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

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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.