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抗生素相关感染的主要原因，抗生素引起的肠道微生物群破坏是感染的先决条件。目前的治疗依赖于少量抗生素，但这些抗生素会对微生物群造成进一步的损害，并且复发是常见的。目前迫切需要一种能够杀死艰难梭菌的物种特异性治疗方法，同时保留肠道微生物群的有益物种。噬菌体有望解决这个棘手的问题。噬菌体是艰难梭菌的高效特异性杀手，可以通过引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，并已经解决了两个完整的收缩噬菌体和一个杀死艰难梭菌的噬菌体尾部样颗粒的近原子分辨率结构。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设计一种噬菌体，以有效地杀死临床上重要的艰难梭菌。我们的重点将放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？我们能否利用杂交受体结合蛋白（rbp）来设计具有更广泛特异性的噬菌体？在穿透宿主细胞包膜的过程中会发生什么结构变化？正是噬菌体纳米机器中组件的精致而复杂的结构安排使其成为如此有效的杀手。我们将开发一种现有的特征良好的噬菌体作为设计更好的杀手的试验台。通过基因修饰和CryoEM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穿透细胞包膜的结构特征。我们还将使用尖端的冷冻电子断层扫描在感染期间对噬菌体进行成像，并确定我们的工程噬菌体在感染模式上的差异。