Bacteriophage engineering as a therapeutic strategy to target antibiotic resistant enterococci

关于该项目该项目将以我们的初步工作为基础，开发针对万古霉素耐药肠球菌(VRE)的噬菌体疗法，VRE是导致社区和医院获得性感染的机会性病原体(Al-zubii等人，2019年)。由于对抗生素产生多重耐药的病原体的出现，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，而不会造成微生物群的失衡。噬菌体识别细菌细胞表面的特定结构，通常显示的宿主范围很窄，有时仅限于几个菌株。为了绕过这个潜在的问题，从环境中分离出来的噬菌体被组合在一起，产生具有更大宿主范围的“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，产生量身定制的治疗方法。该项目的具体目标如下：1)鉴定针对肠球菌的噬菌体受体结合蛋白。我们最近分离出了针对粪肠球菌的强毒噬菌体。3个噬菌体具有较高的序列同源性，但寄主范围不同。有一个噬菌体可以识别肠球菌多糖抗原(EPA)的修饰，不同菌株的修饰不同。利用主要监督员在蛋白质-细菌细胞壁相互作用方面的专业知识，我们建议调查噬菌体受体蛋白如何识别EPA。我们将使用细菌突变体和体外与重组受体结合蛋白和纯化的细胞壁片段的相互作用分析来阐明噬菌体支持表面识别的分子机制。2)改变宿主范围的重组毒力噬菌体。编码不同或结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。利用容易出错的聚合酶链式反应，我们将探索产生具有更大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。将测试噬菌体对浮游细菌和生物膜的抗菌活性。我们还将使用斑马鱼在单一或多个微生物感染期间感染的实验模型，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转型策略，允许生产定制的噬菌体基因组，这些基因组可以使用金门组装进行组装。该项目将产生新的治疗药物来治疗由VRE引起的细菌感染，该细菌感染在医院感染中占很大比例。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。

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.