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

粪肠球菌和粪肠球菌是引起人类疾病的细菌，包括尿路感染、血液感染、心脏内膜和瓣膜炎症，甚至脑膜炎。由于有效抗生素的选择有限，以及对现有抗生素的耐药性不断增加，肠球菌感染可能难以治疗。粪肠球菌多重耐药菌株的出现导致一些医院50%的感染现在对重要的抗生素万古霉素耐药。与敏感菌株相比，这些耐药菌株的死亡率增加了2.5倍，因此针对这些细菌的新抗生素至关重要。目前，估计只有40个靶点被抗生素用于所有细菌病原体，在临床开发的45种新抗生素中，只有11种属于新类别。从历史上看，确定抗生素开发的新目标一直很困难，但确定新目标的一种途径是利用细菌的天然捕食者，即称为噬菌体的病毒。噬菌体是一种能以特定方式杀死细菌的病毒。它们可以很容易地从环境中分离出来，包括从废水和土壤中分离出来，并且与它们感染的细菌不断共同进化。噬菌体首先通过识别细菌外部的特定分子附着在宿主身上，然后将DNA插入细菌中进行复制。这种DNA编码一系列蛋白质，其中一些蛋白质参与劫持细菌，转移能量消耗和资源，以产生更多的病毒颗粒，而不是正常的细胞过程。尽管噬菌体及其编码的蛋白质对劫持细菌很重要，但大约70%的噬菌体编码的蛋白质没有已知的功能。一些噬菌体已经被证明可以产生对细菌有毒的蛋白质，而且，设计用来模仿这些蛋白质功能的小分子也被证明是有毒的。这些噬菌体编码的许多蛋白质通过与宿主细胞内的蛋白质结合而起作用，并阻止或改变其正常功能。因此，噬菌体蛋白的特征及其在肠球菌细胞内形成的相互作用将为开发新型抗生素提供新靶点的信息。在这个研究项目中，我将鉴定能够杀死粪肠球菌和粪肠球菌的噬菌体蛋白。这将与用x射线和高能电子（cryoEM）绘制这些噬菌体蛋白的三维原子排列相结合。这种结合的方法将揭示噬菌体蛋白杀死细菌的机制，并用于寻找抗生素开发的新靶点。谢菲尔德大学最近投资了1000万英镑用于成像基础设施，包括最先进的冷冻电子显微镜（cryoEM）和光显微镜设备，这将使我能够以前所未有的细节研究噬菌体蛋白。新开发的技术，结合冷冻电镜的“分辨率革命”，允许在接近天然的环境中研究这些蛋白质，在这种环境中，被噬菌体感染的细胞被破坏，细胞内容物被用作结构研究的基础。除了利用我在小分子抑制剂靶向蛋白质方面的专业知识为新型抗生素的设计和工程奠定坚实的基础外，更多关于没有已知功能的噬菌体蛋白的信息也将为整个病毒治疗的噬菌体设计和工程提供信息，通过为我们提供提高病毒感染猎物的能力的新工具。

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.