Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [32P]phosphoric acid to reveal a P3 intermediate. The 32P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis.32P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein. Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [32P]phosphoric acid to reveal a P3 intermediate. The 32P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis.32P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein. N-acetylmuramic acid cell wall sorting signal N-acetylglucosamine [2-(trimethylammonium)ethyl]methanethiosulfonate staphylococcal enterotoxin B tryptic soy broth D-isoglutamine radioimmune precipitation buffer [32P]phosphoric acid-labeled S. aureusRN4220 To mount a successful infection, Gram-positive pathogens display proteins on the bacterial surface that adhere to specific receptors on host tissues or provide for microbial escape from the host's immune response (1Navarre W.W. Schneewind O. Microbiol. Mol. Biol. Rev. 1999; 63: 174-229Crossref PubMed Google Scholar). Protein display on the bacterial surface involves the covalent linkage of polypeptides to the cell wall envelope (2Mazmanian S.K. Ton-That H. Schneewind O. Mol. Microbiol. 2001; 40: 1049-1057Crossref PubMed Scopus (309) Google Scholar). As reported for protein A of Staphylococcus aureus, surface proteins are synthesized as P1 precursor molecules in the bacterial cytoplasm, bearing an N-terminal signal peptide and a C-terminal sorting signal (3Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (433) Google Scholar). The 35-residue sorting signal is composed of a LPXTG motif, a hydrophobic domain, and a tail of positively charged residues (4Schneewind O. Mihaylova-Petkov D. Model P. EMBO. 1993; 12: 4803-4811Crossref PubMed Scopus (357) Google Scholar). After translocation across the cytoplasmic membrane, the N-terminal signal peptide is removed by signal peptidase, thereby generating the P2 precursor (4Schneewind O. Mihaylova-Petkov D. Model P. EMBO. 1993; 12: 4803-4811Crossref PubMed Scopus (357) Google Scholar). The C-terminal sorting signal retains the P2 precursor species within the secretory pathway and permits substrate recognition at the LPXTG motif (4Schneewind O. Mihaylova-Petkov D. Model P. EMBO. 1993; 12: 4803-4811Crossref PubMed Scopus (357) Google Scholar, 5Navarre W.W. Schneewind O. J. Bacteriol. 1996; 178: 441-446Crossref PubMed Google Scholar). Sortase, a membrane-anchored transpeptidase, cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif (6Navarre W.W. Schneewind O. Mol. Microbiol. 1994; 14: 115-121Crossref PubMed Scopus (306) Google Scholar, 7Mazmanian S.K. Liu G. Ton-That H. Schneewind O. Science. 1999; 285: 760-763Crossref PubMed Scopus (766) Google Scholar). Cleaved polypeptides are initially tethered as thioester-linked intermediates to the active site sulfhydryl residue of sortase enzymes (8Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Crossref PubMed Scopus (464) Google Scholar). Nucleophilic attack of the amino group of pentaglycine cross-bridges within the staphylococcal peptidoglycan resolves this acyl-enzyme intermediate (8Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Crossref PubMed Scopus (464) Google Scholar), resulting in the formation of an amide bond that tethers the C terminus of surface protein to the cell wall peptidoglycan (9Schneewind O. Fowler A. Faull K.F. Science. 1995; 268: 103-106Crossref PubMed Scopus (374) Google Scholar, 10Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Ton-That H. Labischinski H. Berger-Bächi B. Schneewind O. J. Biol. Chem. 1998; 273: 29143-29149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1998; 273: 29135-29142Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1999; 274: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).The peptidoglycan of S. aureus is synthesized in three cellular compartments, the cytoplasm, the membrane and the cell wall envelope (14Strominger J.L. Harvey Lectures. 1968; 64: 179-213PubMed Google Scholar). The soluble cytoplasmic peptidoglycan precursor UDP-MurNAc-l-Ala-d-iGln-l-Lys-d-Ala-d-Ala1(Park’s nucleotide) is linked to the membrane lipid undecaprenolphosphate, generating lipid I (undecaprenolpyrophosphate-MurNAc-l-Ala-d-iGln-(NH2)-l-Lys-d-Ala-d-Ala) (15Chatterjee A.N. Park J.T. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 9-16Crossref PubMed Scopus (43) Google Scholar, 16Matsuhashi M. Dietrich C.P. Strominger J.L. J. Biol. Chem. 1967; 242: 3191-3206Abstract Full Text PDF Google Scholar, 17Matsuhashi M. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Biochemical Press, Amsterdam1994: 55-72Google Scholar). Lipid I is modified by the addition of GlcNAc and pentaglycine to yield lipid II (undecaprenolpyrophosphate-MurNAc(-l-Ala-d-iGln-(NH2-Gly5)-l-Lys-d-Ala-d-Ala)-(β1–4)-GlcNAc) (18Petit J.-F. Strominger J.L. Soll D. J. Biol. Chem. 1968; 243: 757-767Abstract Full Text PDF PubMed Google Scholar, 19Matsuhashi M. Dietrich C.P. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 587-594Crossref PubMed Scopus (66) Google Scholar). Lipid II is translocated across the cytoplasmic membrane and functions as a substrate for two cell wall biosynthetic reactions that require mono- or bifunctional transglycosylases and transpeptidases (20Nakagawa J. Tamaki S. Tomioka S. Matsuhashi M. J. Biol. Chem. 1984; 259: 13937-13946Abstract Full Text PDF PubMed Google Scholar). In the transglycosylation reaction, lipid II is polymerized to generate linear peptidoglycan strands with the repeating disaccharide (MurNAc-GlcNAc)n. This reaction is fueled by the hydrolysis of lipid II and by further hydrolysis of the undecaprenolpyrophosphate product, which is translocated across the plasma membrane into the cytoplasm (21Sandermann H. Strominger J.L. J. Biol. Chem. 1972; 247: 5123-5131Abstract Full Text PDF PubMed Google Scholar). Linear peptidoglycan strands are cross-linked by transpeptidases that cleave murein pentapeptides (l-Ala-d-iGln-(NH2-Gly5)-l-Lys-d-Ala-d-Ala) and synthesize an amide bond between the carboxyl group ofl-Ala-d-iGln-(NH2-Gly5)-l-Lys-d-Ala-COOH and the amino group of pentaglycine cross-bridges (NH2-Gly5) within neighboring peptidoglycan strands (22Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (626) Google Scholar, 23Izaki K. Matsuhashi M. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 656-663Crossref PubMed Scopus (108) Google Scholar). Together the transglycosylation and transpeptidation reactions generate the three-dimensional network of mature peptidoglycan, which in staphylococci contains less than 1% of free (non-cross-linked) amino groups (NH2-Gly5) and glycan chains that are 12–60 sugar residues in length (24Giesbrecht P. Kersten T. Maidhof H. Wecke J. Microbiol. Mol. Biol. Rev. 1998; 62: 1371-1414Crossref PubMed Google Scholar,25Labischinski H. Maidhof H. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Biochemical Press, Amsterdam1994: 23-38Google Scholar).Treatment of staphylococci with the strong nucleophile hydroxylamine releases surface protein acyl-intermediate from sortase into the extracellular medium (8Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Crossref PubMed Scopus (464) Google Scholar). The released surface proteins bear a C-terminal threonine hydroxamate. These results suggest that the active site of sortase enzymes in staphylococci may be generally occupied with cleaved polypeptides. Thus, the rate-limiting step in surface protein anchoring appears to be the nucleophilic attack of the peptidoglycan substrate that regenerates the active site sulfhydryl of sortase (26Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). What is the peptidoglycan substrate that performs the nucleophilic attack? Previous work addressed this question using two experimental approaches. By following [35S]methionine-labeled polypeptides over time, it was determined that surface protein cleavage at the LPXTG motif occurred both in intact bacteria and in staphylococcal protoplasts, cells in which the peptidoglycan envelope had been removed by enzymatic digestion (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The second approach tested inhibitors of cell wall synthesis for their effect on surface protein anchoring. Vancomycin binds to thed-Ala-d-Ala moiety of lipid II (28Bugg T.D.H. Wright G.D. Dutka-Malen S. Arthur M. Courvalin P. Walsh C.T. Biochemistry. 1991; 30: 10408-10415Crossref PubMed Scopus (511) Google Scholar, 29Handwerger S. Pucci M.J. Volk K.J. Liu J. Lee M.S. J. Bacteriol. 1992; 174: 5982-5984Crossref PubMed Google Scholar) and prevents both transglycosylase and transpeptidase reactions (30Tipper D.J. Strominger J.L. J. Biol. Chem. 1968; 243: 3169-3179Abstract Full Text PDF PubMed Google Scholar). In contrast, moenomycin is an inhibitor of transglycosylation alone (31van Heijenoort Y. Leduc M. Singer H. van Heijenoort J. J. Gen. Microbiol. 