Protein sorting to the cell wall envelope of Gram-positive bacteria

The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria.     

Functional analysis of the sortase YhcS in Bacillus subtilis

Sortases of Gram-positive bacteria catalyze the covalent C-terminal anchoring of proteins to the cell wall. Bacillus subtilis, a well-known host organism for protein production, contains two putative sortases named YhcS and YwpE. The present studies were aimed at investigating the possible sortase function of these proteins in B. subtilis. Proteomics analyses revealed that sortase-mutant cells released elevated levels of the putative sortase substrate YfkN into the culture medium upon phosphate starvation. The results indicate that YfkN required sortase activity of YhcS for retention in the cell wall. To analyze sortase function in more detail, we focused attention on the potential sortase substrate YhcR, which is co-expressed with the sortase YhcS. Our results showed that the sortase recognition and cell-wall-anchoring motif of YhcR is functional when fused to the Bacillus pumilus chitinase ChiS, a readily detectable reporter protein that is normally secreted. The ChiS fusion protein is displayed at the cell wall surface when YhcS is co-expressed. In the absence of YhcS, or when no cell-wall-anchoring motif is fused to ChiS, the ChiS accumulates predominately in the culture medium. Taken together, these novel findings show that B. subtilis has a functional sortase for anchoring proteins to the cell wall.     

Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence

During infection of their hosts, Gram-positive bacteria express surface proteins that serve multiple biological functions. Surface proteins harbouring a C-terminal sorting signal with an LPXTG motif are covalently linked to the cell wall peptidoglycan by a transamidase named sortase. Two genes encoding putative sortases, termed srtA and srtB, were identified in the genome of the intracellular pathogenic bacterium Listeria monocytogenes. Inactivation of srtA abolishes anchoring of the invasion protein InlA to the bacterial surface. It also prevents the proper sorting of several other peptidoglycan-associated LPXTG proteins. Three were identified by a mass spectrometry approach. The DeltasrtA mutant strain is defective in entering epithelial cells, similar to a DeltainlA mutant. In contrast to a DeltainlA mutant, the DeltasrtA mutant is impaired for colonization of the liver and spleen after oral inoculation in mice. Thus, L. monocytogenes srtA is required for the cell wall anchoring of InlA and, presumably, for the anchoring of other LPXTG-containing proteins that are involved in listerial infections.     

Anchor structure of staphylococcal surface proteins. V. Anchor structure of the sortase B substrate IsdC

Staphylococcus aureus sortase A cleaves surface protein precursors bearing C-terminal LPXTG motif sorting signals between the threonine and glycine residues. Using lipid II precursor as cosubstrate, sortase A catalyzes the amide linkage between the carboxyl group of threonine and the amino group of pentaglycine cross-bridges, thereby tethering C-terminal ends of surface proteins to the bacterial cell wall envelope. Staphylococcal sortase B also anchors its only known substrate, the IsdC precursor with a C-terminal NPQTN motif sorting signal, to the cell wall envelope. Herein, we determined the cell wall anchor structure of IsdC. The sorting signal of IsdC is cleaved between threonine and asparagine of the NPQTN motif, and the carboxyl group of threonine is amide-linked to the amino group of pentaglycine crossbridges. In contrast to sortase A substrates, the anchor structure of IsdC displays shorter glycan strands and significantly less cell wall cross-linking. A model is proposed whereby sortases A and B recognize unique features of sorting signals and peptidoglycan substrates to deposit proteins with distinct topologies in the cell wall envelope.     

Anchor structure of cell wall surface proteins in Listeria monocytogenes

Many surface proteins of Gram-positive bacteria are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. In Staphylococcus aureus, surface proteins are cleaved between the threonine and the glycine of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the amino group of the pentaglycine cell wall crossbridge. Here we investigated the anchor structure of surface proteins in Listeria monocytogenes. A methionine and six histidines (MH(6)) were inserted upstream of the LPXTG motif of internalin A (InlA), a cell-wall-anchored surface protein of L. monocytogenes. The engineered protein InlA-MH(6)-Cws was found anchored in the bacterial cell wall. After peptidoglycan digestion with phage endolysin, InlA-MH(6)-Cws was purified by affinity chromatography. COOH-terminal peptides of InlA-MH(6)-Cws were obtained by cyanogen bromide cleavage followed by purification on a nickel-nitriloacetic acid column. Analysis of COOH-terminal peptides with Edman degradation and mass spectrometry revealed an amide linkage between the threonine of the cleaved LPXTG motif and the amino group of the m-diaminopimelic acid crossbridge within the listerial peptidoglycan. These results reveal that the cell wall anchoring of surface proteins in Gram-positive bacteria such as S. aureus and L. monocytogenes occurs by a universal mechanism.     

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

The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.     

Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria.

The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.     

The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing

Many surface proteins of pathogenic gram-positive bacteria are linked to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins of Streptococcus pneumoniae harbor another motif, YSIRK-G/S, which is positioned within signal peptides. The signal peptides of some, but not all, of the 20 surface proteins of Staphylococcus aureus carry a YSIRK-G/S motif, whereas those of surface proteins of Listeria monocytogenes and Bacillus anthracis do not. To determine whether the YSIRK-G/S motif is required for the secretion or cell wall anchoring of surface proteins, we analyzed variants of staphylococcal protein A, an immunoglobulin binding protein with an LPXTG sorting signal. Deletion of the YSIR sequence or replacement of G or S significantly reduced the rate of signal peptide processing of protein A precursors. In contrast, cell wall anchoring or the functional display of protein A was not affected. The fusion of cell wall sorting signals to reporter proteins bearing N-terminal signal peptides with or without the YSIRK-G/S motif resulted in hybrid proteins that were anchored in a manner similar to that of wild-type protein A. The requirement of the YSIRK-G/S motif for efficient secretion implies the existence of a specialized mode of substrate recognition by the secretion pathway of gram-positive cocci. It seems, however, that this mechanism is not essential for surface protein anchoring to the cell wall envelope.     

Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display

Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis suggests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a β-lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR. Covalent anchoring of this fusion protein to the cell wall was confirmed by using the eight-protease-deficient B. subtilis strain WB800 as the host. Inactivation of yhcS abolished the cell wall anchoring reaction. The amounts of fusion protein anchored to the cell wall were proportional to the levels of YhcS. These data demonstrate that YhcS and YhcR are the sortase and sortase substrate, respectively, in B. subtilis. Furthermore, yhcS is not essential for the survival of B. subtilis under the cultivation condition tested. YhcR fusions were distributed helically in the lateral cell wall. Interestingly, when viewed with an epifluorescence microscope, YhcS also appeared to form short helical arcs. This is the first report to illustrate such distribution of sortases in a rod-shaped bacterium. Models for the spatial distribution of both the sortase and its substrate are discussed. The amount of the reporters displayed on the surface was unambiguously quantified via a unique strategy. Under optimal conditions with the overproduction of YhcS, 47,300 YhcR fusions could be displayed per cell. Displayed reporters were biologically functional and surface accessible. Characterization of the sortase-substrate system allowed the successful development of a YhcR-based covalent surface display system. This system may have various biotechnological applications.     

Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable alpha-amylases in Bacillus subtilis and Escherichia coli cells

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable alpha-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.     

Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis

Sortases are enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria by cleaving a sorting motif located in the C-terminus of the protein substrate. The best-characterized motif is LPXTG, which is cleaved between the T and G residues. In this study, a non-gel proteomic approach was used to identify surface proteins recognized by the two sortases of Listeria monocytogenes, SrtA and SrtB. Material containing peptidoglycan and strongly associated proteins was purified from sortase-defective mutants, digested with trypsin, and the resulting peptide mixture analysed by two-dimensional nano-liquid chromatography coupled to ion-trap mass spectrometry. Unlike enzymes involved in peptidoglycan metabolism, other surface proteins displayed uneven distribution in the mutants. A total of 13 LPXTG-containing proteins were identified exclusively in strains having a functional SrtA. In contrast, two surface proteins, Lmo2185 and Lmo2186, were identified only when SrtB was active. The analysis of the peptides identified in these proteins suggests that SrtB of L. monocytogenes may recognize two different sorting motifs, NXZTN and NPKXZ. Taken together, these data demonstrate that non-gel proteomics is a powerful technique to rapidly identify sortase substrates and to gain insights on potential sorting motifs.     

The structure of sortase B, a cysteine transpeptidase that tethers surface protein to the Staphylococcus aureus cell wall

Many surface proteins of Gram-positive bacteria, which play important roles during the pathogenesis of human infections, are anchored to the cell wall envelope by a mechanism requiring sortases. Sortase B, a cysteine transpeptidase from Staphylococcus aureus, cleaves the C-terminal sorting signal of IsdC at the NPQTN motif and tethers the polypeptide to the pentaglycine cell wall cross-bridge. During catalysis, the active site cysteine of sortase and the cleaved substrate form an acyl intermediate, which is then resolved by the amino group of pentaglycine cross-bridges. We report here the crystal structures of SrtBDeltaN30 in complex with two active site inhibitors, MTSET and E64, and with the cell wall substrate analog tripleglycine. These structures reveal, for the first time, the active site disposition and the unique Cys-Arg catalytic machinery of the cysteine transpeptidase, and they also provide useful information for the future design of anti-infective agents against sortases.     

