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Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

Department of Microbiology,² Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637¹

SUMMARY

INTRODUCTION

SURFACE PROTEINS, THE SUBSTRATES OF SORTASE

Staphylococcus aureus Surface Proteins and Their Functions

Signal Peptides and Cell Wall Sorting Signals

Anchor Structure of Staphylococcal Surface Proteins

S. AUREUS SORTASE A

Molecular Genetic Analysis of Sortase A (srtA) Function

Sortase A Structure

Biochemistry of the Sortase A Reaction

Lipid II, the Peptidoglycan Substrate of Sortase A

Sortase A Inhibitors

Applications of the Sortase A Reaction

S. AUREUS SORTASE B

IsdC and Sortase B Contribute to Heme-Iron Transport

Molecular Genetic Analysis of Sortase B (srtB) Function

Biochemistry of the Sortase B Reaction

Sortase B Positions IsdC within the Cell Wall Envelope

SORTASE-CATALYZED POLYMERIZATION OF PILI

Actinomyces naeslundii

Corynebacterium diphtheriae

Streptococcus agalactiae

SORTASE AND SURFACE PROTEIN FUNCTION IN SELECT GRAM-POSITIVE BACTERIA

Listeria monocytogenes

Streptococcus pyogenes

Oral Streptococci

Streptococcus pneumoniae: Surface Proteins and Pili

Streptococcus suis

Bacillus anthracis

Hyphal Development in Streptomyces coelicolor

BIOINFORMATIC ANALYSIS OF SORTASES AND SUBSTRATES

CONCLUSIONS AND FUTURE DIRECTIONS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element for the physical integrity of microbes but also promotes interactions between bacteria and their environment (60). Most importantly for bacterial pathogens, as environments are subject to change, microbes respond with alterations in envelope structure and function. Thus, one should consider the cell wall envelope a dynamic organelle, one that is continuously assembled from precursor molecules and disassembled into individual constituents.

Bacterial cell wall assembly requires peptidoglycan precursors that together form a single large macromolecule, the murein sacculus, encircling the microbial cell with a 20- to 100-nm-thick wall structure (61). Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provide bacterial envelopes with species- and strain-specific properties that, for pathogens, contribute greatly to bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the roles that these enzymes play in bacterial physiology and pathogenesis. Interested readers are referred to other excellent reviews that have examined in depth the structure and assembly of peptidoglycan, teichoic acids, and polysaccharides or proteins that are noncovalently associated with the cell wall envelope (136, 139, 144, 187).

In Staphylococcus aureus, peptidoglycan precursor molecules are fabricated from N-acetylmuramic acid (MurNAc) and L- as well as D-stereoisomer amino acids in the bacterial cytoplasm to yield a soluble intermediate, Park's nucleotide (UDP-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala-D-Ala) (24) (Fig. 1). The precursor is tethered via phosphodiester linkage to a bactoprenol carrier, generating lipid I (C₅₅-PP-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala-D-Ala) in the membrane (24, 117, 118). Further modification with N-acetylglucosamine (GlcNAc) and cross bridge decoration at the -amino of L-Lys (pentaglycine or Gly₅ in staphylococci) generates lipid II {C₅₅-PP-MurNAc-[L-Ala-D-isoGln-L-Lys(Gly₅)-D-Ala-D-Ala]-ß(1-4)-GlcNAc)}. Lipid II is translocated across the cell membrane (133), where it becomes a substrate for penicillin binding proteins (PBPs) that catalyze transglycosylation and transpeptidation reactions. Transglycosylation polymerizes MurNAc-GlcNAc subunits into repeating disaccharide chains, also called glycan strands (194). Transpeptidation involves first cleavage of the pentapeptide precursor [L-Ala-D-isoGln-L-Lys(Gly₅)-D-Ala-D-Ala] at the terminal D-Ala and then formation of an amide bond between the carboxyl group of D-Ala at position four and the amino groups of pentaglycine cross bridges in other wall peptides (85). PBPs use these two reactions together to form a single large macromolecule that displays rigid exoskeletal functions and that serves as a scaffold for the incorporation of other molecules that can be attached to cross bridges, wall peptides, or glycan strands. Peptidoglycan biosynthesis in other bacteria follows a similar scheme, with two exceptions. First, D-isoGlu at position two of wall peptides is typically not amidated. Second, L-Lys, the diamino acid at position three of wall peptides, can be substituted with m-diaminopimelic acid, and the attached cell wall cross bridges can vary in chemical nature between different bacterial species (170).

View larger version (28K): [in this window] [in a new window]   FIG. 1. Peptidoglycan synthesis in S. aureus. Park's nucleotide, a soluble nucleotide precursor, originates in the bacterial cytoplasm by successive addition of L-stereoisomer amino acids (L-Ala and L-Lys) as well as D-stereoisomer amino acids (D-isoglutamine [D-iGln] and D-Ala) to UDP-N-acetylmuramic acid (UDP-NM). Precursor transfer to undecaprenol pyrophosphate, a bacterial membrane carrier, generates lipid I and removes UMP nucleotide. Lipid I modification with N-acetylglucosamine (GN) and pentaglycine cross bridge formation at the -amino of L-Lys with tRNAGly substrate generates lipid II. Following translocation across the cytoplasmic membrane, lipid II serves as substrate for PBPs that catalyze three reactions: transglycosylation, transpeptidation, and carboxypeptidation. Transglycosylases polymerize MN-GN subunits into repeating disaccharide chains, the glycan strands. Transpeptidases cleave the amide bond of the terminal D-Ala in pentapeptide precursors and generate an amide bond between the carboxyl group of D-Ala at position four and the amino group of pentaglycine cross bridges in wall peptides. Carboxypeptidases hydrolyze the C-terminal D-Ala of most non-cross-linked pentapeptides to yield mature peptidoglycan.

Recent discoveries have shown that sortases catalyze diverse transpeptidation reactions using specific polypeptide or peptidoglycan substrates. Further, sortases can target unique domains of the bacterial cell wall envelope and can even promote the assembly of pili in gram-positive bacteria. These discoveries are discussed here in the context of current research frontiers. The underlying contributions of surface proteins and sortases to the pathogenesis of bacterial infections have been revealed in animal models of disease, and these findings may be exploited for the implementation of new therapeutic strategies.

SURFACE PROTEINS, THE SUBSTRATES OF SORTASE

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View larger version (21K): [in this window] [in a new window]   FIG. 2. Sortase A-dependent surface display of staphylococcal proteins. Sortase is responsible for the anchoring of 20 different surface proteins to the cell wall of S. aureus strain Newman. One of these surface proteins, protein A, binds to the Fc terminus of mammalian immunoglobulins in a nonimmune fashion, causing decoration of the staphylococcal surface with antibody. Using Cy3-conjugated immunoglobulin and S. aureus strain Newman, protein A display on the bacterial surface was revealed with phase-contrast microscopy and fluorescence microscopy. Protein A display on the staphylococcal surface is abrogated in the srtA mutant strain (SKM3).

Using cell wall sorting signals as queries in bioinformatic searches, 18 to 22 genes encoding putative sortase-anchored surface proteins were identified in the genomes of S. aureus, varying with the strain under investigation (see Table 1 for a listing of 22 surface proteins) (62, 122, 123, 162). Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are bacterial elements of tissue adhesion and immune evasion (53). The study of several staphylococcal proteins has helped lay the foundation of our current understanding of these molecules, and these include the fibronectin binding proteins FnbpA and FnbpB (52, 89, 166, 178). Both proteins encompass a large N-terminal domain (about 500 amino acid residues) followed by four or five 50-residue repeat domains responsible for binding the N-terminal domain of fibronectin. FnbpA/FnbpB interactions with fibronectin involve structural rearrangements that lead to the ordering of the Fnbp repeat domains upon ligand binding (89, 162, 212). As fibronectin is found in extracellular matrices of most tissues as well as in soluble form within body fluids, staphylococci can adhere to virtually all tissues or serum-coated foreign bodies (151). With such widespread binding potential, one important aspect of staphylococcal binding to fibronectin is the invasion of host cells and subsequent intracellular replication (8, 44).

S. aureus strains clump in the presence of plasma. This phenomenon, which has been exploited for diagnostic purposes, is the product of a molecular interaction between two MSCRAMMs, clumping factors A and B (ClfA and ClfB), and fibrinogen (54, 124, 141). ClfA and ClfB are structurally related and comprise a large N-terminal A domain and a repeat domain (R domain) which is composed exclusively of serine-aspartate repeats (69, 90). The ligand binding sites of ClfA and ClfB have been mapped to residues 220 to 559 (125), which assume an immunoglobulin G (IgG)-like fold (37, 125, 153, 209). An elegant molecular mechanism of fibrinogen substrate binding, coined "dock, lock, and latch," has recently been demonstrated for SdrG, a fibrinogen binding Staphylococcus epidermidis MSCRAMM that also encompasses repeat domains (156). A cleft of 30 Å in length between two IgG-like folds of SdrG constitutes the fibrinogen binding site, with at least 62 contacts between the two molecules that occlude the cleavage sites for thrombin. Both S. aureus and S. epidermidis strains encode multiple cell wall-anchored surface proteins with large serine-aspartate repeat (Sdr) domains (69, 90, 156). Other surface proteins containing Sdr domains include the aforementioned ClfA and ClfB but also SdrC, SdrD, and SdrE. The B domains of Sdr proteins contain high-affinity calcium binding sites which adopt an EF hand fold, a common structure observed in other calcium binding proteins (91, 205). Although it seems likely that these proteins are involved in binding host factors, such interactions have thus far not been demonstrated for the majority of the Sdr proteins.

S. aureus protein A (Spa) binds to the Fc termini of mammalian immunoglobulins in a nonimmune fashion, resulting in the uniform coating of staphylococci with antibodies (86). The protein A amino acid sequence, gene sequence, and three-dimensional nuclear magnetic resonance and X-ray diffraction structures revealed a molecule comprised of five nearly identical immunoglobulin binding domains (36, 65, 179, 206). Mutations in the protein A gene (spa) cause significant defects in the pathogenesis of S. aureus infections. For example, reduced bacterial survival in blood or in the presence of macrophages is likely due to the inability of these variants to sequester immunoglobulin via Fc binding (149). However, the observed phenotypes may also be attributed to defects in the binding of protein A to von Willebrand factor, a serum polypeptide that promotes physiological homeostasis of human or animal blood, or to protein A binding to tumor necrosis factor receptor 1, a signaling molecule involved in proinflammatory cytokine responses and innate immunity (64, 70).

Four Isd proteins (iron-regulated surface determinants) are involved in binding heme or hemoproteins and appear to play a role in iron scavenging during staphylococcal host infection. HarA/IsdH is encoded by a gene outside the isd locus (see below) and has been shown to bind haptoglobin/hemoglobin complexes (42). IsdB, on the other hand, binds to hemoglobin, and four proteins, i.e., IsdA, IsdB, IsdC, and IsdH/HarA, bind heme (121, 182). It has been proposed that these proteins are involved in capturing hemoproteins on the bacterial surface, liberating heme, and promoting heme transport across the bacterial cell wall envelope (182). The functions of twelve S. aureus surface proteins with C-terminal sorting signals, i.e., SasA, SasB, SasC, SasD, SasF, SasG, SasH, SasK, SdrC, SdrD, SdrE, and Pls, are not yet known. Table 1 summarizes the current knowledge about S. aureus surface proteins.

The C-terminal cell wall sorting signal of staphylococcal protein A encompasses a 35-residue peptide with an LPXTG motif, followed by a hydrophobic domain and a positively charged tail (173). Mutations that truncate the sorting signal cause the secretion of mutant protein A into the extracellular medium. In contrast, mutations that delete or substitute residues within the LPXTG motif abolish sortase-mediated cell wall linkage without secretion of mutant protein A (174). The cell wall sorting signal alone is sufficient to cause cell wall anchoring of other polypeptides that are initiated into the secretory pathway of S. aureus via an N-terminal signal peptide (38, 134, 135, 195). Moreover, sorting signals from one species can be functional in another microorganism (173). When the sorting function fails, mutations that either alter the distance between the LPXTG motif and the charged tail or affect residues within the two parts of the sorting signal repair the lack of function of the heterologous cell wall sorting signal (173).

