Specificity of L,D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes
2007; Elsevier BV; Volume: 282; Issue: 18 Linguagem: Inglês
10.1074/jbc.m610911200
ISSN1083-351X
AutoresSophie Magnet, Ana Arbeloa, Jean‐Luc Mainardi, Jean‐Emmanuel Hugonnet, Martine Fourgeaud, Lionel Dubost, Arul Marie, V. Delfosse, Claudine Mayer, Louis B. Rice, Michel Arthur,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoWe report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the l,d-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldtfm from Enterococcus faecium, was previously shown to bypass the d,d-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and β-lactam antibiotics. Ldtfm homologues from Bacillus subtilis (LdtBs) and E. faecalis (Ldtfs) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP3) and l-Lys3-l-Ala-l-Ala at the third position of the stem peptide, respectively, instead of l-Lys3-d-iAsn in E. faecium. Ldtfs differed from Ldtfm and LdtBs by its capacity to hydrolyze the l-Lys3-d-Ala4 bond of tetrapeptide (l,d-carboxypeptidase activity) and pentapeptide (l,d-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldtfs, which was also specific for its cognate acyl donor, Ldtfm tolerated substitution of l-Lys3-d-iAsn by l-Lys3-l-Ala-l-Ala. Likewise, LdtBs tolerated substitution of mesoDAP3 by l-Lys3-d-iAsn and l-Lys3-l-Ala-l-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the l,d-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving l,d-transpeptidases. We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the l,d-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldtfm from Enterococcus faecium, was previously shown to bypass the d,d-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and β-lactam antibiotics. Ldtfm homologues from Bacillus subtilis (LdtBs) and E. faecalis (Ldtfs) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP3) and l-Lys3-l-Ala-l-Ala at the third position of the stem peptide, respectively, instead of l-Lys3-d-iAsn in E. faecium. Ldtfs differed from Ldtfm and LdtBs by its capacity to hydrolyze the l-Lys3-d-Ala4 bond of tetrapeptide (l,d-carboxypeptidase activity) and pentapeptide (l,d-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldtfs, which was also specific for its cognate acyl donor, Ldtfm tolerated substitution of l-Lys3-d-iAsn by l-Lys3-l-Ala-l-Ala. Likewise, LdtBs tolerated substitution of mesoDAP3 by l-Lys3-d-iAsn and l-Lys3-l-Ala-l-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the l,d-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving l,d-transpeptidases. The bacterial cell wall peptidoglycan is a net-like macromolecule that surrounds the cytoplasmic membrane (1Holtje J.V. Microbiol. Mol. Biol. Rev. 1998; 62: 181-203Crossref PubMed Google Scholar). The polymer is essential because it supplies the cell with mechanical protection against the osmotic pressure of the cytoplasm. The peptidoglycan subunit contains β-1,4-linked N-acetylglucosamine (GlcNAc) 2The abbreviations used are: GlcNAc, N-acetylglucosamine; PBP, penicillin-binding protein; d-iAsn, d-iso-asparagine; d-iAsp, d-iso-aspartic acid; mesoDAP, meso-diaminopimelic acid; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MurNAc, N-acetylmuramic acid; RP-HPLC, reverse phase high pressure liquid chromatography. 2The abbreviations used are: GlcNAc, N-acetylglucosamine; PBP, penicillin-binding protein; d-iAsn, d-iso-asparagine; d-iAsp, d-iso-aspartic acid; mesoDAP, meso-diaminopimelic acid; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MurNAc, N-acetylmuramic acid; RP-HPLC, reverse phase high pressure liquid chromatography. and N-acetylmuramic acid (MurNAc) substituted by a peptide stem (Fig. 1A) (2van Heijenoort J. Glycobiology. 2001; 11: 25R-36RCrossref PubMed Google Scholar). Assembly of the subunit at the cell surface is performed by glycosyltransferases that polymerize the glycan strands by formation of β-1,4 bonds and d,d-transpeptidases that cross-link glycan strands (2van Heijenoort J. Glycobiology. 2001; 11: 25R-36RCrossref PubMed Google Scholar). The latter reaction is catalyzed by penicillin-binding proteins (PBPs) that cleave the d-Ala4-d-Ala5 bond of a donor stem pentapeptide and link the carbonyl of d-Ala4 to the amino group of the side chain carried by the third residue of an acceptor stem peptide (Fig. 1B). This two-step reaction involves formation of a covalent adduct between the β-hydroxyl of the active site serine of the PBPs and the carbonyl of d-Ala4 of the donor stem (3Jamin M. Wilkin J.M. Frère J.-M. Essays Biochem. 1995; 29: 1-24PubMed Google Scholar). The glycosyltransferase and d,d-transpeptidase activities of the multimodular peptidoglycan polymerases are the targets of the two major classes of antibiotics available to treat severe infections due to Gram-positive bacteria, the β-lactams and the glycopeptides, that act by different mechanisms. The β-lactams are structural analogues of d-Ala4-d-Ala5 and act as suicide substrates of the d,d-transpeptidase module of the PBPs. The glycopeptides bind to the peptidyl-d-Ala4-d-Ala5 extremity of peptidoglycan precursors and block by steric hindrance both the transglycosylation and transpeptidation reactions (4Reynolds P.E. Eur. J. Clin. Microbiol. Infect. Dis. 1989; 8: 943-950Crossref PubMed Scopus (529) Google Scholar).The assembly pathway of peptidoglycan precursors and its mode of polymerization are generally highly conserved in eubacteria. Variations in the structure of peptidoglycan subunits involve mainly the fifth (C-terminal) and third positions of the pentapeptide stem (5Schleifer K.H. Kandler O. Bacteriol. Rev. 1972; 36: 407-477Crossref PubMed Google Scholar). The specificity of peptidoglycan cross-linking enzymes for the donor is the bottleneck that limits emergence of resistance to glycopeptides because the modifications of the precursors that prevent drug binding should be tolerated by these enzymes (4Reynolds P.E. Eur. J. Clin. Microbiol. Infect. Dis. 1989; 8: 943-950Crossref PubMed Scopus (529) Google Scholar, 6al-Obeid S. Billot-Klein D. van Heijenoort J. Collatz E. Gutmann L. FEMS Microbiol. Lett. 1992; 70: 79-84PubMed Google Scholar). Successful modifications that have spread in Gram-positive pathogens under the selective pressure of glycopeptides include incorporation of d-lactate or d-Ser instead of d-Ala at the fifth position of pentapeptide stems (7Arthur M. Reynolds P. Courvalin P. Trends Microbiol. 1996; 4: 401-407Abstract Full Text PDF PubMed Scopus (314) Google Scholar). Strikingly, production of d-lactate-ending precursors can also have an impact on the activity of β-lactams in the enterococci and staphylococci, presumably because low affinity PBPs responsible for β-lactam resistance cannot function with modified precursors (6al-Obeid S. Billot-Klein D. van Heijenoort J. Collatz E. Gutmann L. FEMS Microbiol. Lett. 1992; 70: 79-84PubMed Google Scholar, 8Severin A. Tabei K. Tenover F. Chung M. Clarke N. Tomasz A. J. Biol. Chem. 2004; 279: 3398-3407Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Total elimination of d-Ala5 by hydrolysis of the C-terminal residue of pentapeptide stems is an alternative mechanism of glycopeptide resistance in mutants of Enterococcus faecium selected in laboratory conditions (9Cremniter J. Mainardi J.L. Josseaume N. Quincampoix J.C. Dubost L. Hugonnet J.E. Marie A. Gutmann L. Rice L.B. Arthur M. J. Biol. Chem. 2006; 281: 32254-32262Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Because PBPs cannot function with tetrapeptide donors, peptidoglycan cross-linking in these mutants requires an l,d-transpeptidase (Ldtfm) that cleaves the l-Lys3-d-Ala4 peptide bond of the donor and links the carboxyl of l-Lys3 to the side chain amine of the acceptor (Fig. 1B). This mode of peptidoglycan cross-linking has been originally identified as a bypass of the PBPs that confers high level β-lactam resistance (10Mainardi J.