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J. Biol. Chem., Vol. 280, Issue 46, 38146-38152, November 18, 2005

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A Novel Peptidoglycan Cross-linking Enzyme for a -Lactam-resistant Transpeptidation Pathway^*

Jean-Luc Mainardi1, Martine Fourgeaud, Jean-Emmanuel Hugonnet, Lionel Dubost¶, Jean-Paul Brouard¶, Jamal Ouazzani||, Louis B. Rice**, Laurent Gutmann, and Michel Arthur

From the INSERM, U655-Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Pierre et Marie Curie (UPMC Paris 6), Faculté deMédecine, Université Paris-Descartes, and Centre de Recherches Biomédicales des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France, Faculté deMédecine, Université Paris-Descartes, AP-HP Hôpital Européen Georges Pompidou, 20 rue Leblanc, 75908 Paris Cedex 15, France, ^¶Plateforme de Spectrométrie de Masse et de Protéomique du Muséum, Département Recherche Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, USM0502-CNRS UMR8041, 75005 Paris, France, ^||Laboratoire de Microbiologie Appliquée, Institut de Chimie des Substances Naturelles, CNRS, 91190 Gif-sur-Yvette, France, and ^**Medical and Research Services, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106

Received for publication, July 7, 2005 , and in revised form, September 1, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The -lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the -lactam ring and the D-alanyl⁴-D-alanine⁵ extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the DD-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in a-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldt_fm, catalyzes in vitro the cross-linking of peptidoglycan subunits in a -lactam-insensitive LD-transpeptidation reaction. The specificity of Ldt_fm for the L-lysyl³-D-alanine⁴ peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic -lactams in contrast to D-alanyl⁴-D-alanine⁵ in the pentapeptide donors required for DD-transpeptidation. Ldt_fm homologues are encountered sporadically among taxonomically distant bacteria, indicating that LD-transpeptidase-mediated resistance may emerge in various pathogens.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
More than 60 years after the introduction of penicillin, the class of -lactam antibiotics remains the most commonly used to treat severe infections despite the emergence of various resistance mechanisms, including drug detoxification by production of -lactamases (1), decreased affinity of the target (2), and decreased permeability (3). The success story of -lactams involves the development of several generations of -lactams to defeat the resistance mechanisms and their association with inhibitors of -lactamases (4, 5). All -lactams have a common mechanism of action, as they share structural similarity with bacterial cell wall precursors. These drugs act as suicide substrates of the DD-transpeptidase catalytic domain of the penicillin-binding proteins (PBPs)² responsible for the last cross-linking step of cell wall assembly (Fig. 1A) (2, 6, 7).

Among the DD-transpeptidases of Enterococcus faecium, the low affinity PBP5 is unessential for growth but responsible for intrinsic low level resistance to ampicillin (8, 9), the first-line drug used at high dosage to treat severe enterococcal infections. In clinical isolates, higher levels of resistance to ampicillin are commonly associated with multiple amino acid substitutions in the transpeptidase domain of PBP5 (10, 11). We have shown previously that even higher levels of resistance to ampicillin can be achieved by a novel mechanism in the absence of PBP5 (12). Analysis of the peptidoglycan of the in vitro selected -lactam-resistant mutant E. faecium M512 revealed that the D-Ala⁴D-iAsn-L-Lys³ cross-link had been replaced by a novel type of cross-link between the L-Lys³ carboxyl group of a donor and the side-chain D-iAsn of an acceptor (12) (Fig. 1B). In the presence of ampicillin, the novel L-Lys³D-iAsn-L-Lys³ cross-links were exclusively found in the peptidoglycan of E. faecium M512, indicating that the DD-transpeptidase activity of the PBPs did not participate in the formation of the cross-links. Selection of E. faecium M512 from E. faecium D344S in five consecutive steps on increasing concentrations of ampicillin led to the gradual activation of the LD-transpeptidation pathway because the proportion of L-Lys³D-iAsn-L-Lys³ was 3.1% for D344S and increased at each selection step (13). Activation of the resistance pathway was associated with the production of a DD-carboxypeptidase that generated precursors containing a tetrapeptide stem in the cytoplasm of E. faecium M512 (13). Based on these observations, we have speculated that precursors containing a tetrapeptide stem are essential for the formation of the L-Lys³D-iAsn-L-Lys³ cross-links.

In the present work, we have identified in E. faecium an LD-transpeptidase as the key enzyme of this alternate transpeptidation pathway and have shown that its substrate does not bear structural similarity to -lactams. The bypass mechanism could invalidate the -lactams as the major class of antibiotics.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Purification of the LD-Transpeptidase and N-terminal Sequencing— The LD-transpeptidase from E. faecium (Ldt_fm) was partially purified in four chromatographic steps using a radioactive exchange assay (13) to detect active fractions. Briefly, E. faecium M512 (12) was grown to an A₆₅₀ of 0.7 in 24 liters of brain heart infusion broth (Difco), harvested by centrifugation, and washed twice in 10 mM sodium phosphate (pH 7.0). Bacteria were disrupted with glass beads in a cell disintegrator (The Mickle Laboratory Engineering Co., Gromshall, UK) for 2 h at 4 °C. The extract was centrifuged (5,000 x g for 10 min at 4 °C) to remove cell debris, and the supernatant was ultracentrifuged at 100,000 x g for 30 min at 4 °C. Soluble proteins (1 g) were loaded onto an anion exchange column (Hi-Load^TM 26/10 Q-Sepharose^TM HP, Amersham Biosciences) equilibrated with 25 mM sodium cacodylate buffer (pH 7.86) (buffer A). Elution was performed with a linear 0–2 M NaCl gradient in buffer A. Active fractions were pooled (30 mg of proteins), concentrated by ultrafiltration (Centricon YM10, Millipore, Saint-Quentin-en-Yve-lines, France), and loaded onto a gel filtration column (Superdex 75 HR26/60, Amersham Biosciences) equilibrated with buffer A containing 0.3 M NaCl. Active fractions (1 mg of proteins) were loaded onto a weak anion exchange column (HiTrap^TM DEAE fast flow^TM 1 ml, Amersham Biosciences) equilibrated with buffer A. Proteins (300 µg) eluting between 0.2 and 0.3 M NaCl were concentrated by ultrafiltration (Amicon ultra-4, Millipore) and loaded onto a gel filtration column (Superdex 200 PC 3.2/30, Amersham Biosciences) equilibrated with buffer A containing 0.3 M NaCl. Active fractions (70 µg of proteins) were concentrated (Amicon ultra-4) and analyzed by SDS-PAGE revealing a major 48-kDa protein band that was transferred onto a polyvinylidene difluoride membrane (Problott, Applied Biosystems, Framingham, MA) by passive adsorption (14). N-terminal Edman sequencing was performed on an Applied Biosystems Procise 494HT instrument with reagents and methods recommended by the manufacturer. The open reading frame for the LD-transpeptidase was identified by similarity searches between the N-terminal sequence of the 48-kDa protein (AEKQEIDPVSQNHQKLDTTV) and the partial genome sequence of E. faecium using the software tBLAST at the National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov/). The partially purified 48-kDa protein corresponded to a proteolytic fragment because the sequence encoding its N terminus was not preceded by a translation initiation codon. The upstream sequence contained a single likely translation initiation site (ACTTAAggagTTGTCGATatg), consisting of an ATG codon preceded by a putative ribosome binding site (lowercase).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Cross-links generated by transpeptidation in E. faecium. A, D-Ala4D-iAsn-L-Lys3 cross-links generated by the DD-transpeptidase activity of the PBPs. In E. faecium, the peptidoglycan subunit contains , 1–4-linked N-acetylglucosamine and N-acetylmuramic acid (GlcNAc-MurNAc, data not shown) substituted by a branched stem pentapeptide (L-alanyl1-D-isoglutamyl2-L-(N-D-isoasparaginyl)lysyl3-D-alanyl4-D-alanine5). The PBPs cleave the C-terminal residue (D-Ala5) of the first substrate (pentapeptide donor) and link the carboxyl of the penultimate residue (D-Ala4) to the amino group of the second substrate (the acceptor). B, L-Lys3D-iAsn-L-Lys3 cross-links generated by the LD-transpeptidase of E. faecium (Ldtfm). The enzyme cleaves the L-Lys3-D-Ala4 bond of the donor and links the carboxyl of L-Lys3 to the side chain of the acceptor. Bypass of the -lactam-sensitive DD-transpeptidase in the mutant E. faecium M512 also requires the production of a DD-carboxypeptidase, which cleaves the C-terminal D-alanine residue (D-Ala5) of the pentapeptide stem, to generate the tetrapeptide donor of the LD-transpeptidase.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Identification and characterization of the LD-transpeptidase (Ldtfm) of E. faecium M512. A, partial purification of Ldtfm (lane 2) from an E. faecium M512 extract (lane 1). B, amino acid sequence of Ldtfm. The N terminus of the partially purified 48-kDa protein is underlined. The cluster of hydrophobic residues at positions 13–28 (italicized) could correspond to a membrane anchor. The conserved C-terminal domain (positions 341–466) is indicated in bold. Asterisks indicate the positions of Ser439 and Cys442 that were modified by site-directed mutagenesis. C, purification of Ldtfm produced in E. coli. D, HPLC analysis of the exchange reaction. cts, counts. E, effect of ampicillin on Ldtfm activity. F, fragmentation of Ac2-L-Lys-D-Met (m/z of 362.24) obtained by incubating Ldtfm with Ac2-L-Lys-D-Ala and D-Met. Loss of a C-terminal D-Met and of additional H2O led to ions at m/z 213.14 and 185.14, respectively. Ions at m/z 84.10 and 126.11 correspond to the immonium of L-Lys and its acetylated form, respectively.