Cell wall sorting signals are functional even if they do not reside at the C-terminal end of the polypeptide chain (135). Nevertheless, sorting signal function absolutely requires an upstream signal peptide. Positioning the cell wall sorting signal in the middle of an engineered polypeptide, flanked at its N-terminal side by the signal peptide-bearing reporter staphylococcal enterotoxin B (Seb) and at its C-terminal border with the mature domain of ß-lactamase (BlaZ), generates a hybrid precursor that is cleaved at the N-terminal signal peptide and initiated into the secretory pathway (135). The precursor is then cleaved between the threonine and the glycine of the LPXTG motif, and the N-terminal portion of the precursor is tethered to the cell wall envelope. In contrast, the C-terminal portion of the precursor with the remainder of the cleaved cell wall sorting signal resides in the bacterial cytoplasm.

Sorting signals have been observed in a plethora of predicted gene products, most of which were identified via genome sequencing of gram-positive bacteria (12, 31, 51, 122, 136, 148). While the great majority of these sorting signals carry the LPXTG motif, others harbor variations of this sequence (Table 2) (see below). If a surface protein gene that contains such variation resides in the same transcriptional unit with a sortase gene, it is generally presumed that the two genes encode an enzyme-substrate pair, i.e., that the sortase specifically recognizes and cleaves the sorting signal of the cotranscribed substrate. This conjecture has been experimentally confirmed for Corynebacterium diphtheriae spa loci (204), S. aureus isd-srtB (123), and Listeria monocytogenes svpA-srtB (10) (see below).

Muramidases cleave the glycan strands of staphylococcal peptidoglycan and release protein A as a spectrum of molecules with different masses. In contrast, lysostaphin releases protein A species with smaller masses (173). C-terminal anchor structures of protein A were deduced by analyzing engineered surface protein sortase substrates. The protein A cell wall sorting signal was fused to the C-terminal end of Escherichia coli maltose binding protein (MalE) (171). Cell wall-anchored MalE was released with lysostaphin from the staphylococcal envelope, purified, and cleaved with trypsin, and C-terminal peptides were analyzed by Edman degradation and mass spectrometry, which revealed the sequence LPET-Gly₄, LPET-Gly₃, and LPET-Gly₂ (171). As the cell wall sorting signal of protein A is cleaved between the threonine and glycine residues of the LPXTG motif, addition of glycine residues to the carboxyl-terminal end of protein A must be due to amide linkage of surface protein to the cell wall cross bridge of staphylococci, and this pentaglycine is cleaved by lysostaphin at positions 2, 3, and 4.

The complete anchor structure of surface proteins in staphylococci was determined after solubilization of peptidoglycan with muramidase, amidase, D-Ala-Gly endopeptidase, and lysostaphin (137, 138, 195). Seb-MHis₆-Cws, an engineered reporter comprised of Seb fused to the protein A cell wall sorting signal (Cws) via a methionyl-six-histidyl linker (MHis₆), can be solubilized from the peptidoglycan via cleavage with muralytic enzymes, purified by affinity chromatography on nickel-nitrilotriacetic acid resin, and then cleaved with cyanogen bromide at methionyl residues. C-terminal anchor peptides are purified by a second round of affinity chromatography and analyzed by mass spectrometry and Edman degradation. Using this technology, surface proteins were found to be linked to the cell wall cross bridges of cross-linked peptidoglycan units, comprised predominantly of murein tetrapeptides {MurNAc-[L-Ala-D-isoGln-L-Lys-(Gly₅)-D-Ala-]-GlcNAc}, and only rarely to murein-pentapeptides {MurNAc-[L-Ala-D-isoGln-L-Lys-(Gly₅)-D-Ala-D-Ala]-GlcNAc} that were released by muramidase cleavage of glycan strands or amidase cleavage of cell wall peptides. The overall picture that emerged from these studies indicates that surface proteins are embedded in peptidoglycan and occupy any position along glycan strands that are comprised of 2 to 11 disaccharide units and at any position along tetrapeptide cross-links with 1 to 15 wall peptide units (Fig. 3).

View larger version (17K): [in this window] [in a new window]   FIG. 3. Cell wall anchor structure of staphylococcal surface proteins. The C-terminal threonine of surface proteins, generated by sortase A-mediated cleavage between the threonine and the glycine of the LPXTG motif, is amide linked to the pentaglycine cross bridge of S. aureus cell wall peptidoglycan. Treatment of the staphylococcal peptidoglycan with lysostaphin (glycyl-glycine endopeptidase), mutanolysin [N-acetylmuramidase that cleaves the ß(1-4) O-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (GN)], amidase (N-acetylmuramoyl-L-Ala amidase), or 11 hydrolase (N-acetylmuramoyl-L-Ala amidase and D-Ala-Gly endopeptidase) releases surface protein with the predicted C-terminal cell wall anchor structures.

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Expression of several surface protein genes appears to be dramatically reduced in S. aureus srtA mutants, and the molecular mechanisms underlying this regulatory phenomenon have not yet been explored (S. K. Mazmanian and O. Schneewind, unpublished observation). Overexpression of plasmid-encoded surface protein genes or reporter genes encoding secreted proteins with C-terminal sorting signals greatly reduces the viability of staphylococci carrying srtA deletions (123). It seems plausible that srtA mutations cause the accumulation of surface proteins within the secretory pathway, which has recently been dubbed the ex-portal for Streptococcus pyogenes (163), a pathogen that is closely related to staphylococci. As these polypeptides cannot be cleaved in the absence of sortase and therefore cannot advance along the sorting pathway, it seems likely that they may block the ex-portal.

