Deciphering the modular structure of phage depolymerase: a dual-action strategy for biofilm degradation and targeted Pseudomonas aeruginosa binding

Abstract

ABSTRACT Pseudomonas aeruginosa is a critical ESKAPE pathogen that can form strong biofilms, which protect it from antibiotics and the host’s immune system. Enzymes from bacteriophages, called depolymerases, are often found in tail fiber proteins and provide a useful way to break down these biofilms. In this work, we studied ORF55, a tail fiber protein from Pseudomonas phage phiPA1-3 that contains an SGNH hydrolase domain. Using AlphaFold2 for structural modeling and modular dissection, we identified the specific functions of its N-terminal and C-terminal parts. We found that the full protein (SGNH55_Full) broke down easily. However, a shorter version that kept the SGNH hydrolase domain (SGNH55Δtail_619) was much more stable and better at degrading biofilms. This truncated enzyme worked well against biofilms from clinical isolates, including those resistant to phages. This shows it has broad potential for treatment that does not depend on the virus itself infecting the bacteria. At the same time, we identified the N-terminal domain (TFP55) as the part responsible for binding to the bacterial receptor. We used TFP55 to develop a fast latex agglutination test to detect P. aeruginosa. This test was very specific and sensitive, with a limit of detection of 10 CFU, and it stayed stable for 6 months. Our results show that breaking down phage tail fibers into modules is a powerful strategy. It provides a clear path for creating both effective anti-biofilm agents and stable tools for diagnosis. IMPORTANCE Biofilm-forming Pseudomonas aeruginosa poses severe clinical challenges, and full-length phage depolymerases, while promising, are often unstable. This study overcomes this limitation through the structure-guided modular dissection of the phage tail fiber protein SGNH55. By decoupling its catalytic and host-binding domains, we generated two highly stable tools: a potent enzyme to eradicate multidrug-resistant biofilms and a sensitive diagnostic probe for rapid pathogen detection. This dual-utility approach provides an innovative therapeutic-diagnostic strategy and establishes a universal framework for engineering complex phage proteins. Biofilm-forming Pseudomonas aeruginosa poses severe clinical challenges, and full-length phage depolymerases, while promising, are often unstable. This study overcomes this limitation through the structure-guided modular dissection of the phage tail fiber protein SGNH55. By decoupling its catalytic and host-binding domains, we generated two highly stable tools: a potent enzyme to eradicate multidrug-resistant biofilms and a sensitive diagnostic probe for rapid pathogen detection. This dual-utility approach provides an innovative therapeutic-diagnostic strategy and establishes a universal framework for engineering complex phage proteins.