Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning.
Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning.
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DOI：
10.7554/elife.63775
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发表时间：
2021-03-09
期刊：
影响因子：
7.7
通讯作者：
Raman S
中科院分区：
文献类型：
--
作者：
Huss P;Meger A;Leander M;Nishikawa K;Raman S
噬菌体与宿主之间的相互作用是由噬菌体受体结合蛋白（RBP）介导的。尽管RBP在控制噬菌体活性和宿主范围方面起着重要作用，但其功能的分子规则仍然是一个谜。在这里，我们通过开发一种高通量、位点特异性的噬菌体工程方法，系统地剖析了T7噬菌体RBP（1660个变体）尖端区域中每个残基的功能作用。这个丰富的数据集使我们能够交叉比较不同宿主的功能概况，以精确识别功能重要的区域，其中许多区域以前是未知的。替代模式显示了突变的位置和物理化学性质在宿主特异性上的差异，揭示了个体宿主的分子适应性。我们发现了针对抗性宿主的功能获得变体和消除某些宿主的宿主收缩变体。为了证明治疗效用，我们设计了高活性的T7变体来对抗尿路病原体。我们的方法为许多噬菌体-细菌系统中序列-功能关系的表征提供了一个广义框架。细菌可以引起疾病，但它们也要与自己的微观敌人作战：一组被称为噬菌体的病毒。例如，T7噬菌体捕食大肠杆菌的各种菌株，大肠杆菌是一种常见于人类肠道的细菌。虽然许多大肠杆菌菌株是无害的，甚至对人体健康有益，但有些菌株可能是致命的。找到一种方法来杀死有害的菌株，同时保留有益的菌株，这将是对医学工具包的有益补充。噬菌体通过一种被称为受体结合蛋白（RBP）的结构识别并与它们的特定靶标相互作用。然而，目前尚不清楚RBP是如何帮助病毒识别感染哪种细菌的。在这里，Huss等人开始绘制和修改T7噬菌体的这种结构，这样病毒就能更有效和更具体地杀死哪一株大肠杆菌。首先，仔细分析了RBP尖端各组成部分的作用；这产生了对大量不同的T7噬菌体进行基因工程所需的知识，每种噬菌体的RBP都有轻微的变化。然后将这些病毒暴露于不同菌株的细菌中。对感染不同大肠杆菌菌株后存活和繁殖最多的噬菌体进行监测，揭示了哪些RBP构建块对效率和特异性很重要。然后通过设计高活性的T7噬菌体变体来对抗引起尿路感染的大肠杆菌菌株，证实了这一点。这些发现表明，即使对噬菌体进行很小的改变，也会对它们感染猎物的能力产生很大的影响。Huss等人开发的方法有助于准确理解RBP如何允许病毒感染特定类型的细菌；有一天，这可能会为利用这些病毒对抗越来越耐药的细菌感染的新疗法铺平道路。
The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP (1660 variants) by developing a high-throughput, locus-specific, phage engineering method. This rich dataset allowed us to cross compare functional profiles across hosts to precisely identify regions of functional importance, many of which were previously unknown. Substitution patterns showed host-specific differences in position and physicochemical properties of mutations, revealing molecular adaptation to individual hosts. We discovered gain-of-function variants against resistant hosts and host-constricting variants that eliminated certain hosts. To demonstrate therapeutic utility, we engineered highly active T7 variants against a urinary tract pathogen. Our approach presents a generalized framework for characterizing sequence–function relationships in many phage–bacterial systems. Bacteria can cause diseases, but they also battle their own microscopic enemies: a group of viruses known as bacteriophages. For instance, the T7 bacteriophage preys on various strains of Escherichia coli, a type of bacteria often found in the human gut. While many E. coli strains are inoffensive or even beneficial to human health, some can be deadly. Finding a way to kill harmful strains while sparing the helpful ones would be a helpful addition to the medicine toolkit. Bacteriophages identify and interact with their specific target through a structure known as the receptor binding protein, or RBP. However, it is still unclear exactly how RBP helps the viruses recognize which type of bacteria to infect. Here, Huss et al. set to map out and modify this structure in T7 bacteriophage so the virus is more efficient and specific about which strain of E. coli it kills. First, the role of each building block in the tip of RBP was meticulously dissected; this generated the knowledge required to genetically engineer a large number of different T7 bacteriophages, each with a slightly variation in their RBP. These viruses were then exposed to various strains of bacteria. Monitoring the bacteriophages that survived and multiplied the most after infecting different strains of E. coli revealed which RBP building blocks are important for efficiency and specificity. This was then confirmed by engineering highly active T7 bacteriophage variants against an E. coli strain that causes urinary tract infections. These findings demonstrate that even small changes to the bacteriophages can make a big difference to their ability to infect their preys. The approaches developed by Huss et al. help to understand exactly how the RBP allows a virus to infect a specific type of bacteria; this could one day pave the way for new therapies that harness those viruses to fight increasingly resistant bacterial infections.