Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7

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.

Engineering bacteriophages for enhanced host range and efficacy: insights from bacteriophage-bacteria interactions

“…In a study conducted by Avramucz et al, the gp17 gene (TF gene) from bacteriophage K1F, which targets E. coli K1, was inserted into the gp17 gene of bacteriophage T7, which targets E. coli K12. The resulting engineered phage exhibited unstable infectivity toward E. coli K1, leading to insufficient replication capacity ( Avramucz et al, 2021 ). Similarly, Hoshiga et al (2019) integrated several RBPs genes from E. coli bacteriophage PP01 into the corresponding genes of bacteriophage T2 of E. coli .…”

Tailed Lytic Bacteriophages of Soft Rot Pectobacteriaceae

“…Phages can adapt their specificity to such mutants naturally, using horizontal gene transfer, as seen for different Studiervirinae phages using almost identical TSP [135], or through the acquisition of Ackermannviridae-like TSP by phage Sucellus. Due to the unified composition of their adsorption apparatus, Podophages and Myophages are often considered as a subject for directed gene engineering, adopting them for a broader range of host bacteria [191][192][193][194]. This approach is often criticised for the addition of a "genetically modified organism" status to the complicated goal of defining bacteriophages as therapeutic drugs [192].…”