Structural Characterisation of Bacteriophage Proteins Involved in Host Hijacking of Enterococcus Species

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Enterococcus faecalis and Enterococcus Faecium are bacteria that cause human disease, including urinary tract infections, bloodstream infections, inflammation of the heart lining and valves, and even meningitis. Enterococcal infections can be hard to treat, due to limited choice of effective antibiotics, and increasing resistance to those antibiotics that are available. The emergence of multi-drug resistant strains of Enterococcus faecium has led to ~50% of infections in some hospitals now being resistant to the important antibiotic vancomycin. These resistant strains are associated with 2.5-fold increased mortality compared with sensitive strains, so new antibiotics targeting these bacteria are critical. Currently, only an estimated 40 targets are exploited by antibiotics towards all bacterial pathogens, and of 45 new antibiotics in clinical development, only 11 belong to novel classes. Identifying new targets for antibiotic development has been historically difficult, but one avenue for identifying new targets is to harness natural predators of bacteria, viruses called bacteriophage.Bacteriophages are viruses that can kill bacteria in a very specific manner. They can be isolated readily from the environment, including from wastewater and the soil, and are constantly co-evolving alongside the bacteria they infect. Bacteriophages first attach to their host by recognising specific molecules on the outside of the bacteria and then insert DNA into the bacteria in order to replicate. This DNA encodes for an array of proteins, some of which are involved in hijacking the bacteria to divert energy expenditure and resources to produce more virus particles rather than normal cellular processes. Despite the importance of bacteriophages and their encoded proteins for hijacking bacteria, around 70% of proteins encoded by bacteriophages have no known function. Some bacteriophages have been shown to produce proteins that are toxic to bacteria, and further, small molecules designed to mimic the function of these proteins have been shown to also be toxic. Many proteins encoded by these phage function by binding to proteins within the host cell, and preventing or redirecting their normal function. Therefore, the characterisation of phage proteins and the interactions they form within Enterococcus cells will provide information about new targets available for the development of new classes of antibiotics.In this research programme, I will identify phage proteins that are able to kill Enterococcus faecalis and Enterococcus Faecium. This will be combined with mapping out the 3D atomic arrangements of these phage proteins with X-rays and high-energy electrons (cryoEM). This combined approach will inform the mechanism by which phage proteins can kill bacteria, and be used in order to find new targets for antibiotic development. The University of Sheffield's recent £10M investment in imaging infrastructure, including state-of-the-art cryoelectron microscopy (cryoEM) and light microscopy facilities, will allow me to study phage proteins in unprecedented detail. Newly developed techniques, combined with the 'resolution revolution' in cryoEM allow for these proteins to be studied in a near-native environment, where cells infected with phage are broken, and the cellular contents are used as a basis for structural study. As well as forming a solid foundation for the design and engineering of novel antibiotics using my established expertise in targeting proteins with small molecule inhibitors, more information about phage proteins with no known function will also inform the design and engineering of bacteriophage for whole virus treatments, by giving us new tools for improving the virus's ability to infect its prey.