The ester bond crosslink discovered within bacterial adhesin proteins offers a captivating insight into the convergent evolution of enzyme-like machinery. Crystal structures reveal a putative catalytic triad comprising an acid-base-nucleophile combination and an oxyanion-like site that suggests a serine protease-like mechanism drives the crosslinking process. We now provide confirmation of the mechanism, revealing functional catalytic dyads or triads, and the recapitulation of protease machinery from a Pseudomonas bacterium and a human cytomegalovirus related only by convergent evolution. Molecular dynamics simulations suggest how a conservative threonine-to-serine mutation of the nucleophile induces hydrolysis and eliminates the ester bond crosslink. Collectively, our structural, functional, and computational efforts detail the molecular intricacies of intramolecular ester bond formation and underscore the convergent evolutionary adaptations of bacteria in exploiting enzyme-like machinery to protect essential adhesin proteins from the mechanical, biological, and chemical hostilities of their replicative niche.

Microbial degradation can provide an avenue for the remediation of plastic pollution, contributing to the urgent environmental problem of global plastic waste. We demonstrate the degradation of polycaprolactone (PCL) by Clonostachys rosea and elucidate its underlying molecular mechanisms. We constructed the genome of this fungal strain and monitored changes in gene expression when exposed to PCL. Twelve genes linked to PCL degradation were found in the genome of C. rosea, and some of them were upregulated in the presence of the plastic, including genes coding for two cutinases. We heterologously expressed the enzymes coded by both genes and confirmed their activity against PCL polymers. We also demonstrate that one of the enzymes was active against polyethylene terephthalate polymers. Glucose inhibited the expression of both genes, completely halting the plastic biodegradation process, possibly serving as a preferred and readily metabolizable carbon source compared with PCL. We confirm the presence of key metabolic pathways linked to PCL degradation in C. rosea, including fatty acid degradation, providing further evidence of the mechanisms central to plastic biodegradation.IMPORTANCEPlastic pollution is one of our most pressing environmental challenges, with billions of tons of plastic waste accumulating in our ecosystems. While recycling helps, it cannot fully address this crisis, making it crucial to find new solutions. Our study reveals how a common soil fungus, Clonostachys rosea, can break down certain plastics, specifically polycaprolactone and polyethylene terephthalate. We identified the exact genes and enzymes responsible for this ability and showed how different environmental conditions affect the fungus's plastic-degrading capabilities. Notably, we discovered that adding glucose completely stops the fungus from breaking down plastic, suggesting that careful control of growth conditions is essential for effective plastic degradation. These findings are significant because they provide a detailed blueprint for optimizing plastic biodegradation using fungi, potentially leading to more effective ways to tackle plastic pollution. This research represents a crucial step toward developing practical, environmentally friendly solutions for plastic waste management.

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F 420 . Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F 420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L -glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

The COVID-19 pandemic caused by the virus SARS-CoV-2 was the greatest global threat to human health in the last three years. The most widely used methodologies for the diagnosis of COVID-19 are quantitative reverse transcription polymerase chain reaction (RT-qPCR) and rapid antigen tests (RATs). PCR is time-consuming and requires specialized instrumentation operated by skilled personnel. In contrast, RATs can be used in-home or at point-of-care but are less sensitive, leading to a higher rate of false negative results. In this work, we describe the development of a disposable, electrochemical, and laser-scribed graphene-based biosensor strips for COVID-19 detection that exploits a split-ester bond ligase system (termed ‘EsterLigase’) for immobilization of a virus-specific nanobody to maintain the out-of-plane orientation of the probe to ensure the efficacy of the probe-target recognition process. An anti-spike VHH E nanobody, genetically fused with the EsterLigase domain, was used as the specific probe for the spike receptor-binding domain (SP-RBD) protein as the target. The recognition between the two was measured by the change in the charge transfer resistance determined by fitting the electrochemical impedance spectroscopy (EIS) spectra. The developed LSG-based biosensor achieved a linear detection range for the SP-RBD from 150 pM to 15 nM with a sensitivity of 0.0866 [log(M)]−1 and a limit of detection (LOD) of 7.68 pM.

Heterobimetallic cages built from Pd and either octahedral Ru or square-planar Pt moieties and bridged by ligands with H bonding-accepting or -donating properties are reported. They showed stimulus-responsive dis- and reassembly, while guest binding was found to be dependent on the complementary properties of the guest to the host in terms of charge, size and H bonding properties.

