Turning of (Ph)age: Expanding the therapeutic potential of our viral allies

Abstract

Novel strategies for treating bacterial infections are needed to combat the growing threat of antibiotic resistance. Here we engineered P4-like particles for bacterial-culture proof-of-concept experiments in two antimicrobial modes: a single-action design intended to deliver model payloads into bacteria without programmed lysis, and a dual-action design intended to lyse target bacteria while releasing a secondary antigenic payload, exemplified here by model antigens. Using a P2 helper-free P4-like particle production platform, we designed, produced and tested P4-mediated single-and dual-action antimicrobial prototypes. After completing bacterial-culture proof-of-concept experiments, we optimized early-stage bioprocessing for future studies, leading to 1011 plaque forming units (PFU) per mL and 0.25 endotoxin units (EU) per 109 PFU. We also challenged the P4 viral-vector packaging limit by deleting sid to favour packaging into P2-sized capsids ([~]25.8 kb estimated cargo capacity). Importantly, repressing payload expression during particle production improved viral titers by about 2 logs, reduced detected cargo-sequence alterations from 12/20 to 0/20 sequenced post-transduction isolates and enabled higher-dose transduction with stronger detectable antigen signal. Altogether, this study supports P4-derived phage-like particles as antimicrobial prototypes that extend phage function beyond bacterial elimination and provides a basis for future validation in pathogen and animal models of infection.