Bacterial Transpeptidase Research Articles

Overexpression, purification and biochemical studies of Sortase A from Enterococcus faecalis (Ef) and its inhibition studies with Aloenin

Sortase A (SrtA) is a bacterial transpeptidase that garnishes the bacterial surface by adding various virulent factors or proteins by cleaving the LPXTG-specific motif between T and G amino acids. These virulence factors assist in the attachment of host cells, which are essential for bacterial virulence. Enterococcus species are among the multidrug-resistant bacteria that cause nosocomial infections; they have drawn a lot of attention recently. SrtA from E. faecalis (Ef) plays a critical role in pathogenesis, making it a suitable target for the development of antibacterial agents. Since SrtA is not involved in bacterial growth and is present on the surface of bacteria, the probability of developing antibiotic resistance is minimal. In this work, we have done cloning, expression and purification of Ef-SrtA using IMAC (Immobilised Metal Affinity Chromatography) followed by Gel filtration chromatography. Purified Ef-SrtA showed maximum activity at pH-8 and temperature between 45 and 55 °C. The fluorescent assay for kinetic studies of Ef-SrtA showed Vmax 3.852 µM.min−1 and kcat 7.7 × 10−2s−1 for the hydrolysis of substrate using Abz-LPETG-K(Dnp)-NH2. We have selected fifteen Aloe vera extracted compounds and performed virtual screening and docking experiments to identify potential inhibitors against Ef-SrtA. Among fifteen molecules, Aloenin-a which was bound to the active site with a binding energy of -6.1 kcal/mol, interacted with the active site residues, Arg139, Pro105, Leu39, Ala46, and Cys126. Aloenin-a showed a significant inhibitory effect against Ef-SrtA, with an IC50 value of 20.68 µM. Aloenin-a inhibits biofilm formation at concentrations of 20–250 µg/mL. The fibrinogen assay showed adherence to fibrinogen was reduced in the presence of Aloenin-a for E. faecalis. The results demonstrated that Aloe vera extracts containing Aloenin-a can be a significant antagonist of Ef-SrtA.

Evaluation of the Antibacterial Activities of Mangrove Honeybee Propolis Extract and the Identification of Transpeptidase and Transglycosylase as Targets for New Antibiotics Using Molecular Docking.

Developing new antibiotics is a critical area of research that grows as a result of the increasing problem of antibiotic resistance. Scientists search for new antibiotics by screening natural sources such as soil, plants, and marine environments. One of the iconic plants in the marine environment is the mangrove, which is a source of honeybee propolis. Propolis collected from the grey mangrove Avicennia marina on Tarout Island, the Eastern Province of Saudi Arabia, was used to evaluate antibacterial activities against three pathogenic bacteria: gram-negative Enterobacter cloacae (RCMB 001(1) ATCC® 23355TM), gram-positive methicillin-resistant Staphylococcus aureus (clinical isolate), and Streptococcus mutans Clark (RCMB 017(1) ATCC® 25175TM). The results indicate the effectiveness of the methanolic extract of such propolis. The chemical composition of this extract was analyzed using LC-MS, and four compounds were identified (alginic acid, carrageenan, fucoxanthin, cycloeudesmol). Their modes of action were evaluated against bacterial cell walls. Bacterial transpeptidase and transglycosylase on the surface are basic for cell divider amalgamation, and numerous antimicrobials have been created to target these compounds. Molecular docking was employed to predict the interactions of four compounds and S. aureus to predict interaction. Alginic acid was found to be the best interaction with a score of -7.44 Kcal/mol with distance ranges between 2.86 and 3.64 and RMSD refined below 2 Å. Carrageenan with -6.64 Kcal/mol and a distance of 3.05 and 2.87 came second. Then, fucoxanthin with -6.57 Kcal/mol and a distance of 1.4. Finally, cycloeudesmol with a score of -4.6 Kcal/mol and a distance of 2.87 showed the least activity. The first three compounds interacted effectively and could form very promising chemicals that could be used one day against pathogenic bacteria in the future.

Sortase E-mediated site-specific immobilization of green fluorescent protein and xylose dehydrogenase on gold nanoparticles

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV–vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

Substrate-derived Sortase A inhibitors: targeting an essential virulence factor of Gram-positive pathogenic bacteria.

