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Abstract Given concerning trends in antibiotic resistance, phages have been increasingly explored as promising antimicrobial agents. However, a major problem with phage therapy is the overly high specificity of phages for their hosts, which is currently addressed by a personalized approach involving screening a bank of wild-type phages against each clinical isolate. To shorten this process, we propose that a focused library of synthetic phages could be rapidly selected for a member binding to a given clinical isolate. We created libraries of recombinant M13 phages expressing receptor-binding proteins based on the collective metagenome of inovirus phages, a diverse group whose members appear to infect nearly all bacterial phyla. Using two rounds of a pull-down selection, phage variants were identified against several Gram-negative pathogens, including a variant (M13PAB) that bound to several Pseudomonas aeruginosa strains, including clinical isolates. To confer bactericidal activity to the nonlytic phage, a last-line but nephrotoxic lipopeptide, colistin, was cross-linked to the M13PAB virions. The colistin-M13PAB phage conjugate lowered the minimal inhibitory concentration of colistin by 1–2 orders of magnitude for multiple strains of P. aeruginosa and showed a lack of hemolytic or cytotoxic activity in vitro, suggesting high potency combined with low toxicity. Thus, a metagenome-inspired library displayed on the M13 phage scaffold, when subjected to a short selection for binding to a bacterial clinical isolate, could yield a phage variant that targets the specified strain. This approach may improve the speed, consistency, and cost-effectiveness of personalized phage therapy.
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A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages
The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains ofP. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages. IMPORTANCE Pseudomonas aeruginosais a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.
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- Award ID(s):
- 2021900
- PAR ID:
- 10535230
- Editor(s):
- Andam, Cheryl P
- Publisher / Repository:
- American Society for Microbiology
- Date Published:
- Journal Name:
- Microbiology Spectrum
- Volume:
- 12
- Issue:
- 3
- ISSN:
- 2165-0497
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1128/spectrum.02897-23