The contribution of S. aureus srtA to the pathogenesis of staphylococcal disease was examined in several different animal model systems of infection. S. aureus strain Newman, a human clinical isolate, was used as a parent, and the srtA gene was replaced with the erythromycin resistance cassette (119). Compared to the wild-type parent, sortase mutants displayed a 1.5-log-unit increase in the 50% lethal dose (LD₅₀) measured after intraperitoneal injection of staphylococci into mice, indicating a reduction in the virulence of the srtA strain. This defect may not seem large, especially compared to virulence genes in microbes that are particularly prone to causing lethal infections in mice, such as Yersinia pestis (155). However, the LD₅₀ for S. aureus strain Newman is already high, requires about 10⁷ CFU (119). Any reduction in virulence of staphylococci beyond 1 to 2 log units is concealed by an experimental ceiling with a lethal dose of about 10⁸ to 10⁹ CFU for any bacterial organism (dead or alive), because massive induction of innate inflammatory responses by bacterial extracts is rapidly fatal.

An organ abscess model has provided greater insight into the contribution of sortase A to the pathogenesis of staphylococcal disease. Following injection of a sublethal dose of 10⁶ CFU of S. aureus strain Newman into the bloodstream, about 1 to 2 log units of staphylococci are rapidly killed by phagocytic cells (112). Those microbes that escape phagocytosis by adherence to specific tissues or invasion of cells can seed abscesses in virtually all organ tissues of mice (104). Abscesses mature within 4 to 5 days and harbor several log units of viable staphylococci, which are then cleared over a period of 5 to 10 days (3). Removal of organ tissue from infected animals and anatomical analysis or enumeration of viable staphylococci can be used as a measure of virulence and pathogenesis. Compared to the wild-type parent strain Newman, srtA mutants display a 3-log-unit reduction in bacterial growth within abscesses in multiple different organs, consistent with the notion that surface proteins of staphylococci are required to resist phagocytic clearance and to escape innate immune responses by directing bacteria to various organ tissues (119).

The septic arthritis model was developed by Bremell et al. (15, 16). Following intravenous injection, staphylococci replicate in joints, causing infectious arthritis, bone destruction, and deformation during wound healing in addition to weight loss. The severity of the infectious arthritis can be quantified by analyzing pathological anatomical lesions after excision of joints. Again sortase A mutants displayed a large reduction in virulence in this animal model system (87, 88).

Staphylococcal endocarditis occurs mainly as infectious foci on heart valves, and damaged valve tissue with fibrin-covered lesions represent a risk factor. This important clinical infection can be recapitulated in rats by first introducing valve tissue lesions with fibrin and platelet deposits via an intravenous polyethylene catheter (130). After the catheter is implanted, animals are challenged with staphylococcal infection, which causes formation of infectious thrombi and deposits of staphylococci on valve lesions followed by tissue destruction. Two days after infection, the hearts are aseptically removed and bacterial titers are determined as CFU. In this experiment, srtA mutants displayed a 2-log-unit reduction in virulence compared with the wild-type parent strain S. aureus Newman (213).

The complete spectrum of molecular mechanisms whereby surface proteins contribute to the pathogenesis of S. aureus infectious diseases cannot yet be appreciated. In fact, only recently have we learned about the contribution of these few surface proteins to pathogenesis, and much work is required to gain a better understanding. Nevertheless, the overall contribution of these surface molecules to staphylococcal pathogenesis can be measured by comparing wild-type and srtA mutant strains in infectious disease models. As is reviewed in detail above, srtA is a key virulence factor of staphylococci. In light of the rising number of antibiotic-resistant S. aureus strains (13), the sortase enzyme has become an important target for the treatment of staphylococcal disease. Additionally, surface proteins must be considered for therapeutic and preventive strategies to combat the tide of infections with this microbe.

In order to obtain soluble enzyme for in vitro activity assays and structural analysis, the N-terminal signal peptide/membrane anchor of sortase A was replaced with a six-histidyl tag and recombinant protein was purified (84, 197). Preliminary examination of the NOESY (nuclear Overhauser effect spectroscopy) signals of sortase nuclear magnetic resonance (NMR) spectra suggested that the enzyme folds into a predominantly ß-strand structure (83). This conjecture was corroborated by determining the three-dimensional structure of sortase by NMR spectroscopy (84) and X-ray crystallography (227). The enzyme assumes a unique fold, consisting of an eight-stranded ß-barrel that includes one or two helices and several loops (Fig. 4). Strands ß7 and ß8 form the floor of a hydrophobic depression where the active site is located. The NMR structure showed that the absolutely conserved Cys¹⁸⁴ and His¹²⁰ residues of sortases reside within the active site (84). While Cys¹⁸⁴ is anchored in ß7, His¹²⁰ is located within a helical region that connects ß2 and ß3, with its imidazole group in the vicinity of the sulfhydryl side chain of Cys¹⁸⁴. The NMR structure showed Asn⁹⁸ anchored at the C-terminal end of ß4 and also protruding near the active site. Asn⁹⁸ is only poorly conserved among sortases. Further, all three aforementioned residues were positioned in a configuration similar to that of the Cys²⁵-His¹⁵⁹-Asn¹⁷⁵ triad of cysteine proteases in the papain family (84, 210). X-ray crystallography data suggest, however, that Asn⁹⁸ and His¹²⁰ are not in the same close proximity as is observed for papain-type proteases and that sortase-mediated catalysis at Cys¹⁸⁴ may occur by another mechanism (227). Arg¹⁹⁷, anchored in ß8, is located in close proximity and parallel to the active-site cysteine (227) (see below). The significance of these structural observations was addressed by measuring the activity of mutant enzymes bearing alanine substitutions of critical residues (see below). Replacement of either Cys¹⁸⁴ or His¹²⁰ completely abolished sortase activity both in vivo and in vitro (197, 200, 201), and replacement of Arg¹⁹⁷ greatly reduced the enzymatic activity (116). In contrast, replacement of Asn⁹⁸ with alanine had no effect on sortase activity (116).

View larger version (69K): [in this window] [in a new window]   FIG. 4. Structure of S. aureus sortase A bound to the LPETG substrate. Sortase folds into an eight-stranded ß-barrel structure. The active site resides in a depression formed by ß7 and ß8 strands. The side chains of His120, Cys184, and Arg197, all of which are absolutely conserved among sortases and are required for activity, as well as the LPETG substrate are drawn with ball-and-stick structures. Cys184 performs a nucleophilic attack on the peptide bond between the threonine and the glycine residues of the substrate, resulting in the formation of an acyl intermediate with the carboxyl group of the C-terminal threonine thioester linked to the sulfur of Cys184. This intermediate is resolved by a second nucleophilic attack on the thioester bond, which results in the release of the reaction products (the structure was generated from atomic coordinates deposited in Protein Data Bank, PDB ID 1T2P) (227).

In the NMR structure, the ß3-ß4 and ß6-ß7 loops contain a set of acidic residues involved in calcium binding (84, 131). This cation, present in millimolar amounts in host tissues, activates sortase activity eightfold (84). Analysis of the sortase NMR spectra in the presence and absence of calcium revealed that Glu¹⁰⁵, Glu¹⁰⁸, and Asp¹⁰⁸ side chains of the ß3-ß4 loop interact with the cation. In contrast, the ß6-ß7 loop forms a flap that is disordered in the absence of calcium (131, 227). As a result of metal binding, slow-motion conformational changes were detected by which Glu¹⁷¹, positioned in the ß6-ß7 loop, transiently interacts with calcium and drives the flap to a closed state (131). This motion primarily affects the wall of the groove that forms the active site, which adopts a conformation better suited for the binding of the LPXTG peptide. Therefore, the binding of calcium ions activates sortase by a mechanism that may facilitate substrate binding (84, 131).

N

Sortase activity can be assessed in vivo by following the maturation of pulse-labeled surface protein, for example, the Seb-Spa_CWS reporter (202). Three species can be distinguished after labeling with [³⁵S]methionine: the full-length precursor (P1); the P2 intermediate, with cleaved a N-terminal signal peptide but still harboring the C-terminal sorting signal; and the mature (M) anchored polypeptide, in which the N-terminal signal peptide and the C-terminal sorting signal have been removed (see below and Fig. 5). The P2/M ratio is a measure of in vivo sortase activity. Using a srtA mutant strain and plasmids encoding sortase variants with amino acid substitutions, the contributions of individual amino acids to in vivo catalysis can be determined (116, 201).

View larger version (20K): [in this window] [in a new window]   FIG. 5. Cell wall sorting pathway of surface proteins in gram-positive bacteria. Surface proteins are first synthesized in the bacterial cytoplasm as full-length precursors (P1) containing an N-terminal signal sequence and a C-terminal sorting signal. The signal sequence directs the cellular export of the polypeptide through the Sec system and, upon translocation, is cleaved by signal peptidase. The product of this reaction, the P2 precursor harboring only the C-terminal sorting signal, is retained within the secretory pathway via its C-terminal hydrophobic domain (black box) and positively charged tail (+). Sortase, a membrane-anchored transpeptidase with active-site cysteine, cleaves the peptide bond between the threonine (T) and the glycine (G) of the LPXTG motif, generating an acyl intermediate (AI). Lipid II, the peptidoglycan biosynthesis precursor, and its pentaglycine cross bridge (Gly5) amino group attack the acyl intermediate, linking the C-terminal threonine of the surface protein to lipid II (P3 precursor) and regenerating the active site of sortase. The P3 precursor functions as a substrate for penicillin binding proteins and is incorporated into the cell wall envelope to generate mature anchored surface protein (M), which is also displayed on the bacterial surface. This pathway is universal in many gram-positive bacteria, and the functional elements of cell wall cross bridges, LPXTG motif, sortase, and penicillin binding proteins are conserved.

Analysis of the kinetic parameters of the transpeptidation reaction indicates that it may resemble a ping-pong mechanism, whereby the binding and cleavage of the LPXTG is followed by the incorporation of the pentaglycine substrate into the active site for the separation of the acyl intermediate (77, 200). Each of these reactions appears to harbor a distinct limiting step, with that of the acylation step during transpeptidation and that of the deacylation step during hydrolysis (77).

The mechanism whereby Cys¹⁸⁴ performs nucleophilic attack at the scissile peptide bond is not yet clear. Reagents that specifically react with sulfhydryl but not with thiolate groups such as iodoacetamide and iodoacetic acid do not inhibit sortase (202), consistent with the notion that the sortase sulfhydryl must be ionized. The NMR structure of the enzyme showed the presence of a histidine residue (His¹²⁰) (see above) located in the active site of the enzyme (84). The residue is absolutely conserved and essential for sortase activity, both in vivo and in vitro (201). This result prompted the hypothesis that sortase would form an imidazolium-thiolate ion pair, mimicking active-site ionization of cysteine proteases (186). In this model, the positively charged imidazol group of His¹²⁰ stabilizes the formation of a thiolate in Cys¹⁸⁴ and acts as a proton donor/acceptor during acylation and deacylation steps (201). In papain, the Cys²⁵-His¹⁵⁹-Asn¹⁷⁵ triad comprises the active site (210). While cysteine and histidine form a thiolate-imidazolium ion pair that is fundamental for papain catalysis, the asparagine side chain positions His¹⁵⁹ in a favorable orientation towards Cys²⁵ through hydrogen bonding. In sortase, two residues, Trp¹⁹⁴ and Asn⁹⁸, that could play a role similar to that of Asn¹⁷⁵ are positioned near His¹²⁰; however, these amino acids are not conserved among sortases. While the replacement of Asn⁹⁸ with alanine or glutamine does not affect sortase activity (116), mutation of Trp¹⁹⁴ to alanine reduced the enzyme's activity both in vitro and in vivo (201). Thus, Trp¹⁹⁴ could play a role in positioning His¹²⁰ in the proper orientation to achieve catalysis.

