Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the β-lactam imipenem

Abstract

The β-lactam antibiotics mimic the D-alanyl4-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 (Ldtfm) leads to high level resistance to ampicillin. Ldtfm is specific for the L-lysyl3-D-alanine4 bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine5. This specificity was proposed to account for resistance, because the substrate of Ldtfm does not mimic β-lactams in contrast to the D-alanyl4-D-alanine5 extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a β-lactam of the carbapenem class, totally inhibited Ldtfm at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldtfm is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldtfm by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldtfm 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 Ldtfm is likely to involve rupture of the β-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of β-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.