Cysteine Transpeptidases Research Articles

Halenaquinol Blocks Staphylococcal Protein A Anchoring on Cell Wall Surface by Inhibiting Sortase A in Staphylococcus aureus.

Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal protein A (SpA), to the peptidoglycan cell wall. Six pentacyclic polyketides (1-6) were isolated from the marine sponge Xestospongia sp., and their structures were elucidated using spectroscopic techniques and by comparing them to previously reported data. Among them, halenaquinol (2) was found to be the most potent SrtA inhibitor, with an IC50 of 13.94 μM (4.66 μg/mL). Semi-quantitative reverse transcription PCR data suggest that halenaquinol does not inhibit the transcription of srtA and spA, while Western blot analysis and immunofluorescence microscopy images suggest that it blocks the cell wall surface anchoring of SpA by inhibiting the activity of SrtA. The onset and magnitude of the inhibition of SpA anchoring on the cell wall surface in S. aureus that has been treated with halenaquinol at a value 8× that of the IC50 of SrtA are comparable to those for an srtA-deletion mutant. These findings contribute to the understanding of the mechanism by which marine-derived pentacyclic polyketides inhibit SrtA, highlighting their potential as anti-infective agents targeting S. aureus virulence.

Structure-Guided Design, Synthesis, and Antivirulence Assessment of Covalent Staphylococcus aureus Sortase A Inhibitors.

Sortase A (SrtA) is a membrane-associated cysteine transpeptidase required for bacterial virulence regulation and anchors surface proteins to cell wall, thereby assisting biofilm formation. SrtA is targeted in antivirulence treatments against Gram-positive bacterial infections. However, the development of potent small-molecule SrtA inhibitors is constrained owing to the limited understanding of the mode of action of inhibitors in the SrtA binding pocket. Herein, we designed and synthesized a novel class of covalent SrtA inhibitors based on the binding mode detailed in the X-ray crystal structure of the ML346/Streptococcus pyogenes SrtA complex. ML346 analog Y40 exhibited 2-fold increased inhibitory activity on Staphylococcus aureus SrtA and showed superior inhibitory effects on biofilm formation in vitro. Y40 protected Galleria mellonella larvae fromS. aureusinfections in vivo while minimally attenuating staphylococcal growth in vitro. Our study indicates that the covalent SrtA inhibitor Y40 is an antivirulence agent that is effective againstS. aureusinfections.

A unique binding mode of P1' Leu-containing target sequences for Streptococcus pyogenes sortase A results in alternative cleavage.

Sortase enzymes are cysteine transpeptidases that attach environmental sensors, toxins, and other proteins to the cell surface in Gram-positive bacteria. The recognition motif for many sortases is the cell wall sorting signal (CWSS), LPXTG, where X = any amino acid. Recent work from ourselves and others has described recognition of additional amino acids at a number of positions in the CWSS, specifically at the Thr (or P1) and Gly (or P1') positions. In addition, although standard cleavage occurs between these two residues (P1/P1'), we previously observed that the SrtA enzyme from Streptococcus pneumoniae will cleave after the P1' position when its identity is a Leu or Phe. The stereochemical basis of this alternative cleavage is not known, although homologs, e.g., SrtA from Listeria monocytogenes or Staphylococcus aureus do not show alternative cleavage to a significant extent. Here, we use protein biochemistry, structural biology, and computational biochemistry to predict an alternative binding mode that facilitates alternative cleavage. We use Streptococcus pyogenes SrtA (spySrtA) as our model enzyme, first confirming that it shows similar standard/alternative cleavage ratios for LPATL, LPATF, and LPATY sequences. Molecular dynamics simulations suggest that when P1' is Leu, this amino acid binds in the canonical S1 pocket, pushing the P1 Thr towards solvent. The P4 Leu (L̲PATL) binds as it does in standard binding, resulting in a puckered binding conformation. We use P1 Glu-containing peptides to support our hypotheses, and present the complex structure of spySrtA-LPALA to confirm favorable accommodation of Leu in the S1 pocket. Overall, we structurally characterize an alternative binding mode for spySrtA and specific target sequences, expanding the potential protein engineering possibilities in sortase-mediated ligation applications.

Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography.

Gram-positive bacteria display pili whose protein components (pilins) are covalently crosslinked by pilus-specific sortase enzymes. These cysteine transpeptidase enzymes catalyze a transpeptidation reaction that joins the pilins together via lysine isopeptide bonds. The crosslinking reaction that builds the SpaA pilus in Corynebacterium diphtheriae is mediated by the SrtA sortase (CdSrtA) and has been reconstituted in vitro. Here, we present a protocol that can be used to measure the kinetics of CdSrtA-catalyzed crosslinking using high-performance liquid chromatography (HPLC). In principle, this biochemical procedure can be used to measure the in vitro crosslinking activity of any pilus-specific sortase.