The bacterial transpeptidase Sortase A (SrtA) is a surface enzyme of Gram-positive pathogenic bacteria. It has been shown to be an essential virulence factor for the establishment of various bacterial infections, including septic arthritis. However, the development of potent Sortase A inhibitors remains an unmet challenge. Sortase A relies on a five amino acid sorting signal (LPXTG), by which it recognizes its natural target. We report the synthesis of a series of peptidomimetic inhibitors of Sortase A based on the sorting signal, supported by computational binding analysis. By employing a FRET-compatible substrate, our inhibitors were assayed in vitro. Among our panel, we identified several promising inhibitors with IC50 values below 200 μM, with our strongest inhibitor - LPRDSar - having an IC50 of 18.9 μM. Furthermore, it was discovered that three of our compounds show an effect on growth and biofilm inhibition of pathogenic Staphylococcus aureus, with the inclusion of a phenyl ring seemingly key to this effect. The most promising compound in our panel, BzLPRDSar, could inhibit biofilm formation at concentrations as low as 32 μg mL-1, manifesting it as a potential future drug lead. This could lead to treatments for MRSA infections in clinics and diseases such as septic arthritis, which has been directly linked with SrtA.

The l,d-Transpeptidase LdtAb from Acinetobacter baumannii Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture.

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of β-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of LdtAb from the clinically important pathogen, Acinetobacter baumannii. We show that A.baumannii survives inactivation of LdtAb alone or in combination with PBP1b or PBP2, while simultaneous inactivation of LdtAb and PBP1a is lethal. Minimal inhibitory concentrations (MICs) of all 13 β-lactam antibiotics tested decreased 2- to 8-fold for the LdtAb deletion mutant, while further decreases were seen for both double mutants, with the largest, synergistic effect observed for the LdtAb + PBP2 deletion mutant. Mass spectrometry experiments showed that LdtAb forms complexes in vitro only with carbapenems. However, the acylation rate of these antibiotics is very slow, with the reaction taking longer than four hours to complete. Our X-ray crystallographic studies revealed that LdtAb has a unique structural architecture and is the only known LDT to have two different peptidoglycan-binding domains.

Enzyme- and UV-Mediated Double-Network Hybrid Hydrogels for 3D Cell Culture application.

Three-dimensional (3D) cell culture using hydrogel scaffolds can closely resemble the natural extracellular matrix (ECM), which offers appropriate mechanical support for cells and regulates cellular behavior. In this study, a bacterial transpeptidase sortase A (SA) is used to prepare enzymatically cross-linked methacrylated hyaluronic acid (HA) peptides (HAMA-P) hydrogel, which reveals fast gel kinetics under high SA cross-linking concentrations and can be used as an injection hydrogel for tissue repair or extrusive 3D bioprinting. Furthermore, methacrylated gelatin (GelMA) is introduced to build the hybrid hydrogel (HAMA-P-GelMA) with double cross-linking of enzyme-UV, which has shown proper physical properties (mechanical properties, swelling, degradation rate, etc.) of the hydrogel matrix, and displayed desirable effects on cell viability, adhesion, and cell spreading, when compared to GelMA or HAMA-P single-network hydrogels. The HAMA-P-GelMA hybrid hydrogels provide a favorable 3D milieu for cell growth and can be used as a 3D bio-ink or a carrier of stem cells/cytokines for injectable tissue repair and filling.

A Review on Ceftazidime and Avibactum

Ceftazidime is a semi synthetic, broad-spectrum, beta-lactam, third-generation cephalosporin antibiotic useful for the treatment of many bacterial infections like joint infections, meningitis, pneumonia, sepsis, urinary tract infections, malignant otitis externa, and vibrio infection. It is synthesized chemically using ethyl acetoacetate, sodium nitrite and hydrochloric acid. The Structural analogues of Ceftazidime are better in terms of activity, potency and stability. Cefmenoxime has increased stability to beta-lactamases, the syn isomer of Ceftizoxime is more potent, Moxalactam has enhanced activity against Pseudomonas, Ceftriaxone has higher serum peak level and a prolonged half-life. Ceftazidime acts by inhibiting bacterial transpeptidases and preventing the synthesis of bacterial cell wall. It is assayed by Infrared spectroscopy. Stability studies showed that the drug degrades extensively on reconstitution in aqueous solution on exposures to heating at 45ºC and UV/Visible radiation, while the drug in solid state is stable. The drug is identified by carbonate or sodium tests and tested for its pH, clarity of solution, loss on drying and bacterial endotoxins. In 2015, USFDA approved a combination of Ceftazidime with Avibactam, a non-β-lactam β-lactamase inhibitor which protects ceftazidime from hydrolysis by a wide range of serine β-lactamases and expands its spectrum of activity to Enterobacteriaceae resistant to ceftazidime, and is used for the treatment of complicated urinary tract infections and intra-abdominal infections. Five new ceftazidime derivatives have improved acid stability and increased spectrum of ceftazidime. All the Schiff bases showed good antimicrobial activity especially against G(+) bacteria. Compounds 2 and 3 showed broader antibacterial spectrum against both G(+) and G(-) bacteria.

Abstract PO007: Tumor-immune communication via extracellular vesicles

Abstract Emerging evidence suggests that tumor-derived extracellular vesicles (tEVs) can influence immune cell behavior both locally within the tumor microenvironment and remotely by accessing the lymphatic and systemic circulation. The in vivo effects of such interactions before and after immunotherapy remain largely unappreciated. In this study, we aimed to develop a sensitive but stringent approach to identify host cells that interact with tEVs, with the ultimate goal of studying how tEV influence immune cells in their native microenvironment. Investigation of tEVs’ roles in cancer has been hampered by the need to isolate them before intravenous reinfusion in animals, which can introduce biases such as homogenization of tEV diversity, judgement of amount to reinfuse and assumptions on blood vs lymph biodistribution. To circumvent these issues, we and others pioneered the use of genetically engineered tumor cells to express membrane-bound reporters, which allow to label untouched, endogenously released tEVs and follow their interactions with immune cells. Still, the amount of reporter proteins that tEVs can carry is relatively small and thus the sensitivity of the approach is suboptimal. Here we addressed this issue by engineering tEVs to display a membrane-bound form of Sortase A, a bacterial transpeptidase that catalyzes the transfer of reporter proteins on the much bigger surface of tEV-binding cells. SrtA catalyzes the formation of a peptide bond between a consensus peptide and an N-terminal glycine of a nearby cell surface proteins (e.g. MHC-I, MHC-II, VE-Cadherin, CD19, integrins). We tested 4 different SrtA protein designs and selected the best performing construct for tEV studies. We genetically encoded SrtA in parental tumor cell lines, which then release SrtA as a tEV-bound transmembrane protein. Once a SrtA+ tEV interacts with a target cell in the presence of the consensus peptide (conjugated to a fluorescent reporter), SrtA catalyzes the formation of a covalent bond between the reporter and surface proteins of the tEV-binding cell. As expected, we observed significant increase in labeling when target cells were engineered to express a surface protein carrying 5 N-terminal glycine residues, which confirms that labeling of tEV-binding cells is due to actual transfer of fluorescent reporter – and not due to acyl intermediate formation. As compared to indirect labeling of EV-binding cells (e.g. using the EV marker CD63 fused to GFP), SrtA-based approach shows 1-2 log increase in sensitivity, depending on the EV-producing cell type. Overall, the catalytic nature of our EV reporter system lowers the amount of tEVs a host cell needs to bind before being detectable, thereby increasing sensitivity. We are currently testing our SrtA-based approach in vivo, where endogenous tEVs mainly accumulate in sentinel lymph nodes. We expect to unearth the full set of lymph node immune cells interacting with native tEVs and to identify whether immunecheckpoint blockade therapy affects tEV tropism toward immune cells. Citation Format: Ferdinando Pucci, Nicklas Hamilton, Natalie Claudio, Randall Armstrong. Tumor-immune communication via extracellular vesicles [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO007.

Laboratory evolution of a sortase enzyme that modifies amyloid-β protein.

Epitope-specific enzymes are powerful tools for site-specific protein modification, but generally require genetic manipulation of the target protein. Here, we describe the laboratory evolution of the bacterial transpeptidase sortase A to recognize the LMVGG sequence in endogenous Aβ protein. Using a yeast display selection for covalent bond formation, we evolved a sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved sortase to label endogenous Aβ in human cerebrospinal fluid, enabling detection of Aβ with sensitivities rivaling those of commercial assays. The evolved sortase can conjugate a hydrophilic peptide to Aβ42, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes towards endogenous targets as a strategy for site-specific protein modification without target gene manipulation, and enable potential future applications of sortase-mediated labeling of Aβ peptides.

1,2,4-Oxadiazole topsentin analogs as staphylococcal biofilm inhibitors targeting the bacterial transpeptidase sortase A.

