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.

关于对抗生素的抗药性是当今人类面临的最大健康挑战之一。梭状芽胞杆菌艰难梭菌是英国医院抗生素相关感染的主要原因，抗生素引起的肠道菌群破坏是感染的先决条件。当前的治疗依赖于少数抗生素，但这些抗生素对微生物群造成了进一步的损害，并且复发是常见的。迫切需要特定物种的治疗剂，这些疗法可以杀死艰难梭菌，同时保留肠道菌群的有益物种。噬菌体是解决这个棘手问题的有前途的解决方案。阶段是艰难梭菌的有效且特定的杀手，可以通过指导的基因工程进一步完善。我们已经开发了一种简化的冷冻管道，用于噬菌体的结构和机械分析，并已经解决了两个完整的收缩噬菌体的近原子分辨率结构和一个杀死艰难梭菌的噬菌体尾巴样粒子。我们还表明，S层是大多数艰难梭菌噬菌体的主要受体（1，2），并解决了该细胞表面结构的结构（3）。在该项目中，我们旨在利用从结构分析中获得的见解来工程噬菌体，以有效地杀死艰难梭菌的临床重要血统。我们的重点将是回答三个关键问题：1。有效包膜穿透的最佳噬菌体尾部长度和收缩率是多少？2。我们可以使用杂交受体结合蛋白（RBP）的噬菌体以更广泛的特异性来设计噬菌体？3。宿主细胞包膜渗透过程中发生了什么结构变化？这是噬菌体纳米机械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良好特征的噬菌体之一，作为工程床的测试床，是一个更好的杀手。通过遗传修饰和冷冻的迭代过程，我们将了解有助于细菌受体识别和在感染过程中细胞膜穿透的结构特征。我们还将使用尖端冷冻电子层析成像在感染过程中对噬菌体进行图像图像，并确定我们的工程噬菌体在感染模式下如何不同。