New polyphenolic glycosides from the stems of Caesalpinia cucullata and their inhibitory effect on methicillin-resistant Staphylococcus aureus with different ways

Anti-virulence strategy represents an emerging alternative strategy in the war against increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) due to its milder selection pressure on bacterial resistance. Sortase A (SrtA), as an important virulence factor, is a membrane-localized cysteine transpeptidase which anchors cell surface proteins to the cell wall. Natural products in medicinal plants are the source of targeting bacterial virulence factors. Here, we found polyphenolic glycosides (1–15), including thirteen new derivatives isolated from the stems of Caesalpinia cucullata, exhibited weak to moderate SrtA inhibitory activity without affecting the growth of MRSA, and compound 7 (53.7 % inhibition at 100 μM) was superior to the positive control curcumin. Meanwhile, compounds 2, 4 and 8 could effectively reduce the dose of ceftiofur in combination in vitro with fractional inhibitory concentration index (FICI) ranging from 0.188 to 0.375, which meant polyphenolic glycosides have got antibacterial activity with different ways. Here, we reported all new compounds structures determined by spectroscopy methods and their antibacterial activities, together, the relationship between structures with the inhibitory efficiency. The results indicated that polyphenolic glycosides could be used as promising therapeutic agents to prevent resistance development for S. aureus infections.

Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition

The cell wall is a critical extracellular barrier for bacteria and many other organisms. In bacteria, this structural layer consists of peptidoglycan, which maintains cell shape and structural integrity and provides a scaffold for displaying various protein factors. To attach proteins to the cell wall, Gram-positive bacteria utilize sortase enzymes, which are cysteine transpeptidases that recognize and cleave a specific sorting signal, followed by ligation of the sorting signal–containing protein to the peptidoglycan precursor lipid II (LII). This mechanism is the subject of considerable interest as a target for therapeutic intervention and as a tool for protein engineering, where sortases have enabled sortase-mediated ligation or sortagging strategies. Despite these uses, there remains an incomplete understanding of the stereochemistry of substrate recognition and ligation product formation. Here, we solved the first structures of sortase A from Streptococcus pyogenes bound to two substrate sequences, LPATA and LPATS. In addition, we synthesized a mimetic of the product of sortase-mediated ligation involving LII (LPAT-LII) and solved the complex structure in two ligand conformations. These structures were further used as the basis for molecular dynamics simulations to probe sortase A-ligand dynamics and to construct a model of the acyl–enzyme intermediate, thus providing a structural view of multiple key states in the catalytic mechanism. Overall, this structural information provides new insights into the recognition of the sortase substrate motif and LII ligation partner and will support the continued development of sortases for protein engineering applications.

Cyanidin chloride protects mice from methicillin-resistant Staphylococcus aureus-induced pneumonia by targeting Sortase A

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) has been developing rapidly in recent years. It poses a severe peril to global health care, and the new strategies to against the MRSA is urgently needed. Sortase A (SrtA) regulates the anchoring of many surface proteins. Compounds repress Staphylococcus aureus (S. aureus) cysteine transpeptidase SrtA are considered adequate potent virulence inhibitors. Then, we describe the identification of an effective SrtA inhibitor, cyanidin chloride, a bioflavonoid compound isolated from various plants. It has a reversible inhibitory effect on SrtA activity at an IC50 of 21.91 μg/mL. As a SrtA inhibitor, cyanidin chloride antagonizes SrtA-related virulence phenotypes due to its breadth and specificity, including fibrinogen adhesion, A549 cell invasion, biofilm formation, and surface protein (SpA) anchoring. Subsequently, molecular docking and fluorescence quenching revealed that SrtA and cyanidin chloride had robust mutual affinity. Further mechanistic studies revealed that Arg-197, Gly-167, and Sep-116 were the key-binding sites mediating the interaction between SrtA and cyanidin chloride. Notably, a significant therapeutic effect of cyanidin chloride in vivo was also observed on the mouse pneumonia model induced by MRSA. In conclusion, our study indicates that cyanidin chloride potentially represents a new candidate SrtA inhibitor for S. aureus and potentially be developed as a new antivirulence agent.

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes.

Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram-positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative β7-β8 loop-mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1' position of the substrate motif, underlined in LPXTG, in contrast to the strict P1' Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of β7-β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7-β8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

Covalent sortase A inhibitor ML346 prevents Staphylococcus aureus infection of Galleria mellonella.

