Anchor Structure of Staphylococcal Surface Proteins

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the pentaglycyl cross-bridge in the staphylococcal peptidoglycan. Here, we asked whether altered peptidoglycan cross-bridges interfere with the sorting reaction and investigated surface protein anchoring in staphylococcalfem mutants. S. aureus strains carrying mutations in the femA, femB, femAB, or the femAX genes synthesize altered cross-bridges, and each of these strains displayed decreased sorting activity. Characterization of cell wall anchor structures purified from thefem mutants revealed that surface proteins were linked to cross-bridges containing one, three, or five glycyl residues, but not to the ε-amino of lysyl in muropeptides without glycine. When tested in a femAB strain synthesizing cross-bridges with mono-, tri-, and pentaglycyl as well as tetraglycyl-monoseryl, surface proteins were found anchored mostly to the five-residue cross-bridges (pentaglycyl or tetraglycyl-monoseryl). Thus, although wild-type peptidoglycan appears to be the preferred substrate for the sorting reaction, altered cell wall cross-bridges can be linked to the COOH-terminal end of surface proteins.

Gram-positive bacteria display proteins on their surface as a means to interact with host tissues and to establish human infections (1,2). The mechanism of surface protein anchoring to the bacterial cell wall has recently been established for protein A of Staphylococcus aureus. After synthesis in the cytoplasm, protein A is first initiated into the secretory pathway by an NH 2 -terminal signal (leader) peptide (3). A 35-residue COOHterminal sorting signal is necessary and sufficient for the anchoring of protein A and functions to first retain the polypeptide within the secretory pathway (4). This allows proteolytic cleavage between the threonine and the glycine of the conserved LPXTG motif (5). The liberated carboxyl of threonine is amide linked to the amino of the pentaglycyl cross-bridge of the staphylococcal peptidoglycan, thereby tethering the COOH-terminal end of protein A to the bacterial cell wall (6,7). This amide bond exchange mechanism displays striking similarity to the penicillin-sensitive transpeptidation reaction (8), during which the cell wall pentapeptide precursor is cleaved at the peptide bond between D-alanyl-D-alanine, while the liberated carboxyl of D-alanyl is amide linked to the free amino of the peptidoglycan cross-bridge (9), pentaglycyl in S. aureus (10). Elements involved in transpeptidation and the sorting reaction are conserved in Gram-positive bacteria (6,11). Thus, it seems likely that cell wall sorting is a universal mechanism for the anchoring of surface proteins (6). If so, sortase, the enzymatic activity that is thought to catalyze this reaction, might also be found conserved in many different bacterial species and could provide a target for an antibacterial therapy that interferes with surface protein anchoring (6).
Although the presence of free amino groups is a common feature in the cross-bridges of bacterial peptidoglycans, the overall chemical nature of this structure varies between different Gram-positive bacteria (11). In staphylococci, the crossbridge is composed of five glycyl, whereas in some streptococci and listerial species it consists of two alanyl and meso-diaminopimelic acid, respectively (11). Genetic analysis of staphylococcal methicillin resistance has provided insights into the synthesis of peptidoglycan cross-bridges. Staphylococcal strains expressing the penicillin binding protein PBP2a (PBP2Ј) are resistant to most ␤-lactam antibiotics including methicillin (12)(13)(14)(15)(16). Genetic screens designed to identify elements that are also necessary for methicillin resistance yielded mutations in approximately ten different fem (aux) genes (17)(18)(19)(20)(21). Some of these genes are involved in the synthesis of the pentaglycyl cross-bridge (22)(23)(24)(25)(26) or the amidation of D-iso-glutamyl within the wall peptide (27,28). The precise biochemical defect of other fem mutations is still unknown (29). Presently available staphylococcal strains harboring mutations in the femA, femB, and femX genes synthesize altered cell wall crossbridges with either three glycyl (femB), one glycyl (femA), or a combination of no or one glycyl (29). The latter phenotype has been reported for a mutant with a combination of a femA mutation and a second one leading to a partial non-functional FemX protein (hereafter called femAX) (29).
Biochemical studies on the synthesis of staphylococcal peptidoglycan revealed that the pentaglycyl cross-bridge is synthesized via modification of the lipid II precursor (undecaprenylpyrophosphate-MurNAc(-L-Ala-D-iGln-L-Lys-D-Ala-D-Ala)-(␤1-4)-GlcNAc) (30 -32). Three glycyl tRNA species are dedicated to this biosynthetic pathway (33)(34)(35)(36). After being charged with amino acid, these tRNAs are thought to serve as substrate in a sequence of reactions that successively add glycine either directly to the ⑀-amino of lysyl or to the amino of another glycyl (31,34). It seems likely that the femA, femB, and femX genes specify enzymatic activities that catalyze these reactions (29). In this model, one enzyme, presumably FemX, adds the first glycine to the ⑀-amino side chain of lysyl within lipid II (29). FemA and FemB are thought to each add two additional glycines, thereby synthesizing cross-bridges with three and five glycyl, respectively (29). It is conceivable that each Fem factor might recognize one of the three glycyl tRNA species (35,36). If so, one would predict that mutations in any one of the three glycyl tRNA genes should cause the same phenotype as the corresponding fem mutants.
Here, we asked whether the sorting reaction of surface proteins can proceed in fem mutant staphylococci. As compared with wild-type cells, the half-life of surface protein precursor molecules was increased in fem mutant strains, suggesting that the sorting reaction is partially hindered. Characterization of anchor structures revealed that surface proteins were linked to peptidoglycan with cross-bridges harboring mono-, tri-, or pentaglycyl as well as tetraglycyl-monoseryl. No surface protein was found attached to the ⑀-amino of lysyl, suggesting that the sorting reaction discriminates between certain peptidoglycan cross-bridges.
Pulse-Chase Analysis of Seb-Cws-BlaZ Processing-Staphylococcal strains harboring pSeb-Cws-BlaZ were grown overnight in tryptic soy broth supplemented with chloramphenicol (10 g/ml), diluted 1:20 into fresh medium, and grown with shaking at 37°C until A 600 0.6. Cells from 1 ml of culture were sedimented by centrifugation at 8000 ϫ g for 2 min and washed with 1 ml of prewarmed minimal medium (3). The cells were suspended in 1 ml of minimal medium, and newly synthesized polypeptide was labeled with 100 Ci of [ 35 S]Promix (Amersham) for 1 min. Labeling was quenched by the addition of an excess nonradioactive amino acid (50-l chase (100 mg/ml casamino acids, 20 mg/ml methionine)). At timed intervals after the addition of the chase, 0, 2, 5, and 10 min, 250-l aliquots were removed, and protein was precipitated by the addition of 250 l of 10% trichloroacetic acid. The precipitate was sedimented by centrifugation 15,000 ϫ g for 10 min, washed with 1 ml of acetone, and dried. Samples were suspended in 1 ml of 0.5 M Tris-HCl, pH 6.8, and staphylococcal peptidoglycan was digested by adding 50 l of mutanolysin (5000 units/ml, Sigma) and incubating 4 h at 37°C. Proteins were again precipitated with trichloroacetic acid, washed with acetone, and subjected to immunoprecipitation with ␣-BlaZ (38) followed by SDS-PAGE and PhosphorImager analysis.
Purification of Anchor Peptides-Staphylococcal strains harboring pHTT4 were grown overnight in tryptic soy broth supplemented with 10 g/ml chloramphenicol, diluted 1:40 into 4 liters of fresh medium, and grown with 250 rpm shaking for 5 h at 37°C. Cells were sedimented by centrifugation at 8000 ϫ g for 15 min. Pellets were suspended in 100 ml of water, extracted with 100 ml of ethanol-acetone (1:1), and incubated for 30 min on ice. The cells were collected by centrifugation, washed with 300 ml of ice-cold water, and suspended in 30 ml of 0.1 M Tris-HCl, pH 7.5. The peptidoglycan was digested by adding 210 g of purified 11 hydrolase for 16 h at 37°C. Seb-MH 6 -Cws was purified as described previously (7). Briefly, the digested cell wall was centrifuged at 17,000 ϫ g for 15 min to remove insoluble material, and the supernatant was subjected to affinity chromatography. 2 ml of Ni-NTA Sepharose (Qiagen) were washed with equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5), loaded with the supernatant of the 11 hydrolase digest, washed first with 30 ml of wash buffer (50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 7.5) and then with 30 ml of equilibration buffer. Bound protein was eluted with 0.5 M imidazole in wash buffer. Seb-MH 6 -Cws was precipitated with 7% trifluoroacetic acid (v/v), washed with acetone, dried under vacuum, and dissolved in 600 l of 70% formic acid. A crystal of CnBr was added, and the cleavage reaction was incubated for 16 h at room temperature. The reaction mixture was dried under vacuum, washed with water, and dissolved in 1 ml of buffer A (6 M guanidine hydrochloride, 0.1 M NaH 2 PO 4 , 0.01 M Tris-HCl, pH 8.0). The sample was subjected to 1 ml Ni-NTA Sepharose column pre-equilibrated with buffer A, washed with 10 ml of buffer A, 10 ml of buffer B (6 M urea, 0.1 M NaH 2 PO 4 , 0.01 M Tris-HCl, pH 8.0), and 10 ml of buffer C (same as buffer B, but pH 6.3). Anchor peptides were eluted with 2 ml of 0.5 M acetic acid, desalted over C18 cartridge (Analtech), and subjected to MALDI-MS.

Sorting of Surface Protein Precursor in Staphylococcal fem
Mutants-Previous work characterized the processing of Seb-Cws-BlaZ, harboring a central sorting signal flanked by NH 2terminal Seb and COOH-terminal BlaZ domains (5). Seb-Cws-BlaZ is exported from the cytoplasm by an NH 2 -terminal signal peptide and cleaved between the threonine and the glycine of its LPXTG motif (5). The NH 2 -terminal Seb fragment is linked to the staphylococcal peptidoglycan, whereas the COOH-terminal BlaZ domain remains in the bacterial cytosol (5). All surface protein that is cleaved at the LPXTG motif is also anchored to the cell wall (5). Hence, the rate of cleavage at the LPXTG motif of pulse-labeled Seb-Cws-BlaZ is a measure for the rate of surface protein anchoring in various staphylococcal fem mutant strains.
Staphylococci were pulse labeled with [ 35 S]methionine for 1 min. At timed intervals during chase, culture aliquots were precipitated with trichloroacetic acid and washed in acetone. The staphylococcal peptidoglycan was digested with mutanolysin, and all protein was again precipitated with trichloroacetic acid prior to immunoprecipitation with ␣-BlaZ and SDS-PAGE. The amount of Seb-Cws-BlaZ precursor and BlaZ cleavage fragment were determined by PhosphorImager analysis. Fig.  1B shows the autoradiogram of a typical experiment that measured the processing of Seb-Cws-BlaZ in S. aureus BB308 (fe-mAB). In wild-type cells the half-life of the precursor was 0.53 min for S. aureus OS2 (spa Ϫ , erm r ) and 1.4 min for BB270 (Mc r ) . All S. aureus fem strains displayed an increased half-life of the Seb-Cws-BlaZ precursor: 2.12 min (UK17 (femA of BB270)) , 2.58 min (UT34 -2 (femB:Tn551 of BB270)), 2.38 min (BB308 (femA:Tn551 of BB270)), and 2.25 min (UK31 (femAX of BB270)). Together, these results suggested that the sorting reaction of surface proteins is significantly slowed in the fem mutant staphylococci.
