Anchor Structure of Staphylococcal Surface Proteins
2005; Elsevier BV; Volume: 280; Issue: 16 Linguagem: Inglês
10.1074/jbc.m500071200
ISSN1083-351X
AutoresLuciano A. Marraffini, Olaf Schneewind,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoStaphylococcus 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. 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 (1Foster T.J. Höök M. Trends Microbiol. 1998; 6: 484-488Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar, 2Navarre W.W. Schneewind O. Microbiol. Mol. Biol. Rev. 1999; 63: 174-229Crossref PubMed Google Scholar, 3Rivas J.M. Speziale P. Patti J.M. Hook M. Curr. Opin. Drug Discovery Dev. 2004; 7: 223-227PubMed Google Scholar). 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 (4Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (438) Google Scholar, 5Schneewind O. Mihaylova-Petkov D. Model P. EMBO J. 1993; 12: 4803-4811Crossref PubMed Scopus (360) Google Scholar, 6Cossart P. Jonquieres R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5013-5015Crossref PubMed Scopus (167) Google Scholar). These surface proteins are synthesized as precursors with N-terminal signal peptides in the bacterial cytoplasm (P1 precursor) (7Abrahmsen L. Moks T. Nilsson B. Hellman U. Uhlen M. EMBO J. 1985; 4: 3901-3906Crossref PubMed Scopus (70) Google Scholar, 8Bae T. Schneewind O. J. Bacteriol. 2003; 185: 2910-2919Crossref PubMed Scopus (85) Google Scholar). After membrane translocation and signal peptide cleavage, the Cterminal sorting signal first retains the polypeptide in the cytoplasmic membrane (P2 precursor) (4Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (438) Google Scholar). Membrane-anchored sortase A then cleaves the sorting signal between the threonine and glycine residues (9Navarre W.W. Schneewind O. Mol. Microbiol. 1994; 14: 115-121Crossref PubMed Scopus (310) Google Scholar, 10Mazmanian S.K. Liu G. Jensen E.R. Lenoy E. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5510-5515Crossref PubMed Scopus (382) Google Scholar), generating a thioester-linked acylenzyme between the sortase active-site thiol of Cys184 and the C-terminal carboxyl group of threonine (11Ton-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 (475) Google Scholar, 12Ton-That H. Mazmanian S.K. Alksne L. Schneewind O. J. Biol. Chem. 2002; 277: 7447-7452Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). 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 (13Schneewind O. Fowler A. Faull K.F. Science. 1995; 268: 103-106Crossref PubMed Scopus (375) Google Scholar, 14Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 15Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1998; 273: 29135-29142Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The cell wall of Gram-positive bacteria is composed of peptidoglycan, a heteropolymeric macromolecule encompassing glycan strands and attached wall peptides (16Ghuysen J-M. Bacteriol. Rev. 1968; 32: 425-464Crossref PubMed Google Scholar, 17Strominger J.L. Harvey Lect. 1968; 64: 179-213PubMed Google Scholar). Glycan strands, which consist of the repeating disaccharide MurNAc 1The abbreviations used are: MurNAc, N-acetylmuramic acid; d-iGln, d-isoglutaminyl; Isd, iron-regulated surface determinant; Fur, ferric uptake repressor; SEB, staphylococcal enterotoxin B; MH6, Met-His6; CWS, cell wall sorting signal; Spa, staphylococcal protein A; Ni-NTA, nickel-nitrilotriacetic acid; PVDF, polyvinylidene difluoride; RP-HPLC, reversed-phase high performance liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MALDI-TOF/TOF-MS, MALDI-TOF tandem mass spectrometry; CID, collision-induced dissociation. (β1–4)GlcNAc (18Ghuysen J-M. Strominger J.L. Biochemistry. 1963; 2: 1119-1125Crossref PubMed Scopus (64) Google Scholar), vary in length and contain up to 30 subunits, with a predominant length of 3–10 and an average of six disaccharide subunits (19Boneca I.G. Huang Z.H. Gage D.A. Tomasz A. J. Biol. Chem. 2000; 275: 9910-9918Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A short peptide component (l-Ala-d-iGln-(Gly5)-l-Lys-d-Ala) is attached via an amide bond between the lactyl moiety of MurNAc and the amino group of l-Ala (20Tipper D.J. Biochemistry. 1968; 7: 1441-1449Crossref Scopus (72) Google Scholar, 21Tipper D.J. Ghuysen J-M. Strominger J.L. Biochemistry. 1965; 4: 468-473Crossref Scopus (30) Google Scholar, 22Tipper D.J. Strominger J.L. Ensign J.C. Biochemistry. 1967; 6: 906-920Crossref PubMed Scopus (41) Google Scholar, 23Munoz E. Ghuysen J-M. Lehy-Bouille M. Petit J.-F. Heymann H. Bricas E. Lefrancier P. Biochemistry. 1966; 5: 3748-3764Crossref Scopus (29) Google Scholar). About 80–95% of the wall peptides of the assembled peptidoglycan are cross-linked, i.e. the amino groups of cross-bridges (pentaglycine (NH2-Gly5) in staphylococci) are amide-linked to the carboxyl groups of d-Ala within neighboring wall peptides (24Strominger J.L. Izaki K. Matsuhashi M. Tipper D.J. Fed. Proc. 1967; 26: 9-18PubMed Google Scholar, 25Snowden M.A. Perkins H.R. Wyke A.W. Hayes M.V. Ward J.B. J. Gen. Microbiol. 