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.

摘要