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation of bacterial virulence and pathogenicity. Therefore, SrtA is considered to be an ideal target for antivirulence therapy. In this study, we report that ML346, a compound with a barbituric acid and cinnamaldehyde scaffold, functions as an irreversible inhibitor of Staphylococcus aureus SrtA (SaSrtA) and Streptococcus pyogenes SrtA (SpSrtA) in vitro at low micromolar concentrations. According to our X-ray crystal structure of the SpSrtAΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the thiol group of Cys208 in the active site of SpSrtA. Importantly, ML346 significantly attenuated the virulence phenotypes of S. aureus and exhibited inhibitory effects on Galleria mellonella larva infection caused by S. aureus. Collectively, our results indicate that ML346 has potential for development as a covalent antivirulence agent for treating S. aureus infections, including methicillin-resistant S. aureus.

SortPred: The first machine learning based predictor to identify bacterial sortases and their classes using sequence-derived information

Sortase enzymes are cysteine transpeptidases that embellish the surface of Gram-positive bacteria with various proteins thereby allowing these microorganisms to interact with their neighboring environment. It is known that several of their substrates can cause pathological implications, so researchers have focused on the development of sortase inhibitors. Currently, six different classes of sortases (A-F) are recognized. However, with the extensive application of bacterial genome sequencing projects, the number of potential sortases in the public databases has exploded, presenting considerable challenges in annotating these sequences. It is very laborious and time-consuming to characterize these sortase classes experimentally. Therefore, this study developed the first machine-learning-based two-layer predictor called SortPred, where the first layer predicts the sortase from the given sequence and the second layer predicts their class from the predicted sortase. To develop SortPred, we constructed an original benchmarking dataset and investigated 31 feature descriptors, primarily on five feature encoding algorithms. Afterward, each of these descriptors were trained using a random forest classifier and their robustness was evaluated with an independent dataset. Finally, we selected the final model independently for both layers depending on the performance consistency between cross-validation and independent evaluation. SortPred is expected to be an effective tool for identifying bacterial sortases, which in turn may aid in designing sortase inhibitors and exploring their functions. The SortPred webserver and a standalone version are freely accessible at: https://procarb.org/sortpred.

Discovery of Sortase A covalent inhibitors with benzofuranene cyanide structures as potential antibacterial agents against Staphylococcus aureus

Sortase A (SrtA) is a cysteine transpeptidase of most gram-positive bacteria that is responsible for the anchoring of many surface protein virulence factors to the cell wall. SrtA ablation has demonstrated to alleviate the infection without affecting the viability of bacteria. Herein, a series of benzofuran cyanide derivatives were synthesized and evaluated. Several compounds exhibited excellent inhibitory activity against SrtA with IC50 values from 3.3 μM to 21.8 μM compared with the known SrtA inhibitor pHMB (IC50 = 130 μM). Ⅲ-1, Ⅲ-15, Ⅲ-34 and V-1 showed potent inhibitory effects on biofilm formation with IC50 values from 2.1 μM to 54.2 μM. Invasion assays showed the four compounds caused a decrease of 4%–24.0% in the uptake of the S. aureus strain by 293T cells. Further assay showed that compound Ⅲ-15 decreased the amount of cell wall-associated protein A by 26.5%. Structure-activity relationship and docking studies demonstrated that the acrylonitrile moiety of the compounds played an important role in enhancing the activity. When the double bond of acrylonitrile changed to single bond, the activity was decreased significantly. This indicates that acrylonitrile, which is a Michael receptor, can inhibit the activity of SrtA by covalent binding effectively to the thiol group of Cys184.

Taxifolin, an Inhibitor of Sortase A, Interferes With the Adhesion of Methicillin-Resistant Staphylococcal aureus.

The evolution and spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant hidden risk to human public health. The majority of antibiotics used clinically have become mostly ineffective, and so the development of novel anti-infection strategies is urgently required. Since Staphylococcus aureus (S. aureus) cysteine transpeptidase sortase A (SrtA) mediates the surface-anchoring of proteins to its surface, compounds that inhibit SrtA are considered potential antivirulence treatments. Herein, we report on the efficacy of the potent SrtA inhibitor taxifolin (Tax), a flavonoid compound isolated from Chinese herbs. It was able to reversibly block the activity of SrtA with an IC50 of 24.53 ± 0.42 μM. Tax did not display toxicity toward mammalian cells or S. aureus at a concentration of 200 μM. In addition, Tax attenuated the virulence-related phenotype of SrtA in vitro by decreasing the adherence of S. aureus, reducing the formation of a biofilm, and anchoring of S. aureus protein A on its cell wall. The mechanism of the SrtA-Tax interaction was determined using a localized surface plasmon resonance assay. Subsequent mechanistic studies confirmed that Asp-170 and Gln-172 were the principal sites on SrtA with which it binds to Tax. Importantly, in vivo experiments demonstrated that Tax protects mice against pneumonia induced by lethal doses of MRSA, significantly improving their survival rate and reducing the number of viable S. aureus in the lung tissue. The present study indicates that Tax is a useful pioneer compound for the development of novel agents against S. aureus infections.

