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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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2018-09-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.00705-18 |
| _version_ | 1798032255731367936 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-11T20:09:52Z |
| format | Article |
| id | doaj.art-f49832bad53f46aca881d478eabf86c3 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-11T20:09:52Z |
| publishDate | 2018-09-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-f49832bad53f46aca881d478eabf86c32022-12-22T04:05:10ZengAmerican Society for MicrobiologymBio2150-75112018-09-019410.1128/mBio.00705-18Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in <italic toggle="yes">Escherichia coli</italic> O157Lauren A. Cowley0Alison S. Low1Derek Pickard2Christine J. Boinett3Timothy J. Dallman4Martin Day5Neil Perry6David L. Gally7Julian Parkhill8Claire Jenkins9Amy K. Cain10Gastrointestinal Bacterial Reference Unit, Public Health England, London United KingdomDivision of Immunity and Infection, the Roslin Institute and Royal (Dick) School of Veterinary Studies, the University of Edinburgh, Midlothian, United KingdomWellcome Trust Sanger Institute, Hinxton, Cambridge United KingdomWellcome Trust Sanger Institute, Hinxton, Cambridge United KingdomGastrointestinal Bacterial Reference Unit, Public Health England, London United KingdomGastrointestinal Bacterial Reference Unit, Public Health England, London United KingdomGastrointestinal Bacterial Reference Unit, Public Health England, London United KingdomDivision of Immunity and Infection, the Roslin Institute and Royal (Dick) School of Veterinary Studies, the University of Edinburgh, Midlothian, United KingdomWellcome Trust Sanger Institute, Hinxton, Cambridge United KingdomGastrointestinal Bacterial Reference Unit, Public Health England, London United KingdomWellcome Trust Sanger Institute, Hinxton, Cambridge United KingdomABSTRACT 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.https://journals.asm.org/doi/10.1128/mBio.00705-18bacteriophageGram-negative bacteriamutagenesistransposonswhole-genome sequencing |
| spellingShingle | 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 mBio bacteriophage Gram-negative bacteria mutagenesis transposons whole-genome sequencing |
| 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_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_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_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 |
| topic | bacteriophage Gram-negative bacteria mutagenesis transposons whole-genome sequencing |
| url | https://journals.asm.org/doi/10.1128/mBio.00705-18 |
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