Engineering P4 satellite phage particles with single and dual antimicrobial actions

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.