Cell-surface proteins known as adhesins enable bacteria to colonize particular environments, and in Gram-positive bacteria often contain autocatalytically formed covalent intramolecular cross-links. While investigating the prevalence of such cross-links, a remarkable example was discovered in Mobiluncus mulieris, a pathogen associated with bacterial vaginosis. This organism encodes a putative adhesin of 7651 residues. Crystallography and mass spectrometry of two selected domains, and AlphaFold structure prediction of the remainder of the protein, were used to show that this adhesin belongs to the family of thioester, isopeptide and ester-bond-containing proteins (TIE proteins). It has an N-terminal domain homologous to thioester adhesion domains, followed by 51 immunoglobulin (Ig)-like domains containing ester- or isopeptide-bond cross-links. The energetic cost to the M. mulieris bacterium in retaining such a large adhesin as a single gene or protein construct suggests a critical role in pathogenicity and/or persistence.

Group A Streptococcus (GAS) is a globally important pathogen causing a broad range of human diseases. GAS pili are elongated proteins with a backbone comprised repeating T-antigen subunits, which extend from the cell surface and have important roles in adhesion and establishing infection. No GAS vaccines are currently available, but T-antigen-based candidates are in pre-clinical development. This study investigated antibody-T-antigen interactions to gain molecular insight into functional antibody responses to GAS pili. Large, chimeric mouse/human Fab-phage libraries generated from mice vaccinated with the complete T18.1 pilus were screened against recombinant T18.1, a representative two-domain T-antigen. Of the two Fab identified for further characterization, one (designated E3) was cross-reactive and also recognized T3.2 and T13, while the other (H3) was type-specific reacting with only T18.1/T18.2 within a T-antigen panel representative of the major GAS T-types. The epitopes for the two Fab, determined by x-ray crystallography and peptide tiling, overlapped and mapped to the N-terminal region of the T18.1 N-domain. This region is predicted to be buried in the polymerized pilus by the C-domain of the next T-antigen subunit. However, flow cytometry and opsonophagocytic assays showed that these epitopes were accessible in the polymerized pilus at 37°C, though not at lower temperature. This suggests that there is motion within the pilus at physiological temperature, with structural analysis of a covalently linked T18.1 dimer indicating "knee-joint" like bending occurs between T-antigen subunits to expose this immunodominant region. This temperature dependent, mechanistic flexing provides new insight into how antibodies interact with T-antigens during infection.

Streptococcus pyogenes, a leading human pathogen, is responsible for a wide range of diseases, including skin and soft tissue infections and severe invasive diseases. S. pyogenes produces a large arsenal of virulence factors, including several immune evasion factors. We have identified an open reading frame (spy0136) in the S. pyogenes SF370 genome encoding a protein of unknown function. Using recombinant Spy0136 in a pull-down assay with human plasma and ELISA, we have identified four complement proteins (C1r, C1s, C3, and C5) as binding partners. Treatment of the complement proteins with PNGase F abrogated binding to C1s, C3, and C5, indicating glycan-dependent interactions. rSpy0136 inhibited complement-mediated hemolysis and interfered with all three complement pathways in a Wieslab complement assay. Furthermore, rSpy0136 inhibited deposition of the C3b opsonin and the membrane attack complex (MAC) on the surface of S. pyogenes. We therefore named the previously unknown protein ‘complement evasion factor’ (CEF).An S. pyogenes Δspy0136/cef deletion mutant showed decreased virulence in an in-vitro whole blood killing assay and a Galleria mellonella (wax moth) infection model. Furthermore, an L. lactis spy0136/cef gain-of-function mutant showed increased survival during growth in whole human blood. Analysis of serum samples from patients with invasive S. pyogenes revealed Spy0136/CEF sero-conversion indicating expression during disease. In summary, we have identified a novel S. pyogenes immune evasion factor that binds to several complement proteins to interfere with complement function. This is the first example of a S. pyogenes virulence factor binding to several different target proteins via glycan-dependent interactions.

