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Journal of Bacteriology, September 2004, p. 5865-5875, Vol. 186, No. 17
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.17.5865-5875.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Novel Sortase, SrtC2, from Streptococcus pyogenes Anchors a Surface Protein Containing a QVPTGV Motif to the Cell Wall

Timothy C. Barnett, Aman R. Patel, and June R. Scott*

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia

Received 26 March 2004/ Accepted 27 May 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Streptococcus pyogenes (group A streptococcus, GAS), is an important gram-positive human pathogen capable of causing a wide variety of diseases (11). The majority of these are mild, self-limiting, suppurative infections of the throat (pharyngitis) and skin (impetigo and pyoderma). However, these infections may lead to serious poststreptococcal sequelae, including rheumatic fever, glomerulonephritis, and reactive arthritis. In addition, GAS have become notorious in recent years for their ability to cause severe, invasive diseases with high mortality rates. These diseases include streptococcal toxic shock syndrome, septicemia, and the "flesh-eating disease," necrotizing fasciitis. The ability of these organisms to cause such a diverse disease spectrum is attributed, in part, to their production of a wide array of extracellular virulence factors. For example, the toxins and superantigens which are secreted by GAS are responsible for many of the symptoms characteristic of different GAS-associated diseases. In addition, cell-associated proteins that are covalently attached to the bacterial surface are important for adherence to, and interaction with, the human host.

Surface proteins of gram-positive bacteria are usually secreted through the cytoplasmic membrane via the Sec system following cleavage of an N-terminal signal sequence (34). These proteins are then either released into the extracellular milieu or attached to the bacterial cell surface. Such surface proteins may be noncovalently associated with other surface molecules such as teichoic acids and lipoteichoic acids, anchored directly to the cytoplasmic membrane by hydrophobic membrane-spanning domains or as lipoproteins, or covalently attached to the cell wall cross-bridge by sortase enzymes (10).

Proteins anchored by sortase contain a cell wall-anchoring domain near the C terminus. This consists of an LPXTG motif followed by a hydrophobic stretch of amino acids and a short, positively charged tail (29, 39, 46). During secretion via the Sec system, the hydrophobic region and charged tail retard translocation across the cytoplasmic membrane (33, 45). The membrane-associated sortase enzyme recognizes the LPXTG motif and anchors it to the cell wall by a two-step transpeptidation reaction (28, 56, 57). This involves recognition of the LPXTG motif and cleavage between the threonine and glycine residues, followed by the covalent linkage of the carboxyl group of threonine to an amino group of the cell wall precursor lipid II (40, 44). In this reaction, the carboxyl group of threonine is covalently attached to the amino group of the pentaglycine branch of lipid II. The resulting molecule is thereby incorporated covalently into the mature cell wall. The universal nature of this reaction in gram-positive organisms is reflected by the ability of Staphylococcus aureus to recognize the cell wall-anchoring domains of proteins from several other gram-positive bacteria, including S. pyogenes, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus sobrinus (45).

Sortase enzymes are characterized by the presence of a conserved histidine (S. aureus SrtA histidine 120) and a conserved cysteine residue (S. aureus SrtA cysteine 184) in a TLXTC signature motif (23, 36). These residues have been demonstrated to be essential for the activity of S. aureus SrtA (55). All gram-positive bacteria examined to date possess a major housekeeping sortase (SrtA), identified by either mutagenesis screens (1, 28), PCR (7, 35), or BLAST searches (5, 18, 25, 26, 36). In S. aureus (27, 28, 30), L. monocytogenes (5, 18), S. pyogenes (1), Streptococcus gordonii (7), Streptococcus mutans (26), Streptococcus suis (35), and Streptococcus pneumoniae (25), this sortase has been shown to be responsible for anchoring the majority of LPXTG-containing proteins to the cell surface.

Analysis of the available genome sequences suggested that most gram-positive bacteria encode one or more additional sortases (36). In many cases, these are encoded close to genes encoding potential sortase substrates, as identified by a C-terminal LPXTG motif followed by a hydrophobic region and charged tail. The anchoring of proteins encoded by genes clustered with an accessory sortase gene has been verified experimentally in four cases (1, 4, 30, 58). We previously showed that some strains of GAS encode two sortases, designated SrtA and SrtB (1). These enzymes recognize different subsets of proteins containing an LPXTG motif. A srtA null mutant is defective in the surface localization of several proteins containing LPXTG followed by an acidic amino acid. SrtB, on the other hand, is required for the surface display of T6 protein, which contains the sequence LPSTG followed by a serine rather than an acidic residue. While the srtA gene is present in all GAS strains examined, srtB is present in only a limited number of strains. This agrees with the results of Bessen and Kalia (3), who showed that the sequence of the region of the GAS genome containing srtB differs among different strains. This region was named the FCT (fibronectin and collagen binding and T antigen) region for the proteins that it encodes.

Using S. aureus SrtA as a BLAST query, Pallen et al. (36) identified two other potential sortase genes (slp3 and slp4) in the genome sequences of GAS. In this study, we searched for these sortase homologs in each of the GAS genome sequences and found that slp3 was encoded in the FCT region of the M1 strain SF370, while slp4 was present in the FCT region of strains belonging to serotypes M3, M5, and M18 (Fig. 1A). Further analysis revealed that slp3 and slp4 were likely to be two different alleles of the same gene, which we have designated srtC1 and srtC2, respectively (Fig. 1B). These genes appear to lie in an operon that also encodes one potential substrate for SrtA and two potential cell wall-anchoring substrates with a motif that differs from LPXTG. We show here that SrtC2 from an M3 strain is required for anchoring of one of these proteins (SPyM3_100), which has a QVPTG motif followed by a hydrophobic region and charged tail. We also demonstrate that substituting LPSTG for QVPTG in this protein prevented its anchoring by either SrtC2 or SrtA.



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  		FIG. 1. FCT regions of different GAS strains. (A) Genomic organization of the FCT regions from different GAS strains. In M1 strains, srtC1 is located in a potential operon with genes encoding a putative signal peptidase (sipA1) and three potential sortase substrate proteins, cpa, SPy128, and SPy130. A similar gene arrangement is seen in other strains. A divergently transcribed gene encoding a potential regulator of this operon (rofA and nra) is located upstream of cpa. Homologous genes are shaded alike. Potential sortase recognition motifs are given in parentheses after gene names. The sequences for M1 (13), M3 (2), M5 (genome sequence at http://www.sanger.ac.uk, GenBank accession no. NC_002958), M6 (3), M12 (3), M18 (49), and M49 (3, 42) have been published. The star denotes that the srtB gene from M12 contains an internal stop codon (codon 102). (B) Multiple sequence alignment of SrtC amino acid sequences from the above strains. Alignment was performed with ClustalW software.

 

   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and plasmids. GAS strain JRS4 is a spontaneous streptomycin-resistant derivative of the serotype M6 strain D471 (48). JRS758 is a srtA mutant derivative of JRS4 (1). GAS strain AM3 (52) was used as a source of DNA for PCR amplification of srtC2-specific sequences. For expression in GAS, DNA fragments were cloned into plasmid pNZ276 under the Lactococcus lactis lacA promoter (12, 41). Escherichia coli strain C600 was used as a host for molecular cloning experiments.

Culture conditions. GAS strains were grown in Todd-Hewitt medium supplemented with 0.2% yeast extract (Difco), and E. coli strains were grown in Luria broth (LB) medium (47) at 37°C. For E. coli and GAS strains harboring pNZ276 derivatives, chloramphenicol was added to the medium at concentrations of 20 and 3 µg/ml, respectively. When appropriate, 1% (wt/vol) glucose was added to LB medium to repress expression from the lacA promoter by catabolite repression.

DNA manipulations. Chromosomal DNA was isolated from GAS strains with the MasterPure DNA extraction kit (Epicentre). Plasmid DNA was isolated with anion exchange columns or Qiaprep spin columns (Qiagen).

Survey for srtA, srtB, srtC1, and srtC2. Internal fragments of sortase genes were amplified with primers srtA-F6 and srtA-R7 (srtA), srtB-F3 and srtB-R3 (srtB), srtC1-F12 and srtC1-R12 (srtC1), and srtC2-F11 and srtC2-R11 (srtC2) (Table 1). PCR was performed with HotStart Taq (Qiagen) according to the manufacturer's protocol. For Southern hybridization analysis (50), PCR products from SF370 (srtC1) and AM3 (srtC2) were labeled with [{alpha}-32P]dATP (Decaprime labeling kit; Ambion) and used to probe HindIII-digested DNA.


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  		TABLE 1. Primers used in this study

 
Construction of bacterial strains expressing Orf100HA and SrtC2. A sequence encoding a hemagglutinin (HA) tag (Sigma) was introduced into the SPyM3_0100 open reading frame (hereafter orf100) by a two-step overlapping PCR protocol with the high-fidelity enzyme Herculase (Stratagene). Primers Cpa-F4 and OrfA-HA-R (Table 1) were used to amplify the 5' 297 bp of orf100 and 2,214 bp of upstream sequence containing sipA2. Similarly, primers OrfA-HA-F and OrfB-R1 (Table 1) were used to amplify the 3' 735 bp of orf100 and 2,317 bp of downstream sequence containing srtC2. Primers OrfA-HA-F and OrfA-HA-R contain additional 27-bp sequences that encode a 9-amino-acid HA tag and also form overlapping regions between the two PCR products. The PCR products were gel purified, combined, and used as the template for second-round reactions.

A DNA fragment containing sipA2-orf100HA was amplified with nested PCR primers LepA-PstI-F and OrfA-R3 (Table 1), digested with PstI and SmaI, and ligated into pNZ276 digested with PstI and MscI to create pJRS1316. Similarly, a fragment containing sipA2-orf100HA-srtC2 was amplified with primers LepA-PstI-F and SrtC2-EcoRV-R (Table 1), digested with PstI and EcoRV (pJRS1317), and ligated into PstI- and MscI-digested pNZ276 to produce pJRS1317.

Construction of strains expressing Orf100HA-LPSTGE. The sequence of orf100 encoding the QVPTGV motif in plasmids pJRS1316 and pJRS1317 was changed to a sequence encoding the LPSTGE motif of the M6 protein (21) to create plasmids pJRS1329 and pJRS1330, respectively. Mutagenesis was performed with the QuikChange mutagenesis system (Stratagene) according to the manufacturer's protocol, along with mutagenic primers OrfA-LPSTGE-F and OrfA-LPSTGE-R (Table 1).

Cell fractionation and immunoblot studies. Cell wall and culture supernatant fractions were prepared by the method of Pancholi and Fischetti (37). Immunoprecipitation of cell wall extracts was performed with anti-HA affinity matrix (Roche) according to the manufacturer's protocol. Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels or 4 to 12% gradient gels (Invitrogen) and transferred to a nitrocellulose membrane (Nitrobind; Osmonics Inc.) for immunoblot analysis.

Whole-cell immunoblots and detection of proteins with antiserum were performed as described previously (6). The HA-7 monoclonal antibody (Sigma) was used at a 1:1,000 dilution. The 10B6 monoclonal antibody (24) was used at a 1:2,000 dilution.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of an operon containing a third sortase homolog from GAS. To determine whether the two sortase gene homologs (slp3 and slp4) identified by Pallen et al. (36) encode sortases that anchor proteins to the cell wall, we searched for both genes in each of the available GAS genome sequences. We found that the genome of the M1 strain SF370 contains slp3, while slp4 is present in the sequences of the strains belonging to serotypes M3, M5, M12, M18, and M49. Furthermore, the slp homolog is always located in the FCT region (3), defined as the region between genes homologous to Spy0123 and SPy0136 of the M1 strain SF370 (Fig. 1A). Further analysis revealed that with the exception of the M6 strain JRS4, all six strains for which the FCT region sequence is available have a slp3 or slp4 homolog.

Alignment of the amino acid sequences encoded by slp3 and slp4 indicated that they are likely to be two different alleles of the same gene. We have named them srtC1 and srtC2, respectively (Fig. 1B). The M1 strain contains srtC1, while the other sequenced strains have srtC2. The srtC1 gene from SF370 encodes a protein predicted to be 46.5% identical and 66.4% similar to the protein encoded by srtC2 from MGAS315, 12.4% identical and 24.6% similar to SrtA from SF370, 15% identical and 26.8% similar to SrtB from the M6 strain JRS4, and 27.8% identical and 49.6% similar to SrtB from S. aureus. SrtC1 and SrtC2 both possess a conserved histidine residue (at positions 149 and 151, respectively) and a conserved cysteine residue in a region that resembles the TLXTC signature sequence characteristic of sortase enzymes (ALSTC for SrtC1 and AFSTC for SrtC2).

In the SF370 (M1) genome, srtC1 appears to lie in an operon containing three genes (cpa, Spy128, and Spy130) that encode proteins with potential cell wall-anchoring domains and a putative signal peptidase gene (sipA1) (Fig. 1A). The C terminus of Cpa from SF370 contains the sequence VVPTG followed by a hydrophobic region and a charged tail. Similarly, the C terminus of Spy128 from SF370 contains the sequence EVPTG, while Spy130 contains LPSTGE preceding a hydrophobic region and a charged tail. Spy130 is predicted to be a substrate for SrtA because it contains an LPXTG motif followed by an acidic residue (1). However, based on our previous results, Cpa and Spy128 are unlikely to be substrates for SrtA or SrtB because they do not contain an LPXTG motif preceding the hydrophobic region. Instead, these predicted proteins contain related sequences that could serve as substrates for another sortase, possibly SrtC1, which is encoded in the same operon.

Upstream of these genes and divergently transcribed from them is a positive regulatory gene, rofA (17). Downstream of Spy130 are two genes transcribed in the other direction that exhibit homology to IS66 family transposases (SPy131 and SPy133), and these are followed by srtB (1).

The arrangement of genes in the FCT region in strains containing srtC2 is similar to that in the M1 strain SF370 (Fig. 1A). For example, in the M3 strain MGAS315, srtC2 appears to lie in an operon encoding three potential sortase substrates that exhibit homology to proteins encoded by M1 genes cpa (SPyM3_0098; 48.5% identity, 60.4% similarity), SPy0128 (SPyM3_100; 36.4% identity, 47.7% similarity), and SPy0130 (SPyM3_0102; 28.8% identity, 45% similarity) as well as a signal peptidase (sipA2; 39.4% identity, 51.6% similarity). Upstream of cpa is a divergently transcribed gene that has homology to nra, which encodes a negative regulator of cpa in an M49 strain (42). Cpa and Spy128 homologs from strains encoding srtC2 all contain the sequences VPPTG and QVPTG, respectively, followed by a hydrophobic region and a charged tail, and are possible substrates for SrtC2. A homolog of Spy130 from these strains contains the sequence LPLAGE, which could be a substrate for SrtA. However, the genes downstream of SPyM3_0102 are different from those downstream of SPy0130 in SF370. Downstream of SPyM3_0102 is SPyM3_0103, encoding a putative AraC-type regulator, and prtF2, encoding a fibronectin binding protein (20).

To determine the relative distribution of srtC1 and srtC2 among different strains of GAS, we developed a PCR assay with specific primers (srtC1-F12 and srtC1-R12; srtC2-F11 and srtC2-R11) (see Materials and Methods). The results of this analysis are summarized in Table 2. Confirming and extending the analysis of available sequences above, the PCR results showed that the srtC1 gene is present in and unique to M1 strains, while srtC2 is present in strains belonging to serotypes M2, M3, M5, M12, M18, M22, M49, and M50. These strains include representatives that are opacity factor positive and negative and include some that are very genetically distant, as determined by multilocus enzyme electrophoresis (31). Strains belonging to serotypes M4 and M6 and strains 64/14 and MGAS273 do not contain a srtC1 or srtC2 allele, as determined by PCR analysis (Table 2).


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  		TABLE 2. Distribution of sortase genes in different GAS strains

 
To rule out the possibility that these strains possessed a srtC1 or srtC2 gene that was not able to be amplified with the PCR primers used above due to sequence divergence, we examined each of these strains by Southern hybridization with probes internal to srtC1 from strain SF370 and srtC2 from strain AM3. Under high-stringency hybridization conditions (50% formamide, 42°C), the srtC1 and srtC2 probes hybridized to DNA from SF370 and AM3, respectively, but not to DNA from any of the strains that were negative for these genes by PCR (data not shown). We did not observe cross-hybridization between srtC1 and srtC2. However, under stringency conditions low enough to detect cross-hybridization between srtC1 and srtC2 (20% formamide, 37°C), we did observe hybridization of the srtC1 probe to a {approx}3.0-kb HindIII fragment from strains 64/14 and MGAS273 (data not shown). This is unlikely to be caused by hybridization to srtA- or srtB-specific sequences because these genes are encoded on 1.1-kb and 5.0-kb HindIII fragments, respectively, in strain 64/14 (1). Furthermore, no hybridization of the srtC1 probe to DNA from M6 strains JRS4 and MGAS303, both of which possess srtA and srtB (1), was detected. Therefore, strains 64/14 and MGAS273 may encode an additional uncharacterized sortase related to srtC1 and srtC2.

Anchoring of Orf100 to the cell wall of GAS requires SrtC2. We reasoned that the protein encoded by SPyM3_0100 (hereafter called orf100) could be a substrate for SrtC2 because it contains the sequence QVPTG in place of an LPXTG motif near its C terminus. To detect this protein (Orf100), we introduced a sequence encoding an HA tag into an orf100 gene on a plasmid, creating orf100HA. Because the N terminus of Orf100 is probably required for secretion through the cell membrane and the C terminus is expected to be needed for cell wall anchoring, we added the tag at an internal region of the protein that we identified as likely to be accessible to detection with an anti-HA monoclonal antibody (HA-7). We placed the HA tag in a region containing a stretch of hydrophilic amino acids that is predicted to have a high probability of surface localization, between amino acids 99 and 100 of the protein encoded by orf100.

To assess the role of SrtC2 in the anchoring of Orf100HA to the cell surface, we constructed two plasmids that differ only in the presence of srtC2 (Fig. 2A). Plasmid pJRS1316 contains the signal peptidase gene sipA2 and orf100HA, while pJRS1317 contains sipA2, orf100HA, and srtC2, cloned into pNZ276 under the lacA promoter from Lactococcus lactis (12, 41). These plasmids were transformed into strain JRS4, a serotype M6 strain that lacks the srtC2 locus.



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  		FIG. 2. Anchoring of Orf100HA to the cell surface requires SrtC2. (A) Regions of the srtC2 operon cloned into pNZ276 under the Plac promoter. Plasmid pJRS1316 contains sipA2 and orf100HA, while pJRS1317 contains sipA2, orf100HA, and srtC2. (B) Whole-cell immunoblot analysis for surface localization of Orf100HA in JRS4 strains harboring pNZ276, pJRS1316, and pJRS1317. Strains were grown overnight and resuspended in saline at an optical density at 600 nm of 2.0. Serial twofold dilutions were spotted onto a nitrocellulose membrane and reacted with monoclonal antibody HA-7. (C) Western immunoblot of cell wall and culture supernatant extracts of JRS4 containing pJRS1316 or pJRS1317 reacted with HA-7 monoclonal antibody. Cell wall extracts were prepared with phage lysin (14) and immunoprecipitated prior to SDS-PAGE. Proteins were separated by SDS-PAGE on 4 to 12% gradient gels, transferred to nitrocellulose, and detected with HA-7 monoclonal antibody. The sizes of molecular mass standards (in kilodaltons) are indicated to the left. The heavy (H) and light (L) chains of the antibody used for immunoprecipitation are also indicated.

 
The presence of Orf100HA on the surface of cells of strain JRS4 containing either pJRS1316 or pJRS1317 was first investigated with a whole-cell immunoblot assay, which we have used previously to determine cell wall anchoring of proteins by SrtA and SrtB (1). The monoclonal anti-HA antibody reacted with whole cells of JRS4/pJRS1317, which encodes srtC2, whereas no HA was detected in whole cells of strain JRS4/pJRS1316, which lacks srtC2, or strain JRS4/pNZ276 (Fig. 2B).

To confirm this result and to determine whether Orf100HA is covalently attached to the cell wall, cells were fractionated into cell wall and culture supernatant fractions, boiled in SDS-PAGE sample buffer, and examined by Western immunoblot (Fig. 2C). The cell wall fractions were obtained by digestion of the cells with phage lysin, an amidase that cleaves the amide bond between N-acetylmuramic acid and L-alanine in the peptidoglycan cross bridge (14) (Fig. 2C), or by digestion of the cells with the muramidases mutanolysin and lysozyme (data not shown). The supernatant was then immunoprecipitated with an anti-HA affinity matrix (Roche). Cell wall extraction with phage lysin or mutanolysin/lysozyme releases peptidoglycan fragments of various sizes (presumably due to partial digestion) attached to covalently anchored cell wall proteins (15). Thus, following SDS-PAGE analysis, a cell wall-anchored protein appears as a ladder of fragments migrating more slowly than expected for the unattached mature protein.

Immunoprecipitated phage lysin extracts (Fig. 2C) and mutanolysin/lysozyme extracts (data not shown) from JRS4/pJRS1317 (containing srtC2) showed the expected ladder of proteins that react with anti-HA, while extracts of JRS4/pJRS1316 (without srtC2) did not. This suggests that Orf100HA is covalently anchored to the GAS cell wall only when SrtC2 is present. A similar ladder of proteins was observed following extraction of cell wall material from the M3 strain AM3 containing pJRS1317 (data not shown). The mobility of this ladder of HA-containing bands corresponds to molecular masses ranging from about 80 to >220 kDa. In addition, near the top of the gel, a smear of very high molecular weight material that reacted with the HA antiserum is visible in the cell wall fraction of JRS4/pJRS1317. The nature of this material was not investigated further, although it was also present in the supernatant of this strain in either the exponential or stationary phase of growth (Fig. 2C and data not shown).

Phage lysin extracts of both JRS4/pJRS1316 and JRS4/pJRS1317 contained a major anti-HA reactive band at the location expected for a protein of about 37 kDa. We believe this to represent an intermediate step in the cell wall attachment process probably detected because orf100HA is overproduced in our strains. Because it is overproduced, some might appear in the cell wall fraction. This phenotype is similar to that seen with S. aureus protein A from mutants that lack an LPXTG motif (46) and for protein A in an S. aureus srtA mutant (27). In both of these cases, protein A is not recognized by SrtA and is missorted into the cytoplasm, membrane, and cell wall fractions.

Altering the QVPTGV motif near the C terminus prevents anchoring of Orf100 to the cell wall by SrtC2. The cell wall-anchoring motif recognized by sortase usually consists of LPXTG followed by a hydrophobic stretch of amino acids and a positively charged tail. Orf100 lacks the LPXTG sequence and instead contains QVPTGV followed by a hydrophobic region and a charged tail. To test whether the QVPTGV sequence is necessary for cell wall anchoring by SrtC2, this sequence was replaced with the LPSTGE sequence of protein M6, a GAS protein whose cell wall anchoring is SrtA dependent (1). For this purpose, the sequence encoding the QVPTGV sequence encoded in plasmids pJRS1316 and pJRS1317 was replaced with a sequence encoding LPSTGE, creating plasmids pJRS1329 (no srtC2) and pJRS1330 (with srtC2), respectively. The sequences of the orf100HA mutant genes encoding the LPSTGE motif in pJRS1329 and pJRS1330 were verified by DNA sequencing (data not shown). These plasmids were transformed into JRS4 and its srtA derivative JRS758 (1).

The whole-cell immunoblot assay was used to examine surface display of Orf100HA and its mutant derivative containing an LPSTGE motif, as described above (Fig. 3A). As seen above, the Orf100HA protein with the native QVPTGV sequence was attached to the cell wall only in the presence of srtC2 (Fig. 3A, rows 5 and 6 versus 7 ad 8). However, the Orf100HA with the LPSTGE motif in place of QVPTGV was not detected in significant amounts on the GAS surface, even when the srtC2 gene was present (Fig. 3A, rows 3 and 4). This indicates that changing the QVPTG motif prevents srtC2-dependent anchoring of Orf100HA.



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  		FIG. 3. Role of QVPTG motif in SrtC2-dependent anchoring of Orf100HA to the cell surface. Strains JRS4 and JRS758 were transformed with pJRS1329, pJRS1330, pJRS1317, and pJRS1316 and assayed for the presence of Orf100HA as described for Fig. 2. (A) Whole-cell immunoblot analysis. (B) Western immunoblot of cell wall extracts. (C) Western immunoblot of culture supernatant fractions. Lanes: 1, JRS4/pJRS1329; 2, JRS758/pJRS1329; 3, JRS4/pJRS1330; 4, JRS758/pJRS1330; 5, JRS4/pJRS1317; 6, JRS758/pJRS1317; 7, JRS4/pJRS1316; 8, JRS758/pJRS1316. The sizes of molecular mass standards (in kilodaltons) are indicated to the left.

 
The effect on cell wall anchoring by SrtA as well as the effect of SrtC2 was examined with the srtA mutant strain JRS758. The LPSTGE version of Orf100HA was not found in significant amounts on the GAS surface in either the SrtA-producing strain JRS4 or the srtA mutant strain JRS758 (Fig. 3A, rows 1 and 3 and rows 2 and 4). Thus, the LPSTGE motif is not sufficient for SrtA anchoring, even of a protein in which it precedes a hydrophobic region and charged tail that is recognized by SrtC2. It was also noticeable that more Orf100HA appeared to be anchored by SrtC2 in an srtA mutant than in an srtA+ background (compare rows 5 and 6) (see below and Discussion).

To determine where Orf100HA and its mutant form were located in each strain tested above, cell wall (Fig. 3B) and culture supernatant (Fig. 3C) fractions were prepared, boiled in SDS-PAGE sample buffer, and examined for the presence of Orf100HA by Western immunoblot. Orf100HA containing its native QVPTGV motif appeared as a ladder of bands, characteristic of cell wall-attached proteins in the presence of srtC2 (Fig. 3B, lanes 5 and 6) and not in the absence of srtC2 (Fig. 3B, lanes 7 and 8). As seen previously, the Orf100HA ladder ranged in apparent size from {approx}80 to >220 kDa (Fig. 3B, lanes 5 and 6).

When the QVPTGV sequence was replaced with LPSTGE, we detected no Orf100HA attached to cell wall fragments in the presence of either srtC2 (Fig. 3B, compare lanes 3 and 5 or 4 and 6) or srtA (Fig. 3B, lanes 1 and 3). In the cell wall extracts, the large amount of Orf100HA that migrated as a band of about 37 kDa (seen previously; Fig. 2B) was visible in all cases except in the extracts from strains containing srtA. Whether or not srtC2 was present, in the presence of srtA this HA-containing band was found instead in the culture supernatant (Fig. 3B and C, lanes 1 and 3).

To test whether secretion of this protein was due to recognition and cleavage of the LPSTGE motif by SrtA, we examined each of the cell wall and culture supernatant fractions from strains JRS4/pJRS1330 and JRS758/pJRS1330 by SDS-PAGE and Western immunoblot (Fig. 4). The cell wall-associated protein in JRS758/pJRS1330 had an apparent molecular mass of approximately 37 kDa, while the secreted protein in JRS4/pJRS1330 had a slightly lower molecular mass ({approx}35 kDa). This is consistent with the interpretation that the species released into the supernatant by SrtA was cleaved at the LPSTGE motif, removing the {approx}3-kDa hydrophobic region and charged tail.



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  		FIG. 4. Orf100HA containing an LPSTGE motif is processed by SrtA. Cell wall and culture supernatant fractions from strains JRS4/pJRS1330 and JRS758/pJRS1330 were separated by SDS-12% PAGE and assayed for the presence of Orf100HA by Western immunoblot. The sizes of molecular mass standards (in kilodaltons) are indicated to the left.

 
As seen previously (Fig. 2C) in the Western immunoblot analysis of Orf100HA (Fig. 3B and 3C), some of this protein was present as a very large species. This material was apparent in both cell wall and culture supernatant fractions but only from strains containing the native Orf100HA (QVPTGV motif) and only in the presence of srtC2 (Fig. 3B and 3C, lanes 5 and 6). This suggests that SrtC2 has attached the Orf100HA protein to a large cell wall component which is partitioned into both the cell wall and culture supernatant fractions.

As seen in the whole-cell immunoblots, more Orf100HA was detected in a srtA mutant than a srtA+ background (Fig. 3B, compare lanes 5 and 6). We reasoned that this might be due to an increased amount of cell wall precursor available to SrtC2 in the absence of SrtA-anchored cell wall proteins. If SrtA and SrtC2 compete for available cell wall precursors, overexpression of orf100HA and srtC2 in a srtA+ strain might result in decreased amounts of a SrtA-anchored protein on the cell surface. To test this, JRS4 strains containing plasmids pNZ276, pJRS1316, and pJRS1317 were examined for the presence of M protein on the cell surface by whole-cell immunoblot (Fig. 5). As expected, the srtA mutant strain JRS758 exhibited no surface-associated M protein. A similar amount of M protein was detected on the surface of the srtC2 mutant strain JRS4/pJRS1316 as in JRS4/pNZ276 (vector control). In contrast, the srtC2+ strain JRS4/pJRS1317 showed much less M protein on the cell surface. Thus, overexpression and anchoring of Orf100HA to the cell wall inhibit the efficient cell wall anchoring of M6 protein.



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  		FIG. 5. Effect of SrtC2-dependent anchoring of Orf100HA on the surface display of M6 protein. Whole-cell immunoblots of JRS4 strains containing plasmids pNZ276, pJRS1316 (srtC2 mutant), and pJRS1317 (srtC2+) and strain JRS758 (srtA mutant) were prepared as described for Fig. 2 and reacted with monoclonal antibody 10B6.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Some gram-positive bacteria have multiple sortases with nonredundant specificities. Analysis in silico identified multiple sortase gene homologs in the genomes of most gram-positive bacteria (36). The housekeeping sortase (SrtA) seems to anchor most cell wall proteins, while a limited number of proteins that are not anchored by SrtA are anchored by accessory sortases. Usually, these accessory sortases are encoded in an operon along with the proteins they are thought to anchor. This has been shown for S. aureus SrtB (30), S. pyogenes SrtB (1), Corynebacterium diphtheriae SrtA (58), and L. monocytogenes SrtB (4). In this work, we have characterized a third sortase from GAS that was identified with the S. aureus SrtA sequence as a BLAST search query (36). This sortase (SrtC2) is encoded in an operon along with three potential cell wall-anchored proteins, Cpa, Orf100, and SPyM3_0102. We have shown here that the presence of srtC2 is required for cell wall anchoring of one of these proteins, Orf100.

