Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157

ABSTRACT Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase,...

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Autores principales:	Lauren A. Cowley, Alison S. Low, Derek Pickard, Christine J. Boinett, Timothy J. Dallman, Martin Day, Neil Perry, David L. Gally, Julian Parkhill, Claire Jenkins, Amy K. Cain
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	bacteriophage Gram-negative bacteria mutagenesis transposons whole-genome sequencing Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/f49832bad53f46aca881d478eabf86c3
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spelling	oai:doaj.org-article:f49832bad53f46aca881d478eabf86c32021-11-15T16:00:15ZTransposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O15710.1128/mBio.00705-182150-7511https://doaj.org/article/f49832bad53f46aca881d478eabf86c32018-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00705-18https://doaj.org/toc/2150-7511ABSTRACT Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens. IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication.Lauren A. CowleyAlison S. LowDerek PickardChristine J. BoinettTimothy J. DallmanMartin DayNeil PerryDavid L. GallyJulian ParkhillClaire JenkinsAmy K. CainAmerican Society for MicrobiologyarticlebacteriophageGram-negative bacteriamutagenesistransposonswhole-genome sequencingMicrobiologyQR1-502ENmBio, Vol 9, Iss 4 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	bacteriophage Gram-negative bacteria mutagenesis transposons whole-genome sequencing Microbiology QR1-502
spellingShingle	bacteriophage Gram-negative bacteria mutagenesis transposons whole-genome sequencing Microbiology QR1-502 Lauren A. Cowley Alison S. Low Derek Pickard Christine J. Boinett Timothy J. Dallman Martin Day Neil Perry David L. Gally Julian Parkhill Claire Jenkins Amy K. Cain Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157
description	ABSTRACT Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens. IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication.
format	article
author	Lauren A. Cowley Alison S. Low Derek Pickard Christine J. Boinett Timothy J. Dallman Martin Day Neil Perry David L. Gally Julian Parkhill Claire Jenkins Amy K. Cain
author_facet	Lauren A. Cowley Alison S. Low Derek Pickard Christine J. Boinett Timothy J. Dallman Martin Day Neil Perry David L. Gally Julian Parkhill Claire Jenkins Amy K. Cain
author_sort	Lauren A. Cowley
title	Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157
title_short	Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157
title_full	Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157
title_fullStr	Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157
title_full_unstemmed	Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157
title_sort	transposon insertion sequencing elucidates novel gene involvement in susceptibility and resistance to phages t4 and t7 in <italic toggle="yes">escherichia coli</italic> o157
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/f49832bad53f46aca881d478eabf86c3
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Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157

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