Bacteria must maintain the ability to modify and repair the peptidoglycan layer without jeopardising its essential functions in cell shape, cellular integrity and intermolecular interactions. A range of new experimental techniques is bringing an advanced understanding of how bacteria regulate and achieve peptidoglycan synthesis, particularly in respect of the central role played by complexes of Sporulation, Elongation or Division (SEDs) and class B penicillin-binding proteins required for cell division, growth and shape. In this review we highlight relationships implicated by a bioinformatic approach between the outer membrane, cytoskeletal components, periplasmic control proteins, and cell elongation/division proteins to provide further perspective on the interactions of these cell division, growth and shape complexes. We detail the network of protein interactions that assist in the formation of peptidoglycan and highlight the increasingly dynamic and connected set of protein machinery and macrostructures that assist in creating the cell envelope layers in Gram-negative bacteria.

Growing antibiotic resistance is rapidly threatening the efficacy of treatments for Gram-negative infections. Bicycle molecules, constrained bicyclic peptides from diverse libraries generated by bacteriophage display that bind with high affinity to a chosen target are a potential new class of antibiotics. The generally impermeable bacterial outer membrane currently limits the access of peptides to bacteria. The conjugation of membrane active peptides offers an avenue for outer membrane penetration. Here, we investigate which physicochemical properties of a specific membrane active peptide (MAP), derived from ixosin-B, could be tweaked to enhance the penetration of conjugates by generating multiple MAP-Bicycle conjugate variants. We demonstrate that charge and hydrophobicity are important factors, which enhance penetration and, therefore, antimicrobial potency. Interestingly, we show that induction of secondary structure, but not a change in amphipathicity, is vital for effective penetration of the Gram-negative outer membrane. These results offer insights into the ways vectors could be designed to deliver Bicycle molecules (and other cargos) through biological membranes.

Bacteriophages are bacterial viruses that provide alternatives to small-molecule drugs to combat infections by antibiotic-resistant bacteria. To infect a bacterial host, a phage needs to bind to the bacterial surface via receptor binding proteins (RBPs), which are critical for determining host specificity. For functionally important receptors, the RBP-receptor interaction could be exploited via phage steering, where emerging bacterial resistance due to receptor modification could make bacteria less fit or virulent. Despite this, relatively little is known about RBP-receptor interactions. Here we build on the recent discovery of coliphages that have the outer membrane (OM) lipopolysaccharide translocon LptDE as their terminal receptor and show via cryogenic electron microscopy (cryoEM) that, surprisingly, the RBP of the small siphophage Oekolampad binds to a hitherto unobserved, open state of LptDE. The open lateral gate of LptD is occupied by a β-strand peptide originating from the degraded N-terminal jellyroll domain of LptD, suggesting the possibility of LptD inhibition via peptidomimetics. A structure of LptDE in complex with the superinfection exclusion (SE) protein Rtp45 of the Oekolampad-related phage Rtp shows a mechanism of SE where Rtp45-induced conformational changes in LptD resulting from steric clashes preclude RBP binding. Finally, analysis of spontaneous Oekolampad-resistant E. coli mutants identifies mutations in LptD that abolish the LptDE-RBP interaction in vitro . SDS-EDTA sensitivity assays of the mutants show no major defects in LptDE function, suggesting that phage steering via LptDE might be challenging. Significance: The outer membrane (OM) of Gram-negative bacteria is a protective barrier generated by lipopolysaccharide (LPS), a complex glycolipid that makes up the outer leaflet of the OM. Following its synthesis and transport from the inner membrane, LPS is inserted into the OM by LptDE, an OM protein complex essential for most Gram-negative bacteria. LPS insertion requires a hitherto unobserved, open state of LptDE. Here we report the unexpected finding that the receptor binding protein (RBP) of a bacteriophage binds to an open state of LptDE, allowing isolation and visualisation via cryogenic electron microscopy. The lateral gate of the LptDE-RBP complex is occupied by a β-strand peptide, suggesting that the LptDE of pathogenic bacteria could be inhibited by peptidomimetics.

The lipopolysaccharide (LPS) transport (Lpt) system in Gram-negative bacteria maintains the integrity of the asymmetric bacterial outer membrane (OM). LPS biogenesis systems are essential in most Gram-negative bacteria, with LptDE responsible for the delivery of LPS to the outer leaflet of the OM. As an externally accessible, essential protein, LptDE offers a promising target for inhibitor development without the need for cellular penetration. However, there are no direct inhibitors of E. coli LptDE, and drug discovery is made challenging since it is a membrane target without a conventional active site. Here, the bicycle phage display platform was used in combination with cryogenic-electron microscopy (cryo-EM) and surface plasmon resonance to identify and map bicyclic peptide binders to Shigella flexneri LptDE (SfLptDE). Four distinct epitopes with unique bicycle molecule binding motifs were identified across the SfLptD β-barrel. This method represents a streamlined workflow for the identification and prioritization of hit molecules against LptDE.

Penicillin binding proteins (PBPs) are well validated antimicrobial targets, but the prevalence of β-lactamase driven resistance and, more rarely, target-based mutations, necessitates new classes of PBP-targeting drugs. Here we describe the discovery and optimisation of novel, bicyclic peptide (Bicycle ^® ) inhibitors of E. coli PBP3 ( Ec PBP3) using a proprietary phage display platform, and their conjugation to linear antimicrobial peptides to confer outer membrane permeation. These molecules exhibited high-affinity binding to E. coli PBP3 and a viable spectrum of killing activity against clinically relevant species of the Enterobacterales. X-ray crystallography was used to explore the mode of binding to PBP3, enabling increased target affinity and improvement of in vitro stability. These compounds bind to the transpeptidase active site cleft of PBP3 and represent a novel non-β-lactam chemical class of high affinity, non-covalent penicillin binding protein inhibitors.

α-Hemolysin (Ahly) is a major Staphylococcus aureus virulence determinant implicated in tissue injury and immune dysregulation; antibody inhibitors have reached clinical trials but alternatives with improved ease of manufacture and tissue penetration are desirable. Here we demonstrate that phage-derived bicyclic peptides can serve as compact, chemically tractable Ahly neutralisers. Using TATB-scaffolded M13 phage libraries we identified WNP-motif containing bicyclic binders, with a lead hit of Peptide 14 (K _D = 1792nM) and progressed the lead by iterative affinity maturation to Peptide 20 (K _D = 609 nM) and by incorporation of strategically chosen non-canonical amino acids to yield Peptide 88 (KD = 96 nM). A 2.2 Å co-crystal structure with AhlyH35A locates the binding footprint on the rim domain and explains the critical role of the WNP motif in target engagement. Functional assays show that the Peptide 88 blocks Ahly mediated hemolysis, inhibits Ahly driven ADAM10 activation, and elucidate its inhibitory mechanism of preventing Ahly binding to human A549 epithelial cells. Peptide 88 protects A549 cells from recombinant toxin and attenuates cytotoxicity in S. aureus co-culture experiments, whilst showing no toxicity to A549 cells. Bicyclic peptides thus represent a new and promising anti-virulence modality: small, synthetically accessible molecules that mimic antibody recognition, with therapeutic potential against S. aureus infections.