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识别其宿主细胞 蛋白质（RBP）从噬​​菌体的宿主附着细胞器“尾巴”中散发出来。 rbps要么很长， 没有酶活性的细长纤维或可以消化或修改的较短且较短的尾巴 从或覆盖细菌细胞表面延伸或覆盖的多糖分子。在主机附件上， 尾巴在噬菌体衣壳和宿主细胞细胞质之间形成导管，允许噬菌体DNA和蛋白质 将其输送到单元格中。这个过程的几个方面，尤其是那些关注从 不可逆依恋的最初识别事件仍然了解不足。作为噬菌体的组成部分 初始识别和不可逆的附件都需要粒子，尾尖端RBP。但是，孤立 Tailspike RBP破坏了细胞表面受体，并使携带这些RBP的噬菌体具有抗性。 此外，尾纤维RBP与宿主细胞结合弱，但这种结合触发了构象变化 粒子将其归于不可逆的附件。我们的目标是描述这种过渡和相关的 细菌g7c病毒颗粒的结构转化，一种感染大肠杆菌和大肠杆菌的病毒 Shigella dysenteriae。 G7C有两种不同类型的短尾巴，24个尾巴rbps，并包含几个大型 衣壳内的蛋白质。在AIM 1中，我们将研究两个尾式rbps的不同域中的作用 宿主细胞识别和依恋。我们将测量G7C RBP与O-抗原结合的能量 基材。我们还将建立每个感染所需的RBP的数量。我们将发展一个 荧光/相对比成像和计算机处理的协议 单个单元格和集合模式中的主机单元。在AIM 2中，我们将研究 噬菌体粒子在不可逆的附着时，借助冷冻电子显微镜，冷冻电子断层扫描， 和X射线晶体学。在AIM 3中，我们将确定导致打开的G7C的外膜受体 尾通道和DNA的释放，并检查与该受体结合的G7C的结构。总而言之， 该提案的总体目标是定量描述噬菌体如何致力于 不可逆转的附件，涉及哪种因素，激活粒子的能源是什么 用于不可逆的依恋。拟议工作的结果将为定量描述奠定基础 在其他噬菌体中附着，并可能在其他病毒中附着。