Group A Streptococcus (GAS) is a major human pathogen responsible for superficial infections through to life-threatening invasive disease and the autoimmune sequelae acute rheumatic fever (ARF). Despite a significant global economic and health burden, there is no licensed vaccine available to prevent GAS disease. Several pre clinical vaccines that target conserved GAS antigens are in development. Assays that measure antigen-specific antibodies are essential for vaccine research. The aim of this study was to develop a multiplex beadbased immunoassay that can detect and quantify antibody responses to multiple GAS antigen targets in small volume blood samples. This builds on our existing triplex assay comprised of antigens used in clinical serology for the diagnosis of ARF (SLO, DNase B and SpnA). Five additional conserved putative GAS vaccine antigens (Spy0843, SCPA, SpyCEP, SpyAD and the Group A carbohydrate), were coupled to spectrally unique beads to form an 8-plex antigen panel. After optimisation of the assay protocol, standard curves were generated, and assessments of assay specificity, precision and reproducibility were conducted. A broad range of antibody (IgG) titres were able to be quickly and accurately quantified from a single serum dilution. Assay utility was assessed using a panel of 62 clinical samples including serum from adults with GAS bacteraemia and children with ARF. Circulating IgG to all eight antigens was elevated in patients with GAS disease (n = 23) compared to age-matched controls (n = 39) (P < 0.05). The feasibility of using dried blood samples to quantify antigen-specific IgG was also demonstrated. In summary, a robust and reproducible 8-plex assay has been developed that simultaneously quantifies IgG antibodies to GAS vaccine and diagnostic antigens.

Intramolecular isopeptide bonds, formed autocatalytically between Lys and Asn/Asp side chains, are widely present in the immunoglobulin-like domains of Gram-positive bacterial adhesins, including Group A Streptococcus, and confer considerable mechanical and chemical stability. These properties make them attractive for applications in biotechnology. Here, we detail the practical considerations that are involved in engineering isopeptide bonds into Ig-like proteins, including the choice of a site where bond-forming residues could be introduced and the appropriate methodology for mutagenesis. We specify how to determine whether an isopeptide bond has formed, what strategies can be adopted to overcome problems, and how to monitor the stability of the engineered protein.

Molecular superglues covalently ligate two or more macromolecules together into super stable, covalently linked assemblies. The discovery of intramolecular isopeptide and ester bond crosslinks in bacterial adhesin proteins, inspired the development of two distinct protein ligating technologies based on split protein domains. These chemically distinct technologies could be combined as orthogonal (non-cross-reacting) technologies to make complex assemblies. Here we provide simple practical instructions in the discovery, characterisation, and application of orthogonal ester bond crosslinks as molecular superglues. A large toolkit of diverse, orthogonal molecular superglues will expand our assembly repertoire, and afford increasingly more complex one-, two-, and three-dimensional protein nanomaterials with exquisite control over the final molecular architecture.

Group A Streptococcus (GAS) (Streptococcus pyogenes) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines.

Group A Streptococcus (GAS) is a globally important human pathogen that causes a broad spectrum of disease ranging from mild superficial infections to severe invasive diseases with high morbidity and mortality. Currently, there is no vaccine available for human use. GAS produces a vast array of virulence factors including multiple adhesin molecules. These mediate binding of the bacteria to host tissues and are essential in the initial phases of infection. Prophylactic vaccination with adhesins is a promising vaccine strategy and many GAS adhesins are currently in development as vaccine candidates. The most advanced candidates, having entered clinical trials, are based on the M protein, while components of the pilus and a number of fibronectin-binding proteins are in pre-clinical development. Adhesin-based vaccines aim to induce protective immunity via two main mechanisms: neutralisation where adhesin-specific antibodies block the ability of the adhesin to bind to host tissue and opsonisation in which adhesin-specific antibodies tag the GAS bacteria for phagocytosis. This review summarises our current knowledge of GAS adhesins and their structural features in the context of vaccine development.

The discovery of isopeptide bonds in the surface protein of the Gram-positive bacterium Streptococcus pyogenes changed this simplistic view (Kang et al. 2007). Isopeptide bonds are formed auto-catalytically between lysine and asparagine or an aspartate residues that are brought together in a hydrophobic environment during protein folding. More recently, new intramolecular crosslinks formed between threonine and glutamine side chains (ester bonds) were discovered in the surface protein Cpe0147 of another Gram-positive bacterium, Clostridium perfringens (Kwon et al. 2014).

The overall stability of globular protein structures is marginal, a balance between large numbers of stabilizing non-covalent interactions and a destabilizing entropic term. Higher stability can be engineered by introduction of disulfide bonds, provided the redox environment is controlled. The discovery of stabilizing isopeptide bond crosslinks, formed spontaneously between lysine and asparagine (or aspartic acid) side chains in certain bacterial cell-surface proteins suggests that such bonds could be introduced by protein engineering as an alternative protein stabilization strategy. We report the first example of an isopeptide bond engineered de novo into an immunoglobulin-like protein, the minor pilin FctB from Streptococcus pyogenes . Four mutations were sufficient; lysine, asparagine and glutamic acid residues were introduced for the bond-forming reaction, with a fourth Val/Phe mutation to help steer the lysine side chain into position. The spontaneously-formed isopeptide bond was confirmed by mass spectrometry and X-ray crystallography, and was shown to increase the thermal stability by 10 °C compared with the wild type protein. This novel method for increasing the stability of IgG-like proteins has potential to be adopted by the field of antibody engineering, which share similar β-clasp Ig-type domains.