-L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar).Variation at the third position of the peptide stem concerns both the nature of the diamino acid present at this position, e.g. l-Lys or meso-diaminopimelic acid (mesoDAP), and the presence or absence of a side chain comprising from one to five amino acids (5Schleifer K.H. Kandler O. Bacteriol. Rev. 1972; 36: 407-477Crossref PubMed Google Scholar) (Fig. 1A). Glycine and l-amino acids are incorporated into the side chain of peptidoglycan precursors by transferases of the Fem family that use aminoacyl-tRNAs as the substrate (11Plapp R. Strominger J.L. J. Biol. Chem. 1970; 245: 3667-3674Abstract Full Text PDF PubMed Google Scholar). d-Aspartic acid is activated as β-aspartyl-phosphate and ligated to the precursors by ATP-dependent ligases belonging to the ATP-Grasp superfamily (12Bellais S. Arthur M. Dubost L. Hugonnet J.E. Gutmann L. van Heijenoort J. Legrand R. Brouard J.P. Rice L. Mainardi J.L. J. Biol. Chem. 2006; 281: 11586-11594Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Synthesis of complete side chains is essential for β-lactam resistance mediated by low affinity PBPs in Gram-positive bacteria, in particular methicillin resistance mediated by PBP2a in Staphylococcus aureus (13Stranden A.M. Ehlert K. Labischinski H. Berger-Bachi B. J. Bacteriol. 1997; 179: 9-16Crossref PubMed Scopus (164) Google Scholar, 14Rohrer S. Berger-Bachi B. Antimicrob. Agents Chemother. 2003; 47: 837-846Crossref PubMed Scopus (89) Google Scholar) and penicillin resistance-mediated PBP2X in Streptococcus pneumoniae (15Laible G. Spratt B.G. Hakenbeck R. Mol. Microbiol. 1991; 5: 1993-2002Crossref PubMed Scopus (212) Google Scholar). For this reason, Fem transferases are considered as attractive targets for the development of novel antibiotics active against β-lactam-resistant pathogens (16Kopp U. Roos M. Wecke J. Labischinski H. Microb. Drug Resist. 1996; 2: 29-41Crossref PubMed Scopus (77) Google Scholar, 17Benson T. Prince D. Mutchler V. Curry K. Ho A. Sarver R. Hagadorn J. Choi G. Garlick R. Structure (Camb). 2002; 10: 1107-1115Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The antibacterial activity of such Fem inhibitors will ultimately depend upon the incapacity of cross-linking enzymes to use precursors with incomplete side chains.Interaction of the peptidoglycan cross-linking enzymes with their substrates has not been extensively investigated, despite its pivotal role for drug development and for our understanding of the mechanisms of resistance to glycopeptides and β-lactams. In this report, the specificity of Ldtfm and of the PBPs was compared based on heterospecific expression of a Fem transferase of E. faecalis in E. faecium and search for modified stem peptides containing l-Lys3-l-Ala instead of l-Lys3-d-iAsn in the donor and acceptor positions of dimers generated in vivo by l,d-transpeptidation and d,d-transpeptidation. The specificity of Ldtfm was also studied in vitro by directly testing the cross-linking of peptidoglycan fragments isolated from three bacterial species (E. faecium, Bacillus subtilis, and E. faecalis) representative of the structural variability at the third position of the stem peptides (Fig. 1A). Finally, Ldtfm homologues (Fig. 1C) were purified from E. faecalis and B. subtilis to compare the specificity of enzymes from bacteria producing peptidoglycan of different chemotypes. This analysis represents the first direct assessment of the specificity of peptidoglycan cross-linking enzymes because the PBPs are generally inactive in vitro. In addition, characterization of Ldtfs revealed that members of the l,d-transpeptidase family can also display l,d-carboxypeptidase and l,d-endopeptidase activities.EXPERIMENTAL PROCEDURESProduction and Purification of l,d-Transpeptidases—A catalytically active fragment of Ldtfm from E. faecium M512 (residues 119–466) was produced in E. coli and purified by affinity, anion exchange, and size exclusion chromatographies, as previously described (18Mainardi J. Fourgeaud M. Hugonnet J.E. Dubost L. Brouard J.P. Ouazzani J. Rice L.B. Gutmann L. Arthur M. J. Biol. Chem. 2005; 280: 38146-38152Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). A fragment of the open reading frame encoding residues 136–474 of the l,d-transpeptidase of E. faecalis strain JH2-2 (19Jacob A.E. Hobbs S.J. J. Bacteriol. 