X-ray structures of Clostridium perfringens sortase C with C-terminal cell wall sorting motif of LPST demonstrate role of subsite for substrate-binding and structural variations of catalytic site

Pili of Gram-positive bacteria are flexible rod proteins covalently attached to the bacterial cell wall, that play important roles in the initial adhesion of bacterial cells to host tissues and bacterial colonization. Pili are formed by the polymerization of major and minor pilins, catalyzed by class C sortase (SrtC), a family of cysteine transpeptidases. The Gram-positive bacterium Clostridium perfringens has a major pilin (CppA), a minor pilin (CppB), and SrtC (CpSrtC). CpSrtC recognizes the C-terminal cell wall sorting signal motifs with five amino acid residues, LPSTG of CppA and LPETG of CppB, for the polymerization of pili. Here, we report biochemical analysis to detect the formation of Clostridium perfringens pili in vivo, and the X-ray structure of a novel intermolecular substrate-enzyme complex of CpSrtC with a sequence of LPST at the C-terminal site. The results showed that CpSrtC has a subsite for substrate-binding to aid polymerization of pili, and that the catalytic site has structural variations, giving insights into the enzyme catalytic reaction mechanism and affinities for the C-terminal cell wall sorting signal motif sequences.

Eriodictyol as a Potential Candidate Inhibitor of Sortase A Protects Mice From Methicillin-Resistant Staphylococcus aureus-Induced Pneumonia.

New anti-infective approaches are urgently needed to control multidrug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Sortase A (SrtA) is a membrane-bound cysteine transpeptidase that plays an essential role in the catalysis of covalent anchoring of surface proteins to the cell wall of Staphylococcus aureus (S. aureus). The present study reports identification of a flavonoid, eriodictyol, as a reversible inhibitor of SrtA with an IC50 of 2.229 ± 0.014 μg/mL that can be used as an innovative means to counter both resistance and virulence. The data indicated that eriodictyol inhibited the adhesion of the bacteria to fibrinogen and reduced the formation of biofilms and anchoring of staphylococcal protein A (SpA) on the cell wall. The results of fluorescence quenching experiments demonstrated a strong interaction between eriodictyol and SrtA. Subsequent mechanistic studies revealed that eriodictyol binds to SrtA by interacting with R197 amino acid residue. Importantly, eriodictyol reduced the adhesion-dependent invasion of A549 cells by S. aureus and showed a good therapeutic effect in a model of mouse pneumonia induced by S. aureus. Overall, the results indicated that eriodictyol can attenuate MRSA virulence and prevent the development of resistance by inhibiting SrtA, suggesting that eriodictyol may be a promising lead compound for the control of MRSA infections.

Prospecting Potential Inhibitors of Sortase A from Enterococcus faecalis: A Multidrug Resistant Bacteria, through In-silico and In-vitro Approaches.

Enterococcus faecalis (Ef) infections are becoming dreadfully common in hospital environments. Infections caused by Ef are difficult to treat because of its acquired resistance to different class of antibiotics, making it a multidrug resistant bacteria. Key pathogenic factor of Ef includes its ability to form biofilm on the surface of diagnostic and other medical devices. Sortase A (SrtA) is a cysteine transpeptidase which plays a pivotal role in the formation of biofilm in Ef, hence, it is considered as an important enzyme for the pathogenesis of Ef. Thus, inhibition of (SrtA) will affect biofilm formation, which will reduce its virulence and eventually Ef infection will be abridged. To find potential inhibitors of Enterococcus faecalis Sortase A (EfSrtA) through insilico and in-vitro methods. Gene coding for EfSrtA was cloned, expressed and purified. Three-dimensional model of EfSrtA was created using Swiss-Model workspace. In-silico docking studies using Autodock vina and molecular dynamics simulations of the modelled structures using Gromacs platform were performed to explore potential lead compounds against EfSrtA. In-vitro binding experiments using spectrofluorometric technique was carried out to confirm and validate the study. In-silico docking and in-vitro binding experiments revealed that curcumin, berberine and myricetin bound to EfSrtA at nanomolar concentrations with high affinity. This is a first structural report of EfSrtA with curcumin, berberine and myricetin. Taking in account the herbal nature of these compounds, the use of these compounds as inhibitors will be advantageous. This study validated curcumin, berberine and myricetin as potential inhibitors of EfSrtA.

Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB-substrate complexes. Furthermore, we also demonstrated that residues 163-168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

Genome-based analysis for the bioactive potential of Streptomyces yeochonensis CN732, an acidophilic filamentous soil actinobacterium

BackgroundAcidophilic members of the genus Streptomyces can be a good source for novel secondary metabolites and degradative enzymes of biopolymers. In this study, a genome-based approach on Streptomyces yeochonensis CN732, a representative neutrotolerant acidophilic streptomycete, was employed to examine the biosynthetic as well as enzymatic potential, and also presence of any genetic tools for adaptation in acidic environment.ResultsA high quality draft genome (7.8 Mb) of S. yeochonensis CN732 was obtained with a G + C content of 73.53% and 6549 protein coding genes. The in silico analysis predicted presence of multiple biosynthetic gene clusters (BGCs), which showed similarity with those for antimicrobial, anticancer or antiparasitic compounds. However, the low levels of similarity with known BGCs for most cases suggested novelty of the metabolites from those predicted gene clusters. The production of various novel metabolites was also confirmed from the combined high performance liquid chromatography-mass spectrometry analysis. Through comparative genome analysis with related Streptomyces species, genes specific to strain CN732 and also those specific to neutrotolerant acidophilic species could be identified, which showed that genes for metabolism in diverse environment were enriched among acidophilic species. In addition, the presence of strain specific genes for carbohydrate active enzymes (CAZyme) along with many other singletons indicated uniqueness of the genetic makeup of strain CN732. The presence of cysteine transpeptidases (sortases) among the BGCs was also observed from this study, which implies their putative roles in the biosynthesis of secondary metabolites.ConclusionsThis study highlights the bioactive potential of strain CN732, an acidophilic streptomycete with regard to secondary metabolite production and biodegradation potential using genomics based approach. The comparative genome analysis revealed genes specific to CN732 and also those among acidophilic species, which could give some insights into the adaptation of microbial life in acidic environment.

Sortases from a Probiotic Lactobacillus rhamnosus GG: Cloning, Expression, Purification, Crystallization and Preliminary X-Ray Diffraction Study

Sortases are membrane-associated cysteine transpeptidases found mainly in Gram-positive bacteria. They have been intensely studied in pathogenic strains as they play a key role in covalent attachment of various virulence-associated surface proteins including pili to the cell wall and also in biogenesis of pili. The recent identification of sortase genes in some members of gut microbiota indicates their likely role in establishing interaction with the human gastrointestinal tract for probiotic traits. While the structural information about sortases thus far is limited to pathogenic bacteria, here we have the produced recombinant form of housekeeping (SrtA) and pilin-specific (SrtC1) sortases from gut-adapted probiotic strain Lactobacillus rhamnosus GG and crystallized them. The crystals of SrtA and SrtC1 diffracted to a resolution of 1.9 A and belonged to monoclinic and tetragonal crystal forms respectively.

Structures of major pilins in Clostridium perfringens demonstrate dynamic conformational change.

Pili in Gram-positive bacteria are flexible rod proteins associated with the bacterial cell surface, and they play important roles in the initial adhesion to host tissues and colonization. The pilus shaft is formed by the covalent polymerization of major pilins, catalyzed by sortases, a family of cysteine transpeptidases. Here, X-ray structures of the major pilins from Clostridium perfringens strains 13 and SM101 and of sortase from strain SM101 are presented with biochemical analysis to detect the formation of pili in vivo. The major pilin from strain 13 adopts an elongated structure to form noncovalently linked polymeric chains in the crystal, yielding a practical model of the pilus fiber structure. The major pilin from strain SM101 adopts a novel bent structure and associates to form a left-handed twist like an antiparallel double helix in the crystal, which is likely to promote bacterial cell-cell interactions. A modeling study showed that pilin with a bent structure interacts favorably with sortase. The major pilin from strain SM101 was considered to be in an equilibrium state between an elongated and a bent structure through dynamic conformational change, which may be involved in pili-mediated colonization and sortase-mediated polymerization of pili.

A comprehensive in silico analysis of sortase superfamily.

Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Gram-positive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.