Cell wall anchor structure of BcpA pili in Bacillus anthracis

Assembly of pili in Gram-positive bacteria and their attachment to the cell wall envelope are mediated by sortases. In Bacillus cereus and its close relative Bacillus anthracis, the major pilin protein BcpA is cleaved between the threonine and the glycine of its C-terminal LPXTG motif sorting signal by the pilin-specific sortase D. The resulting acyl enzyme intermediate is relieved by the nucleophilic attack of the side-chain amino group of lysine within the YPKN motif of another BcpA subunit. Cell wall anchoring of assembled BcpA pili requires sortase A, which also cleaves the LPXTG sorting signal of BcpA between its threonine and glycine residues. We show here that sortases A and D require only the C-terminal sorting signal of BcpA for substrate cleavage. Unlike sortase D, which accepts the YPKN motif as a nucleophile, sortase A forms an amide bond between the BcpA C-terminal carboxyl group of threonine and the side-chain amino group of diaminopimelic acid within the cell wall peptidoglycan of bacilli. These results represent the first demonstration of a cell wall anchor structure for pili, which are deposited by sortase A into the envelope of many different microbes.     

Bacillus anthracis sortase A (SrtA) anchors LPXTG motif-containing surface proteins to the cell wall envelope

Cell wall-anchored surface proteins of gram-positive pathogens play important roles during the establishment of many infectious diseases, but the contributions of surface proteins to the pathogenesis of anthrax have not yet been revealed. Cell wall anchoring in Staphylococcus aureus occurs by a transpeptidation mechanism requiring surface proteins with C-terminal sorting signals as well as sortase enzymes. The genome sequence of Bacillus anthracis encodes three sortase genes and eleven surface proteins with different types of cell wall sorting signals. Purified B. anthracis sortase A cleaved peptides encompassing LPXTG motif-type sorting signals between the threonine (T) and the glycine (G) residues in vitro. Sortase A activity could be inhibited by thiol-reactive reagents, similar to staphylococcal sortases. B. anthracis parent strain Sterne 34F(2), but not variants lacking the srtA gene, anchored the collagen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) BasC (BA5258/BAS4884) to the bacterial cell wall. These results suggest that B. anthracis SrtA anchors surface proteins bearing LPXTG motif sorting signals to the cell wall envelope of vegetative bacilli.     

Secretion activities of Bacillus subtilis alpha-amylase signal peptides of different lengths in Escherichia coli cells

The B. subtilis alpha-amylase promoter and signal peptide are functional in E. coli cells. DNA fragments coding for signal peptides with different lengths (28, 31, 33 and 41 amino acids from the translation initiator Met) were prepared and fused with the E. coli beta-lactamase structural gene. In B. subtilis cells, the sequences of 31, 33 and 41 amino acids were able to secrete beta-lactamase into the surrounding media, but the 28 amino acid sequence was not. In contrast, all of the four sequences were able to export beta-lactamase into the periplasmic space of E. coli cells. Thus, the recognition of the B. subtilis alpha-amylase signal peptide in E. coli cells seems to be different from that in B. subtilis cells.     

Sortase B, a new class of sortase in Listeria monocytogenes

Sortases are transamidases that covalently link proteins to the peptidoglycan of gram-positive bacteria. The genome of the pathogenic bacterium Listeria monocytogenes encodes two sortases genes, srtA and srtB. The srtA gene product anchors internalin and some other LPXTG-containing proteins to the listerial surface. Here, we focus on the role of the second sortase, SrtB. Whereas SrtA acts on most of the proteins in the peptidoglycan fraction, SrtB appears to target minor amounts of surface polypeptides. We identified one of the SrtB-anchored proteins as the virulence factor SvpA, a surface-exposed protein which does not contain the LPXTG motif. Therefore, as in Staphylococcus aureus, the listerial SrtB represents a second class of sortase in L. monocytogenes, involved in the attachment of a subset of proteins to the cell wall, most likely by recognizing an NXZTN sorting motif. The DeltasrtB mutant strain does not have defects in bacterial entry, growth, or motility in tissue-cultured cells and does not show attenuated virulence in mice. SrtB-mediated anchoring could therefore be required to anchor surface proteins involved in the adaptation of this microorganism to different environmental conditions.     

Characterization of the sortase repertoire in Bacillus anthracis

LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss), bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins.     

Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.     

In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus

Many surface proteins of Gram-positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C-terminal sorting signal. To achieve cell-wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell-wall-spanning region and cell-wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell-wall anchoring of enzymatically active lipase. The cell-wall-immobilized lipase (approximately 10000 molecules per cell) retained more than 80% of the specific activity, compared to the C-terminally unmodified S. hyicus lipase secreted by S. carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the influence of the distance between the cell-wall sorting signal and the C-terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the pep-tidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coliβ-lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S, carnosus. These results demonstrate that it is possible to immobilize normally soluble enzymes on the cell wall of S. carnosus - without radically altering their catalytic activity - by fusing them to a cell-wall-immobilization unit, consisting of a suitable cellwall-spanning region and a standard cell-wall sorting signal.