Tong, E. J. | bioRxiv Science

bioRxiv2026

Lipopolysaccharide truncation and restoration drives a trade-off in resistance to two phages in Pseudomonas aeruginosa

Phage therapy is a promising treatment for multidrug resistant bacterial infections, and for patients no longer able to tolerate antibiotic treatments. A major challenge for phage therapy is emergent phage resistance, which target bacteria acquire by structurally modifying or masking phage receptors to prevent adsorption. Functionally diverse phage cocktails that target a broad range of receptors are less prone to resistance as there is a higher fitness cost associated with modifying multiple receptors. Expanding phage libraries with well-characterised phages that target a broad range of receptors would aid in timely and strategic design of functionally diverse phage cocktails. Here, we aimed to isolate phages targeting novel receptors by enriching wastewater samples on a Pseudomonas aeruginosa PAO1 {Delta}pilA {Delta}galU unmarked deletion mutant lacking O-antigen, outer core lipopolysaccharide (LPS) and type IV pili (T4P) - the three most common Pseudomonas phage receptors. This led to the isolation of a novel phage, named Vale. Vale was predicted to bind the LPS inner core as it could only infect strains with truncated LPS, suggesting that the outer core LPS blocks Vale from accessing the inner core. We identified a trade-oM in resistance to Vale and another phage, Tor, that targets the LPS outer core, mediated by host-derived LPS modifications. The PAO1 host evolved resistance to Tor by 100-200kb genomic deletions, which resulted in LPS truncation and sensitivity to Vale. Complete LPS restoration in the {Delta}pilA {Delta}galU mutant conferred resistance to Vale and sensitivity to Tor in two out of three replicates. Combined treatment with Tor and Vale delayed the emergence of resistance in PAO1 for at least three times longer than individual phage treatments. This study provides an example of how using phage receptors to strategically design phage cocktails can minimise the likelihood of emergent phage resistance. Graphical abstractA summary of the LPS modifications, genomic mutations and phage susceptibilities of Tor and Vale resistant mutants. "Parent strain" refers to PAO1 {Delta}hsdR. {Delta}pilA {Delta}galU refers to PAO1 {Delta}hsdR {Delta}pilA {Delta}galU. The parent strain gains resistance to Tor via LPS truncation associated with 100-200 kb genomic deletions, resulting in sensitivity to Vale. {Delta}pilA {Delta}galU gains resistance to Vale by restoring its LPS, conferring sensitivity to Tor in two out of three repeats. As resistance to one phage sensitises bacteria to the other, combined treatment with both phages suppresses phage resistance for longer than individual treatments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/700494v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1efee0forg.highwire.dtl.DTLVardef@f64bfeorg.highwire.dtl.DTLVardef@1f71130org.highwire.dtl.DTLVardef@1897bd6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Eliminating the type I restriction endonuclease from Pseudomonas aeruginosa PAO1 for optimised phage isolation

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 idealised system would use a highly promiscuous bacterial host to isolate a broader range of phages and streamline optimised phage production. Anti-phage defences constrain bacterial host promiscuity, such as restriction-modification systems that recognise 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 and yielded seven times more phages from freshwater samples than wildtype PAO1 - an important step in producing an optimised P. aeruginosa strain for isolating and propagating phages for clinical phage therapy. Graphical abstract(Created in BioRender.com) [A] The type I R-M system comprises three subunits HsdS, HsdM and HsdR. One HsdS and two HsdM subunits form the methyltransferase, and the addition of two HsdR subunits to this complex forms the restriction endonuclease. [B] In the wildtype PAO1, the type I R-M system destroys phage DNA while protecting the host. The HsdS subunit of the restriction endonuclease binds to unmethylated recognition sequences. Then, HsdR translocates the DNA, pulling it together in both directions until it collides with another restriction endonuclease, cleaving the DNA and preventing phage proliferation. The methyltransferase protects the host DNA through the methylation of recognition sequences, preventing the restriction endonuclease from binding. [C] In {Delta}hsdR, the restriction endonuclease cannot form, preventing phage DNA cleavage and increasing phage proliferation. The methyltransferase is still active, so both host and phage DNA are methylated, providing phage progeny with protection from R-M systems in future hosts. O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/656992v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@9418f8org.highwire.dtl.DTLVardef@f3703dorg.highwire.dtl.DTLVardef@4dcde6org.highwire.dtl.DTLVardef@8d6fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