The hydrolysis potential of ester bonds in covalently cross-linking proteins is captured in our novel protein ligation technology. This new type of "molecular superglue" based on the spontaneously-formed Thr-Gln ester bonds found in cell-surface adhesins, affords a unique mechanism to both rationally assemble and disassemble complex protein nanomaterials.

Staphylococcus aureus is an opportunistic pathogen that produces many virulence factors. Two major families of which are the staphylococcal superantigens (SAgs) and the Staphylococcal Superantigen-Like (SSL) exoproteins. The former are immunomodulatory toxins that induce a Vβ-specific activation of T cells, while the latter are immune evasion molecules that interfere with a wide range of innate immune defences. The superantigenic properties of Staphylococcal enterotoxin-like X (SElX) have recently been established. We now reveal that SElX also possesses functional characteristics of the SSLs. A region of SElX displays high homology to the sialyl-lactosamine (sLacNac)-specific binding site present in a sub-family of SSLs. By analysing the interaction of SElX with sLacNac-containing glycans we show that SElX has an equivalent specificity and host cell binding range to the SSLs. Mutation of key amino acids in this conserved region affects the ability of SElX to bind to cells of myeloid origin and significantly reduces its ability to protect S. aureus from destruction in a whole blood killing (WBK) assay. Like the SSLs, SElX is up-regulated early during infection and is under the control of the S. aureus exotoxin expression (Sae) two component gene regulatory system. Additionally, the structure of SElX in complex with the sLacNac-containing tetrasaccharide sialyl Lewis X (sLeX) reveals that SElX is a unique single-domain SAg. In summary, SElX is an 'SSL-like' SAg.

Gram-positive bacteria are decorated by many surface proteins including single molecule wide, elongated adhesins that form projections from the bacterial surface with "sticky" ends to provide adherence to host cells, other bacteria, and extracellular surfaces.The C. perfringens adhesin Cpe0147 contains 11 repeat Ig-like domains that form a long stalk that projects a "sticky" adhesin domain from the bacterium surface.Each of the repeat domains contains an intramolecular ester bond formed spontaneously between the side chains of threonine and a glutamine residues [1,2].These cross-links provide greatly enhanced mechanical, thermal, and often proteolytic stability to the entire adhesin.We have previously shown, using thermal stability experiments and circular dichroism, that single domain mutants unable to form cross-links, appear to be either unfolded or highly dynamic in solution.This poses a conundrum: how can these critical intramolecular ester bonds form if the protein domains are inherently unstable and unfolded until the bond is formed?Our working hypothesis is that while single domains are inherently unfolded or dynamic, when part of the full polymer, adjacent domains cooperatively induce folding and bond formation even though they are separated in space by ~20 Å [1].We are examining a combination of domain constructs using size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS), differential scanning calorimetry, nuclear magnetic resonance, small-angle X-ray scattering (Fig. 1), and X-ray crystallography experiments to understand the potential for cooperative folding in these elongated, multi-domain surface proteins that allow bacteria to exist and flourish in a harsh host environment.

Bacterial type I signal peptidases (SPases) are membrane-anchored serine proteases that process the signal peptides of proteins exported via the Sec and Tat secretion systems. Despite their crucial importance for bacterial virulence and their attractiveness as drug targets, only one such enzyme, LepB from Escherichia coli, has been structurally characterized, and the transient nature of peptide binding has stymied attempts to directly visualize SPase-substrate complexes. Here, the crystal structure of SpsB, the type I signal peptidase from the Gram-positive pathogen Staphylococcus aureus, is reported, and a peptide-tethering strategy that exploits the use of carrier-driven crystallization is described. This enabled the determination of the crystal structures of three SpsB-peptide complexes, both with cleavable substrates and with an inhibitory peptide. SpsB-peptide interactions in these complexes are almost exclusively limited to the canonical signal-peptide motif Ala-X-Ala, for which clear specificity pockets are found. Minimal contacts are made outside this core, with the variable side chains of the peptides accommodated in shallow grooves or exposed faces. These results illustrate how high fidelity is retained despite broad sequence diversity, in a process that is vital for cell survival.

Staphylococcus aureus infections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets. S. aureus has a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, the spsB gene from S. aureus Newman strain was cloned and overexpressed in Escherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space group P2(1) and diffracted to 2.05 Å resolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases.