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Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences
The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichi...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9221748/ https://www.ncbi.nlm.nih.gov/pubmed/35735615 http://dx.doi.org/10.3390/cimb44060174 |
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author | Ludwig, Tobias Hoffmann, Ralf Krizsan, Andor |
author_facet | Ludwig, Tobias Hoffmann, Ralf Krizsan, Andor |
author_sort | Ludwig, Tobias |
collection | PubMed |
description | The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures. |
format | Online Article Text |
id | pubmed-9221748 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-92217482022-06-24 Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences Ludwig, Tobias Hoffmann, Ralf Krizsan, Andor Curr Issues Mol Biol Article The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures. MDPI 2022-06-01 /pmc/articles/PMC9221748/ /pubmed/35735615 http://dx.doi.org/10.3390/cimb44060174 Text en © 2022 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 Ludwig, Tobias Hoffmann, Ralf Krizsan, Andor Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences |
title | Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences |
title_full | Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences |
title_fullStr | Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences |
title_full_unstemmed | Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences |
title_short | Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences |
title_sort | construction and characterization of t7 bacteriophages harboring apidaecin-derived sequences |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9221748/ https://www.ncbi.nlm.nih.gov/pubmed/35735615 http://dx.doi.org/10.3390/cimb44060174 |
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