van Raaij, M. J. | Search bioRxiv

bioRxiv2026

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 tailspikes 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 tailspikes, 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.

biochemistry↗

bioRxiv2022

Monomodular Pseudomonas aeruginosa phage JG004 lysozyme (Pae87) contains a bacterial surface-active antimicrobial peptide-like region and a possible substrate-binding subdomain

Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy for introducing a more precise approach in the way we apply antimicrobial therapy. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had been previously identified as an antimicrobial agent candidate, able to interact with the Gram-negative surface and disrupt it. Further evidence is hereby provided on this matter, based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18 [A] away from the catalytic site and located at the opposite side of the lysin molecule. This substrate binding region was conserved among phylogenetically related lysins lacking an additional cell wall binding domain, but not among those containing such a module. Two glutamic acids were identified as relevant for the peptidoglycan degradation activity, although Pae87 antimicrobial activity was seemingly unrelated to it. In contrast, an antimicrobial peptide-like region within Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and have antibacterial activity by itself. Therefore, we propose an antimicrobial mechanism for Pae87 in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell wall function, either with or without the participation of Pae87 catalytic activity. SynopsisThe structure of the monomodular Pseudomonas aeruginosa bacteriophage JG004 lysin Pae87 is presented and investigated in relationship with its function repurposed as an antimicrobial agent. The structure with its peptidoglycan ligand revealed a possible cell wall binding region. A C-terminal antimicrobial peptide-like region is shown to be important for disrupting the bacterial cell wall.

molecular biology↗