The inhibition or prevention of biofilm formation represents an emerging strategy in the war against antibiotic resistance, interfering with key players in bacterial virulence. This approach includes the inhibition of the catalytic activity of transpeptidase sortase A (Srt A), a membrane enzyme responsible for covalently attaching a wide variety of adhesive matrix molecules to the peptidoglycan cell wall in Gram-positive strains. A new series of seventeen 1,2,4-oxadiazole derivatives was efficiently synthesized and screened as potential new anti-virulence agents. The ability of inhibiting biofilm formation was evaluated against both Gram-positive and Gram-negative pathogens. Remarkably, all these compounds inhibited S.aureus and/or P.aeruginosa biofilm formation in a dose dependent manner, with 50% biofilm inhibitory concentrations (BIC50s) below 10μM for the most active compounds. Inhibition of SrtA was validated as one of the possible mechanisms of action of these new 1,2,4-oxadiazole derivatives, in the tested Gram-positive pathogen, using a specific enzymatic assay for a recombinant S.aureus SrtA. The three most active compounds, eliciting BIC50 values for S.aureus ATCC 25923 between 0.7 and 9.7μM, showed a good activity toward the enzyme eliciting IC50 values ranging from 2.2 to 10.4μM.

Cell Surface Labeling by Engineered Extracellular Vesicles.

Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically engineering membrane-bound reporters into parental cells. Still, the amount of reporter molecules that EVs can carry is relatively small, and thus, the sensitivity of the approach is suboptimal. This work addresses this issue by engineering EVs to display a membrane-bound form of Sortase A (SrtA), a bacterial transpeptidase that can catalyze the transfer of reporter molecules on the much bigger surface of EV-binding cells. SrtA design and reaction requirements are optimized and validated. Efficient in vitro labeling of EV-binding cells is achieved, even in the presence of only one N-terminal glycine on cell surface proteins. As compared to indirect labeling of EV-binding cells (e.g., using CD63-GFP fusion), the SrtA-based approach shows 1-2 log increase in sensitivity, depending on the EV source. This novel approach will be useful to identify and study the full set of host cells interacting with native EVs in vivo.

D-Amino Acid Derivatives as in Situ Probes for Visualizing Bacterial Peptidoglycan Biosynthesis.

The bacterial cell wall is composed of membrane layers and a rigid yet flexible scaffold called peptidoglycan (PG). PG provides mechanical strength to enable bacteria to resist damage from the environment and lysis due to high internal turgor. PG also has a critical role in dictating bacterial cell morphology. The essential nature of PG for bacterial propagation, as well as its value as an antibiotic target, has led to renewed interest in the study of peptidoglycan biosynthesis. However, significant knowledge gaps remain that must be addressed before a clear understanding of peptidoglycan synthesis and dynamics is realized. For example, the enzymes involved in the PG biosynthesis pathway have not been fully characterized. Our understanding of PG biosynthesis has been frequently revamped by the discovery of novel enzymes or newly characterized functions of known enzymes. In addition, we do not clearly know how the respective activities of these enzymes are coordinated with each other and how they control the spatial and temporal dynamics of PG synthesis. The emergence of molecular probes and imaging techniques has significantly advanced the study PG synthesis and modification. Prior efforts utilized the specificity of PG-targeting antibiotics and proteins to develop PG-specific probes, such as fluorescent vancomycin and fluorescent wheat germ agglutinin. However, these probes suffer from limitations due to toxic effects toward bacterial cells and poor membrane permeability. To address these issues, we designed and introduced a family of novel molecular probes, fluorescent d-amino acids (FDAAs), which are covalently incorporated into PG through the activities of endogenous bacterial transpeptidases. Their high biocompatibility and PG specificity have made them powerful tools for labeling peptidoglycan. In addition, their enzyme-mediated incorporation faithfully reflects the activity of PG synthases, providing a direct in situ method for studying PG formation during the bacterial life cycle. In this Account, we describe our efforts directed at the development of FDAAs and their derivatives. These probes have enabled for the first time the ability to visualize PG synthesis in live bacterial cells and in real time. We summarize experimental evidence for FDAA incorporation into PG and the enzyme-mediated incorporation pathway. We demonstrate various applications of FDAAs, including bacterial morphology analyses, PG growth model studies, investigation of PG-enzyme correlation, in vitro PG synthase activity assays, and antibiotic inhibition tests. Finally, we discuss the current limitations of the probes and our ongoing efforts to improve them. We are confident that these probes will prove to be valuable tools that will enable the discovery of new antibiotic targets and expand the available arsenal directed at the public health threat posed by antibiotic resistance.

