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Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7

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,...

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Autores principales: Avramucz, Ákos, Møller-Olsen, Christian, Grigonyte, Aurelija M., Paramalingam, Yanahan, Millard, Andrew, Sagona, Antonia P., Fehér, Tamás
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234382/
https://www.ncbi.nlm.nih.gov/pubmed/34202963
http://dx.doi.org/10.3390/biology10060556
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author Avramucz, Ákos
Møller-Olsen, Christian
Grigonyte, Aurelija M.
Paramalingam, Yanahan
Millard, Andrew
Sagona, Antonia P.
Fehér, Tamás
author_facet Avramucz, Ákos
Møller-Olsen, Christian
Grigonyte, Aurelija M.
Paramalingam, Yanahan
Millard, Andrew
Sagona, Antonia P.
Fehér, Tamás
author_sort Avramucz, Ákos
collection PubMed
description 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. ABSTRACT: 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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spelling pubmed-82343822021-06-27 Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7 Avramucz, Ákos Møller-Olsen, Christian Grigonyte, Aurelija M. Paramalingam, Yanahan Millard, Andrew Sagona, Antonia P. Fehér, Tamás Biology (Basel) Article 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. ABSTRACT: 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. MDPI 2021-06-20 /pmc/articles/PMC8234382/ /pubmed/34202963 http://dx.doi.org/10.3390/biology10060556 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Avramucz, Ákos
Møller-Olsen, Christian
Grigonyte, Aurelija M.
Paramalingam, Yanahan
Millard, Andrew
Sagona, Antonia P.
Fehér, Tamás
Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7
title Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7
title_full Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7
title_fullStr Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7
title_full_unstemmed Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7
title_short Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7
title_sort analysing parallel strategies to alter the host specificity of bacteriophage t7
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234382/
https://www.ncbi.nlm.nih.gov/pubmed/34202963
http://dx.doi.org/10.3390/biology10060556
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