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[Display omitted] • Phages use structurally diverse receptor binding proteins (RBPs) to target different cell wall structures. • RBPs play a critical role in spatial and temporal positioning of the tail apparatus to the bacterial surface during infection. • High-resolution structures of RBPs can be used as 'blueprints' to guide the precise engineering of RBP binding range. Bacteriophages (phages) use specialized tail machinery to deliver proteins and genetic material into a bacterial cell during infection. Attached at the distal ends of their tails are receptor binding proteins (RBPs) that recognize specific molecules exposed on host bacteria surfaces. Since the therapeutic capacity of naturally occurring phages is often limited by narrow host ranges, there is significant interest in expanding their host range via directed evolution or structure-guided engineering of their RBPs. Here, we describe the design principles of different RBP engineering platforms and draw attention to the mechanisms linking RBP binding and the correct spatial and temporal attachment of the phage to the bacterial surface. A deeper understanding of these mechanisms will directly benefit future engineering of more effective phage-based therapeutics. [ABSTRACT FROM AUTHOR]

Bacteriophages are the most abundant biological entity on earth, acting as the predators and evolutionary drivers of bacteria. Owing to their inherent ability to specifically infect and kill bacteria, phages and their encoded endolysins and receptor-binding proteins (RBPs) have enormous potential for development into precision antimicrobials for treatment of bacterial infections and microbial disbalances; or as biocontrol agents to tackle bacterial contaminations during various biotechnological processes. The extraordinary binding specificity of phages and RBPs can be exploited in various areas of bacterial diagnostics and monitoring, from food production to health care. We review and describe the distinctive features of phage RBPs, explain why they are attractive candidates for use as therapeutics and in diagnostics, discuss recent applications using RBPs, and finally provide our perspective on how synthetic technology and artificial intelligence-driven approaches will revolutionize how we use these tools in the future. [ABSTRACT FROM AUTHOR]

Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections. [ABSTRACT FROM AUTHOR]

Bacteriophages provide excellent tools for diagnostics, remediation, and targeted microbiome manipulation, yet isolating viruses with suitable host specificity remains challenging. Using Listeria phage PSA, we present a synthetic biology blueprint for host-range engineering through targeted modification of serovar-specific receptor binding proteins (RBPs). We identify Gp15 as the PSA RBP and construct a synthetic phage library featuring sequence-randomized RBPs, from which host range mutants are isolated and subsequently integrated into a synthetic, polyvalent phage with extended host range. To enable rational design of chimeric RBPs, we determine the crystal structure of the Gp15 receptor-binding carboxyl terminus at 1.7-Å resolution and employ bioinformatics to identify compatible, prophage-encoded RBPs targeting different Listeria serovars. Structure-guided design enables exchange of heterologous RBP head, neck, or shoulder domains to generate chimeric phages with predictable and extended host ranges. These strategies will facilitate the development of phage biologics based on standardized virus scaffolds with tunable host specificities. • Adaptation of Listeria phage serovar specificity through targeted RBP variations • High-resolution crystal structure of a Listeria phage receptor binding protein • Structure-guided design of RBP chimeras yields phages with predictable host ranges • Synthetic RBPs extend phage binding specificity (from SV 4b to 4a, 4b, 4d, 5, 6b) Receptor binding proteins (RBPs) are key determinants of bacteriophage host range. Dunne et al. solve the RBP crystal structure of Listeria phage PSA and combine synthetic biology with structure-guided approaches to re-program RBP specificity. Using the temperate, narrow host range PSA scaffold, they construct virulent phages with extended and defined host specificities. [ABSTRACT FROM AUTHOR]

Summary The ability of phages to infect specific bacteria has led to their exploitation as bio-tools for bacterial remediation and detection. Many phages recognize bacterial hosts via adhesin tips of their long tail fibers (LTFs). Adhesin sequence plasticity modulates receptor specificity, and thus primarily defines a phage's host range. Here we present the crystal structure of an adhesin (gp38) attached to a trimeric β-helical tip (gp37) from the Salmonella phage S16 LTF. Gp38 contains rare polyglycine type II helices folded into a packed lattice, herein designated "PG II sandwich." Sequence variability within the domain is limited to surface-exposed helices and distal loops that form putative receptor-binding sites. In silico analyses revealed a prevalence of the adhesin architecture among T-even phages, excluding the archetypal T4 phage. Overall, S16 LTF provides a valuable model for understanding binding mechanisms of phage adhesins, and for engineering of phage adhesins with expandable or modulated host ranges. Graphical Abstract Highlights • Crystal structure of Salmonella phage S16 long tail fiber adhesin tip (gp38-gp37) • Gp38 features rare polyglycine type II (PG II) helices folded into a PG II sandwich • Exposed residues of the PG II sandwich are determinants of host binding • S16 adhesin architecture is prevalent among the important T-even phage family Dunne et al. present the crystal structure of the adhesin tip (gp38) of the Salmonella bacteriophage S16 long tail fiber. Gp38 contains rare polyglycine type II (PG II) helices arranged as a three-layered "PG II sandwich." Surface residues on the PG II sandwich are key determinants of phage-host interaction. [ABSTRACT FROM AUTHOR]