The structure of the Salmonella phage epsilon15 tailspike reveals multiple O-antigen binding sites and a protruding esterase domain

Many bacteriophages use tailspikes to degrade host bacterial polysaccharides, facilitating access to the outer membrane. The homotrimeric tail spikes of the Salmonella phage epsilon15 feature a slender phage-binding domain, a kink, and a barrel-shaped section with three petal-like protrusions. Here, we present the crystal structures of the monomeric protruding petal domain alone and of the trimeric barrel-shaped section with three petal domains. The barrel-shaped section includes a trimeric beta-helix, typical of phage tail spikes, alongside a trimeric beta-sandwich domain. The petal domain exhibits a fold characteristic of the serine-glycine-asparagine-histidine (SGNH) esterase family. Co-crystallisation with O-antigen fragments identified four binding sites on the tailspike: two adjacent sites on the surface of the triple beta-helix, one in the beta-sandwich domain and a fourth near the petal esterase site. These binding sites align with the expected orientation of the phage just before DNA transfer. Nuclear magnetic resonance spectroscopy and site-directed mutagenesis revealed an endorhamnosidase activity, showed that the reaction mechanism proceeds by inversion of the configuration and revealed that the active site is located at the junction of the two beta-helix binding sites. Analogous experiments also revealed an esterase site in the petal domain. Together, the structural and functional insights suggest a dual role for the phage epsilon15 tailspike: de-acetylation of the O-antigen, potentially affecting the local structure and lipopolysaccharide flexibility, plus cleavage of the O-antigen, enabling the phage to approach the bacterial membrane.