Balance between Two Transpeptidation Mechanisms Determines the Expression of β-Lactam Resistance in Enterococcus faecium

Abstract

The synthesis of bacterial cell wall peptidoglycan is a two-stage process. First, the disaccharide peptide monomer unit is assembled in a series of cytoplasmic and membrane reactions (1). In Enterococcus faecium, the resulting unit is composed of N-acetylglucosamine (GlcNAc) 1 and N-acetylmuramic acid (Fig. 1A). In clinical isolates, acquired high-level resistance to these antibiotics is generally associated with increased production of PBP5 or with amino acid substitutions near the conserved motifs of this protein (9 -13). Recently, we searched for other resistance mechanisms and obtained after five selection steps a highly ampicillin-resistant mutant, designated D344M512, or briefly M512, from the hypersusceptible E. faecium D344S that does not harbor the pbp5 gene. Analysis of the peptidoglycan structure by reverse-phase HPLC (RP-HPLC) coupled to mass spectrometry revealed substitution of D-Ala 4 3

Specificity of L,D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes

“…In E. faecium, D-Asp is added to the precursors by the Asl fm ligase and subsequently partially amidated (12). The bppA1 gene of E. faecalis was cloned under the control of an inducible promoter to generate plasmid pJEH6 and introduced into E. faecium M512 in order to manipulate the structure of the substrate of the cross-linking reaction that can be catalyzed in this mutant by Ldt fm and by the PBPs (28). The BppA1 transferase efficiently competed with the Asl fm ligase in E. faecium M512/ pJEH6(bppA1) because the main monomers contained L-Ala instead of D-iAsp (Fig.…”

Synthesis of Mosaic Peptidoglycan Cross-bridges by Hybrid Peptidoglycan Assembly Pathways in Gram-positive Bacteria

“…Derivatives of expression vector pNJ2 harboring fem and pbp genes (see below) were introduced into E. faecalis JH2Sm::Tn916 (17) by electroporation and transferred by conjugation to E. faecalis JH2-2 (18), E. faecalis JH2-2⌬bppA2 (14), E. faecalis JH2-2⌬pbp5 (17), E. faecium D344S (19), and E. faecium BM4107 (20), as previously described (17). Spectinomycin (60 g/ml) was used in all experiments to counter select loss of the plasmids.…”

“…mecA, pbp5 fs , and pbp5 fm were cloned into the expression vector pNJ2 and introduced into E. faecalis JH2-2⌬pbp5 and E. faecium D344S. The latter hosts are previously characterized mutants susceptible to ␤-lactams because of deletion of their respective species specific pbp5 genes (17,19). Ceftriaxone, a third generation cephalosporin, was used to monitor expression of ␤-lactam resistance, because deletion of the pbp5 genes produced large decreases (Ͼ1000-fold) in the minimal inhibitory concentration of this antibiotic both in E. faecium and E. faecalis.…”

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

“…Ldt fm exclusively uses donor substrate carrying a tetrapeptide stem (16 (14,16). Full elimination of pentapeptide stems ending in D-Ala 5 by this DDcarboxypeptidase leads to high level cross-resistance to a second family of antibiotics, the glycopeptides, that bind to the peptidyl-D-Ala 4 -D-Ala 5 extremity of peptidoglycan precursors (19).…”

“…Acquisition of higher levels of resistance to this drug in clinical isolates results from overproduction of PBP5 fm and from amino acid substitutions in the DD-transpeptidase domain of the protein that further decrease the interaction of the protein with ampicillin (11, 12). We have previously described a novel mechanism of ␤-lactam resistance in a mutant of E. faecium selected in vitro in five consecutive steps on increasing concentrations of ampicillin (13,14 (Fig. 1A).…”