1989; 135: 3015-3022PubMed Google Scholar, 26Gally D. Archibald A.R. J. Gen. Microbiol. 1993; 139: 1907-1913Crossref PubMed Scopus (21) Google Scholar). During cell wall synthesis, soluble nucleotide-linked Mur-NAc pentapeptide precursor (UDP-MurNAc-l-Ala-d-iGln-l-Lys-d-Ala-d-Ala) is assembled in the cytoplasm and transferred to the bactoprenyl membrane carrier, thereby generating lipid I(C55-PP-MurNAc-l-Ala-d-iGln-l-Lys-d-Ala-d-Ala) (27Chatterjee A.N. Park J.T. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 9-16Crossref PubMed Scopus (43) Google Scholar, 28Matsuhashi M. Dietrich C.P. Strominger J.L. J. Biol. Chem. 1967; 242: 3191-3206Abstract Full Text PDF Google Scholar, 29Matsuhashi M. Ghuysen J-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishers B.V., Amsterdam1994: 55-72Google Scholar). After disaccharide formation and addition of the pentaglycine cross-bridge, lipid II (C55-PP-MurNAc(l-Ala-d-iGln-(NH2-Gly5)-l-Lys-d-Ala-d-Ala)(β1–4)GlcNAc), the biosynthetic substrate of extracellular cell wall assembly (30Petit J-F. Strominger J.L. Soll D. J. Biol. Chem. 1968; 243: 757-767Abstract Full Text PDF PubMed Google Scholar, 31Matsuhashi M. Dietrich C.P. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 587-594Crossref PubMed Scopus (66) Google Scholar), is translocated across the bacterial membrane (32Nakagawa J. Tamaki S. Tomioka S. Matsuhashi M. J. Biol. Chem. 1984; 259: 13937-13946Abstract Full Text PDF PubMed Google Scholar). The peptidoglycan is finally polymerized via transglycosylation (33Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (637) Google Scholar) and transpeptidation (34Izaki K. Matsuhashi M. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 656-663Crossref PubMed Scopus (110) Google Scholar) reactions catalyzed by penicillin-binding proteins. Several lines of evidence indicate that lipid II functions as the cell wall substrate for sortase A, generating C55-PP-MurNAc(l-Ala-d-iGln-(surface protein-Gly5)-l-Lys-d-Ala-d-Ala)(β1–4)-GlcNAc (11Ton-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 (475) Google Scholar, 35Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 36Perry A.M. Ton-That H. Mazmanian S.K. Schneewind O. J. Biol. Chem. 2002; 277: 16241-16248Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 37Ruzin A. Severin A. Ritacco F. Tabei K. Ingh SG. Bradford P.A. Siegel M.M. Projan S.J. Shlaes D.M. J. Bacteriol. 2002; 184: 2141-2147Crossref PubMed Scopus (74) Google Scholar). It is believed that subsequent peptidoglycan polymerization would result in the incorporation of the surface protein into the cell wall. 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 (38Mazmanian S.K. Ton-That H. Su K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2293-2298Crossref PubMed Scopus (309) Google Scholar), a DNA-binding protein with affinity for canonical DNA sites (Fur boxes) (39Xiong A. Singh V.K. Cabrera G. Jayaswal R.K. Microbiology. 2000; 146: 659-668Crossref PubMed Scopus (91) Google Scholar). 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 (40Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachimiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar, 41Skaar E.P. Gaspar A.H. Schneewind O. J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). 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 (40Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachimiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar). 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 (40Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachimiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar). 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 (38Mazmanian S.K. Ton-That H. Su K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2293-2298Crossref PubMed Scopus (309) Google Scholar). 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. Bacterial Strains and Plasmids—S. aureus strains Newman (42Duthie E.S. Lorenz L.L. J. Gen. Microbiol. 1952; 6: 95-107Crossref PubMed Google Scholar), RN4220 (43Kreiswirth B.N. Lofdahl S. Betley M.J. O'Reilly M. Schlievert P.M. Bergdoll M.S. Novick R.P. Nature. 1983; 305: 709-712Crossref PubMed Scopus (1009) Google Scholar), and SKM15 (RN4220 fur::tet) (38Mazmanian S.K. Ton-That H. Su K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2293-2298Crossref PubMed Scopus (309) Google Scholar) have been described previously. To construct pAMP2 (encoding SEB-MH6-CWSIsdC), DNA sequences specifying CWSIsdC were amplified by PCR using S. aureus Newman chromosomal DNA as template and primers 6HisdCsig (5′-AAGGGTACCATGCATCACCATCACCATCACAAAGTAGAAAATCCACAAACAAAT-3′) and SasK-3-B (5′AAGGATCCTTATTCCACATTGCCTTTAG-3′). The PCR product was cut with KpnI and BamHI and inserted into pHTT4 (14Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) cut with the same enzymes. This cloning step replaced the MH6-CWSSpa coding sequences fused downstream of the seb gene in pHTT4 with those of MH6-CWSIsdC, thereby creating pA-MP1. The srtA promoter-srtB coding sequence fusion was obtained by digestion of pSM75 (38Mazmanian S.K. Ton-That H. Su K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2293-2298Crossref PubMed Scopus (309) Google Scholar) with EcoRI. The fragment was ligated to Eco-RI-linearized pAMP1, thereby generating pAMP2. Escherichia coli DH5α was used as host for DNA transformation. The construct was verified by restriction mapping and sequencing and was then transformed into S. aureus SKM15 (44Mazmanian S.K. Schneewind O. Sonenshine A. Losick R. Hoch J. Bacillus subtilis and Its Closest Relatives. 2nd Ed. American Society for Microbiology, Washington, D. C.2002: 57-70Google Scholar). The antibiotics used in the selective medium were ampicillin (100 μg/ml for E. coli) and chloramphenicol (10 μg/ml for S. aureus). Purification of Φ11 Hydrolase and Φ11Δ d-Ala-Gly Endopeptidase— Φ11 hydrolase and Φ11Δ d-Ala-Gly endopeptidase were purified by Ni-NTA affinity chromatography (Qiagen Inc.) from E. coli BL21(DE3) cells harboring pHTT2 and pWil54, respectively. The detailed procedure is described elsewhere (45Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1999; 274: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Enzyme concentration was measured by a modified biuret reaction (BCA kit, Pierce) against a standard of bovine serum albumin. Enzyme preparations were stored at –20 °C. Purification of Staphylococcal Cell Walls—Cell wall preparations were obtained as described previously (15Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1998; 273: 29135-29142Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, colonies were inoculated into tryptic soy broth containing 10 μg/ml chloramphenicol, and S. aureus SKM15 (pAMP2) was grown overnight at 37 °C. Cells were diluted 1:50 into fresh medium, grown to A600 = 0.8, and harvested by centrifugation at 10,000 × 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 × g for 15 min to sediment cell wall sacculi and membranes. Sediment was suspended in 100 ml of 100 mm KH2PO4 (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 × 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 –80 °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 2,2′-dipyridyl. Staphylococcal cultures were diluted 1:30 into 2 ml of the same medium and grown to A600 = 0.8, and cells were harvested by centrifugation at 10,000 × 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, Φ11 hydrolase, and Φ11Δ digestions. Cell wall components were solubilized with 700 units/ml mutanolysin (Sigma) and 70 μg/ml lysostaphin, Φ11 hydrolase, or Φ11Δ. Insoluble material was removed by centrifugation at 15,000 × g for 5 min, and proteins in the supernatant were precipitated by addition of 1 ml of 1:5 chloroform/methanol solution (46Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3170) Google Scholar), washed with 1 ml of methanol, and suspended in 100 μlof loading buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 0.5 m β-mercpatoethanol, 20% glycerol, 0.5 mg/ml bromophenol blue). Five μl of each sample was subjected to SDS-PAGE and electrotransferred to a PVDF membrane. IsdC was detected by immunoblotting using rabbit antiIsdC polyclonal serum. The membrane was blocked in the presence of human IgG (12.5 μg/ml; Sigma) to prevent the non-immune binding of protein A. Proteinase K Digestion of Surface Proteins—S. aureus RN4220 (pHTT4, encoding SEB-MH6-CWSSpa) and SKM15 (pAMP2, encoding SEB-MH6-CWSIsdC) cultures were grown in tryptic soy broth containing 10 μg/ml chloramphenicol to A600 ∼ 1.0. Cells were sedimented by centrifugation at 6000 × g, washed twice with TSM buffer (50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 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 μg 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 ×g for 5 min; the supernatant removed; and staphylococci were suspended in 1 ml of TSM buffer containing 20 μgof lysostaphin (Ambi) and incubated for 20 min at 37 °C. Staphylococcal protoplasts were sedimented by centrifugation at 12,000 ×g for 5 min, and 1-ml supernatants were transferred to fresh tubes and either treated with 150 μg 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 μl 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-MH6-CWSIsdC—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, Φ11 hydrolase, and Φ11Δ digestions. To each cell wall suspension were added 20,000 units of mutanolysin, 2 mg of lysostaphin, 2 mg of Φ11 hydrolase, or 2 mg of Φ11Δ. 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 × g for 15 min. SEB-MH6-CWSIsdC 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-MH6-CW-SIsdC 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 NaH2PO4, 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 NaH2PO4, 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-MH6-CWSIsdC, N-acetylmuramic acid residues were reduced with sodium borohydride to generate N-acetylmuramitol, thereby facilitating HPLC separation and mass spectrometric analysis (47Arbeloa A. Hugonnet J.E. Sentilhes A.C. Josseaume N. Dubost L. Monsempes C. Blanot D. Brouard J.P. Arthur M. J. Biol. Chem. 2004; 279: 41546-41556Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Finally, peptides were subjected to RP-HPLC on a Hypersil C18 column (2 × 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% H2O (0.1% trifluoroacetic acid) to 99% CH3CN (0.1% trifluoroacetic acid) over 100 min. Elution of peptides was monitored at 215 nm, and 1-min fractions were collected. MALDI Mass Spectrometry—Dried HPLC fractions containing peptides of interest were suspended in 15 μl of CH3CN/water/trifluoroacetic acid (30:70:0.1). MALDI mass spectra were obtained on a Reflectron time-of-flight instrument (ABI Biosystems) in the reflection mode. Samples (0.5 μl) were co-spotted with 0.5 μl of matrix (10 mg/ml α-cyano-4-hydroxy-cinnamic acid (Sigma) in CH3CN/water/trifluoroacetic acid (70:30:0.1)). All samples were externally calibrated to a standard of bovine insulin. 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 (38Mazmanian S.K. Ton-That H. Su K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2293-2298Crossref PubMed Scopus (309) Google Scholar). Using signal peptide algorithms and canonical sorting signal cleavage site predictions, Ala29 was identified as the N-terminal residue and Thr192 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 2,2′-dipyridyl, an iron-chelating reagent, thereby activating isd expression (38Mazmanian S.K. Ton-That H. Su K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2293-2298Crossref PubMed Scopus (309) Google Scholar). 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) (48Schindler C.A. Schuhardt V.T. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 414-421Crossref PubMed Scopus (323) Google Scholar), solubilized IsdC as a single species of 19 kDa (Fig. 1B). Φ11 hydrolase cleaves the amide bond of MurNAc– l-Ala as well as the d-Ala–Gly peptide bond of the S. aureus peptidoglycan (45Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1999; 274: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and released IsdC as a species with a uniform 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 (49Calandra G.B. Cole R. Infect. Immun. 1980; 28: 1033-1037Crossref PubMed Google Scholar, 50Yokogawa K. Kawata S. Nishimura S. Ikeda Y. Yoshimura Y. Antimicrob. Agents Chemother. 1974; 6: 156-165Crossref PubMed Scopus (81) Google Scholar), solubilized IsdC as a spectrum of seven fragments with different masses. The fastest migrating species displayed a mass similar to that of Φ11 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 [35S]methionine was used to release SEB-CWSIsdC, a hybrid containing the IsdC sorting signal (CWSIsdC) fused to the SEB polypeptide. The solubilized fusion proteins were captured by immunoprecipitation and detected by autoradiography (40Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachimiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar). In contrast to our observations reported here, a single mutanolysin-released SEB-CWSIsdC species was found (40Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachimiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar). This apparent discrepancy appears to be due to the pulse-labeling technique, as immunoprecipitation and autoradiography detected only the most abundant SEB-CWSIsdC species (40Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachimiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar). The Φ11Δ enzyme cleaves the peptide bond of d-Ala–Gly, but not the amide bond of MurNAc–l-Ala (45Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1999; 274: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Treatment with Φ11Δ released three IsdC fragments from the cell wall, all of which migrated more slowly than the Φ11 hydrolase- and lysostaphin-released counterparts. Thus, it appears that IsdC is linked to the pentaglycine cross-bridges of the staphylococcal cell wall, as only treatment with lysostaphin and Φ11 hydrolase, i.e. enzymes that directly cut cross-bridges or excise cross-bridges with wall peptides, released IsdC with a uniform mass. Cleavage of only glycan strands (mutanolysin) or cross-bridges (Φ11Δ) 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-MH6-CWSIsdC and SEB-MH6-CWSSpa—Fusion of sorting signal sequences to the C-terminal end of SEB, a highly expressed exoprotein, generates cell wall-anchored hybrids (14Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 38Mazmanian S.K. Ton-That H. Su K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2293-2298Crossref PubMed Scopus (309) Google Scholar). 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 (40Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachimiak A. Missiakas D.M. Schneewind
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