The observed pK_as for the side chains of both Cys¹⁸⁴ and His¹²⁰ preclude the possibility of a thiolate-imidazolium ion pair within the sortase active site (32). Using an inhibitor of sortase obtained after the replacement of the T-G peptide bond with a vinyl sulfone, which reacts with cysteine thiolate, the investigators examined inhibition as a function of proton concentration. While the K_i, a value that reflects the binding of the inhibitor to the enzyme, remained constant, the k_i, a measure of the effectiveness of the inhibitor, increased only beyond pH 9.4 (32). This argues in favor of the presence of a thiol group in the sortase active site at physiological pH. The pK_a for the imidazol group of His¹²⁰ was determined by NMR following the chemical shifts of ¹H-1 and ¹H-1 atoms of this residue as a function of pH. The titration suggested a pK_a of approximately 7.0 (32). Again, this indicates that at pH 7.5 the imidazol group of His¹²⁰ would be only partially protonated. Moreover, the observed pK_a is independent of Cys¹⁸⁴, as the titration curve for a sortase Cys¹⁸⁴Ala mutant did not change (32). Together these experiments suggest that sortase catalysis cannot occur via a mechanism involving the thiolate-imidazolium ion pair, as originally proposed (84, 201).

Analysis of the X-ray crystallographic structure of sortase A with LPETG peptide led to the formulation of a new hypothesis. As is pointed out above, this structure revealed the presence of Arg¹⁹⁷ in the active site (227). This residue is absolutely conserved among sortases and is positioned in front of and parallel to Cys¹⁸⁴. Replacement of Arg¹⁹⁷ with alanine, lysine, or histidine greatly impaired sortase activity, both in vivo and in vitro (116). Because the guanidinium group of Arg¹⁹⁷ interacts with the carbonyl group of the scissile bond in the X-ray structure, it was proposed that Arg¹⁹⁷ forms an oxyanion hole that may stabilize the acylated adduct (227). This hypothesis was corroborated by an experiment in which hydroxylamine was unable to resolve the acyl intermediate when Arg¹⁹⁷ was replaced by alanine or lysine, indicating that in the absence of the guanidinium group, the thioacyl intermediate is not formed (116). These results suggest that the sortase active site may comprise a cysteine-arginine dyad (225, 228). It is important to note that sortases display absolute conservation of several residues. Two of these, Leu⁹⁷ and Tyr¹⁵³, have been replaced by alanine in order to assess their importance for the enzyme's activity. Despite their conservation, these residues were not required for sortase activity either in vitro or in vivo (201). The contribution of other conserved amino acids to sortase catalysis remains unknown.

The specificity of sortase A for different pentapeptide motifs was studied by determining the in vitro activity of the enzyme towards a peptide library with 18 amino acid substitutions in every position (99). This study confirmed bioinformatic analysis of sortase substrates, which indicate that the enzyme recognizes LPXTG sequences. Not surprisingly, initial-velocity analysis showed that only leucine is tolerated in position 1 in XPETG peptides and only proline is tolerated in position 2 in LXETG peptides, whereas any residue is tolerated in position 3 in LPXTG peptides. Only threonine in position 4 in LPEXG peptides is accepted as a substrate, and only glycine is accepted in position 5 in the LPETX peptide library. The enzyme's residues involved in this specificity were detected by comparing NMR signals of bound versus unbound sortase (see above) (108). Besides those in Cys¹⁸⁴ and Arg¹⁹⁷, chemical shift changes in Thr¹⁸⁰ and Ala¹¹⁸ (absolutely conserved residues) and Ile¹⁸² (partially conserved) were also detected. Mutation of these residues significantly impaired sortase activity in vitro (108). Whether these residues contribute to the substrate specificity of sortase remains to be assessed, and it would be interesting to screen the peptide library and determine whether peptides with sequences differing from LPXTG can be substrates of these mutants.

Another important aspect of the sortase reaction is the interaction of the enzyme with its cell wall substrate, the pentaglycine cross bridge. In vivo, sortase can catalyze the transpeptidation of surface proteins to cell wall cross bridges containing one, three, and five glycine residues, but not to the -NH₂ group of the L-lysine residue of wall peptides. This conclusion was reached following analysis of the anchor structure of surface proteins generated by S. aureus fem mutants defective in cross bridge biosynthesis (196). At least three Fem factors (factors essential for methicillin resistance) are required for the addition of glycine residues to the cross bridge of S. aureus peptidoglycan (101). FemX is responsible for the addition of the first glycine residue to the L-lysine of the wall peptide, while FemA adds the second and third glycine residues and FemB completes the cross bridge by incorporating the fourth and fifth glycine. Therefore, femB mutants synthesize Gly₃ cross bridges, femA mutants synthesize Gly₁ cross bridges, and a partial femAX mutant either carries Gly₃ cross bridges or completely lacks cross bridge (97). The cell wall anchor structure of 11-hydrolase-released Seb-Spa_CWS, which is expressed in each of these fem mutants (see above), revealed that sortase can link surface protein to Gly₅, Gly₃, and Gly₁ cross bridges in wild-type, femB, femA, and femAX strains but failed to anchor protein to the -amino of L-Lys (196). Nevertheless, the velocity of the sorting reaction is diminished in fem mutants compared to the wild type (196), indicating that sortase prefers pentaglycine as a cell wall substrate. This conjecture was corroborated in vitro by the observation that Gly, Gly₂, and Gly₃ can be used as nucleophiles by the enzyme and are linked to the threonine of LPETG peptides (77, 200). Diglycyl-histidine and diglycyl-leucine can also be used in the in vitro transpeptidation reaction, although the binding is decreased (as deduced from the apparent K_m values). Glycyl-alanine and glycyl-valine also retain substrate properties, but their binding is reduced by 10-fold. In contrast, alanyl-glycine and valyl-alanine cannot be used as substrates for the transpeptidation reaction (77). Thus, cell wall substrate recognition of sortase tolerates only glycine as the N-terminal residue and strongly prefers another glycine at the second position. While the enzyme's constraints for the selection of a cell wall substrate are being delineated, the actual binding site for peptidoglycan substrate remains unknown. Gly₃ substrate was modeled into the crystal structure of sortase (227). It was speculated that Gly₃ may be positioned in the loop that connects ß7 and ß8, replacing a water molecule that otherwise contacts the backbone atoms of this loop. Nevertheless, experimental data are needed to reveal the peptidoglycan binding site of sortase.

