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

摘要 细菌病毒(噬菌体)通过特殊的受体结合来识别它们的宿主细胞。 从噬菌体的宿主附着细胞器‘尾’发出的蛋白质(RBPs)。RBP要么很长，要么 没有酶活性的细长纤维或可以消化或修饰的更短更结实的尾穗 从细菌细胞表面延伸出来或覆盖在细菌细胞表面的多糖分子。在连接主机时， Tail在噬菌体衣壳和宿主细胞细胞质之间建立了一条管道，允许噬菌体DNA和蛋白质 输送到细胞内。这一进程的几个方面，特别是涉及从 对不可逆依恋的最初认知事件，目前仍知之甚少。作为噬菌体的组成部分 颗粒、尾峰限制性商业惯例对于初始识别和不可逆附着都是必需的。然而，孤立的 尾峰限制性商业惯例破坏细胞表面受体，使细胞对携带限制性商业惯例的噬菌体产生抵抗力。 此外，尾部纤维RBPs与宿主细胞结合较弱，但这种结合触发了 粒子将其附着到不可逆的位置。我们的目标是描述这种过渡和相关的 噬菌体G7C病毒颗粒的结构转化 志贺氏菌痢疾。G7C有一条短尾，有两种不同类型的24个尾尖RBP，包含几个大的 衣壳内的蛋白质。在目标1中，我们将研究两个尾峰限制性商业惯例的不同结构域在 宿主细胞的识别和附着。我们将测量G7C限制性商业惯例与其O抗原的结合能 底物。我们还将确定感染所需的每种颗粒的限制性商业惯例数量。我们将开发一种 荧光/相衬成像和计算机处理噬菌体与 宿主细胞在单细胞和系综模式下。在目标2中，我们将研究 在冷冻电子显微镜、冷冻电子断层扫描的帮助下， 和X射线结晶学。在目标3中，我们将确定导致开放的G7C的外膜受体 并检查与该受体结合的G7C的结构。总而言之， 这项提案的首要目标是定量描述噬菌体如何致力于 不可逆转的依附，涉及到什么样的因素，激活粒子的能量来源是什么 不可逆的依恋。该工作的结果将为定量化描述奠定基础。 附着在其他噬菌体上，也可能附着在其他病毒上。

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.