Towards phage therapy: combining genetics and cutting edge CryoEM to optimise a bacterial virus to kill a superbug

关于该项目对抗生素的耐药性增加是当今人类面临的最大健康挑战之一。艰难梭状芽胞杆菌是英国医院抗生素相关感染的主要原因，抗生素诱导的肠道微生物区系破坏是感染的先决条件。目前的治疗方法依赖于少量的抗生素，但这些会对微生物区系造成进一步的破坏，复发很常见。迫切需要一种针对特定物种的疗法，既能杀死艰难梭菌，又能保护肠道微生物区系中的有益物种。噬菌体是解决这一棘手问题的一个有希望的解决方案。噬菌体是艰难梭菌的有效和特异的杀手，并可以通过引导基因工程进一步提炼。我们已经开发了一条用于噬菌体结构和机械分析的流线型低温EM管道，并已经解决了杀死艰难梭菌的两个完整可收缩噬菌体和一个噬菌体尾状颗粒的近原子分辨结构。我们还发现S层是大多数艰难梭菌噬菌体(1，2)的主要受体，并解决了这种细胞表面结构(3)的结构。在这个项目中，我们的目标是利用从结构分析中获得的见解来设计一种能够有效杀灭艰难梭菌临床上重要谱系的噬菌体。我们的重点将集中在回答三个关键问题上：1.有效穿透包膜的最佳噬菌体尾长和收缩比是多少？2.我们能否利用杂交受体结合蛋白(RBPs)来设计具有更广泛特异性的噬菌体？3.在穿透宿主细胞包膜的过程中发生了什么结构变化？正是噬菌体纳米机器中组件的精致和复杂的结构排列使其成为如此有效的杀手。我们将开发一种现有的特性良好的噬菌体，作为设计更好的杀手的试验台。通过遗传修饰和低温EM的迭代过程，我们将了解在感染过程中有助于细菌受体识别和细胞膜穿透的结构特征。我们还将使用尖端的冷冻电子断层扫描技术对感染期间的噬菌体进行成像，并确定我们的工程噬菌体在感染模式上的不同之处。

About the ProjectIncreasing resistance to antibiotics is one of the greatest health challenges facing humanity today. Clostridioides difficile is the primary cause of antibiotic-associated infections in UK hospitals and antibiotic-induced disruption of the gut microbiota is a prerequisite for infection. Current treatments rely on a small number of antibiotics but these cause further damage to the microbiota and relapse is common. There is an urgent need for species specific therapeutics that can kill C. difficile while sparing the beneficial species of the gut microbiota. Bacteriophage are a promising solution to this tricky problem.Phage are efficient and specific killers of C. difficile and could be further refined through guided genetic engineering. We have developed a streamlined CryoEM pipeline for the structural and mechanistic analysis of phage and have already solved the near atomic resolution structures of two complete contractile phages and one phage tail-like particle that kill C. difficile. We have also shown that the S-layer is the major receptor for the majority of C. difficile phage (1, 2) and have solved the structure of this cell surface structure (3).In this project we aim to use the insights gained from structural analysis to engineer a phage to efficiently kill clinically important lineages of C. difficile. Our focus will be on answering three key questions:1. What is the optimum phage tail length and contraction ratio for effective envelope penetration?2. Can we engineer phage with wider specificity using hybrid receptor binding proteins (RBPs)?3. What structural changes occur during penetration of the host cell envelope?It is the exquisite and intricate structural arrangement of components in the phage nanomachine that makes it such an effective killer. We will develop one of our existing well-characterised phage as a test-bed for engineering a better killer. Through an iterative process of genetic modification and CryoEM we will understand the structural features that contribute to bacterial receptor recognition and penetration of the cell envelope during infection. We will also use cutting edge cryo-electron tomography to image phage during infection and determine how our engineered phages differ in modes of infection.