Additional evidence for lipid II as the peptidoglycan substrate for surface protein anchoring was garnered with in vitro reactions. LPXTG peptide is linked to lipid II by purified sortase A, and vancomycin can block this reaction (165). Analysis of surface protein anchoring in protoplasts promoted the notion that the sorting reaction does not require mature, assembled peptidoglycan (202). The cell wall envelope of S. aureus was removed by digestion with muralytic enzyme, protoplasts were pulse-labeled with [³⁵S]methionine, and the radiolabeled surface protein was immunoprecipitated. Protoplasts catalyzed surface protein precursor cleavage at the LPXTG motif at a rate similar to that for staphylococci with intact cell wall envelopes. A unique surface protein sorting intermediate was detected in protoplast membranes. Further evidence for a linkage between surface proteins and lipid II in vivo was obtained by labeling staphylococci with [³²P]phosphoric acid, which is incorporated into lipid II molecules (154). Following removal of the cell wall envelope with muramidase, which cannot cleave lipid II, labeled polypeptides were immunoprecipitated and detected by autoradiography. ³²P-labeled surface protein species were identified, and their synthesis required sortase A activity. Radiolabeled lipid II could be removed from surface protein by lysostaphin cleavage at pentaglycine cross bridges, whereas muramidase, which cannot cleave lipid II, displayed no effect. Treatment of staphylococci with tunicamycin, an inhibitor of phosphor-N-acetylmuramyl-pentapeptide translocase (the enzyme required for formation of lipid I and lipid II [191]) abolished sortase A-dependent biosynthesis of ³²P-labeled surface protein. The C-terminal anchor of immunoprecipitated ³²P-labeled surface protein was analyzed by thin-layer chromatography and observed to bind nisin (154), an antibiotic that specifically interacts with lipid II (214). Thus, the cell wall sorting intermediate P3 is comprised of surface protein linked to lipid II (154). A model that emerged from these studies suggests that P3 not only is the product of the sortase reaction but also serves as a substrate for the transglycosylation and transpeptidation reactions of cell wall biosynthesis, similar to the case for lipid II (Fig. 5). Obviously, the amino group of the pentaglycine cross bridge of P3 is already engaged in an amide bond and cannot perform the nucleophilic attack at PBP acyl enzyme intermediates with cleaved wall peptides. Nevertheless, the pentapeptide structure permits PBP cleavage at the D-Ala-D-Ala of P3 and attachment of other pentaglycine cross bridges from neighboring wall peptides at this site. In this manner, the P3 sorting intermediate can be fully incorporated into the three-dimensional network of staphylococcal peptidoglycan.

The first search for sortase inhibitors occurred even before the enzyme was identified (202). Methane-thiosulfonates such as MTSET and (2-sulfonatoethyl)methane-thiosulfonate inhibit sortase in vivo and in vitro, with MTSET achieving complete inhibition. The mercurial p-hydroxymercuribenzoic acid could also inhibit sortase. All of these compounds react with the catalytic Cys¹⁸⁴ and prevent formation of acyl intermediates. In contrast, sulfhydryl alkylating agents such as iodoacetamide, N-ethylmaleimide, or iodoacetic acid do not inhibit sortase. While these reagents proved useful to elucidate the catalytic mechanism of the enzyme, nondiscriminate interactions of thiol-reactive molecules renders these compounds useless for therapeutic studies because of their associated toxicity in mammalian organisms.

Several recent efforts have examined natural or chemical compounds for the property of inhibiting sortase A in vitro. For example, extracts from 80 medicinal plants were tested and those obtained from Cocculus trilobus, Fritillaria verticillata, Liriope platyphylla, and Rhus verniciflua displayed inhibitory activity (95). The extract from Fritillaria verticillata bulbs was subjected to silica gel chromatography, and a fraction with potent inhibitory effects on sortase was isolated. The constituent of this fraction was identified by NMR as glucosylsterol ß-sitosterol-3-O-glucopyranol (93). As sitosterol alone does not inhibit sortase, it was concluded that the inhibitory effect must reside within the glucopyranoside moiety of the molecule. A similar experimental approach for extracts of Coptis chinensis identified the isoquinoline alkaloid berberine chloride as a sortase inhibitor (94). Both compounds exhibit a lower MIC than p-hydroxymercuribenzoic acid (see above) and were able to inhibit binding of S. aureus to fibronectin-coated surfaces (143), an interaction mediated by the sortase A substrates fibronectin binding proteins A and B (FnbpA and FnbpB) (see above). However, the k_i values for these inhibitors have not been obtained, precluding their comparison with other known sortase inhibitors.

Another strategy for the development of inhibitors employed modifications to the scissile bond of LPXTG peptides. In the first of these studies, the threonine-glycine peptide bond was substituted by moieties known to alkylate the active-site thiol of cysteine proteases. These included peptidyl-diazomethane (LPAT-CHN₂) and peptidyl-chloromethane (LPAT-CH₂Cl) (176). Both compounds successfully inhibited sortase activity in vitro, with a k_i/K_i of 2.2 x10⁴ M^–1 · min^–1 (k_i = 5.8 x 10^–3 min^–1) for LPAT-CHN₂ and a k_i/K_i of 2.1 x10⁴ M^–1 · min^–1 (k_i = 1.1 x 10^–2 min^–1) for LPAT-CH₂Cl. In a second study, the scissile bond was replaced with vinyl sulfone [LPAT-SO₂(Ph)], a moiety known to covalently modify the active-site thiolate of cysteine proteases via formation of a thioether adduct (32). Due to the requirement for ionization of the thiol group of Cys¹⁸⁴, this modified peptide achieved maximal inhibition at pHs greater than 8.0. As expected, inhibition was irreversible, and at pH 7.0 the k_i/K_i was measured to be 44.4 M^–1 · min^–1 (k_i = 4 x 10^–4 min^–1). Different types of vinyl sulfones, i.e., di-, ethyl-, methyl-, and phenyl vinyl sulfones, all inhibited sortase A. Phenyl vinyl sulfone (PVS) displayed the greatest effect, with a k_i/K_i of 20.1 M^–1 · min^–1 (55). Interestingly, PVS-treated S. aureus cells failed to bind to a fibronectin-coated surface, suggesting that PVS can inhibit the sortase-dependent surface display of fibronectin binding proteins in vivo. However, additional studies documenting the effects of PVS on mammalian cell viability and on other steps of the sorting reaction are required for a clearer understanding and confirmation of this inhibition.

