Sortase Enzymes Research Articles

Protocol for sortase-mediated construction of DNA–protein hybrids and functional nanostructures

Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA–protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA–peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA–protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein–DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA–protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes.

Virtual screening of LPXTG competitive SrtA inhibitors targeting signal transduction mechanism in Bacillus anthracis: a combined experimental and theoretical study

Members of the sortase enzyme super family decorate the surfaces of Bacillus anthracis cell wall with proteins that play key roles in microbial pathogenesis and its biofilm formation. Bacillus anthracis Sortase-A (Ba-SrtA) is a potential target for new therapeutics as it is required for B. anthracis survival and replication within macrophages. An understanding of the binding site pocket and substrate recognition mechanism by SrtA enzymes may serve to be beneficial in the rational development of sortase inhibitors. Here, the LPXTG signal peptide-based competitive inhibitors are screened against the Ba-SrtA and compounds with reasonable inhibition, specificity, and mechanisms of inactivation of SrtA have been covered. The screened compounds are experimentally validated against the phylogenetically similar Gram-positive pathogen B. cereus. In situ microscopic visualizations suggest that these screened compounds showed the microbial and biofilm inhibitory activity against B. cereus. It facilitates the further development of these molecules into useful anti-infective agents to treat infections caused by B. anthracis and other Gram-positive pathogens. These results provide insight into basic design principles for generating new clinically relevant lead molecules. It also provides an alternative strategy where a screened ligand molecule can be used in combination to battle increasingly against the Gram-positive pathogens.

Extracellular Nucleotide Catabolism by the Group B Streptococcus Ectonucleotidase NudP Increases Bacterial Survival in Blood

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

Force Spectroscopy of Tip Link Proteins: A Study of Inner-Ear Biophysics

Hair cells in the inner ear convert mechanical stimuli, in the vestibular and auditory systems, into electrical signals which can be processed by the brain. Hair cells are highly polarized with a unique elaboration of modified microvilli at their apical surface known as stereo cilia. Sensory stimuli such as sound waves of a particular frequency will result in oscillations of the stereociliary bundle at that frequency. Movements of this bundle couple mechanically to the channel. This force acts to gate the channel in turn resulting in a mechano-electrical transduction. The force is relayed to the channel by filaments known as tip links. Tip links are made of two atypical cadherins, protocadherin-15 and cadherin-23 at the lower and upper ends respectively. The tip link is held together by a non-covalent interaction between two anti-parallel pairs of EC domains. Although much has been discerned about the biophysics of this system the lack of adequate technology has prevented the direct measurement of many of these properties. We have developed molecular tools to facilitate the single molecule study of these properties. Self-assembled DNA nanoswitches are functionalized with protocadherin 15 and cadherin 23 fragments using the enzyme sortase. In order to preserve protein function, protein-DNA coupling is performed under physiological conditions. In this two-step process, a small synthetic peptide is first coupled to a DNA oligo. Next, utilizing the enzyme sortase, the protein is coupled to the DNA-peptide chimera under physiological conditions. This strategy frontloads all of the protein-incompatible chemistry so that it is performed on an oligo and a synthetic peptide, which are far more tolerant of non-physiological conditions. Once assembled, the nanoswitches are then used in an optical tweezer system to measure the rupture forces of the tip link under different conditions.

Computational Studies of the Catalytic Mechanism of the Staphylococcus Aureus Sortase a Enzyme

When surface proteins are attached to the cell wall of Gram-positive bacteria, they play important roles in multiple pathogens such as pneumonia, meningitis, osteomyelitis, and so on. Class A sortase (SrtA) enzymes play a key role in the insertion of surface proteins into the cell wall. StrA recognizes an LPXTG sorting signal motif in a target protein and catalyzes a transpeptidation reaction that joins it to a Lipid II molecules that will be embedded into the cell wall. Bacteria strains that lack SrtA are incapable of placing surface proteins, suggesting that SrtA may be a novel drug target for therapeutics against Gram-positive bacteria. However, the precise mechanism of transpeptidation is not well understood because of the unstable catalytic intermediates. Here we report on a series of molecular dynamics simulations using a hybrid quantum mechanical/molecular mechanics (QM/MM) approach to investigate the catalytic mechanism of SrtA.

Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.

Focal targeting by human β-defensin 2 disrupts localized virulence factor assembly sites in Enterococcus faecalis

Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human β-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.

Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step.

