Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7.
Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7.
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DOI：
10.3390/biology10060556
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发表时间：
2021-06-20
期刊：
影响因子：
4.2
通讯作者：
Fehér T
中科院分区：
文献类型：
--
作者：
Avramucz Á;Møller-Olsen C;Grigonyte AM;Paramalingam Y;Millard A;Sagona AP;Fehér T
抗生素耐药性问题突出，需要新的抗生素替代品。噬菌体是一种以宿主细菌为目标的病毒，由于其抗菌特性可以有效地利用，以解决抗微生物药物耐药性问题。在本研究中，我们将探索如何对T7噬菌体进行基因修饰，使其趋向性更广，从而使其能够攻击更多种类的细菌。我们正在使用不同的方法来实现这一目标，其中使用电穿孔DNA （BRED）进行噬菌体重组似乎是最有效的。噬菌体对宿主细菌的识别和结合通常是通过高度特异性的受体-配体类型的相互作用实现的，而噬菌体的受体结合蛋白（rbp）是宿主特异性的主要决定因素。特异性修饰rbp可以改变或扩展噬菌体的宿主范围，否则会表现出所需的表型特性。本研究采用两种不同的策略对通常感染共生K12大肠杆菌菌株的T7噬菌体进行重编程，使其感染病原体相关的表达k1胶囊的菌株。该策略基于基于质粒的同源重组或利用电穿孔DNA （BRED）的噬菌体重组。我们的工作是构建两种基因设计：一种是替换T7的gp17基因，另一种是替换T7的gp11、gp12和gp17基因，用它们的K1F对应基因。通过PCR筛选，这两种策略都成功地将K1F序列整合到T7基因组中。使用多种方法来选择或富集仅含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，上述策略均导致噬菌体在新宿主上的繁殖能力较差，这表明嵌合噬菌体比野生型更不适合，不能促进持续的自主繁殖。然而，从BRED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，表明存在K1F和T7表型。这项研究表明，BRED可以作为一种工具，在不需要设计可持续复制噬菌体的情况下，快速获取新的RBP构建体的潜力。此外，我们表明，在某些情况下，仅仅重新利用原代RBP不足以产生可行的嵌合噬菌体。
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.
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影响因子：
4.7
作者：
Grigonyte, Aurelija M.;Harrison, Christian;Millard, Andrew
通讯作者：
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影响因子：
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作者：
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影响因子：
48
作者：
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影响因子：
4.7
作者：
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通讯作者：
Fauconnier, Alan
DOI：
10.1073/pnas.1520040113
发表时间：
2016-03-01
影响因子：
11.1
作者：
Nyerges, Akos;Csoergo, Balint;Pal, Csaba
通讯作者：
Pal, Csaba