Overcoming the Limitations of Sortase with Proximity-Based Sortase-Mediated Ligation (PBSL).

S. aureus sortase A (SrtA), a calcium-dependent bacterial transpeptidase, is commonly used to site-specifically label proteins containing a LPXTG SrtA recognition motif with a wide array of chemical moieties. A major limitation of sortase-mediated labeling, however, is SrtA's poor binding affinity to its recognition motif, resulting in long reaction times and poor ligation efficiencies. Here we describe proximity-based sortase-mediated ligation (PBSL), which utilizes the SpyTag-SpyCatcher peptide-protein pair to tether target proteins with a SrtA recognition motif to SrtA, dramatically increasing their local concentrations and overcoming this limitation.

Engineering Antibodies with C-Terminal Sortase-Mediated Modification for Targeted Nanomedicine.

The current advances in nanoengineered materials coupled with the precise targeting capability of recombinant antibodies can create nanoscale diagnostics and therapeutics which show enhanced accumulation and extended retention at a target tissue. Smaller antibodies such as single-chain variable fragments (scFv) preserve the selective and strong binding of their parent antibody to their antigen with the benefits of low immunogenicity, more efficient tissue penetration and easy introduction of functional residues suitable for site-specific conjugation. This is of high importance as nonspecific antibody modification often involves attachment to free cysteine or lysine amino acids which may reside in the active site, leading to reduced antigen binding.In this chapter, we outline a facile and versatile chemoenzymatic approach for production of targeted nanocarrier scFv conjugates using the bacterial trans-peptidase Sortase A (Srt A). Srt A efficiently mediates sequence-specific peptide ligation under mild conditions and has few undesirable side reactions. We first describe the production, purification and characterization of Srt A enzyme and a scFv construct which targets activated platelets, called scFvanti-GPIIb/IIIa. Following this, our protocol illustrates the chemoenzymatic modification of the antibody at the C-terminus with an orthogonal click chemistry linker. This avoids any random attachment to the biologically active antigen binding site of the antibody. Finally, we describe the modification of a nanoparticle surface with scFv attachment via two methods: (1) direct Sortase-mediated conjugation; or (2) a two-step system which consists of scFv Sortase-mediated conjugation followed by strain promoted azide-alkyne cycloaddition. Finally, methodology is described to assess the successful assembly of targeted particles.

CRISPR/Cas9-Mediated Genetic Engineering of Hybridomas for Creation of Antibodies that Allow for Site-Specific Conjugation.

Covalent conjugation of chemical moieties to antibodies has numerous applications, including antibody-drug conjugates, antibody conjugation for diagnostics, and more. Most nonspecific chemical conjugation methods ligate onto any of a number of sites on the antibody, leading to multiple conjugated species, many of which perturb antibody function. To solve these problems, we used CRISPR/Cas9-edited hybridomas to introduce a Sortase tag (LPXTG) and a Flag tag at the 3' end of the CH3 heavy chain region of a mouse monoclonal antibody. The Flag tag allows easy purification of the antibody, while the LPXTG is then acted on by the bacterial transpeptidase Sortase to site-specifically add on any of a number of chemical moieties that possess a triglycine repeat. This technique thus allows rapid production of an antibody onto which a wide array of chemical moieties can be site-specifically conjugated.

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting.

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

A versatile papaya mosaic virus (PapMV) vaccine platform based on sortase-mediated antigen coupling

BackgroundFlexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles—a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated.ResultsWe have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection.ConclusionsThis technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines.

Proximity‐Based Sortase‐Mediated Ligation

AbstractProtein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site‐specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity‐based sortase‐mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein to SrtA using the SpyTag–SpyCatcher peptide–protein pair. By expressing the target protein with SpyTag C‐terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher–SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.

Proximity-Based Sortase-Mediated Ligation.

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site-specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity-based sortase-mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein to SrtA using the SpyTag-SpyCatcher peptide-protein pair. By expressing the target protein with SpyTag C-terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher-SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.

Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide.

Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide κ-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native κ-PVIIA, cyclic κ-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated κ-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of κ-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules. Biotechnol. Bioeng. 2016;113: 2202-2212. © 2016 Wiley Periodicals, Inc.