾纤维 RBP 与宿主细胞的结合较弱，但这种结合会引发宿主细胞的构象变化。 粒子使其不可逆转地附着。我们的目标是描述这种转变以及相关的 噬菌体 G7C（一种感染大肠杆菌的病毒）的病毒颗粒的结构转化 志贺氏痢疾杆菌。 G7C有一个短尾，24个两种不同类型的尾刺RBP，并包含几个大的 衣壳内的蛋白质。在目标 1 中，我们将研究两个尾尖 RBP 的不同域在 宿主细胞识别和附着。我们将测量 G7C RBP 与其 O 抗原的结合能量 基材。我们还将确定感染所需的每个颗粒的 RBP 数量。我们将开发一个 用于荧光/相差成像和噬菌体附着的计算机处理的方案 宿主细胞有单细胞和群体模式。在目标 2 中，我们将研究经济结构的转型 借助冷冻电子显微镜、冷冻电子断层扫描不可逆附着的噬菌体颗粒， 和X射线晶体学。在目标 3 中，我们将识别导致开放的 G7C 外膜受体 尾通道和 DNA 释放，并检查与该受体结合的 G7C 的结构。综上所述， 该提案的总体目标是定量描述噬菌体如何致力于 不可逆附着，涉及哪些因素，激活粒子的能量来源是什么 用于不可逆转的附着。所提出的工作结果将为定量描述奠定基础 附着在其他噬菌体中，也可能附着在其他病毒中。