1974; 117: 360-372Crossref PubMed Google Scholar), Ldtfs, was amplified with primers 5′-AACCATGGGGAGTATCCGTCGAGGCAATGG-3′ and 5′-AAGGATCCTACTTCTTCGCCGTAATCTA-3′. The PCR product was digested with NcoI and BamHI (underlined) and cloned into pET2818, a derivative of pET2816 (18Mainardi J. Fourgeaud M. Hugonnet J.E. Dubost L. Brouard J.P. Ouazzani J. Rice L.B. Gutmann L. Arthur M. J. Biol. Chem. 2005; 280: 38146-38152Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) lacking the sequence specifying the thrombin cleavage site. The resulting plasmid, pET2818Ωldtfs, was introduced in E. coli BL21(DE3) harboring pREP4GroESL (20Amrein K.E. Takacs B. Stieger M. Molnos J. Flint N.A. Burn P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1048-1052Crossref PubMed Scopus (150) Google Scholar), and bacteria were grown at 37 °C to an optical density at 600 nm of 0.8 in brain heart infusion broth (Difco, Elancourt, France) containing ampicillin (100 μg/ml). Isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.5 mm, and incubation was continued for 17 h at 16 °C. The cells were disrupted by sonication in 50 mm Tris-HCl, pH 7.5, containing 300 mm NaCl, and cell debris were removed by centrifugation. Ldtfs was purified from the resulting clarified lysate by affinity chromatography on Ni2+-nitrilotriacetate-agarose resin (Qiagen GmbH, Hilden, Germany). Proteins eluted with 200 mm imidazole were dialyzed against 50 mm Tris-HCl, pH 8.0, containing 60 mm NaCl, loaded onto an anion exchange column (MonoQ HR5/5, Amersham Biosciences) equilibrated with the same buffer. Ldtfs, eluting at ∼300 mm NaCl, was further purified by size exclusion chromatography on a Superdex HR10/30 column (Amersham Biosciences) equilibrated with 50 mm Tris-HCl, pH 7.5, containing 300 mm NaCl. The protein was obtained with an overall yield of 3 mg/liter of culture, as estimated by the Bio-Rad protein assay using bovine serum albumin as a standard. The protein was stored at –80 °C in 50 mm Tris-HCl, pH 7.5, containing 300 mm NaCl.The open reading frame encoding the l,d-transpeptidase of B. subtilis strain 168, LdtBs, previously named ykuD (21Bielnicki J. Devedjiev Y. Derewenda U. Dauter Z. Joachimiak A. Derewenda Z. Proteins Struct. Funct. Genet. 2005; 62: 144-151Crossref Scopus (78) Google Scholar), was amplified with primers 5′-AACCATGGGGCTGCTTACGTACCAGGTGAAGC-3′ and 5 ′-TTGGATCCCCGGTTAATCGTGACTCTCGT-3′. The PCR product digested with NcoI and BamHI (underlined) was cloned into pET2818, and LdtBs was produced in E. coli BL21(DE3)/pREP4GroESL using the inducing conditions described above for the l,d-transpeptidase of E. faecalis. LdtBs was purified in one step by affinity chromatography on Ni2+-nitrilotriacetate-agarose resin (Qiagen), dialyzed against 50 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, and stored at –20 °C in the same buffer. 10 mg of protein were obtained per liter of culture.l,d-Transpeptidase Assays—The source of the disaccharide-peptides used as substrates was as follows. The disaccharide-tetrapeptide substituted by a d-iso-asparagine residue (l-Lys3-d-iAsn) was purified from the peptidoglycan of E. gallinarum strain SC1 (22Grohs P. Gutmann L. Legrand R. Schoot B. Mainardi J.L. J. Bacteriol. 