Eliminating the type I restriction endonuclease from Pseudomonas aeruginosa PAO1 for optimized phage isolation
Ellie J Tong1, Kate A Bickerton1, Alina J Creber1
1Biosciences, Faculty of Health and Life Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
Abstract:
Phage therapy is a promising treatment for multidrug-resistant bacterial infections. Due to their high host specificity, phages must be matched to the target clinical strains. Efficiently identifying appropriate phages and producing sufficient titres for clinical use requires comprehensive phage libraries and multiple propagation hosts. An idealized system would use a highly promiscuous bacterial host to isolate a broader range of phages and streamline optimized phage production. Anti-phage defences constrain bacterial host promiscuity, such as restriction-modification systems that recognize and cleave foreign DNA. Here, the type I restriction endonuclease, HsdR, was deleted from Pseudomonas aeruginosa PAO1 to make a more promiscuous phage isolation and propagation host. Removal of this endonuclease more than doubled the efficiency of phage propagation on solid media, improved yields from hard-to-propagate phages in liquid bulk-ups and yielded seven times more phages from freshwater samples than wild-type PAO1 - an important step in producing an optimized P. aeruginosa strain for isolating and propagating phages for clinical phage therapy.
Insights
Researchers enhanced phage therapy by engineering a Pseudomonas aeruginosa host. Deleting the HsdR enzyme increased phage isolation and production efficiency, crucial for treating multidrug-resistant infections.
Area of Science:
- Microbiology
- Bacteriology
- Virology
Background:
- Phage therapy offers a promising alternative for combating multidrug-resistant bacterial infections.
- High host specificity of bacteriophages necessitates matching phages to specific bacterial strains for effective treatment.
- Current methods for phage isolation and production require extensive phage libraries and multiple bacterial hosts.
Purpose of the Study:
- To develop a more promiscuous bacterial host for improved bacteriophage isolation and propagation.
- To enhance the efficiency of phage therapy by optimizing the bacterial host system.
Main Methods:
- Deletion of the type I restriction endonuclease (HsdR) gene from *Pseudomonas aeruginosa* PAO1.
- Evaluation of the modified *P. aeruginosa* strain as a host for phage isolation and propagation on solid and liquid media.
- Quantification of phage yields from environmental samples using wild-type and modified PAO1 strains.
Main Results:
- The HsdR-deleted *P. aeruginosa* strain demonstrated more than double the efficiency of phage propagation on solid media.
- Improved yields of difficult-to-propagate phages were achieved in liquid culture using the modified host.
- The engineered strain yielded seven times more phages from freshwater samples compared to the wild-type strain.
Conclusions:
- Engineering *Pseudomonas aeruginosa* by deleting HsdR significantly enhances its utility as a host for bacteriophage isolation and propagation.
- This optimized bacterial host is a critical advancement for developing robust phage libraries and streamlining phage production for clinical phage therapy.
- The findings pave the way for more efficient and effective phage-based treatments against bacterial infections.
