Bacteriophage engineering as a therapeutic strategy to target antibiotic resistant enterococci

关于该项目该项目将建立在我们的初步工作，以开发靶向万古霉素耐药肠球菌（VRE）的噬菌体治疗剂，VRE是引起社区和医院获得性感染的机会性病原体（Al-zubidi et al.，2019年）。由于病原体对抗生素多重耐药的出现，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，而不会导致微生物组失衡。噬菌体识别细菌细胞表面的特定结构，通常显示出狭窄的宿主范围，有时仅限于少数菌株。为了规避这个潜在的问题，从环境中分离的噬菌体被组合以产生具有扩展的宿主范围的“噬菌体鸡尾酒”。另一种方法是利用噬菌体工程技术对受体结合蛋白进行修饰，从而产生针对性的治疗药物。我们最近分离出了针对粪肠球菌的毒性大肠杆菌。三个引物显示出高序列同一性，但不同的主机范围。一个噬菌体显示出识别肠球菌多糖抗原（EPA）的装饰，菌株之间的变化。利用蛋白质-细菌细胞壁相互作用的主要主管的专业知识，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和重组受体结合蛋白和纯化的细胞壁片段的体外相互作用试验，以阐明支撑表面识别的分子机制。2)工程改造具有改变的宿主范围的重组毒性大肠杆菌。编码不同或组合的受体结合蛋白的重组cDNA将在体外组装并使用金门组装“重启”以修饰或扩大其宿主范围。利用易错PCR技术，我们将探索产生具有更大宿主范围的ESTs的可能性。3）探索重组ESTs的治疗潜力。将测试抗微生物剂的抗微生物活性。我们还将研究如何在宿主-病原体相互作用的背景下，使用斑马鱼实验模型的感染，在单或多微生物infections.This工作将利用最近描述的变革策略，允许生产定制的噬菌体基因组，可以使用金门组装。该项目将产生新的治疗药物来治疗由VRE引起的细菌感染，占医院感染的很大比例。该项目将涉及多学科方法，包括微生物学，细胞壁生物化学，合成生物学和宿主-病原体相互作用。

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.