Tail-Engineered Phage P2 Enables Delivery of Antimicrobials into Multiple Gut Pathogens

Abstract

Bacteriophages can be reprogrammed to deliver antimicrobials for therapeutic and biocontrol purposes and are a promising alternative treatment to antimicrobial-resistant bacteria. Here, we developed a bacteriophage P4 cosmid system for the delivery of a Cas9 antimicrobial into clinically relevant human gut pathogens Shigella flexneri and Escherichia coli O157:H7. Our P4 cosmid design produces a high titer of cosmid-transducing units without contamination by a helper phage. Further, we demonstrate that genetic engineering of the phage tail fiber improves the transduction efficiency of cosmid DNA in S. flexneri M90T as well as allows recognition of a nonnative host, E. coli O157:H7. We show that the transducing units with the chimeric tails enhanced the overall Cas9-mediated killing of both pathogens. This study demonstrates the potential of our P4 cas9 cosmid system as a DNA sequence-specific antimicrobial against clinically relevant gut pathogenic bacteria.

P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri

“… 62 However, our previous study indicated a lower transduction efficiency of cosmid DNA into S. flexneri 5a M90T by P2 when compared to P1 infection, despite both phages having broad host ranges. 6250 We are currently developing a P4 cosmid system which could produce pure cosmid transducing particles, using the mutant strain of P2 lysogen described by Tridgett et al ( 62 ) Replacing the host range determining region of the P2 tail fiber with that of P1 may improve the transduction efficiency of cosmid DNA into S. flexneri 5a M90T. Comparisons between P1 phagemid lysates and P4 cosmid lysates should provide insights into the effects of using pure cosmid transducing particles on the non-Cas9 killing of S. flexneri .…”

Engineering of the temperate Burkholderia phage Milagro for use as a therapeutic

“…Multiple approaches have been proposed to modify phage tail fibers, one of which is a "domain swapping" strategy in which the C-terminal portion of a phage tail fiber is replaced by the C-terminal portion of a tail fiber taken from a heterologous phage (Dunne et al, 2021). This approach has been shown to function in multiple systems including Listeria phage PSA (Dunne et al, 2019), coliphages P2 (Fa-arun et al, 2023) and T2 (Mahichi et al, 2009), R-type pyocin (Williams et al, 2008), and T5-like phages infecting Salmonella (Zhang et al, 2022).…”