Project description:Bacteriophages are potent therapeutics against biohazardous bacteria that are rapidly acquiring multidrug resistance. However, routine administration of bacteriophage therapy is currently impeded by a lack of safe phage production methodologies and insufficient phage characterization. We thus developed a versatile cell-free platform for host-independent production of phages targeting gram-positive and gram-negative bacteria. A few microliters of a one-pot reaction produces effective doses of phages against potentially antibiotic-resistant bacteria such as enterohemorrhagic E. coli (EAEC) and Yersinia pestis, which also possibly pose threats as biological warfare agents. We also introduce a method for transient, non-genomic phage engineering to safely confer additional functions, such as a purification tag or bioluminescence for host detection, for only one replication cycle. Using high-resolution and time-resolved mass spectrometry, we validated the expression of 40 hypothetical proteins from two different phages (T7 and CLB-P3) and identified genes in the genome of phage T7 that express exceptionally late during phage replication. Our comprehensive methodology thus allows for accelerated reverse and forward phage engineering as well as for safe and customized production of clinical-grade therapeutic bacteriophages.
Project description:Temperate bacteriophages play a pivotal role in the biology of their bacterial host. Of particular interest are bacteriophages infecting enterohemorrhagic E. coli (EHEC) due to their significant contribution in the pathogenicity of these pathogens, most notably by encoding the key virulence factor of this pathogen, the Shiga toxin. To better understand the role of EHEC phages on the functionality of its host, we isolated eight temperate phages from clinical EHEC isolates and characterized their genomic composition, morphology and receptor targeting. Morphological analysis identified one long-tailed member from the Siphoviridae family, targeting the OmpC receptor for host recognition, while the other seven phages are short-tailed (Podoviridae) and target the essential BamA protein. Genomic characterization revealed significant variation between the long- and short-tailed phages. Five of the eight isolated phages encode the potent Shiga toxin. Comparative analysis displays the typical lambdoid mosaicism, indicative of horizontal gene transfer driving evolution. These findings provide insights into the genetic and morphologic diversity and receptor specificity of EHEC phages, highlighting their role in evolution and pathogenicity of clinical EHEC strains
