Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem.
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.
Mainardi
Jean-Luc
JL
INSERM, U872, LRMA Pôle 4, Equipe 12 F-75006 Paris, France.
Hugonnet
Jean-Emmanuel
JE
Rusconi
Filippo
F
Fourgeaud
Martine
M
Dubost
Lionel
L
Moumi
Angèle Nguekam
AN
Delfosse
Vanessa
V
Mayer
Claudine
C
Gutmann
Laurent
L
Rice
Louis B
LB
Arthur
Michel
M
D000215:Acylation / Q000187:drug effects; D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000667:Ampicillin / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016984:Enterococcus faecium / Q000201:enzymology* / Q000254:growth & development; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000494:pharmacology; D016908:Gram-Positive Bacterial Infections / Q000188:drug therapy / Q000201:enzymology; D015378:Imipenem / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D013058:Mass Spectrometry; D013379:Substrate Specificity / Q000187:drug effects; D014357:Trypsin / Q000737:chemistry; D047090:beta-Lactams / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004151:Dipeptides; D004792:Enzyme Precursors; D047090:beta-Lactams; D015378:Imipenem; D000667:Ampicillin; D019881:Aminoacyltransferases; C022330:peptidoglycan transpeptidase; D014357:Trypsin