2000; 182: 6228-6232Crossref PubMed Scopus (32) Google Scholar). The disaccharide-pentapeptide and the disaccharide-tetrapeptide substituted by an l-Ala-l-Ala side chain (l-Lys3-l-Ala-l-Ala) were purified from E. faecalis JH2-2 (19Jacob A.E. Hobbs S.J. J. Bacteriol. 1974; 117: 360-372Crossref PubMed Google Scholar) and from a derivative of strain JH2-2 harboring the vanA glycopeptide resistance gene cluster (23Bouhss A. Josseaume N. Severin A. Tabei K. Hugonnet J.E. Shlaes D. Mengin-Lecreulx D. Van Heijenoort J. Arthur M. J. Biol. Chem. 2002; 277: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), respectively. The disaccharide-tetrapeptide containing meso-diaminopimelic acid (mesoDAP3) was purified from E. coli strain ATCC 25113. The procedures used for peptidoglycan preparation, digestion with muramidases, and reduction of MurNAc to muramitol with sodium borohydride have been previously described for enterococci (24Arbeloa 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 (57) Google Scholar) and E. coli (25Glauner B. Anal. Biochem. 1988; 172: 451-464Crossref PubMed Scopus (347) Google Scholar). The resulting muropeptides were separated by RP-HPLC in acetonitrile gradients containing trifluoroacetic acid (24Arbeloa 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 (57) Google Scholar) and identified by mass spectrometry (MS). The concentration of the muropeptides was estimated by amino acid analysis after acidic hydrolysis with a Hitachi autoanalyzer (26Mengin-Lecreulx D. Falla T. Blanot D. van Heijenoort J. Adams D.J. Chopra I. J. Bacteriol. 1999; 181: 5909-5914Crossref PubMed Google Scholar).In vitro formation of muropeptide dimers was tested in 10 μl of phosphate buffer (20 mm, pH 7.0) containing the l,d-transpeptidase (7 μm) from E. faecium (Ldtfm), E. faecalis (Ldtfs), or B. subtilis (LdtBs) and a combination of three reduced disaccharide-tetrapeptides (200 μm each) containing l-Lys3-d-iAsn, l-Lys3-l-Ala-l-Ala, or mesoDAP3 at the third position of a tetrapeptide stem. The reaction was incubated for 2 h at 37°C, desalted using a micro column (ZipTipC18; Millipore, Saint Quentin-en-Yvelines, France), and analyzed by nanoelectrospray MS in the positive mode (Qstar Pulsar I; Applied Biosystem, Courtaboeuf, France). The sequence of the cross-links in dimers generated in vitro was determined by tandem mass spectrometry (MS/MS). Briefly, dimers were generated in vitro as described above except that the disaccharide-peptides used as substrates were not reduced. The reaction mixture was treated with ammonium hydroxide, desalted using a micro column (ZipTipC18, Millipore), and the resulting lactoyl-peptides were analyzed by nanoelectrospray MS/MS using N2 as the collision gas (24Arbeloa 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 (57) Google Scholar).The l,d-transpeptidase activity of Ldtfs was also tested by using the dipeptides l-Ala-l-Ala and d-Ala-d-Ala as acyl acceptors. The assay performed in 10 μl of 20 mm potassium phosphate buffer, pH 7.0, contained Ldtfs (7 μm), the cognate disaccharide-tetrapeptide containing an l-Ala-l-Ala side chain as the acyl donor (200 μm), and 1 mml-Ala-l-Ala or d-Ala-d-Ala (Sigma). The products of the reactions were identified by MS and MS/MS, as previously described (18Mainardi J. Fourgeaud M. Hugonnet J.E. Dubost L. Brouard J.P. Ouazzani J. Rice L.B. Gutmann L. Arthur M. J. Biol. Chem. 2005; 280: 38146-38152Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar).Expression of the bppA1 Gene of E. faecalis in E. faecium M512 and Analysis of Peptidoglycan Structure—The bppA1 gene of plasmid pDA15 (27Bouhss A. Josseaume N. Allanic D. Crouvoisier M. Gutmann L. Mainardi J.-L. Mengin-Lecreulx D. van Heijenoort J. Arthur M. J. Bacteriol. 2001; 183: 5122-5127Crossref PubMed Scopus (35) Google Scholar) was subcloned under the control of the inducible promoter of pJEH4 (12Bellais S. Arthur M. Dubost L. Hugonnet J.E. Gutmann L. van Heijenoort J. Legrand R. Brouard J.P. Rice L. Mainardi J.L. J. Biol. Chem. 2006; 281: 11586-11594Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) using XbaI and KpnI. The resulting plasmid, pJEH6(bppA1), was introduced by electroporation into E. faecium M512 (10Mainardi J.-L. Legrand R. Arthur M. Schoot B. van Heijenoort J. Gutmann L. J. Biol. Chem. 2000; 275: 16490-16496Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The recombinant strain was grown in brain heart infusion broth or agar (Difco) containing spectinomycin (120 μg/ml) to counter select loss of pJEH6(bppA1). Induction of the bppA1 gene was performed with 0.3 mm isopropyl-β-d-thiogalactopyranoside at an optical density at 600 nm of 0.02. Incubation was continued at 37 °C until the optical density reached 0.6, and bacteria were collected by centrifugation. Peptidoglycan was extracted with boiling SDS and digested with mutanolysin and lysozyme (Sigma-Aldrich) (24Arbeloa 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 (57) Google Scholar). The resulting muropeptides were cleaved under alkaline conditions to generate lactoyl-peptides, separated by RP-HPLC, and analyzed by MS and MS/MS (24Arbeloa 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 (57) Google Scholar).RESULTS AND DISCUSSIONProbing the Substrate Specificity of Ldtfm in Vivo—Synthesis of the side chain of peptidoglycan precursors is catalyzed in E. faecalis by two members of the Fem family, BppA1 and BppA2, that sequentially add two l-Ala residues (Fig. 1A) (23Bouhss A. Josseaume N. Severin A. Tabei K. Hugonnet J.E. Shlaes D. Mengin-Lecreulx D. Van Heijenoort J. Arthur M. J. Biol. Chem. 2002; 277: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In E. faecium, d-Asp is added to the precursors by the Aslfm ligase and subsequently partially amidated (12Bellais S. Arthur M. Dubost L. Hugonnet J.E. Gutmann L. van Heijenoort J. Legrand R. Brouard J.P. Rice L. Mainardi J.L. J. Biol. Chem. 2006; 281: 11586-11594Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The bppA1 gene of E. faecalis was cloned under the control of an inducible promoter to generate plasmid pJEH6 and introduced into E. faecium M512 in order to manipulate the structure of the substrate of the cross-linking reaction that can be catalyzed in this mutant by Ldtfm and by the PBPs (28Mainardi J.L. Morel V. Fourgeaud M. Cremniter J. Blanot D. Legrand R. Frehel C. Arthur M. Van Heijenoort J. Gutmann L. J. Biol. Chem. 2002; 277: 35801-35807Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The BppA1 transferase efficiently competed with the Aslfm ligase in E. faecium M512/pJEH6(bppA1) because the main monomers contained l-Ala instead of d-iAsp (Fig. 2, peaks 1 and 2). The free side chains in the major dimers generated by l,d- and d,d-transpeptidation also contained l-Ala, indicating that Ldtfm, as the PBPs, had catalyzed cross-link formation using donors containing this residue (Fig. 2, peaks 3–5). The cross-links of dimers generated by l,d-transpeptidation exclusively contained d-iAsp, whereas d-iAsp or l-Ala was found in cross-links generated by d,d-transpeptidation. Thus, Ldtfm tolerated the substitution only in the donor substrate, in contrast to the PBPs that catalyzed peptidoglycan cross-linking with modified donor and acceptor substrates. Modifications of the side chain of peptidoglycan precursors were also shown to be tolerated by PBPs of E. faecalis and S. aureus in previous studies (12Bellais S. Arthur M. Dubost L. Hugonnet J.E. Gutmann L. van Heijenoort J. Legrand R. Brouard J.P. Rice L. Mainardi J.L. J. Biol. Chem. 2006; 281: 11586-11594Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 24Arbeloa 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 (57) Google Scholar).FIGURE 2Peptidoglycan composition of E. faecium M512 producing BppA1 from E. faecalis. A, RP-HPLC profile of muropeptides from E. faecium M512/pJEH6(bppA1). Bacteria were grown in the presence of spectinomycin (120 μg/ml) to counter select loss of plasmid