Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium.
The d,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel beta-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting from l,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated with no modification of the level of production of the PBPs or of their affinity for beta-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A beta-lactam-insensitive l,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a beta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys(3) --> d-Asx-Lys(3) cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the l,d-transpeptidase.
Mainardi
Jean-Luc
JL
INSERM EMI-U 0004 Laboratoire de Recherche Moléculaire sur les Antibiotiques, UFR Broussais-Hôtel Dieu, Université Paris VI, 75270 Paris, France. jlmainar@bhdc.jussieu.fr
Morel
Véronique
V
Fourgeaud
Martine
M
Cremniter
Julie
J
Blanot
Didier
D
Legrand
Raymond
R
Frehel
Claude
C
Arthur
Michel
M
Van Heijenoort
Jean
J
Gutmann
Laurent
L
D000409:Alanine / Q000737:chemistry; D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry; D004351:Drug Resistance; D016984:Enterococcus faecium / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008954:Models, Biological; D002267:Muramoylpentapeptide Carboxypeptidase / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D010458:Peptidyl Transferases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013997:Time Factors; D047090:beta-Lactams / Q000378:metabolism
D000900:Anti-Bacterial Agents; D003432:Cross-Linking Reagents; D004151:Dipeptides; D010457:Peptidoglycan; D047090:beta-Lactams; D000667:Ampicillin; D010458:Peptidyl Transferases; D002267:Muramoylpentapeptide Carboxypeptidase; D008239:Lysine; D000409:Alanine