Dataset Information

Control of λ Lysogenic Escherichia coli Cells by Synthetic λ Phage Carrying cI^antisense.

ABSTRACT: Enterobacterial phage λ is a temperate phage that infects Escherichia coli and has a lytic-lysogenic life cycle. CI, a λ repressor, regulates the expression of lytic transcripts and acts as a major genetic switch that determines the lysogenic state. To manipulate the genome of phage λ, the CRISPR-Cas9 genome editing system was constructed in lysogenic E. coli MG1655 cells. For instance, we successfully changed cI⁸⁵⁷ to cI^WT in the phage genome through Cas9-mediated single-nucleotide editing. A lytic phage was prepared by introducing an amber mutation in the middle of the cI gene, but it could not lyse lysogenic MG1655 cells. We prepared a phage expressing cI antisense mRNA by reverse substitution of the cI gene. Lysis of λ cI⁸⁵⁷ lysogenic cells occurred by the infection of the λ cI^antisense. These results suggest an effective lysogenic cell control method by a synthetic phage expressing antisense mRNA of the genetic switch gene. It is expected to be applied as a tool to control harmful lysogenic microorganisms.

SUBMITTER: Lee HJ

PROVIDER: S-EPMC9680875 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Publications

Control of λ Lysogenic Escherichia coli Cells by Synthetic λ Phage Carrying cIantisense.

Lee Ho Joung HJ Kim Hyun Ju HJ Lee Sang Jun SJ

ACS synthetic biology 20221103 11

Enterobacterial phage λ is a temperate phage that infects Escherichia coli and has a lytic-lysogenic life cycle. CI, a λ repressor, regulates the expression of lytic transcripts and acts as a major genetic switch that determines the lysogenic state. To manipulate the genome of phage λ, the CRISPR-Cas9 genome editing system was constructed in lysogenic E. coli MG1655 cells. For instance, we successfully changed cI857 to cIWT in the p ...[more]

PMID: 36326101

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Project description:Bacteriophages (phages) are ideal alternatives to traditional antimicrobial agents in a world where antimicrobial resistance (AMR) is emerging and spreading at an unprecedented speed. In addition, due to their narrow host ranges, phages are also ideal tools to modulate the gut microbiota in which alterations of specific bacterial strains underlie human diseases, while dysbiosis caused by broad-spectrum antibiotics can be harmful. Here, we engineered a lambda phage (Eλ) to target enterohemorrhagic Escherichia coli (EHEC) that causes a severe, sometimes lethal intestinal infection in humans. We enhanced the killing ability of the Eλ phage by incorporating a CRISPR-Cas3 system into the wild-type λ (wtλ) and the specificity by introducing multiple EHEC-targeting CRISPR spacers while knocking out the lytic gene cro. In vitro experiments showed that the Eλ suppressed the growth of EHEC up to 18 h compared with only 6 h with the wtλ; at the multiplicity of infection (MOI) of 10, the Eλ killed the EHEC cells with ~100% efficiency and did not affect the growth of other laboratory- and human-gut isolated E. coli strains. In addition, the EHEC cells did not develop resistance to the Eλ. Mouse experiments further confirmed the enhanced and strain-specific killing of the Eλ to EHEC, while the overall mouse gut microbiota was not disturbed. Our methods can be used to target other genes that are responsible for antibiotic resistance genes and/or human toxins, engineer other phages, and support in vivo application of the engineered phages. IMPORTANCE Pathogenic strains of Escherichia coli are responsible for 0.8 million deaths per year and together ranked the first among all pathogenic species. Here, we obtained, for the first time, an engineered phage, Eλ, that could specifically and efficiently eliminate EHEC, one of the most common and often lethal pathogens that can spread from person to person. We verified the superior performance of the Eλ over the wild-type phage with in vitro and in vivo experiments and showed that the Eλ could suppress EHEC growth to nondetectable levels, fully rescue the EHEC-infected mice, and rescore disturbed mouse gut microbiota. Our results also indicated that the EHEC did not develop resistance to the Eλ, which has been the biggest challenge in phage therapy. We believe our methods can be used to target other pathogenic strains of E. coli and support in vivo application of the engineered phages.

Dataset Information

Control of λ Lysogenic Escherichia coli Cells by Synthetic λ Phage Carrying cI^antisense.

Publications

Control of λ Lysogenic <i>Escherichia coli</i> Cells by Synthetic λ Phage Carrying <i>cI</i><sup><i>antisense</i></sup>.

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