Abstract
In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.
Highlights
In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem
Considering that a very large proportion of phage genomes encode for hypothetical proteins with unknown functions[7], a possible approach to get some space for genome engineering would be the precise removal of those genes from viral genomes
We envisioned that synthetic phages carrying a minimal number of genes essential for their replication and host killing, would be more accepted for therapy and a minimal genome concept would facilitate the integration of extra functions into the phage genome to improve its performance
Summary
In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. Since P. aeruginosa is currently considered by the World Health Organization as one of the top priority bacterial pathogens urgently requiring new treatments[13], in this study we isolated a new P. aeruginosa phage and used it as a model to design our synthetic phages From our knowledge, this is the first study focused on minimizing phage genomes and understanding its impact on phages’ performance
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