Pilin regions that select for the small RNA phages in Pseudomonas aeruginosa type IV pilus

Type IV pili (TFPs) serve as the primary receptor for phages in Pseudomonas aeruginosa (PA), playing a key role in phage selection and determining the phage host range. We previously demonstrated that a fiersphage Pepevirus rubrum (PP7) selectively infects PA strains with group II (G2) TFP pilin. In this study, we expanded on this by profiling the host range of another fiersphage, Pepevirus spumicola (LeviOr01), which was able to infect several PA strains including PMM23. Analysis of 47 in-house PA strains showed that PMM23, along with nine other LeviOr01-susceptible strains and four resistant strains, possessed group I (G1) TFP pilin. This suggests that LeviOr01 targets specific regions of G1 pilin for phage-receptor interaction. Comparative sequence analysis revealed significant differences in the amino acid composition of the αβ and β1-β2 loop regions between susceptible and resistant G1-pilin strains. Pilin switching experiments using a PA surrogate strain (PAK) lacking endogenous pilin confirmed that G1 pilin is essential for LeviOr01 infection, while the accessory gene (tfpO) associated with G1 pilin was not involved in phage susceptibility. Structural predictions and comparisons between G1 and G2 pilins revealed conformational similarities but distinct differences at the αβ loop, which is likely critical for interacting with the phage virions. These findings suggest that the αβ loop region plays a pivotal role in determining RNA phage selectivity, offering new insights into the molecular mechanisms governing phage-host interactions and the host tropism of the RNA phages in various PA strains.IMPORTANCEThe host range of bacteriophages (phages) is crucial for both fundamental research and practical applications, particularly when targeting bacterial pathogens like Pseudomonas aeruginosa (PA). Previous studies have shown that the RNA phage PP7 (Pepevirus rubrum) binds to the αβ loop region of group II (G2) pilin. In this study, we demonstrate that another RNA phage, LeviOr01 (Pepevirus spumicola), also relies on the same region in group I (G1) pilins for infection. Computational modeling and structural comparisons between G1 and G2 pilins suggest that variations in the αβ loop region determine the selectivity of RNA phage binding, with similar interactions observed in both pilin groups. These findings enhance our understanding of the molecular interactions between RNA phages and their pilin receptors, offering valuable insights for developing RNA phage-based therapeutic strategies to combat PA infections.