The different sortases encoded in the genomes of gram-positive bacteria exhibit different specificities for the proteins they anchor. While all cell wall-anchored proteins contain a hydrophobic region followed by a charged C terminus, the motifs preceding this region appear to be sortase specific. Consequently, when more than one sortase has been characterized from a single organism, there is usually no overlap in the proteins anchored. For example, the S. aureus housekeeping sortase (SrtA) recognizes and anchors proteins with an LPXTG motif (27, 28, 30), while the accessory sortase (SrtB) anchors a single protein with an NPQTN motif (30). Similarly, some GAS strains possess two sortases, SrtA and SrtB, which anchor different subsets of proteins with an LPXTG motif (1). SrtA anchors the majority of these proteins, which contain an LPXTG motif followed by an acidic residue, while SrtB anchors a protein containing the sequence LPSTG followed by a serine and does not appear to anchor SrtA-dependent surface proteins.

The SrtC2-anchored protein (Orf100) investigated in this study possesses a unique cell wall-anchoring domain, which consists of a QVPTG motif followed by a hydrophobic region and charged tail. We have demonstrated that SrtC2 specifically recognizes this motif, since substituting the SrtA-dependent LPXTG motif for the QVPTG motif abolishes SrtC2 anchoring of Orf100. Thus, the multiple sortases encoded in the genomes of S. aureus and GAS exhibit specificity for the proteins they anchor. Therefore, the multiple sortase homologs encoded in the genomes of gram-positive bacteria that have been characterized to date appear to have unique functions.

The two steps of the cell wall-anchoring reaction require different signals. The cell wall-anchoring process involves a two-step transpeptidation reaction (28, 56, 57). First, the sortase recognizes the LPXTG motif of the protein to be anchored and cleaves it between the threonine and glycine residues, forming an acyl-enzyme intermediate. In the second step, the protein intermediate is transferred to the cell wall precursor lipid II (40, 44) and subsequently incorporated into the mature cell wall. From our work, it appears that the ability of SrtA to transfer the intermediate to the cell wall precursor requires signals present in the protein being anchored in addition to the LPXTG motif. In this work, we found that the hydrophobic and charged regions of the Orf100 cell wall-anchoring domain together with the preceding QVPTG sequence were sufficient to allow it to be anchored by SrtC2 (Fig. 6, part I). In contrast, a mutant version of Orf100 in which the QVPTGV sequence was replaced with the SrtA recognition sequence LPSTGE was not anchored to the cell wall. Instead, this protein was secreted into the culture supernatant by a mechanism that requires SrtA (Fig. 6, part II). It appears that Orf100HA-LPSTGE is recognized and cleaved by SrtA, since the secreted protein had a slightly smaller apparent molecular mass than the nonsecreted form found in the srtA mutant strain. However, the cleaved Orf100HA-LPSTGE protein is not attached to the cell wall by SrtA. Thus, the second step in the SrtA-catalyzed transpeptidation reaction (transfer to the cell wall intermediate) may require a signal present N-terminal to the LPXTG motif, and this signal is not needed for the first step (recognition and cleavage of the LPXTG motif).



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  		FIG. 6. Fates of the different Orf100 proteins investigated in this study. (A) Orf100 is synthesized in the cytoplasm and transported to the cytoplasmic membrane, where the signal peptide (Sig) is removed by a mechanism that may require SipA2. (B) Orf100 is tethered to the membrane by its C-terminal hydrophobic region and charged tail. (C) SrtC2 recognizes and cleaves the QVPTG motif. (D) SrtC2 links the cleaved protein to the cell wall precursor. (E) Same as steps A and B. (F) SrtA recognizes and cleaves the LPSTGE motif, but other inherent properties of Orf100HA-LPSTGE prevent it from being linked to the cell wall precursor. (G) Instead, this protein is released into the culture supernatant. This figure was adapted from that of Navarre and Schneewind (34) and modified to include the results from this study.

 
In S. aureus, it has also been observed that, for some proteins, cell wall anchoring by SrtA may require a signal N-terminal to the LPXTG motif (45), although this study did not determine whether this signal was required for one or both steps of the transpeptidation reaction. Proteins constructed with fusions of the cell wall-anchoring domains of E. faecalis PrgB, S. pyogenes T6, and L. monocytogenes InlA to protein A were efficiently anchored to the S. aureus cell wall, while proteins with the same cell wall-anchoring domains fused to enterotoxin B were anchored with much lower efficiency (PrgB and T6) or not anchored at all (InlA). Therefore, a signal present N-terminal of the LPXTG motif in protein A but absent from enterotoxin B is required for these proteins to be anchored to the staphylococcal cell wall by SrtA.

SrtA and SrtC2 probably anchor proteins to the same cell wall substrate. Evidence from this study suggests that anchoring of proteins to the GAS cell wall is inefficient and might be limited by the availability of the cell wall substrate. Although both SrtC2 and Orf100 were overproduced in our strain, most of the Orf100 was not anchored to the cell wall but instead was present as a band similar in size to that observed in the absence of SrtC2 ({approx}37 kDa) (Fig. 2C and 3B). This implies that the anchoring process is inefficient. Similarly, a significant amount of M protein produced by GAS is not anchored to the cell wall by SrtA but is found instead in the culture medium (1). Thus, it appears that anchoring by both SrtA and SrtC is inefficient in GAS.