LD-Transpeptidase Assays—The exchange reaction assays for the capacity of the enzyme to catalyze cleavage of the L-Lys-D-Ala peptide bond of the model donor dipeptide substrate Ac₂-L-Lys-D-Ala and formation of a peptide bond between Ac₂-L-Lys and D-[¹⁴C]Ala (13, 22). The standard assay (50 µl) contained Ac₂-L-Lys-D-Ala (5 mM), D-[¹⁴C]Ala (0.15 mM; 2.0 GBq/mmol, ICN Pharmaceuticals, Orsay, France), 10 mM sodium cacodylate buffer (pH 6.0), and 0.1% Triton X-100 (v/v). The reaction products were separated by reverse phase HPLC and detected by scintillation with a radioflow detector (LB508, PerkinElmer Life Sciences) coupled to the HPLC device (13). To assay for inhibition by ampicillin, Ldt_fm (3 µg) was incubated with Ac₂-L-Lys-D-Ala (0.3 mM), D-[¹⁴C]Ala (0.15 mM), and various concentrations of the drug. To test different donors, 3 µg of Ldt_fm were incubated for 60 min in the same conditions, except that Ac₂-L-Lys-D-Ala was replaced by UDP-MurNAc-tetrapeptide (2.5 mM), UDP-MurNAc-pentapeptide (2.5 mM), tetrapeptide (2.5 mM), pentapeptide (2.5 mM), GlcNAc-MurNAc-tetrapeptide-iAsn (1 mM), and GlcNAc-MurNAc-pentapeptide-iAsn (1 mM).

View larger version (30K): [in this window] [in a new window]   FIGURE 3. In vitro formation of dimers by Ldtfm. A, combinations of donor and acceptor substrates used by Ldtfm. The substrates contained the disaccharide GlcNAc-MurNAc substituted by L-Ala--D-iGln-L-(N-D-iAsn)Lys-D-Ala (tetrapeptide-iAsn), L-AlaD-iGln-L-(N-D-iAsn)Lys (tripeptide-iAsn), and L-Ala-D-iGln-L-Lys-D-Ala (tetrapeptide). B, fragmentation of the ion at m/z 1118.5 obtained by ammonium hydroxide treatment of the dimer with a monoisotopic mass of 1927.88. Boxes indicate ions generated by cleavage at single peptide bonds. D-Lac, D-lactoyl. C, structure of the dimer and inferred fragmentation pattern.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Identification of the LD-Transpeptidase of E. faecium M512 (Ldt_fm)— We identified the gene encoding the LD-transpeptidase for the formation of the L-Lys³D-iAsn-L-Lys³ responsible cross-links in E. faecium M512 by partial purification of the enzyme (48 kDa), sequencing of its N terminus, and similarity searches in the partial genome sequence of E. faecium (Fig. 2, A and B). The partially purified protein was a proteolytic fragment lacking the 118 N-terminal residues, including a putative membrane anchor. The portion of the open frame encoding the proteolytic fragment was expressed in Escherichia coli for large scale protein purification (Fig. 2C). The protein was active in an exchange assay (Fig. 2D) and was not inhibited by ampicillin (Fig. 2E), indicating that the gene encoding the LD-transpeptidase of E. faecium M512 (Ldt_fm) had been successfully identified.

