Anchor Structure of Staphylococcal Surface Proteins

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.

The cell wall envelope of staphylococci and other Grampositive bacteria functions as a surface organelle for microbial interaction with host tissues during infection. Many pathogenic strategies of staphylococci require the function of surface proteins that interact with extracellular matrices, specific host molecules, or target cells, thereby enabling bacterial adherence to tissues, target cell invasion, or evasion of immune responses (1)(2)(3). Many, but not all, surface proteins of staphylococci or other Gram-positive bacteria are anchored to the cell wall envelope by a mechanism requiring a C-terminal 35-amino acid sorting signal with an LPXTG motif (4 -6). These surface proteins are synthesized as precursors with N-terminal signal peptides in the bacterial cytoplasm (P1 precursor) (7,8). After membrane translocation and signal peptide cleavage, the Cterminal sorting signal first retains the polypeptide in the cytoplasmic membrane (P2 precursor) (4). Membrane-anchored sortase A then cleaves the sorting signal between the threonine and glycine residues (9,10), generating a thioester-linked acyl-enzyme between the sortase active-site thiol of Cys 184 and the C-terminal carboxyl group of threonine (11,12). Sortase acylenzyme intermediates are resolved by nucleophilic attack of the amino group of pentaglycine cross-bridges within wall peptides, thereby anchoring the C terminus of surface proteins to the cell wall envelope of staphylococci (13)(14)(15).
The cell wall of Gram-positive bacteria is composed of peptidoglycan, a heteropolymeric macromolecule encompassing glycan strands and attached wall peptides (16,17). Glycan strands, which consist of the repeating disaccharide MurNAc 1 (␤1-4)GlcNAc (18), vary in length and contain up to 30 subunits, with a predominant length of 3-10 and an average of six disaccharide subunits (19). A short peptide component (L-Ala-D-iGln-(Gly 5 )-L-Lys-D-Ala) is attached via an amide bond between the lactyl moiety of MurNAc and the amino group of L-Ala (20 -23). About 80 -95% of the wall peptides of the assembled peptidoglycan are cross-linked, i.e. the amino groups of cross-bridges (pentaglycine (NH 2 -Gly 5 ) in staphylococci) are amide-linked to the carboxyl groups of D-Ala within neighboring wall peptides (24 -26).
The Staphylococcus aureus isd locus is thought to be composed of three transcriptional units (isdA, isdB, and isdCDEF srtB isdG), each of which is regulated by Fur (38), a DNAbinding protein with affinity for canonical DNA sites (Fur boxes) (39). The isd locus is involved in bacterial heme iron uptake and specifies heme-binding proteins. IsdA, IsdB, and IsdC are cell wall-anchored proteins; IsdD, the IsdE lipoprotein, and the IsdF ATP-binding cassette transporter are membrane proteins; and IsdG is a cytoplasmic heme-cleaving enzyme (40,41). IsdA and IsdB are sortase A (srtA)-anchored proteins with C-terminal LPXTG motif sorting signals. They are displayed on the staphylococcal surface and are accessible to extracellular protease (40). In contrast, IsdC is a sortase B (srtB)-anchored protein with a C-terminal NPQTN motif sorting signal. It is shielded from extracellular proteinase digestion by the cell wall envelope and is therefore not displayed on the staphylococcal surface (40).
Previous work showed that purified sortase B cleaves peptides bearing an NPQTN motif in vitro, but left unresolved the IsdC cleavage site, IsdC anchor structure, and anchoring mechanism of sortase B (38). Here, we report the cell wall anchor structure of IsdC. In contrast to sortase A substrates, the anchor structure of IsdC displays shorter glycan strands and significantly less cell wall cross-linking. We discuss a model whereby sortases A and B recognize unique features of sorting signals and peptidoglycan substrates to deposit surface proteins with distinct topologies in the cell wall envelope.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Plasmids-S. aureus strains Newman (42), RN4220 (43), and SKM15 (RN4220 fur::tet) (38) have been described previously. To construct pAMP2 (encoding SEB-MH 6 -CWS IsdC ), DNA sequences specifying CWS IsdC were amplified by PCR using S. aureus Newman chromosomal DNA as template and primers 6HisdCsig (5Ј-A-AGGGTACCATGCATCACCATCACCATCACAAAGTAGAAAATCCAC-AAACAAAT-3Ј) and SasK-3-B (5ЈAAGGATCCTTATTCCACATTGCCT-TTAG-3Ј). The PCR product was cut with KpnI and BamHI and inserted into pHTT4 (14) cut with the same enzymes. This cloning step replaced the MH 6 -CWS Spa coding sequences fused downstream of the seb gene in pHTT4 with those of MH 6 -CWS IsdC , thereby creating pA-MP1. The srtA promoter-srtB coding sequence fusion was obtained by digestion of pSM75 (38) with EcoRI. The fragment was ligated to Eco-RI-linearized pAMP1, thereby generating pAMP2. Escherichia coli DH5␣ was used as host for DNA transformation. The construct was verified by restriction mapping and sequencing and was then transformed into S. aureus SKM15 (44). The antibiotics used in the selective medium were ampicillin (100 g/ml for E. coli) and chloramphenicol (10 g/ml for S. aureus).
Purification of Staphylococcal Cell Walls-Cell wall preparations were obtained as described previously (15). Briefly, colonies were inoculated into tryptic soy broth containing 10 g/ml chloramphenicol, and S. aureus SKM15 (pAMP2) was grown overnight at 37°C. Cells were diluted 1:50 into fresh medium, grown to A 600 ϭ 0.8, and harvested by centrifugation at 10,000 ϫ g for 10 min. Sedimented cells were washed once with 100 ml of 50 mM Tris-HCl (pH 7.5) and suspended in 50 ml of the same buffer supplemented with 5 mM phenylmethanesulfonyl fluoride. Cell walls were broken in a Bead-Beater instrument (Biospec Products Inc.) by 15 pulses of 1 min, followed by 5 min of incubation on ice. The crude lysate was decanted to remove the glass beads and centrifuged at 33,000 ϫ g for 15 min to sediment cell wall sacculi and membranes. Sediment was suspended in 100 ml of 100 mM KH 2 PO 4 (pH 7.5), 1% Triton X-100, and 1 mM phenylmethanesulfonyl fluoride and incubated for 3 h at 4°C with stirring to extract membrane lipids. Murein sacculi were again sedimented by centrifugation at 33,000 ϫ g for 15 min, washed three times with 100 ml of 100 mM sodium phosphate (pH 6.0), suspended in 50 ml of the same buffer, and stored at Ϫ80°C.
Solubilization and Detection of IsdC-S. aureus RN4220 was grown overnight at 37°C in tryptic soy broth in either the presence or absence of 1 mM 2,2Ј-dipyridyl. Staphylococcal cultures were diluted 1:30 into 2 ml of the same medium and grown to A 600 ϭ 0.8, and cells were harvested by centrifugation at 10,000 ϫ g for 10 min. Cells were suspended in 1 ml of 100 mM sodium phosphate (pH 6.0) and 1 mM phenylmethanesulfonyl fluoride for mutanolysin digestion or 1 ml of equilibration buffer (Tris-HCl (pH 7.5) and 150 mM NaCl) for lysostaphin, ⌽11 hydrolase, and ⌽11⌬ digestions. Cell wall components were solubilized with 700 units/ml mutanolysin (Sigma) and 70 g/ml lysostaphin, ⌽11 hydrolase, or ⌽11⌬. Insoluble material was removed by centrifugation at 15,000 ϫ g for 5 min, and proteins in the supernatant were precipitated by addition of 1 ml of 1:5 chloroform/methanol solution (46), washed with 1 ml of methanol, and suspended in 100 l of loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 0.5 M ␤-mercpatoethanol, 20% glycerol, 0.5 mg/ml bromophenol blue). Five l of each sample was subjected to SDS-PAGE and electrotransferred to a PVDF membrane. IsdC was detected by immunoblotting using rabbit anti-IsdC polyclonal serum. The membrane was blocked in the presence of human IgG (12.5 g/ml; Sigma) to prevent the non-immune binding of protein A.
Proteinase K Digestion of Surface Proteins-S. aureus RN4220 (pHTT4, encoding SEB-MH 6 -CWS Spa ) and SKM15 (pAMP2, encoding SEB-MH 6 -CWS IsdC ) cultures were grown in tryptic soy broth containing 10 g/ml chloramphenicol to A 600 ϳ 1.0. Cells were sedimented by centrifugation at 6000 ϫ g, washed twice with TSM buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , and 0.5 M sucrose), and suspended in the same buffer. Three 1-ml aliquots of staphylococcal suspensions were dispensed into reaction vials. One sample was treated with 150 g of proteinase K (Roche Applied Science), and the three samples were incubated for 8 h at 37°C with mild shaking. Cells were collected by centrifugation at 12,000 ϫg for 5 min; the supernatant removed; and staphylococci were suspended in 1 ml of TSM buffer containing 20 g of lysostaphin (Ambi) and incubated for 20 min at 37°C. Staphylococcal protoplasts were sedimented by centrifugation at 12,000 ϫg for 5 min, and 1-ml supernatants were transferred to fresh tubes and either treated with 150 g of proteinase K for 2 h at 37°C or left untreated. Proteins in all samples were precipitated with 7.5% trichloroacetic acid, washed with ice-cold acetone, and suspended in 100 l of loading buffer. Proteins were separated by 15% SDS-PAGE; transferred to a PVDF membrane; and analyzed by staining with nickel-horseradish peroxidase conjugate (Pierce), followed by chemiluminescence development.
Purification of Cell Wall-anchored SEB-MH 6 -CWS IsdC -Cell walls isolated from 2 liters of S. aureus SKM15 (pAMP2) culture were washed with 100 ml of 100 mM sodium phosphate (pH 6.0) and suspended in 30 ml of the same buffer supplemented with 1 mM phenylmethanesulfonyl fluoride for mutanolysin digestion or in 30 ml of equilibration buffer for lysostaphin, ⌽11 hydrolase, and ⌽11⌬ digestions. To each cell wall suspension were added 20,000 units of mutanolysin, 2 mg of lysostaphin, 2 mg of ⌽11 hydrolase, or 2 mg of ⌽11⌬. Digestion proceeded for 16 h at 37°C with mild agitation. Solubilized cell wall fragments and surface proteins were separated from undigested insoluble cell walls by centrifugation at 33,000 ϫ g for 15 min. SEB-MH 6 -CWS IsdC was purified from the lysate supernatant by affinity chromatography using 1 ml of Ni-NTA pre-equilibrated with 10 ml of equilibration buffer. The column was successively washed with 10 ml of equilibration buffer, 10 ml of wash buffer (Tris-HCl (pH 7.5), 150 mM NaCl, and 10% glycerol) supplemented with 10 mM imidazole, and 10 ml of wash buffer. Finally, proteins were eluted by a step gradient of imidazole from 50 to 500 mM.
Preparation of C-terminal Anchor Peptides-Purified SEB-MH 6 -CW-S IsdC was methanol/chloroform-precipitated, dried under vacuum in a SpeedVac concentrator (Savant), and suspended in 3 ml of 70% formic acid. One g of cyanogen bromide was added, and cleavage reactions were incubated for 16 h at room temperature in the dark. The cleaved peptides were dried under vacuum, washed twice with water, and suspended in 1 ml of buffer A (10 mM Tris-HCl, 100 mM NaH 2 PO 4 , 6 M guanidine hydrochloride, pH 8.0). Samples were loaded onto a column packed with 1 ml of Ni-NTA pre-equilibrated with 10 ml of buffer A. The column was washed with 10 ml each of buffer A, buffer B (8 M urea, 100 mM NaH 2 PO 4 , and 10 mM Tris-HCl (pH 8.0)), and buffer B at pH 6.3. Elution was carried out with 2 ml of buffer A at pH 4.3. When peptides were derived from mutanolysin-solubilized SEB-MH 6 -CWS IsdC , N-acetylmuramic acid residues were reduced with sodium borohydride to generate N-acetylmuramitol, thereby facilitating HPLC separation and mass spectrometric analysis (47). Finally, peptides were subjected to RP-HPLC on a Hypersil C 18 column (2 ϫ 250 mm; Keystone Scientific Inc.). Separation was carried out at a flow rate of 0.2 ml/min with a linear gradient starting 10 min after injection from 99% H 2 O (0.1% trifluoroacetic acid) to 99% CH 3 CN (0.1% trifluoroacetic acid) over 100 min. Elution of peptides was monitored at 215 nm, and 1-min fractions were collected.

Solublization of IsdC from the Staphylococcal Cell Wall with Murein
Hydrolases-IsdC is synthesized in the cytoplasm as a 227-amino acid precursor with an N-terminal signal peptide and a C-terminal NPQTN-type sorting signal (38). Using signal peptide algorithms and canonical sorting signal cleavage site predictions, Ala 29 was identified as the N-terminal residue and Thr 192 as the C-terminal residue of mature IsdC. The calculated molecular mass of predicted mature IsdC is 18,140 Da. To analyze the cell wall anchor structure of IsdC, we sought to solubilize the polypeptide from the staphylococcal peptidoglycan with murein hydrolases. Staphylococci were grown in tryptic soy broth with or without 2,2Ј-dipyridyl, an iron-chelating reagent, thereby activating isd expression (38). Staphylococci were harvested by centrifugation, and their peptidoglycan was digested with murein hydrolases. Proteins in cell wall lysates were precipitated with chloroform/methanol and separated by 15% SDS-PAGE, and IsdC revealed by immunoblotting (Fig. 1).
Mock treatment of staphylococci without murein hydrolase did not release IsdC from the cell wall envelope (data not shown). Lysostaphin, a glycylglycine endopeptidase that cleaves pentaglycine cross-bridges of staphylococcal cell walls ( Fig. 1A) (48), solubilized IsdC as a single species of 19 kDa (Fig. 1B). ⌽11 hydrolase cleaves the amide bond of MurNAc-L-Ala as well as the D-Ala-Gly peptide bond of the S. aureus peptidoglycan (45) and released IsdC as a species with a uni-form mass, migrating somewhat more slowly upon SDS-PAGE than the lysostaphin-solubilized counterpart. These observations suggest that IsdC must be linked to the staphylococcal cell wall, as murein hydrolase treatment was required to release IsdC. Cell wall digestion with mutanolysin, a muramidase that cuts the repeating disaccharide MurNAc-GlcNAc (49,50), solubilized IsdC as a spectrum of seven fragments with different masses. The fastest migrating species displayed a mass similar to that of ⌽11 hydrolase-released IsdC, whereas the other six muramidase-released species migrated more slowly and with decreasing intensity upon SDS-PAGE (Fig.  1B). In a previous report, mutanolysin treatment of staphylococci pulse-labeled with [ 35 S]methionine was used to release SEB-CWS IsdC , a hybrid containing the IsdC sorting signal (CWS IsdC ) fused to the SEB polypeptide. The solubilized fusion proteins were captured by immunoprecipitation and detected by autoradiography (40). In contrast to our observations reported here, a single mutanolysin-released SEB-CWS IsdC species was found (40). This apparent discrepancy appears to be due to the pulse-labeling technique, as immunoprecipitation and autoradiography detected only the most abundant SEB-CWS IsdC species (40).
The ⌽11⌬ enzyme cleaves the peptide bond of D-Ala-Gly, but not the amide bond of MurNAc-L-Ala (45). Treatment with ⌽11⌬ released three IsdC fragments from the cell wall, all of which migrated more slowly than the ⌽11 hydrolase-and lysostaphin-released counterparts. Thus, it appears that IsdC is linked to the pentaglycine cross-bridges of the staphylococcal  (14) and is anchored to the cell wall envelope by sortase A; the SEB-MH 6 -CWS IsdC chimera is encoded by pAMP2 and is anchored to the cell wall envelope by sortase B. LPXTG and NPQTN motifs and signal peptide and sorting signal cleavage sites are indicated. Black boxes depict hydrophobic domains, and the boxed plus signs represent the positively charged tails. B, proteinase K digestion of staphylococcal surface proteins. Staphylococcal cells were washed, suspended in buffer, and dispensed into three reaction vials as indicated (lanes 1-6). One sample was treated with proteinase K (lanes 3 and 6), and all three samples were incubated at 37°C. Cells were collected by centrifugation; the supernatant was removed; and staphylococci were suspended in buffer containing 20 g of lysostaphin and incubated at 37°C. Staphylococcal protoplasts were sedimented by centrifugation, and supernatants were transferred to fresh tubes and either treated with proteinase K (lanes 2 and 5) or left untreated. Proteins in all samples were precipitated with 7.5% trichloroacetic acid, separated by SDS-PAGE, transferred to a PVDF membrane, and analyzed by staining with nickel-horseradish peroxidase conjugate.  (38). Solubilized surface proteins were precipitated with chloroform/methanol, suspended in loading buffer, separated by SDS-PAGE, and electrotransferred to a PVDF membrane. IsdC was detected with rabbit anti-IsdC polyclonal antiserum and chemiluminescence staining. cell wall, as only treatment with lysostaphin and ⌽11 hydrolase, i.e. enzymes that directly cut cross-bridges or excise crossbridges with wall peptides, released IsdC with a uniform mass. Cleavage of only glycan strands (mutanolysin) or cross-bridges (⌽11⌬) each released distinct spectra of IsdC fragments, consistent with the hypothesis that the polypeptides must be linked to polymerized MurNAc-GlcNAc chains as well as wall peptides with some degree of cross-linking.
Surface Display of SEB-MH 6 -CWS IsdC and SEB-MH 6 -CWS Spa -Fusion of sorting signal sequences to the C-terminal end of SEB, a highly expressed exoprotein, generates cell wallanchored hybrids (14,38). We wondered whether SEB hybrids anchored via NPQTN sorting signals display similar properties as wild-type IsdC. Previous work showed that IsdC is sequestered in the staphylococcal envelope and protected from extracellular protease, whereas sortase A-anchored surface proteins (IsdA and IsdB) are not (40). To test whether SEB hybrids exhibit a similar phenotype, two plasmids encoding SEB-MH 6 -CWS Spa and SEB-MH 6 -CWS IsdC were transformed into staphylococci. SEB-MH 6 -CWS Spa represents a hybrid with a C-terminal LPXTG motif sorting signal (45), whereas SEB-MH 6 -CWS IsdC is anchored to the envelope via an NPQTN motif sorting signal (see below) ( Fig. 2A).
Staphylococci were washed and suspended in sucrose buffer to stabilize bacterial protoplasts without cell walls that were generated in the course of this experiment. Staphylococcal suspensions were dispensed into three equal 1-ml aliquots. One sample was treated with proteinase K to remove surface-displayed polypeptides, whereas the other two samples served as controls and were left untreated. After incubation, all cells were sedimented by centrifugation and suspended in buffer lacking proteinase K. The cell walls of staphylococci in all three samples were then cleaved with lysostaphin. The resulting protoplasts were sedimented by centrifugation, and the supernatants (containing solubilized cell wall proteins) were transferred to new reaction vials. One of the two control samples was treated with proteinase K as a measure for protease sensitivity, whereas the other was left untreated. Proteins in all three samples were precipitated with 7.5% trichloroacetic acid, washed with acetone, and separated by SDS-PAGE. After electrotransfer to a PVDF membrane, SEB hybrids were detected by staining with nickel-horseradish peroxidase conjugate. Fig. 2B shows that sortase A-anchored hybrids (SEB-MH 6 -CWS Spa ) were completely digested by extracellular proteinase K treatment (lane 3), whereas sortase B-anchored SEB-MH 6 -CWS IsdC was not (lane 6). However, SEB-MH 6 -CWS IsdC is not intrinsically resistant to protease digestion, as treatment of lysostaphin-solubilized cell wall proteins with proteinase K degraded the polypeptide (lanes 2 and 5). Thus, SEB-MH 6 -CWS Spa hybrids are displayed on the staphylococcal surface, whereas SEB-MH 6 -CWS IsdC is protected from extracellular protease and buried within the cell wall envelope. It therefore appears that SEB-MH 6 -CWS IsdC is anchored to the envelope in a similar fashion as full-length IsdC and represents a useful surrogate to study the cell wall anchor structure of IsdC. Solublization of SEB-MH 6 -CWS IsdC from the Staphylococcal Cell Wall with Murein Hydrolases-Previous work reported that SEB-CWS IsdC , a hybrid generated by fusion of the C- terminal end of enterotoxin B to the cell wall sorting signal of IsdC, is anchored to the staphylococcal cell wall (38,40). To analyze the anchor structure of IsdC, we generated SEB-MH 6 -CWS IsdC , a hybrid polypeptide with an engineered insertion of a Met-His 6 sequence upstream of the IsdC cell wall sorting signal. Our overall experimental strategy resembles that used for LPXTG anchor structure determination (14,15), whereby cell wall-anchored SEB-MH 6 -CWS IsdC can be solubilized with different murein hydrolases and then purified by affinity chromatography on Ni-NTA. Eluted proteins are cleaved at methionyl residues with CNBr (51), and C-terminal peptides are purified by a second round of affinity chromatography on Ni-NTA. Eluted NPQTN anchor peptides can then be subjected to mass spectrometric analysis. To achieve a high efficiency of anchoring of the SEB-MH 6 -CWS IsdC chimera, we overexpressed it along with sortase B and thus generated pAMP2 (Fig. 3A). The plasmid was transformed into the fur mutant strain S. aureus SKM15 (38) to mimic iron starvation conditions.
To test whether SEB-MH 6 -CWS IsdC is anchored to the staphylococcal peptidoglycan in a similar manner as full-length IsdC, the hybrids were solubilized with different murein hydrolases, purified by Ni-NTA affinity chromatography, and analyzed on Coomassie Blue-stained SDS-polyacrylamide gel (Fig. 3B). Lysostaphin, a glycylglycine endopeptidase that cleaves S. aureus pentaglycine cross-bridges (Fig. 1A) (48), solubilized SEB-MH 6 -CWS IsdC from isolated staphylococcal murein sacculi as two species that migrated at 28 and 30 kDa, respectively. The faster migrating species is composed of cell wall-anchored products (see below), whereas the slower migrating species represents the P2 precursor with an uncleaved sorting signal, which was also found in cell wall lysates generated with other murein hydrolases (Fig. 3B, arrows). The hypothesis of P2 accumulation was first proposed upon overexpression of SEB-CWS IsdC , as its P2 precursor absolutely requires sortase B to generate the faster migrating mature anchor species (38). This notion was corroborated here by mass spectrometric analysis of purified peptide fragments generated via CNBr cleavage of the purified SEB-MH 6 -CWS IsdC P2 precursor, which revealed uncleaved sorting signal sequence (data not shown).
⌽11 hydrolase cleaves the peptidoglycan by hydrolyzing the MurNAc-L-Ala as well as D-Ala-Gly amide bonds (Fig. 1A) (45). This enzyme released SEB-MH 6 -CWS IsdC as a 28-kDa species (Fig. 3B). These observations suggest that the faster migrating SEB-MH 6 -CWS IsdC species is indeed linked to the staphylococcal cell wall, as murein hydrolase treatment was required to release the polypeptide. Cell wall digestion with mutanolysin, a muramidase that cuts the repeating disaccharide MurNAc-GlcNAc (49,50), solubilized SEB-MH 6 -CWS IsdC as a spectrum of seven fragments with different masses. The fastest migrating species displayed a mass similar to that of ⌽11 hydrolasereleased IsdC, whereas the other six muramidase species migrated more slowly and with decreasing intensity upon SDS-PAGE. The ⌽11⌬ enzyme cleaves the peptide bond of D-Ala-Gly, but not the amide bond of MurNAc-L-Ala (45). Treatment with ⌽11⌬ released three SEB-MH 6 -CWS IsdC fragments from the cell wall. Thus, it appears that, similar to IsdC, SEB-MH 6 -CWS IsdC is linked to the pentaglycine cross-bridges of the staphylococcal cell wall, as only treatment with lysostaphin and ⌽11 hydrolase, enzymes that directly cut cross-bridges, released SEB-MH 6 -CWS IsdC with a uniform mass. Cleavage of only glycan strands or wall peptides each released distinct spectra of SEB-MH 6 -CWS IsdC fragments, consistent with the hypothesis that the polypeptides must be linked to polymerized MurNAc-GlcNAc chains as well as wall peptides with a modest degree of cross-linking. Cell Wall Anchor Structure of Lysostaphin-solubilized SEB-MH 6 -CWS IsdC -Isolated staphylococcal cell walls harboring anchored SEB-MH 6 -CWS IsdC were treated with lysostaphin to cleave the bacterial peptidoglycan, and cell wall lysates were subjected to affinity chromatography on Ni-NTA. After elution with imidazole, SEB-MH 6 -CWS IsdC was precipitated with chloroform/methanol, washed with methanol, and dried. Protein samples were suspended in formic acid, cleaved at methionyl residues with cyanogen bromide, washed, and dried prior to a second round of affinity purification. C-terminal anchor peptides were eluted from Ni-NTA with a declining pH gradient and subjected to RP-HPLC on a C 18 column. Anchor peptides that eluted at 14% CH 3 CN and 0.01% trifluoroacetic acid were subjected to MALDI-TOF-MS, revealing a predominant ion signal at m/z 1809.02 (Fig. 4). This measurement is consistent with the structure of a C-terminal SEB-MH 6 -CWS IsdC peptide that terminates at Thr 192 and is amide-linked to three glycines (calculated m/z 1809.92). The second most abundant ion signal at m/z 1752.00 was interpreted as a C-terminal anchor peptide linked to two glycines (Fig. 4). To test whether m/z 1809.02 encompasses the predicted sequence H 6 KVENPQT-Gly 3 , the ion signal was subjected to collision-induced dissociation (CID), and daughter ion spectra were collected via MALDI-TOF/ TOF-MS (Fig. 4C). The observed daughter ions are listed in Table I together with their predicted m/z values and CID fragmentation patterns, which confirmed the peptide sequence H 6 KVENPQT-Gly 3 . Several other ions in the spectrum of Fig.  4A were identified by MALDI-TOF/TOF-MS as N-terminal amino-formylated or amino-carbamylated anchor peptides carrying two or three C-terminal glycines (data not shown). Formylation and carbamylation of anchor peptides occur during CNBr cleavage (carried out in 70% formic acid) and affinity chromatography (performed with urea-containing buffers) of anchor peptides, respectively (15). Cell Wall Anchor Structure of ⌽11 Hydrolase-solubilized SEB-MH 6 -CWS IsdC -Isolated staphylococcal cell walls harboring anchored SEB-MH 6 -CWS IsdC were digested with ⌽11 hydrolase, and SEB-MH 6 -CWS IsdC was purified from cell wall lysates as described above. After cleavage with CNBr, C-terminal peptides were repurified and subjected to RP-HPLC on a C 18 column. ⌽11 hydrolase-released anchor peptides eluted at 41% CH 3 CN and 0.01% trifluoroacetic acid and were subjected to MALDI-TOF-MS (Fig. 5A) (Fig. 5B). The presence of cell wall pentapeptides indicates lack of cross-linking with other murein subunits. To test the predicted structure, the parent ion at m/z 2321.17 was subjected to CID, and daughter ion spectra were collected (Fig. 5C). Table II  The predominant ion signal of the second cluster encompassed m/z 3004.49, 3032.48, 3075.52, and 3103.52. Assuming that these compounds represent anchor peptides with incompletely cleaved D-Ala-Gly peptidoglycan bonds, an average cell wall tetrapeptide mass of 683.32 Da was added to m/z 2321.17, which explained m/z 3004.59 as an anchor peptide linked to a tetrapeptide-tetrapeptide. Furthermore, m/z 3075.59 was interpreted as an anchor peptide linked to a tetrapeptide-pentapeptide and therefore suggests the absence of cross-linking to a third murein subunit. The signals at m/z 3032.48 and 3103.52 represent formylated compounds. The same interpretation was applied to the third and least abundant cluster of ion signals, including m/z 3687.79, 3716.78, 3758.85, and 3786.81. The predicted masses of cell wall anchor structures linked to a tetrapeptide-tetrapeptide-tetrapeptide and a tetrapeptide-tetrapeptide-pentapeptide matched the observed m/z 3687.79 and 3758.82, respectively, whereas m/z 3716.78 and 3786.81 were again explained as formylated compounds.
Cell Wall Anchor Structure of Muramidase-solubilized SEB-MH 6 -CWS IsdC -The glycan strands of the isolated staphylococcal peptidoglycan were cut with mutanolysin at the (␤1-4)glycosidic bond of the repeating disaccharide MurNAc-GlcNAc.
SEB-MH 6 -CWS IsdC was purified from cell wall lysates and cleaved with CNBr, and C-terminal peptides were affinitypurified. The amino sugars of the C-terminal peptides were reduced by sodium borohydride treatment, and glycopeptides were separated by RP-HPLC on a C 18 column. Mutanolysinreleased anchor peptides eluted at 33% CH 3 CN and 0.01% trifluoroacetic acid and were subjected to MALDI-TOF-MS (Fig. 6A). The compound at m/z 2872.44 represented the most FIG. 5. Mass spectrometric analysis of ⌽11 hydrolase-released anchor peptides. A, MALDI-TOF mass spectrum of SEB-MH 6 -CW-S IsdC anchor peptides released from staphylococcal murein sacculi. The cell wall preparation was treated with ⌽11 hydrolase, and the lysate was subjected to Ni-NTA affinity chromatography. Purified SEB-MH 6 -CWS IsdC was digested with CNBr to generate C-terminal anchor peptides, which were isolated by a second chromatography on Ni-NTA and then separated by RP-HPLC. Anchor peptides eluted at 41% CH 3  abundant muramidase-released ion, and its structure was explained as a C-terminal anchor peptide linked to the murein disaccharide pentapeptide (GlcNAc(␤1-4)MurNAc-(L-Ala-D-iGln-L-Lys-(NH 2 -H 6 KVENPQT-Gly 5 )-D-Ala-D-Ala-CO 2 H), calculated m/z 2872.02) (Fig. 6B). To test this prediction, the compound was subjected to CID in a MALDI-TOF/TOF-MS experiment. Consistent with a previous report using mass spectrometry to analyze the structure of peptidoglycan fragments (47), the predominant CID daughter ions resulted from the breakage of the (␤1-4)-glycosidic bond of GlcNAc-MurNAc (observed m/z 2668.94, calculated m/z 2667.81) (Table III). However, many other, less abundant ions were detected and could be structurally assigned to N-or C-terminal CID fragments of the structure depicted in Fig. 6 (Table III).
The MALDI-TOF mass spectrum in Fig. 6A reveals a second cluster of muramidase-released anchor peptides with linked peptidoglycan. Three ion signals at m/z 4035.96, 4063.99, and 4078.02 were interpreted as C-terminal anchor peptides linked to the murein disaccharide tetrapeptide-disaccharide pentapeptide (calculated m/z 4034.21) and its amino-formylated (calculated m/z 4062.20) and amino-carbamylated (calculated m/z 4077.21) species, respectively. Although several attempts were made to identify ⌽11 hydrolase-or muramidase-released anchor peptides with higher degrees of murein cross-linking, these experiments failed to reveal higher degrees of crosslinked peptidoglycan fragments tethered to IsdC anchor peptides. Furthermore, sortase B-anchored SEB-MH 6 -CWS IsdC was linked to N,O-6-diacetylated murein subunits (data not shown), a modification that is known to occur at approximately half of all MurNAc residues in the staphylococcal peptidoglycan (25,52). Thus, unlike sortase A-anchored surface proteins, the cell wall anchor structure of IsdC reveals predominantly short glycan strands and a non-cross-linked peptidoglycan. DISCUSSION Staphylococcal sortases anchor proteins to the bacterial cell wall envelope. Sortase A recognizes its substrates by the presence of an LPXTG motif, a site that is subsequently cleaved between the threonine and glycine residues (11). The enzyme performs a transpeptidation reaction whereby the FIG. 6. Mass spectrometry of mutanolysin-released anchor peptides. A, MALDI-TOF mass spectrum of SEB-MH 6 -CWS IsdC anchor peptides released from staphylococcal murein sacculi. The cell wall preparation was treated with mutanolysin, and the lysate was subjected to Ni-NTA affinity chromatography. Purified SEB-MH 6 -CWS IsdC was digested with CNBr to generate C-terminal anchor peptides, which were isolated by a second chromatography on Ni-NTA and then separated by RP-HPLC. Anchor peptides eluted at 33% CH 3    c The nomenclature used refers to the N-and C-terminal cleavage fragments according to Biemann (61); an exception is indicated below. Ion number was calculated considering the histidine tail as the main N terminus. d Immonium ion.