Purification of Surface Proteins from 11 Hydrolase-digested Peptidoglycan-To characterize the anchor structure of surface proteins in fem mutant staphylococci, we employed another hybrid protein. Seb-MH 6 -Cws is composed of enterotoxin B carrying an NH 2 -terminal signal peptide and a COOH-terminally fused sorting signal of protein A. At the fusion joint between Seb and the sorting signal, a methionine followed by six histidines is inserted. When expressed in staphylococci, this protein is exported and linked to the bacterial cell wall. 11 hydrolase cleaves the staphylococcal peptidoglycan at the peptide bonds between N-acetylmuramyl-L-alanyl (amidase) and D-alanyl-glycyl. 2 After peptidoglycan solubilization with the 11 hydrolase, Seb-MH 6 -Cws was affinity purified on nickel Sepharose, cleaved at methionyl with CnBr, and COOH-terminal anchor peptides were purified by another round of chromatography on Ni-NTA Sepharose. Fig. 2 shows that purified Seb-MH 6  Cell Wall Anchor Structure of Surface Proteins in femAB Mutant Staphylococci-Strain BB308 carries a transposon insertion in the promoter region of the femAB operon, a mutation which is known to decrease the expression of both genes (18,23). In contrast to the femA and femB mutants analyzed above, strain BB308 synthesizes cross-bridges containing pentaglycyl, tetraglycyl-monoseryl (Gly 4 -Ser 1 ), monoglycyl, and small amounts of triglycyl (26,29). When subjected to MALDI-MS, anchor peptides purified from 11 hydrolase-digested peptidoglycan of strain BB308 yielded four main ion clusters. For better analysis of the signals at m/z 2000-3000, the spectrum was drawn on an expanded scale in Fig. 5B Cell Wall Anchor Structure of Surface Proteins in femAX Mutant Staphylococci-The femAX mutant strain UK31 has been generated by chemical mutagenesis of strain UK14 (femA) (29). The peptidoglycan of S. aureus UK31 displayed reduced peptidoglycan cross-linking as compared with its UK14 parent (63% as compared with 67% for UK14 and 76% for wild-type strain BB270) (29). In contrast to the cell wall of strain UK14, in which all cross-bridges consist of monoglycyl, about half of all peptidoglycan monomer isolated from S. aureus UK31 contains cross-bridges without glycine, in which the ⑀-amino of lysyl is directly linked to the D-alanyl at position four of a neighboring cell wall subunit. The remaining 50% is composed of monoglycyl cross-bridges, similar to the UK17 parent (29).

DISCUSSION
All bacterial peptidoglycan is synthesized from lipid-linked precursor molecules (10,11). Penicillin binding proteins have evolved to cleave these precursors at the amide bond of Dalanyl-D-alanine (9). Even when compared with penicillin binding protein homologs from distantly related species, these enzymes share significant sequence and structural similarity (9). In contrast to the conservation of D-alanyl-D-alanine, the chemical nature of cross-bridges within bacterial peptidoglycans is highly variable. Nevertheless, the presence of a free amino group is a common feature (11), and this element functions as a nucleophile to attack the acyl intermediate formed between the carbonyl of D-alanyl and the active site serine residue of PBPs. The nucleophilic attack results in the formation of a peptide bond between D-alanyl and the peptidoglycan crossbridge as well as the regeneration of the hydroxyl at the active site serine (9). In vitro, when tested with purified pentapeptide substrate and enzyme, several different nucleophiles can substitute for the free amino of the peptidoglycan cross-bridge, including hydroxylamine, alanine, glycine, and others (40). However, in vivo the transpeptidation reaction is known to proceed with great specificity, cross-linking only neighboring wall peptides (41). These observations suggest that the fidelity of the transpeptidation reaction may depend at least in part on FIG. 2. Purification of cell wall-anchored surface proteins from 11 hydrolase-digested peptidoglycan. A, structure of Seb-MH 6 -Cws harboring an NH 2 -terminal signal (leader) peptide with signal peptidase cleavage site as well as a COOH-terminally fused cell wall sorting signal consisting of the LPXTG motif, hydrophobic domain (black box), and positively charged tail (boxed ϩ). Upstream of the LPXTG motif, a methionine followed by a histidine tag was inserted that allowed purification of the recombinant Seb-MH 6 -Cws protein on Ni-NTA Sepharose. Surface proteins were solubilized from the staphylococcal peptidoglycan by treatment with 11 hydrolase, purified on Ni-NTA Sepharose, separated on 12% SDS-PAGE, and stained with Coomassie Brilliant Blue. The SDS-PAGE shows Seb-MH 6 -Cws purified from S. aureus strains OS2 (wild type, WT), UK17 (femA), UT34 -2 (femB), BB308 (femAB), and UK31 (femAX). The migration of molecular size markers is indicated in kDa. B, structure of the staphylococcal peptidoglycan. Arrows point to the peptide cleavage sites for 11 hydrolase. Gly n indicates the variable number of glycyl within the crossbridges of staphylococcal fem mutants. a unique environment in which the free amino of the crossbridge is the only available nucleophile.
