A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages

Abstract

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 of P. 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.IMPORTANCEPseudomonas aeruginosa is 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.

Keywords: Pseudomonas aeruginosa; phage reboot; phage therapy; synthetic biology.

Fig 1

Schematic representation of the experimental procedure. Using direct extraction of the phage genome or the construction of overlapping fragments amplified by PCR, we were able to clone or construct the phage genome in yeast and maintain it using yeast elements. Next, extraction of yeast DNA and digestion by restriction enzymes allowed us to obtain full-length phage DNA that is free from yeast elements. Finally, PA transformation permitted us to obtain rebooted phage particles.

Fig 2

Analysis of JG024 genome. (A) Overview of assembly results compared to the reference genome from NCBI; assembly was performed using Unicycler, Trycycler, Flye, and PhageTerm. (B) Visual representation of overlapping fragments used for the amplification of the full JG024 genome. (C) Coverage by position (with colors representing base calls: A, green; T, red; G, orange; and C, blue) and location of long reads (>30,000 bp) mapped to the JG024 reference genome using IGV. (D) Representation of the expected digestion sites from a circular JG024 genome using XbaI; fragments marked with a green “V” are observed on the corresponding agarose gel. (E) Agarose gel of XbaI digestion; ND undigested JG024 genome; XbaI, digestion of JG024 genome with XbaI; and ScaI digestion of JG024 genome with ScaI.

Fig 3

Phage yield (in titer) following infection or reboot under different experimental conditions. (A) PA14 was infected with JG024 with and without chloroform treatment to assess sensitivity. Chloroform was found to have a significant effect on phage titer (analysis of variance, ANOVA; P = 0.004) and decreased the phage titer. (B) Following infection, PA14 was allowed to recover 3 or 24 h, and phages were collected either from the cell pellet (C) or supernatant (S). A total of 100 ng of gDNA from this recovered phage solution was then electroporated into PA14 to determine the impact of recovery time (3 vs 24 h) and phage release (C vs S) on yield. The phage titer of the phages found in the supernatant was 11-fold higher than the phages released from chloroform extraction (ANOVA; P = 0.01). C- JG024 was rebooted using different starting amounts of phage gDNA in PA14. The quantity of JG024 gDNA was found to have a significant effect on the phage titer (ANOVA; P = 0.0003) as only gDNA quantities of at least 100 ng resulted in consistent plaques. There was no significant difference in phage titer between 100 and 500 ng of gDNA (ANOVA; P > 0.05) with phage titer reaching an average of 1.8 × 10¹⁰ PFU/mL for both DNA quantities.

Fig 4

Reassembly of the JG024 genome in yeast using TAR cloning. (A) Schematic of TAR-cloning procedure. (B) Cloning efficiency of the entire JG024 genome and two smaller parts in yeast. Simplex PCR consists of one PCR that amplifies a single region of the genome. Recombination PCR involves the amplification of recombination scars, and multiplex PCR uses a set of several primers to amplify multiple regions around the phage genome (in this case, 10). (C) Representation of the three batches of PCR done to validate the cloning of the phage genome in yeast. (D) Example agarose gel of multiplex PCR products performed to validate phage genome integrity in clones. Expected bands were produced from the intact genome and Half 2, but Half 1 only yielded bands corresponding to untransformed yeast controls.

Fig 5

Construction of synthetic phage DNA in yeast. (A) Visual representation of the three-fragment PCR design for JG024 genome amplification. (B) Agarose gel of PCR fragments (10, 11, and 12 in panel A) obtained for JG024 cloning in yeast. (C) Cloning efficiency of assembled JG024 fragments in yeast. Simplex and multiplex PCR are as described in Fig. 4. (D) Agarose gel of multiplex PCR (as described in Fig. 4) of the JG024 genome obtained successively after 10 passages in yeast demonstrating the stability of the construct.

Fig 6

Comparison of DMS3 reboot to JG024 in PA14 and PAO1. (A) Cloning efficiency of DMS3 in yeast. Simplex PCR consists of one PCR that amplifies a single region of the genome. Recombination PCR involves amplification of recombination scars, and multiplex PCR uses a set of several primers to amplify multiple regions around the phage genome (in this case, six). (B) Example reboot result from yeast DNA obtained for linearized DMS3 and JG024 genomes in PA14 and PAO1. (C) Reboot of linear DMS3 phage DNA from yeast in wild-type PA14 and PAO1, as well as PA mutants lacking CRISPR (∆CRISPR), restriction-modification (∆RE), and Wadjet (∆Wadjet) defense systems. The knockout strains had a significant effect on the phage titer (analysis of variance; P = 0.007). The PAO1∆RE strain had a 42-fold higher phage titer than the PAO1 WT strain (t-test; P = 0.0001). Individual P-values represent the results of t-tests between incremental defense system removals (e.g., ∆CRISPR and ∆CRISPR∆RM). There was no significant difference in phage titer between PA14 and PA14∆CRISPR. (D) Example reboot result using linearized DMS3 genomes from yeast in PAO1, PA14, and a PA14 mutant lacking CRISPR and restriction-modification defense systems.

Fig 7

Phage reboot from yeast DNA in PA mutants. (A) Reboot of linearized JG024 phage DNA from yeast in wild-type PA14 and PAO1, as well as PA mutants lacking CRISPR (∆CRISPR), restriction-modification (∆RE), and Wadjet (∆Wadjet) defense systems. Individual P-values represent the results of t-tests between incremental defense system removals (e.g., ∆CRISPR and ∆CRISPR∆RM). N.S, not significant (P > 0.05). (B) Example reboot result of linearized JG024 genomes obtained from yeast in PA14 and PAO1 mutants, denoted as in panel A.