中文摘要：抗生素耐药性问题十分严峻，需要新的抗生素替代品。噬菌体是一种以宿主细菌为靶标的病毒，其抗菌特性可有效地解决细菌耐药性问题。本研究探索了基因修饰T7噬菌体的方法，使其宿主范围更广，从而能够攻击更多种类的细菌。为此试验了不同的方法，发现电穿孔DNA (BRED)的噬菌体重组工程最有效。噬菌体对宿主细菌的识别和结合通常通过高度特异的受体-配体相互作用实现，噬菌体的受体结合蛋白（RBPs）是宿主特异性的主要决定因素。特异性修饰RBPs可以改变或扩展噬菌体的宿主范围，否则表现出所需的表型特性。本研究采用两种不同的策略，基于质粒的同源重组或利用电穿孔DNA (BRED)的噬菌体重组，对通常感染K12大肠杆菌共生菌株的T7噬菌体进行修饰，用以感染与病原体相关的K1荚膜表达菌株。本研究构建了两个基因设计：一个替换T7的gp17基因，另一个将T7的gp11、gp12和gp17与它们的K1F对应物进行替换。这两种方法都成功地将K1F序列整合到T7基因组中，并通过了PCR筛选检测。使用多种方法选择或富集仅结合K1F gp17的嵌合噬菌体，包括trxA、宿主特异性和基于CRISPR-Cas的选择。无论采用何种选择方法，嵌合噬菌体在新宿主上繁殖的重复性较差，表明嵌合噬菌体在促进持续的自主繁殖方面不如野生型。然而，从BRED中获得的含有gp11-12和gp17的嵌合噬菌体均表现出2期感染模式，表明K1F和T7表型均存在。可见，无需构建可持续复制的噬菌体，BRED可以作为一种快速获取新的RBP构建物潜力的工具。此外，还发现在某些情况下仅仅利用原RBP不足以产生可行的嵌合噬菌体。
外文摘要：Simple Summary The problem of antimicrobial resistance is prominent and new alternatives to antibiotics are necessary. Bacteriophages are viruses that target host bacteria and can be used efficiently for their antibacterial properties to solve the problem of antimicrobial resistance. In this study, we explore ways to genetically modify T7 bacteriophage and make its tropism broader, so that it can attack a higher variety of bacteria. We are using different methodologies to achieve this, among of those bacteriophage recombineering using electroporated DNA (BRED), which seems to be the most efficient. The recognition and binding of host bacteria by bacteriophages is most often enabled by a highly specific receptor-ligand type of interaction, with the receptor-binding proteins (RBPs) of phages being the primary determinants of host specificity. Specifically modifying the RBPs could alter or extend the host range of phages otherwise exhibiting desired phenotypic properties. This study employed two different strategies to reprogram T7 phages ordinarily infecting commensal K12 Escherichia coli strains to infect pathogen-associated K1-capsule-expressing strains. The strategies were based on either plasmid-based homologous recombination or bacteriophage recombineering using electroporated DNA (BRED). Our work pursued the construction of two genetic designs: one replacing the gp17 gene of T7, the other replacing gp11, gp12, and gp17 of T7 with their K1F counterparts. Both strategies displayed successful integration of the K1F sequences into the T7 genome, detected by PCR screening. Multiple methods were utilised to select or enrich for chimeric phages incorporating the K1F gp17 alone, including trxA, host-specificity, and CRISPR-Cas-based selection. Irrespective of the selection method, the above strategy yielded poorly reproducible phage propagation on the new host, indicating that the chimeric phage was less fit than the wild type and could not promote continual autonomous reproduction. Chimeric phages obtained from BRED incorporating gp11-12 and gp17, however, all displayed infection in a 2-stage pattern, indicating the presence of both K1F and T7 phenotypes. This study shows that BRED can be used as a tool to quickly access the potential of new RBP constructs without the need to engineer sustainably replicating phages. Additionally, we show that solely repurposing the primary RBP is, in some cases, insufficient to produce a viable chimeric phage.
外文关键词：bacteriophage；phage；host specificity；host range；RBP；tail fibres；BRED
作者：Avramucz, A；Moller-Olsen, C；Grigonyte, AM；Paramalingam, Y；Millard, A；Sagona, AP；Feher, T
作者单位：Eotvos Lorand Res Network ELKH；Univ Szeged；Univ Warwick；Univ Leicester
期刊名称：BIOLOGY-BASEL
期刊影响因子：3.796
出版年份：2021
出版刊次：6
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