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.