Substrate peptides have been generated with the expectation of mimicking the transition state for the formation of sortase acyl intermediates. In order to obtain such an inhibitor, the threonine residue of an LPETG peptide was replaced by a phosphinate group (LPE{PO₂H-CH₂}G) (98), a peptide modification that has been successfully used for the design of zinc protease inhibitors. As the tetravalent coordination of the phosphorous atom imitates the acyl intermediate transition state, this modified peptide should compete with LPETG substrate for the sortase active site. Inhibition was achieved with the phosphinate compound and was therefore exploited to determine different kinetic parameters of the sortase reaction (98).

Another strategy for the discovery of sortase inhibitors has been to screen libraries of small-molecule compounds (142). One thousand compounds were tested for their ability to inhibit sortase in vitro, and the initial hits were subjected to successive structural and chemical modifications with the goal of achieving more pronounced inhibitory effects on sortase activity. This resulted in the isolation of a set of substituted (Z)-diarylacrylonitriles that exhibit potent inhibition towards sortase. Most of the compounds described here were tested only in vitro and typically require micromolar or low millimolar concentrations for inhibition of sortase. Much work still needs to be done before one can analyze compounds with K_i at low micromolar or nanomolar concentrations and with inhibitory specificity that permits testing in animal models of S. aureus pathogenesis.

Another application of the sortase reaction is the generation of self-cleavable chimeras for one-step purification of recombinant proteins (113). The concept relies on the expression and purification of a recombinant His₆-sortase-LPETG-target protein fusion that cleaves itself once the enzyme has been activated by the addition of calcium and triglycine. The transpeptidation product, i.e., nontagged target protein, can be eluted in a single chromatography step, with glycine as the only modification introduced by the purification procedure. The sortase strategy differs from other systems employing N-terminal carriers that are cleaved off from the target protein by the addition of a protease, in which the separation of the target protein from the protease requires additional chromatography steps. The approach was tested for the purification of GFP, Cre, and p27 proteins (113). In all cases, the presence of an N-terminal sortase carrier increased the expression and solubility of the recombinant protein. Importantly, neither autocleavage nor transpeptidation with E. coli proteins containing an N-terminal glycine was observed during expression. Following affinity chromatography of cleared cell lysate on Ni-nitrilotriacetic acid Sepharose and several washes, charged resin was incubated in buffer containing calcium and Gly₃. Concentrated and 98% pure target protein was recovered from the supernatants, indicating that sortase-based protein purification provides a simple and effective method that may be generally applicable to many proteins.

S. AUREUS SORTASE B

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Sortase homologs have been revealed in every gram-positive bacterium for which genome sequences are available, and most species encode more than one sortase (148). S. aureus encodes two sortases, and the second enzyme has been named sortase B (122, 123). The structural gene for sortase B (srtB) is part of the isd (iron-regulated surface determinant) locus, which is comprised of three transcriptional units, isdA, isdB, and isdCDEF-srtB-isdG (Fig. 6A) (123). IsdA, IsdB, and IsdC are cell wall-anchored proteins. IsdD is thought to be inserted into the plasma membrane. IsdE lipoprotein and the IsdF ATP binding cassette (ABC) transporter presumably function as heme-iron transporters in the plasma membrane. IsdG is located in the cytoplasm, and it cleaves the heme tetrapyrrol ring and liberates iron for staphylococcal growth. IsdA, IsdB, IsdC, IsdD, IsdE, and IsdG all bind heme-iron (121). Additionally, IsdB (but not IsdA or IsdC) binds hemoglobin (121). Two additional Isd proteins are encoded elsewhere in the genome of S. aureus: IsdH (HarA), a haptoglobin binding, cell wall-anchored protein (42), and IsdI, an IsdG homolog with heme oxygenase activity (181). A fur box (46), i.e., a DNA sequence to which the ferric uptake repressor binds and inhibits transcription when staphylococci grow in iron-replete conditions (76, 215), is present in the promoter regions of all of these genes. Thus, the Isd proteins and sortase B are expressed only under conditions when iron is limiting (121, 123). However, fur mutant staphylococci express the isd locus and srtB in a constitutive fashion (123).

View larger version (47K): [in this window] [in a new window]   FIG. 6. Isd-mediated heme-iron uptake in S. aureus. A. The isd locus is comprised of isdA, isdB, and isdC, which encode cell wall-anchored proteins carrying LPKTG, LPQTG, and NPQTN motifs in their respective sorting signals. Located elsewhere in the S. aureus genome, isdH and isdI encode a fourth LPKTG surface protein and a heme oxygenase, respectively. All isd genes are regulated by the ferric uptake repressor (Fur), which represses transcription under iron-replete conditions by binding to fur boxes present in promoter regions (shaded boxes). Arrows indicate the direction of transcription. B. A model for Isd-mediated heme-iron transport across the cell wall of S. aureus. IsdA, IsdB, and IsdH are anchored to the cell wall by sortase A and function as receptors for hemoprotein ligands, including haptoglobin (Hpt), hemoglobin (Hb), or heme. Upon binding to Isd receptors, heme is released from the hemoproteins by an as-yet-undefined mechanism and passaged through the cell wall in an IsdC-dependent manner. Treatment of staphylococcal cells with extracellular proteinase K completely degrades IsdB, only partially digests IsdA, and leaves IsdC intact, suggesting different degrees of surface exposure for each of these cell wall proteins. The heme molecule is then transported through the membrane transport system composed of IsdDEF into the cytoplasm. Upon entry into the cytoplasm, heme is degraded by IsdG and IsdI heme monooxygenases. This leads to the release of free iron for use by the bacterium as a nutrient source. (Adapted from reference 182 with permission from Elsevier.)