Emslander, Q., Vogele, K., Braun, P., Stender, J., Willy, C., Joppich, M., Hammerl, J.A., Abele, M., Meng, C., Pichlmair, A., Ludwig, C., Bugert, J.J., Simmel, F.C., and Westmeyer, G.G.
Cell Chem Biol, 2022, online ahead of print.
Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria
Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.