View larger version (100K): [in this window] [in a new window]   FIGURE 4. Alignment of the deduced sequence of the L, D-transpeptidase from E. faecium (Ldtfm) with close homologues from Gram-positive bacteria. L. plant, Lactobacillus plantarum WCFS1; C. aceto, Clostridium acetobutylicum ATCC: 824; E. faeca, Enterococcus faecalis V583; B. anthr, Bacillus anthracis Ames (positions 341–466). The sequence of the consensus of pfam PF03734 is shown below the alignment. The related C-terminal domain of Ldtfm (residues 341–466) contains Ser439 and Cys442 that were modified by site-directed mutagenesis (indicated by asterisks).

View this table: [in this window] [in a new window]   TABLE ONE Exchange reaction (Ac2-L-Lys-D-Ala + X Ac2-L-Lys-X + D-Ala) catalyzed by Ldtfm between Ac2-L-Lys-D-Ala and various acceptors Ac2-L-Lys-D-Ala (0.3 mM) was incubated with Ldtfm (3 µg) and various D-2-amino acids (0.3 mM) or D-2-hydroxyacid (0.3 mM) acceptors for 1 h at 37 °C. Products were detected by mass spectrometry, and the structure was confirmed by tandem mass spectrometry.

Cross-linking of Peptidoglycan Subunits in Vitro—Because Ldt_fm had all the characteristics expected for a peptidoglycan cross-linking enzyme, we investigated the formation of L-Lys³D-iAsn-L-Lys³ cross-links with substrates closely mimicking the natural peptidoglycan precursors. Such substrates were prepared from the peptidoglycan of E. gallinarum, as it contains large amounts of uncross-linked monomers containing a tetrapeptide iAsn stem (19). LD-Transpeptidation was assayed with a reconstituted pool of three muropeptides to simultaneously test six combinations of donors and acceptors (Fig. 3A). Mass spectrometric analysis of the reaction products revealed formation of dimers with two types of donors (tetrapeptide and tetrapeptide-iAsn) and two types of acceptors (tetrapeptide-iAsn and tripeptide-iAsn) in the four possible combinations. The muropeptide containing an unsubstituted tetrapeptide stem was not used as an acceptor, indicating that the side-chain iAsn is essential. Accordingly, direct Lys³L-Lys³ cross-links were not detected in the peptidoglycan of E. faecium M512 (12). To confirm the structure of the dimers obtained in vitro, the reaction was scaled up and treated with ammonium hydroxide to cleave the ether link internal to MurNAc. This treatment produced lactoyl peptides, which are more amenable to sequencing by tandem mass spectrometry than disaccharide peptides (21). This treatment was also found to convert D-iAsn into D-isoaspartyl. The fragmentation patterns (Fig. 3, B and C) demonstrated the in vitro formation of L-Lys³D-iAsn-L-Lys³ cross-links by Ldt_fm. Of note, dimer formation has not been obtained in the case of purified DD-transpeptidase (PBPs), except in very special cases involving highly reactive artificial substrates (e.g. thioester) or atypical enzymes (e.g. the soluble R61 DD-peptidase from Streptomyces spp.) (see Ref. 24 for a recent discussion). Thus, Ldt_fm differs from the PBPs in its capacity to function in a soluble acellular system, a feature that could be exploited to design screens for the identification of cross-linking inhibitors.

