Bellis, N. | Search bioRxiv

bioRxiv2025

Structural atlas of Pakpunavirus P7-1 reveals determinants of virion stability and genome ejection

Bacteriophages of the Pakpunavirus genus exhibit broad host range and potent bacteriolytic activity, making them promising candidates for clinical use. Here, we present a structural atlas of the therapeutic phage Pakpunavirus P7-1, a component of a phage cocktail targeting Pseudomonas aeruginosa that has undergone Phase 1/2 clinical trials. We determined the near-atomic structure of the extended virion and obtained a medium-resolution reconstruction of the contracted tail. Atomic models were built for 20 structural proteins comprising the icosahedral capsid, neck, contractile tail, and baseplate. We identified six upward-pointing Short Tail Fibers that stabilize the extended sheath and six highly flexible Long Tail Fibers likely involved in host recognition. Ordered fragments of the Tape Measure Protein revealed six copies inside the tail tube, forming a 3-helix cork at the tail tip. Sheath contraction repositions the baseplate, projecting all twelve tail fibers outward, yet contraction alone is insufficient to trigger genome ejection.

biochemistry↗

bioRxiv2025

High-resolution cryo-EM analysis of the therapeutic Pseudomonas phage Pa223

Cryogenic electron microscopy (cryo-EM) analysis of bacteriophages is a valuable method for deciphering virus composition and conformational plasticity. In this study, we present a high-resolution structural atlas of the Pseudomonas virus Pa223, a phage from the Bruynoghevirus genus that has recently been used in clinical cocktails for treating cystic fibrosis and non-cystic fibrosis bronchiectasis, as well as for compassionate care. By combining bioinformatics, proteomics, cryo-EM single particle analysis, and localized reconstruction, we annotated and built atomic models for eight structural polypeptide chains that form the icosahedral capsid and noncontractile tail. We discovered that the Pa223 capsid is decorated by a spike protein that features a unique triple-{beta} helix fold with no structural homologs in the database. The Pa223 tail features six trimeric tail fibers extending upwards, similar to, but shorter than, those found in phage T7. Unlike T7, the Pa223 tail is extended by two head-to-tail adaptors and sealed by a trimeric tail needle, similar to P22-like phages. We identified a protein bound around the outer perimeter of the portal protein, positioned similarly to the ejection protein gp72, which was identified in the Pseudomonas phage DEV, a Litunavirus phage and member of the reclassified Schitoviridae family. This structural hint led us to identify the Pa223 ejection proteins gp53, gp54, and gp56, which bioinformatically resemble those of T7-like phages more closely than Schitoviridae. Thus, phage Pa223 contains diverse structural elements found in P22-like, T7-like, and Litunavirus phages, providing a framework for understanding the diversification and evolution of ejection proteins in Bruynogheviruses. HighlightsO_LIThe high-resolution structure of Bruynoghevirus Pa223 reveals hybrid structural features that are shared among P22-like, T7-like, and Litunavirus phages. C_LIO_LIThe Pa223 capsid is decorated with a trimeric spike asymmetrically bound at the icosahedral 3-fold axes. C_LIO_LIThe Pa223 tail features two quasi-equivalent conformations of the head-to-tail adaptor protein arranged into two coaxial rings. C_LIO_LIIdentification of the ejection protein gp54 through structural similarity to gp72 from the Litunavirus DEV. C_LIO_LIBioinformatic mapping of the Pa223 ejection proteins gp53 and gp56 validated through mass spectrometry analysis of infectious virions. C_LI

biochemistry↗