Reprogramming bacteriophage host range: design principles and strategies for engineering receptor binding proteins

Abstract

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.

Figure 1.. Minimal and evolved baseplates of two contractile tail nanomachines.

Shown are the structure of the P. aeruginosa R2 pyocin baseplate in the pre-attachment state (A) and the E. coli phage T4 baseplate in pre- and post-attachment states (B). Orthologues are shown in the same colors. A mutant used for the determination of the T4 baseplate structure contained no sheath. In the contracted state of the baseplate, the tube does not interact with it and dissociates away from it. The short tail fibers are disordered, and their electron density averages out in the cryoEM reconstruction. Figures generated using UCSF Chimera [84].

Figure 2.. A selection of structurally distinct tail fiber binding tips.

The distal ends of tail fibers are shown as ribbon representations (top) and with sequence diversity mapped onto surface representations (bottom) for E. coli phage T4 gp37 (A) [11], P. aeruginosa pyocin R2 (B) [13], Salmonella phage S16 (C) [14] and E. coli phage T7 (D) [12]. Sequence variable regions are colored cyan as indicated in the color bars provided for each panel. In panel (C), AD stands for attachment domain. E) The variable region of a human antibody (light chain, purple; heavy chain, green) is shown. Highlighted are the sequence-variable complementarity determining regions (CDRs; red) that closely resemble the distal binding loops of phage tail fibers. For panels (A)-(D), the first 250 most similar sequences found by a BLAST [85] search were aligned by COBALT [86] and mapped on the molecular surface of the protein using UCSF Chimera [84]. Panel E was generated using PyMOL Molecular Graphics System, Version 1.4 Schrödinger, LLC.

Figure 3.. Representative RBPs used by phages to target different cell wall ligands.

Ribbon diagrams are shown for: (A) TSP3 (orf212/gp164) of E. coli phage CBA120 that features a middle β-helical glycosidase domain that digests the O77 O-antigen of E. coli [26,59], (B) the capsule-degrading TSP (gp38) of Klebsiella phage KP32 [52], (C) the RBP (gp15) of Listeria phage PSA that recognizes serovar 4b wall teichoic acids via the head binding domain [33], and (D) the large multidomain TSP of Staphylococcus phage Φ11 that binds α- or β-N-acetylglucosamine moieties of S. aureus wall teichoic acids via the central propeller domain [87]. CBM, carbohydrate binding module. Figures produced using PyMOL Molecular Graphics System, Version 1.4 Schrödinger, LLC.