Unexpected Inhibition of Peptidoglycan LD-Transpeptidase from Enterococcus faecium by the β-Lactam Imipenem

Angèle Nguekam Moumi,Lionel Dubost,Filippo Rusconi,Martine Fourgeaud,Claudine Mayer,Laurent Gutmann,Louis B Rice,Jean-Emmanuel Hugonnet,Vanessa Delfosse,Michel Arthur,Jean-Luc Mainardi

doi:10.1074/jbc.m704286200

Abstract

The beta-lactam antibiotics mimic the D-alanyl(4)-D-alanine(5) extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the dd-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high level resistance to ampicillin. Ldt(fm) is specific for the L-lysyl(3)-D-alanine(4) bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine(5). This specificity was proposed to account for resistance, because the substrate of Ldt(fm) does not mimic beta-lactams in contrast to the D-alanyl(4)-D-alanine(5) extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a beta-lactam of the carbapenem class, totally inhibited Ldt(fm) at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt(fm) is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt(fm) by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt(fm) containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt(fm) is likely to involve rupture of the beta-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of beta-lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.

Highlights

␤1– 4-linked N-acetylglucosamine and N-acetylmuramic acid, a linear stem pentapeptide (L-Ala1-D-iGln2-L-Lys3-D-Ala4-DAla5) linked to the lactoyl group of N-acetylmuramic acid by an amide bond, and a side-chain D-isoasparagynyl or D-isoaspartyl (D-iAsx)2 residue linked to the ⑀ amino group of L-Lys3 [1, 2]
␤-Lactam Resistance Profile Resulting from Activation of the LD-Transpeptidation Pathway in E. faecium M512—Because ampicillin was the only ␤-lactam studied in previous reports [14, 16], antibiotic susceptibility testing was performed with 22
These results indicate that the susceptibility of M512 to imipenem is due to inhibition of the cross-linking activity of LD-transpeptidase of Enterococcus faecium (Ldtfm) by this antibiotic

Summary

The abbreviations used are

D-iAsx, D-isoasparagynyl or D-isoaspartyl; PBP, penicillin-binding protein; MIC, minimum inhibitory concentration; rp-HPLC, reversed-phase high-performance liquid chromatography; Ldtfm, LD-transpeptidase from E. faecium. Analysis of the peptidoglycan structure of the resulting mutant, E. faecium M512, indicated that ampicillin resistance was due to target substitution, because the D-Ala43D-iAsx-L-Lys cross-links generated by the DD-. Faecium, Ldtfm, is the first functionally characterized representative of a conserved family of active site cysteine peptidase [16, 17] that include enzymes involved in peptidoglycan cross-linking [15] and in the anchoring of lipoproteins to the peptidoglycan of Escherichia coli [18]. We show that Ldtfm is the target of the carbapenems in vivo and that these drugs inactivate the enzyme by acylation of the catalytic cysteine These results extend the diversity of the targets of ␤-lactams to include active-site cysteine peptidase of the LD-specificity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION