Finnerty, R.; Lim, C.; Secor, P. R.; Marshall, C. W.

Bacteria often evolve resistance to phage infection by altering the cell-surface structures required for viral adsorption. However, the role extracellular metabolites play in influencing phage susceptibility and the evolution of phage resistance remains unclear. Here, we evaluated whether sustained exposure to putrescine, a polyamine released during phage-mediated cell lysis, alters susceptibility of the pathogen Pseudomonas aeruginosa to the type IV pili-dependent phage DMS3vir. Using adaptive laboratory evolution over ~66 generations, we evolved P. aeruginosa with or without putrescine and with or without DMS3vir. As expected, direct phage exposure rapidly led to complete phage resistance. Interestingly, populations exposed to putrescine also developed phage resistance by the end of the experiment, despite having never encountered the phage. Whole-population genome sequencing revealed parallel mutations in genes associated with type IV pili and the global transcriptional regulator mexT. Using transposon insertion mutants in the type IV ATPases pilT and pilB, we confirmed that disruption of these genes leads to DMS3vir phage resistance. We also used a type IV pilus biogenesis factor fimV transposon mutant, which showed a putrescine-dependent reduction in phage susceptibility. These findings show that sustained exposure to a host-derived metabolite can drive the evolution of phage resistance through modification of key phage-adsorption sites and regulatory genes. Our work identifies elevated polyamine exposure as a selective pressure that promotes type IV pili-mediated phage resistance, even in the absence of phage exposure.