More SrtC2-anchored Orf100 was detectable on the GAS cell surface of a srtA mutant strain than a srtA+ strain (Fig. 3A). This did not appear to result from an increase in our ability to detect Orf100 in the absence of surface proteins anchored by SrtA because we observed an increased association of Orf100 with cell wall fragments following extraction of cell wall material (Fig. 3B). Thus, we suggest that SrtA may compete with SrtC for cell wall precursors to which proteins can be covalently attached. The converse experiment also agrees with this idea. There was less detectable SrtA-anchored M protein on the GAS surface in the strain overexpressing SrtC2 and Orf100 than in its parental strain (Fig. 5). Because it appears that SrtC2 competes with SrtA to anchor surface proteins, it seems likely that they anchor proteins to the same cell wall substrate.

Possible functions of genes encoded in the SrtC1 and SrtC2 operons. Although not all strains of GAS encode a SrtC, those strains most commonly isolated both from invasive clinical specimens and from cases of pharyngitis encode either SrtC1 (M1 strains) or SrtC2 (M3 and M12). Thus, it is plausible that SrtC plays an important role in GAS pathogenesis for the strains that are currently predominant.

For the GAS strains whose sequences are available, srtC is closely linked to, and probably cotranscribed with, several other genes (Fig. 1A). The first gene is cpa, and, because this encodes a protein that has a hydrophobic C-terminal region followed by a charged tail, it is likely to be cell wall anchored. However, in place of an LPXTG motif, it contains the sequence V(P/V)PTG and therefore may be anchored by SrtC instead of SrtA or SrtB. Following cpa is a gene (sipA) with homology to the signal peptidase (lepA) of GAS. Many gram-positive bacteria contain several sip homologs (8, 38, 54). Sometimes these have apparently redundant functions, while in other cases their functions appear to be unique. It seems possible that sipA of GAS may be involved in secretion through the cytoplasmic membrane of the cell wall-anchored proteins encoded in this operon. Following sipA lies orf100, the product of which we have now demonstrated to be anchored to the cell wall by SrtC2. Immediately downstream of orf100 is srtC, followed by a gene (SPyM3_0102) that encodes a protein with an LPX(T/A)G motif followed by an acidic residue that is likely to be anchored by SrtA.

The proteins encoded by orf100 and SPyM3_0102 do not exhibit homology to any characterized proteins in the GenBank database. However, in an M49 strain, Cpa, encoded by the first gene in this locus, was shown to bind collagen (42). Furthermore, another adhesin, protein F (20), which is produced only in some GAS strains (32), is also encoded nearby. In M12 strains, protein F1 may be encoded in the same operon as srtC2. Thus, the FCT region seems to encode surface proteins, some of which have been shown to adhere to extracellular matrix proteins. This locus may also encode proteins needed for secretion and cell wall attachment to these GAS surface proteins. Consequently, it seems possible that this locus may have an important role in the early steps of GAS infection involving binding to and colonization of the host. The homologous proteins encoded in the SrtC1 and SrtC2 operons found in different GAS strains are likely to have related functions. However, their differences may allow the different GAS strains to colonize different host microenvironments, such as different types of host cells (skin or respiratory epithelium, for example), or to specifically colonize individual hosts with differences in their extracellular matrix proteins or epithelial surface proteins.

Regulation of the SrtC operons. Although SrtA, which anchors most cell wall proteins in gram-positive bacteria, is usually expressed constitutively, secondary sortases are often found in operons that are expressed only under specific environmental conditions. For example, expression of SrtB in S. aureus is dependent on the iron concentration (30). In GAS, the arrangement of srtC in an operon along with the proteins it anchors to the cell wall should allow coordinated control of the production and surface display of these proteins under appropriate conditions.

The SrtC1 and SrtC2 operons (Fig. 1) appear to be differentially regulated by RofA and Nra, respectively. RofA is a positive regulator of protein F under anaerobic conditions in the M6 strain JRS4 (16, 17), and under aerobic conditions, expression of protein F is induced by a RofA-independent mechanism (17). In an M49 strain, Nra has been reported to be a negative regulator of Cpa expression (42). Therefore, although the signals required for expression of the SrtC1 and SrtC2 operons may differ, both operons appear to be regulated. Consequently, expression of these genes is likely to occur only under appropriate environmental conditions. For example, this operon may be expressed in the high oxygen tension present at the host surface but not in deep tissues. This would be advantageous if the proteins encoded by the operon are needed for colonization but not for spread of the GAS. Identification of the signals required for induction of the srtC1 and srtC2 operons and the function of the encoded cell wall-anchored proteins should give clues to the role of these genes in the biology of GAS.


   ACKNOWLEDGMENTS
 
This work was supported by grant AI055605 from the National Institutes of Health.

We thank Vince Fischetti for supplying strains, phage lysin, and monoclonal antibody 10B6.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, Rollins Research Center, Atlanta, GA 30322. Phone: (404) 727-0402. Fax: (404) 727-8999. E-mail: scott{at}microbio.emory.edu. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, September 2004, p. 5865-5875, Vol. 186, No. 17
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.17.5865-5875.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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