Physiological Function of Ldt_fm—Similar Ldt_fm activity is present in crude extracts from the ampicillin-resistant E. faecium mutant M512 and from the susceptible parental strain D344S (13). Accordingly, Western blot analysis of crude extracts from E. faecium D344S and M512 revealed similar amounts of the same form of Ldt_fm corresponding to the full-length protein (data not shown). The identification of the corresponding gene, ldt_fm, allowed us to confirm that its sequence was identical in both strains and in the E. faecium genome data base. These observations indicate that activation of the LD-transpeptidation pathway (Fig. 1B) does not involve modification of the activity of Ldt_fm per se but that of the supply of the appropriate tetrapeptide donor substrate for the cross-linking reaction. The physiological role of the LD-transpeptidase in -lactam-susceptible E. faecium is unknown. Previous analyses of the peptidoglycan structure in E. coli revealed that a small proportion of the cross-links is generated by LD-transpeptidation during the exponential phase of growth (5.8%) (25). An increase of their abundance during the stationary phase (11.3%) was attributed to a short supply of peptidoglycan subunits containing the pentapeptide required for DD-transpeptidation (25). Because the mature peptidoglycan of E. faecium contains virtually no pentapeptide stems (12), we propose that Ldt_fm may have a role in the maintenance of peptidoglycan structure because the enzyme can catalyze new cross-links without de novo incorporation of pentapeptide-containing subunits.

Distribution of Ldt_fm—Sequence comparisons indicated that Ldt_fm is the first representative of a novel family of proteins that is sporadically distributed among taxonomically distant bacteria. Close homologues (Fig. 4) were detected in pathogenic Gram-positive bacteria including Bacillus anthracis and Enterococcus faecalis but not in S. aureus and Streptococcus pneumoniae. Sequence similarity restricted to the C terminus of Ldt_fm was also detected in proteins of unknown functions from other Gram-negative and Gram-positive bacteria, but the architecture and the domain composition of the proteins were different. This domain is related to 341 sequences from eubacteria appearing in the Protein Families Data Base of Alignments under the pfam accession number PF03734. Members of the family appear nearly ubiquitous, but none of them has been functionally characterized, and their putative involvement in -lactam resistance has not been evaluated. Highly conserved residues of the C-terminal domain included Ser and Cys, present at positions 439 and 442 of Ldt_fm (Fig. 4) as potential catalytic residues. Site-directed mutagenesis of Ldt_fm led to an inactive protein for the C442A substitution. The mutant protein with the S439A substitution retained 2% of the activity of the wild-type enzyme. These results suggest that Cys⁴⁴² could be the catalytic residue of Ldt_fm. In contrast, the PBPs possess an essential active site serine, which is acylated by their substrate and -lactams.

In conclusion, Ldt_fm is the first characterized representative of a novel type of peptidoglycan transpeptidase. The wide distribution of Ldt_fm homologues indicates that -lactam resistance by the LD-transpeptidase bypass mechanism can potentially emerge in various pathogenic bacteria. The specificity of the Ldt_fm for tetrapeptide substrates implies the lack of the -lactam ring-associated activity of the most broadly used class of antimicrobial agents. This also implies that strategies applied previously to defeat other -lactam resistance mechanisms, in particular by modification or replacement of the structures attached to the -lactam ring, cannot be transposed to the development of LD-transpeptidase inhibitors.

	FOOTNOTES

 
^* This work was supported by the European Community (Combating Resistance to Antibiotics, contract Life Science Health Medicine Contract-2003-503335, 6th Programme Cadre de Recherche et de Développement Technologique) and by the NIAID, National Institutes of Health Grant R01 AI45626. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 33-1-42-34-68-62; Fax: 33-1-43-25-68-12; E-mail: jlmainar{at}bhdc.jussieu.fr.

² The abbreviations used are: PBP, penicillin-binding protein; Ac₂-L-Lys-D-Ala, N,N-diacetyl-L-lysyl-D-alanine; iAsn, isoasparaginyl; Ldt_fm, LD-transpeptidase of Enterococcus faecium; MurNAc, N-acetylmuramic acid; HPLC, high pressure liquid chromatography; iGlu, isoglutamyl; iGln, isoglutaminyl.

	ACKNOWLEDGMENTS

 
We thank D. Blanot for synthesis of Ac₂-L-Lys-D-Ala, J. Cremniter and S. Mesnage for the production of the rabbit antiserum against Ldt_fm and J. van Heijenoort, R. Menard, and F. Rusconi for critical reading of the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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