Abstract
BACKGROUND: Bacteriophages and bacteria frequently occupy the same ecological niches, driving complex and dynamic host-virus interactions. In Pseudomonas aeruginosa, phages from the Migulavirinae subfamily, tail fibre proteins (TFPs) are crucial to host recognition. These proteins, located within the phage tail structure, are subject to frequent recombination and may play a key role in shaping host range. This study investigates the molecular basis of host specificity in Litunavirus and Luzseptimavirus phages, focusing on the structure and variation of their TFPs. RESULTS: Host spectrum analysis divided phages into three categories; however, contrary to expectations, no direct correlation was found between TFP recombination history and host range, most likely because subsequent single amino acid changes in the pyocin knob regions, critical for adsorption, altered the host spectrum after the recombination event. Notably, phages sharing highly similar pyocin knob 2 domain architectures displayed identical host spectra, suggesting a strong link between this region and host specificity. Despite high sequence variability, all TFPs adopted a conserved trimeric fold with five regions: N-terminal, GrpE-like, GDSL-like with a carbohydrate-binding module, pyocin knob, and C-terminal. Structural similarities to bacterial PilA and pyocins were noted. Variation in the pyocin knob region, especially substitutions involving polar residues, was partially correlated with host range, likely via hydrogen bonding with the O-antigen. The GrpE-like domain resembled type IV pili, suggesting a role in reversible attachment, while the GDSL-like domain may support enzymatic processing of the O-antigen. CONCLUSIONS: Our findings support a multi-step adsorption mechanism of Migulavirinae phages, initiated by random encounters with the bacterial surface, followed by specific, stable interactions between the pyocin knob region and the bacterial lipopolysaccharide (LPS) O-antigen. Final stabilization involves additional interactions with the LPS core region. While the GrpE-like domain may contribute to transient stabilization near the surface, its structural similarity to PilA suggests a possible evolutionary convergence rather than a direct pilus-binding function. Despite high sequence variability, TFPs maintain conserved structural features, allowing for modular adaptations that precisely adjust host specificity. Importantly, the lack of a direct link between TFP recombination and host range suggests that factors beyond recombination influence phage host specificity.