The sorting reaction of Gram-positive bacteria displays similarity to the penicillin-sensitive transpeptidation reaction (6). Here, the substrate for proteolytic cleavage is the LPXTG motif of surface proteins, which is cleaved between the threonine (T) and the glycine (G) (5). The nucleophilic amino group donor of the sorting reaction is identical to that of the transpeptidation reaction, i.e., the peptidoglycan cross-bridge. Because the LPXTG motif is found in sorting signals of many different surface proteins (42), it seems likely that sortase, the enzyme proposed to catalyze this reaction, is structurally conserved between different Gram-positive bacteria. Here, we asked whether mutationally altered cross-bridges of the staphylococcal peptidoglycan can serve as substrates for the sorting reaction. The rate of surface protein precursor cleavage at the LPXTG motif was employed to measure this reaction. S. aureus strains carrying mutations in the fem genes displayed a decreased rate of precursor cleavage as compared with the wildtype strains, suggesting that the altered cross-bridges slowed the anchoring of surface proteins.
We also revealed here the anchor structures of surface proteins expressed in S. aureus strains that carry mutations in various fem genes. The results showed that surface protein was linked to tri-and monoglycyl cross-bridges of peptidoglycan isolated from femB and femA mutant staphylococci, respectively. However, no surface protein was found linked directly to the ⑀-amino of lysyl within the cell wall of the femAX strain UK31, indicating that not all cross-bridges serve as a substrate for the sorting reaction. S. aureus BB308 carries a transposon insertion in the promoter of the femAB operon (18). Peptidoglycan analysis of this strain revealed the presence of pentaglycyl, tetraglycyl-monoseryl, and monoglycyl as well as small amounts of triglycyl cross-bridges. Analysis of anchor peptides purified from the peptidoglycan of S. aureus BB308 showed that surface proteins were mostly linked to tetraglycylmonoseryl as well as pentaglycyl. Although monoglycyl containing murein is known to be the most abundant species in strain BB308, we observed little surface protein anchoring to this cross-bridge, suggesting that the sortase activity of S. aureus preferred cross-bridges containing five residues. BB308 also contains small amounts of peptidoglycan with triglycyl cross-bridges; however, we could not identify surface protein linked to this species. This is likely due to the low abundance of triglycyl murein subunits in the peptidoglycan BB308 since the results from strain UT34 -2 (femB) indicate that surface proteins can be linked to triglycyl amino donors.
The loss of ␤-lactam resistance in fem mutants of strain BB270 suggests that the PBP2Ј enzyme cannot efficiently recognize the altered peptidoglycan cross-bridges (20). This is corroborated by electron microscopic studies of fem strains that revealed gross defects in morphology as well as cell lysis (29). Furthermore, all fem strains contain reduced amounts of crosslinked peptidoglycan (29). Introduction of the femA or femB mutation into strains that do not express the PBP2Ј enzyme also caused significant changes in the amount of peptidoglycan cross-linking as well as cell wall physiology. Thus, similar to the sorting reaction, staphylococcal PBPs can employ different  amino group donors for transpeptidation, although these crossbridges cannot fully substitute for the wild-type pentaglycyl substrate.