C-terminal threonine of the LPXT-bearing polypeptide is amide-linked to pentaglycine cross-bridges (13)(14)(15). Several lines of evidence suggest that sortase A utilizes lipid II as a peptidoglycan substrate and that proteins linked to lipid II are incorporated into the growing cell wall envelope via transpeptidation and transglycosylation reactions (35,36). Consistent with the view that sortase A and cell wall biosynthetic enzymes use the same substrates, anchored proteins are tethered to a highly cross-linked peptidoglycan, embedded in chains of glycan strands of five or more MurNAc-GlcNAc disaccharides in length and cross-linked to as many as 11 cell wall peptides (15). Sortase A-anchored polypeptides are displayed on the bacterial surface and evenly distributed within the staphylococcal envelope (53,54). Because cell wall synthesis and sortase A anchoring may be constitutive and coordinated events in growing staphylococci, continuous incorporation of proteins into the cell wall could result in the even distribution of these very abundant polypeptides. Sortase B seems to be expressed only when staphylococci enter mammalian hosts, i.e. when availability of nutrient iron is limited, causing derepression of Fur-regulated genes such as the isd cluster. In contrast to sortase A, which anchors 20 different polypeptides, sortase B appears to recognize only one substrate, IsdC, which is synthesized as a precursor bearing an N-terminal signal peptide and a C-terminal sorting signal with an NPQTN motif. In contrast to sortase A-anchored, Fur-regulated IsdA, IsdB, and IsdH, IsdC does not appear to be displayed on the staphylococcal surface (40). Previous work showed also that, when transplanted to the C terminus of enterotoxin B, the IsdC sorting signal mediates cell wall anchoring of the hybrid protein in a sortase B-dependent manner (38,40). Although the earlier results suggested that anchored IsdC may not be tethered to a cross-linked peptidoglycan, the sortase B cleavage site of IsdC and the nature of its cell wall anchor were not revealed.
Here, we have reported the IsdC anchor structure. First, we demonstrated that SEB-MH 6 -CWS IsdC , but not SEB-MH 6 -CWS Spa , is protected from extracellular protease and buried in the cell wall envelope (Fig. 2). This result validated the use of SEB-MH 6 -CWS IsdC as a surrogate substrate to analyze the otherwise non-abundant IsdC anchor peptides. When comparing the mobility of IsdC and SEB-MH 6 -CWS IsdC upon SDS-PAGE, we observed that sortase B substrates required cell wall digestion for solubility and that lysostaphin and ⌽11 hydrolase, but not muramidase or ⌽11⌬, released IsdC protein with uniform mobility upon SDS-PAGE (Figs. 1 and 3). Furthermore, muramidase-released IsdC displayed a low degree of cross-linked peptidoglycan fragments (four to seven species upon SDS-PAGE), much less than sortase A-anchored proteins (15 or more species) (15). The simplest explanation for these results is that IsdC is linked to the pentaglycine cross-bridge of peptidoglycan fragments with relatively little cross-linking. This hypothesis could be validated by mass spectrometric analysis of purified anchor peptides (Figs. 4 -6). Our results demonstrate that sortase B cleaves IsdC between the threonine and asparagine residues of the NPQTN motif and tethers the carboxyl group of threonine to the pentaglycine cross-bridge. Anchor peptides linked to the murein tetrapeptide and pentapeptide were observed. In addition, we identified small amounts of tetrapeptide-pentapeptide and even tetrapeptide-tetrapeptidepentapeptide anchor structures. However, peptidoglycan fragments with a higher order of cross-linking could not be detected.
In this work, we have also shown that burial of IsdC and SEB-MH 6 -CWS IsdC within the staphylococcal envelope appears to be a property of the sorting signal and sortase B, but not of the mature polypeptide chain. The cell wall envelope of staphylococci is 40 -100 nm in diameter (55), a distance that can clearly accommodate folded or even unfolded proteins. Why then are some polypeptides displayed, whereas others are not? Furthermore, can this phenomenon be explained on the basis of their cell wall anchor structure? Although we do not yet fully understand the molecular mechanisms that dictate the travels of proteins within the envelope, we believe that the cell wall substrates of sorting reactions may determine the subsequent location of proteins in the envelope. For example, if sortase A used lipid II as a substrate in vivo, thereby ensuring uniform distribution of anchored proteins in the envelope, sortase B would then employ a different peptidoglycan substrate, probably not a biosynthetic precursor, but an assembled cell wall,  thereby restricting the topology of anchored IsdC. One possible substrate may be linear polymerized glycan strands with a non-cross-linked peptidoglycan. In such a model, unique molecular properties of sortase B would have to be responsible for selecting polymerized peptidoglycan substrates that presumably can no longer be redirected within the envelope. The presumed properties of sortases in substrate selection may be gleaned from bioinformatic or structural comparison of the two enzymes. The polypeptide chain of sortase B is longer than that of sortase A. Although both enzymes largely fold into a very similar structure, two short ␣-helices at the N terminus (␣1 and ␣2) and a long ␣-helix (␣5) in the direct vicinity of the active site may determine the unique substrate properties of sortase B (56,57). If so, maybe future experiments would allow the engineering of sortases to assume different substrate specificities, thereby advancing our appreciation of enzyme/substrate interactions as well as our general understanding of cell wall envelope assembly. The findings reported here are likely not unique to staphylococcal sortases A and B. Listeria monocytogenes and Bacillus anthracis also express sortases A and B. The L. monocytogenes sortase B substrate SvpA, but not the sortase A substrate InlA (58), seems to be unevenly distributed throughout the bacterial envelope (59), consistent with the notion that these sortases may also recognize distinct cell wall substrates to anchor their proteins.