Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria. Issue 9 (15th September 2022)

• Record Type: Journal Article
• Authors: Emslander, Quirin; Vogele, Kilian; Braun, Peter; Stender, Jana; Willy, Christian; Joppich, Markus; Hammerl, Jens A.; Abele, Miriam; Meng, Chen; Pichlmair, Andreas; Ludwig, Christina; Bugert, Joachim J.; Simmel, Friedrich C.; Westmeyer, Gil G.
• Is Part Of: Cell chemical biology. Volume 29:Issue 9(2022)
• Page Start: 1434
• Page End: 1445.e7
• Publication Date: 2022-09-15
• Subjects: cell-free production -- therapeutic bacteriophages -- multidrug-resistant bacteria -- personalized medicine -- time-resolved proteomics -- non-genomic phage engineering -- phage therapy -- non-structural phage proteins -- biosafety; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2022.06.003
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: 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. Graphical abstract: Highlights: Personalized and cell-free production of phages against multidrug-resistant bacteria Extending cell-free production to phages targeting gram-positive bacteria Transient, non-genomic engineering of bacteriophages Time-resolved proteomics during phage assembly and validation of phage proteins Abstract : Emslander, Vogele et al. demonstrate the personalized production of therapeutic bacteriophages in a cell-free system. This approach is faster and safer than the conventional method based on cultivating host bacteria. Cell-free production also allows for time-resolved proteome analysis during phage assembly and biosafe, non-genomic engineering of single-use phages targeting multidrug-resistant bacteria.