Balance between Two Transpeptidation Mechanisms Determines the
Expression of 
-Lactam Resistance in Enterococcus
faecium*
Jean-Luc
Mainardi
§,
Véronique
Morel,
Martine
Fourgeaud,
Julie
Cremniter,
Didier
Blanot¶,
Raymond
Legrand
,
Claude
Fréhel**,
Michel
Arthur,
Jean
van
Heijenoort¶, and
Laurent
Gutmann
From the INSERM EMI-U 0004 Laboratoire de Recherche
Moléculaire sur les Antibiotiques, UFR Broussais-Hôtel
Dieu, Université Paris VI, 75270 Paris, France, the
¶ Enveloppes Bactériennes et Antibiotiques, UMR 8619, CNRS,
91405 Orsay, France, the Physics Department, Hoechst Marion
Roussel, 93235 Romainville, France, and ** INSERM U-411,
Faculté de Médecine Necker Enfants Malades,
75730 Paris cedex 15, France
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
-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-Ala4 
D-Asx-Lys3) by cross-links resulting from
L,D-transpeptidation (L-Lys3 D-Asx-Lys3). This was associated with no
modification of the level of production of the PBPs or of their
affinity for -lactams, indicating that altered PBP activity was not
required for ampicillin resistance. A -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 -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-Lys3
D-Asx-Lys3 cross-links showed that the
enzyme was activating the resistance pathway by generating the
substrate for the
L,D-transpeptidase.
*
This work was supported by a Medical School Grant
from Merck Sharp and Dohme-Chibret Laboratoires and by National
Institutes of Health Grant R01AI-45626-01.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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