Engineering of Salmonella Phages into Novel Antimicrobial Tailocins

Abstract

Due to the extensive use of antibiotics, the increase of infections caused by antibiotic-resistant bacteria is now a global health concern. Phages have proven useful for treating bacterial infections and represent a promising alternative or complement to antibiotic treatment. Yet, other alternatives exist, such as bacteria-produced non-replicative protein complexes that can kill their targeted bacteria by puncturing their membrane (Tailocins). To expand the repertoire of Tailocins available, we suggest a new approach that transforms phages into Tailocins. Here, we genetically engineered the virulent Ackermannviridae phage S117, as well as temperate phages Fels-1, -2 and Gifsy-1 and -2, targeting the food pathogen Salmonella, by deleting the portal vertex or major capsid gene using CRISPR-Cas9. We report the production of Tailocin particles from engineered virulent and temperate phages able to kill their native host. Our work represents a steppingstone that taps into the huge diversity of phages and transforms them into versatile puncturing new antimicrobials.

The branched receptor binding complex ofAckermannviridaephages promotes adaptative host recognition

“…Homologous recombination was promoted by infecting Salmonella Typhimurium (LT2c ΔStyLTI), carrying the homologous template and the CRISPR-Cas9 system with phage S117. Salmonella Typhimurium LT2c previously deleted for the StyLTI restriction-modification system was used to maintain the two plasmids in the host 17 . Subsequently, single plaques were picked and screened by PCR for the presence of the tsp3 or tsp4 genes of CBA120, respectively.…”

“…Genetic engineering of phages was performed through CRISPR/Cas9, as previously described 17 . Briefly, we used a recently developed two plasmids system, pEcCas (addgene #73227) and pEcgRNA (addgene #166581), where Cas9 was encoded by the pEcCas plasmid, and the Cas9 RNA guide could be cloned into the pEcgRNA plasmid as well as the recombinant template used to modify the phage genomes.…”