Efficient labeling of protein-based targeting ligands with various cargos (drugs, imaging agents, nanoparticles, etc.) is essential to the fields of molecular imaging and targeted therapeutics. Many common bioconjugation techniques, however, are inefficient, nonstoichiometric, not site-specific, and/or incompatible with certain classes of protein scaffolds. Additionally, these techniques can result in a mixture of conjugated and unconjugated products, which are often difficult to separate. In this study, a bacterial sortase enzyme was utilized to condense targeting ligand purification and site-specific conjugation at the C-terminus into a single step. A model was produced to determine optimal reaction conditions for high conjugate purity and efficient utilization of cargo. As proof-of-principle, the sortase-tag expressed protein ligation (STEPL) technique was used to generate tumor-specific affinity ligands with fluorescent labels and/or azide modifications at high purity (>95%) such that it was not necessary to remove unconjugated impurities. Click chemistry was then used for the highly efficient and site-specific attachment of the azide-modified targeting ligands onto nanoparticles.

Role of sortase in Streptococcus mutans under the effect of nicotine

Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen I/II. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/II-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased (P≤0.05) biofilm metabolic activity (2- to 3-fold increase) as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation (2- to 3-fold increase) as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen I/II was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.

Receptor-Directed Chimeric Toxins Created by Sortase-Mediated Protein Fusion

Chimeric protein toxins that act selectively on cells expressing a designated receptor may serve as investigational probes and/or antitumor agents. Here, we report use of the enzyme sortase A (SrtA) to create four chimeric toxins designed to selectively kill cells bearing the tumor marker HER2. We first expressed and purified: (i) a receptor recognition-deficient form of diphtheria toxin that lacks its receptor-binding domain and (ii) a mutated, receptor-binding-deficient form of anthrax-protective antigen. Both proteins carried at the C terminus the sortase recognition sequence LPETGG and a H₆ affinity tag. Each toxin protein was mixed with SrtA plus either of two HER2-recognition proteins--a single-chain antibody fragment or an Affibody--both carrying an N-terminal G₅ tag. With wild-type SrtA, the fusion reaction between the toxin and receptor-recognition proteins approached completion only after several hours, whereas with an evolved form of the enzyme, SrtA*, the reaction was virtually complete within 5 minutes. The four fusion toxins were purified and shown to kill HER2-positive cells in culture with high specificity. Sortase-mediated ligation of binary combinations of diverse natively folded proteins offers a facile way to produce large sets of chimeric proteins for research and medicine.

Site-specific N-terminal labeling of proteins using sortase-mediated reactions

This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.

Sortase A as a Tool to Functionalize Surfaces

A widely accepted approach to combat surface fouling is based on the prevention of biofoulants to attach to a surface by the functionalization with poly(ethylene glycol) (PEG). The goal of this study was to generate a proof of concept for the enzymatic coupling of PEG to a peptide precoated surface by using the enzyme Sortase A (SrtA). A hydrophobic polystyrene surface was primed with anchoring peptide P3 equipped with a pentaglycine acceptor motif for SrtA, to enable subsequent transpeptidation with either biotin or a PEG-tail containing the sortase recognition motif LPETG. High levels of surface-bound biotin were detected only in cases with biotin-LPETG and SrtA. Little if any reactivity was detected in wells treated with the SrtA scrambled motif EGLTP, or in the absence of SrtA. Conjugation of PEG resulted in a significant decrease of bacterial adherence to the surface.

Relevance and application of sortase and sortase-dependent proteins in lactic acid bacteria

Lactic acid bacteria (LAB) are a diverse group of Gram-positive bacteria found in a vast array of environments including dairy products and the human gastrointestinal tract (GIT). In both niches, surface proteins play a crucial role in mediating interactions with the surrounding environment. The sortase enzyme is responsible for covalently coupling a subset of sortase-dependent proteins (SDPs) to the cell wall of Gram-positive organisms through recognition of a conserved C-terminal LPXTG motif. Genomic sequencing of LAB and annotation has allowed for the identification of sortase and SDPs. Historically, sortase and SDPs were predominately investigated for their role in mediating pathogenesis. Identification of these proteins in LAB has shed light on their important roles in mediating nutrient acquisition through proteinase P as well as positive probiotic attributes including adhesion, mucus barrier function, and immune signaling. Furthermore, sortase expression signals in LAB have been exploited as a means to develop oral vaccines targeted to the GIT. In this review, we examine the collection of studies which evaluate sortase and SDPs in select species of dairy-associated and health promoting LAB.

The binding mechanism, multiple binding modes, and allosteric regulation of Staphylococcus aureus Sortase A probed by molecular dynamics simulations

Sortase enzymes are vitally important for the virulence of gram-positive bacteria as they play a key role in the attachment of surface proteins to the cell wall. These enzymes recognize a specific sorting sequence in proteins destined to be displayed on the surface of the bacteria and catalyze the transpeptidation reaction that links it to a cell wall precursor molecule. Because of their role in establishing pathogenicity, and in light of the recent rise of antibiotic-resistant bacterial strains, sortase enzymes are novel drug targets. Here, we present a study of the prototypical sortase protein Staphylococcus aureus Sortase A (SrtA). Both conventional and accelerated molecular dynamics simulations of S. aureus SrtA in its apo state and when bound to an LPATG sorting signal (SS) were performed. Results support a binding mechanism that may be characterized as conformational selection followed by induced fit. Additionally, the SS was found to adopt multiple metastable states, thus resolving discrepancies between binding conformations in previously reported experimental structures. Finally, correlation analysis reveals that the SS actively affects allosteric pathways throughout the protein that connect the first and the second substrate binding sites, which are proposed to be located on opposing faces of the protein. Overall, these calculations shed new light on the role of dynamics in the binding mechanism and function of sortase enzymes.

Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity

Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded β-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.

Beyond conventional antibiotics for the future treatment of methicillin-resistantStaphylococcus aureusinfections: two novel alternatives

The majority of antibiotics currently used to treat methicillin-resistant Staphylococus aureus (MRSA) infections target bacterial cell wall synthesis or protein synthesis. Only daptomycin has a novel mode of action. Reliance on limited targets for MRSA chemotherapy, has contributed to antimicrobial resistance. Two alternative approaches to the treatment of S.aureus infection, particularly those caused by MRSA, that have alternative mechanisms of action and that address the challenge of antimicrobial resistance are cationic host defence peptides and agents that target S.aureus virulence. Cationic host defence peptides have multiple mechanisms of action and are less likely than conventional agents to select resistant mutants. They are amenable to modifications that improve their stability, effectiveness and selectivity. Some cationic defence peptides such as bactenecin, mucroporin and imcroporin have potent in vitro bactericidal activity against MRSA. Antipathogenic agents also have potential to limit the pathogenesis of Saureus. These are generally small molecules that inhibit virulence targets in S.aureus without killing the bacterium and therefore have limited capacity to promote resistance development. Potential antipathogenic targets include the sortase enzyme system, the accessory gene regulator (agr) and the carotenoid biosynthetic pathway. Inhibitors of these targets have been identified and these may have potential for further development.

Bio‐Click Chemistry: Enzymatic Functionalization of PEGylated Capsules for Targeting Applications

All sorted: The enzyme Sortase A was used to catalyze functionalization of PEGylated capsules with an activation-specific anti-platelet single-chain antibody (scFv). This enzymatic method allows fast, covalent, and site-directed functionalization of delivery vehicles under mild conditions. Activation-specific anti-platelet scFv-coated PEGylated capsules exhibited a high level of selective binding to thrombi, thus suggesting their potential for thrombosis therapy.

Protein secretion and surface display in Gram-positive bacteria

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions.

Cellular aspects of the distinct M protein and SfbI anchoring pathways in Streptococcus pyogenes

Wall-anchored surface proteins are critical for the in vivo survival of Streptococcus pyogenes. Cues in the signal sequence direct the membrane translocation of surface proteins: M protein to the septum, and SfbI to the poles. Both proteins are subsequently anchored to the wall by the membrane bound enzyme sortase A. However, the cellular features of these pathways are not fully understood. Here we show that M protein and SfbI are anchored simultaneously throughout the cell cycle. M protein is rapidly anchored at the septum, and in part of the cell cycle, is anchored simultaneously at the mother and daughter septa. Conversely, SfbI accumulates gradually on peripheral peptidoglycan, resulting in a polar distribution. Sortase is not required for translocation of M protein or SfbI at their respective locations. Methicillin-induced unbalanced peptidoglycan synthesis diminishes surface M protein but not SfbI. Furthermore, overexpression of the division regulator DivIVA also diminishes surface M protein but increases SfbI. These results demonstrate a close connection between the regulation of cell division and protein anchoring. Better understanding of the spatial regulation of surface anchoring may lead to the identification of novel targets for the development of anti-infective agents, given the importance of surface molecules for pathogenesis.

Aerial development in Streptomyces coelicolor requires sortase activity

Streptomyces coelicolor is a multicellular bacterium whose life cycle encompasses three differentiated states: vegetative hyphae, aerial hyphae and spores. Among the factors required for aerial development are the 'chaplins', a family of eight secreted proteins that coat the surface of aerial hyphae. Three chaplins (the 'long' chaplins, ChpA, B and C) possess an LAXTG-containing C-terminal sorting signal and are predicted sortase substrates. The five remaining 'short' chaplins are presumed to be associated with the cell surface through interactions with the long chaplins. We show here that two sortase enzymes, SrtE1 and SrtE2, cleave LAXTG-containing peptides at two distinct positions in vitro, and are required for cell wall anchoring of ChpC in vivo. srtE1/E2 double mutants are delayed in aerial hyphae formation, do not sporulate and fail to display all short chaplins on their aerial surfaces. Surprisingly, these mutant characteristics were not shared by a long chaplin mutant, which exhibited only modest delays in aerial development, leading us to revise the current model of chaplin-mediated aerial development. The sortase mutant phenotype, instead, appears to stem from an inability to transcribe aerial hyphae-specific genes, whose products have diverse functions. This suggests that sortase activity triggers an important, and previously unknown, developmental checkpoint.