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: 10650340
• 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/10650340
• 关键词: Antibiotic Therapy; Antibiotics; 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; Mass Spectrum Analysis; Measures; Medical; Membrane; Membrane Proteins; Modeling; Molecular Conformation; Motion; N-terminal; Nature; O Antigens; Organelles; Pathway interactions; Phase; Polysaccharides; Process; Proteins; Protocols documentation; Regulation; Resistance; Resolution; Role; Rotation; Shigella dysenteriae; Side; Signal Transduction; Structure; Surface; System; Tail; Tertiary Protein Structure; Therapeutic; VWA7 gene; Vertebral column; Virion; Virus; Virus Activation; West Nile; Work; X-Ray Crystallography; ZIKA; antibiotic resistant infections; antimicrobial; contrast imaging; fluorescence imaging; gut microbiota; human pathogen; improved; instrument; microcalorimetry; mutant; particle; public health relevance; rational design; receptor; receptor binding; small molecule; sugar; tool; transforming virus

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.

摘要 细菌病毒（噬菌体）在特异性受体结合的帮助下识别其宿主细胞。 从噬菌体的宿主附着细胞器“尾部”发出的蛋白质（RBP）。RBP要么很长， 没有酶活性的细长纤维或更短更粗壮的尾穗，可以消化或改变 从细菌细胞表面延伸或覆盖细菌细胞表面的多糖分子。主机连接后， 尾部在噬菌体衣壳和宿主细胞质之间形成了一个管道， 被输送到细胞中。这一进程的几个方面，特别是那些涉及从 不可逆依恋的初始识别事件仍然知之甚少。作为噬菌体的组成部分 粒子，尾钉RBP是需要的初始识别和不可逆的连接。然而，孤立 尾刺RBP破坏细胞表面受体并使细胞对携带那些RBP的噬菌体具有抗性。 此外，尾纤维RBP与宿主细胞的结合较弱，但这种结合引发了细胞内构象的变化。 粒子使其不可逆地附着。我们的目标是描述这种转变和相关的 噬菌体G7 C的病毒颗粒的结构转化，噬菌体G7 C是一种感染大肠杆菌的病毒， 志贺氏菌G7 C有一个短尾，24尾穗两种不同类型的RBP，并含有几个大的 衣壳内的蛋白质在目标1中，我们将研究两个尾峰RBP的不同结构域在 宿主细胞识别和附着。我们将测量G7 C RBPs与其O-抗原结合的能量 印刷受体.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/相衬成像和噬菌体附着到细胞的计算机处理的方案 单细胞中宿主细胞和系综模式。在目标2中，我们将研究 在冷冻电子显微镜，冷冻电子断层扫描， 和X射线晶体学在目标3中，我们将鉴定引起开放的G7 C的外膜受体 尾通道和DNA的释放，并检查与该受体结合的G7 C的结构。总而言之， 该提案的首要目标是定量描述噬菌体如何致力于 不可逆转的附着，涉及哪些因素，激活粒子的能量来源是什么 不可逆的连接研究结果将为定量描述 在其他病毒中也可能存在。