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

迈向噬菌体疗法：结合遗传学和尖端冷冻电镜来优化细菌病毒以杀死超级细菌

• 批准号: 2902040
• 金额: --
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2902040
• 关键词: Towards; phage; therapy; combining; genetics

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.

抗生素耐药性的增加是当今人类面临的最大健康挑战之一。艰难梭菌是英国医院中疟疾相关感染的主要原因，疟疾诱导的肠道微生物群破坏是感染的先决条件。目前的治疗方法依赖于少量抗生素，但这些抗生素会对微生物群造成进一步损害，复发很常见。目前迫切需要一种能杀死C.艰难的，同时保留肠道微生物群的有益物种。噬菌体是一种高效、特异的杀灭C.艰难的，并可以通过指导基因工程进一步完善。我们已经开发了一个流线型的CryoEM管道用于噬菌体的结构和机制分析，并且已经解决了两个完全收缩的噬菌体和一个杀死C的噬菌体尾状颗粒的近原子分辨率结构。很难我们还表明，S层是大多数C的主要受体。艰难梭菌噬菌体（1，2），并解决了这种细胞表面结构的结构（3）。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工程化噬菌体，以有效地杀死临床上重要的C.很难我们的重点将是回答三个关键问题：1。什么是最佳的噬菌体尾部长度和收缩比有效的信封穿透？2.我们能用杂交受体结合蛋白（RBP）设计具有更广泛特异性的噬菌体吗？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。我们将开发我们现有的一种特征良好的噬菌体，作为设计更好的杀手的试验台。通过遗传修饰和CryoEM的迭代过程，我们将了解在感染期间有助于细菌受体识别和穿透细胞包膜的结构特征。我们还将使用最先进的冷冻电子断层扫描技术在感染过程中对噬菌体进行成像，并确定我们的工程化噬菌体在感染模式上的差异。