Targeting multidrug-resistant bacteria with genetic-information-free protein-only phages

Abstract

Bacteriophages offer advantages over small-molecule antibiotics, including host specificity and general compatibility with the human phagenome. However, their evolvability as replicating biological entities introduces therapeutic unpredictability and risks of phage-bacteria co-evolution. Here, we retain the targeting benefits of phages while avoiding genetic replication by engineering genetic-information-free, protein-only phages (POPs). These genome-free particles self-assemble in a cell-free protein synthesis system from modular, de novo gene fragments encoding only structural and antimicrobial proteins. Using Enterobacteria phage T7 and its susceptible bacterial host as a model, we test the hypothesis that POPs stochastically encapsulate small antimicrobial proteins during self-assembly and deliver them into bacteria during adsorption via an ejectome-mediated injection mechanism. A computational survey of T7 small proteins revealed early and mid-genome enrichments of hypothetical proteins and capsid volume sufficient to accommodate multiple small proteins in the absence of the [~]40-kb genome. In time-series antimicrobial susceptibility assays (48-72 h), POPs produced initial growth inhibition comparable to wild-type T7 at the highest doses with a linear dose-effect relationship and a minimum inhibitory concentration-like threshold. These results establish the feasibility of genetic-information-free POPs as protein-based antimicrobials that couple phage receptor specificity with minimal biosafety risks, supporting the development of more stable and predictable phage-inspired therapeutics.