Designing P. aeruginosa synthetic phages with reduced genomes

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.

Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation

“…Such combinatorial phage treatment approach would likely benefit from selection of strictly lytic phages (over lysogenic ones) that would ensure a higher efficiency of bacterial lysis, while reducing the risk of horizontal gene transfer of toxins or ARGs into bacterial chromosomes by lysogeny (Sulakvelidze et al, 2001). Additionally, genomic phage manipulation may enable the conversion of lysogenic phages into strictly lytic phages by the deletion of their integrase genes or alteration of natural phage specificity toward pre-determined identification of new hosts without impacting their antimicrobial properties (Pires et al, 2021). In addition to its therapeutic potential, such phage combination treatment could be highly advantageous in microbiota research, by enabling the testing of putative disease-causing commensals.…”

Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages

“…The bacterial cultures were grown at 37 °C in lysogeny broth (LB, NZytech, Lisbon, Portugal), LB agar or LB soft agar overlays containing 1.5% ( w / v ) or 0.6% ( w / v ) of agar, respectively. The two P. aeruginosa phages used in this work were previously isolated and characterized as vB_PaeM_CEB_DP1 (short name DP1) [ 29 ] and vB_PaeP_E3 (short name PE3) [ 30 ].…”

A Phage Foundry Framework to Systematically Develop Viral Countermeasures to Combat Antibiotic-Resistant Bacterial Pathogens

“…The ability of phages to rapidly evolve to evade target pathogen resistance can be exploited using in vitro directed evolution to “train” libraries of phages against panels of targets to create banks of complementary phage antimicrobial agents for cocktails ( Rohde et al., 2018 ; Burrowes et al., 2019 ; Abdelsattar et al., 2021 ; Borin et al., 2021 ; Eskenazi et al., 2022 ; Torres-Barceló et al., 2022 ). The small genomic size of phages enable both full genome synthesis and possibly “booting” (producing viable phage particles from synthetic DNA) when isolation is difficult as well as efficient engineering of designed genetic changes ( Chan et al., 2005 ; Ando et al., 2015 ; Pires et al., 2016 , 2021a , 2021b ; Kilcher et al., 2018 ; Lemire et al., 2018 ; Dunne et al., 2019 ; Kilcher and Loessner, 2019 ; Weynberg and Jaschke, 2020 ; Lenneman et al., 2021 ). Designer changes include engineered genetic payloads that increase toxicity, counteract defenses, and potentially suppress horizontal gene transfer of resistance genes by, for example, degrading the target bacterial genome rapidly ( Lu and Collins, 2007 ; Yosef et al., 2015 , 2017 ; Barbu et al., 2016 ; Dunne et al., 2019 ; Kilcher and Loessner, 2019 ; Yehl et al., 2019 ; Lenneman et al., 2021 ).…”