pJEH6(bppA1) and of isopropyl-β-d-thiogalactopyranoside (0.3 mm) to induce the bppA1 gene encoding a transferase of the Fem family for incorporation of l-Ala into the side chain of peptidoglycan precursors. Peptidoglycan was extracted with SDS and treated with ammonium hydroxide, and the resulting lactoyl-peptides were separated by RP-HPLC. mAU, absorbance unit × 103 at 210 nm. B, structure of muropeptides. The relative abundance (%) of the material in peaks 1 to 5 was calculated by integration of the absorbance at 210 nm. The retention time (RT) is indicated for minor muropeptides that could not be assigned to specific peaks because of their low abundance. The structure of lactoyl-peptides was deduced from the observed monoisotopic mass (Mass) and confirmed by tandem mass spectrometry for most monomers and dimers (indicated by a star). The observed and calculated mass differed at the maximum by 0.2. The stem peptide of monomers and the acceptor stem of dimers consisted of the tripeptide l-Ala1-d-iGln2-l-Lys3 (Tri), the tetrapeptide l-Ala1-d-iGln2-l-Lys3-d-Ala4 (Tetra), and the pentapeptide l-Ala1-d-iGln2-l-Lys3-d-Ala4-d-Ala5 (Penta). In certain muropeptides of low abundance, the C-terminal d-Ala4 was replaced by a glycyl residue (Gly C-ter). Cross-links generated by d,d-transpeptidases (DD) contained d-iAsp or l-Ala (d-Ala4→d-iAsp-l-Lys3 and d-Ala4→l-Ala-l-Lys3). Cross-links generated by l,d-transpeptidation (LD) exclusively contained d-iAsp (l-Lys3→d-iAsp-l-Lys3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ldtfm Is Also Specific for d-iAsn-substituted Acceptors in Vitro—The specificity of Ldtfm was analyzed by incubating the enzyme with three disaccharide-tetrapeptides obtained by digestion of peptidoglycan of three different chemotypes with muramidases. The substrates were representative of the main variations found at the third position of peptidoglycan precursors of Gram-positive bacteria including the absence of a side chain (mesoDAP3 in B. subtilis) and presence of a side chain consisting of d and l amino acids (l-Lys3-d-iAsn in E. faecium and l-Lys3-l-Ala-l-Ala in E. faecalis) (Fig. 1A). With these three substrates, the cross-linking reaction can potentially lead to the formation of nine dimers, including three homodimers, if the same disaccharide-peptide is used as the donor and the acceptor substrate, and six heterodimers, if different disaccharide-peptides are used in all possible combinations (Table 1). Mass spectrometry analyses of the reaction products indicated that Ldtfm catalyzed formation of a homodimer containing d-iAsn-substituted acceptor and donor stem peptides and of a heterodimer containing stem peptides substituted by l-Ala-l-Ala and d-iAsn (Fig. 3). Tandem mass spectrometry was performed to determine whether the l-Ala-l-Ala-substituted disaccharide-peptide had been used as a donor or an acceptor substrate in the formation of the heterodimer (Fig. 4). Fragmentation was performed on lactoyl-peptides obtained by cleavage of the disaccharide-peptides by alkaline treatment because amino acid sequencing of peptidoglycan dimers is more efficient in the absence of the disaccharide moiety of the molecules (24Arbeloa 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 (57) Google Scholar). This treatment also converts d-iAsn into d-iAsp (24Arbeloa 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 (57) Google Scholar). As detailed in Fig. 4, fragmentation of the heterodimer allowed assigning l-Ala-l-Ala to the free side chain of the donor stem and d-iAsp to the cross-link. Thus, Ldtfm tolerated presence of l-Ala in the donor but not in the acceptor substrate of the cross-linking reaction. The specificity of Ldtfm observed in vitro in the absence of any other cell wall biosynthesis enzyme accounts for the in vivo selection of the acceptor and donor substrates in th
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