Strategies for developing phages into novel antimicrobial tailocins.

Tailocins are high-molecular-weight bacteriocins produced by bacteria to kill related environmental competitors by binding and puncturing their target. Tailocins are promising alternative antimicrobials, yet the diversity of naturally occurring tailocins is limited. The structural similarities between phage tails and tailocins advocate using phages as scaffolds for developing new tailocins. This article reviews three strategies for producing tailocins: disrupting the capsid-tail junction of phage particles, blocking capsid assembly during phage propagation, and creating headless phage particles synthetically. Particularly appealing is the production of tailocins through synthetic biology using phages with contractile tails as scaffolds to unlock the antimicrobial potential of tailocins.

Diverse bacteriophages for biocontrol of ESBL- and AmpC-ß-lactamase-producing E. coli.

Novel solutions are needed to reduce the risk of transmission of extended spectrum ß-lactamase (ESBL) and AmpC ß-lactamase producing Escherichia coli (ESBL/AmpC E. coli) from livestock to humans. Given that phages are promising biocontrol agents, a collection of 28 phages that infect ESBL/AmpC E. coli were established. Whole genome sequencing showed that all these phages were unique and could be assigned to 15 different genera. Host range analysis showed that 82% of 198 strains, representing the genetic diversity of ESBL/AmpC E. coli, were sensitive to at least one phage. Identifying receptors used for phage binding experimentally as well as in silico predictions, allowed us to combine phages into two different cocktails with broad host range targeting diverse receptors. These phage cocktails efficiently inhibit the growth of ESBL/AmpC E. coli in vitro, thus suggesting the potential of phages as promising biocontrol agents.

A hybrid receptor binding protein enables phage F341 infection of Campylobacter by binding to flagella and lipooligosaccharides.

Flagellotropic bacteriophages are interesting candidates as therapeutics against pathogenic bacteria dependent on flagellar motility for colonization and causing disease. Yet, phage resistance other than loss of motility has been scarcely studied. Here we developed a soft agar assay to study flagellotropic phage F341 resistance in motile Campylobacter jejuni. We found that phage adsorption was prevented by diverse genetic mutations in the lipooligosaccharides forming the secondary receptor of phage F341. Genome sequencing showed phage F341 belongs to the Fletchervirus genus otherwise comprising capsular-dependent C. jejuni phages. Interestingly, phage F341 encodes a hybrid receptor binding protein (RBP) predicted as a short tail fiber showing partial similarity to RBP1 encoded by capsular-dependent Fletchervirus, but with a receptor binding domain similar to tail fiber protein H of C. jejuni CJIE1 prophages. Thus, C. jejuni prophages may represent a genetic pool from where lytic Fletchervirus phages can acquire new traits like recognition of new receptors.

Agtrevirus phage AV101 recognizes four different O-antigens infecting diverse E. coli.

Bacteriophages in the Agtrevirus genus are known for expressing multiple tail spike proteins (TSPs), but little is known about their genetic diversity and host recognition apart from their ability to infect diverse Enterobacteriaceae species. Here, we aim to determine the genetic differences that may account for the diverse host ranges of Agrevirus phages. We performed comparative genomics of 14 Agtrevirus and identified only a few genetic differences including genes involved in nucleotide metabolism. Most notably was the diversity of the tsp gene cluster, specifically in the receptor-binding domains that were unique among most of the phages. We further characterized agtrevirus AV101 infecting nine diverse Extended Spectrum ß-lactamase (ESBL) Escherichia coli and demonstrated that this phage encoded four unique TSPs among Agtrevirus. Purified TSPs formed translucent zones and inhibited AV101 infection of specific hosts, demonstrating that TSP1, TSP2, TSP3, and TSP4 recognize O8, O82, O153, and O159 O-antigens of E. coli, respectively. BLASTp analysis showed that the receptor-binding domain of TSP1, TSP2, TSP3, and TSP4 are similar to TSPs encoded by E. coli prophages and distant related virulent phages. Thus, Agtrevirus may have gained their receptor-binding domains by recombining with prophages or virulent phages. Overall, combining bioinformatic and biological data expands the understanding of TSP host recognition of Agtrevirus and give new insight into the origin and acquisition of receptor-binding domains of Ackermannviridae phages.

Engineering of Salmonella Phages into Novel Antimicrobial Tailocins.

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.

Subtypes of tail spike proteins predicts the host range of Ackermannviridae phages.

Phages belonging to the Ackermannviridae family encode up to four tail spike proteins (TSPs), each recognizing a specific receptor of their bacterial hosts. Here, we determined the TSPs diversity of 99 Ackermannviridae phages by performing a comprehensive in silico analysis. Based on sequence diversity, we assigned all TSPs into distinctive subtypes of TSP1, TSP2, TSP3 and TSP4, and found each TSP subtype to be specifically associated with the genera (Kuttervirus, Agtrevirus, Limestonevirus, Taipeivirus) of the Ackermannviridae family. Further analysis showed that the N-terminal XD1 and XD2 domains in TSP2 and TSP4, hinging the four TSPs together, are preserved. In contrast, the C-terminal receptor binding modules were only conserved within TSP subtypes, except for some Kuttervirus TSP1s and TSP3s that were similar to specific TSP4s. A conserved motif in TSP1, TSP3 and TSP4 of Kuttervirus phages may allow recombination between receptor binding modules, thus altering host recognition. The receptors for numerous uncharacterized phages expressing TSPs in the same subtypes were predicted using previous host range data. To validate our predictions, we experimentally determined the host recognition of three of the four TSPs expressed by kuttervirus S117. We confirmed that S117 TSP1 and TSP2 bind to their predicted host receptors, and identified the receptor for TSP3, which is shared by 51 other Kuttervirus phages. Kuttervirus phages were thus shown encode a vast genetic diversity of potentially exchangeable TSPs influencing host recognition. Overall, our study demonstrates that comprehensive in silico and host range analysis of TSPs can predict host recognition of Ackermannviridae phages.