1987; 133: 667-674PubMed Google Scholar). Addition of vancomycin caused peptidoglycan synthesis inhibition and a steady accumulation of P2 precursor, indicating that this compound causes a reduction of surface protein anchoring (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). A similar effect was observed when moenomycin was added to staphylococcal cultures (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Together these results suggest that sortase utilizes a peptidoglycan precursor, but not mature assembled cell wall, as a substrate for surface protein anchoring.In this report we have labeled S. aureus cells with [32P]phosphoric acid and revealed the P3 intermediate of surface protein anchoring. The P3 intermediate likely represents surface protein linked to lipid II and functions as a substrate for the transglycosylation and transpeptidation reactions that incorporate surface protein into the peptidoglycan envelope.DISCUSSIONSeveral recent studies focused on characterizing the peptidoglycan substrate of the sortase-catalyzed anchoring reaction. By measuring the processing of pulse-labeled surface proteins, it was determined that both whole cells and staphylococcal protoplasts are capable of anchoring surface proteins (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Furthermore, antibiotic inhibition ofde novo bacterial peptidoglycan synthesis inhibits surface protein anchoring (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Both results are consistent with the view that sortase utilizes the peptidoglycan precursor lipid II but not mature assembled cell walls as substrates for its transpeptidation reaction (2Mazmanian S.K. Ton-That H. Schneewind O. Mol. Microbiol. 2001; 40: 1049-1057Crossref PubMed Scopus (309) Google Scholar). Another argument in favor of lipid II is the notion that the amide bond between the threonine and the glycine of surface proteins is identical for the substrate (LPXTG motif) and the product (LPXT-Gly5) of the sorting reaction. Thus, if sortase were to interact with assembled peptidoglycan and if sortase catalyzed both forward and reverse transpeptidation reactions, the enzyme would in fact cut cell wall-anchored surface protein. This notion is not supported by our in vivo labeling experiments, revealing that sortase rapidly and efficiently anchors surface proteins to the cell wall (3Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (433) Google Scholar). We presume that surface protein linked to lipid II is rapidly incorporated into the cell wall and that this mechanism prevents sortase from catalyzing a reversible reaction.Purified sortase catalyzes an in vitro transpeptidation reaction of surface protein anchoring using LPXTG peptides and NH2-Gly, NH2-Gly2, NH2-Gly3, NH2-Gly4, or NH2-Gly5 as peptidoglycan substrates (26Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Previous work used in vitro as well as in vivotechniques to determine that the pentaglycine cross-bridges (NH2-Gly5) are better substrates than shorter cross-bridges for the sorting reaction (11Ton-That H. Labischinski H. Berger-Bächi B. Schneewind O. J. Biol. Chem. 1998; 273: 29143-29149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). However, this work still left unresolved whether murein tetra- or pentapeptides with or without linked undecaprenol and disaccharide (lipid II) are the preferred substrate for the in vitro sorting reaction. To identify the peptidoglycan substrate of the sorting reaction in vivo we entertained the possibility that surface proteins can be labeled with [32P]phosphoric acid to generate the P3 intermediate. Such a species could indeed be observed. The following arguments suggest that P3 intermediates represent surface protein linked to lipid II. (i) 32P-Labeled surface protein (44 kDa) migrates more slowly than the 35S-labeled mature species (30 kDa). The predicted mass of the P3 precursor is about 33 kDa. Assuming that undecaprenolpyrophosphate does not separate on SDS-PAGE in the same manner as polypeptides without lipid decoration, the slower mobility of P3 suggest that lipid is indeed attached to surface protein. (ii) The 32P-label of surface proteins can be removed by lysostaphin digestion but not by muramidase treatment. (iii) The formation of 32P-labeled P3 precursor absolutely requires sortase as treatment with MTSET or deletion of the sortase gene abolishes its appearance. (iv) Inhibition of peptidoglycan synthesis with antibiotics interferes with the biosynthesis of32P-labeled P3 precursor. (v) Lysostaphin treatment of P3 intermediates results in the release of 32P-labeled anchor species that can be separated on TLC plates. (vi) Nisin, a lantibiotic that is known to bind lipid II, also forms a complex with 32P-labeled anchor species. Together these data suggest that 32P-labeled P3 precursor likely represents surface protein linked to lipid II. It is anticipated that P3 intermediates are present in very small numbers in living cells, because lipid I and lipid II molecules are the least abundant intermediates of peptidoglycan biosynthesis (46van Heijenoort J. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Biochemical Press, Amsterdam1994: 39-54Google Scholar). It seems certain that the purification and analysis strategies established for surface proteins anchored to cell wall can not be used for the isolation of P3 (10Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). To mount a successful infection, Gram-positive pathogens display proteins on the bacterial surface that adhere to specific receptors on host tissues or provide for microbial escape from the host's immune response (1Navarre W.W. Schneewind O. Microbiol. Mol. Biol. Rev. 1999; 63: 174-229Crossref PubMed Google Scholar). Protein display on the bacterial surface involves the covalent linkage of polypeptides to the cell wall envelope (2Mazmanian S.K. Ton-That H. Schneewind O. Mol. Microbiol. 2001; 40: 1049-1057Crossref PubMed Scopus (309) Google Scholar). As reported for protein A of Staphylococcus aureus, surface proteins are synthesized as P1 precursor molecules in the bacterial cytoplasm, bearing an N-terminal signal peptide and a C-terminal sorting signal (3Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (433) Google Scholar). The 35-residue sorting signal is composed of a LPXTG motif, a hydrophobic domain, and a tail of positively charged residues (4Schneewind O. Mihaylova-Petkov D. Model P. EMBO. 1993; 12: 4803-4811Crossref PubMed Scopus (357) Google Scholar). After translocation across the cytoplasmic membrane, the N-terminal signal peptide is removed by signal peptidase, thereby generating the P2 precursor (4Schneewind O. Mihaylova-Petkov D. Model P. EMBO. 1993; 12: 4803-4811Crossref PubMed Scopus (357) Google Scholar). The C-terminal sorting signal retains the P2 precursor species within the secretory pathway and permits substrate recognition at the LPXTG motif (4Schneewind O. Mihaylova-Petkov D. Model P. EMBO. 1993; 12: 4803-4811Crossref PubMed Scopus (357) Google Scholar, 5Navarre W.W. Schneewind O. J. Bacteriol. 1996; 178: 441-446Crossref PubMed Google Scholar). Sortase, a membrane-anchored transpeptidase, cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif (6Navarre W.W. Schneewind O. Mol. Microbiol. 1994; 14: 115-121Crossref PubMed Scopus (306) Google Scholar, 7Mazmanian S.K. Liu G. Ton-That H. Schneewind O. Science. 1999; 285: 760-763Crossref PubMed Scopus (766) Google Scholar). Cleaved polypeptides are initially tethered as thioester-linked intermediates to the active site sulfhydryl residue of sortase enzymes (8Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Crossref PubMed Scopus (464) Google Scholar). Nucleophilic attack of the amino group of pentaglycine cross-bridges within the staphylococcal peptidoglycan resolves this acyl-enzyme intermediate (8Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Crossref PubMed Scopus (464) Google Scholar), resulting in the formation of an amide bond that tethers the C terminus of surface protein to the cell wall peptidoglycan (9Schneewind O. Fowler A. Faull K.F. Science. 1995; 268: 103-106Crossref PubMed Scopus (374) Google Scholar, 10Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Ton-That H. Labischinski H. Berger-Bächi B. Schneewind O. J. Biol. Chem. 1998; 273: 29143-29149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1998; 273: 29135-29142Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1999; 274: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The peptidoglycan of S. aureus is synthesized in three cellular compartments, the cytoplasm, the membrane and the cell wall envelope (14Strominger J.L. Harvey Lectures. 1968; 64: 179-213PubMed Google Scholar). The soluble cytoplasmic peptidoglycan precursor UDP-MurNAc-l-Ala-d-iGln-l-Lys-d-Ala-d-Ala1(Park’s nucleotide) is linked to the membrane lipid undecaprenolphosphate, generating lipid I (undecaprenolpyrophosphate-MurNAc-l-Ala-d-iGln-(NH2)-l-Lys-d-Ala-d-Ala) (15Chatterjee A.N. Park J.T. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 9-16Crossref PubMed Scopus (43) Google Scholar, 16Matsuhashi M. Dietrich C.P. Strominger J.L. J. Biol. Chem. 1967; 242: 3191-3206Abstract Full Text PDF Google Scholar, 17Matsuhashi M. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Biochemical Press, Amsterdam1994: 55-72Google Scholar). Lipid I is modified by the addition of GlcNAc and pentaglycine to yield lipid II (undecaprenolpyrophosphate-MurNAc(-l-Ala-d-iGln-(NH2-Gly5)-l-Lys-d-Ala-d-Ala)-(β1–4)-GlcNAc) (18Petit J.-F. Strominger J.L. Soll D. J. Biol. Chem. 1968; 243: 757-767Abstract Full Text PDF PubMed Google Scholar, 19Matsuhashi M. Dietrich C.P. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 587-594Crossref PubMed Scopus (66) Google Scholar). Lipid II is translocated across the cytoplasmic membrane and functions as a substrate for two cell wall biosynthetic reactions that require mono- or bifunctional transglycosylases and transpeptidases (20Nakagawa J. Tamaki S. Tomioka S. Matsuhashi M. J. Biol. Chem. 1984; 259: 13937-13946Abstract Full Text PDF PubMed Google Scholar). In the transglycosylation reaction, lipid II is polymerized to generate linear peptidoglycan strands with the repeating disaccharide (MurNAc-GlcNAc)n. This reaction is fueled by the hydrolysis of lipid II and by further hydrolysis of the undecaprenolpyrophosphate product, which is translocated across the plasma membrane into the cytoplasm (21Sandermann H. Strominger J.L. J. Biol. Chem. 1972; 247: 5123-5131Abstract Full Text PDF PubMed Google Scholar). Linear peptidoglycan strands are cross-linked by transpeptidases that cleave murein pentapeptides (l-Ala-d-iGln-(NH2-Gly5)-l-Lys-d-Ala-d-Ala) and synthesize an amide bond between the carboxyl group ofl-Ala-d-iGln-(NH2-Gly5)-l-Lys-d-Ala-COOH and the amino group of pentaglycine cross-bridges (NH2-Gly5) within neighboring peptidoglycan strands (22Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (626) Google Scholar, 23Izaki K. Matsuhashi M. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 656-663Crossref PubMed Scopus (108) Google Scholar). Together the transglycosylation and transpeptidation reactions generate the three-dimensional network of mature peptidoglycan, which in staphylococci contains less than 1% of free (non-cross-linked) amino groups (NH2-Gly5) and glycan chains that are 12–60 sugar residues in length (24Giesbrecht P. Kersten T. Maidhof H. Wecke J. Microbiol. Mol. Biol. Rev. 1998; 62: 1371-1414Crossref PubMed Google Scholar,25Labischinski H. Maidhof H. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Biochemical Press, Amsterdam1994: 23-38Google Scholar). Treatment of staphylococci with the strong nucleophile hydroxylamine releases surface protein acyl-intermediate from sortase into the extracellular medium (8Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Crossref PubMed Scopus (464) Google Scholar). The released surface proteins bear a C-terminal threonine hydroxamate. These results suggest that the active site of sortase enzymes in staphylococci may be generally occupied with cleaved polypeptides. Thus, the rate-limiting step in surface protein anchoring appears to be the nucleophilic attack of the peptidoglycan substrate that regenerates the active site sulfhydryl of sortase (26Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). What is the peptidoglycan substrate that performs the nucleophilic attack? Previous work addressed this question using two experimental approaches. By following [35S]methionine-labeled polypeptides over time, it was determined that surface protein cleavage at the LPXTG motif occurred both in intact bacteria and in staphylococcal protoplasts, cells in which the peptidoglycan envelope had been removed by enzymatic digestion (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The second approach tested inhibitors of cell wall synthesis for their effect on surface protein anchoring. Vancomycin binds to thed-Ala-d-Ala moiety of lipid II (28Bugg T.D.H. Wright G.D. Dutka-Malen S. Arthur M. Courvalin P. Walsh C.T. Biochemistry. 1991; 30: 10408-10415Crossref PubMed Scopus (511) Google Scholar, 29Handwerger S. Pucci M.J. Volk K.J. Liu J. Lee M.S. J. Bacteriol. 1992; 174: 5982-5984Crossref PubMed Google Scholar) and prevents both transglycosylase and transpeptidase reactions (30Tipper D.J. Strominger J.L. J. Biol. Chem. 1968; 243: 3169-3179Abstract Full Text PDF PubMed Google Scholar). In contrast, moenomycin is an inhibitor of transglycosylation alone (31van Heijenoort Y. Leduc M. Singer H. van Heijenoort J. J. Gen. Microbiol. 1987; 133: 667-674PubMed Google Scholar). Addition of vancomycin caused peptidoglycan synthesis inhibition and a steady accumulation of P2 precursor, indicating that this compound causes a reduction of surface protein anchoring (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). A similar effect was observed when moenomycin was added to staphylococcal cultures (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Together these results suggest that sortase utilizes a peptidoglycan precursor, but not mature assembled cell wall, as a substrate for surface protein anchoring. In this report we have labeled S. aureus cells with [32P]phosphoric acid and revealed the P3 intermediate of surface protein anchoring. The P3 intermediate likely represents surface protein linked to lipid II and functions as a substrate for the transglycosylation and transpeptidation reactions that incorporate surface protein into the peptidoglycan envelope. DISCUSSIONSeveral recent studies focused on characterizing the peptidoglycan substrate of the sortase-catalyzed anchoring reaction. By measuring the processing of pulse-labeled surface proteins, it was determined that both whole cells and staphylococcal protoplasts are capable of anchoring surface proteins (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Furthermore, antibiotic inhibition ofde novo bacterial peptidoglycan synthesis inhibits surface protein anchoring (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Both results are consistent with the view that sortase utilizes the peptidoglycan precursor lipid II but not mature assembled cell walls as substrates for its transpeptidation reaction (2Mazmanian S.K. Ton-That H. Schneewind O. Mol. Microbiol. 2001; 40: 1049-1057Crossref PubMed Scopus (309) Google Scholar). Another argument in favor of lipid II is the notion that the amide bond between the threonine and the glycine of surface proteins is identical for the substrate (LPXTG motif) and the product (LPXT-Gly5) of the sorting reaction. Thus, if sortase were to interact with assembled peptidoglycan and if sortase catalyzed both forward and reverse transpeptidation reactions, the enzyme would in fact cut cell wall-anchored surface protein. This notion is not supported by our in vivo labeling experiments, revealing that sortase rapidly and efficiently anchors surface proteins to the cell wall (3Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (433) Google Scholar). We presume that surface protein linked to lipid II is rapidly incorporated into the cell wall and that this mechanism prevents sortase from catalyzing a reversible reaction.Purified sortase catalyzes an in vitro transpeptidation reaction of surface protein anchoring using LPXTG peptides and NH2-Gly, NH2-Gly2, NH2-Gly3, NH2-Gly4, or NH2-Gly5 as peptidoglycan substrates (26Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Previous work used in vitro as well as in vivotechniques to determine that the pentaglycine cross-bridges (NH2-Gly5) are better substrates than shorter cross-bridges for the sorting reaction (11Ton-That H. Labischinski H. Berger-Bächi B. Schneewind O. J. Biol. Chem. 1998; 273: 29143-29149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). However, this work still left unresolved whether murein tetra- or pentapeptides with or without linked undecaprenol and disaccharide (lipid II) are the preferred substrate for the in vitro sorting reaction. To identify the peptidoglycan substrate of the sorting reaction in vivo we entertained the possibility that surface proteins can be labeled with [32P]phosphoric acid to generate the P3 intermediate. Such a species could indeed be observed. The following arguments suggest that P3 intermediates represent surface protein linked to lipid II. (i) 32P-Labeled surface protein (44 kDa) migrates more slowly than the 35S-labeled mature species (30 kDa). The predicted mass of the P3 precursor is about 33 kDa. Assuming that undecaprenolpyrophosphate does not separate on SDS-PAGE in the same manner as polypeptides without lipid decoration, the slower mobility of P3 suggest that lipid is indeed attached to surface protein. (ii) The 32P-label of surface proteins can be removed by lysostaphin digestion but not by muramidase treatment. (iii) The formation of 32P-labeled P3 precursor absolutely requires sortase as treatment with MTSET or deletion of the sortase gene abolishes its appearance. (iv) Inhibition of peptidoglycan synthesis with antibiotics interferes with the biosynthesis of32P-labeled P3 precursor. (v) Lysostaphin treatment of P3 intermediates results in the release of 32P-labeled anchor species that can be separated on TLC plates. (vi) Nisin, a lantibiotic that is known to bind lipid II, also forms a complex with 32P-labeled anchor species. Together these data suggest that 32P-labeled P3 precursor likely represents surface protein linked to lipid II. It is anticipated that P3 intermediates are present in very small numbers in living cells, because lipid I and lipid II molecules are the least abundant intermediates of peptidoglycan biosynthesis (46van Heijenoort J. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Biochemical Press, Amsterdam1994: 39-54Google Scholar). It seems certain that the purification and analysis strategies established for surface proteins anchored to cell wall can not be used for the isolation of P3 (10Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Several recent studies focused on characterizing the peptidoglycan substrate of the sortase-catalyzed anchoring reaction. By measuring the processing of pulse-labeled surface proteins, it was determined that both whole cells and staphylococcal protoplasts are capable of anchoring surface proteins (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Furthermore, antibiotic inhibition ofde novo bacterial peptidoglycan synthesis inhibits surface protein anchoring (27Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Both results are consistent with the view that sortase utilizes the peptidoglycan precursor lipid II but not mature assembled cell walls as substrates for its transpeptidation reaction (2Mazmanian S.K. Ton-That H. Schneewind O. Mol. Microbiol. 2001; 40: 1049-1057Crossref PubMed Scopus (309) Google Scholar). Another argument in favor of lipid II is the notion that the amide bond between the threonine and the glycine of surface proteins is identical for the substrate (LPXTG motif) and the product (LPXT-Gly5) of the sorting reaction. Thus, if sortase were to interact with assembled peptidoglycan and if sortase catalyzed both forward and reverse transpeptidation reactions, the enzyme would in fact cut cell wall-anchored surface protein. This notion is not supported by our in vivo labeling experiments, revealing that sortase rapidly and efficiently anchors surface proteins to the cell wall (3Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (433) Google Scholar). We presume that surface protein linked to lipid II is rapidly incorporated into the cell wall and that this mechanism prevents sortase from catalyzing a reversible reaction. Purified sortase catalyzes an in vitro transpeptidation reaction of surface protein anchoring using LPXTG peptides and NH2-Gly, NH2-Gly2, NH2-Gly3, NH2-Gly4, or NH2-Gly5 as peptidoglycan substrates (26Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Previous work used in vitro as well as in vivotechniques to determine that the pentaglycine cross-bridges (NH2-Gly5) are better substrates than shorter cross-bridges for the sorting reaction (11Ton-That H. Labischinski H. Berger-Bächi B. Schneewind O. J. Biol. Chem. 1998; 273: 29143-29149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). However, this work still left unresolved whether murein tetra- or pentapeptides with or without linked undecaprenol and disaccharide (lipid II) are the preferred substrate for the in vitro sorting reaction. To identify the peptidoglycan substrate of the sorting reaction in vivo we entertained the possibility that surface proteins can be labeled with [32P]phosphoric acid to generate the P3 intermediate. Such a species could indeed be observed. The following arguments suggest that P3 intermediates represent surface protein linked to lipid II. (i) 32P-Labeled surface protein (44 kDa) migrates more slowly than the 35S-labeled mature species (30 kDa). The predicted mass of the P3 precursor is about 33 kDa. Assuming that undecaprenolpyrophosphate does not separate on SDS-PAGE in the same manner as polypeptides without lipid decoration, the slower mobility of P3 suggest that lipid is indeed attached to surface protein. (ii) The 32P-label of surface proteins can be removed by lysostaphin digestion but not by muramidase treatment. (iii) The formation of 32P-labeled P3 precursor absolutely requires sortase as treatment with MTSET or deletion of the sortase gene abolishes its appearance. (iv) Inhibition of peptidoglycan synthesis with antibiotics interferes with the biosynthesis of32P-labeled P3 precursor. (v) Lysostaphin treatment of P3 intermediates results in the release of 32P-labeled anchor species that can be separated on TLC plates. (vi) Nisin, a lantibiotic that is known to bind lipid II, also forms a complex with 32P-labeled anchor species. Together these data suggest that 32P-labeled P3 precursor likely represents surface protein linked to lipid II. It is anticipated that P3 intermediates are present in very small numbers in living cells, because lipid I and lipid II molecules are the least abundant intermediates of peptidoglycan biosynthesis (46van Heijenoort J. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Biochemical Press, Amsterdam1994: 39-54Google Scholar). It seems certain that the purification and analysis strategies established for surface proteins anchored to cell wall can not be used for the isolation of P3 (10Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar).