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

参与肠球菌宿主劫持的噬菌体蛋白的结构表征

• 批准号: BB/Z515188/1
• 负责人: Jason Wilson
• 金额: $ 53.5万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FZ515188%2F1
• 关键词: Structural; Characterisation; Bacteriophage; Proteins; Involved

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.

粪肠球菌和肠球菌是引起人类疾病的细菌，包括尿路感染，血流感染，心脏内衬和瓣膜的感染，甚至是脑膜炎。由于有效的抗生素选择有限，并且对那些可用的抗生素的耐药性增加，因此很难治疗肠球菌感染。粪肠球菌多药抗性菌株的出现导致某些医院的感染约50％，现在对重要的抗生素万古霉素具有抗药性。与敏感菌株相比，这些抗性菌株与死亡率增加了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个靶标被抗生素对所有细菌病原体所利用，而在临床发育中的45种新抗生素中，只有11个属于新的类别。从历史上看，确定抗生素开发的新靶标一直很困难，但是鉴定新靶标的途径是利用细菌的天然捕食者，称为细菌的病毒。细菌性噬菌体是可以以非常具体的方式杀死细菌的病毒。它们可以轻松地与环境中的环境分离，包括从废水和土壤中分离出来，并不断与他们感染的细菌共同发展。噬菌体首先通过在细菌外部识别特定分子，然后将DNA插入细菌以复制。该DNA编码一系列蛋白质，其中一些涉及劫持细菌以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。尽管细菌及其编码蛋白对于劫持细菌的重要性，但大约70％的由细菌编码的蛋白质尚无已知功能。一些细菌噬细胞已被证明会产生对细菌有毒的蛋白质，此外，旨在模仿这些蛋白质功能的小分子也证明也有毒。通过这些噬菌体功能编码的许多蛋白质通过与宿主细胞中的蛋白质结合，并防止或重定向其正常功能。因此，噬菌体蛋白质及其在肠球菌细胞中形成的相互作用的表征将提供有关可用于开发新类别抗生素的新目标的信息。在本研究计划中，我将确定能够杀死粪肠球菌和粪肠球菌的噬菌体蛋白。这将结合绘制这些噬菌体蛋白与X射线和高能电子（Cryoem）的3D原子排列。这种合并的方法将为噬菌体蛋白杀死细菌的机制提供信息，并用于寻找抗生素发育的新靶标。谢菲尔德大学（University of Sheffield）最近在成像基础设施上进行了1000万英镑的投资，包括最先进的冷冻电子显微镜（Cryoem）和光学显微镜设施，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，结合了冷冻中的“分辨率革命”，可以在近乎本地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破裂，细胞含量被用作结构研究的基础。除了使用我既定的专业知识在具有小分子抑制剂靶向蛋白质方面的新型抗生素的设计和工程中构成稳固的基础外，还提供有关噬菌体蛋白质具有无知功能的噬菌体蛋白质的更多信息，还将为整个病毒治疗的细菌设计和工程提供，从而为整个病毒治疗的设计和工程提供了改善病毒感染能力的新工具。