Bacteriophage engineering as a therapeutic strategy to target antibiotic resistant enterococci

噬菌体工程作为针对抗生素耐药性肠球菌的治疗策略

• 批准号: 2902045
• 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-2902045
• 关键词: Bacteriophage; engineering; therapeutic; strategy; target

About the ProjectThis project will build on our preliminary work to develop phage therapeutics targeting Vancomycin-Resistant-Enterococci (VRE), which are opportunistic pathogens causing both community and hospital-acquired infections (Al-zubidi et al., 2019). Due to the emergence of pathogens multi-resistant to antibiotics, there is a pressing need for novel antimicrobial strategies. Bacteriophages are highly suitable as therapeutic agents because they target specific pathogens within complex bacterial communities without causing a microbiome imbalance. Bacteriophages recognise specific structures at the bacterial cell surface and usually display a narrow host range, sometimes limited to a few strains. To circumvent this potential issue, phages isolated from the environment are combined to produce "phage cocktails" with an extended host range. An alternative approach is to carry out phage engineering to modify the receptor binding proteins, to generate tailored therapeutics.The specific objectives of the project are the following:1) Characterising the receptor binding proteins of phages targeting enterococci. We recently isolated virulent phages targeting Enterococcus faecalis. Three phages displayed high sequence identity but distinct host ranges. One phage was shown to recognise decorations of the Enterococcal Polysaccharide Antigen (EPA), variable between strains. Using the expertise of the primary supervisor in protein-bacterial cell wall interactions, we propose to investigate how phage receptor proteins recognise the EPA. We will use both bacterial mutants and in vitro interaction assays with recombinant receptor binding proteins and purified cell wall fragments to elucidate the molecular mechanism underpinning surface recognition by phages. 2) Engineering recombinant virulent phages with altered host range. Recombinant phages encoding distinct or combined receptor binding proteins will be assembled in vitro and "rebooted" using golden gate assembly to modify or expand their host range of. Using error-prone PCR, we will explore the possibility to generate phages with extended host range.3) Exploring the therapeutic potential of recombinant phages. The antimicrobial activity of phages against planktonic cultures and biolfilms will be tested. We will also investigate how phages eradicate infections in the context of host-pathogen interaction using the zebrafish experimental model of infection during mono-or polymicrobial infections.This work will exploit the transformative strategy recently described that allows the production of tailored-made phage genomes that can be assembled using golden gate assembly. The project will generate new therapeutic agents to treat bacterial infection caused by VRE, accounting for a large proportion of nosocomial infections.The project will involve a multidisciplinary approach encompassing microbiology, cell wall biochemistry, synthetic biology and host-pathogen interactions.

关于该项目该项目将建立在我们开发针对万古霉素耐药肠球菌 (VRE) 的噬菌体疗法的初步工作的基础上，VRE 是导致社区和医院获得性感染的机会性病原体（Al-zubidi 等人，2019）。由于对抗生素具有多重耐药性的病原体的出现，迫切需要新的抗菌策略。噬菌体非常适合作为治疗剂，因为它们针对复杂细菌群落中的特定病原体，而不会引起微生物组失衡。噬菌体识别细菌细胞表面的特定结构，通常表现出狭窄的宿主范围，有时仅限于少数菌株。为了规避这个潜在问题，从环境中分离出的噬菌体被组合起来产生具有扩展宿主范围的“噬菌体鸡尾酒”。另一种方法是进行噬菌体工程来修饰受体结合蛋白，以产生定制的治疗药物。该项目的具体目标如下：1）表征针对肠球菌的噬菌体的受体结合蛋白。我们最近分离出了针对粪肠球菌的剧毒噬菌体。三种噬菌体表现出较高的序列同一性，但宿主范围不同。一种噬菌体能够识别肠球菌多糖抗原 (EPA) 的修饰，该抗原在菌株之间存在差异。利用主要主管在蛋白质-细菌细胞壁相互作用方面的专业知识，我们建议研究噬菌体受体蛋白如何识别 EPA。我们将使用细菌突变体以及重组受体结合蛋白和纯化细胞壁片段的体外相互作用测定来阐明噬菌体表面识别的分子机制。 2) 改造宿主范围改变的重组剧毒噬菌体。编码不同或组合受体结合蛋白的重组噬菌体将在体外组装，并使用金门组装“重新启动”，以修改或扩大其宿主范围。使用易错PCR，我们将探索产生具有扩展宿主范围的噬菌体的可能性。3) 探索重组噬菌体的治疗潜力。将测试噬菌体针对浮游培养物和生物膜的抗菌活性。我们还将研究噬菌体如何在单一或多微生物感染期间使用斑马鱼感染实验模型来研究噬菌体如何在宿主-病原体相互作用的背景下根除感染。这项工作将利用最近描述的变革策略，该策略允许生产定制的噬菌体基因组，可以使用金门组件进行组装。该项目将产生新的治疗药物来治疗由 VRE 引起的细菌感染，这种细菌感染占医院感染的很大一部分。该项目将涉及涵盖微生物学、细胞壁生物化学、合成生物学和宿主-病原体相互作用的多学科方法。