Lahane, T. S. | Search bioRxiv

bioRxiv2026

Identification and characterization of new P. aeruginosa phages using a surface receptor platform

Bacteriophages are of increasing interest for their potential use as alternatives to antibiotics for treating drug-resistant infections. Phage steering, specifically receptor-based steering, involves use of phages that bias the development of resistance in bacteria towards phenotypes such as reduced fitness. Phage therapy typically involves administering cocktails of phages with different targets to reduce the likelihood of resistance emerging. To ensure effective steering and cocktail design, there is a need to identify the receptors used by phages of interest, and to isolate phages that use diverse receptors. Here we describe a SURFAce Receptor (SURFAR) platform consisting of a set of defined Pseudomonas aeruginosa strains and mutants for rapid characterization of phage receptors. This platform can also be used to enrich for and isolate diverse phages from environmental samples, yielding novel phage genomes from wastewater. Using the SURFAR platform, two new phages were isolated and characterized: Dalewood, which targets both flagella and the conserved LPS outer core, and Bowman, a biofilm targeting phage capable of supressing growth of a subset of carbapenem-resistant clinical isolates. This tool helps to standardize the laborious but important task of phage receptor characterization, with the goal of expanding the known repertoire of P. aeruginosa phage receptors and phages.

microbiology↗

bioRxiv2025

The repertoire of resistance mutations selected by a Pseudomonas aeruginosa type IV pilus-targeting lytic bacteriophage

P. aeruginosa type IV pili (T4P) are complex surface-exposed nanomachines used for twitching motility and biofilm formation. Exposure to bacteriophages that use T4P as their primary receptor can select for resistance mutations that reduce or abolish pilus expression. Since T4P are important virulence factors, such loss can affect pathogenicity and fitness, desirable outcomes for phage therapy. Here, we evaluated the repertoire of mutations in P. aeruginosa PAO1 exposed to the pilus-specific lytic phage PO4 and their ability to revert after removal of phage. Resistant isolates ranged from hyperpiliated to bald, but none retained twitching motility. We focused on isolates that still expressed surface pili, including one with an internal 4-amino acid duplication in the essential prepilin peptidase/methyltransferase, PilD. This duplication, distal to the predicted active sites, delayed prepilin processing. Accumulation of unprocessed subunits suppressed expression of new prepilins via inhibition of PilSR two-component system activity, restricting availability of functional subunits and conferring phage resistance. Introduction of a PilS point mutation that makes cells insensitive to accumulation of unprocessed pilins restored motility and phage susceptibility. Of the mutants tested, only those with a duplication in pilD recovered wild-type motility following removal of phage pressure, likely through slip-strand mispairing as a codon-modified variant did not revert. This work shows that resistance to pilus-specific phages does not require loss of pilus expression, and certain mutations can allow bacteria to regain pilus function. Characterizing spontaeous mutations selected by phages can help to define the function and vulnerabilities of the type IV pilus system. SIGNIFICANCEAs use of phages to treat antibiotic-resistant pathogens such as Pseudomonas aeruginosa increases, it is important to understand potential outcomes of phage exposure. Most therapeutic P. aeruginosa phages use lipopolysaccharides (LPS) or T4P as primary receptors. Studying the properties of strains resistant to T4P-targeting phages can guide the design of phage cocktails to mitigate treatment resistance. We show that depending on the mutation, some phage-resistant strains can revert to wild-type sequences, emphasizing the importance of combining diverse phages to supress resurgence. By characterizing mutations that confer resistance, we can better understand whether pilus structural or regulatory components are more likely to be lost. Using phages to select for loss of pilus function represents an unbiased approach to identify new mutations in pilus-related proteins, shedding light on understudied components. Building a database of such mutations will help guide strategies to target and disarm this key P. aeruginosa virulence factor.

microbiology↗